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A study of the biology and ecology of selected predators of phytophagous mites on Florida citrus

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Title:
A study of the biology and ecology of selected predators of phytophagous mites on Florida citrus
Creator:
Villanueva, Raul Timoteo
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English
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xii, 155 leaves : ill. ; 29 cm.

Subjects

Subjects / Keywords:
Eggs ( jstor )
Female animals ( jstor )
Grapefruits ( jstor )
Leaves ( jstor )
Mites ( jstor )
Orange fruits ( jstor )
Orchards ( jstor )
Pollen ( jstor )
Predators ( jstor )
Species ( jstor )
Biological pest control agents ( lcsh )
Citrus -- Diseases and pests -- Biological control ( lcsh )
Dissertations, Academic -- Entomology and Nematology -- UF ( lcsh )
Entomology and Nematology thesis, Ph. D ( lcsh )
Mites -- Biological control ( lcsh )
City of Gainesville ( local )
Genre:
bibliography ( marcgt )
theses ( marcgt )
non-fiction ( marcgt )

Notes

Thesis:
Thesis (Ph. D.)--University of Florida, 2002.
Bibliography:
Includes bibliographical references (leaves 138-154).
General Note:
Printout.
General Note:
Vita.
Statement of Responsibility:
by Raul Timoteo Villanueva.

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A STUDY OF THE BIOLOGY AND ECOLOGY OF SELECTED PREDATORS OF
PHYTOPHAGOUS MITES ON FLORIDA CITRUS












By

RAUL TIMOTEO VILLANUEVA














A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2002



























This thesis is dedicated to my parents Rosa Angele and Rafael M. Villanueva; to my sister Filca; my brothers Luis; Julio; and Rafael; and to Lucia Fabiola because she represents the next generation.





























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ACKNOWLEDGMENTS

This work was completed with the contribution of many people. First I wish to thank my supervisor; Dr. Carl C. Childers for his guidance, suggestions, and financial assistance; and for giving me the opportunity to develop this study with independence. I also want to thank Dr. J.P. Michaud for his friendship, suggestions and for our discussions in biology and ecology, and for letting me use a space in his office. I thank Jose Carlos Rodrigues, because without his persistence the predatory behavior of Cryptothelea gloverii would not have been discovered. Robin Stuart also gave me some profitable suggestions and advice. Belen Belliure and Zenaida Viloria helped me on several occasions and were my confidants. I want to thank people that I met at Lake Alfred. Many became my friends and we shared some good times: Angie Grant, Elisabeth Knapp, John Jiffon, Joaquin Gonzales de la Fuente, Shachindra Mondal, Zeynel Dalkillic and Hugo Aguilar. In Gainesville, Drs. J. Capinera and Maria Gallo-Meagher provided me with lab space to work there. Hector Cabrera was my friend, and colleague, and let me stay at his house many times. I specially thank Mrs. Percivia Mariner for slide mounting many phytoseiid specimens and for her support in maintaining the mite colonies. The following people helped by giving advice or by lending me equipment: Drs. H. Nigg, C. McCoy, J. Syvertsen, G. Albrigo, Pete Timmer. Mrs. Pam Russ was a great support in the library and Ms. Beatriz Nielsen, who provided citrus plants for my experiments. Diann Achor and Gretchen Baut gave me suggestions on taking photographs for this thesis. I offer thanks to my father Rafael Maglorio for his example in life, and to my brothers Lucho, Julio (and his wife Marianna) and Rafa for their unconditional friendship, support and love.



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TABLE OF CONTENTS
page

ACKN OW LEDGM ENTS ........................................... ................................................. ii

LIST O F TA BLES ................................................ ......................................................... v

LIST OF FIGURES ........................................ vii

IN TR O D U C TIO N ................................................ .........................................................1

G eneral Introduction ....................................................................................................... 1
Review of Literature ................................................................................................ 4


PHYTOSEIID DISTRIBUTION ON LEAVES SAMPLED THROUGHOUT THE DAY AND EFFECTS OF LEAF POSITION IN THE CITRUS CANOPY ........................... 14

Introduction ................................................................................................................... 14
M aterials and M ethods......................................... ................................................... 16
R esults............. ....................................................................................................... . 17
D iscussion ....................................................................................................... ............ 19


LEAVES DAMAGED BY THE CITRUS LEAFMINER PROVIDE REFUGES FOR PREDATORY MITES IN CITRUS ...................................................... 32

Introduction................ ............................................................................................. 32
M aterial and M ethods ........................................................................................... 36
R esults............. ....................................................................................................... . 38
D iscussion .......................................................................................................... . 40


COMPARATIVE DEVELOPMENT OF IPHISEIODES QUADRIPILIS (BANKS) (ACARI: PHYTOSEIIDAE) ON VARIOUS NATURAL DIETS AND PREDATION ON ACULOPS PELEKASSI(KEIFER) (ACARI: ERIOPHYIDAE) IN THE LA BO RA TO RY ..........................................................................................................55

Introduction ............................................................................. ................................ 55
M aterials and M ethods................................................................................................ 58
A ssays w ith natural diets................................................................................ . 58
Predation of SBM deutonymphs and females on pink rust mite (PRM)............. 59
R esults............. .............................................. .............. ..................................... . 61
A ssays w ith natural diets................................................................................ . 61
Predation of SBM deutonymphs and females on PRM ..................................... 61
Discussion ................................................................................... 62
EVIDENCE FOR HOST PLANT PREFERENCE IN A PREDACIOUS MITE IPHISEIODES QUADRIPILIS (ACARI: PHYTOSEIIDAE) ........................................... 72


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Introduction ................................................................................................................... 72
M aterials and M ethods......................................... ................................................... 74
Abundance of Iphiseiodes quadripilis on the field ........................................ 74
Direct contact choice experiment on 12 by 2 mm leaf strips .............................. 75
Y- Tube olfactometer .................................................................................. 76
R esults........................................ ............................................................................. 77
Abundance of Iphiseiodes quadripilis in the field ....................................... 77
Direct contact choice experiment on 12 by 2 mm leaf strips........................... 79
Y- Tube olfactometer .................................................................................. 79
D iscussion .................................................. 80


ECOLOGY AND FEEDING BEHAVIOR OF TWO NEW SPECIES OF CECIDOMYIIDAE PREDACIOUS ON RUST MITES AND THE MICROLEPIDOPTERA CRYPTOTHELEA GLOVERII (PSYCHIDAE), A PREDATOR AND PEST ON FLORIDA CITRUS .................................................... 92

Introduction ................................................................................................................... 92
M aterials and M ethods......................................... ................................................... 96
Cecidomyiid Midges Studies ....................................................................... 96
Cecidomyiid collection ....................................................... 96
Rearing of cecidomyiids ...................................................... 96
Predation by cecidomyiids on citrus rust mites. ..............................................97
Orange Bagworm (OBW) Cryptothelea gloverii............................ ........ 97
Mite predation by OBW.................................................... 97
Assessment of OBW damage to orange and grapefruit leaves and fruits......100
R esults............. .................................................................................................... . . 101
Cecidomyiid Midges ........................................ 101
Orange Bagworm ........................................ 102
D iscussion .................................................................................................. . 104
Cecidom yiid M idges ......................................................................................... 104
O range Bagw orm .............................................................................................. 107


SUMMARY AND CONCLUSIONS .......................................................................122

MIXED ANALYSIS OF VARIANCE OF MITES FOUND IN 4 SITES WITH ADJACENT ORCHARDS OF ORANGE AND GRAPEFRUIT ................................1...32

LIST O F REFEREN CES .............................................................................................. 138

BIOGRAPHICAL SKETCH ........................................................................................ 155









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LIST OF TABLES

Table page

Table 2.1 Agricultural practices employed in the Bader grove in Highland City ..................25

Table 2.2. Two-way analysis of variance for data on number of phytoseiids found on the east, inner and west sides of the tree canopy at different sampling times on indicated dates during the year. ....................................................... ................................................26

Table 3.1. Analysis of variance (first seven rows) and least significant difference (LSD) analysis (last four rows) for mean numbers of Eutetranychus banksi eggs on healthy mature, leafminer-damaged and young leaves ................................................46

Table 3.2. Analysis of variance (first seven rows) and least significant difference (LSD) analysis (last four rows) for mean numbers of Eutetranychus banksi motiles on healthy mature, leafminer-damaged and young leaves ................................................47

Table 3.3. Analysis of variance (first seven rows) and least significant difference (LSD) analysis (last four rows) for mean numbers of Eotetranychus sexmaculatus eggs on healthy mature, leafminer-damaged and young leaves. ..................................... ....48

Table 3.4. Analysis of variance (first seven rows) and least significant difference (LSD) analysis (last four rows) for mean numbers of Eotetranychus sexmaculatus motiles on healthy, leafmniner-damaged and young leaves. ...............................................49

Table 3.5. Analysis of variance (first seven rows) and least significant difference (LSD) analysis (last four rows) for mean numbers of citrus rust mite motiles on healthy, leafminer-damaged and young leaves .......................................................50

Table 3.6. Analysis of variance (first seven rows) and least significant difference (LSD) analysis (last four rows) for mean numbers of phytoseiid mite motiles on healthy, leafminer-damaged and young leaves .......................................................51

Table 3.7. Analysis of variance (first seven rows) and least significant difference (LSD) analysis (last four rows) for mean numbers of stigmaeid motiles on healthy, leaf minerdam aged and young leaves ..............................................................................................52

Table 5.1 Total number of Phytoseiids found in 4 sites on 2 sample dates by type of fruit and phytoseiid species. Significant differences calculated with the test of proportions are indicated in each column and row.................................................................................. 83

Table 5.2. Mixed analysis of variance for Iphiseiodes quadripilis numbers found in adjacent rows of grapefruit and orange at 4 sites sampled on two dates each ....................83

Table 5.3. Mixed analysis of variance for the total number of 'other phytoseiids' (phytoseiids other than I. quadripilis) found in adjacent rows of grapefruit and orange at
4 sites sampled on two dates each.................................................................................. 83


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Table 5.4. Mixed analysis of variance for the total number of phytoseiids found in adjacent rows of grapefruit and orange at 4 sites sampled on two dates each ....................84

Table 5.5. Mixed analysis of variance for the total number of Eutetranychus banksi eggs found in adjacent rows of grapefruit and orange at 4 sites sampled on two dates each.........84

Table 5.6. Mixed analysis of variance for the total number of Eutetranychus banksi motiles found in adjacent rows of grapefruit and orange at 4 sites sampled on two dates each. ............................................................................................ .....................................84

Table 5.7. Analysis of variance for the total number of Iphiseiodes quadripilis motiles found in adjacent rows of orange and grapefruit in Block 22 sampled on four dates............85

Table A. 1. Transformed means, standard error of means (SEM), and + 95% confidence interval limits for total numbers of Iphiseiodes quadripilis motiles............................. 133

Table A2. Transformed means, standard error of means (SEM), and � 95% confidence interval limits for total numbers of other phytoseiid motiles (phytoseiids other than I. quadripilis).......................................................................................................................... ..... 134

Table A3. Transformed means, standard error of means (SEM), and � 95% confidence interval limits for total numbers of phytoseiid motiles (. quadripilis + other phytoseiids)...135

Table A.4. Transformed means, standard error of means (SEM), and � 95% confidence interval limits for total numbers of Eutetranychus banksi eggs.................................. 136

Table A.4. Transformed means, standard error of means (SEM), and + 95% confidence interval limits for total numbers of Eutetranychus banksi eggs.................................. 136

Table A.5. Transformed means, standard error of means (SEM), and � 95% confidence interval limits for total numbers of Eutetranychus banksi motiles ....................................137

Table A.5. Transformed means, standard error of means (SEM), and � 95% confidence interval limits for total numbers ofEutetranychus banksi motiles .................................... 137
















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LIST OF FIGURES

Figure pa

Figure 2.1. Daily average relative humidity (%), temperature (o C) and light intensity (Lumens) on 14, 20 and 28 October 1999, 9 and 16 March 2000, and 17 and 24 August 2000 on the east, west and inside areas of the tree canopy at the Bader orchard in H ighland City, Florida. ..................................................... ...............................................27

Figure 2.2. Mean number of phytoseiid motiles � SEM for 14, 20 and 28 October 1999. Values bearing the same letter were not significantly different using the HSD Tukey's test (P < 0.05).........................................................................................................................28

Figure 2.3. Mean number of phytoseiid motiles � SEM for 9 and 16 March 2000. Values bearing the same letter were not significantly different using the HSD Tukey's test (P <
0 .0 5). ......................................................................................................................................2 9

Figure 2.4. Mean number of phytoseiid motiles + SEM for 17 and 24 August 2000. Values bearing the same letter were not significantly different using the HSD Tukey's test (P < 0.05)................ .................. ........ .................................................................... 30

Figure 2.5. Percentage of phytoseiids identified from 30% of the total numbers found on October 1999, March and August 2000. Text box number indicates the total number of phytoseiids identified each m onth.......................................................................................... 31

Figure 3.1. Mean numbers (� SEM) of Eutetranychus banksi eggs on healthy mature, leafminer-damaged and young leaves. Values bearing the same letter were not significantly different in an ANOVA or LSD test (P > 0.05) on a given sampling date. ......46

Figure 3.2. Mean numbers (� SEM) of Eutetranychus banksi motiles on healthy mature, leafminer-damaged and young leaves. Values bearing the same letter were not significantly different in an ANOVA or LSD test (P > 0.05) on a given sampling date. ......47

Figure 3.3. Mean numbers (� SEM) of Eotetranychus sexmaculatus eggs on healthy mature, leafminer-damaged and young leaves. Values bearing the same letter were not significantly different in an ANOVA or LSD test (P > 0.05) on a given sampling date ......48

Figure 3.4. Mean numbers (� SEM) of Eotetranychus sexmaculatus motiles on healthy mature, leafminer-damaged and young leaves. Values bearing the same letter were not significantly different in an ANOVA or LSD test (P > 0.05) on a given sampling date. ......49

Figure 3.5. Mean numbers (� SEM) of citrus rust mite motiles on healthy mature, leafminer-damaged and young leaves. Values bearing the same letter were not significantly different in an ANOVA or LSD test (P > 0.05) on a given sampling date......50

Figure 3.6. Mean numbers (� SEM) of phytoseiid motiles on healthy mature, leafminerdamaged and young leaves. Values bearing the same letter were not significantly different in an ANOVA or LSD test (P > 0.05) on a given sampling date ........................51


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Figure 3.7. Mean numbers (+ SEM) of stigmaeid motiles on healthy mature, leafminerdamaged and young leaves. Values bearing the same letter were not significantly different in an ANOVA or LSD test (P > 0.05) on a given sampling date .........................52

Figure 3.8. Relative abundance of Euseius mesembrinus, Iphiseiodes quadripilis and Typhlodromus. peregrinus on healthy mature, leafminer damaged leaves by Phyllocnistis citrella and young grapefruit leaves on three dates with the highest numbers of phytoseiids registered ...................................................... ................................................53

Figure 3.9. Relationship between phytoseiids (� SEM) and pollen grains (� SEM) found on grapefruit leaves from 2 February to 16March, 2001. .......................................53

Figure 3.10. Temperature and rainfall recorded on the Florida automated weather network CREC-Lake Alfred Station from 31 December 2000 to 20 May 2001.................54

Figure 4.1. Orange fruit arena: design and components. ..................................... .....67

Figure 4.2. PRM (Aculops pelekassi) and CRM (Phyllocoptruta oleivora) main taxonomic differences. PRM: long dorso-lateral setae (lds), rounded dorsum and distinctive dorsal shield. CRM: short dorso-lateral setae (sds), dorsum with trough present and dorsal shield with well defined hexagon (hx)..................................................68

Figure 4.3. Determination of time interval for predation of eriophyids by Iphiseiodes quadrip ilis (SB M ) ......................................................... ..................................................69

Figure 4.4. Duration of female Iphiseiodes quadripilis life stages provided individual diets. Different letter between diets in the same instar indicates significant differences (LSD -test, P < 0.05) ....................................................... ................................................70

Figure 4.5: Consumed and encountered eriophyids by SBM deutonymphs and adult females after starvation. Different letter between the numbers of eriophyids encountered or consumed within the same time of starvation and instar indicates significant differences (t-test, P < 0.05) ...................................................................................................71

Figure 5.2. Y-tube olfactometer used for the choice test. Leaf disks of grapefruit (GF) or orange (OR) ca. 3.5 cm diameter were the sources of plant volatiles.................................86

Figure 5.3. Numbers of I. quadripilis per 10 leaves and other phytoseiids other than I. quadripilis, found on the sample at 4 adjacent orchards of orange and grapefruit on the
(A) first sampling dates and (B) second sampling dates......................................87

Figure 5.4. Total numbers of phytoseiids (Iphiseiodes quadripilis + phytoseiids other than I. quadripilis) found on grapefruit and orange in adjacent rows of grapefruit and orange orchards. No significant differences were found (P >0.05) ...................................................88

Figure 5.5 Numbers of Eutetranychus banksi + SEM on the second sampling dates (A) TCM eggs and, (B) TCM motiles .................................................................................. 89



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Fig. 5.7 Contact choice test of 10 Iphiseiodes quadripilis females for leaf strips of orange or grapefruit. Different letter in each replicate indicates significance (P < 0.05) .................91

Figure. 5.8. Choice test on leaf disks in the olfactometer for Iphiseiodes quadripilis. Each column represents the type of fruit where female I quadripilis were collected. Different letter in each column indicates significance (P < 0.05)..........................................91

Figure 6.1. Arena used to quantify OBW predation of Phyllocoptruta oleivora. The area confined by the transparent plastic cup contained the OBW .................................................14

Figure 6.2. Damage caused by Cryptothelea gloverii larvae feeding on russeted and healthy 'Hamlin' oranges. C. gloverii is more commonly found on russeted fruits ........115

Figure 6.3. Adult Feltiella n. sp. predator of the citrus rust mite Phyllocoptruta oleivora obtained in the laboratory, vertical bars beneath insect are on millimeters .......................1....16

Figure 6.4. Two distinct larval types of two new species of cecidomyiids surrounded by their citrus rust mite Phyllocoptruta oleivora prey. (A). Feltiella n. sp. and (B) a species near Lestodiplosis n. sp. Note ring around the anterior end of the larva........................... 117

Figure 6.5. Mean numbers � SEM of different citrus rust mite (Phyllocoptruta oleivora) stages consumed by larvae of Feltiella n sp. and a species near Lestodiplosis n. sp.............l 18

Figure 6.6. A Cryptothelea gloverii larva preying on the citrus rust mite Phyllocoptruta oleivora .... ............................................................................................................... 119

Figure 6.7. Hole created by Cryptothelea gloverii larvae into the rind of a 'Hamlin' orange. C. gloverii larvae penetrate the rind but do not feed on the fruit sacs. ..................1...19

Figure 6.8. Damage produced by a Cryptothelea gloverii larva on a citrus leaf. C. gloverii can grasp the outer layer of the upper or lower epidermis without penetrating through the leaf and m aking a hole ..................................................................................... 120

Figure 6.9. Penicillium contamination associated with feeding damage caused by Cryptothelea gloverii. The bag of C. gloverii (arrow) is often found on this type of fruit or close by and larval feeding on fungi was observed. ..................................... .....120

Figure 6.10. Winged male Cryptothelea gloverii. Scale beside the microlepidopteran is in m illim eters ...................................................................................................... ............... 121

Figure 7.1. Prey-predator relationships of the citrus-acari-insects system on Florida citrus. Predation is indicated by arrows. Broken lines are not well studied relationships, blue lines are relationships observed in this study. (Green) = Natural enemies, (Red) = Phytophagous, (Light blue) = Undefined roles, (Orange) omnivores, and (Yellow) pollen. The (+) and (-) signs indicate the relationship respect to plants. (?) indicates poorly studied relationship......................................................................................................... 130




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Figure 7.2. Ecological relationships that enhance focusing on phytoseiid mites on citrus. Various relationships were observed in this study. Some were observed in other studies on different crops. Continuous arrows indicate relationships observed in this study. Broken line arrow indicates relationships that require further study. Red line indicates the presence of these organisms on citrus leaves ...................................... 131














































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Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy

A STUDY OF THE BIOLOGY AND ECOLOGY OF SELECTED PREDATORS OF PHYTOPHAGOUS MITES IN FLORIDA CITRUS By

Raul Timoteo Villanueva

April 2002

Chairman: Carl C. Childers
Major Department: Entomology and Nematology


Phytoseiid mites were found in higher numbers on leaves within the canopy of

grapefruit trees than on outer leaves. No differences in phytoseiid abundance were found among leaf samples collected at different hours throughout the day between 0600-2200 h. Mite abundance on grapefruit trees was compared among young-leaves, healthy-mature leaves, and mined-leaves by Phyllocnistis citrella Stainton. Predacious phytoseiids and stigmaeids and Eotetranychus sexmaculatus (Riley) were significantly more abundant on mined leaves than on young or healthy-mature leaves in weekly samples taken between February and April from an unsprayed orchard. Eutetranychus banksi (McGregor), and Phyllocoptruta oleivora (Ashmead) did not differ in abundance among the three types of leaves.

Development and reproduction of Iphiseiodes quadripilis (Banks) was evaluated on diets of Malephora crocea Jacquin and Quercus sp pollens and, E. banksi, Panonychus



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citri (McGregor), and P. oleivora at 280+1 OC, 14:10 (L:D) daylength and 45%-RH. Iphiseiodes quadripilis completed its life cycle and oviposited viable eggs on exclusive diets of M. crocea, Quercus sp. pollens or E. banksi or P. citri eggs and motiles. Panonychus citri stages were acceptable as prey to I. quadripilis but survival of larvae to adulthood was significantly lower than on Quercus sp. pollen. Iphiseiodes quadripilis larvae provided water, or P. oleivora alone, developed to protonymphs and then died. Starved I. quadripilis nymphs and adult females preyed on Aculops pelekassi (Keifer), but not on P. oleivora. Iphiseiodes quadripilis was significantly more abundant on grapefruit leaves than on orange leaves in adjacent citrus orchards at four sites in central Florida. This disparity was consistent whether samples were taken from border or interior trees. Total numbers of phytoseiids were not significantly different between citrus species in most cases. Eutetranychus banksi was significantly more abundant on orange than on grapefruit, suggesting that biological control by I. quadripilis was better on grapefruit trees. Iphiseiodes quadripilis preferred grapefruit leaves over orange leaves in olfactometer experiments and in contact choice tests using leaf discs of the two citrus species.

Cryptothelea gloverii (Packard) (Lepidoptera) was identified as a predator of both P. oleivora and P. citri, although it also feeds on fruit and leaves of citrus. In the laboratory, C. gloverii preferred to feed on oranges russeted by P. oleivora than on fruit without russeting. Feltiella sp. and a Lestodiplosis sp. two undescribed Cecidomyiidae were highly specialized predators feeding on P. oleivora eggs, nymphs, and adults.








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CHAPTER 1
INTRODUCTION

General Introduction



The citrus rust mite (CRM), Phyllocoptruta oleivora (Ashmead), and the pink citrus rust mite (PRM) Aculops pelekassi (Keifer) are the key pests of Florida citrus fruits (Childers and Achor 1999). These species are found in all citrus-growing regions of the world, especially in areas of high humidity. CRM was first recorded in Florida in the early 1880's (Hubbard 1883, Yothers 1914,Yothers and Mason 1930). Citrus rust mites cause direct damage to citrus by feeding on the epidermal cells of leaves, green twigs and fruit (Albrigo et al. 1981, McCoy 1976). Both the CRM and the PRM have probably been present in Florida since the middle 1800's but because of their small size, they were not properly identified until 1961 (Denmark 1963). Russeting of fruit surfaces reduces the value of fruit destined for the fresh market. Direct damage to fruit can also result in increased alcohol or phenol concentrations, reduction of fruit size, increased fruit drop, and increased water loss relative to undamaged fruit (Yothers 1918, Ismail 1971, Allen 1978, 1979, Yang et al. 1995). A positive correlation between CRM damage and the incidence of leaf infection by greasy spot, Mycosphaerella citri Whiteside, has also been observed (van Brussel 1975, Timmer et al. 1980), although both are favored by high humidity.

Acaricides are frequently applied for CRM control and expenditures range from 75100 million dollars annually in Florida alone (Childers 1994a). Use of pesticides to

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control rust mites is a temporary solution because CRM has developed resistance to several compounds such as zineb (Voss 1988, Childers 1994b) and dicofol (Omoto et al. 1995). CRM hormoligosis has been observed in response to various copper formulations (Childers 1994b). Similar phenomena have been reported for different mite pests in diverse cropping systems, i.e. Panonychus ulmi on apple (Villanueva and Harmsen 1998), Tetranychus urticae (Koch) on roses and tree fruits (Goka 1999), and on strawberry and pear (Croft et al. 1984). Other phytophagous mites of importance in the citrus orchard ecosystem of Florida include the Texas citrus mite (TCM), Eutetranychus banksi (McGregor), and the citrus red mite (CReM), Panonychus citri (McGregor). Although the latter species are more sporadic pests than CRM and PRM, in some instances, they require acaricide applications.

One possible way to break this cycle is through the use of selected pesticides that do not cause negative effects on natural enemies. Also, further research on natural enemies is needed to improve understanding of their ecological requirements and optimize their survival to the point where they might reduce CRM and PRM populations below economic levels. Predacious mites in the families Phytoseiidae and Stigmaeidae and certain predatory insects (cecidomyiid midges, neuropteran lacewings, coccinellid ladybeetles and several thrips species) have been identified as natural enemies that feed on citrus rust mites (Hubbard 1883, Muma et al. 1961, Pefia 1992, Childers 1994a). While natural enemies of phytophagous mites in citrus are abundant, few have been well studied and their biology and behavior in relation to eriophyid (CRM and PRM) pests remain largely unknown. In this study, I examined various aspects of selected natural enemies of the eriophyid and tetranychid mites including their distribution, food sources,



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host plant preferences and behavior. The natural enemies studied here included phytoseiid mites and insects (two cecidomyiids and a lepidopteran). The objectives pursued in this study were:

1) To evaluate the distribution of selected phytoseiid mite species between outer

and inner leaves of the tree canopy to determine whether diurnal migration

within the tree occurs (Chapter 2).

2) To compare the numbers of predacious and phytophagous mites between

leaves damaged by Phyllocnistis citrella Stainton (Lepidoptera:

Gracillariidae), healthy mature leaves and young leaves. Also, the

relationship between numbers of phytoseiids and the presence of pollen

grains on citrus leaves was examined (Chapter 3).

3) To evaluate the development and reproduction of Iphiseiodes quadripilis

(Banks) on different diets in the laboratory and determine the acceptability of the citrus rust mite and the pink citrus rust mite (PRM) as food sources

for different I. quadripilis life stages (Chapter 4).

4) To determine if I. quadripilis expresses a preference for grapefruit leaves over

sweet orange leaves. (Chapter 5)

5) To characterize two cecidomyiid midge species and a predatory moth,

Cryptothelea gloverii (Packard) (Lepidoptera: Psychidae), that have been

observed preying upon citrus rust mite and describe their ecological

relationships with pest mites and citrus plants (Chapter 6).







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4


Review of Literature

A diverse fauna of predacious mites inhabit Florida citrus orchards including at least 33 different species of Phytoseiidae (Childers and Denmark, unpublished). Although these mites are important natural enemies of many pests within the citrus ecosystem, their role in controlling phytophagous mites is poorly understood. Many of these predators feed on pollen, some are generalist predators, and other species may have little or no association with pest mites (Muma et al. 1961, McMurtry 1977, Pefia 1992, Childers 1994a). Furthermore, insects such as coccinellids, chrysopids and cecidomyiids have been described as predators of phytophagous mites, further complicating the ecological relationships between pest mites and their predators in the citrus ecosystem.

The economically important tetranychid mites on Florida citrus include the citrus red mite (CReM) P. citri and the Texas citrus mite (TCM) E. banksi. Both TCM and CReM prefer dry conditions and are more abundant in the citrus-growing regions of California and Texas, respectively than in Florida. Both spider mite species can emerge as important pests in Florida from March through June (Childers et al. 1991; Childers 1994a). TCM has a tan to brownish-green color with dark green to black spots on the lateral sides of the body and its eggs are flat and vary from yellow to green in color. All instars of CReM and eggs are reddish in color. Both mites feed on green twigs and fruit and cause stippling on the leaves and mesophyll collapse (Albrigo et al. 1981). When TCM feeding injury follows dry weather conditions in Texas, it may result in leaf drop in the tops of citrus trees (Dean 1980). TCM is presently the most common spider mite on Florida citrus of these two spider mites (Childers et al. 1996).





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CRM and the PRM mite are the most abundant and important eriophyid species on

citrus in Florida (Childers 1994a, Childers and Achor 1999). The citrus bud mite (CBM) Aceria sheldoni (Ewing) also occurs in Florida but is not an important pest (Childers and Achor 1999). In contrast, A. sheldoni is an important pest in California. Denmark (1963) provided a key to separate these three eriophyid mite species. CRM has been reported on Florida citrus since the mid 1800 (Yothers and Mason 1930). Both CRM and PRM are found in all citrus-growing areas of Florida (Childers and Achor 1999).

CRM and PRM are frequently misidentified, partly due to their small size. However, differences in their phenology, oviposition sites, body shape and coloration have been observed. PRM migrates earlier than CRM from leaves to fruit, sometime between April and May (Childers and Achor 1999). CRM eggs are spherical and tend to be deposited in depressions on the fruit surface including the oil glands, whereas PRM eggs are translucent and are deposited indiscriminately on the fruit surface (Childers and Achor 1999). CRM have a convex ophisthosoma (dorsal area) and CRM have a concave ophisthosoma (Denmark 1963). Body coloration is yellow to straw for the CRM and pink to reddish for the PRM, although this character alone is not conclusive for distinguishing the species. CRM and PRM feeding results in direct damage to epidermal cells of leaves, green twigs and russeting on the rind surface of fruit (Allen 1979, Albrigo et al. 1981, McCoy 1996). Heavily damaged fruit are discolored and have a reduced value in the fresh market (Albrigo and McCoy 1974).

The conventional method to control citrus rust mites is with acaricides (Childers et al. 1996). Hubbard (1885) reported the application of sulphur powder to control CRM but this had negative impacts on phytoseiids and other natural enemies. Fungicides used for



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control of pathogens also have been used to control citrus rust mites but resistance to many of these products has developed (Childers 1990). In addition, fungicides have a negative impact on the pathogenic fungus Hirsutella thompsoni Fisher, that attacks citrus rust mites at high population densities (Fisher and Griffiths 1950). Petroleum oils in combination with certain insecticides or acaricides (i.e. abamectin and ethion) also have been used to control rust mites and these mixes improved the residual activity of the acaricides (Childers et al. 1996). However, petroleum oil applications initially were restricted from the mid-summer to mid fall period due to tree and fruit injury factors (Carman 1977). New oil formulations with additives such as oxidation inhibitors prevented phytotoxicity (Kipiani 1976). Today, oil sprays are becoming increasingly important in integrated pest management on Florida citrus (Childers et al. 1996) and other parts of the world: Matsumoto et al. (1981 a, b) in Australia and Japan, Walker and Aitken (1996) in California, Rae et al. in China (2000). Factors such low mammalian toxicity, low cost, and the additive residual activity when combined with certain acaricides, contribute to petroleum oil use. Further CRM has developed resistance to pesticides such as: diflubenzuron (Knapp et al. 1988), zineb (Voss 1988), fenbutatin oxide (Childers 1994b), dicofol (Omoto et al. 1995), fenpropathrin (French and Villarreal 1990). On the other hand, natural enemies (phytoseiid) populations are significantly lower in orchards that received heavy sprays of acaricides, insecticides and fungicides. Pesticides also induced resurgence of CRM populations (Voss 1988, Childers 1994b); due to hormoligosis (Dittrich 1987), and potentially leading to a pesticide treadmill effect. Moreover, costs for chemical control of citrus rust mites are at least 75 million dollars annually in Florida (Childers 1994a) but may be as high as 100 million (McCoy 1996).



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In addition to these pests, there are phytophagous mites that are considered of secondary importance and include the six-spotted mite (6SM), Eotetranychus sexmaculatus (Riley), the two-spotted spider mite (2SSM), Tetranychus urticae Koch, the broad mite, Polyphagotarsonemus latus (Banks) (Tarsonemidae), and a species complex in the genus Brevipalpus (Tenuipalpidae). These pest mites can cause serious localized damage in citrus orchards or in greenhouses on occasion. Special attention should be given to the Brevipalpus species because they transmit citrus leprosis virus in citrus and cause considerable losses in those countries where the disease occurs. The virus does not occur in Florida today (Childers et al. 2001) but it has been previously reported and referred to as nailhead rust or scaly bark in Pinellas County (Fawcett and Lee 1926)

Phytoseiid mites are considered effective biological control agents of spider mites in numerous cropping systems (grapes - Baillod et al. 1982; apples- Childers and Enns 1975, Bostanian and Coulombe 1986; greenhouses - Sabelis 1985a and Brodsgaard and Hansen 1992, De Moraes and McMurtry 1986; strawberry - Easterbrook 1998; cassava Yaninek et al. 1998). There have been several studies on phytoseiid mites on Florida citrus, but the abundance, biology and predatory capacity of several species are inconclusive or unknown. Muma (1964) reported the abundance of phytoseiid mites on citrus leaves in a survey on north, central and south Florida as follows: T peregrinus ( Amblyseius peregrinus) 62.9 %, I. quadripilis (= Amblyseius quadripilis) 15.3 %, Typhlodromips simplicissimus (De Leon) (= Amblyseius simplicissimus) 5.3%, Euseius hibisci (Chant) (= Amblyseius hibisci) 4.5%, Typhlodromina subtropica Muma and Denmark (= T. conspicua) 2.9 %, Galendromus alveolus (= G. floridanus) 2.8 %, and 10 other phytoseiid species represented 6.3 %. Pefia et al. (1989) sampling for predators of



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P. latus in southern Florida lime trees found that T. peregrinus accounted for 72 % of all the predacious mites followed by T. dentilis (15%). Childers (1994a) reported that the most abundant phytoseiid mite on citrus was Euseius mesembrinus (Dean), followed by Typhlodromalus peregrinus (Muma), and Iphiseiodes quadripilis (Banks). The discrepancy between Childers (1994), and Muma (1964) and Pefia et al. (1989) on the abundance of E. mesembrinus and T. peregrinus can be due to sampling location and/or time differences. Also, E. mesembrinus is a relatively new species in Florida, and first reported by McCoy and Rakha in 1985.

Research on E. mesembrinus has demonstrated that it preys on tetranychids and also completes its life cycle on the pollen of various plants (Abou-Setta and Childers 1987, Badii and Hernandez 1993). However, studies using eriophyids as prey have produced unsatisfactory results. Abou-Setta (1988) stated that P. oleivora was not a suitable prey for E. mesembrinus. Badii and Flores (personal communication, Universidad Autonoma de Nuevo Leon, Mexico) could not rear E. mesembrinus on CRM alone, and even when E. mesembrinus was starved for 24 h and then placed in arenas with CRM, predation was not observed (personal observation). Muma (1967a) noted that T. peregrinus reproduces and develops on P. citri, E. sexmaculatus, E. banksi, white fly eggs and Parlatoria pergandei (Comstock) eggs and crawlers. Pefia (1992) showed that adult T. peregrinus did not reject P. oleivora as a prey when it was provided as a food source but favored Polyphagotarsonemus latus when the latter was present. Fouly et al. (1995) reported that T. peregrinus completed its life cycle on diets of T. urticae, P. citri, and pollens of Malephora crocea Jacquin (ice plant), Quercus spp. (oak) and Typha latifolia L. (cattail).





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The shiny button mite (SBM), Iphiseiodes quadripilis is presently the third most

abundant phytoseiid on Florida citrus (Childers and Denmark, unpublished data). The SBM has been described as a facultative generalist predator and it can feed on eriophyids, tetranychids, honey alone, citrus pollen and other less economically important citrus mites (Muma, 1971). However, little is known about the life cycle of SBM, its predatory behavior, or the ecological factors that influence its development. This is the least studied phytoseiid of the three previously discussed. The SBM can be easily recognized with the naked eye and it is distinguishable from other phytoseiid mites by its shiny, dark, mahogany-red color and its round, almost hemispherical body shape as an adult (Muma 1975). SBM can be misidentified with the velvet mite Phytoscutus sexpilis (Muma) in Florida, because the latter has a velvety, rose-red body color and similar body shape. However, under the dissecting stereomicroscoscope, four long slender setae project from the distal end of the body whereas the SBM has only two setae projected backwards (Muma and Denmark 1971).

Stigmaeid mites are predacious on many pest mites but are less abundant than phytoseiids in citrus orchards (Childers 1994a). Muma et al. (1961) described five species of Stigmaeidae, Rakha and McCoy (1984) described an additional species, and recently Ueckerman and Childers discovered two more (personal communication). Agistemusfloridanus Gonzalez is the most common and feeds on P. citri, E. sexmaculatus and Tydeus gloveri (Ashmead) (Tydeidae). Agistemusfloridanus also feeds on CRM and A. floridanus adults were maintained on an exclusive diet of CRM alone by Muma and Selhime (1971). This report, although largely descriptive and without statistical analysis, indicated that A. floridanus had a long generation time and a



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low reproductive rate. Yue and Childers (1994) noted that the stigmaeid A. exertus Gonzales found on citrus had a lower prey consumption rate when compared with the phytoseiid Phytoseiulus persimilis Athias-Henriot. The significance of stigmaeids is discussed here because these predacious mites are important within the citrus acarine system and can prey on pest mites. Their relationship with phytoseiids has not been studied. Stigmaeids are commonly found in the apple orchards of southern Ontario and northeastern New York and the stigmaeid Zetzellia mali (Ewing) contributes to the biological control of the apple rust mite, Aculus schlechdentali Nalepa and the European red mite, Panonychus citri Koch (Villanueva 1997). Also, Z. mali feeds on the eggs of the phytoseiid Typhlodromus caudiglans (Schuster), but phytoseiids were unable to consume Z mali eggs (Clements and Harmsen 1990). MacRae and Croft (1996) found a similar behavior with Z. mali in Oregon although in this case the phytoseiids Metaseiulus occidentalis Nesbitt and Typhlodromus pyri (Schueten) consumed a cumulative mean of < 1 Z mali egg or immature after 6 days. Both phytoseiids and stigmaeids can contribute to pest mite control under high prey densities. The competitive relationship between these two families of predacious mites was described in apple orchards (Clements and Harmsen 1990) and can occur on Florida citrus.

The acarine fauna on citrus orchards in Florida includes a diverse group of species

from different families. Most do not damage citrus and their potential capacity as natural enemies is poorly understood. These mites include different species in the following predacious families: Anystidae, Bdellidae, Eupalopsellidae, Cheyletidae, and Cunaxidae. Also, there are mite species that can be an alternative prey. The latter group includes the





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Tydeidae, Tarsonemidae, and Acaridae (fungus mites). These two groups have not been well studied and their relationships with phytoseiids are unclear.

McMurtry (1992) noted that generalist phytoseiids predominate in citrus in California. These species consume a variety of foods, including some of plant origin such as pollen (McMurtry and Croft 1997). It is possible to hypothesize that the feeding and attacks on CRM by phytoseiids may be lessened due to: 1) the presence of alternative prey that are larger or more abundant than CRM. This alternative prey present on Florida citrus orchards includes tetranychids, tydeids, tarsonemids and fungal mites. 2) The alternative prey also can be a more suitable prey and enhance phytoseiid reproductive capacity; and 3) generalist phytoseiids rather than specialist predators are more abundant in citrus, which was noted by McMurtry (1992) in California. McMurtry and Croft (1997) defined 'generalists' as those species that consume a variety of foods including some of plant origin such as pollen in some perennial plants. Two of the most abundant phytoseiids of Florida, E. mesembrinus and T. peregrinus fall within this category. Either one or all of these factors may contribute to lessen the impact of phytoseiids on eriophyids in Florida citrus.

In addition to the mite and insect predators of phytophagous mites described above, the fungus H. thompsonii was first reported by Yothers and Mason (1930) in association with P. oleivora. Subsequent evaluations of H. thompsonii have been made by several authors (Muma 1955a, McCoy et al. 1971, McCoy and Couch 1982). However, to be effective H. thompsonii requires high CRM population densities and high relative humidity to reach epizootic levels. McCoy (1985) was unable to reduce CRM populations by spraying H. thompsonii formulations.



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Insects also contribute to reducing phytophagous mite populations and four orders of insects are known to prey on CRM on citrus. Hubbard (1883) found a midge larva (Diptera: Cecidomyiidae, formerly Itonididae) feeding on CRM and later Muma et al. (1961) collected Itonidini species within P. oleivora infestations. Yothers and Mason (1930) described Stethorus nanus LeConte (Coleoptera: Coccinelidae) and Chrysopa sp. (Neuroptera: Chysopidae) preying upon CRM. Muma (1958, 1967a; Muma et al. 1961) also mentioned Coniopteryx vicina Hagen (Neuroptera: Coniopterygidae) preying upon CRM. The thrips Scolothrips sexmaculatus (Pergande) and Leptothrips mali (Hunter) (Thysanoptera) have been reported as predators of CRM by Bailey (1939) and Muma (1955b), respectively. This study focuses only on cecidomyiids and Cryptothelea gloverii (Packard) (Lepidoptera; Psychidae) found on citrus and their predacious behavior toward the citrus rust mites.

The genus Feltiella has a worldwide distribution (Gagne 1989) and all described

species form a distinctive group associated exclusively with spider mites (Tetranychidae) Gagne (1995). Feltiella acarisuga (Vallot) has been reported preying on tetranychid mites in various crops such as vegetables (Opit et al. 1997, Gillespie et al. 2000), and strawberries (Croft et al. 1998, Easterbrook 1998, Enkegaard et al. 2000). However, there are few examples of predation by cecidomyiids on eriophyid mites. The eriophyid Aceria litchii Keifer is a serious pest of Litchi chinensis Sonnerat (Sapindaceae) in Australia and China and is preyed on by a cecidomyiid larva Arthrocnodax sp. (Waite and Gerson 1994). Medetera species (Diptera: Dolichopodidae) have been observed preying on Aculus schlechtendali Nalepa on apple in Washington (Rathman et al. 1988). Cecidomyiids have been reported feeding on CRM in California (Hubbard 1883) and



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Florida (Muma et al. 1961) but these reports were vague and the dipteran species were not identified.

The orange bagworm (OBW) Cryptothelea gloverii (Packard) (Lepidoptera: Psychidae) was first described in Florida in 1858 (Hubbard 1885). The larvae of bagworms can be easily recognized by their portable case or bag in which they reside. It is constructed of dense silk and then covered with bits of dry leaves, bark and other scraps (Quayle 1938). The bag has an irregular conical shape and, when the larva is fully grown, it is approximately 1.8 and 1.3 cm long for females and males, respectively (Hubbard, 1885). Hunt (1923) described an outbreak of OBW on orange in Sebring, Florida, and reported damage on fruit, growing twigs, and mature leaves. The OBW was also described as a predator of the camphor scale Pseudaonidia duplex Cockerell, the soft brown scale Coccus hesperidum L, a species of Ceroplastes, and black scale Saissetia olea (Olivier) (Plank and Cressman 1933). No further information on this insect is available on citrus.





















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CHAPTER 2
PHYTOSEIID DISTRIBUTION ON LEAVES SAMPLED THROUGHOUT
THE DAY AND EFFECTS OF LEAF POSITION IN THE CITRUS CANOPY



Introduction

The small size of predatory phytoseiids (and many other mites i.e. stigmaeids) is a factor that makes mite field studies more laborious. In addition, phytoseiids are scarcer than their prey, but they are more active and faster than stigmaeids (Muma and Selhime 1971, Chant 1985). These predators are continuously moving when they are foraging for prey or food (Sabelis 1985b). This foraging behavior, however, may not only depend upon prey availability but also on abiotic factors such relative humidity, temperature and light intensity. Muma (1967b, 1975) observed that T. peregrinus is active at night and prefers shaded leaves during the day. Allen and McCoy (1979) also noticed that the distribution of citrus rust mites on individual fruit and in the whole citrus tree suggests an avoidance of direct sunlight. Villanueva and Childers (unpublished) also noticed analogous responses ofP. citri and E. banksi on citrus. Other researchers studying other species of phytoseiids (C.C. Childers; R. Harmsen; personal communications) have observed similar behavior.

This type of behavior can be described as negative photo-taxis (Fraenkel and Gunn 1961) and may have evolved as a defense mechanism against high temperatures and low humidity on external leaves during daylight hours. Indeed, the large body surface area and small size of these mites results in increased water loss potential when exposed to



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light. Therefore, phytoseiids may find better micro-environmental conditions on internal leaves rather than on outer leaves (Allen and Syvertsen 1981). Time of the day for sampling may influence phytoseiid counts because most of the orchards in Florida are set north-south (Wheaton et al. 1978). This is because phytoseiids may be affected by the direct incidence of sunlight that can increase the temperature of the eastern leaves in the morning and the western leaves in the afternoon. Hence phytoseiids can migrate from the east to the interior leaves in the morning and in the afternoon they may return when these leaves become shaded. Similar movement of phytoseiids may occur on the western side but in the opposite direction.

Temperature and relative humidity in the interior leaves of the tree canopy are more constant compared with conditions on the outer leaves (Ferro et al. 1979) and these factors indubitably affect mite populations. Moreover, we must consider the existence of a boundary layer on fruit or leaf surfaces. The boundary layer is a thin space above the leaf surface up to 1000 um deep, where temperature, and %RH are more constant than the surrounding environment (Allen and Syvertsen 1981). The thickness of the boundary layer decreases with high winds (Gates 1968). This boundary layer is the result of leaf or fruit transpiration and provides unique micro-environmental conditions on the surface with different gradients of temperature and humidity than their surroundings. Small mites such as eriophyids are able to survive and flourish in this highly localized microenvironment. The boundary layer also provides resistance to water vapor diffusion and resistance to heat transfer between a leaf and its surroundings (Martin et al. 1999). The east side of the tree canopy receives direct solar radiation during the morning hours; whereas the west side of the tree canopy is affected in the afternoon. Solar radiation





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would be significantly different in small body sizes and it may be possible that this affects phytoseiid densities depending on how the solar radiation is directed. The aim of this chapter is to determine whether there are any differences in phytoseiid populations on the east, west or interior leaves of the tree canopy depending on time of sampling. In addition, the distribution of phytoseiids within the tree canopy was assessed to determine if time of sampling resulted in phytoseiids density changes.



Materials and Methods

This experiment was conducted in a grapefruit orchard in Highland City, Polk County, Florida. The trees were 20 years old and the orchard was selected because of the occurrence of high numbers of phytoseiid mites based on previous experiments. The trees were minimally sprayed. Four 'Marsh' grapefruit trees were selected, flagged and marked. All trees were in the same row and separated from each other by 8 to 10 trees. Leaves were collected randomly from the tree canopy between I and 2 m. per sample type. Forty leaves were collected from each of the interior, east and west sides of each of four trees. Each 40-leaf sample was placed in an individual labeled jar containing between 0.2 to 0.3 liters of 80% ethanol and taken to the laboratory. In the laboratory, leaves were rinsed in water over a No. 400 sieve with an average opening of 38 um. The residues including mites, small organic particles, soil and spider webbing remaining in the sieve were placed in separate labeled jars with 80% ethanol for each sample. Later all of the phytoseiids found in each jar were sorted and counted using a dissecting stereomicroscope. The phytoseiids from each sample were then placed in a small vial containing 80% ethanol and labeled for eventual slide preparation. Mites were slidemounted in Hoyer's medium (Krantz 1978), oven dried and later identified to species.





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Samples were collected on 14, 20 and 28 October 1999 at 2 h intervals (0600, 0800, 1000, 1200, 1400, 1600, 1800, 200 and 2200 h). Similar types of samples were collected on 9 and 16 March and on 17 and 24 August 2000. However, in the last two months, the leaves were collected at 3 h intervals (0600, 0900, 1200, 1500, 1800 and 2100 h.). All dates were selected when the weather forecast indicated clear and sunny days with low probabilities of rain.

To record environmental conditions, three HOBO@ data loggers (Onset Computer

Corporation, Bourne, MA) were used during each of the sampling dates and placed on the east, west and interior parts of the tree canopy. The HOBO data loggers registered temperature in Celsius degrees, % relative humidity and light intensity in lumens. Phytoseiid data were transformed as J(X + 0.5), where X is the number of phytoseiids found on each 40-leaf sample. A two-way analysis of variance was performed on all data followed by Tukey's Honest Significant Difference (HSD) test for separation of means using Statistica @ (Stat Soft, Inc. 2000). Figures presented show original mean values of data without transformation � standard error of means (SEM).



Results

Cultural practices such as disking, hedging, mowing, application of fertilizers and pesticides for 1999 and 2000 are presented in Table 2.1. Environmental conditions for the sampled dates are presented in Fig. 2.1. Environmental data for August showed that mean %RH inside the tree canopy was 7 to 11% higher than the outside canopy (east and west) %RH. Temperatures were 3.3 to 4.70 C lower inside the tree canopy than outside and the light intensity was 400 lumens lower on the shaded side versus the sunny side of





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the outer canopy. Environmental data for October 1999 and February 2000 showed similar trends (Fig. 2.1). The environmental conditions inside the canopy might be more favorable for phytoseiid development than the outer canopy.

The two-way analyses of variance for all the sampled dates are presented in Table 2.2. Position of leaf sampling (east, west, or inside tree canopy) showed significant differences (P < 0.05) in the number of phytoseiids but effects of sampling time throughout the day and the interaction were not significant on any date (P > 0.05). Grouping the total number of phytoseiids found for all the three dates in October, 1999 yielded 346, 366, and 248 phytoseiids in each of the 1,440 leaf samples. Similarly, for March, 2000 the numbers were 599, 1,964, 613 on the east, inner and west sides, respectively, for each of the 960 leaf samples. For August 2000, the numbers were 69, 435, and 37 on the east, inner and west side of the tree canopy, respectively, for each of the 960 leaf samples. More phytoseiids were collected in the March 2000 samples (mean = 1.10 phytoseiids/leaf) than in October 1999 or August 2000 (mean = 0.16 and mean =

0.19 phytoseiids/leaf, respectively).

No significant differences were obtained for any of the samples in October 1999 using Tukey's honest significant differences (HSD) test (Fig. 2.2). In contrast, samples taken in March 2000 resulted in the highest numbers of phytoseiids collected (Fig 2.3). Samples taken on 16 March at 1200 h had the highest number with 60 + 14.12 (mean number + SEM) phytoseiids /40 leaves. Significantly higher numbers ofphytoseiids (P <

0.05, Tukey's HSD test) were obtained from the inner leaves than from either east or west leaf samples at 1200,1500,1800 and 2100 h on 9 March 2000 and during all the times throughout the day on 16 March (Fig. 2.3) using HSD Tukey's test. The numbers





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of phytoseiids counts in the August samples were very low. However, the trend was similar to the March samples. Most of the predators were found on the inner than outer leaves (Fig. 2.4). Significant differences were found (P < 0.05, Tukey's HSD test) at 0600, 1200, and 2100 h on 17 August; and again at 0600, 1200, 1500 h on 24 August.

The phytoseiid species found in the orchard were: Typhlodromalusperegrinus (n = 870), Iphiseiodes quadripilis (n = 282), Euseius mesembrinus (n = 184), Typhlodromina subtropica Muma and Denmark (n = 21), Typhlodromips dentilis (De Leon) (n = 20), Clavidromus transvaalensis (Nesbitt) (n = 2), Galendromus helveolus (n = 1), Typhlodromalus sp. (n = 1) and Phytoscutus sexpilis (n = 1). Their distributions in the tree canopy are presented in Fig. 2.5. The most abundant species were T. peregrinus (42.1%) and I. quadripilis (50.4%) on October 1999. The most abundant species was T. peregrinus (76.2%) in March 2000 and, E. mesembrinus was the most abundant (54.1% of all identified species) in August 2000.



Discussion

The relative abundance of phytoseiids during the three periods of sampling (October 1999, March 2000 and August 2000) was dissimilar and the cause of this can be explained by the phenology of these mites. Generally phytoseiid species are more abundant from March to May (Childers 1994a). Muma (1967b) referring to T. peregrinus (then the most abundant phytoseiid in Central Florida) stated that its population was larger during late winter and early spring. This coincides with the data presented here and also can be explained by the presence of pollens (Citrus sp., Quercus sp, Pinus sp., and various weed species) that are abundant in March (see Chapter 3).





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No significant differences in abundance of phytoseiids (P>0.05) were obtained for the sampling times throughout the day. This latter result is unexpected because the initial hypothesis for sampling throughout the day was that direct solar radiation on the east sided leaves during the morning would initiate a migration of phytoseiids from the east to the inner leaves. Also, it may be possible that phytoseiids would be foraging on the west side because this direct sunlight increases temperatures and reduces %RH on the east side. Likewise the opposite would happen on the west side; west sided leaves would have fewer mites at mid afternoon than the eastern leaves for similar reasons. This result can be explained by the preference of phytoseiids for the lower leaf surface and the boundary layer of the leaf surface that acts like a buffer producing more stable environmental conditions. In addition, phytoseiids can also rest in crevices and bark of the citrus trunk during times of high solar radiation. Despite the solar radiation the eastsided leaves received during the morning, the environmental conditions between these leaves and the west-sided leaves might be similar within the space enclosed by the boundary layer compared to their surroundings (Gates 1968, Ferro et al. 1999). Still, there are studies that reported phytoseiid differences in sampling times throughout the day in citrus. Garcia Mari et al. (1985) reported that Euseius stipulatus ((Athias-Henriot) was more abundant at night between 2200 and 2400 h than sampling during the daylight on outer leaves of 'Washington' navel in Spain. Muma (1967b) also described a similar nocturnal abundance of T. peregrinus in Central Florida. Garcia Mari et al. (1985) and Muma (1967b) explained this behavior of phytoseiids as a search for prey. Phytoseiids moved from mature inner leaves to the outer young leaves at night because these leaves had more tetranychid prey. McMurtry et al. (1970) explained this behavior as a search





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for shaded areas and Ferragut et al. (1988) explained that phytoseiids preferred trees with abundant foliage. On other plants, phytoseiids also used domatia structures as a refuge site (O'Dowd and Willson 1997, Walter 1996, Roda et al. 2000, Norton et al. 2001). Recently, Onzo et al. (2002 In Press) observed that the phytoseiid Typhlodromalus aripo De Leon rested in domatia structures during the day on cassava and searched for the green cassava mite Mononychellus tanajoa (Bondar) on leaves at night. Domatia do not exist on citrus leaves but phytoseiids use mines created by the leafminer Phyllocnistis citrella Stainton (Gracillariidae) in a similar way to domatia structures since they provided shelter to the predator (see chapter 4).

No significant differences (P > 0.05) were obtained for the interaction between

sampling hours and sampling side (Table 2.2). However, significant differences (P < 0.05) occurred with the two-way ANOVA on the numbers of phytoseiids obtained in sampling position (east, inside or west side of tree canopy) for all dates (Table 2.2), but these differences did not occur (P > 0.05) with the Tukey's HSD post-hoc test of means for all dates sampled on October 1999 (Fig. 2.2). The highest number of phytoseiids found during October was 10.0 + 1.43 (mean � SEM) motiles/40 leaves from samples collected on 20 October at 0800 h (Fig. 2.2). This number is very small (0.25 phytoseiids/leaf) if we extrapolate the number to phytoseiids/leaf. Hence the low number of phytoseiids found during this month produced greater variability between the sampling sides and this reduced the probabilities to observe differences. These lower phytoseiid numbers may be affected negatively by the herbicide glyphosate (Roundup @) and the insecticide chlorpyrifos (Lorsban �) sprayed on 13 September 1999 just one month before the October 1999 sample and the petroleum oil spray on 17 July 2000 one month





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before the August 2000 sample. Chlorpyrifos is relatively non-toxic to E. mesembrinus but has a repellent effect and petroleum oils are moderately toxic to non-toxic to this phytoseiid species (Childers et al. 2001).

The samples collected in March and August 2000 showed that higher numbers of

phytoseiids were present on the inner leaves (Figs. 2.3 and 2.4) than the outer leaves of the tree canopy. Significant differences (P > 0.05, Tukey's HSD test) were found for many of the hours sampled. In March, this is apparent because the numbers of mites were the highest observed (0.6, 2.0, 0.6 phytoseiids/leaf on the east, inner and west side of the tree canopy, respectively). Although phytoseiid numbers in August were lower than in March, the phytoseiids showed a similar trend. More phytoseiids were found in the inner canopy compared to leaves collected from the east and west sides (0.4, 0.07 and

0.03 phytoseiids/leaf, respectively). Phytoseiid numbers on the inner leaves showed considerable variance but were significantly different (P < 0.05) when compared to outer leaf samples mainly because the outer east and west leaves of the tree canopy were very low (Figs. 2.3 and 2.4). This latter observation can be explained as an adaptation of phytoseiid mites to inner leaves that work like a refuge, protecting them from harsher environmental conditions that occur on outer leaves exposed to higher temperatures and lower humidity conditions (Fig. 2.1). Muma (1967b) observed similar results with T. peregrinus. However, he provided only mean numbers without statistical analysis or time of the year that experiments took place.

Although Fig. 2.5 can be misleading because it is expressed in percentages;

numerically the most ubiquitous and abundant phytoseiid was T. peregrinus during the three month sampling periods. Also, in Fig. 2.5 it is evident that both I. quadripilis and





23


T. peregrinus were equally abundant in October 1999, and T. peregrinus was the most abundant in March 2000, whereas E. mesembrinus was the most abundant phytoseiid in August 2000. Muma (1964) observed similar density patterns in the winter and summer months with T. peregrinus. It is possible that the observed densities of E. mesembrinus were due to the mite's phenological cycle. There is not enough evidence to confirm this categorically due to the low numbers observed in the summer months. However, I. quadripilis was found throughout the year (Muma and Denmark 1971) and this corresponds to the results observed here. Childers and Denmark (unpublished) found similar seasonal abundance trends with these species, although they noted (personal communication) that E. mesembrinus was most abundant followed by T. peregrinus and I quadripilis. Their study also was more extensive and intensive. It included 7 orchards in central and south Florida and there were monthly samples taken from 1994 to1996. Data presented here however, coincides with Pefia et al. (1989). They found that T. peregrinus was the most abundant phytoseiid in southern Florida with 72.4 % although the total numbers of predators identified were only 472. The rest of the phytoseiid species identified are found in Florida although they are less common and poorly studied (Muma 1964, Muma and Denmark 1971).

March and August 2000 (Figs. 2.3 and 2.4) data have shown that phytoseiid mites are more abundant on the inner leaves of the tree canopy. However, numbers of phytoseiids obtained in October 1999 (Fig. 2.2) were low, and this may be the reason why significant differences were not observed. Phytoseiid abundance on Florida citrus is generally observed during the months of March to May. This is one reason why the March data resulted in higher phytoseiid numbers and not to the sampling technique employed. This





24


peak of phytoseiid abundance coincides with the pollen season of orange and grapefruit and several trees (oak, pines and various weed species) and with tetranychid mites abundance in Florida. An explanation to the increase in the phytoseiid populations between March and May is the presence of abundant pollen during these months. Evidence of pollen feeding by the three most abundant phytoseiids was presented in studies by Abou-Setta and Childers (1987) on E. mesembrinus, Pefia (1992) on T. peregrinus and in chapter 4 for I. quadripilis. All three species completed their life cycle and reproduced on different pollen diets alone in the laboratory. From results presented here we confirm that sampling time through the day did not affect phytoseiid densities and that there is no evidence that phytoseiids move from the inner leaves to the outer leaves during the night. However, the data presented confirm that phytoseiids prefer inner leaves compared to outer leaves of citrus. This can be due to more adequate microenvironmental conditions (temperatures and relative humidity) in inner than outer leaves.





25



Table 2.1 Agricultural practices employed in the Bader grove in Highland City.

Date Material / Practice Product Name Rate / Hectare

1 Mar 1999 Disking 1 way

24 Mar 1999 Hedging
Herbicide spray Glyphosate 2.8 1 10 May 1999 Herbicide spray Simazine 4.7 1 Insecticide Chlorpyrifos 0.47 1 Fertilizer Techmangam 14.0 Kg 30 May 1999 Zinc 7.0 Kg Insecticide spray Soluble oil 58.4 1 28 Jul 1999 Disking 1 way

31 Jul 1999 Fungicide spray Soluble oil 88.8 1

13 Sep 1999 Herbicide spray Glyphosate 2.8 1 Insecticide spray Chlorpyrifos 0.471 23 Sep 1999 Mowing
Fertilizer Dolomite 2.47 Ton 2 Nov 1999 15-2-15 0.50 Ton Disking 1 way
29 Feb, 2000 Fertilizer 16-0-16 0.61 Ton

9 May 2000 Disking 1 way

30 May 2000 Herbicide spray Glyphosate 2.80 1 Insecticide spray Chlorpyrifos 0.47 1 Fertilizer
1 Jul 2000 Fertilizer 15-2-15 0.61 Ton Disking I way
Insecticide spray Petroleum oil 56.1 1 17 Jul 2000 Fertilizer Techmangam 12.0 Kg Zinc 5.6 Kg
2 Nov 2000 Fertilizer 15-2-15 0.61 Ton

18 Nov 2000 Disking 1 way






26

Table 2.2. Two-way analysis of variance for data on numbers of phytoseiids found on the east, inner and west sides of the tree canopy at different sampling times on indicated dates during the year.
Date Source df MS Effect F value P-value
Side 2, 81 0.754 3.348 0.040 14 Oct 1999 Hours 8, 81 0.628 2.789 0.008
Interaction 16, 81 0.376 1.671 0.069 Side 2, 81 1.066 3.253 0.043 20 Oct 1999 Hours 8, 81 0.401 1.224 0.295
Interaction 16, 81 0.286 0.875 0.598 Side 2, 81 101.009 13.423 < 0.001 28 Oct 1999 Hours 8, 81 8.363 1.111 0.364
Interaction 16, 81 6.1039 0.811 0.669 Side 2, 54 38.616 34.216 < 0.001 9 Mar 2000 Hours 5, 54 1.502 1.330 0.265
Interaction 10, 54 0.882 0.781 0.645 Side 2, 54 99.328 55.948 < 0.001 16 Mar 2000 Hours 5, 54 2.799 1.576 0.182
Interaction 10, 54 1.382 0.778 0.648 Side 2, 54 38.616 34.216 < 0.001 17 Aug 2000 Hours 5, 54 1.502 1.330 0.265
Interaction 10, 54 0.882 0.781 0.645 Side 2, 54 99.328 55.948 < 0.001 24 Aug 2000 Hours 5, 54 2.799 1.576 0.182
Interaction 10,54 1.382 0.778 0.648









27
















100 100 100 Aut2000 80 so 10 10 10
10 __0 70










ousHria Hou
0 n0 60 150 50 50


20 20 20 10 10 10

6:00 9:00 12:00 15:00 18:00 21:00 6800 900 1200 1500 18:00 21:00 6:00 9:00 12:00 150 186:00 21:00









50 50 50



301 30 20 20 09 20 10 10 10 0 0 0----6:00 9:00 12:00 15:00 16:00 21:00 6:00 900 2:00 1500 18:00 21:00 6:00 9:00 12:00 1500 18:00 21:00







Hou Hours Hour


(Lumens) on 14, 20 and 28 October 1999, 9 and 16 March 2000, and 17 and 24 August
700 70 '-'i \

__ S oo 6100 400 600





6:00 9:00 1200 15:00 18:00 21:00 :00 9,00 12:00 10:00 16100 21:00 6:00 9:00 12:00 15&0 16:00 21:00

Hours Hours Hours




Figure 2.1. Daily average relative humidity (%), temperature (0 C) and light intensity (Lumens) on 14, 20 and 28 October 1999, 9 and 16 March 2000, and 17 and 24 August 2000 on the east, west and inside areas of the tree canopy at the Bader orchard in Highland City, Florida.







28





14 Oct 1999 12 - EAST
+ INSIDE +-- WEST
9






3



6:00 9:00 12:00 15:00 18:00 21:00



15
20 Oct 1999

12


0T











6:00 9:00 12:00 15:00 1:00 21:00



Is




















Time Figure 2.2. Mean number of phytoseiid motiles 28SEM for 14, 20 and 28 October 1999. Values bearing the same letter were not significantly different using the HSD Tukey's test (P < 0.05).







29



70
9 Mar 2000

6[ -- EAST m -0- INSIDE
- WEST 40

b a




(a


a a
0
6:00 9:00 12:00 15:00 18:00 21:00




80
16 Mar 2000 70 b


40


ro



30


.20



a a a
0 ~----6:00 9:00 12:00 5:00 100 200
Time

Figure 2.3. Mean number of phytoseiid motiles � SEM for 9 and 16 March 2000. Values bearing the same letter were not significantly different using the HSD Tukey's test (P <
0.05).






30



25
17 Aug 2000 + EAST S--o- INSIDE > -*- WEST o 15




Ia b


S0a






25
24 Aug 2000 2O



5
0











a a
6:00 9:00 12:00 15:00 18:00 21:00

Time

Figure 2.4. Mean number of phytoseiid motiles SEM for 17 and 24 August 2000. Values bearing the same letter were not significantly different using the HSD Tukey's test (P < 0.05). test (P < 0.05).






31






60
S n=228 n n=951 n n=209

50
N T. peregrinus " 40 01. quadripilis
0
E [ E. mesembrinus & 30 i �Other Phyto


0

o




East I Inside West East Inside West East Inside West

Oct-99 Mar-00 I Aug-00


Figure 2.5. Percentage of phytoseiids identified from 30% of the total numbers found on October 1999, March and August 2000. Text box number indicates the total number of phytoseiids identified each month.





32









CHAPTER 3
LEAVES DAMAGED BY THE CITRUS LEAFMINER PROVIDE REFUGES FOR PREDATORY MITES IN CITRUS



Introduction

Topographical or morphological changes on the leaf surface due to feeding and/or damage by pests may produce changes in small arthropod populations including mites. One of the pests that causes change of leaf surfaces is the citrus leafminer (CLM) Phyllocnistis citrella Stainton (Lepidoptera: Gracillariidae). The CLM is a recently introduced pest into the USA. It was reported for the first time in Dade County, Florida in 1993 followed by rapid spread throughout the rest of Florida (Heppner 1993) and neighboring states including: Alabama, Louisiana and Texas in August 1994 (Legaspi et al. 1999). The CLM was initially considered a serious pest in Florida but is currently under control by native natural enemies (Amalin et al. 2001), introduced parasitoids (Hoy and Nguyen 1997) and IPM programs. Today, the CLM is considered a secondary pest problem (Browning et al. 2001). Adult CLM females deposit eggs singly on young citrus foliage (flush) preferring the midrib on the abaxial surface, and occasionally on green twigs and young fruits. Immediately after hatching, the CLM larva bores through the leaf epidermis and forms a mine due to the ingestion of the outer epidermal cells. Feeding by the larva leaves the outer epidermal wall and cuticle intact to form the outer wall of the mine (Sohi and Verma 1965, Achor et al. 1997). Later, chlorotic leaf patches are





33

observed where the larva mined. The mine is serpentine in form and each succeeding larval instar widens the tunnel. Mines caused by CLM larvae prevent young leaves from expanding uniformly with folding and/or twisting of leaves eventually occurring. The third larval instar causes most of the damage and this prepupal larva prepares a pupal chamber by folding the edge of the leaf down or upwards and binding it together with silk (Hoy and Nguyen, 1997). This damage facilitates other insects such as aphids and mealybugs to continue feeding on the leaf and the citrus plant becomes increasingly vulnerable to infection by Asiatic citrus canker caused by Xanthomonas axonopodis pv. citri (Hasse). Large amounts of inoculum are produced in these mined areas that promote the spread of the bacterium by rain splash (Gottwald et al. 1997).

Phylloplane fungi may have better growth in mined leaves than in healthy leaves

(Timmer, personal communication). Phylloplane epiphytic fungi grow externally on a leaf surface without parasitizing it. During the winter months, these fungi can develop better in mined leaves than in healthy leaves due to the sheltered microenvironment provided by the damaged leaves. Most mined leaves maintain their photosynthetic capacity although it is reduced during the remainder of their development. Wang et al. (1999) identified 68 different isolate species of phylloplane epiphytic fungi on citrus in China.

Leaves folded at the edge by third instar CLM are sheltered spaces that provide refugia to other small arthropods. These spaces may provide more favorable microenvironment conditions than leaves without CLM damage. The sheltered spaces resemble structures called domatia that are present on leaves of many tropical and subtropical plants (O'Dowd and Wilson 1989). Domatia are located on the lower surface





34

of the leaves of many plants and occur in many different forms. Domatia are located at the junction of the primary and secondary veins and sometimes are protected by leaf hairs (O'Dowd and Willson 1997, Norton et al. 2001). Domatia are classified in one of the following types: pockets, cavities beneath expanded veins, pits, cavities which sink into the mesophyll, dense hair tuffs; and rolled leaf margins, which are less common (Pemberton and Turner 1989)

The physiological functions of domatia are unknown but it is hypothesized that they have a co-evolutionary development with natural enemies, as a protective association against pests (O'Dowd and Willson 1989, Agrawal 1997, Agrawal and Karban 1997). Various authors have demonstrated that when these structural openings are covered or blocked the populations of predacious phytoseiid mites diminish, while pest mites increase. When artificial domatia were placed on leaves by attaching small pockets on lower leaf surface, phytoseiid mite numbers increased and pest numbers decreased (Walter 1996). A grapefruit leaf or any citrus leaf of commercial varieties is smooth and lacks domatia. In contrast, leaves with domatia often times have a rougher surface with veins protruding and/or hairs occurring around the axes of the veins.

Phytoseiid mites were classified into four categories by McMurtry and Croft (1997): Type I, specialized predators of Tetranychus species represented by Phytoseiulus species; Type II, selective predators of tetranychids mites represented by Galendromus, some Neoseiulus and a few Typhlodromus species; Type 1E generalist predators represented by most Typhlodromus and Amblyseius species; and Type IV specialized pollen feeders/generalist predators represented by Euseius species. The three most prevalent phytoseiids on Florida citrus are: Euseius mesembrinus (Dean), Typhlodromalus





35

peregrinus (Muma) (Childers 1994a) and Iphiseiodes quadripilis (Banks) (See Chapter 2 and Childers and Denmark unpublished). All three species completed their life cycles and reproduced on pollen (Abou-Setta 1987, Pefia 1992, Badii and Hernandez 1993, McMurtry and Croft 1997, see chapter 4 for I. quadripilis). Similar studies have been shown with other phytoseiids in different parts of the world (Castagnoli et al. 1999, van Rijn and Tanigoshi 1999). It is apparent that many of the phytoseiids in Florida belong to either type III and IV species. Furthermore, one of the phytoseiid peaks in Florida coincides with citrus, Quercus sp. and Pinus sp. flowering and the species involved are: E. mesembrinus, T, peregrinus and I. quadripilis (Childers and Abou-Setta, 1999). Quercus sp. and Pinus sp. are commonly found around citrus orchards as wind breaks. Pollen grains from these plants and other species of weeds and shrubs accumulate on the adaxial surface of citrus leaves and almost certainly the three phytoseiids forage for pollen to complement their diet breadth. Studies between the density of pollen grains and phytoseiid abundance were completed in apple orchards in Canada with Typhlodromus pyri Scheuten (Addison et al. 2000), and on citrus in South Africa with Euseius addoensis addoensis (van der Merwe and Ryke) (Grout and Richards 1992a, b). These studies demonstrated a high correlation between early pollen availability and phytoseiid abundance in the field. For convenience and considering that this study was developed during the time of the year with the highest pollen abundance (Citrus sp., Quercus sp. and Pinus sp. flowering occurs by the end of February to middle March); the relationship between pollen grains deposited on the adaxial leaf surface and phytoseiid abundance on leaves was studied.





36

Two main objectives are pursued in this chapter. First, to compare the relative

abundance of phytophagous and predatory mite populations between CLM damaged leaves, mature healthy leaves and young leaves. Second, the relationship between numbers of phytoseiids and pollen grains found on sampled leaves in the same orchard for the duration of this study was assessed.

Material and Methods

Weekly samples were taken from eleven-year-old 'Ruby red' grapefruit trees at the Citrus Research and Education Center in Lake Alfred. All ten sample trees were in one row and selected at random. Samples were taken from 2 February to 13 April 2001 between 1000 and 1100 hours and consisted of terminals with 5 leaves. Terminals were of three types: healthy mature terminals without CLM damage and terminals with leaves that had suffered CLM damage and young terminals with 5 leaves. The first two types of terminals had leaves that flushed the previous year while the third type of terminals were sampled from 23 March through 13 April. These latter terminals started to flush around mid-March 2001. This grove had not received any pesticide sprays since September of the previous year. On 5 April, the grove was sprayed with carbaryl (Sevin@ 4F) at a rate of 94.1 kg/ha in 2.8 Kl of water to control the eastern lubber grasshopper Romalea microptera (Beauvois) (= R. guttata) (Orthoptera: Acrididae). The spray was applied using a handgun directed to the ground and up to I m high on the trees because the flightless grasshopper nymphs concentrated there.

Pollen counts were taken from 2 February to 16 March (during grapefruit flowering in this orchard). One healthy leaf from each of the same 10 trees sampled was collected for pollen counts. In the laboratory, immediately after sampling, a 5 to 7 cm long transparent adhesive tape was placed along the middle vein on the upper leaf surface of each leaf and





37

left there for 3 to 5 minutes, then removed, placed in a wax paper, labeled and stored in the refrigerator until processed. Slides were prepared from 1 cm2 sample areas of the adhesive tape. These 1 cm2 areas of tape were placed individually on the slide with the adhesive part up and then a drop of dye was added followed by placement of the cover slide (Addison et al. 2000). The dye was prepared with 0.2 g of Trypan Blue in 200 ml of 50% glycerol. This dye is used for ascospore staining (Skaria and Tao 1996) and glycerol will cause pollen grains to slightly swell (Addison et al. 2000). A contrast microscope was used in the laboratory to count pollen grains, with a magnification of 400X. The area for counting pollen grains (field of view) was approximately 1.130 mm2. All pollen grains in this field of view were counted and the area within the field of view was calculated with a Hemacytometer Bright Line � (Reichert, Buffalo, NY). Pollen counts were completed as follows. The first grain of pollen found searching the slide was centered in the middle of the field of view and then all the pollen grains in that view were counted.

The three types of terminals were taken to the laboratory and counts of phytophagous and predatory mite eggs and motiles were taken using a stereomicroscope. Data were transformed using X + 0.5 , where X is the number of eggs or motile mites per 5-leaf terminals. ANOVA was used to analyze the data and the means for the last three sample dates were compared using an LSD test (Multiple Range Test for separation of means using Statistica @ (Stat Soft, Inc. 2000). Means � SEM values shown here are untransformed data. Pollen counts and the total number of phytoseiids present from leaf samples on the indicated dates were evaluated to determine correlations between the two factors.





38



Results

Pests mites found in this study included: Eutetranychus banksi (TCM), Eotetranychus sexmaculatus (6-SSM), citrus rust mites (CRM and PRM) and a few Brevipalpus sp., while not a single Panonychus citri was found. The phytoseiids: E. mesembrinus, T. peregrinus and I quadripilis; the stigmaeid Agistemus sp., and a few Stethorus sp. (Coccinellidae) larvae and adults were also found. Stethorus sp. is a predator associated with E. sexmaculatus (Muma et al. 1961). Tydeids mites were also present in this orchard, but their populations were low with no significant differences found among the three types of terminals (P > 0.05).

Tables 3.1 and 3.2 present the ANOVA and the least significant difference (LSD) tests for E. banksi eggs and motiles, respectively. Figures 3.1 and 3.2 present the means (� SEM) for E. banksi eggs and motiles, respectively. Significant differences (P < 0.05) were found in the numbers of E. banksi eggs on 23 March and 13 April, when E. banksi preferred to oviposit on the new young leaves (Fig. 3.1). However, no differences were found for E. banksi motiles on any of the dates (Fig. 3.2).

Tables 3.3 and 3.4 present the ANOVA and the LSD tests for E. sexmaculatus eggs and motiles, respectively. Figures 3.3 and 3.4 present the means (+ SEM) for E. sexmaculatus eggs and motiles, respectively. Significantly (P < 0.01) higher numbers of both E. sexmaculatus eggs and motiles were found on mined leaves than on healthy leaves. Significant differences in mean numbers of E. sexmaculatus eggs were found on 2, 8, 23 and 31 March and 13 April (Fig. 3.3). Significantly higher numbers of E. sexmaculatus motiles were obtained on leafminer leaves than on healthy mature and young leaves on 23 February; 2, 8, 23 and 31 March; and 7 and 13 April (Fig. 3.4).





39

No significant differences (P > 0.05) were found for citrus rust mites on any of the dates sampled (Table 3.5 and Fig. 3.5). During this period, the peak citrus rust mite population occurred in mid February (c.a. 100 RM / 5 leaves on healthy mature leaves and 75 RM / 5 leaves on leafminer damaged leaves). This population decreased to its lowest levels on 9 April (<5 RM / 5 leaves on all three types of leaves). Phytoseiid numbers increased from 8 February until the end of March with the largest numbers of phytoseiids occurring on mined leaves (P < 0.05). Significantly higher numbers of phytoseiids were found on 2 February, 2, 8, 16, 23 and 31 March, and 7 April (Fig. 3.6). The highest numbers of phytoseiids occurred on 23 March with 8.9 � 2.0 (mean + SEM) motiles / 5 leaves on leafminer damaged leaves, whereas for healthy mature leaves there were 1.9 � 0.6 phytoseiid motiles / 5 leaves. Table 3.7 and Fig. 3.7 present the ANOVA and the LSD tests, and means (� SEM) for motile stigmaeids. Although high numbers of stigmaeid predators were found on mined leaves they were not significantly different due to the patchy distribution of these mites with one exception on 23 February (Fig. 3.7). Permanent slides were prepared and identifications were made with most of the phytoseiids found on 16, 23 and 31 March. They were identified as Iphiseiodes quadripilis (n = 139), Typhlodromalusperegrinus (n = 122), Euseius mesembrinus (n = 18), and 1 each of Phytoscutus sexpilis and Galendromus helveolus (Fig. 3.8).

Pollens from different types of plants were found on the grapefruit leaves, including Quercus sp, Pinus sp., citrus, and unrecognized pollen types. There were no differences in the types of pollen found but the total number of pollen grains found on the field of view increased as the plants started flowering. Grapefruit trees in this orchard bloomed between 2 March and 16 March with most of the flowers already shed by 16 March. The





40

correlation between the number of pollen grains in the field of view and phytoseiid mites was positive and highly significant (P = 0.004) using the Pearson correlation coefficient with a R2 = 0.83 (Fig. 3.9) (Statistica, 2000). Temperatures (0 C) and rainfall (cm) records for the duration of this study are presented in Figure 3.9.



Discussion

Numbers of phytophagous mites varied on the three types of leaves sampled. No preference for leaf type was shown by E. banksi, but E. sexmaculatus preferred mined leaves. E. banksi produce little webbing compared to E. sexmaculatus, and as expected, they should have exploited the healthy mature or young leaves. However, on the 23 March and 13 April samples, the numbers of oviposited E. banksi eggs were higher on young flushes than on healthy mature or leafminer damaged leaves. Henderson and Holloway (1942) reported a similar preference of oviposition on new leaves by P. citri. The reason for this was that younger leaves might be more nutritious than older leaves. The younger leaves were also expanding when carbaryl was sprayed on 5 April. The pesticide might not have contacted most of these younger leaves on the sample of 13 April thus new expanding leaves could be used by E. banksi to oviposit eggs on leaves less affected by the pesticide toxicity. It has been reported that carbaryl has little effect on tetranychids but on occasions can stimulate population increases (Dittrich et al. 1974). It is possible that carbaryl has an apparent short negative effect on E. banksi populations because the number of eggs and motiles decreased after the spray (Fig. 3.1 and 3.2) and similarly the physical effect of the hydraulic spray by itself has resulted in temporary reduction of E. banksi. Also, it is likely that the high numbers of E. sexmaculatus eggs were the result of a stimulatory effect by carbaryl.





41

Although E. sexmaculatus is seasonally more abundant following cold winter months and prefers older, harder leaves (Childers, 1994a), the mean numbers of E. sexmaculatus eggs and motiles were significantly more abundant on mined leaves than on healthy mature leaves (Figs. 3.3 and 3.4). As expected, the mined leaves provided increased spaces for webbing for this mite. An important feature here is that we observed the sheltered effect of the mined leaves on E. sexmaculatus. Eotetranychus sexmaculatus had a very moderate decrease in its population after carbaryl was applied but immediately increased by the following sample date (13 April). This increase may not only be due to the protective affect of the mines but also to a hormoligosis effect by the pesticide as explained for many other tetranychid pests. Hormoligosis is described as stimulation by small quantities of a stressor that enhances fertility (Dittrich 1987). Dittrich et al. (1974) demonstrated that the Female:Male ratio on Tetranychus urticae Koch increased significantly after these mites were left on leaves treated with carbaryl.

Phytoseiids and stigmaeids were found in higher numbers on leafminer damaged

leaves than on healthy mature and younger leaves (Figs 3.6 and 3.7). Mean numbers of phytoseiids were significantly (P < 0.05) different, with numbers ranging from 2 to 5 times greater on leafminer damaged leaves versus healthy mature leaves. Mined leaves not only provided protective spaces for mites but also more prey and other types of food. (Villanueva and Harmsen 1996, Villanueva 1997) observed similar effects on mines produced by the spotted tentiform leafminer Phyllonorycter blancardella Fabricius (Gracillariidae) on apple in Ontario. The type of mines formed on apple was different from CLM mines. Both stigmaeids (Zetzellia mali (Ewing)) and phytoseiids (Typhlodromus caudiglans Schuster and Neoseiulusfallacis (Garman)) were found on old





42

mines on apple feeding on Panonychus ulmi and tarsonemids (Villanueva and Harmsen 1996, 1998). The tarsonemids oviposited higher numbers of eggs around the frass and exuviae inside the mines on apple. Frass and exuviae are also found in mines produced by CLM and unpublished observations by Villanueva (unpublished) in the laboratory showed that I. quadripilis and E. mesembrinus feed on phylloplane fungi that grow more abundantly on mined leaves than on healthy leaves.

Here phylloplane is used in reference to a leaf as a habitat for microorganisms. More importantly, epiphytic fungi had recently been studied for their antagonistic effects to pathogenic fungal diseases such sooty mold on mandarin (Srivastava and Thakre 2000) and citrus canker on lemon (Pabitra et al. 1996). Theoretical approaches to the use of entomopathogens as "body guards" of plants have been formulated and discussed recently (Elliot et al. 2000). Hargreaves and Parappukkaran (2000) noted that most common fungi occurring outdoors are those that live on the surfaces of leaves (phylloplane fungi). A few genera, Cladosporium, Alternaria, Epicoccum and Aureobasidium comprise between 40% and 80% of the propagules in the atmosphere in surveys worldwide. Fateh et al. (1995) noted that species of epiphytic fungi varied depending on season, age of leaves and plant variety on grape. In the acarine system, Belczewski and Harmsen (1997) found that sprays of the phylloplane fungus Alternaria alternata Keissler on apple leaves in the field enhanced population growth of Tetranychus urticae compared to that of Panonychus ulmi. Whereas Zemec and Prenerova (1997) observed that T. pyri fed on the conidia of powdery mildew species Erysiphe orontii Castellani from tobacco and Oidiumfragariae Harz from strawberry in the laboratory. Typhlodromus pyri reproduced and completed its life cycle on E. orontii.





43

The relative abundance of the phytoseiids I. quadripilis and T. peregrinus is shown in Fig. 3.8 on the three types of leaves sampled on 16, 23 and 31 March. These three dates had the highest phytoseiid numbers. Iphiseiodes quadripilis and T. peregrinus combined for more than 90% of the total number of phytoseiids found in this study. This coincides with Childers and Denmark survey (unpublished) where T. peregrinus and I quadripilis are the second and third most abundant phytoseiids in seven central Florida orchards. However, E. mesembrinus was the most abundant phytoseiid in their survey but was not abundant in this study. Iphiseiodes quadripilis and T. peregrinus are generalist predacious mites (Muma 1961) and the former shows preference for grapefruit over orange (see chapter 5). The biology and behavior of T. peregrinus have been studied more than I. quadripilis. Typhlodromus peregrinus fed on pollen, P. latus, P. oleivora and tetranychids (Pefia 1989). It may be reasonable to think that E. mesembrinus was not abundant due to its phenology and may be less fit for the exploitation of mined leaves than I quadripilis and T. peregrinus. Stigmaeids were found regularly during the duration of this study, but their distribution was localized. Hence variability is observed in the high SEM values obtained (Fig. 3.7). Similar observations in patchy distribution of A. floridanus were made by Muma and Selhime (1971).

Both phytoseiid numbers and pollen grains increased between 2 February and 16 March. Similar observations are reported on pollen abundance and increases of phytoseiid populations in many crops. Addison et al. (2000) suggested that T. pyri abundance had a positive correlation with early season pollen density than with the abundance of the mite prey (Aculus schlechtendali Nalepa) in apples. Similarly, when Euseius tularensis Congdon was released into navel orange groves, the phytoseiid





44


exhibited a significantly greater increase in groves with a ground cover crop of mixed leguminous plants over a 4-week period than in groves without ground cover crop in California (Grafton-Cardwell et al. 1999). The results shown here had similar responses and the high correlation coefficient obtained (r = 0.91) may explain I. quadripilis and T. peregrinus abundance during citrus flowering (Fig. 3.9).

This chapter describes two ecological components that affect phytoseiid populations on citrus: the mine caused by P. citrella and pollen deposited on the leaves. The mine provides phytoseiids shelter and food. Phytoseiids utilize mines as refugia where they can increase more than on healthy leaves due to better micro-environmental conditions. The phylloplane fungi developing within the mines and the leafminer frass are also utilized as food sources. This can be important especially during the winter months when prey availability is scarce. Pollen is a proven food source of phytoseiids and pollens of Quercus sp, Pinus sp. and Citrus sp. coincided with increased phytoseiid numbers in the field. Both I quadripilis and T. peregrinus are generalist phytoseiids mites that complete their life cycles and reproduced on pollen diets in the laboratory (Muma 1967b and 1971, Pefia 1992, see Chapter 4). The citrus leafminer, P. citrella is not considered a beneficial insect but changes in leaf morphology produced by this pest are used positively by phytoseiids and thus augment pest mite control. A practical application of this research is that sampling on mined leaves alone will provide increased phytoseiid numbers. However, it also may be possible that only one or more predatory species such I. quadripilis and T. peregrinus exploit this type of habitat while other phytoseiid species avoid it. This study only focused on acarine populations but insects such thrips, psyllids, curculionids, hemipterans and arachnids were observed more frequently on mined leaves





45


than on healthy mature leaves. More research is needed because the mines can affect beneficial arthropod numbers not only on citrus but in other plants too.





46



Table 3.1. Analysis of variance (first seven rows) and least significant difference (LSD) analysis (last four rows) for mean numbers of Eutetranychus banksi eggs on healthy mature, leafminer-damaged and young leaves.
Date df F value P LSD test
02-FEB 1, 18 08-Feb 1, 18 1.44 0.24 16-Feb 1, 18 0.35 0.56 23-Feb 1, 18 0 1 02-Mar 1, 18 0.16 0.69 08-Mar 1, 18 0.26 0.61 16-Mar 1, 18 0.21 0.64 23-Mar 2, 27 4.95 0.01 * 31-Mar 2, 27 2.10 0.14 n.s. 07-Apr 2, 27 4.41 0.02 * 13-Apr 2, 27 5.23 0.01 ** One asterisk (*) means that there are significant differences (P < 0.05) and two asterisks means that differences are highly significant (P < 0.01).
45
40 -r Healthy L. - - -
LM-Leaves
35 1--I Young L.
b
0 30
> b
"25

F 20




~~ a
29-Jan 8-Feb 18-Feb 28-Feb 10-Mar 20-Mar 30-Mar 9-Apr 19-Apr Date
Figure 3.1. Mean numbers (� SEM) of Eutetranychus banksi eggs on healthy mature, leafminer-damaged and young leaves. Values bearing the same letter were not significantly different in an ANOVA or LSD test (P > 0.05) on a given sampling date.





47




Table 3.2. Analysis of variance (first seven rows) and least significant difference (LSD) analysis (last four rows) for mean numbers of Eutetranychus banksi motiles on healthy mature, leafminer-damaged and young leaves.
Date d.f. F value P LSD test 02-Feb 1, 18
08-Feb 1, 18 0.19 0.66 16-Feb 1, 18 1.20 0.28 23-Feb 1, 18 0.91 0.35 02-Mar 1, 18 0.02 0.86 08-Mar 1, 18 0.003 0.95 16-Mar 1, 18 0.12 0.72 23-Mar 2, 27 1.63 0.21 n.s. 31-Mar 2, 27 2.15 0.13 n.s. 07-Apr 2, 27 0.53 0.58 n.s. 13-Apr 2, 27 1.57 0.22 n.s. One asterisk (*) means that there are significant differences (P < 0.05) and two asterisks means that differences are highly significant (P < 0.01).

30

25 --&-Healthy L.
LM-Leaves
g 20

15
0 E


5 1


29-Jan 8-Feb 18-Feb 28-Feb 10-Mar 20-Mar 30-Mar 9-Apr 19-Apr Date
Figure 3.2. Mean numbers (� SEM) of Eutetranychus banksi motiles on healthy mature, leafminer-damaged and young leaves. Values bearing the same letter were not significantly different in an ANOVA or LSD test (P > 0.05) on a given sampling date.





48


Table 3.3. Analysis of variance (first seven rows) and least significant difference (LSD) analysis (last four rows) for mean numbers of Eotetranychus sexmaculatus eggs on healthy mature, leafiminer-damaged and young leaves.
Date d.f. F value P LSD test 02-Feb 1, 18
08-Feb 1, 18 1.21 0.28 16-Feb 1, 18 1.40 0.25 23-Feb 1, 18 4.7 0.04 02-Mar 1, 18 5.63 0.02 08-Mar 1, 18 9.27 0.006 16-Mar 1, 18 0.01 0.92 23-Mar 2, 27 5.53 0.009 ** 31-Mar 2, 27 6.78 0.004 ** 07-Apr 2, 27 2.87 0.07 n.s. 13-Apr 2, 27 7.68 0.002 ** One asterisk (*) means that there are significant differences (P < 0.05) and two asterisks means that differences are highly significant (P < 0.01).
100
90 Healthy L. -
80 -- ___ -- LM-Leaves
-o- Young L.
70








29-Jan 8-Feb 18-Feb 28-Feb 10-Mar 20-Mar 30-Mar 9-Apr 19-Apr Date
Figure 3.3. Mean numbers (+ SEM) of Eotetranychus sexmaculatus eggs on healthy mature, leafminer-damaged and young leaves. Values bearing the same letter were not significantly different in an ANOVA or LSD test (P > 0.05) on a given sampling date.





49


Table 3.4. Analysis of variance (first seven rows) and least significant difference (LSD) analysis (last four rows) for mean numbers of Eotetranychus sexmaculatus motiles on healthy, leafminer-damaged and young leaves.
Date d.f. F value P LSD test 02-Feb 1, 18 0.79 0.38 08-Feb 1, 18 0.90 0.35 16-Feb 1, 18 1.53 0.23 23-Feb 1, 18 5.58 0.02 02-Mar 1, 18 4.83 0.04 08-Mar 1, 18 7.05 0.01 16-Mar 1, 18 0.54 0.46 23-Mar 2, 27 6.12 0.006 ** 31-Mar 2, 27 8.93 < 0.001 ** 07-Apr 2, 27 8.44 < 0.001 ** 13-Apr 2, 27 7.65 0.002 ** One asterisk (*) means that there are significant differences (P < 0.05) and two asterisks means that differences are highly significant (P < 0.01).

45
0LM-Leaves

J--~

b






29-Jan 8-Feb 18-Feb 28-Feb 10-Mar 20-Mar 30-Mar 9-Apr 19-Apr Date

Figure 3.4. Mean numbers (d SEM) of Eotetranychus sexmaculatus motiles on healthy mature, leafminer-damaged and young leaves. Values bearing the same letter were not significantly different in an ANOVA or LSD test (P > 0.05) on a given sampling date.





50



Table 3.5. Analysis of variance (first seven rows) and least significant difference (LSD) analysis (last four rows) for mean numbers of citrus rust mite motiles on healthy, leafminer-damaged and young leaves.
Date d.f. F value P LSD test 02-Feb 1, 18
08-Feb 1, 18 0.167 0.68 16-Feb 1, 18 3.35 0.08 23-Feb 1, 18 0.01 0.90 02-Mar 1, 18 0.86 0.36 08-Mar 1, 18 0.133 0.71 16-Mar 1, 18 2.52 0.12 23-Mar 2, 27 2.34 0.11 n.s. 31-Mar 2, 27 1.32 0.28 n.s. 07-Apr 2, 27 1.06 0.35 n.s. 13-Apr 2, 27 1.39 0.26 n.s. One asterisk (*) means that there are significant differences (P < 0.05) and two asterisks means that differences are highly significant (P < 0.01).

120

100 1 ---.. ..--- -.. .Hea.lthy L
-4-LM-Leaves








29-Jan 8-Feb 18-Feb 28-Feb 10-Mar 20-Mar 30-Mar 9-Apr 19-Apr Date

Figure 3.5. Mean numbers (� SEM) of citrus rust mite motiles on healthy mature, leafminer-damaged and young leaves. Values bearing the same letter were not significantly different in an ANOVA or LSD test (P > 0.05) on a given sampling date.





51


Table 3.6. Analysis of variance (first seven rows) and least significant difference (LSD) analysis (last four rows) for mean numbers of phytoseiid mite motiles on healthy, leafminer-damaged and young leaves.
Date d.f. F value P LSD test 02-Feb 1, 18 5 0.03 08-Feb 1, 18 0 1 16-Feb 1, 18 1.14 0.29 23-Feb 1, 18 3.09 0.09 02-Mar 1, 18 5.26 0.03 08-Mar 1, 18 4.47 0.04 16-Mar 1, 18 28.78 < 0.01 23-Mar 2, 27 14.91 < 0.01 ** 31-Mar 2, 27 17.32 < 0.01 ** 07-Apr 2,27 3.84 0.03 * 13-Apr 2,27 2.39 0.11 n.s. One asterisk (*) means that there are significant differences (P < 0.05) and two asterisks means that differences are highly significant (P < 0.01) on the LSD test.

SH eathy L.
10 LM-Leaves
damaged and young leaves. Values bearing the same letter were n Youn L.




different in an ANOVA or LSD test (P > 0.05




29-Jan a 8-Feb 18-Feb 28-Feb 10-Mar 20-Ma 30-Mar 9-Apr 19-Apr

Figure 3.6. Mean numbers (� SEM) of phytoseiid motiles on healthy mature, leafminerdamaged and young leaves. Values bearing the same letter were not significantly different in an ANOVA or LSD test (P > 0.05) on a given sampling date.





52



Table 3.7. Analysis of variance (first seven rows) and least significant difference (LSD) analysis (last four rows) for mean numbers of stigmaeid motiles on healthy, leaf minerdamaged and young leaves.
Date d.f. MSE F value P LSD test
02-Feb N/A N/A N/A N/A
08-Feb 1, 18 16.2 0.89 0.35 16-Feb 1, 18 3.2 3.64 0.07 23-Feb 1, 18 57.8 5.41 0.03 02-Mar 1, 18 80 1.26 0.27 08-Mar 1, 18 64.8 3.38 0.08 16-Mar 1, 18 28.8 3.62 0.07
23-Mar 2, 27 1.48 2.43 0.10 n.s.
31-Mar 2,27 0.79 1.17 0.32 n.s.
07-Apr 2,27 0.19 2.20 0.12 n.s.
13-Apr 2, 27 0.42 1.65 0.20 n.s.
One asterisk (*) means that there are significant differences (P < 0.05) and two asterisks means that differences are highly significant (P < 0.01).
9
8 10 Healthy L, UPLM-Leaves

6








29-Jan 8-Feb 18-Feb 28-Feb 10-Mar 20-Mar 30-Mar 9-Apr 19-Apr Date

Figure 3.7. Mean numbers (+ SEM) of stigmaeid motiles on healthy mature, leafminerdamaged and young leaves. Values bearing the same letter were not significantly different in an ANOVA or LSD test (P > 0.05) on a given sampling date.






53

f~ ~ ~ ~ ~ ~ ~ ~ ~~~~~~~~~~..... ........................ ...................................... ..... -......

90
O Emesembrinus 80- I. quadipilis 70- T peregrinus

60
o_16 Mar 23 Mar --- 31 Mar


o 40



Z 20-/
-a
0


10


Healthy LM- Young Healthy LM- Young Healthy LM- Young Leaves Leaves Leaves Leaves Leaves Leaves Leaves Leaves Leaves


Figure 3.8. Relative abundance of Euseius mesembrinus, Iphiseiodes quadripilis and Typhlodromus. peregrinus on healthy mature, leafminer damaged leaves by Phyllocnistis citrella and young grapefruit leaves on three dates with the highest numbers of phytoseiids registered.


12

10 y = 0.4707x - 2.5621
* R = 0.834
8 P = 0.004



0r)
S4

2 0
0 5 10 15 20 25 30 Pollen grains / 1.130 mm2

Figure 3.9. Relationship between phytoseiids (+ SEM) and pollen grains (+ SEM) found on grapefruit leaves from 2 February to 16 March 2001.





54




30 - 6 25- 5









10 - Rainfall 2 50 0.-- r---r ----- ---- ---------- --- -- ------ - -- --- --- -t -I -- ---- ------ 4-31-Dec-00 20-Jan-01 9-Feb-01 1-Mar-01 21-Mar-01 10-Apr-01 30-Apr-01 20-May-01


Figure 3.10. Temperature and rainfall recorded on the Florida automated weather network CREC-Lake Alfred Station from 31 December 2000 to 20 May 2001.













CHAPTER 4
COMPARATIVE DEVELOPMENT OF IPHISEIODES QUADRIPILIS (BANKS)
(ACARI: PHYTOSEIIDAE) ON VARIOUS NATURAL DIETS AND PREDATION
ON ACULOPS PELEKASSI (KEIFER) (ACARI: ERIOPHYIDAE) IN THE LABORATORY



Introduction

Phytoseiid mites are considered effective biological control agents on numerous crop systems (Baillod et al. 1982; Bostanian and Coulombe 1986; Brodsgaard and Hansen 1992; De Moraes and McMurtry 1985; and Yaninek et al. 1998). They prey on phytophagous mites in Florida citrus orchards but their potential use in controlling mite pests in orchards under IPM regimes is not well known. Muma (1975) found 19 species of phytoseiids associated with citrus trees but this number is probably low. One of the more abundant phytoseiid mites on Florida citrus is the Shiny button mite (SBM) Iphiseiodes quadripilis (Banks) and it is considered the third most abundant phytoseiid today (Childers and Denmark, unpublished data). The SBM has been described as a general facultative predator that can feed on eriophyids, tetranychids and honey + citrus pollen and other lesser economically important citrus mites (Muma 1971). However, little is known about the life cycle of SBM, its predatory behavior, or the ecological factors that influence its development. The SBM can be easily observed visually in the field by the naked eye and it is distinguishable from most other phytoseiid mites by its shiny, dark, mahogany-red color. The body shape of adults is almost round and swollen to a nearly hemispheric profile (Muma 1975).



55






56

The citrus rust mite (CRM) Phyllocoptruta oleivora (Ashmead) (Eriophyidae) is one of the key pests on Florida citrus and is found in all citrus-growing areas of the world. The CRM can cause considerable economic losses in the citrus industry due to direct feeding damage on green twigs, leaves and fruits and the high cost of acaricides for its control. Heavy infestations produce fruit russeting that decreases fruit quality (Childers 1994a). Pink citrus rust mite (PRM) Aculops pelekassi (Keifer) is another eriophyid found in Florida and is generally misidentified with CRM due to their small size. Although CRM has a yellowish color that is different from the pinkish coloration of PRM, this is distinguishable only at higher magnifications using a dissecting stereomicroscope. Even then coloration is not a clear indicator for species recognition and reliable identification requires slide mounting of specimens (Childers and Achor 1999).

Tetranychidae are common pests on citrus and the two most common species are the Texas citrus mite (TCM) Eutetranychus banksi (McGregor) and citrus red mite (CReM) Panonychus citri (McGregor) (Childers et al. 1991, Childers 1994a). These mites prefer dry conditions and are common in citrus areas in Texas and California, respectively. They are important pests in Florida from March through June or during other extended dry periods throughout the year (Childers 1994a). Both mites cause stippling on the leaves, mesophyll collapse at high densities and can feed on green twigs and fruits (Albrigo et al. 1981). On occasion, leaf drop in the upper portions of trees is associated with TCM feeding injury following dry weather conditions in Texas (Dean 1980)

Pollen can be an important alternative or supplementary component of diet for many predacious mites in the family Phytoseiidae (Abou-Setta and Childers 1987, Badii and





57

Hernandez 1993, Fouly et al. 1995, McMurtry and Croft 1997, Yamamoto 1994). Pollen is found in Florida throughout the year from wild flowers, various annual and perennial flowering plants, shrubs and ground cover plants and weeds. However, the most abundant period of tree pollen release occurs from late February to May when Quercus spp. (oak) (Fagaceae), Pinus elliottii Engelman, P. palustris Miller (Pinaceae), Casuarina equisetifolia J.R. Forsts and G. Forst, C. cunninghamiana Miquel (Casuarinaceae), Taxodium distichum (L.) (Taxodiaeae), Juniperus silicicola (Small) Bailey (Cupressaceae), Myrica cerifera L. (Miricaceae), Broussonetia papyrifera (L.) Vent (Moraceae), and Morus rubra L. (Moraceae) are blooming (Bucholtz et al. 1991). Citrus trees also bloom during this period. Oak trees produce profuse amounts of pollen during flowering and oak pollen is dispersed by wind and deposited on citrus leaves during this period. Alternatively, pollen of ice plant (Malephora crocea Jacquin, family: Aizoaceae) is often used to maintain phytoseiids in laboratory colonies and is sold commercially. Pollen has not been evaluated as a dietary component for SBM. It is not known which life stages feed on pollen or complete development and oviposition with a diet of pollen alone.

The objective of this study was to determine if I. quadripilis can complete its life cycle and oviposit successfully on diets of CRM, CReM, TCM and oak or ice plant pollens in the laboratory. Also, the acceptability of CRM and PRM as food sources to different I. quadripilis life stages was assessed.





58


Materials and Methods


Assays with natural diets

Reproductive SBM females were collected in the field and placed on leaf arenas in the laboratory with eggs and motile stages of CReM and ice plant pollen as food sources. SBM eggs were collected and placed individually onto arenas containing the different diets. Three species of prey mites were used in this experiment: CRM, CReM and TCM, and two types of pollen: ice plant and oak, and a separate treatment of water alone was included. Three types of arenas were employed. The first type was used to test SBM development without food and consisted of a square piece of ceramic tile approximately

5.0 to 7.0 cm in length per side, 1.0 cm thick and surrounded by a wet strip of cotton. The piece of tile was placed in a 15.0 cm diam. x 2.5 cm deep Petri dish filled with water to avoid mite escape and to maintain the desired moisture level on the cotton strips. The tile was beige in color, had a glazed-matte finish, and this surface was used for the arena. Each tile was divided into 16 squares (arenas) using thin wet cotton strips. SBM mites were introduced individually using a 5-0' sable brush.

The second type of arena (Fig. 4.1) was used to test the diet of SBM on CRM only and was prepared on an orange fruit c.a. 4.0 to 6.0 cm diam. Each fruit was placed on a PVC pipe (3.5 cm diam. x 1.5 cm tall) and then placed in a Petri dish (8.5 cm diam. by 1.5 cm deep) half filled with water to maintain moisture on the cotton strips during the duration of the experiment. Each fruit had a circular experimental area approximately 3.5 to 4.0 cm diameter on the upper part of the fruit that was surrounded with a 1.5 cm wide wet cotton strip to confine the mites. The orange fruits were chosen for the high numbers of eriophyid mites each contained (> 200 mites /cm2). All insects and mites other than the





59

citrus rust mites were removed. Most of the eriophyids in each arena were CRM based on the yellowish color of their bodies. One arena of this type was used for each SBM egg.

The third type of arena contained a grapefruit leaf placed in a 15.0 cm diam. x 2.5 cm deep plastic Petri dish. The leaf was placed with its upper surface facing up on the top of a wet foam pad. This arena was used to test the diet of CReM, TCM, or the two types of pollen. Wet cotton strips completely surrounded each leaf to confine the mites. Leaves used to prepare these arenas were cleaned with a solution of 5% sodium hypochlorite (bleach) to remove contaminants. The leaf arenas for the diets using CReM were divided into quarters using wet cotton strips. Within each arena, five female CReM were left for 3 days to lay eggs before placement of the SBM eggs per arena. Similar steps were made for the TCM diet. Arenas for the pollen diet were divided similarly and pollen was added at the same time with one SBM egg. More female CReM, TCM, or one of the two types of pollen were added to each respective arena to maintain development of the SBM during the duration of the experiment. The leaf arenas were replaced periodically before they started to deteriorate and leaves used for this purpose followed similar cleaning and preparation steps as described above.

The mean developmental times of the different SBM stadia (egg, larva, nymphs) and the preoviposition interval were analyzed using ANOVA followed by the LSD test (post hoc comparisons) and % survival to adults was analyzed using the test of difference between proportions (@Statistica 2000).

Predation of SBM deutonymphs and females on pink rust mite (PRM)

'Valencia' orange fruits approximately 4.0 to 6.0 cm diam. were brought to the

laboratory and arenas (ca. 3.5 to 4.0 cm diameter) were prepared within areas containing





60


the highest concentrations of eriophyids found. This arena was similar to the type 2 unit described in the previous assay and was maintained with adequate moisture for 5 to 7 days. Mite species and insects were removed from the experimental arena with only the eriophyids remaining. Pink and yellow colored eriophyids were observed in the arenas, and samples of each were taken for identification. Identification made through permanent slides and electron microscopy photography showed that these mites were PRM and CRM, respectively (Fig 4.2).

The mean numbers of CRM and PRM were 66.2 � 5.63 and 40.4 + 1.57 per cm2, respectively, on the apex the arena. Most of the eriophyids were concentrated in areas nearer to the cotton strips. All arenas contained both PRM and CRM in excess. SBM deutonymphs reared with pollen and CReM in the laboratory were starved for 6 h and SBM adult females were starved for 6 and 24 h. After these starvation periods, the respective stages were transferred individually onto the arenas using a fine No 5-0 sable brush followed by four-minute continuous periods of observation. The number of encounters and consumed eriophyids were recorded during this time. An encounter is defined as the change in the searching behavior of the SBM such as palpation and/or arrestment. The four-minute interval was determined because at this time 100 percent of the SBM females (n= 10 per interval time) placed in the arena consumed 2 or more eriophyids. This time seemed to be appropriate to evaluate and compare eriophyid predation (Fig. 4.3). Comparisons of the means of eriophyids consumed and encountered by SBM deutonymphs and females, respectively, were assessed using the paired t-test (Excel � 2000).





61

Results


Assays with natural diets

The SBM larval stage developed successfully to the protonymphal stage using CRM, CreM, TCM, ice plant or oak pollen diets and there were no significant differences among the diets in this stage (P > 0.05) (Fig. 4.4). However, on water alone and with the CRM only diet, all the SBM protonymphs stopped development and died. Survival of SBM to adults was obtained with oak pollen, ice plant pollen, TCM and CreM and percentage survival rates were of 68, 60, 48, 36 %, respectively. A significant difference in percentage survival of SBM to adults was obtained between the oak pollen alone and CReM alone diets (p < 0.05). The female:male ratios were 5:4 (CReM, 1 female died before laying an egg), 7:5 (TCM, 1 female died before laying an egg), 8:9 (oak, 1 female died before laying egg) and 8:7 (ice plant, 2 females died before laying eggs). SBM females completed their life cycles on CReM, ice plant pollen alone, oak pollen alone and TCM diets in 9.48 � 0.2 (n = 4), 9.44 � 0.5 (n = 6), 9.39 + 0.3 (n = 7), 8.5 � 0.2 (n = 6) days � SE, respectively ((Fig. 4.4).

Predation of SBM deutonymphs and females on PRM

SBM deutonymphs encountered significantly higher mean numbers of CRM (8.3 +

2.45 / 4 min.) than PRM (1.5 � 0.3 / 4 min.) (d.f. = 9, t-critical = 2.262, P < 0.014) after 6 h starvation (Fig. 4.5) This corresponds with the highest number of CRM:PRM proportions in the arenas (0.62 and 0.38, respectively). However, SBM deutonymphs only fed on PRM that were encountered (t-critical = 2.262, P < 0.002) and did not feed on CRM. Similar results were found for SBM adult females (Fig. 4.5), although in this case the number of CRM and PRM encountered by 6 h starved SBM females (d.f. = 11, t-





62

critical = 2.200, P = 0.29) and 24 h starved SBM females (d.f. = 19, t-critical = 2.093, P =

0.709) were not significantly different (t-test, P > 0.05) (Fig. 4.5). SBM females fed on

1.8 + 0.47 and 3.5 � 0.45 PRM / 4 minutes after 6 h (d.f. = 11, t-critical = 3.10) and 24 h (d.f. = 19, t-critical = 2.86) starvation, respectively, and did not feed on CRM. These results were highly significant (P < 0.002 and P < 2.49-7, respectively).

Discussion


SBM did not complete its life cycle on the CRM diet. SBM molted from larva to

protonymph when provided with CRM alone. However, this was not an indication that SBM larvae were preying on CRM since SBM larva maintained on the ceramic tiles with wet cotton strips molted to the protonymphal stage. This result is similar to previous reports where I. zuluagai Denmark and Muma in Brazil, a close relative of I. quadripilis did not eat during the larval stage (Yamamoto, 1994). Lee and Davis (1968) found that Typhlodromus occidentalis (Nesbitt) larvae did not feed. Sabelis (1985a) noted that the larval stage is not influenced by the prey supply for most phytoseiids. Chitteden and Saito (2001) concluded that the non-feeding larval behavior may be an adaptation to avoid sibling cannibalism. They also gave examples of non-feeding larvae of Amblyseius californicus McGregor, A. degenerans (Berlese), A. womersleyi Schicha, Phytoseiulus persimilis Athias-Henriot, and Typhlodromus bambusae Ehara.

There were statistical differences in the duration of the SBM egg stage among the different diets (Fig. 4.4). However, the diet did not have an apparent effect on the total developmental times of SBM females as shown in Fig. 4.4. Diets of oak alone and CReM alone were significantly different on the subsequent duration of the egg stage but total developmental times were not significantly different. Significant differences were





63

obtained in nymphal periods (Fig. 4.4). In this study, the duration of the nymphal stage has a direct relationship with the total duration of the SBM generational time (Fig. 4.4). The shortest nymphal stage was obtained with the TCM diet alone and this was significantly different from the rest of the diets. This short developmental time contributes to the total generation time (P< 0.05) of SBM that was also the shortest and statistically significant (P< 0.05) from the rest of diets except the oak pollen diet (Fig.

4.4). The longest generational time was with the CReM diet alone and this also affected the total generation time of SBM (Fig. 4.4). Both periods of development with the CReM diet alone were significantly greater (P< 0.05) than the rest of the diets tested.

The diets with TCM alone and CReM alone resulted in the longest and shortest

developmental times of SBM, respectively. Diets of tetranychids reportedly affect the duration of different phytoseiid life cycles. Proprioseiopsis asetus Chant (Phytoseiidae) fed on a diet of Eutetranychus orientalis Klein instead of date pollen (Phoenix dactylifera L.) and shortened its generation time (Fouly, 1997). Abou-Setta and Childers (1989) fed Euseius mesembrinus a diet of ice plant and had the shortest generation time (9.6 d) whereas with CReM it was the longest (11.9 d). These authors indicated that when mated E. mesembrinus females were changed from a diet with ice plant to a diet with CReM high mortality resulted. Sabelis (1985a) found that the duration of the nymphal stage and the preoviposition period depend on prey availability. In this case, all the diets provided an abundant food supply, thus differences in developmental time between the tetranychid diets might be explained by two reasons. First, reduced webbing was produced by TCM when compared with CReM. This webbing has been observed disturbing the movement of SBM and increasing the SBM searching area on CReM arenas. Presence of webbing





64

is explained by Gerson (1985) as a defensive mechanism for CReM to protect spider mite eggs. Abou-Setta (1988) also found that E. mesembrinus had similar difficulties when preying on CReM due to the presence of webbing. Euseius mesembrinus became entangled in the CReM webbing. Although CReM is not a prolific web producer as is T. urticae, the presence of webbing had a repellent effect on SBM because many immatures and adults died trying to leave the arenas. This may explain why the lowest percent survival rate was found with the CReM only diet. The second explanation may be justified by possible differences in nutrients provided by the two types of spider mites. TCM may be considered a better food source than CReM. In Florida, similar results were noted for E. mesembrinus. The developmental time was shorter in a diet with TCM than CReM (Abou-Setta and Childers 1989).

No significant differences were found in SBM development times on diets with oak or ice plant pollen alone. However, the survival rates were higher in the pollen diets than with the spider mite diets. SBM may be adapted to feed on pollen and consequently has a preference for pollen because various pollen types are available throughout most of the year in Florida. This also, may be the result of the SBM life history. SBM is a polyphagous predatory mite and it is abundant during the peak pollen season (See Chapters 3 and 4). Muma (1971) described this mite as a fungal feeder and similar behavior was observed during the duration of this study (personal observation). A fungus was observed more frequently on the oak and ice plant pollen arenas than on tetranychid arenas due to deterioration of the pollen. It is possible that SBM had fed on this fungus thus a mixed diet of fungus and pollen can provide an additive nutritional effect for SBM and shortened its life cycle. SBM eggs present a characteristic reddish coloration that is





65


distinct from other phytoseiids on Florida citrus. SBM that fed on the two pollen diets produced eggs with a small pinkish area instead of an intense, larger reddish area observed on SBM reared on CReM or TCM and from eggs collected on the field. This difference in egg color can be an indicator of the varied diet sources of SBM in the field.

The experiment on predation of citrus rust mites by SBM showed a clear preference by SBM for PRM over CRM as a food source. This preference was obtained for SBM nymphs and adult females starved for 6 and 24 hrs since these instars preyed only on PRM and did not prey upon CRM (Fig. 4.5). Encounters of SBM with citrus rust mites (both PRM and CRM) can be described as direct palpation of rust mites nymphs and adults. In some cases, the rust mites were raised by the pedipalps and/or chelicera of SBM. This behavior may be an indicator of attempted predation but all CRM were left alive after a couple of seconds and, in other cases similar behavior was observed toward the PRM. When SBM preyed on PRM it was observed that the SBM completely sucked the body fluids (haemolymph) from PRM and only left the shrunken exoskeleton. This predatory behavior clarifies previous studies about the feeding of citrus rust mites by SBM. Muma (1971) described that CRM was both inadequate and adequate as a food source for SBM. However, he did not distinguish between CRM and PRM. This latter point may be an inaccuracy in previous studies between predatory phytoseiids and rust mites on citrus worldwide if more than one eriophyid species occurs. The inability of SBM to prey on CRM is similar to responses of E. mesembrinus observed by Abou-Setta (1989) and Flores et al. (1996). The reason why SBM did not prey on CRM requires further study and may involve nutritional quality, pheromonal or toxic effects of this mite toward this phytoseiid species. Sabelis (1996) hypothesized that eriophyids had a low





66


nutritional quality and low profitability compared to other prey. In addition, toxic substances within eriophyids may have a repellent affect toward phytoseiids. One or both of these factors may be especially relevant with CRM. It is necessary to make a clear distinction between the two eriophyids found in citrus orchards in Florida because many earlier studies on predatory mites assumed only the presence of CRM. Childers and Achor (1999) stated that in future studies it is important not to lump the rust mites because their development, biology, feeding damage and seasonal activities obviously differ.

In this study, the effects of the diets on oviposition during the entire life span of SBM were not followed. However, this study has provided valuable information on the identification of various food sources, developmental times and behavioral aspects of this important predatory mite in Florida. We learned that SBM is able to feed and complete its life cycle on pollen, CReM and TCM and, it will prey on PRM but not on CRM. This work enhances the importance of identifying correctly CRM from PRM. The small size of the eriophyids makes it more difficult to identify species with a handlens. The only viable way to effectively distinguish these two species is by the pinkish coloration of the pink citrus rust mite (Childers and Achor 1999). However, this requires practice and most importantly the confirmation by slide-mounted specimens.





67





4-- 3.5 cm

I , " Encircled experimental arena Wet cotton strip p Orange (4 to 5 cm diameter) Connecting cotton strip


3.5cm > PVC pipe




Petri dish with water I 8.5 cm


Figure 4.1. Orange fruit arena: design and components.






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PRM CRM











Rounded Trough








Ids


Figure 4.2. PRM (Aculopspelekassi) and CRM (Phyllocoptruta oleivora) main taxonomic differences. PRM: long dorso-lateral setae (ids), rounded dorsum and distinctive dorsal shield. CRM: short dorso-lateral setae (sds), dorsum with trough present and dorsal shield with well defined hexagon (hx).





69





6
100
E% SBM consumed >
2 eriophyids / interval 5
80 -[] No. eriophyids
consumed / interval
4

60




20
M

2


20 1



0 0
1 min 2 min 3 min 4 min 5 min

Figure 4.3. Determination of time interval for predation of eriophyids by Iphiseiodes quadripilis (SBM)






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Nymph


Ice Plant iiiabiiii ab pollen


Oak iii bc
pollen . . abe E. banksi ab c c P. citri a a


0 1 2 3 4 5 6 7 8 9 10 Days





Figure 4.4. Duration of female Iphiseiodes quadripilis life stages provided individual diets. Different letter between diets in the same instar indicates significant differences (LSD -test, P < 0.05)





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12
Deutonymph: Adult Females: Adult Females:

6 Hrs Starvation 6 Hrs Starvation 24 H's Starvation

10
0 No. Encountered a * No. Consume e8
t L

'E
"6



w4
a

b"
02 0 0



0
CRM PRM CRM PRM CRM PRM

Figure 4.5: Consumed and encountered eriophyids by SBM deutonymphs and adult females after starvation. Different letter between the numbers of eriophyids encountered or consumed within the same time of starvation and instar indicates significant differences (t-test, P < 0.05).













CHAPTER 5
EVIDENCE FOR HOST PLANT PREFERENCE IN A PREDACIOUS MITE
IPHISEIODES QUADRIPILIS (ACARI: PHYTOSEIIDAE) Introduction



Price et al. (1980) proposed the three-trophic level theory that establishes the basis for interaction among herbivores (prey), natural enemies (predators) and their host plants. This theory suggests that natural enemies and plants have a mutualistic relationship. Inherent plant defense mechanisms and plant traits that foster natural enemies evolved through natural selection and both are part of a plant defense that results in a negative impact on herbivores. These traits included attractants (nectars, pollen) and structural refuges (leaf domatia) (Walter 1996, O'Dowd and Willson 1997). Research in plant volatiles demonstrated that plants send signals -plant volatiles or infochemicals - to predators and these predators respond to them. Phytoseiulus persimilis Athias-Henriot responded to volatiles emitted by bean plants when they were damaged by punctures that mimicked Tetranychus urticae (Koch) feeding damage (Dicke and Sabelis 1988, Takabayashi and Dicke 1992, Dicke et al. 1998). However, all of these observations were on annual plants in the greenhouse. They are less complex than perennial plants in the field due to greater variables such as the presence of other organisms, environmental conditions and space. Drukker et al. (1997) showed the influence of P. persimilis performance and adaptation on tomato plants. They found that P. persimilis maintained for four generations on tomato exhibited higher survival and reproduction compared to P.



72





73

persimilis reared on bean plants and then transferred directly onto tomato. They explained that there was an intergenerational adjustment in the behavior and physiology of P. persimilis and a selection of P. persimilis genotypes with higher fitness on tomato plants. Similarly, Duso and Vettorazzo (1999) showed that two native phytoseiids in Italian vineyards distinctly preferred different grape (Vitis vinifera L.) varieties as host plants. Amblyseius andersoni (Chant) was more abundant on 'Merlot' grapes that had slight pubescence on the undersurface of its leaves. Phytoseiusfinitimus (Ribaga) was more abundant on 'Prosecco' grapes with a greater under-surface leaf pubescence than glabrous leaves of 'Riesling' grapes.

Iphiseiodes quadripilis is a generalist predatory mite on Florida citrus (chapter 4) that feeds on spider mites, ice plant and oak pollens in the laboratory. However, little is known about its life history and ecology. Abou-Setta et al. (1991) described the postembryonic stages and noted that the I. quadripilis female retained an egg with a welldeveloped larva. Iphiseiodes quadripilis was the most abundant predatory mite on mined leaves of grapefruit damaged by citrus leafminer larvae and exploits this type of leaves better than other phytoseiids (Chapter 3). Observations during my field work led me to assume that I. quadripilis tended to be more abundant on grapefruit than on sweet orange trees. Hence, this question was addressed with both field and laboratory assays. This mechanism implies a tritrophic relationship in a perennial plant that needed to be investigated. The aim of this study was to determine if there is a preference by Iphiseiodes quadripilis for grapefruit compared with sweet orange leaves.





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Materials and Methods

Abundance of Iphiseiodes quadripilis on the field.

Four adjacent orchards of grapefruit and sweet orange were selected for this

experiment. The pairs of adjacent orchards at the four sites received similar types of cultural practices and pesticide regimes. Three are experimental orchards in the vicinity of the Citrus Research and Education Center at Lake Alfred and the fourth site is a commercial orchard in Highland City (Polk County, FL). The first orchard was Block 1 and trees were 3-year-old 'Lang' navel oranges and 'Marsh' grapefruit. Leaves were collected on 12 and 19 April 2001. The second orchard was Block 24B and had 10 rows of 10 different types of citrus trees (mandarins, oranges, grapefruit), with I row per variety and with 8 to 12 trees per row. The trees were planted in 1990. This orchard was adjacent to a large experimental orchard of "Valencia" oranges. The varieties selected here were 'Pineapple' orange and 'Oroblanco' grapefruit and the leaves were collected on 18 and 22 April 2001. The third orchard was Block 22 with 'Hamlin' oranges and 'Flame' grapefruit. Samples were taken on 15 and 20 April 2001 and the trees were planted in 1997. The fourth orchard in Highland City included 20 and 23 years old 'Hamlin' oranges and 'Marsh' grapefruit, respectively. The samples were collected on 17 April and 7 May 2001.

Five border trees of each orange and grapefruit block were selected in each of the sites and 10 mined leaves (mines damaged by the citrus leafminer P. citrella) were taken randomly from each tree. Leafminer damaged leaves were selected because they have higher numbers of phytoseiids (see Chapter 3). The cumulative number of phytoseiids was analyzed using the test of proportions Statistica� (Statsoft 2000) to observe if there were differences between the total numbers of phytoseiids found on grapefruit or orange.





75

A similar test was completed for the total number of . quadripilis and other species of phytoseiids. Also, only data on TCM eggs and motiles, I. quadripilis and the total number of phytoseiid motiles found on the two sample dates per citrus species were analyzed separately with a mixed analysis of variance using the Satterthwaithe method Statistical (Statsoft 2000). the data were transformed using X + 0.5 , where X is the number of mites counted on 5 leaves. Mean numbers + SEM employed in figures and mentioned in the text are untransformed data.

Additional samples were taken from Block 22 on 29 April from the border trees of

both orange and grapefruit and phytoseiids counts were completed. The 12 May samples were taken from the border trees and 5th row inside of each type of citrus fruit and counts of both phytophagous and predatory mite eggs and motiles were completed in the laboratory. This time only 5 trees were sampled and 5-mined leaves were taken from each tree. Data were transformed using X + 0.5 , where Xis the number of mites counted on 5 leaves. Data were analyzed using the analysis of variance for repeated measures followed by a Scheffe-test (Statsoft 2000).

Direct contact choice experiment on 12 by 2 mm leaf strips

Ten female I. quadripilis were collected from each orange and grapefruit block and then placed on an arena. The arenas consisted of 3 alternating strips of orange and grapefruit leaves surrounded by wet cotton strips. The strips were 12 mm long by 2 mm wide (Fig. 5.1). Leaves used for the arenas were previously washed thoroughly with tap water and all mites and insects removed. Observations and counts of mite preferences were completed every 5 minutes for 1 h. Three replicates were conducted for each of the types of plants (orange or grapefruit) where I. quadripilis were obtained. All of the adult





76

female I. quadripilis were placed in alcohol and later slide-mounted in Hoyer's mounting medium and identified. This experiment was completed in the laboratory at 250 C and 55 + 5 % RH during May 2001. Data were analyzed using a paired t-test for comparison of means (Excel @, Microsoft).

Y- Tube olfactometer

Iphiseiodes quadripilis females were collected from orange and grapefruit trees on

several dates during May 2001. The I. quadripilis were collected from orange leaves and then placed on an orange leaf arena. Those collected from grapefruit were placed on a grapefruit leaf arena. Leaves from the same orchards were also collected and returned to the laboratory. These leaves were washed thoroughly with deionized water to remove any prey, scale insects or dust and then air-dried. The Y-tube olfactometer model (Fig.

5.2) was constructed using the design of Sabelis and Van de Baan (1983). Air was pumped at a rate of 500 cm3 of air current per minute and measured by a Gas flowmeter (Dwyer Instruments, Inc. Michigan City, Indiana). The air passed through activated charcoal before entering the odor chambers.

For each plant type, 70 I. quadripilis females were employed and used only once. A single female I quadripilis per trial was placed at the mouth of the Y-tube and left there to make an odor attraction choice of leaf. The leaf sources were in the chambers at the ends of the Y-tube arms and consisted of either a 3.5 cm leaf disk of orange or grapefruit. A 5-minute time limit for each predator was provided to make a choice. The assay for each female ended when the predator entered into one of the two chambers. The chambers of the Y-tube were switch every four trials and the 3.5 cm disks were replaced after each switch. Mites that did not make a choice during this time limit were separated and not considered in the statistical analysis. This experiment was completed in the





77


laboratory at 250 C and 55 � 5 % RH between May and June 2001. The number of predacious mites that chose either grapefruit or orange were counted as well as the time required in each trial. Data for the choice test were analyzed using the X2 test and the time required per predator to reach either chamber was analyzed using the ANOVA (Excel�, Microsoft, 2000).

Results

Abundance of Iphiseiodes quadripilis in the field

The total numbers of phytoseiids by type of fruit and species found at the four sample sites during the two collection dates are shown in Table 5.1. Significantly more I. quadripilis (P < 0.001) mites were found on grapefruit than orange, and the 'other phytoseiids' (phytoseiids other than I. quadripilis) were found in higher numbers on orange than in grapefruit (P < 0.001). The total numbers of phytoseiids were also significantly higher (P < 0.001) on grapefruit than on orange. However, the total numbers of I. quadripilis were not different from the total numbers of other phytoseiids (P > 0.05).

The data for the two sample dates and four sites gave us the results observed in Tables 5.2 to 5.6. A more detailed analysis of these results is presented in the appendix. Iphiseiodes quadripilis was significantly more abundant (P < 0.05) on grapefruit than orange (Tables 5.2 and Figs. 5.3 A and B). The highest mean numbers + (SEM) of I. quadripilis motiles on the first date of samples obtained in Block 24B were 20.0 4- 7.7 motiles / 10 leaves on 'Oroblanco' grapefruit versus 2.8 � 1.4 motiles/ 10 leaves for 'Pineapple' oranges. The lowest mean numbers � (SEM) of I. quadripilis were found at the Bader block with 4.6 � 1.5 motiles / 10 leaves on 'Marsh' grapefruit and 1.6 � 1.0 motiles/ 10 leaves on 'Hamlin' oranges (Fig. 5.3A). The highest mean numbers � (SEM)






78

of L. quadripilis on the second sampling dates were obtained at Block 22 with 22.6 � 4.2 motiles / 10 leaves on 'Flame' grapefruit versus 5.2 � 2.8 motiles/10 leaves on 'Hamlin' oranges. The lowest mean numbers � (SEM) of . quadripilis motiles were found at the Bader block with 1.8 � 0.4 motiles / 10 leaves on 'Marsh' grapefruit and 0.2 - 0.2 motiles/ 10 leaves on 'Hamlin' oranges (Fig 5.3 B).

Phytoseiids other than I. quadripilis are referred here as 'other phytoseiids' and their mean numbers + (SEM) were significantly different in the first sample dates between orchards and between trees. However, no differences (P >0.05) were found for the second sampling dates for both orchards and tree fruit type (Table 5.3 and Fig 5.3A). Also, no significant differences were obtained between the total numbers of phytoseiids (I. quadripilis +"other phytoseiids') found on grapefruit and orange on either of the two sample dates (Table 5.4 and Fig. 5.4). The Texas citrus mite eggs and motiles were very low in the first sampling dates on all orchards except Bader. This was the only significant difference found due to the interaction. There were 9.0 + 3.9 and 21.0 � 7.5 eggs and motiles / 10 leaves on 'Marsh' grapefruit and 122.2 + 41.7 and 140.6 � 47.4 /10 leaves on 'Hamlin' oranges. No significant differences in TCM egg and motile numbers were found between orchards or fruit types. On the second sampling dates, TCM eggs and motiles were high and significant differences occurred among orchard sites and tree types for the numbers of eggs and between tree types for TCM motiles (Tables 5.5 and

5.6 and Figs. 5A and 5B)

Mean numbers � SEM for I. quadripilis on samples of 29 April (1,8 df, F = 37.48, P <

0.01), and 12 May (3,16 df, F = 46.3, P < 0.001), 10 September (1,8df, F = 0.80, P .0.05), and 22 October (3,16 df, F = 5.68, P < 0.01) from Block 22 are presented in Table





79


5.7. Iphiseiodes quadripilis motiles were more abundant on grapefruit than orange during most of the times sampled except on 10 September. Additional samples of fruit on the fifth row within each orchard gave similar results for I. quadripilis motiles but the TCM eggs and motiles were significantly more abundant on orange than grapefruit (Fig.

5.6 A and B).

Direct contact choice experiment on 12 by 2 mm leaf strips

The numbers of . quadripilis females that were collected on grapefruit were

significant (11 df, t-critical = 2.20, P < 0.05) most of the time on grapefruit than on orange (mean � SEM: 5.81 + 0.18 vs. 4.19 �0.18 I. quadripilis females in grapefruit or orange, respectively) leaf strips for all replicates (Table 5.8 and Fig. 5.7). Whereas I. quadripilis females collected from orange did not show a preference for either orange or grapefruit leaf strips (11 df, t-critical = 2.20, P > 0.05) (mean � SEM: 4.9 - 0.26 vs. 5.1 +

0.26, respectively) (Table 5.8 and Fig. 5.7).



Y- Tube olfactometer

Iphiseiodes quadripilis females that were collected from orange or grapefruit showed preference for the grapefruit leaf disk. The number of I. quadripilis from grapefruit that selected grapefruit and orange were 44 and 26, respectively. By coincidence, the number of . quadripilis females collected from orange that preferred grapefruit and orange were also 44 and 26, respectively. In both cases, these numbers were significantly different (1 df and X2 critical =3.84). The times employed for I. quadripilis females collected from orange or grapefruit in making their choice for either leaf disk were 115.0 � 8.91 and 97.6 � 8.2 seconds (mean � SEM), respectively, and these were not significantly different (1, 138 df, F = 2.06 and P = 0.15).





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Discussion

The same species of phytophagous mites affect orange and grapefruit orchards

although some citrus species may tend to be more susceptible to certain kinds of pest mites than others. The broad mite Polyphagotarsonemus latus (Banks) is more commonly found on lemon and lime in south Florida than on grapefruit (Childers 1994a). However, I. quadripilis (SBM) was observed in highest numbers on grapefruit than orange on the border trees of the four adjacent sites. This indicates a preference of I. quadripilis for its host plant. This can be inferred because each pair of adjacent orchards received similar cultural practices and pesticide regimes. Furthermore, prey availability was favorable to orange, although in the first sampling dates there were no differences in E. banksi (TCM) eggs and motiles with the exception of the Bader orchard. During the second sampling, the densities of TCM were observed to be lower on grapefruit than on sweet orange, suggesting that better biological control by I. quadripilis was occurring on the grapefruit varieties. If this was the case, why did I. quadripilis not migrate from grapefruit to orange? Is this a seasonal occurrence or are there other factors that retain I. quadripilis on grapefruit? There must be since I. quadripilis developed and reproduced readily on E. banksi (Chapter 4) and this pest mite was present in greater numbers on orange than on grapefruit. Also, I. quadripilis had the shortest developmental rate on E. banksi compared to P. citri, oak (Quercus sp) or ice plant (Mallephora crocea) pollen diets (Chapter 4).

Thus a mutualistic relationship between grapefruit and I. quadripilis appears likely. If a mutualistic relationship exists, it may be independent of prey availability such as an enhancement of the host plant to foster predators. Other unknown factors may exist that contribute to grapefruit plants maintaining I. quadripilis populations in grapefruit






81


orchards. These can be inherent in the plant or exogenous to it or both factors may apply. Inherent factors may include the production of volatiles and a better nutritional quality of grapefruit pollen than orange pollen. Other inherent characters could include the composition of essential oils in leaves of the two citrus species. Cheng and Lee (1981) completed a study of leaf essential oils in ten citrus species including orange and grapefruit. They found 45 different essential oils and the percentage compositions were similar for most except Sabinene and A3-carene. Sabinene was the most abundant oil in all the species with 49.7% in oranges and 61.9 % in grapefruit and A3-carene was 4.9% in orange and 0.12% in grapefruit. However, studies of these oils have focused primarily for the perfume and cosmetic industries. It would be interesting to know if one or more of these essential oils affect the composition of mite or insect faunas in the different citrus species.

Exogenous factors may include food sources other than prey mites that are available for I. quadripilis such scale insect eggs and/or phylloplane fungi. Muma (1971) found that I. quadripilis preyed on whitefly (Aleurodidae) eggs and larvae and described this food source as both adequate and inadequate. However, Muma (1971) did not indicate the reproductive or life cycle parameters of I. quadripilis with this prey. No heavy infestations of whitefly were observed in any of the four orchards. Also, it might be possible that some specific types of phylloplane fungi may contribute to the preference of I. quadripilis for grapefruit leaves but to this date no studies have been completed on citrus. Effects of phylloplane fungi have been recently studied. Belczewski and Harmsen (2000) found that non-pathogenic phylloplane fungi on beans affect the net reproductive rate of Tetranychus urticae Koch. Similarly, Zemec and Prenerova (1996)





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observed that the phytoseiid Typhlodromus pyri Scheuten feeds on two types of powdery mildew (Ascomycotina: Erysiphales), Erysiphe orontii Castellani on tobacco and Oidium fragaria Harz on strawberry. Mature females T. pyri females successfully reproduced although their oviposition rate was low. Villanueva (unpublished) also observed that I. quadripilis, E. mesembrinus, T. peregrinus and Amblyseius sp. feed on unknown fungi that occur within colonies of phytoseiid mites reared in the laboratory.

The contact and olfactometer assays demonstrate that both factors (a grapefruit substrate and volatiles produced by the plant) may contribute to the preference of . quadripilis for grapefruit. In the contact assay, it was apparent that the origin of the I. quadripilis population has an imprinting effect on the predator because I. quadripilis collected on orange did not show preference for either grapefruit or orange leaf strips. However, I. quadripilis collected on grapefruit showed a significant preference for the grapefruit substrate (P < 0.05). Imprinting can imply the evolution of two lineages of I. quadripilis. This phenomenon was reported on the predatory mite Hypoaspis aculeifer (Canestrini) (Acari: Laelapidae) by Lesna (1998) and Lesna and Sabelis (1999). They showed that H. aculeifer can rapidly exhibit genetic variation in preference for two prey species: the bulb mite Rhizoglyphus robini Claparede or the copra mite Tyrophagous putrescentiae (Schrank) depending where H. aculeifer was reared. It may be possible that I. quadripilis from grapefruit and orange can form two lineages that are very closely related and the only way to differentiate the two would be with molecular analysis rather than by classical taxonomic methods.






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Table 5.1 Total number of Phytoseiids found in 4 sites on 2 sample dates by type of fruit and phytoseiid species. Significant differences calculated with the test of proportions are indicated in each column and row

Iphiseiodes quadripilis Other Phytoseiids Total P
Grapefruit 458 151 609 < 0.001 Orange 114 400 514 < 0.001 Total 572 551 1123 0.25 P < 0.001 < 0.001 0.007




Table 5.2. Mixed analysis of variance for Iphiseiodes quadripilis numbers found in adjacent rows of grapefruit and orange at 4 sites sampled on two dates each.

Factor d.f. F P Orchard Site 3,3 2.44 0.241 1st sample dates Tree type 1,3 15.13 0.030 Orchard*Tree 3,32 1.37 0.267
Orchard Site 3,3 3.96 0.144 2nd sample dates Tree type 1,3 15.28 0.029 Orchard*Tree 3,32 2.13 0.115




Table 5.3. Mixed analysis of variance for the total number of 'other phytoseiids' (phytoseiids other than I. quadripilis) found in adjacent rows of grapefruit and orange at
4 sites sampled on two dates each.

Factor d.f. F P
Orchard Site 3,3 15.19 0.03 1st sample date Tree type 1,3 26.08 0.01
Orchard*Tree 3,32 0.54 0.66
Orchard Site 3,3 1.70 0.34 2"d sample date Tree type 1,3 9.39 0.05
Orchard*Tree 3,32 0.72 0.54





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Table 5.4. Mixed analysis of variance for the total number of phytoseiids found in adjacent rows of grapefruit and orange at 4 sites sampled on two dates each.

Factor d.f. F P
Orchard Site 3,3 1.67 0.34 1st sample date Tree type 1,3 0.37 0.58
Orchard*Tree 3,32 0.75 0.52 Orchard Site 3,3 3.33 0.17 2nd sample date Tree type 1,3 3.91 0.14
Orchard*Tree 3,32 1.17 0.338




Table 5.5. Mixed analysis of variance for the total number of Eutetranychus banksi eggs found in adjacent rows of grapefruit and orange at 4 sites sampled on two dates each.

Factor d.f. F P
Orchard Site 3,3 2.17 0.26 1st sample date Tree type 1,3 1.14 0.36
Orchard*Tree 3,32 10.65 < 0.001
Orchard Site 3,3 16.57 0.02 2"d sample date Tree type 1,3 23.77 0.01
Orchard*Tree 3,32 0.52 0.66




Table 5.6. Mixed analysis of variance for the total number of Eutetranychus banksi motiles found in adjacent rows of grapefruit and orange at 4 sites sampled on two dates each.

Factor d.f. F P
Orchard Site 3,3 2.12 0.24 1st sample date Tree type 1,3 4.00 0.14
Orchard*Tree 3,32 6.54 0.001
Orchard Site 3,3 6.00 0.09 2nd sample date Tree type 1,3 13.79 0.03
Orchard*Tree 3,32 1.67 0.19





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Table 5.7. Analysis of variance for the total number of Iphiseiodes quadripilis motiles found in adjacent rows of orange and grapefruit in Block 22 sampled on four dates.

Orange Grapefruit Statistical 5th Row Border Border 5th Row Test 29 April -0 a 11.0 + 2.9 b ANOVA 12 May 0a 3.6 � 1.1 b 13.2 + 1.4 c 8.4 1.2 c Scheffe 10 Sep - 1.1 � 0.4 a 1.7 � 0.5 a ANOVA 22 Oct 0 a 1.0 � 0.6 a 1.7 + 0.4 b 2.5 + 0.8 b Scheffe




Table 5.8 Mean numbers � SEM of Iphiseiodes quadripilis (n = 10) in a choice test between grapefruit or orange strips during 5-minute observations per 1 hour depending on where they were originally collected
Iphiseiodes Replicate No I. quadripilis N I. quadripilis d.f. P quadripilis on Orange on Grapefruit
1 4.16 �0.36 5.83�0.36 11 0.0043 From Grapefruit 2 4.41 � 0.25 5.58� 0.25 11 0.046 3 4.0+0.30 6.0-0.30 11 0.006 1 5.25�0.35 4.66�0.35 11 0.49 From Orange 2 4.66�0.61 5.33�0.61 11 0.6 3 4.83�0.36 5.16�0.36 11 0.61






86



4 12mm




2 mm


12 mm









WET COTTON Figure 5.1 Arena used for the contact experiments, Brownish dots on strips are female Iphiseiodes quadripilis.



Y-tube olfactometer






g y FILTERS
500 cc of
AIR/min







Figure 5.2. Y-tube olfactometer used for the choice test. Leaf disks of grapefruit (GF) or orange (OR) ca. 3.5 cm diameter were the sources of plant volatiles.






87



28

Block 1 (L.A) Block 24BBock 22 Rogers (C)
24 April 12 (L.A) (L.A) April 17 O Other 20 Phytoseiids
S20
m 1. quadripilis
-J
o 16


12


o.


4



Marsh-GF LangN-OR OroBlan-GF P-apple-OR Flame-GF Hanlin-OR Marsh-GF Hamlin-OR


28

Block 1 (LA) I Block 24B Block 22 (LA) Rogers (HC)
24 Ar 19 (L.A) April 22 May 7

w 20 O Other
Phytoseiids 16 m I. quadripilis

12

0


4

0
Marsh-GF LangN-OR OroBlan-GF P-apple-OR Flarne-GF Hanlin-OR Marsh-GF Hantin-OR
Grapefruit Orange Grapefruit Orange Grapefruit Orange Grapefruit Orange


Figure 5.3. Numbers of . quadripilis per 10 leaves and other phytoseiids other than I. quadripilis, found on the sample at 4 adjacent orchards of orange and grapefruit on the
(A) first sampling dates and (B) second sampling dates.




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A STUDY OF THE BIOLOGY AND ECOLOGY OF SELECTED PREDATORS OF PHYTOPHAGOUS MITES ON FLORIDA CITRUS } ; 7 M i V' I ' i t i > \ : I By ' ; RAUL TIMOTEO VILLANUEVA A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2002

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1 H This thesis is dedicated to my parents Rosa Angeles and Rafael M. Villanueva; to my sister Filca; my brothers Luis; Julio; and Rafael; and to Lucia Fabiola because she represents the next generation. i

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ACKNOWLEDGMENTS This work was completed with the contribution of many people. First I wish to thank my supervisor; Dr. Carl C. Childers for his guidance, suggestions, and financial assistance; and for giving me the opportunity to develop this study with independence. I also want to thank Dr. J. P. Michaud for his friendship, suggestions and for our discussions in biology and ecology, and for letting me use a space in his office. I thank Jose Carlos Rodrigues, because without his persistence the predatory behavior of Cryptothelea gloverii would not have been discovered. Robin Stuart also gave me some profitable suggestions and advice. Belen Belliure and Zenaida Viloria helped me on several occasions and were my confidants. I want to thank people that I met at Lake Alfred. Many became my friends and we shared some good times: Angle Grant, Elisabeth Knapp, John Jiffon, Joaquin Gonzales de la Fuente, Shachindra Mondal, Zeynel Dalkillic and Hugo Aguilar. hi Gainesville, Drs. J. Capinera and Maria Gallo-Meagher provided me with lab space to work there. Hector Cabrera was my friend, and colleague, and let me stay at his house many times. I specially thank Mrs. Percivia Mariner for slide mounting many phytoseiid specimens and for her support in maintaining the mite colonies. The following people helped by giving advice or by lending me equipment: Drs. H. Nigg, C. McCoy, J. Syvertsen, G. Albrigo, Pete Timmer. Mrs. Pam Russ was a great support in the library and Ms. Beatriz Nielsen, who provided citrus plants for my experiments. Diann Achor and Gretchen Baut gave me suggestions on taking photographs for this thesis. I offer thanks to my father Rafael Maglorio for his example in life, and to my brothers Lucho, Julio (and his wife Marianna) and Rafa for their unconditional fiiendship, support and love. I ! 11

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TABLE OF CONTENTS Eage ACKNOWLEDGMENTS ii LIST OF TABLES v LIST OF FIGURES vii INTRODUCTION 1 General Introduction 1 Review of Literature 4 PHYTOSEIID DISTRIBUTION ON LEAVES SAMPLED THROUGHOUT THE DAY AND EFFECTS OF LEAF POSITION IN THE CITRUS CANOPY 14 Introduction 14 Materials and Methods 16 Results 17 Discussion 19 LEAVES DAMAGED BY THE CITRUS LEAFMINER PROVIDE REFUGES FOR PREDATORY MITES IN CITRUS 32 Introduction 32 Material and Methods 36 Results 38 Discussion 40 COMPARATIVE DEVELOPMENT OF IPHISEIODES QUADRIPILIS (BANKS) (ACARI: PHYTOSEHDAE) ON VARIOUS NATURAL DIETS AND PREDATION ON ACULOPS PELEKASSI (KEIFER) (ACARI: ERIOPHYIDAE) IN THE LABORATORY 55 Introduction 55 Materials and Methods 58 Assays with natural diets 58 Predation of SBM deutonymphs and females on pink rust mite (PRM) 59 Results 52 Assays with natural diets 51 Predation of SBM deutonymphs and females on PRM 61 Discussion 52 EVIDENCE FOR HOST PLANT PREFERENCE IN A PREDACIOUS MITE IPHISEIODES QUADRIPILIS (ACARI: PHYTOSEHDAE) 72 iii

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Introduction 72 Materials and Methods 74 Abundance of Iphiseiodes quadripilis on the field 74 Direct contact choice experiment on 12 by 2 mm leaf strips 75 YTube olfactometer 76 Results 77 Abundance of Iphiseiodes quadripilis in the field 77 Direct contact choice experiment on 12 by 2 mm leaf strips 79 YTube olfactometer 79 Discussion 80 ECOLOGY AND FEEDING BEHAVIOR OF TWO NEW SPECIES OF CECIDOMYIIDAE PREDACIOUS ON RUST MITES AND THE MICROLEPIDOPTERA CRYPTOTHELEA GLOVERII (?SYCniDAE), A PREDATOR AND PEST ON FLORIDA CITRUS 92 Introduction 92 Materials and Methods 96 Cecidomyiid Midges Studies 96 Cecidomyiid collection 96 Rearing of cecidomyiids 96 Predation by cecidomyiids on citrus rust mites 97 Orange Bagworm (OBW) Cryptothelea gloverii 97 Mite predation by OBW 97 Assessment of OBW damage to orange and grapefruit leaves and fruits 100 Results 101 Cecidomyiid Midges 101 Orange Bagworm 102 Discussion 104 Cecidomyiid Midges 104 Orange Bagworm 107 SUMMARY AND CONCLUSIONS 122 MIXED ANALYSIS OF VARIANCE OF MITES FOUND IN 4 SITES WITH ADJACENT ORCHARDS OF ORANGE AND GRAPEFRUIT 132 LIST OF REFERENCES '. ] ng BIOGRAPHICAL SKETCH 155 iv

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LIST OF TABLES Table page Table 2.1 Agricultural practices employed in the Bader grove in Highland City 25 Table 2.2. Two-way analysis of variance for data on number of phytoseiids found on the east, inner and west sides of the tree canopy at different sampling times on indicated dates during the year 26 Table 3.1. Analysis of variance (first seven rows) and least significant difference (LSD) analysis (last four rows) for mean numbers of Eutetranychus banksi eggs on healthy mature, leafminer-damaged and young leaves 46 Table 3.2. Analysis of variance (first seven rows) and least significant difference (LSD) analysis (last four rows) for mean numbers of Eutetranychus banksi motiles on healthy mature, leafminer-damaged and young leaves 47 Table 3.3. Analysis of variance (first seven rows) and least significant difference (LSD) analysis (last four rows) for mean numbers of Eotetranychus sexmaculatus eggs on healthy mature, leafminer-damaged and young leaves 48 Table 3.4. Analysis of variance (first seven rows) and least significant difference (LSD) analysis (last four rows) for mean numbers of Eotetranychus sexmaculatus motiles on healthy, leafininer-damaged and young leaves 49 Table 3.5. Analysis of variance (first seven rows) and least significant difference (LSD) analysis (last four rows) for mean numbers of citrus rust mite motiles on healthy, leafininer-damaged and young leaves 50 Table 3.6. Analysis of variance (first seven rows) and least significant difference (LSD) analysis (last four rows) for mean numbers of phytoseiid mite motiles on healthy, leafininer-damaged and young leaves 51 Table 3.7. Analysis of variance (first seven rows) and least significant difference (LSD) analysis (last four rows) for mean numbers of stigmaeid motiles on healthy, leafminerdamaged and young leaves 52 Table 5.1 Total number of Phytoseiids found in 4 sites on 2 sample dates by type of fiuit and phytoseiid species. Significant differences calculated with the test of proportions are indicated in each column and row 83 Table 5.2. Mixed analysis of variance for Iphiseiodes quadripilis numbers found in adjacent rows of grapefiiiit and orange at 4 sites sampled on two dates each 83 Table 5.3. Mixed analysis of variance for the total number of 'other phytoseiids' (phytoseiids other than /. quadripilis) found in adjacent rows of grapefioiit and orange at 4 sites sampled on two dates each 83 V

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Table 5.4. Mixed analysis of variance for the total number of phytoseiids found in adjacent rows of grapefruit and orange at 4 sites sampled on two dates each 84 Table 5.5. Mixed analysis of variance for the total number of Eutetranychus banksi eggs found in adjacent rows of grapefruit and orange at 4 sites sampled on two dates each 84 Table 5.6. Mixed analysis of variance for the total number of Eutetranychus banksi motiles found in adjacent rows of grapefruit and orange at 4 sites sampled on two dates each 84 Table 5.7. Analysis of variance for the total number of Iphiseiodes quadripilis motiles found in adjacent rows of orange and grapefruit in Block 22 sampled on four dates 85 Table A.l. Transformed means, standard error of means (SEM), and ± 95% confidence interval limits for total numbers of Iphiseiodes quadripilis motiles 133 Table A2. Transformed means, standard error of means (SEM), and ± 95% confidence interval limits for total numbers of other phytoseiid motiles (phytoseiids other than /. quadripilis) 134 Table A3. Transformed means, standard error of means (SEM), and ± 95% confidence interval limits for total numbers of phytoseiid motiles (. quadripilis + other phytoseiids). ..135 Table A.4. Transformed means, standard error of means (SEM), and ± 95% confidence interval limits for total numbers of Eutetranychus banksi eggs 136 Table A.4. Transformed means, standard error of means (SEM), and ± 95% confidence interval limits for total numbers of Eutetranychus banksi eggs 136 Table A.5. Transformed means, standard error of means (SEM), and ± 95% confidence interval limits for total numbers of Eutetranychus banksi motiles 137 Table A.5. Transformed means, standard error of means (SEM), and ± 95% confidence interval limits for total numbers of Eutetranychus banksi motiles 137 vi

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LIST OF FIGURES Figure page Figure 2. 1 . Daily average relative humidity (%), temperature (° C) and light intensity (Lumens) on 14, 20 and 28 October 1999, 9 and 16 March 2000, and 17 and 24 August 2000 on the east, west and inside areas of the tree canopy at the Bader orchard in Highland City, Florida 27 Figure 2.2. Mean number of phytoseiid motiles ± SEM for 14, 20 and 28 October 1999. Values bearing the same letter were not significantly different using the HSD Tukey's test (P< 0.05) 28 Figure 2.3. Mean number of phytoseiid motiles ± SEM for 9 and 16 March 2000. Values bearing the same letter were not significantly different using the HSD Tukey's test (P < 0.05) 29 Figure 2.4. Mean number of phytoseiid motiles ± SEM for 17 and 24 August 2000. Values bearing the same letter were not significantly different using the HSD Tukey's test (P < 0.05) 30 Figure 2.5. Percentage of phytoseiids identified from 30% of the total numbers found on October 1999, March and August 2000. Text box number indicates the total number of phytoseiids identified each month 3 1 Figure 3.1. Mean numbers (± SEM) of Eutetranychus banksi eggs on healthy mature, leafminer-damaged and young leaves. Values bearing the same letter were not significantly different in an ANOVA or LSD test (P > 0.05) on a given sampling date 46 Figure 3.2. Mean numbers (± SEM) of Eutetranychus banksi motiles on healthy mature, leafminer-damaged and young leaves. Values bearing the same letter were not significantly different in an ANOVA or LSD test (P > 0.05) on a given sampling date 47 Figure 3.3. Mean numbers (± SEM) of Eotetranychus sexmaculatus eggs on healthy mature, leaftniner-damaged and young leaves. Values bearing the same letter were not significantly different in an ANOVA or LSD test (P > 0.05) on a given sampling date 48 Figure 3.4. Mean numbers (± SEM) of Eotetranychus sexmaculatus motiles on healthy mature, leafminer-damaged and young leaves. Values bearing the same letter were not significantly different in an ANOVA or LSD test (P > 0.05) on a given sampling date 49 Figure 3.5. Mean numbers (± SEM) of citrus rust mite motiles on healthy mature, leafminer-damaged and young leaves. Values bearing the same letter were not significantly different in an ANOVA or LSD test (P > 0.05) on a given sampling date 50 Figure 3.6. Mean numbers (± SEM) of phytoseiid motiles on healthy mature, leafminerdamaged and young leaves. Values bearing the same letter were not significantly different in an ANOVA or LSD test (P > 0.05) on a given sampling date 51 Vll

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Figure 3.7. Mean numbers (± SEM) of stigmaeid motiles on healthy mature, leafminerdamaged and young leaves. Values bearing the same letter were not significantly different in an ANOVA or LSD test (P > 0.05) on a given sampling date 52 Figure 3.8. Relative abundance of Euseius mesembrinus, Iphiseiodes quadripilis and Typhlodromus. peregrinus on healthy mature, leafminer damaged leaves by Phyllocnistis citrella and young grapefruit leaves on three dates with the highest numbers of phytoseiids registered 53 Figure 3.9. Relationship between phytoseiids (± SEM) and pollen grains (± SEM) found on grapefruit leaves from 2 February to 16March, 2001 53 Figure 3.10. Temperature and rainfall recorded on the Florida automated weather network CREC-Lake Alfred Station from 31 December 2000 to 20 May 2001 54 Figure 4. 1 . Orange fruit arena: design and components 67 Figure 4.2. PRM {Aculops pelekassi) and CRM {Phyllocoptruta oleivora) main taxonomic differences. PRM: long dorso-lateral setae (Ids), rounded dorsum and distinctive dorsal shield. CRM: short dorso-lateral setae (sds), dorsum with trough present and dorsal shield with well defined hexagon (hx) 68 Figure 4.3. Determination of time interval for predation of eriophyids by Iphiseiodes quadripilis (SBM) 69 Figure 4.4. Duration of female Iphiseiodes quadripilis life stages provided individual diets. Different letter between diets in the same instar indicates significant differences (LSD -test, P < 0.05) 70 Figure 4.5: Consumed and encountered eriophyids by SBM deutonymphs and adult females after starvation. Different letter between the numbers of eriophyids encountered or consumed within the same time of starvation and instar indicates significant differences (Mest, P < 0.05) 71 Figure 5.2. Y-tube olfactometer used for the choice test. Leaf disks of grapefruit (GF) or orange (OR) ca. 3.5 cm diameter were the sources of plant volatiles 86 Figure 5.3. Numbers of I. quadripilis per 10 leaves and other phytoseiids other than /. quadripilis, found on the sample at 4 adjacent orchards of orange and grapefiiiit on the (A) first sampling dates and (B) second sampling dates 87 Figure 5.4. Total numbers of phytoseiids {Iphiseiodes quadripilis + phytoseiids other than /. quadripilis) found on grapefruit and orange in adjacent rows of grapefinit and orange orchards. No significant differences were found (P >0.05) 88 Figure 5.5 Numbers of Eutetranychus banksi ± SEM on the second sampling dates (A) TCM eggs and, (B) TCM motiles 89 viii

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Fig. 5.7 Contact choice test of 10 Iphiseiodes quadripilis females for leaf strips of orange or grapefruit. Different letter in each replicate indicates significance (P < 0.05) 91 Figure. 5.8. Choice test on leaf disks in the olfactometer for Iphiseiodes quadripilis. Each column represents the type of fruit where female /. quadripilis were collected. Different letter in each column indicates significance (P < 0.05) 91 Figure 6. 1 . Arena used to quantify OBW predation of Phyllocoptruta oleivora. The area confined by the transparent plastic cup contained the OBW 114 Figure 6.2. Damage caused by Cryptothelea gloverii larvae feeding on russeted and healthy 'Hamlin' oranges. C. gloverii is more commonly found on russeted fiiiits 115 Figure 6.3. Adult Feltiella n. sp. predator of the citrus rust mite Phyllocoptruta oleivora obtained in the laboratory, vertical bars beneath insect are on millimeters 116 Figure 6.4. Two distinct larval types of two new species of cecidomyiids surrounded by their citrus rust mite Phyllocoptruta oleivora prey. (A). Feltiella n. sp. and (B) a species near Lestodiplosis n. sp. Note ring around the anterior end of the larva 117 Figure 6.5. Mean numbers ± SEM of different citrus rust mite (Phyllocoptruta oleivora) stages consumed by larvae of Feltiella n sp. and a species near Lestodiplosis n. sp 118 Figure 6.6. A Cryptothelea gloverii larva preying on the citrus rust mite Phyllocoptruta oleivora 11 9 Figure 6.7. Hole created by Cryptothelea gloverii larvae into the rind of a 'Hamlin' orange. C. gloverii larvae penetrate the rind but do not feed on the fruit sacs 119 Figure 6.8. Damage produced by a Cryptothelea gloverii larva on a citrus leaf C. gloverii can grasp the outer layer of the upper or lower epidermis without penetrating through the leaf and making a hole 120 Figure 6.9. Penicillium contamination associated with feeding damage caused by Cryptothelea gloverii. The bag of C. gloverii (arrow) is often found on this type of fruit or close by and larval feeding on fungi was observed 120 Figure 6.10. Winged male Cryptothelea gloverii. Scale beside the microlepidopteran is in millimeters 121 Figure 7.1. Prey-predator relationships of the citrus-acari-insects system on Florida citrus. Predation is indicated by arrows. Broken lines are not well studied relationships, blue lines are relationships observed in this study. (Green) = Natural enemies, (Red) = Phytophagous, (Light blue) = Undefined roles, (Orange) omnivores, and (Yellow) pollen. The (+) and (-) signs indicate the relationship respect to plants. (?) indicates poorly studied relationship ix

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Figure 7.2. Ecological relationships that enhance focusing on phytoseiid mites on citrus. Various relationships were observed in this study. Some were observed in other studies on different crops. Continuous arrows indicate relationships observed in this study. Broken line arrow indicates relationships that require further study. Red line indicates the presence of these organisms on citrus leaves 131 X

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Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy A STUDY OF THE BIOLOGY AND ECOLOGY OF SELECTED PREDATORS OF PHYTOPHAGOUS MITES IN FLORIDA CITRUS By Raul Timoteo Villanueva April 2002 Chairman: Carl C. Childers Major Department: Entomology and Nematology Phytoseiid mites were found in higher numbers on leaves within the canopy of grapefiaiit trees than on outer leaves. No differences in phytoseiid abundance were found among leaf samples collected at different hours throughout the day between 0600-2200 h. Mite abundance on grapefruit trees was compared among young-leaves, healthy-mature leaves, and mined-leaves by Phyllocnistis citrella Stainton. Predacious phytoseiids and stigmaeids and Eotetranychus sexmaculatus (Riley) were significantly more abundant on mined leaves than on young or healthy-mature leaves in weekly samples taken between February and April from an unsprayed orchard. Eutetranychus banksi (McGregor), and Phyllocoptruta oleivora (Ashmead) did not differ in abundance among the three types of leaves. Development and reproduction of Iphiseiodes quadripilis (Banks) was evaluated on diets of Malephora crocea Jacquin and Quercus sp pollens and, E. banksi, Panonychus xi

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citri (McGregor), and P. oleivora at 28°±1 °C, 14: 10 (L:D) daylength and 45%-RH. Iphiseiodes quadripilis completed its life cycle and oviposited viable eggs on exclusive diets of M. crocea, Quercus sp. pollens or E. banksi or P. citri eggs and motiles. Panonychus citri stages were acceptable as prey to /. quadripilis but survival of larvae to adulthood was significantly lower than on Quercus sp. pollen. Iphiseiodes quadripilis larvae provided water, or P. oleivora alone, developed to protonymphs and then died. Starved /. quadripilis nymphs and adult females preyed on Aculops pelekassi (Keifer), but not on P. oleivora. Iphiseiodes quadripilis was significantly more abimdant on ' grapefruit leaves than on orange leaves in adjacent citrus orchards at four sites in central Florida. This disparity was consistent whether samples were taken from border or interior trees. Total numbers of phytoseiids were not significantly different between citrus species in most cases. Eutetranychus banksi was significantly more abundant on orange than on grapefruit, suggesting that biological control by /. quadripilis was better on grapefruit trees. Iphiseiodes quadripilis preferred grapefruit leaves over orange leaves in olfactometer experiments and in contact choice tests using leaf discs of the two citrus species. Cryptothelea gloverii (Packard) (Lepidoptera) was identified as a predator of both P. oleivora and P. citri, although it also feeds on fruit and leaves of citrus. In the laboratory, C. gloverii preferred to feed on oranges russeted by P. oleivora than on fruit without russeting. Feltiella sp. and a Lestodiplosis sp. two undescribed Cecidomyiidae were highly specialized predators feeding on P. oleivora eggs, nymphs, and adults. xii

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CHAPTER 1 INTRODUCTION General Introduction The citrus rust mite (CRM), Phyllocoptruta oleivora (Ashmead), and the pink citrus rust mite (PRM) Aculops pelekassi (Keifer) are the key pests of Florida citrus fruits (Childers and Achor 1999). These species are found in all citrus-growing regions of the world, especially in areas of high humidity. CRM was first recorded in Florida in the early 1880's (Hubbard 1883, Yothers 1914,Yothers and Mason 1930). Citrus rust mites cause direct damage to citrus by feeding on the epidermal cells of leaves, green twigs and fruit (Albrigo et al. 1981, McCoy 1976). Both the CRM and the PRM have probably been present in Florida since the middle 1800's but because of their small size, they were not properly identified until 1961 (Denmark 1963). Russeting of fruit surfaces reduces the value of fruit destined for the fresh market. Direct damage to fruit can also result in increased alcohol or phenol concentrations, reduction of fruit size, increased fruit drop, and increased water loss relative to undamaged fruit (Yothers 1918, Ismail 1971, Allen 1978, 1979, Yang et al. 1995). A positive correlation between CRM damage and the incidence of leaf infection by greasy spot, Mycosphaerella citri Whiteside, has also been observed (van Brussel 1975, Timmer et al. 1980), although both are favored by high humidity. Acaricides are frequently applied for CRM control and expenditures range from 75100 million dollars annually in Florida alone (Childers 1994a). Use of pesticides to 1

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2 control rust mites is a temporary solution because CRM has developed resistance to several compounds such as zineb (Voss 1988, Childers 1994b) and dicofol (Omoto et al. 1995). CRM hormoligosis has been observed in response to various copper formulations (Childers 1994b). Similar phenomena have been reported for different mite pests in diverse cropping systems, i.e. Panonychus ulmi on apple (Villanueva and Harmsen 1998), Tetranychus urticae (Koch) on roses and tree fruits (Goka 1999), and on strawberry and pear (Croft et al. 1984). Other phytophagous mites of importance in the citrus orchard ecosystem of Florida include the Texas citrus mite (TCM), Eutetranychus banksi (McGregor), and the citrus red mite (CReM), Panonychus citri (McGregor). Although the latter species are more sporadic pests than CRM and PRM, in some instances, they require acaricide applications. One possible way to break this cycle is through the use of selected pesticides that do not cause negative effects on natural enemies. Also, further research on natural enemies is needed to improve understanding of their ecological requirements and optimize their survival to the point where they might reduce CRM and PRM populations below economic levels. Predacious mites in the families Phytoseiidae and Stigmaeidae and certain predatory insects (cecidomyiid midges, neuropteran lacewings, coccinellid ladybeetles and several thrips species) have been identified as natural enemies that feed on citrus rust mites (Hubbard 1883, Muma et al. 1961, Peiia 1992, Childers 1994a). While natural enemies of phytophagous mites in citrus are abundant, few have been well studied and their biology and behavior in relation to eriophyid (CRM and PRM) pests remain largely unknown. In this study, I examined various aspects of selected natural enemies of the eriophyid and tetranychid mites including their distribution, food sources. 2

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3 host plant preferences and behavior. The natural enemies studied here included phytoseiid mites and insects (two cecidomyiids and a lepidopteran). The objectives pursued in this study were: 1) To evaluate the distribution of selected phytoseiid mite species between outer and inner leaves of the tree canopy to determine whether diurnal migration within the tree occurs (Chapter 2). 2) To compare the numbers of predacious and phytophagous mites between leaves damaged by Phyllocnistis citrella Stainton (Lepidoptera: Gracillariidae), healthy mature leaves and young leaves. Also, the relationship between numbers of phytoseiids and the presence of pollen grains on citrus leaves was examined (Chapter 3). 3) To evaluate the development and reproduction of Iphiseiodes quadripilis (Banks) on different diets in the laboratory and determine the acceptability of the citrus rust mite and the pink citrus rust mite (PRM) as food sources for different /. quadripilis life stages (Chapter 4). 4) To determine if/, quadripilis expresses a preference for grapefruit leaves over sweet orange leaves. (Chapter 5) 5) To characterize two cecidomyiid midge species and a predatory moth, Cryptothelea gloverii (Packard) (Lepidoptera: Psychidae), that have been observed preying upon citrus rust mite and describe their ecological relationships with pest mites and citrus plants (Chapter 6). 3

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4 Review of Literature A diverse fauna of predacious mites inhabit Florida citrus orchards including at least 33 different species of Phytoseiidae (Childers and Denmark, unpublished). Although these mites are important natural enemies of many pests within the citrus ecosystem, their role in controlling phytophagous mites is poorly understood. Many of these predators feed on pollen, some are generalist predators, and other species may have little or no association with pest mites (Muma et al. 1961, McMurtry 1977, Pena 1992, Childers 1994a). Furthermore, insects such as coccinellids, chrysopids and cecidomyiids have been described as predators of phytophagous mites, further complicating the ecological relationships between pest mites and their predators in the citrus ecosystem. The economically important tetranychid mites on Florida citrus include the citrus red mite (CReM) P. citri and the Texas citrus mite (TCM) E. banksi. Both TCM and CReM prefer dry conditions and are more abundant in the citrus-growing regions of California and Texas, respectively than in Florida. Both spider mite species can emerge as important pests in Florida from March through June (Childers et al. 1991; Childers 1994a). TCM has a tan to brownish-green color with dark green to black spots on the lateral sides of the body and its eggs are flat and vary from yellow to green in color. All instars of CReM and eggs are reddish in color. Both mites feed on green twigs and fruit and cause stippling on the leaves and mesophyll collapse (Albrigo et a/. 1981). When TCM feeding injury follows dry weather conditions in Texas, it may result in leaf drop in the tops of citrus trees (Dean 1980). TCM is presently the most common spider mite on Florida citrus of these two spider mites (Childers et al. 1996). 4

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CRM and the PRM mite are the most abundant and important eriophyid species on citrus in Florida (Childers 1994a, Childers and Achor 1999). The citrus bud mite (CBM) Aceria sheldoni (Ewing) also occurs in Florida but is not an important pest (Childers and Achor 1999). In contrast, A. sheldoni is an important pest in California. Denmark (1963) provided a key to separate these three eriophyid mite species. CRM has been reported on Florida citrus since the mid 1800 (Yothers and Mason 1930). Both CRM and PRM are found in all citrus-growing areas of Florida (Childers and Achor 1999). CRM and PRM are frequently misidentified, partly due to their small size. However, differences in their phenology, oviposition sites, body shape and coloration have been observed. PRM migrates earlier than CRM from leaves to fruit, sometime between April and May (Childers and Achor 1999). CRM eggs are spherical and tend to be deposited in depressions on the fruit surface including the oil glands, whereas PRM eggs are translucent and are deposited indiscriminately on the fruit surface (Childers and Achor 1999). CRM have a convex ophisthosoma (dorsal area) and CRM have a concave ophisthosoma (Denmark 1963). Body coloration is yellow to straw for the CRM and pink to reddish for the PRM, although this character alone is not conclusive for distinguishing the species. CRM and PRM feeding results in direct damage to epidermal cells of leaves, green twigs and russeting on the rind surface of froiit (Allen 1979, Albrigo et al. 1981, McCoy 1996). Heavily damaged fruit are discolored and have a reduced value in the fresh market (Albrigo and McCoy 1974). The conventional method to control citrus rust mites is with acaricides (Childers et al. 1996). Hubbard (1885) reported the application of sulphur powder to confrol CRM but this had negative impacts on phytoseiids and other natural enemies. Fungicides used for

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control of pathogens also have been used to control citrus rust mites but resistance to many of these products has developed (Childers 1990). In addition, fungicides have a negative impact on the pathogenic fiingus Hirsutella thompsoni Fisher, that attacks citrus rust mites at high population densities (Fisher and Griffiths 1950). Petroleum oils in combination with certain insecticides or acaricides (i.e. abamectin and ethion) also have been used to control rust mites and these mixes improved the residual activity of the acaricides (Childers et al. 1996). However, petroleum oil applications initially were restricted from the mid-summer to mid fall period due to tree and fruit injury factors (Carman 1977). New oil formulations with additives such as oxidation inhibitors prevented phytotoxicity (Kipiani 1976). Today, oil sprays are becoming increasingly important in integrated pest management on Florida citrus (Childers et al. 1996) and other parts of the world: Matsumoto et al. (1981a, b) in Australia and Japan, Walker and Aitken (1996) in California, Rae et al. in China (2000). Factors such low mammalian toxicity, low cost, and the additive residual activity when combined with certain acaricides, contribute to petroleum oil use. Further CRM has developed resistance to pesticides such as: diflubenzuron (Knapp et al. 1988), zineb (Voss 1988), fenbutatin oxide (Childers 1994b), dicofol (Omoto et al. 1995), fenpropathrin (French and Villarreal 1990). On the other hand, natural enemies (phytoseiid) populations are significantly lower in orchards that received heavy sprays of acaricides, insecticides and fungicides. Pesticides also induced resurgence of CRM populations (Voss 1988, Childers 1994b); due to hormoligosis (Dittrich 1987), and potentially leading to a pesticide treadmill effect. Moreover, costs for chemical control of citrus rust mites are at least 75 million dollars annually in Florida (Childers 1994a) but may be as high as 100 million (McCoy 1996).

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In addition to these pests, there are phytophagous mites that are considered of secondary importance and include the six-spotted mite (6SM), Eotetranychus sexmaculatus (Riley), the two-spotted spider mite (2SSM), Tetranychus urticae Koch, the broad mite, Polyphagotarsonemus latus (Banks) (Tarsonemidae), and a species complex in the genus Brevipalpus (Tenuipalpidae). These pest mites can cause serious localized damage in citrus orchards or in greenhouses on occasion. Special attention should be given to the Brevipalpus species because they transmit citrus leprosis virus in citrus and cause considerable losses in those countries where the disease occurs. The virus does not occur in Florida today (Childers et al. 2001) but it has been previously reported and referred to as nailhead rust or scaly bark in Pinellas County (Fawcett and Lee 1926) Phytoseiid mites are considered effective biological control agents of spider mites in numerous cropping systems (grapes Baillod et al. 1982; applesChilders and Enns 1975, Bostanian and Coulombe 1986; greenhouses Sabelis 1985a and Brodsgaard and Hansen 1992, De Moraes and McMurtry 1986; strawberry Easterbrook 1998; cassava Yaninek et al. 1998). There have been several studies on phytoseiid mites on Florida citrus, but the abundance, biology and predatory capacity of several species are inconclusive or unknown. Muma (1964) reported the abundance of phytoseiid mites on citrus leaves in a survey on north, central and south Florida as follows: T. peregrinus (= Amblyseius peregrinus) 62.9 %, /. quadripilis (= Amblyseius quadripilis) 15.3 %, Typhlodromips simplicissimus (De Leon) {--Amblyseius simplicissimus) 5.3%, Euseius hibisci (Chant) {= Amblyseius hibisci) 4.5%, Typhlodromina subtropica Muma and Denmark (= T. conspicua) 2.9 %, Galendromus alveolus (= G. floridanus) 2.8 %, and 10 other phytoseiid species represented 6.3 %. Pena et al. (1989) sampling for predators of

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8 P. latus in southern Florida lime trees found that T. peregrinus accounted for 72 % of all the predacious mites followed by T. dentilis (15%). Childers (1994a) reported that the most abundant phytoseiid mite on citrus was Euseius mesembrinus (Dean), followed by Typhlodromalus peregrinus (Muma), and Iphiseiodes quadripilis (Banks). The discrepancy between Childers (1994), and Muma (1964) and Pena et al. (1989) on the abundance of E. mesembrinus and T. peregrinus can be due to sampling location and/or time differences. Also, E. mesembrinus is a relatively new species in Florida, and first reported by McCoy and Rakha in 1985. Research on E. mesembrinus has demonstrated that it preys on tetranychids and also completes its life cycle on the pollen of various plants (Abou-Setta and Childers 1987, Badii and Hernandez 1993). However, studies using eriophyids as prey have produced unsatisfactory results. Abou-Setta (1988) stated that P. oleivora was not a suitable prey for E. mesembrinus. Badii and Flores (personal communication, Universidad Autonoma de Nuevo Leon, Mexico) could not rear E. mesembrinus on CRM alone, and even when E. mesembrinus was starved for 24 h and then placed in arenas with CRM, predation was not observed (personal observation). Muma (1967a) noted that T. peregrinus reproduces and develops on P. citri, E. sexmaculatus, E. banksi, white fly eggs and Parlatoria pergandei (Comstock) eggs and crawlers. Peila (1992) showed that adult T. peregrinus did not reject P. oleivora as a prey when it was provided as a food source but favored Polyphagotarsonemus latus when the latter was present. Fouly et al. (1995) reported that T. peregrinus completed its life cycle on diets of T. urticae, P. citri, and pollens of Malephora crocea Jacquin (ice plant), Quercus spp. (oak) and Typha latifolia L. (cattail). 8

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9 1 The shiny button mite (SBM), Iphiseiodes quadripilis is presently the third most abundant phytoseiid on Florida citrus (Childers and Denmark, unpublished data). The SBM has been described as a facultative generalist predator and it can feed on eriophyids, tetranychids, honey alone, citrus pollen and other less economically important citrus mites (Muma, 1971). However, little is known about the life cycle of SBM, its predatory behavior, or the ecological factors that influence its development. This is the least studied phytoseiid of the three previously discussed. The SBM can be easily recognized with the naked eye and it is distinguishable from other phytoseiid mites by its shiny, dark, mahogany-red color and its round, almost hemispherical body shape as an adult (Muma 1975). SBM can be misidentified with the velvet mite Phytoscutus sexpilis (Muma) in Florida, because the latter has a velvety, rose-red body color and similar body shape. However, under the dissecting stereomicroscoscope, four long slender setae project from the distal end of the body whereas the SBM has only two setae projected backwards (Muma and Denmark 1971). Stigmaeid mites are predacious on many pest mites but are less abundant than phytoseiids in citrus orchards (Childers 1994a). Muma et al. (1961) described five species of Stigmaeidae, Rakha and McCoy (1984) described an additional species, and recently Ueckerman and Childers discovered two more (personal communication). Agistemus floridanus Gonzalez is the most common and feeds on P. citri, E. sexmaculatus and Tydeus gloveri (Ashmead) (Tydeidae). Agistemus floridanus also feeds on CRM and A. floridanus adults were maintained on an exclusive diet of CRM alone by Muma and Selhime (1971). This report, although largely descriptive and without statistical analysis, indicated that A. floridanus had a long generation time and a 9

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10 low reproductive rate. Yue and Childers (1994) noted that the stigmaeid A. exertus Gonzales found on citrus had a lower prey consumption rate when compared with the phytoseiid Phytoseiulus persimilis Athias-Henriot. The significance of stigmaeids is discussed here because these predacious mites are important within the citrus acarine system and can prey on pest mites. Their relationship with phytoseiids has not been studied. Stigmaeids are commonly found in the apple orchards of southern Ontario and northeastern New York and the stigmaeid Zetzellia mali (Ewing) contributes to the biological control of the apple rust mite, Aculus schlechdentali Nalepa and the European red mite, Panonychus citri Koch (Villanueva 1997). Also, Z. mali feeds on the eggs of the phytoseiid Typhlodromus caudiglans (Schuster), but phytoseiids were unable to consume Z. mali eggs (Clements and Harmsen 1990). MacRae and Croft (1996) found a similar behavior with Z. mali in Oregon although in this case the phytoseiids Metaseiulus occidentalis Nesbitt and Typhlodromus pyri (Schueten) consumed a cumulative mean of < 1 Z mali egg or immature after 6 days. Both phytoseiids and stigmaeids can contribute to pest mite control under high prey densities. The competitive relationship between these two families of predacious mites was described in apple orchards (Clements and Harmsen 1990) and can occur on Florida citrus. The acarine fauna on citrus orchards in Florida includes a diverse group of species from different families. Most do not damage citrus and their potential capacity as natural enemies is poorly understood. These mites include different species in the following predacious families: Anystidae, Bdellidae, Eupalopsellidae, Cheyletidae, and Cunaxidae. Also, there are mite species that can be an alternative prey. The latter group includes the 10

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11 Tydeidae, Tarsonemidae, and Acaridae (fungus mites). These two groups have not been well studied and their relationships with phytoseiids are unclear. McMurtry (1992) noted that generalist phytoseiids predominate in citrus in California. These species consume a variety of foods, including some of plant origin such as pollen (McMurtry and Croft 1997). It is possible to hypothesize that the feeding and attacks on CRM by phytoseiids may be lessened due to: 1) the presence of alternative prey that are larger or more abundant than CRM. This alternative prey present on Florida citrus orchards includes tetranychids, tydeids, tarsonemids and fungal mites. 2) The alternative prey also can be a more suitable prey and enhance phytoseiid reproductive capacity; and 3) generalist phytoseiids rather than specialist predators are more abundant in citrus, which was noted by McMurtry (1992) in California. McMurtry and Croft (1997) defined 'generalists' as those species that consume a variety of foods including some of plant origin such as pollen in some perennial plants. Two of the most abundant phytoseiids of Florida, E. mesembrinus and T. peregrinus fall within this category. Either one or all of these factors may contribute to lessen the impact of phytoseiids on eriophyids in Florida citrus. In addition to the mite and insect predators of phytophagous mites described above, the fiingus H. thompsonii was first reported by Yothers and Mason (1930) in association with P. oleivora. Subsequent evaluations of H. thompsonii have been made by several authors (Muma 1955a, McCoy et al. 1971, McCoy and Couch 1982). However, to be effective H. thompsonii requires high CRM population densities and high relative humidity to reach epizootic levels. McCoy (1985) was unable to reduce CRM populations by spraying H. thompsonii formulations. 11

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12 Insects also contribute to reducing phytophagous mite populations and four orders of insects are known to prey on CRM on citrus. Hubbard (1883) found a midge larva (Diptera: Cecidomyiidae, formerly Itonididae) feeding on CRM and later Muma et al. (1961) collected Itonidini species within P. oleivora infestations. Yothers and Mason (1930) described Stethorus nanus LeConte (Coleoptera: Coccinelidae) and Chrysopa sp. (Neuroptera: Chysopidae) preying upon CRM. Muma (1958, 1967a; Muma et al. 1961) also mentioned Coniopteryx vicina Hagen (Neuroptera: Coniopterygidae) preying upon CRM. The thrips Scolothrips sexmaculatus (Pergande) and Leptothrips mali (Hunter) (Thysanoptera) have been reported as predators of CRM by Bailey (1939) and Muma (1955b), respectively. This study focuses only on cecidomyiids and Cryptothelea gloverii (Packard) (Lepidoptera; Psychidae) found on citrus and their predacious behavior toward the citrus rust mites. The genus Feltiella has a woridwide distribution (Gagne 1989) and all described species form a distinctive group associated exclusively with spider mites (Tetranychidae) Gagne (1995). Feltiella acarisuga (Vallot) has been reported preying on tetranychid mites in various crops such as vegetables (Opit et al. 1997, Gillespie et al. 2000), and strawberries (Croft et al. 1998, Easterbrook 1998, Enkegaard et al. 2000). However, there are few examples of predation by cecidomyiids on eriophyid mites. The eriophyid Aceria litchii Keifer is a serious pest of Litchi chinensis Sonnerat (Sapindaceae) in Australia and China and is preyed on by a cecidomyiid larva Arthrocnodax sp. (Waite and Gerson 1994). Medetera species (Diptera: Dolichopodidae) have been observed preying on Aculus schlechtendali Nalepa on apple in Washington (Rathman et al. 1988). Cecidomyiids have been reported feeding on CRM in California (Hubbard 1883) and 12

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13 Florida (Muma et al. 1961) but these reports were vague and the dipteran species were not identified. The orange bagworm (OBW) Cryptothelea gloverii (Packard) (Lepidoptera: Psychidae) was first described in Florida in 1858 (Hubbard 1885). The larvae of bagworms can be easily recognized by their portable case or bag in which they reside. It is constructed of dense silk and then covered with bits of dry leaves, bark and other scraps (Quayle 1938). The bag has an irregular conical shape and, when the larva is fully grown, it is approximately 1 .8 and 1 .3 cm long for females and males, respectively (Hubbard, 1885). Hunt (1923) described an outbreak of OBW on orange in Sebring, Florida, and reported damage on fruit, growing twigs, and mature leaves. The OBW was also described as a predator of the camphor scale Pseudaonidia duplex Cockerell, the soft brown scale Coccus hesperidum L, a species of Ceroplastes, and black scale Saissetia olea (Olivier) (Plank and Cressman 1933). No further information on this insect is available on citrus. 13

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CHAPTER 2 PHYTOSEIID DISTRIBUTION ON LEAVES SAMPLED THROUGHOUT THE DAY AND EFFECTS OF LEAF POSITION IN THE CITRUS CANOPY Introduction , The small size of predatory phytoseiids (and many other mites i.e. stigmaeids) is a factor that makes mite field studies more laborious. In addition, phytoseiids are scarcer than their prey, but they are more active and faster than stigmaeids (Muma and Selhime 1971, Chant 1985). These predators are continuously moving when they are foraging for prey or food (Sabelis 1985b). This foraging behavior, however, may not only depend upon prey availability but also on abiotic factors such relative humidity, temperature and light intensity. Muma (1967b, 1975) observed that T. peregrinus is active at night and prefers shaded leaves during the day. Allen and McCoy (1979) also noticed that the distribution of citrus rust mites on individual fruit and in the whole citrus tree suggests an avoidance of direct sunlight. Villanueva and Childers (unpublished) also noticed analogous responses of P. citri and E. banksi on citrus. Other researchers studying other species of phytoseiids (C.C. Childers; R. Harmsen; personal communications) have observed similar behavior. This type of behavior can be described as negative photo-taxis (Fraenkel and Gunn 1961) and may have evolved as a defense mechanism against high temperatures and low humidity on external leaves during daylight hours. Lideed, the large body surface area and small size of these mites results in increased water loss potential when exposed to 14

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15 > * light. Therefore, phytoseiids may find better micro-environmental conditions on internal leaves rather than on outer leaves (Allen and Syvertsen 1981). Time of the day for sampling may influence phytoseiid counts because most of the orchards in Florida are set north-south (Wheaton et al. 1978). This is because phytoseiids may be affected by the direct incidence of sunlight that can increase the temperature of the eastern leaves in the morning and the western leaves in the afternoon. Hence phytoseiids can migrate from the east to the interior leaves in the morning and in the afternoon they may return when these leaves become shaded. Similar movement of phytoseiids may occur on the western side but in the opposite direction. Temperature and relative humidity in the interior leaves of the tree canopy are more constant compared with conditions on the outer leaves (Ferro et al. 1979) and these factors indubitably affect mite populations. Moreover, we must consider the existence of a boundary layer on fruit or leaf surfaces. The boundary layer is a thin space above the leaf surface up to 1000 um deep, where temperature, and %RH are more constant than the surrounding environment (Allen and Syvertsen 1981). The thickness of the boundary layer decreases with high winds (Gates 1968). This boundary layer is the result of leaf or fruit franspiration and provides unique micro-environmental conditions on the surface with different gradients of temperature and humidity than their surroundings. Small mites such as eriophyids are able to survive and flourish in this highly localized microenvironment. The boundary layer also provides resistance to water vapor diffusion and resistance to heat transfer between a leaf and its surroundings (Martin et al. 1999). The east side of the tree canopy receives direct solar radiation during the morning hours; whereas the west side of the tree canopy is affected in the afternoon. Solar radiation

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16 would be significantly different in small body sizes and it may be possible that this affects phytoseiid densities depending on how the solar radiation is directed. The aim of this chapter is to determine whether there are any differences in phytoseiid populations on the east, west or interior leaves of the tree canopy depending on time of sampling. In addition, the distribution of phytoseiids within the tree canopy was assessed to determine if time of sampling resulted in phytoseiids density changes. Materials and Methods This experiment was conducted in a grapefruit orchard in Highland City, Polk County, Florida. The trees were 20 years old and the orchard was selected because of the occurrence of high numbers of phytoseiid mites based on previous experiments. The trees were minimally sprayed. Four 'Marsh' grapefruit trees were selected, flagged and marked. All trees were in the same row and separated from each other by 8 to 10 trees. Leaves were collected randomly from the tree canopy between 1 and 2 m. per sample type. Forty leaves were collected from each of the interior, east and west sides of each of four frees. Each 40-leaf sample was placed in an individual labeled jar containing between 0.2 to 0.3 liters of 80% ethanol and taken to the laboratory, hi the laboratory, leaves were rinsed in water over a No. 400 sieve with an average opening of 38 urn. The residues including mites, small organic particles, soil and spider webbing remaining in the sieve were placed in separate labeled jars with 80% ethanol for each sample. Later all of the phytoseiids found in each jar were sorted and counted using a dissecting stereomicroscope. The phytoseiids from each sample were then placed in a small vial containing 80% ethanol and labeled for eventual slide preparation. Mites were slidemounted in Hoyer's medium (Krantz 1978), oven dried and later identified to species.

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17 Samples were collected on 14, 20 and 28 October 1999 at 2 h intervals (0600, 0800, 1000, 1200, 1400, 1600, 1800, 200 and 2200 h). Similar types of samples were collected on 9 and 16 March and on 17 and 24 August 2000. However, in the last two months, the leaves were collected at 3 h intervals (0600, 0900, 1200, 1500, 1800 and 2100 h.). All dates were selected when the weather forecast indicated clear and sunny days with low probabilities of rain. To record environmental conditions, three HOBO® data loggers (Onset Computer Corporation, Bourne, MA) were used during each of the sampling dates and placed on the east, west and interior parts of the tree canopy. The HOBO data loggers registered temperature in Celsius degrees, % relative humidity and light intensity in lumens. Phytoseiid data were transformed as ^J{X + 0.5) , where A' is the number of phytoseiids found on each 40-leaf sample. A two-way analysis of variance was performed on all data followed by Tukey's Honest Significant Difference (HSD) test for separation of means using Statistica ® (Stat Soft, Inc. 2000). Figures presented show original mean values of data without transformation ± standard error of means (SEM). K Results Cultural practices such as disking, hedging, mowing, application of fertilizers and pesticides for 1999 and 2000 are presented in Table 2.1. Environmental conditions for the sampled dates are presented in Fig. 2. 1 . Environmental data for August showed that mean %RH inside the tree canopy was 7 to 1 1% higher than the outside canopy (east and west) %RH. Temperatures were 3.3 to 4.7° C lower inside the tree canopy than outside and the light intensity was 400 lumens lower on the shaded side versus the sunny side of

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18 the outer canopy. Environmental data for October 1999 and February 2000 showed similar trends (Fig. 2.1). The environmental conditions inside the canopy might be more favorable for phytoseiid development than the outer canopy. The two-way analyses of variance for all the sampled dates are presented in Table 2.2. Position of leaf sampling (east, west, or inside tree canopy) showed significant differences (P < 0.05) in the number of phytoseiids but effects of sampling time throughout the day and the interaction were not significant on any date (P > 0.05). Grouping the total number of phytoseiids found for all the three dates in October, 1999 yielded 346, 366, and 248 phytoseiids in each of the 1,440 leaf samples. Similarly, for March, 2000 the numbers were 599, 1,964, 613 on the east, inner and west sides, respectively, for each of the 960 leaf samples. For August 2000, the numbers were 69, 435, and 37 on the east, inner and west side of the tree canopy, respectively, for each of the 960 leaf samples. More phytoseiids were collected in the March 2000 samples (mean ==1.10 phytoseiids/leaf) than in October 1999 or August 2000 (mean = 0.16 and mean = 0.19 phytoseiids/leaf, respectively). No significant differences were obtained for any of the samples in October 1999 using Tukey's honest significant differences (HSD) test (Fig. 2.2). hi contrast, samples taken in March 2000 resulted in the highest numbers of phytoseiids collected (Fig 2.3). Samples taken on 16 March at 1200 h had the highest number with 60 ± 14.12 (mean number ± SEM) phytoseiids /40 leaves. Significantly higher numbers of phytoseiids (P < 0.05, Tukey's HSD test) were obtained from the inner leaves than fi-om either east or west leaf samples at 1200,1500,1800 and 2100 h on 9 March 2000 and during all the times throughout the day on 16 March (Fig. 2.3) using HSD Tukey's test. The numbers

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19 of phytoseiids counts in the August samples were very low. However, the trend was similar to the March samples. Most of the predators were found on the inner than outer leaves (Fig. 2.4). Significant differences were found (P < 0.05, Tukey's HSD test) at 0600, 1200, and 2100 h on 17 August; and again at 0600, 1200, 1500 h on 24 August. The phytoseiid species found in the orchard were: Typhlodromalus peregrinus (n = 870), Iphiseiodes quadripilis (n 282), Euseius mesembrinus (n = 184), Typhlodromina subtropica Muma and Denmark (n = 21), Typhlodromips dentilis (De Leon) (n = 20), Clavidromus transvaalensis (Nesbitt) (n = 2), Galendromus helveolus (n = 1), Typhlodromalus sp. (n = 1) and Phytoscutus sexpilis (n = 1). Their distributions in the tree canopy are presented in Fig. 2.5. The most abundant species were T. peregrinus (42.1%) and /. quadripilis (50.4%) on October 1999. The most abundant species was T. peregrinus (76.2%) in March 2000 and, E. mesembrinus was the most abundant (54.1% of all identified species) in August 2000. Discussion The relative abundance of phytoseiids during the three periods of sampling (October 1999, March 2000 and August 2000) was dissimilar and the cause of this can be explained by the phenology of these mites. Generally phytoseiid species are more abundant from March to May (Childers 1994a). Muma (1967b) referring to T. peregrinus (then the most abundant phytoseiid in Central Florida) stated that its population was larger during late winter and early spring. This coincides with the data presented here and also can be explained by the presence of pollens {Citrus sp., Quercus sp, Pinus sp., and various weed species) that are abundant in March (see Chapter 3).

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' ' ' 20 i No significant differences in abundance of phytoseiids {F>0.05) were obtained for the sampling times throughout the day. This latter result is unexpected because the initial hypothesis for sampling throughout the day was that direct solar radiation on the east sided leaves during the morning would initiate a migration of phytoseiids from the east to the inner leaves. Also, it may be possible that phytoseiids would be foraging on the west side because this direct sunlight increases temperatures and reduces %RH on the east side. Likewise the opposite would happen on the west side; west sided leaves would have fewer mites at mid afternoon than the eastern leaves for similar reasons. This result can be explained by the preference of phytoseiids for the lower leaf surface and the boundary layer of the leaf surface that acts like a buffer producing more stable environmental conditions, hi addition, phytoseiids can also rest in crevices and bark of the citrus trunk during times of high solar radiation. Despite the solar radiation the eastsided leaves received during the morning, the environmental conditions between these leaves and the west-sided leaves might be similar within the space enclosed by the boundary layer compared to their surroundings (Gates 1968, Ferro et al. 1999). Still, there are studies that reported phytoseiid differences in sampling times throughout the day in citrus. Garcia Mari et al. (1985) reported that Euseius stipulatus ((Athias-Henriot) was more abundant at night between 2200 and 2400 h than sampling during the daylight on outer leaves of 'Washington' navel in Spain. Muma (1967b) also described a similar nocturnal abundance of T. peregrinus in Central Florida. Garcia Mari et al. (1985) and Muma (1967b) explained this behavior of phytoseiids as a search for prey. Phytoseiids moved from mature inner leaves to the outer young leaves at night because these leaves had more tetranychid prey. McMurtry et al. (1970) explained this behavior as a search

PAGE 34

21 for shaded areas and Ferragut et al. (1988) explained that phytoseiids preferred trees with abundant foliage. On other plants, phytoseiids also used domatia structures as a refuge site (O'Dowd and Willson 1997, Walter 1996, Roda et al. 2000, Norton et al. 2001). Recently, Onzo et al. (2002 In Press) observed that the phytoseiid Typhlodromalus aripo De Leon rested in domatia structures during the day on cassava and searched for the green cassava mite Mononychellus tanajoa (Bondar) on leaves at night. Domatia do not exist on citrus leaves but phytoseiids use mines created by the leafminer Phyllocnistis citrella Stainton (Gracillariidae) in a similar way to domatia structures since they provided shelter to the predator (see chapter 4). > ' " No significant differences (P > 0.05) were obtained for the interaction between sampling hours and sampling side (Table 2.2). However, significant differences {P < 0.05) occurred with the two-way ANOVA on the numbers of phytoseiids obtained in sampling position (east, inside or west side of tree canopy) for all dates (Table 2.2), but these differences did not occur {P > 0.05) with the Tukey's HSD post-hoc test of means for all dates sampled on October 1999 (Fig. 2.2). The highest number of phytoseiids found during October was 10.0 ± 1.43 (mean ± SEM) motiles/40 leaves from samples collected on 20 October at 0800 h (Fig. 2.2). This number is very small (0.25 phytoseiids/leaf) if we extrapolate the number to phytoseiids/leaf Hence the low number of phytoseiids found during this month produced greater variability between the sampling sides and this reduced the probabilities to observe differences. These lower phytoseiid numbers may be affected negatively by the herbicide glyphosate (Roundup ®) and the insecticide chlorpyrifos (Lorsban ®) sprayed on 13 September 1999 just one month before the October 1999 sample and the petroleum oil spray on 17 July 2000 one month

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22 before the August 2000 sample. Chlorpyrifos is relatively non-toxic to E. mesembrinus but has a repellent effect and petroleum oils are moderately toxic to non-toxic to this phytoseiid species (Childers et al. 2001). The samples collected in March and August 2000 showed that higher numbers of phytoseiids were present on the inner leaves (Figs. 2.3 and 2.4) than the outer leaves of the tree canopy. Significant differences (P > 0.05, Tukey's HSD test) were found for many of the hours sampled. In March, this is apparent because the numbers of mites were the highest observed (0.6, 2.0, 0.6 phytoseiids/leaf on the east, inner and west side of the tree canopy, respectively). Although phytoseiid numbers in August were lower than in March, the phytoseiids showed a similar trend. More phytoseiids were found in the inner canopy compared to leaves collected from the east and west sides (0.4, 0.07 and 0.03 phytoseiids/leaf, respectively). Phytoseiid numbers on the inner leaves showed considerable variance but were significantly different {P < 0.05) when compared to outer leaf samples mainly because the outer east and west leaves of the tree canopy were very low (Figs. 2.3 and 2.4). This latter observation can be explained as an adaptation of phytoseiid mites to inner leaves that work like a refuge, protecting them fi^om harsher environmental conditions that occur on outer leaves exposed to higher temperatures and lower humidity conditions (Fig. 2.1). Muma (1967b) observed similar results with T. peregrinus. However, he provided only mean numbers without statistical analysis or time of the year that experiments took place. Although Fig. 2.5 can be misleading because it is expressed in percentages; numerically the most ubiquitous and abundant phytoseiid was T. peregrinus during the three month sampling periods. Also, in Fig. 2.5 it is evident that both /. quadripilis and

PAGE 36

T. peregrinus were equally abundant in October 1999, and T. peregrinus was the most abundant in March 2000, whereas E. mesembrinus was the most abundant phytoseiid in August 2000. Muma (1964) observed similar density patterns in the winter and summer months with T. peregrinus. It is possible that the observed densities of E. mesembrinus were due to the mite's phenological cycle. There is not enough evidence to confirm this categorically due to the low numbers observed in the summer months. However, /. quadripilis was found throughout the year (Muma and Denmark 1971) and this corresponds to the results observed here. Childers and Denmark (unpublished) found similar seasonal abundance trends with these species, although they noted (personal communication) that E. mesembrinus was most abundant followed by T. peregrinus and / quadripilis. Their study also was more extensive and intensive. It included 7 orchards in central and south Florida and there were monthly samples taken from 1994 to 1996. Data presented here however, coincides with Pena et al. (1989). They found that T. peregrinus was the most abundant phytoseiid in southern Florida with 72.4 % although the total numbers of predators identified were only 472. The rest of the phytoseiid species identified are found in Florida although they are less common and poorly studied (Muma 1964, Muma and Denmark 1971). March and August 2000 (Figs. 2.3 and 2.4) data have shown that phytoseiid mites are more abundant on the inner leaves of the tree canopy. However, numbers of phytoseiids obtained in October 1999 (Fig. 2.2) were low, and this may be the reason why significant differences were not observed. Phytoseiid abundance on Florida citrus is generally observed during the months of March to May. This is one reason why the March data resulted in higher phytoseiid numbers and not to the sampling technique employed. This

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24 peak of phytoseiid abundance coincides with the pollen season of orange and grapefruit and several trees (oak, pines and various weed species) and with tetranychid mites abundance in Florida. An explanation to the increase in the phytoseiid populations between March and May is the presence of abundant pollen during these months. Evidence of pollen feeding by the three most abundant phytoseiids was presented in studies by Abou-Setta and Childers (1987) on E. mesembrinus, Pena (1992) on T. peregrinus and in chapter 4 for /. quadripilis. All three species completed their life cycle and reproduced on different pollen diets alone in the laboratory. From results presented here we confirm that sampling time through the day did not affect phytoseiid densities and that there is no evidence that phytoseiids move from the inner leaves to the outer leaves during the night. However, the data presented confirm that phytoseiids prefer inner leaves compared to outer leaves of citrus. This can be due to more adequate microenvironmental conditions (temperatures and relative humidity) in inner than outer leaves.

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25 Table 2.1 Agricultural practices employed in the Bader grove in Highland City. iviaieridi / rrdciicc rrociut/i iNdme Kaie / iieciare 1 Mar 1999 Disking 1 way 24 Mar 1999 Hedging — 10 May 1999 Herbicide spray Herbicide spray Insecticide Glyphosate Simazine Chlorpyrifos 2.8 1 4.7 1 0.471 30 May 1999 Fertilizer Insecticide spray Techmangam Zinc Soluble oil 14.0 Kg 7.0 Kg 58.41 28 Jul 1999 Disking 1 way 31 Jul 1999 OUIUUIC Ull 88 8 1 oo.o 1 1 3
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26 Table 2.2. Two-way analysis of variance for data on numbers of phytoseiids found on the east, inner and west sides of the tree canopy at different sampling times on indicated dates during the year. Source di mh biiect F value n 1 r-value Side 2, 81 0.754 3.348 0.040 1 A r\^41 Ann 14 Oct lyyy Hours 8, 81 0.628 2.789 0.008 Interaction 16, 81 0.376 1.671 0.069 Side 2, 81 1.066 3.253 0.043 2u (Jet lyyy Hours 8, 81 0.401 1.224 0.295 Interaction 16, 81 0.286 0.875 0.598 Side 2, 81 loi.ooy 13.423 < 0.001 /o uct lyyy Hours 8, 81 8.363 1.111 0.364 Interaction 16, 81 6.1039 0.811 0.669 Side 2, 54 38.616 34.216 < 0.001 y Mar zUUU Hours 5, 54 1.502 1.330 0.265 Interaction 10, 54 0.882 0.781 0.645 Side 2, 54 99.328 55.948 < 0.001 10 Mar zUUU Hours 5, 54 2.799 1.576 0.182 hiteraction 10, 54 1.382 0.778 0.648 Side 2, 54 38.616 34.216 < 0.001 17 Aug 2000 Hours 5, 54 1.502 1.330 0.265 Interaction 10, 54 0.882 0.781 0.645 Side 2, 54 99.328 55.948 < 0.001 24 Aug 2000 Hours 5,54 2.799 1.576 0.182 [nteraction 10, 54 1.382 0.778 0.648

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27 Hours Hours Hours Figure 2.1. Daily average relative humidity (%), temperature (°C) and light intensity (Lumens) on 14, 20 and 28 October 1999, 9 and 16 March 2000, and 17 and 24 August 2000 on the east, west and inside areas of the tree canopy at the Bader orchard in Highland City, Florida.

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28 12:00 19:00 »:00 15:00 18:00 21:00 12:00 1S:00 18:00 Time Figure 2.2. Mean number of phytoseiid motiles ± SEM for 14, 20 and 28 October 1999. Values bearing the same letter were not significantly different using the HSD Tukey's test (P < 0.05).

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70 9 Mar 2000

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30 Figure 2.4. Mean number of phytoseiid motiles ± SEM for 17 and 24 August 2000. Values bearing the same letter were not significantly different using the HSD Tukey's test {P < 0.05).

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31 Figure 2.5. Percentage of phytoseiids identified from 30% of the total numbers found on October 1999, March and August 2000. Text box number indicates the total number of phytoseiids identified each month.

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32 CHAPTER 3 LEAVES DAMAGED BY THE CITRUS LEAFMINER PROVIDE REFUGES FOR PREDATORY MITES IN CITRUS Introduction Topographical or morphological changes on the leaf surface due to feeding and/or damage by pests may produce changes in small arthropod populations including mites. One of the pests that causes change of leaf surfaces is the citrus leafininer (CLM) Phyllocnistis citrella Stainton (Lepidoptera: Gracillariidae). The CLM is a recently introduced pest into the USA. It was reported for the first time in Dade County, Florida in 1993 followed by rapid spread throughout the rest of Florida (Heppner 1993) and neighboring states including: Alabama, Louisiana and Texas in August 1994 (Legaspi et al. 1999). The CLM was initially considered a serious pest in Florida but is currently under control by native natural enemies (Amalin et al. 2001), introduced parasitoids (Hoy and Nguyen 1997) and IPM programs. Today, the CLM is considered a secondary pest problem (Browning et al. 2001). Adult CLM females deposit eggs singly on young citrus foliage (flush) preferring the midrib on the abaxial surface, and occasionally on green twigs and young fruits. Immediately after hatching, the CLM larva bores through the leaf epidermis and forms a mine due to the ingestion of the outer epidermal cells. Feeding by the larva leaves the outer epidermal wall and cuticle intact to form the outer wall of the mine (Sohi and Verma 1965, Achor et al. 1997). Later, chlorotic leaf patches are

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33 observed where the larva mined. The mine is serpentine in form and each succeeding larval instar widens the tunnel. Mines caused by CLM larvae prevent young leaves from expanding uniformly with folding and/or twisting of leaves eventually occurring. The third larval instar causes most of the damage and this prepupal larva prepares a pupal chamber by folding the edge of the leaf down or upwards and binding it together with silk (Hoy and Nguyen, 1997). This damage facilitates other insects such as aphids and mealybugs to continue feeding on the leaf and the citrus plant becomes increasingly vulnerable to infection by Asiatic citrus canker caused hy Xanthomonas axonopodis pv. citri (Hasse). Large amounts of inoculum are produced in these mined areas that promote the spread of the bacterium by rain splash (Gottwald et al. 1997). Phylloplane fungi may have better growth in mined leaves than in healthy leaves (Timmer, personal communication). Phylloplane epiphytic fiingi grow externally on a leaf surface without parasitizing it. During the winter months, these fungi can develop better in mined leaves than in healthy leaves due to the sheltered microenvironment provided by the damaged leaves. Most mined leaves maintain their photosynthetic capacity although it is reduced during the remainder of their development. Wang et al. (1999) identified 68 different isolate species of phylloplane epiphytic fungi on citrus in China. Leaves folded at the edge by third instar CLM are sheltered spaces that provide refugia to other small arthropods. These spaces may provide more favorable microenvironment conditions than leaves without CLM damage. The sheltered spaces resemble structures called domatia that are present on leaves of many tropical and subtropical plants (O'Dowd and Wilson 1989). Domatia are located on the lower surface

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34 of the leaves of many plants and occur in many different forms. Domatia are located at the junction of the primary and secondary veins and sometimes are protected by leaf hairs (O'Dowd and Willson 1997, Norton et al. 2001). Domatia are classified in one of the following types: pockets, cavities beneath expanded veins, pits, cavities which sink into the mesophyll, dense hair tuffs; and rolled leaf margins, which are less common (Pemberton and Turner 1989) The physiological functions of domatia are unknown but it is hypothesized that they have a co-evolutionary development with natural enemies, as a protective association against pests (O'Dowd and Willson 1989, Agrawal 1997, Agrawal and Karban 1997). Various authors have demonstrated that when these structural openings are covered or blocked the populations of predacious phytoseiid mites diminish, while pest mites increase. When artificial domatia were placed on leaves by attaching small pockets on lower leaf surface, phytoseiid mite numbers increased and pest numbers decreased (Walter 1996). A grapefruit leaf or any citrus leaf of commercial varieties is smooth and lacks domatia. In contrast, leaves with domatia often times have a rougher surface with veins protruding and/or hairs occurring around the axes of the veins. Phytoseiid mites were classified into four categories by McMurtry and Croft (1997): Type I, specialized predators of Tetmnychus species represented by Phytoseiulus species; Type n, selective predators of tetranychids mites represented by Galendromus, some Neoseiulus and a few Typhlodromus species; Type HI generalist predators represented by most Typhlodromus and Amblyseius species; and Type IV specialized pollen feeders/generalist predators represented by Euseius species. The three most prevalent phytoseiids on Florida citrus are: Euseius mesembrinus (Dean), Typhlodromalus

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35 peregrinus (Muma) (Childers 1994a) and Iphiseiodes qmdripilis (Banks) (See Chapter 2 and Childers and Denmark unpublished). All three species completed their life cycles and reproduced on pollen (Abou-Setta 1987, Pena 1992, Badii and Hernandez 1993, McMurtry and Croft 1997, see chapter 4 for /. quadripilis). Similar studies have been shown with other phytoseiids in different parts of the world (Castagnoli et al. 1999, van Rijn and Tanigoshi 1999). It is apparent that many of the phytoseiids in Florida belong to either type HI and IV species. Furthermore, one of the phytoseiid peaks in Florida coincides with citrus, Quercus sp. and Pinus sp. flowering and the species involved are: E. mesembrinus, T, peregrinus and/, quadripilis (Childers and Abou-Setta, 1999). Quercus sp. and Pinus sp. are commonly found around citrus orchards as wind breaks. Pollen grains from these plants and other species of weeds and shrubs accumulate on the adaxial surface of citrus leaves and almost certainly the three phytoseiids forage for pollen to complement their diet breadth. Studies between the density of pollen grains and phytoseiid abundance were completed in apple orchards in Canada with Typhlodromus pyri Scheuten (Addison et al. 2000), and on citrus in South Africa with Euseius addoensis addoensis (van der Merwe and Ryke) (Grout and Richards 1992a, b). These studies demonstrated a high correlation between early pollen availability and phytoseiid abundance in the field. For convenience and considering that this study was developed during the time of the year with the highest pollen abundance {Citrus sp., Quercus sp. and Pinus sp. flowering occurs by the end of February to middle March); the relationship between pollen grains deposited on the adaxial leaf surface and phytoseiid abundance on leaves was studied.

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36 Two main objectives are pursued in this chapter. First, to compare the relative abundance of phytophagous and predatory mite populations between CLM damaged leaves, mature healthy leaves and young leaves. Second, the relationship between numbers of phytoseiids and pollen grains found on sampled leaves in the same orchard for the duration of this study was assessed. Material and Methods ^ , v., '. : > f / Weekly samples were taken from eleven-year-old 'Ruby red' grapefruit trees at the Citrus Research and Education Center in Lake Alfred. All ten sample trees were in one row and selected at random. Samples were taken from 2 February to 13 April 2001 between 1000 and 1 100 hours and consisted of terminals with 5 leaves. Terminals were of three types: healthy mature terminals without CLM damage and terminals with leaves that had suffered CLM damage and young terminals with 5 leaves. The first two types of terminals had leaves that flushed the previous year while the third type of terminals were sampled from 23 March through 13 April. These latter terminals started to flush around mid-March 2001 . This grove had not received any pesticide sprays since September of the previous year. On 5 April, the grove was sprayed with carbaryl (Sevin® 4F) at a rate of 94.1 kg/ha in 2.8 Kl of water to control the eastern lubber grasshopper /?oma/ea microptera (Beauvois) (= R. guttata) (Orthoptera: Acrididae). The spray was applied using a handgun directed to the ground and up to 1 m high on the frees because the flightless grasshopper nymphs concentrated there. Pollen counts were taken from 2 February to 16 March (during grapefruit flowering in this orchard). One healthy leaf from each of the same 10 frees sampled was collected for pollen counts, hi the laboratory, immediately after sampling, a 5 to 7 cm long fransparent adhesive tape was placed along the middle vein on the upper leaf surface of each leaf and

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37 left there for 3 to 5 minutes, then removed, placed in a wax paper, labeled and stored in the refrigerator until processed. Slides were prepared from 1 cm^ sample areas of the adhesive tape. These 1 cm^ areas of tape were placed individually on the slide with the adhesive part up and then a drop of dye was added followed by placement of the cover slide (Addison et al. 2000). The dye was prepared with 0.2 g of Trypan Blue in 200 ml of 50% glycerol. This dye is used for ascospore staining (Skaria and Tao 1996) and glycerol will cause pollen grains to slightly swell (Addison et al. 2000). A confrast microscope was used in the laboratory to count pollen grains, with a magnification of 400X. The area for counting pollen grains (field of view) was approximately 1.130 mm^. All pollen grains in this field of view were counted and the area within the field of view was calculated with a Hemacytometer Bright Line ® (Reichert, Buffalo, NY). Pollen counts were completed as follows. The first grain of pollen found searching the slide was centered in the middle of the field of view and then all the pollen grains in that view were counted. The three types of terminals were taken to the laboratory and counts of phytophagous and predatory mite eggs and motiles were taken using a stereomicroscope. Data were transformed using + 0.5 , where ^ is the number of eggs or mofile mites per 5-leaf terminals. ANOVA was used to analyze the data and the means for the last three sample dates were compared using an LSD test (Multiple Range Test for separation of means using Statistica ® (Stat Soft, Inc. 2000). Means ± SEM values shown here are untransformed data. Pollen counts and the total number of phytoseiids present from leaf samples on the indicated dates were evaluated to determine correlations between the two factors.

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Results Pests mites found in this study included: Eutetranychus banksi (TCM), Eotetranychus sexmaculatus (6-SSM), citrus rust mites (CRM and PRM) and a few Brevipalpus sp., while not a single Panonychus citri was found. The phytoseiids: E. mesembrinus, T. peregrinus and I quadripilis; the stigmaeid Agistemus sp., and a few Stethorus sp. (Coccinellidae) larvae and adults were also found. Stethorus sp. is a predator associated with E. sexmaculatus (Muma et al. 1961). Tydeids mites were also present in this orchard, but their populations were low with no significant differences found among the three types of terminals {P > 0.05). Tables 3.1 and 3.2 present the ANOVA and the least significant difference (LSD) tests for^". banksi eggs and motiles, respectively. Figures 3.1 and 3.2 present the means (± SEM) for E. banksi eggs and motiles, respectively. Significant differences (P < 0.05) were found in the numbers of E. banksi eggs on 23 March and 13 April, when E. banksi preferred to oviposit on the new young leaves (Fig. 3.1). However, no differences were found for E. banksi motiles on any of the dates (Fig. 3.2). Tables 3.3 and 3.4 present the ANOVA and the LSD tests for £. sexmaculatus eggs and motiles, respectively. Figures 3.3 and 3.4 present the means (± SEM) for E. sexmaculatus eggs and motiles, respectively. Significantly {P < 0.01) higher numbers of both E. sexmaculatus eggs and motiles were found on mined leaves than on healthy leaves. Significant differences in mean numbers of E. sexmaculatus eggs were found on 2, 8, 23 and 31 March and 13 April (Fig. 3.3). Significantly higher numbers ofE. sexmaculatus motiles were obtained on leafininer leaves than on healthy mature and young leaves on 23 February; 2, 8, 23 and 31 March; and 7 and 13 April (Fig. 3.4).

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39 No significant differences (P > 0.05) were found for citrus rust mites on any of the dates sampled (Table 3.5 and Fig. 3.5). During this period, the peak citrus rust mite population occurred in mid February (c.a. 100 RM / 5 leaves on healthy mature leaves and 75 RM / 5 leaves on leafininer damaged leaves). This population decreased to its lowest levels on 9 April (<5 RM / 5 leaves on all three types of leaves). Phytoseiid numbers increased from 8 February until the end of March with the largest numbers of phytoseiids occurring on mined leaves (P < 0.05). Significantly higher numbers of phytoseiids were found on 2 February, 2, 8, 16, 23 and 31 March, and 7 April (Fig. 3.6). The highest numbers of phytoseiids occurred on 23 March with 8.9 ± 2.0 (mean ± SEM) motiles / 5 leaves on leafininer damaged leaves, whereas for healthy mature leaves there were 1.9 ± 0.6 phytoseiid motiles / 5 leaves. Table 3.7 and Fig. 3.7 present the ANOVA and the LSD tests, and means (± SEM) for motile stigmaeids. Although high numbers of stigmaeid predators were found on mined leaves they were not significantly different due to the patchy distribution of these mites with one exception on 23 February (Fig. 3.7). Permanent slides were prepared and identifications were made with most of the phytoseiids found on 16, 23 and 31 March. They were identified as Iphiseiodes quadripilis (n = 139), Typhlodromalus peregrinus (n = 122), Euseius mesembrinus (n = 18), and 1 each of Phytoscutus sexpilis and Galendromus helveolus (Fig. 3.8). Pollens from different types of plants were found on the grapefiiiit leaves, including Quercus sp, Pinus sp., citrus, and unrecognized pollen types. There were no differences in the types of pollen found but the total number of pollen grains found on the field of view increased as the plants started flowering. Grapefiiiit trees in this orchard bloomed between 2 March and 16 March with most of the flowers already shed by 16 March. The

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40 correlation between the number of pollen grains in the field of view and phytoseiid mites was positive and highly significant (P = 0.004) using the Pearson correlation coefficient with a = 0.83 (Fig. 3.9) (Statistica, 2000). Temperatures (° C) and rainfall (cm) records for the duration of this study are presented in Figure 3.9. Discussion Numbers of phytophagous mites varied on the three types of leaves sampled. No preference for leaf type was shown by E. banksi, but E. sexmaculatus preferred mined leaves. E. banksi produce little webbing compared to E. sexmaculatus, and as expected, they should have exploited the healthy mature or young leaves. However, on the 23 March and 13 April samples, the numbers of oviposited E. banksi eggs were higher on young flushes than on healthy mature or leafminer damaged leaves. Henderson and Holloway (1942) reported a similar preference of oviposition on new leaves by P. citri. The reason for this was that younger leaves might be more nutritious than older leaves. The younger leaves were also expanding when carbaryl was sprayed on 5 April. The pesticide might not have contacted most of these younger leaves on the sample of 13 April thus new expanding leaves could be used by E. banksi to oviposit eggs on leaves less affected by the pesticide toxicity. It has been reported that carbaryl has little effect on tetranychids but on occasions can stimulate population increases (Dittrich et al. 1974). It is possible that carbaryl has an apparent short negative effect on E. banksi populations because the number of eggs and motiles decreased after the spray (Fig. 3.1 and 3.2) and similarly the physical effect of the hydraulic spray by itself has resulted in temporary reduction of £. banksi. Also, it is likely that the high numbers of £. sexmaculatus eggs were the result of a stimulatory effect by carbaryl.

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41 Although E. sexmaculatus is seasonally more abundant following cold winter months and prefers older, harder leaves (Childers, 1994a), the mean numbers of E. sexmaculatus eggs and motiles were significantly more abundant on mined leaves than on healthy mature leaves (Figs. 3.3 and 3.4). As expected, the mined leaves provided increased spaces for webbing for this mite. An important feature here is that we observed the sheltered effect of the mined leaves on E. sexmaculatus. Eotetranychus sexmaculatus had a very moderate decrease in its population after carbaryl was applied but immediately increased by the following sample date (13 April). This increase may not only be due to the protective affect of the mines but also to a hormoligosis effect by the pesticide as explained for many other tetranychid pests. Hormoligosis is described as stimulation by small quantities of a stressor that enhances fertility (Dittrich 1987). Dittrich et al. (1974) demonstrated that the Female: Male ratio on Tetranychus urticae Koch increased significantly after these mites were left on leaves treated with carbaryl. Phytoseiids and stigmaeids were found in higher numbers on leafininer damaged leaves than on healthy mature and younger leaves (Figs 3.6 and 3.7). Mean numbers of phytoseiids were significantly (P < 0.05) different, with numbers ranging fi-om 2 to 5 times greater on leafininer damaged leaves versus healthy mature leaves. Mined leaves not only provided protective spaces for mites but also more prey and other types of food. (Villanueva and Harmsen 1996, Villanueva 1997) observed similar effects on mines produced by the spotted tentiform leafininer Phyllonorycter blancardella Fabricius (Gracillariidae) on apple in Ontario. The type of mines formed on apple was different from CLM mines. Both stigmaeids (Zetzellia mali (Ewing)) and phytoseiids {Typhlodromus caudiglans Schuster and Neoseiulus fallacis (Garman)) were found on old

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42 mines on apple feeding on Panonychus ulmi and tarsonemids (Villanueva and Harmsen 1996, 1998). The tarsonemids oviposited higher numbers of eggs around the frass and exuviae inside the mines on apple. Frass and exuviae are also found in mines produced by CLM and unpublished observations by Villanueva (unpublished) in the laboratory showed that /. quadripilis and E. mesembrinus feed on phylloplane fiingi that grow more abundantly on mined leaves than on healthy leaves. Here phylloplane is used in reference to a leaf as a habitat for microorganisms. More importantly, epiphytic fungi had recently been studied for their antagonistic effects to pathogenic fungal diseases such sooty mold on mandarin (Srivastava and Thakre 2000) and citrus canker on lemon (Pabitra et al. 1996). Theoretical approaches to the use of entomopathogens as "body guards" of plants have been formulated and discussed recently (Elliot et al. 2000). Hargreaves and Parappukkaran (2000) noted that most common fungi occurring outdoors are those that live on the surfaces of leaves (phylloplane fungi). A few genera, Cladosporium, Altemaria, Epicoccum and Aureobasidium comprise between 40% and 80% of the propagules in the atmosphere in surveys worldwide. Fateh et al. (1995) noted that species of epiphytic fungi varied depending on season, age of leaves and plant variety on grape. In the acarine system, Belczewski and Harmsen (1997) found that sprays of the phylloplane fungus Alternaria alternata Keissler on apple leaves in the field enhanced population growth of Tetranychus urticae compared to that of Panonychus ulmi. Whereas Zemec and Prenerova (1997) observed that T. pyri fed on the conidia of powdery mildew species Erysiphe orontii Castellani fi-om tobacco and Oidium fragariae Harz fi-om strawberry in the laboratory. Typhlodromus pyri reproduced and completed its life cycle on E. orontii.

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43 The relative abundance of the phytoseiids /. quadripilis and T. peregrinus is shown in Fig. 3.8 on the three types of leaves sampled on 16, 23 and 31 March. These three dates had the highest phytoseiid numbers. Iphiseiodes quadripilis and T. peregrinus combined for more than 90% of the total number of phytoseiids found in this study. This coincides with Childers and Denmark survey (unpublished) where T. peregrinus and /. quadripilis are the second and third most abundant phytoseiids in seven central Florida orchards. However, E. mesembrinus was the most abundant phytoseiid in their survey but was not abundant in this study. Iphiseiodes quadripilis and T. peregrinus are generalist predacious mites (Muma 1961) and the former shows preference for grapefruit over orange (see chapter 5). The biology and behavior of T. peregrinus have been studied more than /. quadripilis. Typhlodromus peregrinus fed on pollen, P. latus, P. oleivora and tetranychids (Pena 1989). It may be reasonable to think that E. mesembrinus was not abundant due to its phenology and may be less fit for the exploitation of mined leaves than /. quadripilis and T. peregrinus. Stigmaeids were found regularly during the duration of this study, but their distribution was localized. Hence variability is observed in the high SEM values obtained (Fig. 3.7). Similar observations in patchy distribution of A.floridanus were made by Muma and Selhime (1971). Both phytoseiid numbers and pollen grains increased between 2 February and 16 March. Similar observations are reported on pollen abundance and increases of phytoseiid populations in many crops. Addison et al. (2000) suggested that T. pyri abundance had a positive correlation with early season pollen density than with the abundance of the mite prey (Aculus schlechtendali Nalepa) in apples. Similarly, when Euseius tularensis Congdon was released into navel orange groves, the phytoseiid

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44 exhibited a significantly greater increase in groves with a ground cover crop of mixed leguminous plants over a 4-week period than in groves without ground cover crop in California (Grafton-Cardwell et al. 1999). The results shown here had similar responses and the high correlation coefficient obtained (r = 0.91) may explain /. quadripilis and T. peregrinus abundance during citrus flowering (Fig. 3.9). This chapter describes two ecological components that affect phytoseiid populations on citrus: the mine caused by P. citrella and pollen deposited on the leaves. The mine provides phytoseiids shelter and food. Phytoseiids utilize mines as refugia where they can increase more than on healthy leaves due to better micro-environmental conditions. The phylloplane fungi developing within the mines and the leafminer frass are also utilized as food sources. This can be important especially during the winter months when prey availability is scarce. Pollen is a proven food source of phytoseiids and pollens of Quercus sp, Pinus sp. and Citrus sp. coincided with increased phytoseiid numbers in the field. Both /. quadripilis and T. peregrinus are generalist phytoseiids mites that complete their life cycles and reproduced on pollen diets in the laboratory (Muma 1967b and 1971, Pena 1992, see Chapter 4). The citrus leafminer, P. citrella is not considered a beneficial insect but changes in leaf morphology produced by this pest are used positively by phytoseiids and thus augment pest mite control. A practical application of this research is that sampling on mined leaves alone will provide increased phytoseiid numbers. However, it also may be possible that only one or more predatory species such /. quadripilis and T. peregrinus exploit this type of habitat while other phytoseiid species avoid it. This study only focused on acarine populations but insects such thrips, psyllids, curculionids, hemipterans and arachnids were observed more frequently on mined leaves

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45 than on healthy mature leaves. More research is needed because the mines can affect beneficial arthropod numbers not only on citrus but in other plants too.

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46 Table 3.1. Analysis of variance (first seven rows) and least significant difference (LSD) analysis (last four rows) for mean numbers of Eutetranychus banksi eggs on healthy \Ja\C UI F value r LbD test 02-FEB 1, 18 08-Feb 1, 18 1.44 0.24 16-Feb 1, 18 0.35 0.56 • 23-Feb 1, 18 0 1 02-Mar 1,18 : 0.16 0.69 08-Mar 1,18 0.26 0.61 16-Mar 1, 18 0.21 0.64 23-Mar 2, 27 4.95 0.01 * 31 -Mar 2, 27 2.10 0.14 n.s. 07-Apr 2, 27 4.41 0.02 * 13-Apr 2, 27 5.23 0.01 One asterisk (*) means that there are significant differences (P < 0.05) and two asterisks means that differences are highly significant (P < 0.01). 45 40 35 30 25 20 15 10 5 •Healthy L. LM-Leaves -P-Young L. 8-Feb 18-Feb 28-Feb 10-Mar 20-Mar Date 30-Mar 9-Apr 19-Apr Figure 3.1. Mean numbers (± SEM) of Eutetranychus banksi eggs on healthy mature, leafminer-damaged and young leaves. Values bearing the same letter were not significantly different in an ANOVA or LSD test (P > 0.05) on a given sampling date.

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47 Table 3.2. Analysis of variance (first seven rows) and least significant difference (LSD) analysis (last four rows) for mean numbers of Eutetranychus banksi motiles on healthy mature, leafminer-damaged and young leaves. Date d.f F value LSD test 02-Feb 1, 18 08-Feb 1, 18 0.19 0.66 16-Feb 1, 18 1.20 0.28 23-Feb 1, 18 0.91 0.35 02-Mar 1 1 8 1, lo u.uz U.oo 08-Mar 1, 18 0.003 0.95 16-Mar 1, 18 0.12 0.72 23-Mar 2, 27 1.63 0.21 n.s. 31 -Mar 2, 27 2.15 0.13 n.s. 07-Apr 2, 27 0.53 0.58 n.s. 13-Apr 2, 27 1.57 0.22 n.s. One asterisk (*) means that there are significant differences (P < 0.05) and two asterisks means that differences are highly significant (P < 0.01). 30 25 20 £ 15 10 -^Healthy L. ~*~LM-Leaves ~^~Young L. 29-Jan 8-Feb 18-Feb 28-Feb 10-Mar 20-Mar 30-Mar 9-Apr 19-Apr Date Figure 3.2. Mean numbers (± SEM) of Eutetranychus banksi motiles on healthy mature, leafminer-damaged and young leaves. Values bearing the same letter were not significantly different in an ANOVA or LSD test (P > 0.05) on a given sampling date.

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48 Table 3.3. Analysis of variance (first seven rows) and least significant difference (LSD) analysis (last four rows) for mean numbers of Eotetranychus sexmaculatus eggs on Date d.f F value P LSD test 02-Feb 1, 18 08-Feb 1, 18 1.21 0.28 16-Feb 1, 18 1.40 0.25 23-Feb 1, 18 4.7 0.04 02-Mar 1, 18 5.63 0.02 08-Mar 1, 18 9.27 0.006 16-Mar 1, 18 0.01 0.92 23-Mar 2, 27 5.53 0.009 ** 31 -Mar 2, 27 6.78 0.004 ** 07-Apr 2, 27 2.87 0.07 n.s. 13-Apr 2, 27 7.68 0.002 ** 01 One asterisk (*) means that there are significant differences (P < 0.05) and two asterisks means that differences are highly significant (P < 0.01). 100 90 80 « 70 S 60 50 40 30 20 10 0 29-Jan -^Healthy L. — — LM-Leaves —o— Young L. o~. \ ^) 8-Feb 18-Feb 28-Feb 10-Mar Date 20-Mar 30-Mar 9-Apr 19-Apr Figure 3.3. Mean numbers (± SEM) of Eotetranychus sexmaculatus eggs on healthy manire, leafminer-damaged and young leaves. Values bearing the same letter were not significantly different in an ANOVA or LSD test (P > 0.05) on a given sampling date.

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49 Table 3.4. Analysis of variance (first seven rows) and least significant difference (LSD) analysis (last four rows) for mean numbers of Eotetranychus sexmaculatus motiles on healthy, leafininer-damaged and young leaves. Date d.f F value LSD test 02-Feb 1 1 O 1, lo u.yy 0.38 08-Feb 1 1 o 1, lo 0.90 0.35 16-Feb 1 1 O 1, 18 1.53 0.23 23-Feb 1 1 o 1, 18 5.58 0.02 02-Mar 1 1 o 1, lo 4.o3 0.04 Uo-Mar 1, 18 7.05 0 01 16-Mar 1, 18 0.54 0.46 23-Mar 2, 27 6.12 0.006 ** 31 -Mar 2, 27 8.93 < 0.001 07-Apr 2, 27 8.44 < 0.001 13-Apr 2, 27 7.65 0.002 ** One asterisk (*) means that there are significant differences (P < 0.05) and two asterisks means that differences are highly significant (P < 0.01). 30 2S Healthy L. LM-Leaves Young L. b 1 1 1 1=1 — b— ^^'^ a 29-Jan 10-Mar Date 9-Apr 19Apr Figure 3.4. Mean numbers (± SEM) of Eotetranychus sexmaculatus motiles on healthy mature, leaftniner-damaged and young leaves. Values bearing the same letter were not significantly different in an ANOVA or LSD test (P > 0.05) on a given sampling date.

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50 Table 3.5. Analysis of variance (first seven rows) and least significant difference (LSD) analysis (last four rows) for mean numbers of citrus rust mite motiles on healthy, leafminer-damaged and young leaves. Date d.f F value P LSD test 02-Feb 1, 18 08-Feb 1, 18 0.167 0.68 16-Feb 1, 18 3.35 0.08 23-Feb 1, 18 0.01 0.90 02-Mar 1, 18 0.86 0.36 08-Mar 1, 18 0.133 0.71 16-Mar 1, 18 2.52 0.12 23-Mar 2, 27 2.34 0.11 n.s. 31 -Mar 2, 27 1.32 0.28 n.s. 07-Apr 2, 27 1.06 0.35 n.s. 13-Apr 2, 27 1.39 0.26 n.s. One asterisk (*) means that there are significant differences (P < 0.05) and two asterisks means that differences are highly significant (P < 0.01). —A— Healthy L. ~^~"LM -Leaves ~^*~Younfl L. T . . . , . Q ^^^^ a^^^S 18-Feb 28-Feb 10-Mar 20-Mar 30-Maf 9-Apr Oat* Figure 3.5. Mean numbers (± SEM) of citrus rust mite motiles on healthy mature, leafminer-damaged and young leaves. Values bearing the same letter were not significantly different in an ANOVA or LSD test (P > 0.05) on a given sampling date.

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51 Table 3.6. Analysis of variance (first seven rows) and least significant difference (LSD) analysis (last four rows) for mean numbers of phytoseiid mite motiles on healthy, leafminer-damaged and young leaves. Date d.f F value P LSD test 02-Feb 1, 18 5 . 0.03 08-Feb 1, 18 0 1 . . • t ' 16-Feb 1, 18 ' 1.14 0.29 23-Feb 1, 18 3.09 0.09 02-Mar , 1, 18 5.26 0.03 08-Mar 1 1 a 1 , 1 o A Al U.U4 16-Mar 1, 18 28.78 <0.01 23-Mar 2,27 14.91 . <0.01 ** 31 -Mar 2, 27 17.32 <0.01 ** 07-Apr 2, 27 3.84 0.03 * 13-Apr 2, 27 2.39 0.11 n.s. One asterisk (*) means that there are significant differences (P < 0.05) and two asterisks means that differences are highly significant (P < 0.01) on the LSD test. 29-Jan g 8-Feb 18-Feb 28-Feb 10-Mar 20-Ma? 30-Mar 9-Apr 19-Apr Figure 3.6. Mean numbers (± SEM) of phytoseiid motiles on healthy mature, leafminerdamaged and young leaves. Values bearing the same letter were not significantly different in an ANOVA or LSD test (P > 0.05) on a given sampling date.

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52 Table 3.7. Analysis of variance (first seven rows) and least significant difference (LSD) analysis (last four rows) for mean numbers of stigmaeid motiles on healthy, leaf minerdamaged and young leaves. H f U.l. \yf<5F IvloSZ r value n r LciU test 02-Feb N/A N/A N/A N/A 08-Feb 1, 18 16.2 0.89 0.35 16-Feb 1, 18 3.2 3.64 0.07 23-Feb 1, 18 57.8 5.41 0.03 02-Mar 1, 18 80 1.26 0.27 08-Mar 1, 18 64.8 3.38 0.08 16-Mar 1, 18 28.8 3.62 0.07 23-Mar 2, 27 1.48 2.43 0.10 n.s. 31 -Mar 2, 27 0.79 1.17 0.32 n.s. 07-Apr 2,27 0.19 2.20 0.12 n.s. 13-Apr 2, 27 0.42 1.65 0.20 n.s. One asterisk (*) means that there are significant differences (P < 0.05) and two asterisks means that differences are highly significant (P < 0.01). E o s 9-Apr 19-Apr Figure 3.7. Mean numbers (± SEM) of stigmaeid motiles on healthy mature, leafininerdamaged and young leaves. Values bearing the same letter were not significantly different in an ANOVA or LSD test (P > 0.05) on a given sampling date.

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53 Figure 3.8. Relative abundance of Euseius mesembrinus, Iphiseiodes quadripilis and Typhlodromus. peregrinus on healthy mature, leafininer damaged leaves by Phyllocnistis citrella and young grapefruit leaves on three dates with the highest numbers of phytoseiids registered. 12 1 0 -I < 1 , , , -, 0 5 10 15 20 25 30 Pollen grains / 1.130 mm^ Figure 3.9. Relationship between phytoseiids (± SEM) and pollen grains (± SEM) found on grapefruit leaves from 2 February to 16 March 2001.

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54 Figure 3.10. Temperature and rainfall recorded on the Florida automated weather network CREC-Lake Alfred Station from 3 1 December 2000 to 20 May 2001.

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CHAPTER 4 COMPARATIVE DEVELOPMENT OF IPHISEIODES QUADRIPILIS (BANKS) (ACARI: PHYTOSEHDAE) ON VARIOUS NATURAL DIETS AND PREDATION on ACULOPS PELEKASSI {Y^WEK) (ACARI: ERIOPHYIDAE) IN THE LABORATORY Introduction v Phytoseiid mites are considered effective biological control agents on numerous crop systems (Baillod et al. 1982; Bostanian and Coulombe 1986; Brodsgaard and Hansen 1992; De Moraes and McMurtry 1985; and Yaninek et al. 1998). They prey on phytophagous mites in Florida citrus orchards but their potential use in controlling mite pests in orchards under IPM regimes is not well known. Muma (1975) found 19 species of phytoseiids associated with citrus trees but this number is probably low. One of the more abundant phytoseiid mites on Florida citrus is the Shiny button mite (SBM) Iphiseiodes quadripilis (Banks) and it is considered the third most abundant phytoseiid today (Childers and Denmark, unpublished data). The SBM has been described as a general facultative predator that can feed on eriophyids, tetranychids and honey + citrus pollen and other lesser economically important citrus mites (Muma 1971). However, little is known about the life cycle of SBM, its predatory behavior, or the ecological factors that influence its development. The SBM can be easily observed visually in the field by the naked eye and it is distinguishable from most other phytoseiid mites by its shiny, dark, mahogany-red color. The body shape of adults is almost round and swollen to a nearly hemispheric profile (Muma 1975). 55

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56 The citrus rust mite (CRM) Phyllocoptruta oleivora (Ashmead) (Eriophyidae) is one of the key pests on Florida citrus and is found in all citrus-growing areas of the world. The CRM can cause considerable economic losses in the citrus industry due to direct feeding damage on green twigs, leaves and fruits and the high cost of acaricides for its control. Heavy infestations produce fruit russeting that decreases fruit quality (Childers 1994a). Pink citrus rust mite (PRM) Aculops pelekassi (Keifer) is another eriophyid found in Florida and is generally misidentified with CRM due to their small size. Although CRM has a yellowish color that is different from the pinkish coloration of PRM, this is distinguishable only at higher magnifications using a dissecting stereomicroscope. Even then coloration is not a clear indicator for species recognition and reliable identification requires slide mounting of specimens (Childers and Achor 1999). Tetranychidae are common pests on citrus and the two most common species are the Texas citrus mite (TCM) Eutetranychus banksi (McGregor) and citrus red mite (CReM) Panonychus citri (McGregor) (Childers et al. 1991, Childers 1994a). These mites prefer dry conditions and are common in citrus areas in Texas and California, respectively. They are important pests in Florida from March through June or during other extended dry periods throughout the year (Childers 1994a). Both mites cause stippling on the leaves, mesophyll collapse at high densities and can feed on green twigs and fruits (Albrigo et al. 1981). On occasion, leaf drop in the upper portions of trees is associated with TCM feeding injury following dry weather conditions in Texas (Dean 1980) Pollen can be an important alternative or supplementary component of diet for many predacious mites in the family Phytoseiidae (Abou-Setta and Childers 1987, Badii and

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57 Hernandez 1993, Fouly et al. 1995, McMurtry and Croft 1997, Yamamoto 1994). Pollen is found in Florida throughout the year from wild flowers, various annual and perennial flowering plants, shrubs and ground cover plants and weeds. However, the most abundant period of tree pollen release occurs from late February to May when Quercus spp. (oak) (Fagaceae), Pinus elliottii Engelman, P. palustris Miller (Pinaceae), Casuarina equisetifolia J.R. Forsts and G. Forst, C. cunninghamiana Miquel (Casuarinaceae), Taxodium distichum (L.) (Taxodiaeae), Juniperus silicicola (Small) Bailey (Cupressaceae), Myrica cerifera L. (Miricaceae), Broussonetia papyrifera (L.) Vent (Moraceae), and Moms rubra L. (Moraceae) are blooming (Bucholtz et al. 1991). Citrus trees also bloom during this period. Oak trees produce profuse amounts of pollen during flowering and oak pollen is dispersed by wind and deposited on citrus leaves during this period. Alternatively, pollen of ice plant {Malephora crocea Jacquin, family: Aizoaceae) is often used to maintain phytoseiids in laboratory colonies and is sold commercially. Pollen has not been evaluated as a dietary component for SBM. It is not known which life stages feed on pollen or complete development and oviposition with a diet of pollen alone. The objective of this study was to determine if /. quadripilis can complete its life cycle and oviposit successfiilly on diets of CRM, CReM, TCM and oak or ice plant pollens in the laboratory. Also, the acceptability of CRM and PRM as food sources to different /. quadripilis life stages was assessed.

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58 Materials and Methods Assays with natural diets Reproductive SBM females were collected in the field and placed on leaf arenas in the laboratory with eggs and motile stages of CReM and ice plant pollen as food sources. SBM eggs were collected and placed individually onto arenas containing the different diets. Three species of prey mites were used in this experiment: CRM, CReM and TCM, and two types of pollen: ice plant and oak, and a separate treatment of water alone was included. Three types of arenas were employed. The first type was used to test SBM development without food and consisted of a square piece of ceramic tile approximately 5.0 to 7.0 cm in length per side, 1.0 cm thick and surrounded by a wet strip of cotton. The piece of tile was placed in a 15.0 cm diam. x 2.5 cm deep Petri dish filled with water to avoid mite escape and to maintain the desired moisture level on the cotton strips. The tile was beige in color, had a glazed-matte finish, and this surface was used for the arena. Each tile was divided into 16 squares (arenas) using thin wet cotton strips. SBM mites were introduced individually using a 5-0' sable brush. The second type of arena (Fig. 4. 1) was used to test the diet of SBM on CRM only and was prepared on an orange fi^it c.a. 4.0 to 6.0 cm diam. Each fruit was placed on a PVC pipe (3.5 cm diam. x 1.5 cm tall) and then placed in a Petri dish (8.5 cm diam. by 1.5 cm deep) half filled with water to maintain moisture on the cotton strips during the duration of the experiment. Each fioiit had a circular experimental area approximately 3.5 to 4.0 cm diameter on the upper part of the fruit that was surrounded with a 1.5 cm wide wet cotton strip to confine the mites. The orange fiuits were chosen for the high numbers of eriophyid mites each contained (> 200 mites /cm^). All insects and mites other than the

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59 citrus rust mites were removed. Most of the eriophyids in each arena were CRM based on the yellowish color of their bodies. One arena of this type was used for each SBM eggThe third type of arena contained a grapefruit leaf placed in a 15.0 cm diam. x 2.5 cm deep plastic Petri dish. The leaf was placed with its upper surface facing up on the top of a wet foam pad. This arena was used to test the diet of CReM, TCM, or the two types of pollen. Wet cotton strips completely surrounded each leaf to confine the mites. Leaves used to prepare these arenas were cleaned with a solution of 5% sodium hypochlorite . (bleach) to remove contaminants. The leaf arenas for the diets using CReM were divided into quarters using wet cotton strips. Within each arena, five female CReM were left for 3 days to lay eggs before placement of the SBM eggs per arena. Similar steps were made for the TCM diet. Arenas for the pollen diet were divided similarly and pollen was added at the same time with one SBM egg. More female CReM, TCM, or one of the two types of pollen were added to each respective arena to maintain development of the SBM during the duration of the experiment. The leaf arenas were replaced periodically before they started to deteriorate and leaves used for this purpose followed similar cleaning and preparation steps as described above. f s! The mean developmental times of the different SBM stadia (egg, larva, nymphs) and the preoviposition interval were analyzed using ANOVA followed by the LSD test (post hoc comparisons) and % survival to adults was analyzed using the test of difference between proportions (®Statistica 2000). Predation of SBM deutonymphs and females on pink rust mite (PRM) 'Valencia' orange fiaiits approximately 4.0 to 6.0 cm diam. were brought to the laboratory and arenas (ca. 3.5 to 4.0 cm diameter) were prepared within areas containing

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60 the highest concentrations of eriophyids found. This arena was similar to the type 2 unit described in the previous assay and was maintained with adequate moisture for 5 to 7 days. Mite species and insects were removed from the experimental arena with only the eriophyids remaining. Pink and yellow colored eriophyids were observed in the arenas, and samples of each were taken for identification. Identification made through permanent slides and electron microscopy photography showed that these mites were PRM and CRM, respectively (Fig 4.2). The mean numbers of CRM and PRM were 66.2 ± 5.63 and 40.4 ± 1.57 per cm^, respectively, on the apex the arena. Most of the eriophyids were concentrated in areas nearer to the cotton strips. All arenas contained both PRM and CRM in excess. SBM deutonymphs reared with pollen and CReM in the laboratory were starved for 6 h and SBM adult females were starved for 6 and 24 h. After these starvation periods, the respective stages were transferred individually onto the arenas using a fine N° 5-0 sable brush followed by four-minute continuous periods of observation. The number of encounters and consumed eriophyids were recorded during this time. An encounter is ' defined as the change in the searching behavior of the SBM such as palpation and/or arrestment. The four-minute interval was determined because at this time 100 percent of the SBM females (n=10 per interval time) placed in the arena consumed 2 or more eriophyids. This time seemed to be appropriate to evaluate and compare eriophyid predation (Fig. 4.3). Comparisons of the means of eriophyids consumed and encountered by SBM deutonymphs and females, respectively, were assessed using the paired ^test (Excel ® 2000).

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61 Results Assays with natural diets The SBM larval stage developed successfully to the protonymphal stage using CRM, CreM, TCM, ice plant or oak pollen diets and there were no significant differences among the diets in this stage (P > 0.05) (Fig. 4.4). However, on water alone and with the CRM only diet, all the SBM protonymphs stopped development and died. Survival of SBM to adults was obtained with oak pollen, ice plant pollen, TCM and CreM and percentage survival rates were of 68, 60, 48, 36 %, respectively. A significant difference in percentage survival of SBM to adults was obtained between the oak pollen alone and CReM alone diets (p < 0.05). The female:male ratios were 5:4 (CReM, 1 female died before laying an egg), 7:5 (TCM, 1 female died before laying an egg), 8:9 (oak, 1 female died before laying egg) and 8:7 (ice plant, 2 females died before laying eggs). SBM females completed their life cycles on CReM, ice plant pollen alone, oak pollen alone and TCM diets in 9.48 ± 0.2 (n = 4), 9.44 ± 0.5 (n = 6), 9.39 ± 0.3 (n = 7), 8.5 ± 0.2 (n = 6) days ± SE, respectively ((Fig. 4.4). Predation of SBM deutonymphs and females on PRM SBM deutonymphs encountered significantly higher mean numbers of CRM (8.3 ± 2.45 / 4 min.) than PRM (1.5 ±0.3/4 min.) (d.f = 9, ^-critical = 2.262, P < 0.014) after 6 h starvation (Fig. 4.5) This corresponds with the highest number of CRM:PRM proportions in the arenas (0.62 and 0.38, respectively). However, SBM deutonymphs only fed on PRM that were encountered (r-critical = 2.262, P < 0.002) and did not feed on CRM. Similar results were found for SBM adult females (Fig. 4.5), although in this case the number of CRM and PRM encountered by 6 h starved SBM females (d.f = 11,/-

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62 critical = 2.200, P = 0.29) and 24 h starved SBM females (d.f. = 19, ^-critical = 2.093, P = 0.709) were not significantly different (Mest, P > 0.05) (Fig. 4.5). SBM females fed on 1.8 ± 0.47 and 3.5 ± 0.45 PRM / 4 minutes after 6 h (d.f = 11, /-critical = 3.10) and 24 h (d.f = 19, /-critical = 2.86) starvation, respectively, and did not feed on CRM. These results were highly significant (P < 0.002 and P < 2.49"^, respectively). Discussion SBM did not complete its life cycle on the CRM diet. SBM molted from larva to protonymph when provided with CRM alone. However, this was not an indication that SBM larvae were preying on CRM since SBM larva maintained on the ceramic tiles with wet cotton strips molted to the protonymphal stage. This result is similar to previous reports where /. zuluagai Denmark and Muma in Brazil, a close relative of /. quadripilis did not eat during the larval stage (Yamamoto, 1994). Lee and Davis (1968) found that Typhlodromus occidentalis (Nesbitt) larvae did not feed. Sabelis (1985a) noted that the larval stage is not influenced by the prey supply for most phytoseiids. Chitteden and Saito (2001) concluded that the non-feeding larval behavior may be an adaptation to avoid sibling cannibalism. They also gave examples of non-feeding larvae of Amblyseius californicus McGregor, A. degenerans (Berlese), A. womersleyi Schicha, Phytoseiulus persimilis Athias-Henriot, and Typhlodromus bambusae Ehara. There were statistical differences in the duration of the SBM egg stage among the different diets (Fig. 4.4). However, the diet did not have an apparent effect on the total developmental times of SBM females as shown in Fig. 4.4. Diets of oak alone and CReM alone were significantly different on the subsequent duration of the egg stage but total developmental times were not significantly different. Significant differences were

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63 obtained in nymphal periods (Fig. 4.4). In this study, the duration of the nymphal stage has a direct relationship with the total duration of the SBM generational time (Fig. 4.4). The shortest nymphal stage was obtained with the TCM diet alone and this was significantly different from the rest of the diets. This short developmental time contributes to the total generation time (P< 0.05) of SBM that was also the shortest and statistically significant (P< 0.05) from the rest of diets except the oak pollen diet (Fig. 4.4). The longest generational time was with the CReM diet alone and this also affected the total generation time of SBM (Fig. 4.4). Both periods of development with the CReM diet alone were significantly greater (P< 0.05) than the rest of the diets tested. The diets with TCM alone and CReM alone resulted in the longest and shortest developmental times of SBM, respectively. Diets of tetranychids reportedly affect the duration of different phytoseiid life cycles. Proprioseiopsis asetus Chant (Phytoseiidae) fed on a diet of Eutetranychus orientalis Klein instead of date pollen {Phoenix dactylifera L.) and shortened its generation time (Fouly, 1997). Abou-Setta and Childers (1989) fed Euseius mesembrinus a diet of ice plant and had the shortest generation time (9.6 d) whereas with CReM it was the longest (1 1 .9 d). These authors indicated that when mated E. mesembrinus females were changed from a diet with ice plant to a diet with CReM high mortality resulted. Sabelis (1985a) found that the duration of the nymphal stage and the preoviposition period depend on prey availability, hi this case, all the diets provided an abundant food supply, thus differences in developmental time between the tetranychid diets might be explained by two reasons. First, reduced webbing was produced by TCM when compared with CReM. This webbing has been observed disturbing the movement of SBM and increasing the SBM searching area on CReM arenas. Presence of webbing

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64 is explained by Gerson (1985) as a defensive mechanism for CReM to protect spider mite eggs. Abou-Setta (1988) also found that E. mesembrinus had similar difficulties when preying on CReM due to the presence of webbing. Euseius mesembrinus became entangled in the CReM webbing. Although CReM is not a prolific web producer as is T. urticae, the presence of webbing had a repellent effect on SBM because many immatures and adults died trying to leave the arenas. This may explain why the lowest percent survival rate was found with the CReM only diet. The second explanation may be justified by possible differences in nutrients provided by the two types of spider mites. TCM may be considered a better food source than CReM. In Florida, similar results were noted for E. mesembrinus. The developmental time was shorter in a diet with TCM than CReM (Abou-Setta and Childers 1989). No significant differences were found in SBM development times on diets with oak or ice plant pollen alone. However, the survival rates were higher in the pollen diets than with the spider mite diets. SBM may be adapted to feed on pollen and consequently has a preference for pollen because various pollen types are available throughout most of the year in Florida. This also, may be the result of the SBM life history. SBM is a polyphagous predatory mite and it is abundant during the peak pollen season (See Chapters 3 and 4). Muma (1971) described this mite as a flingal feeder and similar behavior was observed during the duration of this study (personal observation). A fungus was observed more frequently on the oak and ice plant pollen arenas than on tetranychid arenas due to deterioration of the pollen. It is possible that SBM had fed on this fungus thus a mixed diet of fungus and pollen can provide an additive nutritional effect for SBM and shortened its life cycle. SBM eggs present a characteristic reddish coloration that is

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65 distinct from other phytoseiids on Florida citrus. SBM that fed on the two pollen diets produced eggs with a small pinkish area instead of an intense, larger reddish area observed on SBM reared on CReM or TCM and from eggs collected on the field. This difference in egg color can be an indicator of the varied diet sources of SBM in the field. The experiment on predation of citrus rust mites by SBM showed a clear preference by SBM for PRM over CRM as a food source. This preference was obtained for SBM nymphs and adult females starved for 6 and 24 hrs since these instars preyed only on PRM and did not prey upon CRM (Fig. 4.5). Encounters of SBM with citrus rust mites (both PRM and CRM) can be described as direct palpation of rust mites nymphs and adults. In some cases, the rust mites were raised by the pedipalps and/or chelicera of SBM. This behavior may be an indicator of attempted predation but all CRM were left alive after a couple of seconds and, in other cases similar behavior was observed toward the PRM. When SBM preyed on PRM it was observed that the SBM completely sucked the body fluids (haemolymph) from PRM and only left the shrunken exoskeleton. This predatory behavior clarifies previous studies about the feeding of citrus rust mites by SBM. Muma (1971) described that CRM was both inadequate and adequate as a food source for SBM. However, he did not distinguish between CRM and PRM. This latter point may be an inaccuracy in previous studies between predatory phytoseiids and rust mites on citrus worldwide if more than one eriophyid species occurs. The inability of SBM to prey on CRM is similar to responses of £'. mesembrinus observed by Abou-Setta (1989) and Flores et al. (1996). The reason why SBM did not prey on CRM requires fiirther study and may involve nutritional quality, pheromonal or toxic effects of this mite toward this phytoseiid species. Sabelis (1996) hypothesized that eriophyids had a low

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66 nutritional quality and low profitability compared to other prey. In addition, toxic substances within eriophyids may have a repellent affect toward phytoseiids. One or both of these factors may be especially relevant with CRM. It is necessary to make a clear distinction between the two eriophyids found in citrus orchards in Florida because many earlier studies on predatory mites assumed only the presence of CRM. Childers and Achor (1999) stated that in future studies it is important not to lump the rust mites because their development, biology, feeding damage and seasonal activities obviously differ. In this study, the effects of the diets on oviposition during the entire life span of SBM were not followed. However, this study has provided valuable information on the identification of various food sources, developmental times and behavioral aspects of this important predatory mite in Florida. We learned that SBM is able to feed and complete its life cycle on pollen, CReM and TCM and, it will prey on PRM but not on CRM. This work enhances the importance of identifying correctly CRM from PRM. The small size of the eriophyids makes it more difficult to identify species with a handlens. The only viable way to effectively distinguish these two species is by the pinkish coloration of the pink citrus rust mite (Childers and Achor 1999). However, this requires practice and most importantly the confirmation by slide-mounted specimens.

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67 Encircled experimental arena Wet cotton strip ^ Orange (4 to 5 cm diameter) Connecting cotton strip < 3.5 cm PVCpipe Petri dish with water 8.5 cm >l Figure 4.1. Orange fruit arena: design and components.

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68 PRM [ ^^^^^^^^^H Trough ^^^^^^^ ''^^Sjll^^^^^S Figure 4.2. PRM (Aculops pelekassi) and CRM {Phyllocoptruta oleivora) main taxonomic differences. PRM: long dorso-lateral setae (Ids), rounded dorsum and distinctive dorsal shield. CRM: short dorso-lateral setae (sds), dorsum with trough present and dorsal shield with well defined hexagon (hx).

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69 1 min 2 min 3 min 4 min 5 min Figure 4.3. Determination of time interval for predation of eriophyids by Iphiseiodes quadripilis (SBM)

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70 Figure 4.4. Duration of female Iphiseiodes quadripilis life stages provided individual diets. Different letter between diets in the same instar indicates significant differences (LSD -test, P < 0.05)

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71 12 10 (A Q 4-1 3 C E D £ a o £ 4 Deutonynrph: 6 Hrs Starvation 0 Adult Females: 6 Hrs Starvation No. Encountered No. Consumed 0 Adult Females: 24 Hrs Starvation 0 CRM PRM CRM PRM CRM PRM Figure 4.5: Consumed and encountered eriophyids by SBM deutonymphs and adult females after starvation. Different letter between the numbers of eriophyids encountered or consumed within the same time of starvation and instar indicates significant differences (/-test, P < 0.05).

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CHAPTER 5 EVIDENCE FOR HOST PLANT PREFERENCE IN A PREDACIOUS MITE IPHISEIODES QUADRIPILIS (ACARI: PHYTOSEHDAE) Introduction Price et al. (1980) proposed the three-trophic level theory that establishes the basis for interaction among herbivores (prey), natural enemies (predators) and their host plants. This theory suggests that natural enemies and plants have a mutualistic relationship. Inherent plant defense mechanisms and plant traits that foster natural enemies evolved through natural selection and both are part of a plant defense that results in a negative impact on herbivores. These traits included attractants (nectars, pollen) and structural refuges (leaf domatia) (Walter 1996, O'Dowd and Willson 1997). Research in plant volatiles demonstrated that plants send signals -plant volatiles or infochemicals to predators and these predators respond to them. Phytoseiulus persimilis Athias-Henriot responded to volatiles emitted by bean plants when they were damaged by punctures that mimicked Tetranychus urticae (Koch) feeding damage (Dicke and Sabelis 1988, Takabayashi and Dicke 1992, Dicke et al. 1998). However, all of these observations I were on annual plants in the greenhouse. They are less complex than perennial plants in the field due to greater variables such as the presence of other organisms, environmental conditions and space. Drukker et al. (1997) showed the influence of P. persimilis performance and adaptation on tomato plants. They found that P. persimilis maintained for four generations on tomato exhibited higher survival and reproduction compared to P. 72

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73 persimilis reared on bean plants and then transferred directly onto tomato. They explained that there was an intergenerational adjustment in the behavior and physiology of P. persimilis and a selection of P. persimilis genotypes with higher fitness on tomato plants. Similarly, Duso and Vettorazzo (1999) showed that two native phytoseiids in Italian vineyards distinctly preferred different grape {Vitis vinifera L.) varieties as host plants. Amblyseius andersoni (Chant) was more abundant on 'Merlot' grapes that had I slight pubescence on the undersurface of its leaves. Phytoseius finitimus (Ribaga) was more abundant on 'Prosecco' grapes with a greater under-surface leaf pubescence than glabrous leaves of 'Riesling' grapes. Iphiseiodes quadripilis is a generalist predatory mite on Florida citrus (chapter 4) that feeds on spider mites, ice plant and oak pollens in the laboratory. However, little is known about its life history and ecology. Abou-Setta et al. (1991) described the postembryonic stages and noted that the /. quadripilis female retained an egg with a welldeveloped larva. Iphiseiodes quadripilis was the most abundant predatory mite on mined leaves of grapefruit damaged by citrus leafminer larvae and exploits this type of leaves better than other phytoseiids (Chapter 3). Observations during my field work led me to assume that /. quadripilis tended to be more abundant on grapefiiiit than on sweet orange trees. Hence, this question was addressed with both field and laboratory assays. This mechanism implies a tritrophic relationship in a perennial plant that needed to be investigated. The aim of this study was to determine if there is a preference by Iphiseiodes quadripilis for grapefiaiit compared with sweet orange leaves.

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74 Materials and Methods Abundance of Iphiseiodes quadripilis on the field. Four adjacent orchards of grapefruit and sweet orange were selected for this experiment. The pairs of adjacent orchards at the four sites received similar types of cultural practices and pesticide regimes. Three are experimental orchards in the vicinity of the Citrus Research and Education Center at Lake Alfred and the fourth site is a commercial orchard in Highland City (Polk County, FL). The first orchard was Block 1 and trees were 3-year-old 'Lang' navel oranges and 'Marsh' grapefruit. Leaves were collected on 12 and 19 April 2001. The second orchard was Block 24B and had 10 rows of 10 different types of citrus trees (mandarins, oranges, grapefruit), with 1 row per variety and with 8 to 12 frees per row. The frees were planted in 1990. This orchard was adjacent to a large experimental orchard of "Valencia" oranges. The varieties selected here were 'Pineapple' orange and 'Oroblanco' grapefruit and the leaves were collected on 18 and 22 April 2001 . The third orchard was Block 22 with 'Hamlin' oranges and 'Flame' grapefruit. Samples were taken on 15 and 20 April 2001 and the frees were planted in 1997. The fourth orchard in Highland City included 20 and 23 years old 'Hamlin' oranges and 'Marsh' grapefruit, respectively. The samples were collected on 17 April and 7 May 2001. Five border frees of each orange and grapefruit block were selected in each of the sites I and 10 mined leaves (mines damaged by the citrus leafminer P. citrella) were taken randomly from each free. Leafminer damaged leaves were selected because they have higher numbers of phytoseiids (see Chapter 3). The cumulative number of phytoseiids was analyzed using the test of proportions Statistica® (Statsoft 2000) to observe if there were differences between the total numbers of phytoseiids found on grapefruit or orange.

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A similar test was completed for the total number of /. quadripilis and other species of phytoseiids. Also, only data on TCM eggs and motiles, /. quadripilis and the total number of phytoseiid motiles found on the two sample dates per citrus species were analyzed separately with a mixed analysis of variance using the Satterthwaithe method I Statistica® (Statsoft 2000). the data were transformed using yJX + 0.5 , where A' is the number of mites counted on 5 leaves. Mean numbers ± SEM employed in figures and mentioned in the text are untransformed data. Additional samples were taken from Block 22 on 29 April from the border trees of both orange and grapefruit and phytoseiids counts were completed. The 12 May samples were taken from the border trees and 5th row inside of each type of citrus fruit and counts of both phytophagous and predatory mite eggs and motiles were completed in the laboratory. This time only 5 trees were sampled and 5-mined leaves were taken from each free. Data were fransformed using 7^^ + 0.5 , where Z is the number of mites counted on 5 leaves. Data were analyzed using the analysis of variance for repeated measures followed by a Scheffe-test (Statsoft 2000). Direct contact choice experiment on 12 by 2 mm leaf strips Ten female /. quadripilis were collected from each orange and grapefruit block and then placed on an arena. The arenas consisted of 3 alternating strips of orange and grapefioiit leaves surrounded by wet cotton strips. The strips were 12 mm long by 2 mm wide (Fig. 5.1). Leaves used for the arenas were previously washed thoroughly with tap water and all mites and insects removed. Observations and counts of mite preferences were completed every 5 minutes for 1 h. Three replicates were conducted for each of the types of plants (orange or grapefruit) where /. quadripilis were obtained. All of the adult

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76 female /. quadripilis were placed in alcohol and later slide-mounted in Hoyer's mounting medium and identified. This experiment was completed in the laboratory at 25° C and 55 ± 5 % RH during May 200 1 . Data were analyzed using a paired f-test for comparison of means (Excel ®, Microsoft). YTube olfactometer Iphiseiodes quadripilis females were collected from orange and grapefruit trees on several dates during May 2001 . The /. quadripilis were collected fi-om orange leaves and then placed on an orange leaf arena. Those collected from grapefruit were placed on a grapefruit leaf arena. Leaves from the same orchards were also collected and returned to the laboratory. These leaves were washed thoroughly with deionized water to remove I any prey, scale insects or dust and then air-dried. The Y-tube olfactometer model (Fig. 5.2) was constructed using the design of Sabelis and Van de Baan (1983). Air was pumped at a rate of 500 cm^ of air current per minute and measured by a Gas flowmeter (Dwyer Instruments, hic. Michigan City, Indiana). The air passed through activated charcoal before entering the odor chambers. For each plant type, 70 /. quadripilis females were employed and used only once. A single female /. quadripilis per trial was placed at the mouth of the Y-tube and left there to make an odor attraction choice of leaf The leaf sources were in the chambers at the ends of the Y-tube arms and consisted of either a 3.5 cm leaf disk of orange or grapefruit. A 5-minute time limit for each predator was provided to make a choice. The assay for each female ended when the predator entered into one of the two chambers. The chambers of the Y-tube were switch every four trials and the 3.5 cm disks were replaced after each switch. Mites that did not make a choice during this time limit were separated and not considered in the statistical analysis. This experiment was completed in the

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: 77 laboratory at 25° C and 55 ± 5 % RH between May and June 2001. The number of predacious mites that chose either grapefruit or orange were counted as well as the time required in each trial. Data for the choice test were analyzed using the test and the time required per predator to reach either chamber was analyzed using the ANOVA (Excel®, Microsoft, 2000). Results Abundance of Iphiseiodes quadripilis in the field The total numbers of phytoseiids by type of fruit and species found at the four sample sites during the two collection dates are shown in Table 5.1. Significantly more /. quadripilis {P < 0.001) mites were found on grapefruit than orange, and the 'other phytoseiids' (phytoseiids other than /. quadripilis) were found in higher numbers on orange than in grapefruit (P < 0.001). The total numbers of phytoseiids were also significantly higher (P < 0.001) on grapefruit than on orange. However, the total numbers of I. quadripilis were not different from the total numbers of other phytoseiids (P>0.05). I The data for the two sample dates and four sites gave us the results observed in Tables 5.2 to 5.6. A more detailed analysis of these results is presented in the appendix. Iphiseiodes quadripilis was significantly more abundant (P < 0.05) on grapefruit than orange (Tables 5.2 and Figs. 5.3 A and B). The highest mean numbers ± (SEM) of/. quadripilis motiles on the first date of samples obtained in Block 24B were 20.0 ± 7.7 motiles / 10 leaves on 'Oroblanco' grapefruit versus 2.8 ± 1.4 motiles/ 10 leaves for I 'Pineapple' oranges. The lowest mean numbers ± (SEM) of I. quadripilis were found at the Bader block with 4.6 ± 1.5 motiles / 10 leaves on 'Marsh' grapefruit and 1.6 ± 1.0 motiles/ 10 leaves on 'Hamlin' oranges (Fig. 5.3A). The highest mean numbers ± (SEM)

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1 78 of /. quadripilis on the second sampling dates were obtained at Block 22 with 22.6 ± 4.2 motiles / 10 leaves on 'Flame' grapefruit versus 5.2 ± 2.8 motiles/10 leaves on 'Hamlin' I oranges. The lowest mean numbers ± (SEM) of /. quadripilis motiles were found at the Bader block with 1.8 ± 0.4 motiles / 10 leaves on 'Marsh' grapefruit and 0.2 ± 0.2 motiles/ 10 leaves on 'Hamlin' oranges (Fig 5.3 B). Phytoseiids other than /. quadripilis are referred here as 'other phytoseiids' and their mean numbers ± (SEM) were significantly different in the first sample dates between orchards and between trees. However, no differences {P >0.05) were found for the second sampling dates for both orchards and tree fruit type (Table 5.3 and Fig 5. 3 A). Also, no significant differences were obtained between the total numbers of phytoseiids (/. quadripilis +"other phytoseiids') found on grapefruit and orange on either of the two I sample dates (Table 5.4 and Fig. 5.4). The Texas citrus mite eggs and motiles were very low in the first sampling dates on all orchards except Bader. This was the only significant difference found due to the interaction. There were 9.0 ± 3.9 and 21.0 ± 7.5 eggs and motiles / 10 leaves on 'Marsh' grapefruit and 122.2 ± 41.7 and 140.6 ± 47.4 /lO leaves on 'Hamlin' oranges. No significant differences in TCM egg and motile numbers were found between orchards or fruit types. On the second sampling dates, TCM eggs and motiles were high and significant differences occurred among orchard sites and tree types for the numbers of eggs and between tree types for TCM motiles (Tables 5.5 and 5.6 and Figs. 5A and 5B) j Mean numbers ± SEM for /. quadripilis on samples of 29 April (1,8 df, F = 37.48, P < 0.01), and 12 May (3,16 df, F = 46.3, P < 0.001), 10 September (l,8df, F = 0.80, P .0.05), and 22 October (3,16 df, F = 5.68, P < 0.01) from Block 22 are presented in Table

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79 5.7. Iphiseiodes quadripilis motiles were more abundant on grapefruit than orange during most of the times sampled except on 10 September. Additional samples of fruit on the fifth row within each orchard gave similar results for /. quadripilis motiles but the TCM eggs and motiles were significantly more abundant on orange than grapefiiiit (Fig. 5.6 A and B). Direct contact choice experiment on 12 by 2 mm leaf strips The numbers of /. quadripilis females that were collected on grapefinit were significant (1 1 df, ^-critical = 2.20, P < 0.05) most of the time on grapefiiiit than on orange (mean ± SEM: 5.81 ± 0.18 vs. 4.19 ±0.18 /. quadripilis females in grapefruit or orange, respectively) leaf strips for all replicates (Table 5.8 and Fig. 5.7). Whereas /. quadripilis females collected from orange did not show a preference for either orange or grapefiuit leaf strips (1 1 df, ^-critical = 2.20, P > 0.05) (mean ± SEM: 4.9 ± 0.26 vs. 5.1 ± 0.26, respectively) (Table 5.8 and Fig. 5.7). YTube olfactometer Iphiseiodes quadripilis females that were collected from orange or grapefiuit showed preference for the grapefioiit leaf disk. The number of /. quadripilis from grapefioiit that selected grapefiiiit and orange were 44 and 26, respectively. By coincidence, the number of I. quadripilis females collected from orange that preferred grapefiiiit and orange were also 44 and 26, respectively, hi both cases, these numbers were significantly different (1 df and^ critical =3.84). The times employed for/, quadripilis females collected from orange or grapefruit in making their choice for either leaf disk were 1 15.0 ± 8.91 and 97.6 ± 8.2 seconds (mean ± SEM), respectively, and these were not significantly different (1, 138 df, F = 2.06 and P = 0.15).

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80 Discussion The same species of phytophagous mites affect orange and grapefruit orchards although some citrus species may tend to be more susceptible to certain kinds of pest mites than others. The broad mite Polyphagotarsonemus latus (Banks) is more commonly found on lemon and lime in south Florida than on grapefruit (Childers 1994a). However, /. quadripilis (SBM) was observed in highest numbers on grapefruit than orange on the border trees of the four adjacent sites. This indicates a preference of /. quadripilis for its host plant. This can be inferred because each pair of adjacent orchards received similar cultural practices and pesticide regimes. Furthermore, prey availability was favorable to orange, although in the first sampling dates there were no differences in E. banksi (TCM) eggs and motiles with the exception of the Bader orchard. During the second sampling, the densities of TCM were observed to be lower on grapefruit than on sweet orange, suggesting that better biological control by /. quadripilis was occurring on the grapefruit varieties. If this was the case, why did /. quadripilis not migrate from grapefruit to orange? Is this a seasonal occurrence or are there other factors that retain /. quadripilis on grapefruit? There must be since /. quadripilis developed and reproduced readily on E. banksi (Chapter 4) and this pest mite was present in greater numbers on orange than on grapefruit. Also, /. quadripilis had the shortest developmental rate on E. banksi compared to P. citri, oak (Quercus sp) or ice plant (Mallephora crocea) pollen diets (Chapter 4). Thus a mutualistic relationship between grapefruit and /. quadripilis appears likely. If a mutualistic relationship exists, it may be independent of prey availability such as an enhancement of the host plant to foster predators. Other unknown factors may exist that contribute to grapefruit plants maintaining /. quadripilis populations in grapefruit

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81 orchards. These can be inherent in the plant or exogenous to it or both factors may apply. hiherent factors may include the production of volatiles and a better nutritional quality of I grapefruit pollen than orange pollen. Other inherent characters could include the composition of essential oils in leaves of the two citrus species. Cheng and Lee (1981) I completed a study of leaf essential oils in ten citrus species including orange and grapefruit. They found 45 different essential oils and the percentage compositions were similar for most except Sabinene and A -carene. Sabinene was the most abundant oil m all the species with 49.7% in oranges and 61.9 % in grapefruit and A^-carene was 4.9% in orange and 0.12% in grapefruit. However, studies of these oils have focused primarily for the perfume and cosmetic industries. It would be interesting to know if one or more of these essential oils affect the composition of mite or insect faunas in the different citrus species. Exogenous factors may include food sources other than prey mites that are available I for /. quadripilis such scale insect eggs and/or phylloplane fungi. Muma (1971) found that /. quadripilis preyed on whitefly (Aleurodidae) eggs and larvae and described this food source as both adequate and inadequate. However, Muma (1971) did not indicate the reproductive or life cycle parameters of/, quadripilis with this prey. No heavy infestations of whitefly were observed in any of the four orchards. Also, it might be I possible that some specific types of phylloplane fungi may contribute to the preference of /. quadripilis for grapefruit leaves but to this date no studies have been completed on citrus. Effects of phylloplane fungi have been recently studied. Belczewski and Harmsen (2000) found that non-pathogenic phylloplane fungi on beans affect the net reproductive rate of Tetranychus urticae Koch. Similarly, Zemec and Prenerova (1996)

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82 observed that the phytoseiid Typhlodromus pyri Scheuten feeds on two types of powdery mildew (Ascomycotina: Erysiphales), Erysiphe orontii Castellani on tobacco and Oidium fragaria Harz on strawberry. Mature females T. pyri females successfully reproduced although their oviposition rate was low. Villanueva (unpublished) also observed that /. quadripilis, E. mesembrinus, T. peregrinus and Amblyseius sp. feed on unknown fungi that occur within colonies of phytoseiid mites reared in the laboratory. I The contact and olfactometer assays demonstrate that both factors (a grapefruit substrate and volatiles produced by the plant) may contribute to the preference of /. quadripilis for grapefruit, hi the contact assay, it was apparent that the origin of the /. quadripilis population has an imprinting effect on the predator because /. quadripilis collected on orange did not show preference for either grapefhiit or orange leaf strips. However, /. quadripilis collected on grapefruit showed a significant preference for the grapefruit substrate (P < 0.05). hnprinting can imply the evolution of two lineages of/. quadripilis. This phenomenon was reported on the predatory mite Hypoaspis aculeifer (Canestrini) (Acari: Laelapidae) by Lesna (1998) and Lesna and Sabelis (1999). They I showed that H. aculeifer can rapidly exhibit genetic variation in preference for two prey species: the bulb mite Rhizoglyphus robini Claparede or the copra mite Tyrophagous putrescentiae (Schrank) depending where H. aculeifer was reared. It may be possible that /. quadripilis from grapefruit and orange can form two lineages that are very closely related and the only way to differentiate the two would be with molecular analysis rather than by classical taxonomic methods.

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83 Table 5. 1 Total number of Phytoseiids found in 4 sites on 2 sample dates by type of fruit and phytoseiid species. Significant differences calculated with the test of proportions are indicated in each column and row Iphiseiodes quadripilis Other Phytoseiids Total P Grapefruit 458 151 609 < 0.001 Orange 114 400 514 < 0.001 Total 572 551 1123 0.25 P < 0.001 < 0.001 0.007 Table 5.2. Mixed analysis of variance for Iphiseiodes quadripilis numbers found in adjacent rows of grapefruit and orange at 4 sites sampled on two dates each. I ^actor d.f F P Orchard Site 1*' sample dates Tree type Orchard*Tree 3,3 1,3 3,32 2.44 15.13 1.37 0.241 0.030 0.267 ( 2"** sample dates 1 ( Drchard Site Tee type Drchard*Tree 3,3 1,3 3,32 3.96 15.28 2.13 0.144 0.029 0.115 Table 5.3. Mixed analysis of variance for the total number of 'other phytoseiids' (phytoseiids other than /. quadripilis) found in adjacent rows of grapefruit and orange at 4 sites sampled on two dates each. Factor d.f F P Orchard Site 3,3 15.19 0.03 1^' sample date Tree type 1,3 26.08 0.01 Orchard*Tree 3,32 0.54 0.66 Orchard Site 2""* sample date Tree type 3,3 1.70 0.34 1,3 9.39 0.05 Orchard*Tree 3,32 0.72 0.54

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84 Table 5.4. Mixed analysis of variance for the total number of phytoseiids found in adjacent rows of grapefhiit and orange at 4 sites sampled on two dates each. Factor d.f F P Orchard Site 3,3 1.67 0.34 1^' sample date Tree type 1,3 0.37 0.58 Orchard*Tree 3,32 0.75 0.52 2"** sample date Orchard Site 3,3 3.33 0.17 Tree type 1,3 3.91 0.14 Orchard*Tree 3,32 1.17 0.338 Table 5.5. Mixed analysis of variance for the total number of Eutetranychus banksi eggs found in adjacent rows of grapefruit and orange at 4 sites sampled on two dates each. Factor d.f F P 1^' sample date Orchard Site Tree type Orchard*Tree 3,3 1,3 3,32 2.17 1.14 10.65 0.26 0.36 < 0.001 2"'' sample date Orchard Site Tree type Orchard*Tree 3,3 1,3 3,32 16.57 23.77 0.52 0.02 0.01 0.66 Table 5.6. Mixed analysis of variance for the total number of Eutetranychus banksi motiles found in adjacent rows of grapefruit and orange at 4 sites sampled on two dates each. I Factor ±t F P 1^' sample date Orchard Site 3,3 2.12 0.24 Tree type 1,3 4.00 0.14 Orchard*Tree 3,32 6.54 0.001 2"** sample date Orchard Site 3,3 6.00 0.09 Tree type 1,3 13.79 0.03 Orchard*Tree 3,32 1.67 0.19

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85 Table 5.7. Analysis of variance for the total number of Iphiseiodes quadripilis motiles found in adjacent rows of orange and grapefruit in Block 22 sampled on four dates. Orange Grapefhait Statistical 5 th Row Border Border 5th Row Test 29 April Oa 11.0±2.9b ANOVA 12 May Oa 3.6± 1.1 b 13.2 ± 1.4 c 8.4 ± 1.2 c Scheffe 10 Sep 1.1 ±0.4 a 1.7 ±0.5 a ANOVA 22 Oct Oa 1.0 ±0.6 a 1.7 ± 0.4 b 2.5 ± 0.8 b Scheffe : ^ Table 5.8 Mean numbers ± SEM of Iphiseiodes quadripilis (n = 10) in a choice test between grapefruit or orange strips during 5 -minute observations per 1 hour depending on where they were originally collected Iphiseiodes quadripilis Replicate N° /. quadripilis on Orange N° /. quadripilis on Grapefruit d.f. P 1 4.16 ±0.36 5.83±0.36 0.0043 From Grapefruit 2 4.41 ±0.25 5.58± 0.25 0.046 3 4.0±0.30 6.0±0.30 0.006 1 5.25±0.35 4.66±0.35 0.49 From Orange 2 4.66±0.61 5.33±0.61 0.6 . 3 4.83±0.36 5.16±0.36 0.61

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86 Figure 5. 1 Arena used for the contact experiments. Brownish dots on strips are female Iphiseiodes quadripilis. Y-tube olfactometer Figure 5.2. Y-tube olfactometer used for the choice test. Leaf disks of grapefruit (GF) orange (OR) ca. 3 .5 cm diameter were the sources of plant volatiles.

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(0 o > 5 28 24 20 16 =S 12 (0 t 0. Block 1 {L.A) April 12 87 Btock 248 (LA) Bbck22 (LA) Other Phytoseiids bIquadripilis Fiogers (HC) April 17 Marsh-GF LangN-OR OroBlan-GF P-apple-OR Flame-GF hIamlin-OR Marsh-GF Hanlin-OR 28 24 (A 0) re 20 16 2 12 0) i 3 Btock 1 (L.A) April 19 Marsh-GF Grapefruit LangN-OR Orange OroBlan-GF P-apple-OR Grapefruit Orange Flame-GF Hamlin-OR Grapefruit Orange Rogers (HC) May 7 Other Phytoseiids I. quadripilis i Marsh-GF Hamlln-OR Grapefruit Orange Figure 5.3. Numbers of I. quadripilis per 10 leaves and other phytoseiids other than /. quadripilis, found on the sample at 4 adjacent orchards of orange and grapefruit on the (A) first sampling dates and (B) second sampling dates.

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88 Figure 5.4. Total numbers of phytoseiids {Iphiseiodes quadripilis + phytoseiids other than /. quadripilis) found on grapefruit and orange in adjacent rows of grapefruit and orange orchards. No significant differences were found (P >0.05).

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89 Marsh { LangNav Block 1 Oroblanco Pineapple Block 24B • Grapefruit Orange Flame Hamlin Block 22 W. Marsh Hamlin Bader 120 Marsh | LangNav [ Oroblanco ! Pineapple Flame | Hamlin W.Marsh | Hamlin Block 1 I Block 248 I Block 22 I Bader Figure 5.5 Numbers of Eutetranychus banksi ± SEM on the second sampling dates (A) TCM eggs and, (B) TCM motiles

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90 2.5 ra 0) (A .2 1.5 (A o Q. 0.5 0 4O Other Phytos. I. quadripilis Hamlin 5thR HamlinBorder FlameBorder Flame5thR 45 40 S 35 » 30 O) O) 25 on 0) 20 (0 15 (0 X « 10 5 0 B Texas Motiles TexasEggs Hamlin 5thR HamlinBorder FlameBorder FlameSthR Figure 5.6. (A) Numbers of /. quadripilis and other phytoseiids and (B) Numbers of Eutetmnychus banksi eggs and motiles and eggs found on the 12 May sample on border trees of adjacent grapefruit and orange orchards and 5 rows into each fruit type.

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91 Rep. 1 Rep. 2 Rep. 3 Rep. 1 Rep. 2 Rep. 3 Fig. 5.7 Contact choice test of 10 Iphiseiodes quadripilis females for leaf strips of orange or grapefruit. Different letter in each replicate indicates significance (P < 0.05) 80 70 60 50 40 1 30 2 20 10 0 1 b (44) b (44) 1^ Grapefruit Orange a (26) a (26) From GF From Orange Figure. 5.8. Choice test on leaf disks in the olfactometer ior Iphiseiodes quadripilis. Each column represents the type of fruit where female /. quadripilis were collected. Different letter in each column indicates significance (P < 0.05).

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CHAPTER 6 ECOLOGY AND FEEDING BEHAVIOR OF TWO NEW SPECIES OF CECIDOMYIIDAE PREDACIOUS ON RUST MITES AND THE MICROLEPIDOPTERA CRYPTOTHELEA GLOVERII {?^YCmDAE), A PREDATOR AND PEST ON FLORIDA CITRUS Introduction Two species of rust mites (RM) are pests on Florida citrus, the citrus rust mite (CRM) Phyllocoptruta oleivora (Ashmead), and the pink citrus rust mite Aculops pelekassi (Keifer) (Acari: Eriophyidae) (Denmark 1963, Childers and Achor 1999). Both species are economically important because they cause fruit russeting. A third species, the citrus bud mite Aceria sheldoni (Ewing), occurs on occasion but is not considered an important pest on Florida citrus (Childers and Achor 1999). Natural enemies of citrus rust mites include predatory phytoseiids and stigmaeid mites (Muma 1961, Pena 1992, Childers 1994a). Insects of four different orders are also known to prey on citrus rust mites. Hubbard (1883) found "a little coral-red maggot" and a yellow midge larva (Diptera: Cecidomyiidae, formerly Itonididae) feeding on CRM. Later, Muma et al. (1961) reported Itonidini species feeding on P. oleivora and Eotetranychus sexmaculatus (Riley) in Lake Alfred, Florida. Yothers and Mason (1930) described Stethonis nanus LeConte (Coleoptera: Coccinellidae) and Chrysopa sp. (Neuroptera: Chysopidae) preying on citrus rust mite and Muma (1958, 1961 and 1967a) mentioned Coniopteryx vicina Hagen (Neuroptera: Coniopterygidae) as another predator. The thrips Scolothrips sexmaculatus (Pergande) (Thysanoptera: Thripidae) and Leptothrips mali (Hunter) (Phlaeothripidae) j j 92

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93 were also reported as predators of the citrus rust mite by Bailey (1939) and Muma (1955), respectively. The genus Feltiella has a worldwide distribution (Gagne 1989) and all described species form a distinctive group associated exclusively with spider mites (Tetranychidae) I Gagne (1995). This genus belongs to the tribe Lestodiplosini that is composed entirely of predators or parasitoids of insects and mites (Gagne 1989, 1994 and 1995). hi Europe, Feltiella species are divided into two genera: Feltiella and Therodiplosis, but Gagne (1995) considered these two genera synonyms. Feltiella acarisuga (Vallot) is a wellknown predator of Tetranychus urticae Koch. Feltiella acarisuga completes its life cycle between 8 to 10 d in Italy (Roberti 1954) and 29 d on average in Israel (Sharaf 1984). These differences in the life cycle probably are temperature dependent. Tetranychus urticae is a polyphagous pest and has many host plants. The crops included in studies withF. acarisuga included apple (Roberti 1954), eggplant (Sharaf 1984), cucumber (Gillespie et al. 1998), strawberry (Easterbrook 1998), greenhouses (Opit et al. 1997, Enkegaard et al. 2000). Feltiella occidentalis (Felt) occurs on strawberry in California (Oatman et al. 1985) and F. minuta has been found on eggplant (Ho and Chen 1998). There are few examples of eriophyid predation by cecidomyiids. Nij veldt (1969) compiled a list of cecidomyiid species and their respective eriophyid prey. Medetera species (Diptera: Dolichopodidae) was reported preying on Aculus schlechtendali Nalepa on apple in Washington (Rathman et al. 1988). The eriophyid Aceria litchii Keifer is a serious pest oi Litchii chinensis Sonnerat in Australia and China and a cecidomyiid larva Arthrocnodax sp. has been observed preying upon A. litchii (Waite and Gerson 1994).

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I 94 The orange bagworm (OBW) Cryptothelea gloverii (Packard) (Lepidoptera: Psychidae) was first described as a hang or drop worm by Glover (1858) in Florida and was originally named Platoeceticus gloverii (Hubbard 1 885). The bag worms are easily identified because they carry a portable bag of dense silk covered with bits of dry leaves, bark, thorns and other debris pointed outward and fastened to the outside of the case (Quayle 1938). The two ends of the bag are opened; the narrowest opening serves to excrete fi-ass or any other material and later this opening is used for mating. The bag is dragged across the substrate at a certain angle and when the larva moves the head I capsule, the thoraxic legs are extended from the widest opening of the bag. The bag has an irregular cone shape and enlarges as the larva grows. When full grown the bag is approximately 1.8 and 1.3 cm long for the female and male, respectively (Glover, 1858, Hubbard, 1 885). The color of the bag is brownish-gray and is easily camouflaged in the field (personal observation). hi general, species of the Psychidae show sexual dimorphism as adults (Holland 1968, Borror et al. 1989). The adult male is a small, darker moth about 1cm in length, with legs, well-developed wings and feather-like antennae while the female is maggot-like, wingless, legless, and pupates within its bag (Glover 1858). A common psychid larva that is a defoliator of a wide variety of trees and bushes, evergreens, and reduces aesthetic quality and survival of many ornamental plants is the evergreen bagworm, Thyridopteryx ephemeraeformis (Haworth). Leonhardt et al. (1983) described the mating behavior of T. ephemeraeformis as an extraordinary event and this behavior may be similar to the OBW. Before pupation, the female T. ephemeraeformis attaches and seals the wider part of the bag to a leaf or twig and then places its body with the capsular head upside down, then

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pheromone laden hairs are dislodged from the female thorax through the narrow unsealed aperture of the bag that attract males. A male attracted by the pheromone inseminates the female by placing his body upside down and enlarging his abdomen pneumatically through the unsealed opening. The abdomen and aedeagus of the male are extended and pass almost the entire female body length to make contact with the caudal genitalia of the female. The female is known to deposit her eggs within her larval skin, which lines the sack in which she developed (Leonhardt et al. 1983). The female is immediately 'parasitized' by her embryos when they start development within the female body and eventually occupy the entire female body. The female body has been described as being a little more than an ovary (Holland 1968, Kaufman 1968). Hunt (1923) reported an outbreak of OBW on orange in Sebring, Florida. Damage was observed on fruits, green twigs and mature leaves. The OBW feeds on mature fruit through the flavedo (outer skin) leaving irregular patches of damaged fiiiit rind. On occasions, the larva penetrates through the epidermis completely but without making entry holes into the fruit sacs. The OBW was described as a predator of various species of scale insects including: the camphor scale Pseudaonidia duplex Cockerell; the soft brown scale Coccus hesperidum L, a species of Ceroplastes and black scale Saissetia olea (Bernard) (Plank and Cressman 1934). OBW are distributed throughout the gulf coast states of Texas, Louisiana, and Florida (Neck 1977). In this chapter the predatory behavior of two cecidomyiid midge species that have been observed preying upon citrus rust mites are described. Also, the ecological relationship of Cryptothelea gloverii (Lepidoptera: Psychidae) with citrus rust mite and orange plants is described.

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96 Materials and Methods I Cecidomyiid Midges Studies Cecidomyiid collection Sampling for cecidomyiid larvae was completed in June and from October to the first week of December 2001 in an experimental orchard of 'Hamlin' orange trees at the CREC, Lake Alfred, Florida. Two distinct types of larvae were observed. One was yellow in color (Fig. 2A) and the other was orange with a white collar behind the mouthparts (Fig. 2B). Larvae were collected from citrus leaves and fruits and transferred individually to a Petri dish with a fine 5-0 sable brush. Fruits with high numbers of citrus rust mites (>100 cm ) were collected to prepare arenas and to provide a food source for rearing the two midge species. Some of the larvae were allowed to complete their development to obtain adults. Others were used for feeding experiments, behavioral I observations and identification of species. Data obtained on individual prey experiments were recorded separately. Rearing of cecidomyiids Rectangular fransparent plastic containers (Pioneer Plastics hic #295C, Eagan, MN) with a semi-tight lid (3 1 cm long by 24 cm wide and 1 1 cm high) served as rearing chambers for the midge larvae. A lightly moistened piece of paper towel was placed on the bottom of each container to provide increased humidity. 'Hamlin' oranges (4 to 6 cm diam.) with high numbers of CRM were collected in the field and other organisms such ants, spiders, phytoseiid, stigmaeid and tydeid mites were removed. Between 6 to 8 oranges were then placed individually on PVC rings (3.5 cm diam. by 1 cm high) in the rearing chambers and 2 or 3 cecidomyiid larvae were added to each fioiit and then the container was covered with the lid. The containers were held in place in the laboratory at

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97 25 ± 1 °C, 60 ± 10% RH under fluorescent lights set to a day length of 14:10 (L:D). The cecidomyiid larvae were observed daily and oranges with CRM were replaced as I required. When a pupa was found it was removed and isolated in a 5 cm diam. Petri dish held under the same environmental conditions. However, detection of pupae was difficult and usually they were not discovered before adult emergency was observed in the rearing chamber. Pupae were found attached to the fruit, on and under the paper towel and on occasions on the plastic walls of the chamber. Predation by cecidomyiids on citrus rust mites. The predatory behavior of cecidomyiids was observed in arenas consisting of 'Hamlin' oranges (ca. 4 to 6 cm diam.) containing high numbers of CRM. Individual oranges were placed on PVC rings (3.5 cm diam. by 1 cm high). Numbers of CRM were verified under the dissecting stereomicroscope, and a cecidomyiid larva between 0.6 to 0.8 mm long was placed on top of the orange dome within the center of the field of view of the stereomicroscope. The larva was monitored while rotating the fruit to constantly follow and focus on the midge's movement. The number of eggs, nymphs and adult CRM stages consumed by individual larvae of the two cecidomyiid species were counted separately during 10 min observation intervals. The experiment consisted of 17 yellow and 12 orange cecidomyiid larvae. It was not possible to estimate larval age and all larvae used in the experiment were of similar length between 6 to 8 mm. Data were analyzed with a single factor analysis of variance (Zar 1984). Orange Bagworm (OBW) Cryptothelea gloverii Mite predation by OBW Citrus rust mites predation: Heavily infested 'Hamlin' oranges with CRM were collected in the field and returned to the laboratory. The oranges were ca. 5 to 6 cm diam

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98 and were placed individually on a PVC ring of 3.5 cm diam by 1 cm high. One early instar OBW larva (ca. 0.4 to 0.5 cm bag length) was then placed on top of the fruit to observe its predatory behavior under a dissecting stereomicroscope. To quantify predation of citrus rust mites, 10 'Hamlin' oranges were selected and arenas were prepared as shown in Fig 6. 1 . This arena consisted of a fruit ca. 5 to 6 cm diam placed on top of a 3.5 cm diam by 1 cm high PVC ring. A 30 ml transparent inverted plastic cup (Jet Plastica Industries hic, Hatfield, PA) was then placed on top of the upper part of the fruit surface. The 4.0 cm diam plastic cup provided the arena area to assess CRM predation by the OBW and to confine the insect. The area outside the plastic cup was used as a control to quantify CRM numbers without OBW predation. A 1x1 cm square was cut on the base of the plastic cup (opposite to the orange surface) and covered with a fine nylon mesh to maintain adequate moisture. The numbers of CRM were counted on two squares with an area of 1 cm^ each within the confined space of the plastic cup. The I Icm area was defined by 4 dots on each comer of the square marked with a permanent marker on the citrus rind. Citrus rust mites were counted within the limits of the squares. The mean number of CRM in the two Icm^ areas inside the plastic cup perimeter was used as a single replicate for statistical analysis. Correspondingly, two squares with an area of 1 cm^ each were marked on the orange surface outside the plastic cup perimeter and the numbers of CRM were counted on each of them. Similarly only the mean of the squares was used as a single replicate for statistical analysis. One OBW per fhiit was placed within the enclosed space of the plastic cup and left for 24 h. A total often OBW was used in this experiment. The numbers of CRM were counted after this time on the Icm areas with and without OBW. All experiments were performed in October 2001 in

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I 99 the laboratory at 25 ± 1° C, with approximately 50 ± 5 % RH and under fluorescent lights set to a day length of 14: 10 (L:D). Data between arenas with and without OBW were compared using the sign test (Zar 1984). Citrus red mites predation: Two experiments were designed to observe predation by OBW on eggs and adult females of the citrus red mite Panonychus citri (McGregor). All experiments were performed in October 2001 in the laboratory at 25 ± 1° C, with approximately 50 % RH and under fluorescent lights set to a day length of 14: 10 (L:D). Grapefruit leaves were washed thoroughly and all mites and insects removed. Arenas were prepared using a single mature grapefruit leaf placed on top of a damp sponge pad in a 13 cm diam. plastic Petri dish. The leaf border was covered with a damp cotton strip to avoid mite escape. Each leaf was divided into two sections using a wet cotton strip that crossed the leaf perpendicularly to the leaf midrib. Thirty female P. citri were left to oviposit for 36 h on each half then removed and the numbers of P. citri eggs were counted. One OBW (n = 6) larva was then placed on the arena and egg consumption was assessed after 24 h. The arena for observing predation of P. citri females consisted of a grapefiiiit leaf disk (2.5 cm diam.) with the abaxial side facing up and placed on a wet cotton pad in a 5.5 cm diam. Petri dish. The Petri dish cover had two 0.5 cm diam. apertures for ventilation; each aperture was covered with a fine nylon mesh to prevent escape of the OBW larva. Twenty P. citri adult females were placed in each arena and one (ca. 0.5 0.6 cm bag length) OBW larva (n = 6) was then added to each arena, Panonychus citri female consumption was assessed after 12 and 24 h. Consumption here is defined as feeding of the entire P. citri female by the OBW. Mites that were found dead or desiccated were not considered consumed because preliminary observations

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100 showed that the OBW completely devoured female P. citri without leaving any body parts or exuviae. Mites that were damaged by the movement of OBW were removed and replaced periodically every 4 h. In addition, an orange fruit was observed with a heavy infestation of a scale insect. This fruit was separated and one OBW was placed to i determine predation. Assessment of OBW damage to orange and grapefruit leaves and fruits. The OBW were found more frequently on russeted orange fruits following CRM feeding injury compared with healthy clean fruits in the field. A choice test assay was designed to evaluate this apparent preference in the laboratory. Rectangular plastic containers (3 1 cm long by 24 cm wide by 1 1 cm high) with a semi-tight cover as described for cecidomyiid midges were used as rearing chambers, observations on behavior, fruit damage by OBW and for the preference assay. Pieces of paper towel moistened with deionized water were placed on the bottom to regulate humidity. The preference assay consisted of placing 4 russeted and 4 healthy mature 'Hamlin' orange > fruit in two rows within a container as above. The two types of fruit were alternately placed within each row (Fig. 6.2) and then one OBW was placed on each fruit and the damage and location (russeted or healthy frnit) of the larva was observed after 1 and 3 ' days following infestation. Four replicates were completed and data were analyzed using a paired Mest (Ott 1993, Excel® 2000). Leaf feeding by OBW was observed on 'Hamlin' orange {Citrus sinensis [L.] Osbeck), 'Flame' grapefruit {Citrus paradisi Macfadyen), 'Volkamer' lemon (C. volkameriana Tenore and Pasquale) and trifoliate orange {Poncirus trifoliate Rafinesque) in the laboratory using terminal shoots each approximately 8 cm long. The terminals had 4 leaves each and were individually placed in a 50 ml Erlenmeyer Pyrex flask that

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101 contained tap water. A cotton pad was placed as a support to hold the shoots vertically. The shoots were then immediately placed in the rectangular plastic containers of 10 cm long, 13 cm wide and 20 cm tall. Four late instar OBW larvae were placed in each container. Damage to the leaves was observed after 24 h. Results Cecidomylid Midges Success in rearing cecidomyiids to the adult stage was obtained with larvae collected in November and December, but not fi-om larvae collected in July and August. Dr. Raymond Gagne (USDA, Systematic Entomology Laboratory, Beltsville, Maryland) identified the adult cecidomyiids (Fig. 6.3). Adult males and females of the yellow type 1 larvae were recovered whereas only two females fi"om the orange type larvae were recovered. The two distinct types of larvae (Fig. 6.4 A and B) were identified as belonging in two different genera. The yellow larvae were an undescribed species of Feltiella and the females obtained from the orange larvae were identified as Lestodiplosis in the broad sense, although they do not really fit in that genus (R. Gagne, personal communication). This would suggest that they represent a new genus of cecidomyiid flies. Permanent slides mounted with the eriophyids collected during this study were identified as the citrus rust mite Phyllocoptruta oleivora (Ashmead). The number of eggs, nymphs and adult citrus rust mites consumed by Feltiella n. sp. and the species near Lestodiplosis during 10 min. observations are shown on Fig. 6.5. There were no significant differences (P > 0.05) for any of the rust mite stages consumed by Feltiella n. sp. or the species near Lestodiplosis. Feltiella n. sp. and the species near Lestodiplosis consumed 33.8 ± 4.6 (mean ± SEM) and 43.0 ± 6.4 (F = 1.99; 1,27 df, P =0.16) citrus

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102 rust mite eggs; 14.2 ± 1.4 and 15.0 ± 2.0 (F = 1.41; 1,27 df; P = 0.24) citrus rust mite nymphs and, 3.0 ± 0.4 and 5.6 ± 0.9 (F = 0.26; 1,27 df; P = 0.60) citrus rust mite adults, respectively. Orange Bagworm Early instars of OBW were observed preying on P. oleivora (Fig. 6.6). The OBW larva appeared to be distinctively associated with high populations of citrus rust mites in the field. In the laboratory, the proportion of citrus rust mites decreased significantly on arenas with OBW (0.64 ± 0.07 per cm ) (P = 0.02) whereas citrus rust mites increased in arenas without OBW (1 . 1 1 ± 0.08 per cm^) but they were not significantly different (P = 0.34). OBW preying on P. citri females was documented on film. OBW consumed 41.8 ± 8.0 (mean ± SEM) P. citri eggs after 24 h and 7.83 ± 1.66 and 10.3 ± 1.0 P. citri females after 12 and 24 h, respectively. In the choice experiment between russeted and healthy fiuit, significantly more OBW larvae were found on russeted fiaiits (5.3 ± 0.9) than healthy fioiits (2.0 ± 0.7) (n =4, t = 3.18, P = 0.03), respectively, after 24 h (Fig. 6.2). After 3 days, the trend remained significant; more OBW larvae were found on russeted fioiits (5.0 ± 0.4) than on healthy fiuits (2.25 ± 0.3) (n =4, t = 3.18, P = 0.01), respectively. Visual examination of the fruits in each container showed that more damage was produced on russeted finits than on clean healthy fioiits although numerical measurements of damage were not recorded. Feeding by the OBW larva occurs on the outer epithelial layer of the orange and this was sometimes deep enough to penetrate the rind completely and produce a hole although feeding into the fioiit sacs was not observed (Fig. 6.7). The scale insect was identified by Dr. Avas Hamon (Florida Department of Agriculture and Consumer Services, Division of Plant Industry, Gainesville) as the purple

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I 103 scale, Lepidosaphes beckii (Newman) (Diaspididae) and OBW was observed feeding on the scale. OBW also attached and carried the scale integuments on its bag. I All OBW collected in this study were only found in 'Hamlin' and 'Valencia' orange orchards that were heavily infested by P. oleivora. OBW was observed in several counties of Florida (Collier, Hardee, Hillsborough, Lake, Lee, and Polk). It likely is distributed throughout the state in different citrus orchards. Neck (1977) noted that OBW occurs along the Atlantic and Gulf coasts from South Carolina to Central America. However, OBW fed on grapefruit fruits and on young and mature leaves of 'Hamlin' orange, 'Flame' grapefruit, 'Volkamer' lemon and frifoliate rootstocks in the laboratory after 24 h. Damage on grapefruit is similar to damage observed on orange fruits. OBW feeds on leaves by starting from the border and eating part of the leaf or by chewing holes I of 0.3 to 0.8 cm diam. when beginning from the center of the leaf (Fig. 6.8). On occasion, an OBW larva may only consume the outer epithelium of either the axial or abaxial surface of the leaf without penetrating through the leaf Leaf damage in the field is difficult to identify but it was observed that young flush terminals were eaten at the leaflet borders and in many instances OBW were found near the damaged areas. Conversely, damage to fhiit can be easily identified in the field with fhiit having one to several holes eaten into the rind or albedo, or fruits may be deformed and flattened on one side due to eariier feeding damage. After initial feeding damage, some citrus fnxiX were found completely colonized by Penicillium sp. (Fungi: Moniliaceae) (Fig. 6.9) and OBW bags were recovered from these fruit. Penicillium colonization of fruit was also observed in the laboratory. In addition, OBW larvae were observed feeding on this fungus. Neonate OBW larvae are approximately 1 mm long and their body color was

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104 smoke-beige with the head capsule black. Later instars turn darker to completely black. Male winged adults (Fig. 6.10) were obtained in the laboratory and identified as Cryptothelea gloverii (Packard) (Lepidoptera: Psychidae) by Dr. J.M. Hefner (Florida Department of Agriculture and Consumer Services, Division of Plant Industry, • Gainesville) and Dr. Hugo Kons from the American Entomological Institute in Gainesville. Two male parasitoids of Chirotica thyridopteryx (Riley) (Hymenoptera: Ichneumonidae) were recovered from one OBW bag in the laboratory and identified by Dr. Lionel Stange (Florida Department of Agriculture and Consumer Services, Division of Plant Industry, Gainesville) and voucher specimens are deposited at the Division of Plant Industry in Gainesville. Discussion Cecidomyiid Midges Predatory reports on P. oleivora or other citrus rust mites by Feltiella n. sp. and/or other cecidomyiid species on citrus are based largely on anecdotal information. To date, there is not a reliable description of feeding and identification of cecidomyiid predators I of citrus rust mites in Florida or any other citrus growing area of the world. McMurtry (1977) and Perring and McMurtry (1996) cited Muma et al. (1961). However, Muma et al. (1961) only mentioned a midge as a predator that was recovered from P. oleivora colonies. There were no empirical quantification or scientific descriptions of the feeding behavior. Furthermore, all citations refer to the original descriptions by Hubbard (1883) where he appeared to describe the two types of larvae (one yellowish and the other orange with a white collar) found in this report. In this study, adults of Feltiella n. sp. (yellow larva) and a species near Lestodiplosis (orange larva with a white collar) were successfully collected, reared and identified to genus. Voucher specimens are deposited

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105 ;; with the USDA, Systematic Entomology Laboratory, Beltsville, MD. In addition, predation by these midges was observed, described and quantified on P. oleivora. Yothers and Mason (1930) and Childers (personal communication) reported that these midges appeared abundant only where high numbers of CRM occurred. This tendency . was observed with F. minuta when its population increased on Tetranychus kanzawai Kishida (Ho and Chen, 1998). It is evident from the consumption data presented here, that Feltiella n. sp. and the species near Lestodiplosis are efficient predators of P. oleivora eggs and nymphs. If we extrapolate the average number of all P. oleivora stages consumed in 10 minutes to 24 h then 622 adults, 2,104 nymphs and 5,535 eggs are consumed per day per midge larva. The efficiency of Feltiella n. sp. and the species near Lestodiplosis surpass the predatory capacity of Iphiseiodes quadripilis, when we compare these numbers with the predation rates of /. quadripilis on Aculops pelekassi (PRM) (Chap. 4). A similar comparison was described with F. minuta with respect to Amblyseius womersleyi Schicha on eggplant (Ho and Chen 1998). Laboratory observations of these predators indicate that they are looking for citrus rust mites eggs most of the time, continuously moving the anterior part of the body to the left and then to the right while moving forward and changing direction. Once an egg is detected it is rapidly consumed giving the appearance that the larva is still in search of prey. Consumption of citrus rust mite eggs by these two cecidomyiid species occurs urmoticed by a non-trained acarologist/entomologist due to the small size of an egg (ca. I 30-40 Mm) and its whitish to transparent color. All of these observations were taken at 40X magnification using a stereomicroscope. It was noted that the presence of P. oleivora eggs are essential for two reasons. First, both types of cecidomyiid larvae were

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106 I frequently found on the bottom of the plastic containers and separated from the whole fruit arenas that were still infested with citrus rust mite motiles. However, these arenas lacked citrus rust mite eggs that were already consumed by the cecidomyiid larvae. When a new fruit with abundant citrus rust mite eggs was provided then the cecidomyiid larva immediately started to feed and remained on the fruit. Second, on many occasions, both types of larva encountered adult citrus rust mites but they were not interested in feeding. Other times, they raised the adult citrus rust mite off the substrate with their mouthparts and appeared to throw the motile mite aside. On occasion, when the attacks on citrus rust mite adults were successfiil, the larval mouthparts were searching or directed to the ventrolateral part of the citrus rust mite body, just behind the second pair of legs. This was not observed when the attacks were directed to nymphs. Nymphs seemed to be more vulnerable and were successftilly eaten when grasped on any part of their bodies. Abundance of cecidomyiids was not recorded in this study because the priority was to identify the midges and evaluate their predation on citrus rust mites. However, most of the larvae collected in the field were more abundant on fruit than leaves during October and November. This apparent abundance of midges on fruits is consistent because citrus rust mites are more abundant on fruits than on leaves especially when fruits are developing as described for A. pelekassi (Childers and Achor 1999). Other mites (tydeids, tarsonemids and fungal mites) were present in this orchard too. However, their numbers were low and predation by this midge on either E. banksi or P. citri, two important tetranychids in Florida may occur. Both mites were essentially absent from this orchard during the time that cecidomyiid larvae were collected.

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107 The search for prey and/or dispersion by these two cecidomyiid species is unclear but it is important to understand their biology and ecology. Observations in both the laboratory and field showed that both species of midges were capable of jumping or springing off the plant substrate. The larva raised the middle part of its body by placing the head to the other end and creating a "loop". Then it would rapidly let go resulting in a leap or jump (personal observation). The sternal spatula or breastbone aided in this movement as a spring-like device. It is not known if this movement is made at random or if it is directed toward a desired direction. If they fall to the ground, then the larvae would likely desiccate and die. From observations in the rearing arenas in the laboratory, it appears that this movement has a determined direction. When the fioiit in the rearing containers had a few citrus rust mite eggs then the cecidomyiid larvae would abandon the fruit and be found on new fruit that had abundant citrus rust mite eggs. The dispersal strategy of these midges and their uimoticed predation on eggs resulted in earlier failed attempts to rear the dipteran larvae. Similar situations have been encountered by Yothers and Mason (1930). They wrote: "These larvae are very small and extremely delicate, and all attempts to rear them to maturity have failed". Few accurate studies on the biology of predacious cecidomyiids and their effects on prey mite populations are available. This is not only the case with cecidomyiids preying on eriophyids but also on tetranychids (Chazeau 1985). Orange Bagworm The OBW was described as a predator of immobile insects or eggs for the first time by Plank and Cressman (1934) in Louisiana, USA. Sands et al. (1986) described a predacious lepidopteran species from South Afiica 52 years later that was released for biological control of the white wax scale Gascardia destructor (Newstead) in Australia.

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I t 108 Patrick (1999) found the case-bearing larvae of Rhathamictis perspera (Meyrick) (Tortricidae) feeding on stationary live prey, including spider eggs, and pupae of Micromus tasmaniae (Walker) and Wesmaelius subnebulosus Stephens (Neuroptera: Hemerobiidae). Patrick (1999) also observed R. perspera feeding on a variety of dead insects in New Zealand. In this study, OBW not only fed on immobile prey such as L. beckii, and P. citri eggs but also on motile prey such as P. citri females. The predation assays described here with P. citri females and P. oleivora motiles were artificial because the arenas did not allow mite escape to avoid predation by OBW larvae. If we note the movement of P. oleivora, it would be very limited due to its small body size (0.127 mm, Muma, 1975) when compared to the movement of OBW that is about 50 times larger than P. oleivora. Thus, it is improbable that P. oleivora can escape predation by OBW. OBW larvae decreased the numbers of P. oleivora per cm^ by 36%. Whereas, P. citri egg and female numbers were reduced 68% (41.8 ± 8.0) and 50% (=10.3 ± 1.0), respectively, in the laboratory after 24 h. OBW predation on E. banksi eggs and the pink citrus rust mite A. pelekassi were not tested. Predation of citrus rust mites can be an adaptation of earlier instars of the OBW for this type of prey due to prey abundance and richer nutrients compared with vegetative tissue. Plant foliage is of low nutritional value compared with insect tissue. For example, the protein content is 2.8 ± 0.6 versus 17.7 ± 1 .4 of fresh weight for plant versus insect tissues, respectively (Coll and Guershon 2002). Furthermore, many omnivores enhance their fitness by shortening their developmental I times thus reducing their vulnerability to predation (Agrawal and Klein 2000). This latter point needs further study. Neonate OBW larvae that hatched in the laboratory (around middle February) and placed on orange fruit and leaves without rust mites were not able

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109 I to survive. This was due either to the absence of rust mites or to inadequate environmental conditions. These conditions were similar to those provided to OBW larvae collected in October 2001 from the field that already had fed on citrus rust mites. Predation by lepidopteran larvae is a rare event, but there are cases such as the carnivorous caterpillars in the genus Eupithecia (Geometridae) from Hawaii (Montgomery 1983 a, b). An Eupithecia caterpillar was observed preying for the fu-st time in 1972 by Montgomery. Since then at least 18 distinct species have been identified including Eupithecia orichloris Meyrick and Eupithecia rhodopyra Meyrick. These predators use different types of perch sites and camouflage to ambush flying and walking prey. The genus Eupithecia is distributed worldwide but only the Hawaiian species are carnivores. However, predation by lepidopteran larvae has been described in places other than Hawaii. In the USA, the only predatory lepidopteran larva known is the butterfly Feniseca tarquinius (Fabricius) [Lycaenidae]. The female lays eggs in colonies of woolly aphids including the genera Neoprociphilus, Pemphigus, Prociphilus, and Schizoneura. The caterpillars prey upon these aphids and sometimes on scale insects, treehoppers and ants (Pierce 1995). In India Spalgis epius Westwood, another lycaenid species, has been described as a predator of the pink hibiscus mealybug Maconellicoccus hirsutus (Green) (Gowda et al.l996). OBW is the second example of a predatory lepidopteran found in the USA and the first report of this type of predation occurring between a lepidopteran species and an acarine prey species. Predation of rust mites was observed by neonate larvae (ca. 1 mm) and in later instars (ca. 4 mm) but the effect of this diet on the OBW needs fiirther study.

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110 It is known that some psychid species feed on lichens and other microorganisms (Wessels and Wessels 1991). OBW larval preference for russeted fruit, over clean healthy orange fruit demonstrated in choice test experiments, is consistent with a higher accumulation of microorganisms such as fungi that begin to degrade necrotic epidermis areas on 'Sunburst' mandarin leaves affected by rust mites (Achor et al. 1991). OBW despite its predatory behavior, should be considered a minor pest of citrus because it feeds on leaves and fruit of orange and grapefruit, and the leaves of two citrus rootstocks (Volkamer lemon and trifoliate). It is also possible that this insect feeds on other species of citrus. Leaf damage by OBW larvae was observed in the laboratory on four different hosts, but identifying this type of damage can be a difficult task to recognize in the field because many other insects can cause similar types of damage on leaves. OBW was not very abundant in the field and the search and collection for OBW larvae can also be difficult. They can be found on leaves, branches and fruits and the coloration of the bag can easily be camouflaged with small branches or viewed as a thorn. However, identifying damage on fruit can facilitate detection of bagworms because OBW larvae I were generally found either on the damaged fiuit or around it. OBW appears to have a low dispersal capacity. Grapefruit damage was obtained only in the laboratory and not a single OBW was found on grapefruit in several fields sampled in the Lake Alfred area. This information coincides with Plank and Cressman (1934) who reported only slight damage on grapefruit in contrast to more severe damage on orange. In the field, fiaiits damaged by OBW larvae have been observed either deformed or with holes in the albedo. On several occasions, OBW were collected from finits that were completely infested with the fiingus Penicillium sp. The low abundance of OBW in the field can be

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Ill related to the effect of pesticides or to the presence of parasitoids. The two male parasitoids (C. thyridopteryx) that were found on OBW are frequently associated with psychids and this parasitoid also affects the evergreen bagworm T. ephemeraeformis (Bersford and Tsao 1975). Damage caused by OBW is an open passage for pathogens that can infect fruit more easily and this was observed in both the laboratory and in the field with fhiit colonized by I a Penicillium fungus after feeding injury by OBW. OBW also fed on Penicillium and could be categorized as being omnivorous. It was observed that when an OBW larva was feeding on leaves that were perpendicular to the ground, it would be a hard task for the insect to carry the bag and feed. However, OBW attached a part of the bag to the leaf with silk threads before initiating feeding. Thus the larva is able to feed and freely remove most of its body from the case, hi the field, an occasional OBW larva was found hanging on trees from silk threads and in the laboratory, neonate OBW were observed in similar situations hanging from the rearing cages, hi comparison, neonate larvae of T. ephemeraeformis were observed suspended from silk threads and dispersed by ballooning I in the wind (Ghent 1999). In the laboratory, OBW larvae were observed laying silk threads in circles around the upper part of oranges within the rearing cages. This behavior may be due to the influence of light or indicates approach of pupation. Debris and frass were observed being excreted through the apical opening of the case. These were small particles of different colors that varied with the food types OBW fed upon. In this study, three insect species were identified as predators of citrus rust mites. The two cecidomyiids Feltiella n sp. and Lestodiplosis are new undescribed species and appeared to be effective natural enemies of citrus rust mites. Both appeared highly

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112 specialized in their abilities to eat citrus rust mite eggs and nymphs. It is possible that differences in behavior and prey type exist between the two cecidomyiids. However, the original assumption was that these two types of larvae were one species. Although reduction of citrus rust mite populations for a current season due to predation by the two midges may be unnoticed, the positive effects of these predators may be obtained during the following growing season due to a high reduction in egg numbers. Cryptothelea gloverii was long ignored as a pest of citrus but its biology is fascinating. The predatory behavior toward mites remains unknown. OBW is predacious on citrus rust mite, but it is also phytophagous and omnivorous. To this point, there is not enough data to define correctly how dependent OBW is on citrus rust mites. It has been observed that earlier larval instars of OBW fed on citrus rust mites, but late instars can feed on citrus rust mites due to its omnivorous feeding. Accepting that a citrus rust mite diet is more nutritious and may possibly shorten its developmental time than a plant diet alone, OBW preying on CRM can be adaptively advantageous to improve OBW fitness. Also, any use of OBW to reduce citrus rust mites or P. citri populations is minimized by its phytophagy. A positive aspect of its phytophagy is that it prefers russeted to healthy finit. Consequently OBW cannot be considered a natural enemy of citrus rust mites for biological control purposes but rather a secondary pest of citrus. Reasons why OBW have not become abundant need to be studied as well as identifying its natural enemies on Florida citrus. Many questions need to be answered about these three insects such as their phenologies, susceptibility to pesticides, dispersal,

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113 and relationships with other organisms. Further studies are needed and should consider alternative prey, host plants, and evaluation of pesticides.

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114 Figure 6. 1 . Arena used to quantify OBW predation of Phyllocoptruta oleivora. The confined by the transparent plastic cup contained the OBW.

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115 Figure 6.2. Damage caused by Cryptothelea gloverii larvae feeding on russeted and healthy 'Hamlin' oranges. C. gloverii is more commonly found on russeted fruits.

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116 Figure 6.3. Adult Feltiella n. sp. predator of the citrus rust mite Phyllocoptruta oleivora obtained in the laboratory, vertical bars beneath insect are in millimeters.

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117 Figure 6.4. Two distinct larval types of two new species of cecidomyiids surrounded by their citrus rust mite Phyllocoptruta oleivora prey. (A). Feltiella n. sp. and (B) a species near Lestodiplosis n. sp. Note ring around the anterior end of the larva.

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118 V) s 3 C 1 O To E 3 (A C o u (0 a> nj 50 40 30 20 ^ 10 E w 3 0 Feltiella n sp. near Lestodplosis nsp. 1 1 i Eggs Nymphs Adults Eggs Nymphs Adults Stages Figure 6.5. Mean numbers ± SEM of different citrus rust mite {Phyllocoptruta oleivora) stages consumed by larvae of Feltiella n sp. and a species near Lestodiplosis n. sp. I

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Figure 6.7. Hole created by Cryptothelea gloverii larvae into the rind of a 'Hamlin' orange. C. gloverii larvae penetrate the rind but do not feed on the fruit sacs.

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120 Figure 6.8. Damage produced by a Cryptothelea gloverii larva on a citrus leaf C. gloverii can grasp the outer layer of the upper or lower epidermis without penetrating through the leaf and making a hole. Figure 6.9. Penicillium contamination associated with feeding damage caused by Cryptothelea gloverii. The bag of C. gloverii (arrow) is often found on or adjacent to this type of fruit and larval feeding on fiingi was observed.

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121

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CHAPTER 7 SUMMARY AND CONCLUSIONS The 'green revolution' with the use of selected seeds for high yields and modem practices in monoculture, such as use of pesticide sprays against arthropod pests and plant pathogens, and improvement in soil nutrients with chemical fertilizers, has significantly improved crop yields. However, this was only a temporary remedy because phytophagous mite and insect pests have rapidly augmented their populations in these I monocultures as a result, in part, due to lower population densities of natural enemies or new pest problems that have emerged. Pesticide resistance development has been an additional problem. Phytophagous mites have this latter response to several pesticides while natural enemies (i.e. phytoseiids) oftentimes are annihilated or greatly reduced because of their lower ability to develop resistance. Hormoligosis in many acarine and insect pests causes increases in their fertility (Voss 1988, Childers 1994b, Omoto et al. 1995). In addition, some pesticides have contributed to contamination of ground water and the environment. Phytoseiid mites are natural enemies that successfijlly control phytophagous mite pests in glasshouses in Europe and their predatory capacity has been proven in the field (i.e. on strawberry. Croft et al. 1998, Easterbrook 1998 and on cassava, Yaninek et al. 1998). In citrus, there is a contribution by natural enemies in reducing mite pests, although pest mites are above economic threshold levels. Research needs remain. For example, of the 33 species of phytoseiids found on Florida citrus alone (Childers and Denmark 122

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123 unpublished) only the biologies of 4 species have been studied in the last 20 years. These species are Euseius mesembrinus (Abou-Setta and Childers 1987), T. peregrinus (Pena 1992, Fouly et al. 1995), Galendromus helveolus (Chant) (Caceres and Childers 1991), and Proprioseiopsis rotendus (Abou-Setta et al. 1997). Therefore, research on integrated pest management in orchards and the evaluation of ecological factors of organisms associated with pest control are worthy efforts from both economical and ecological perspectives. i The citrus-mite-insects system is represented in Fig. 7. 1 . This is a simplified graph without considering pesticides. This system is complex, features such as changes in leaf morphology caused by the citrus leafininer P. citrella or plant volatiles are not represented. However, both affect the population composition of mites or small I arthropods. The positive effect of mined leaves in providing refuge for phytoseiids was established in Chapter 3, whereas the effects of plant volatiles on /. quadripilis were described in Chapter 5. Also, the roles of tydeid mites observed in abundant numbers within citrus orchards are not completely understood. Preliminary reports on their fungivorous habits were recognized by Muma (1975) and the predacious behavior of Lorryia formosa Cooreman was identified by Aguilar (2001). Furthermore, the effect of phylloplane fungi on predacious phytoseiids requires further study because some species feed on fiingi. These fimgi can be an important supplement on their diet breadth when prey are scarce. Antagonistic relationships between natural enemies such as an unknown I neuropteran larva preying on a coccinellid pupa or a cecidomyiid larva were observed. Villanueva et al. (2002) observed that Harmonia axyridis Pallas (Coccinellidae) consumed Ambyseius sp. and E. mesembrinus in the laboratory.

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124 In this study, it was shown that three prevalent phytoseiid species in citrus orchards preferred inner leaves of the tree canopy compared with the outer leaves. This is significant to make density evaluations of the three species in the field and increase the accuracy of mite surveys. However, mite movement was not detected in this study from inner leaves to outer leaves during the night as reported by other authors (Muma 1967b, Garcia Mari et al. 1985). The results obtained by Muma (1967a) showed numerical differences in his data but did not mention if they were significantly different. Garcia Mari et al. (1985) menfioned that they sampled only young leaves. It is possible that these young leaves would have had higher numbers of tetranychids eggs and motiles as observed with E. banksi during April in this study (Chapter 2). However, young leaves were also observed with low numbers of phytoseiids (Chapter 2). The high densities of predacious mite populations on mined leaves damaged by P. citrella is a novel finding since it has not been previously reported. This can be applied to other crops that are affected by leaftniner pests. Villanueva and Harmsen (1997) reported abundant numbers of tarsonemid mites present on apple leaves damaged by the spotted tentiform leafminer Phyllonotycter blancardella (Fabricius) (Lepidoptera: Gracillariidae). This implies that leaf distortion caused by other pest species need to be evaluated because they can have a similar positive effect on phytoseiid or other small arthropod populations. One of these organisms is the Asiatic citrus psyllid Diaphorina citri Kuwayama (Hemiptera: Psyllidae) a recently introduced pest into Florida citrus (Anonymous 1999) that produces changes in young terminal leaf flushes. Higher numbers of phytoseiids were observed on mined leaves than on healthy mature or young leaves (Chapter 3). This result can be usefial to facilitate collections of some

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125 phytoseiid species in the field. Searching for phytoseiids on mined leaves is still time consuming because it is necessary to tease apart the rolled or distorted leaves, open the mines and then examine the leaves under the dissecting stereomicroscope. Comparatively similar numbers of phytoseiids species were recovered from 10-minedleaves compared to a random 40-leaf sample collected into alcohol jars (See Chapters 2 and 3) during March 1999 and 2001 . Research completed on the shiny button mite (SBM) Iphiseiodes quadripilis in Chapters 4 and 5 showed important findings on its biology, behavior and ecology. SBM was able to feed and reproduce on M crocea (ice plant) and Quercus sp. (oak) pollens and on different stages of E. banksi and P. citri. Iphiseiodes quadripilis has the fastest developmental time when provided with E. banksi as a food source. With Quercus sp. pollen, more adults were obtained than the rest of the evaluated diets. It is apparent that both /. quadripilis females and larvae did not affect citrus rust mite, P. oleivora populations, but selectively preyed on the pink citrus rust mite, A. pelekassi. This finding is the first report showing a preference by a phytoseiid species on Florida citrus for one eriophyid species over another. Additional studies with other phytoseiids and nonphytoseiid predators are needed. Preliminary studies with E. mesembrinus and an undescribed species of Amblyseius in the laboratory resulted in similar outcomes (Villanueva unpublished). The reasons for the rejection of P. oleivora are unknown. They may be related with low nutritional quality and toxic substances within P. oleivora (Sabelis 1996) and this can be one of the reasons to explain the high abundance of this pest in the field. Iphiseiodes quadripilis was observed foraging during daylight hours and this is opposite of reports by Muma (1975) and Muma and Denmark (1971) who found

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126 that /. quadripilis was more active after dusk. Also, Muma (1971, 1975) briefly mentioned that this mite was more abundant on grapefruit, but he did present supportive data. In this study, the preference of /. quadripilis for grapefruit leaves over orange leaves was demonstrated. The relative abundance of /. quadripilis in grapefruit orchards in the field was shown and supported in laboratory assays with direct contact and olfactometer tests to support this argument. This preference needs further study and can 1 be explained by the emission of volatiles by grapefruit that attract and retain /. quadripilis in grapefruit orchards despite more abundant prey available in adjacent orange orchards. The establishment of an /. quadripilis colony in the laboratory failed in spite of many attempts using single or mixed diets of pollen, spider mites, and honey. Earlier efforts by M. Abou-Setta met with similar problems (Childers, personal communication). However, it has been reported that I. quadripilis preyed on eggs of whiteflies and scale insects (Muma 1971). In addition, preliminary assays in this study with Penicillium sp. and two citrus pathogens showed that /. quadripilis would feed on fungi (Villanueva unpublished). The tritrophic relationship among plants, herbivores and natural enemies is important and complicated as is shown here. As new research becomes available, the system in Fig 7. 1 will become increasingly more understandable. The two cecidomyiids studied here, Feltiella n. sp. and the species near Lestodiplosis showed that they effectively prey on citrus rust mite eggs, nymphs and adults. They may also prey on A. pelekassi. However, A. pelekassi were not present during the time of the study. Further studies are needed to know if Feltiella n. sp and the species near Lestodiplosis prey upon P. citri ox E. banksi or if they are specific predators of both citrus rust mite species. From this study, we can infer that these midges can cause citrus rust

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127 mite population reductions during the next generation; although Muma (1971) and McMurtry (1977) suggested that cecidomyiids require high densities of citrus rust mites. Compared to the phytoseiids studied, these midges fed voraciously on P. oleivora and potentially the numbers of P. oleivora motiles and eggs consumed per day per midge could be in the thousands, although this is an extrapolation based on 10 minute feeding studies. It can be argued that phytoseiids are more abundant than cecidomyiids and thus they contribute to the reduction of P. oleivora numbers. Yet, these two types of natural enemies and others can have an additive effect in reducing both species of citrus rust mites and should be included in developing integrated pest management practices. The orange bagworm, Cryptothelea gloverii was found to be associated with P. oleivora and it appears that this is an omnivorous insect. Cryptothelea gloverii preying on either P. oleivora or P.citri has not been previously reported although C. gloverii was observed preying upon scale insects (Plank and Cressman 1934). The impact of C. t gloverii on P. oleivora populations can be minimal because very low numbers of this insect were found. However, P. oleivora may have a positive impact on the fitness of C. I gloverii especially on the first larval instar. This needs further research. Cryptothelea gloverii fed on leaves of different varieties of citrus included grapefinit and orange. However, C. gloverii were only found on orange orchards with high densities of citrus rust mites across several counties of central Florida counties. The use of C. gloverii for biological control in citrus can be inadequate due to the phytophagous behavior of the insect and its ability to damage fiaiit and leaves. Fruit damaged by C. gloverii were easily colonized by pathogens in the field and laboratory.

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c^^Wi--'-^v;•"'•'^:'!^»^^j^^^^' All of these relationships described above can be observed in Fig. 7.2. This figure focuses on phytoseiids and other organisms that can enhance their populations or be antagonistic to them. These relationships are complex and some of them need further study. For example, Kumar and Mishra (1991) observed that pollen grains on rice favored the development of both pathogenic and saprophytic fungi. Belczewski and Harmsen (1997) sprayed an apple orchard with an epiphytic fungus that favored development of T. urticae whereas low numbers of P. citri were observed. The antagonistic relationship of spider mites and eriophyids also was observed on both citrus and apple. Finally, plant volatiles have been studied recently and the effects appear favorable toward some natural enemies. Here /. quadripilis was observed remaining on grapefruit in greater numbers than on orange despite greater E. banksi abundance on the I orange varieties. It is possible that plant volatiles not only attract /. quadripilis but also retain the predator. hi this thesis we observed that: 1. Phytoseiids preferred inner leaves of the tree canopy to outer leaves. 2. Leaves damaged by the leafminer P. citrella are used as refugia for phytoseiid mites and these leaves had higher numbers of phytoseiids compared with healthy mature leaves or young leaves. 3. A high correlation was found between phytoseiid abundance and pollen grain counts on grapefruit leaves during flowering. 4. Iphiseiodes quadripilis developed and reproduced on pollens of M. crocea and Quercus sp and on E. banksi and P. citri. After starvation /. quadripilis only preyed on Aculops pelekassi but did not prey on Phyllocoptruta oleivora.

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129 5. Iphiseiodes quadripilis numbers were more abundant on grapefruit than orange leaves in the field and this mite preferred grapefruit over orange based on olfactometer choice tests. However, the plant type where /. quadripilis was collected appeared to have an imprint influence on the type of plant the predatory mite chose in the contact choice test. 6. Feltiella n. sp. and a species near Lestodiplosis were identified and reared on P. oleivora. Feltiella sp. larva is yellow in color and the species near Lestodiplosis is orange with a white band close to the mouthparts. They prey on eggs, nymphs and adults of P. oleivora. 7. Cryptothelea gloverii (Psychidae), a microlepidopteran, was found in orange orchards. It preyed on P. oleivora motiles and P. citri eggs and females but also caused feeding damage on both citrus fruit and leaves.

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130 OBW Figure 7. 1 . Prey-predator relationships of the citrus-acari-insects system on Florida citrus. Predation is indicated by arrows. Broken lines are not well studied relationships, blue lines are relationships observed in this study, ((ji ecn) = Natural enemies, (Red) = Phytophagous, (Light blue) = Undefined roles, ( ) omnivores, and ( ) pollen. The (+) and (-) signs indicate the relationship to plants. (?) indicates a poorly studied relationship.

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131 Figure 7.2. Ecological relationships that enhance focusing on phytoseiid mites on citrus. Various relationships were observed in this study. Some were observed in other studies on different crops. Solid black arrows indicate relationships observed in this study. Broken line arrows indicate relationships that require fiirther study. Red line indicates the presence of these organisms on citrus leaves.

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APPENDIX MIXED ANALYSIS OF VARIANCE OF MITES FOUND IN 4 SITES WITH ADJACENT ORCHARDS OF ORANGE AND GRAPEFRUIT 132

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133 Table A.l. Transformed means, standard error of means (SEM), and ± 95% confidence interval limits for total numbers of Iphiseiodes quadripilis motiles 1" sample dates Factor Factor n mean SEM -95% +95% CnfLmt CnfLmt Total 40 2.30 0.16 1.97 2.64 {l}Blockl Bl 10 1.87 0.33 1.19 2.54 {l}Block24B B24B 10 2.93 0.33 2.25 3.60 {l}Block22 B22 10 2.71 0.33 2.04 3.38 {IjBader BAD 10 1.71 0.33 1.04 2.38 {2}Grapefruit GF 20 3.06 0.23 2.58 3.53 b {2}0range OR 20 1.55 0.23 1.08 2.03 a 1*2 Bl GF 5 2.40 0.47 1.46 3.35 1*2 Bl OR 5 1.33 0.47 0.38 2.27 1*2 B24B GF 5 4.23 0.47 3.28 5.18 1*2 B24B OR 5 1.62 0.47 0.67 2.57 1*2 B22 GF 5 3.42 0.47 2.48 4.37 1*2 B22 OR 5 1.99 0.46 1.05 2.94 1*2 BAD GF 5 2.16 0.46 1.21 3.10 1*2 BAD OR 5 1.26 0.46 0.31 2.21 2"" sample dates Factor Factor n mean SEM -95% +95% CnfLmt CnfLmt Total 40 2.35 0.17 2.01 2.69 {l}Blockl Bl 10 2.05 0.33 1.37 2.72 {l}Block24B B24B 10 2.81 0.33 2.14 3.49 {l}Block22 B22 10 3.38 0.33 2.71 4.06 {l}Bader BAD 10 1.16 0.33 0.48 1.84 {2}Grapefruit GF 20 3.30 0.23 2.82 3.78 b {2} Orange OR 20 1.40 0.23 0.93 1.88 a 1*2 Bl GF 5 3.39 0.47 2.43 4.34 1*2 Bl OR 5 0.71 0.47 -0.25 1.66 1*2 B24B GF 5 3.57 0.47 2.61 4.52 1*2 B24B OR 5 2.06 0.47 1.10 3.01 1*2 B22 GF 5 4.72 0.47 3.77 5.68 1*2 B22 OR 5 2.04 0.47 1.09 3.00 1*2 BAD GF 5 1.51 0.47 0.55 2.47 1*2 BAD OR 5 0.81 0.47 -0.14 1.77

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134 Table A2. Transformed means, standard error of means (SEM), and ± 95% confidence interval limits for total numbers of other phytoseiid motiles (phytoseiids other than /. quadripilis) 1^' sample dates Factor Factor n mean SEM 95 % + 95% CnfLmt CnfLmt Total 40 2.53 0.23 2.07 2.99 {l}Block 1 Bl 10 2.59 0.43 1.62 3.57 a {IjBlock 24B B24B 10 1.99 0.41 1.05 2.93 ab {l}Block22 B22 10 1.70 0.30 1.01 2.38 a {IjBader BAD 10 3.85 0.38 2.99 4.70 b {2} Grapefruit GF 20 1.91 0.26 1.36 2.46 a {2} Orange OR 20 3.16 0.32 2.49 3.82 b 1*2 Bl GF 5 1.68 0.13 1.33 2.04 1*2 Bl OR 5 3.50 0.64 1.73 5.27 1*2 B24B GF 5 1.25 0.37 0.23 2.27 1*2 B24B OR 5 2.73 0.60 1.06 4.40 1*2 B22 GF 5 1.33 0.29 0.52 2.14 1*2 B22 OR 5 2.07 0.51 0.65 3.48 1*2 BAD GF 5 3.37 0.58 1.75 4.99 1*2 BAD OR 5 4.33 0.43 3.14 5.53 2"*^ sample dates Factor Factor n mean SEM 95 % CnfLmt + 95% CnfLmt Total 40 2.19 0.20 1.78 2.59 {l}Block 1 Bl 10 2.58 0.61 1.20 3.96 {l}Block 24B B24B 10 2.39 0.38 1.53 3.25 {l}Block22 B22 10 1.62 0.27 1.00 2.24 iUBader BAD 10 2.16 0.22 1.67 2.66 {2} Grapefruit GF 20 1.70 0.19 1.29 2.10 {2} Orange OR 20 2.68 0.32 2.01 3.34 1*2 Bl GF 5 1.64 0.32 0.75 2.53 1*2 Bl OR 5 3.52 1.06 0.56 6.47 1*2 B24B GF 5 2.18 0.58 0.58 3.79 1*2 B24B OR 5 2.60 0.54 1.10 4.09 1*2 B22 GF 5 1.28 0.34 0.35 2.22 1*2 B22 OR 5 1.96 0.41 0.83 3.09 1*2 BAD GF 5 1.69 0.25 1.01 2.37 1*2 BAD OR 5 2.63 0.21 2.05 3.22

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135 Table A3. Transformed means, standard error of means (SEM), and ± 95% confidence interval limits for total numbers of phytoseiid motiles (. quadripilis + other phytoseiids). 1*' sample dates F actor Factor n mean SEM 95 % CnfLmt + 95% CnfLmt Total 40 4.84 0.24 4.35 5.32 B 1 10 4.46 0.48 3.49 5.43 1 HRlnck 24R B24B 10 4.92 0.48 3.94 5.89 {l}Block22 B22 10 4.41 0.48 3.44 5.38 {IjBader BAD 10 5.56 0.48 4.59 6.53 {2} Grapefruit GF 20 4.96 0.34 4.28 5.65 {2} Orange OR 20 4.71 0.34 4.02 5.40 1*2 Bl GF 5 4.09 0.67 2.71 5.46 1*2 Bl OR 5 4.83 0.67 3.46 6.20 1*2 B24B GF 5 5.48 0.67 4.11 6.86 1*2 B24B OR 5 4.35 0.67 2.98 5.72 1*2 B22 GF 5 4.75 0.67 3.38 6.12 1*2 B22 OR 5 4.06 0.67 2.69 5.43 1*2 BAD GF 5 5.52 0.67 4.15 6.90 1*2 BAD OR 5 5.59 0.67 4.22 6.97 2" sample dates Factor Factor n mean SEM 95 % + 95% CnfLmt CnfLmt Total 40 4.54 0.21 4.10 4.97 {IjBlock 1 B 1 10 4.63 0.43 3.76 5.50 {l}Block 24B B24B 10 5.20 0.43 4.33 6.07 {l}Block22 B22 10 5.00 0.43 4.13 5.87 { IjBader BAD 10 3.32 0.43 2.45 4.19 {2} Grapefruit GF 20 5.00 0.30 4.38 5.61 {2} Orange OR 20 4.08 0.30 3.46 4.69 1*2 Bl GF 5 5.03 0.60 3.80 6.26 1*2 Bl OR 5 4.22 0.60 2.99 5.45 1*2 B24B GF 5 5.75 0.60 4.52 6.98 1*2 B24B OR 5 4.65 0.60 3.42 5.88 1*2 B22 GF 5 6.01 0.60 4.78 7.24 1*2 B22 OR 5 4.00 0.60 2.77 5.23 1*2 BAD GF 5 3.20 0.60 1.97 4.43 1*2 BAD OR 5 3.44 0.60 2.21 4.67

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136 Table A.4. Transformed means, standard error of means (SEM), and ± 95% confidence interval limits for total numbers of Eutetranychus banksi eggs. 1^* samnle dates Factor Factor fj mean SEM 95 % Pnf T mt + 95% Tntnl 1 ULal 40 Z-tJ 0 90 1 87 % 0"^ j.uj \ I JDIOCK 1 R 1 0 71 0 S7 U.J / 0 Af\ 1 87 1 .o / \ I JDIOCK Z.HD R ^zlR in 1 0 ^7 U.J / 0 ^9 U. JZ 9 8^ z.o J \ I j DHJLKZZ R ?'> D-ZZ 1 0 IKJ 0 87 0 ^7 U.J / 0 9Q -U.Z7 9 04 z.ut 1 1 / oaaer RAH 1 n O.J J 0 ^7 U.J / J. JO 7 fiO { 2 } Grapefruit zu 1 .43 n Ac\ U.Oj 7 77 Z.Z / {2} Orange UK zU 1 AA J. 44 n /in U.4U 7 A7 z.oz /I 77 4.Z / d1 ur 5 ATI A O 1 O.ol -u.y4 ^ 1Z Z.JJ a 1 *9 Rl OR 0 71 0 81 -0 94 9 35 o a 1*2 B24B GF 5 1.66 0.81 0.01 3.30 a 1*2 B24B OR 5 1.71 0.81 0.07 3.36 a 1*2 B22 GF 5 0.71 0.81 -0.94 2.35 a 1*2 B22 OR 5 1.03 0.81 -0.61 2.68 a 1*2 BAD GF 5 2.73 0.81 1.09 4.38 a 1*2 BAD OR 5 10.32 0.81 8.67 11.97 b 1*2 Bl GF 2.45 0.29 1.87 3.03 a J. ClV/LVJi mean SEM 95 % Tnf T mt + 95% fnf T mt Total 40 5 43 0 33 4 75 6 1 1 b 1 1 \Rlnrk 1 Rl 10 1 \J 6 X4 0 67 S 48 8 91 h u R74R 10 u.ou 0 ftl u.u / S "^0 J. JU 8 03 O.UJ u / 1 lRlnrk-79 R77 DZZ 1 0 9 '^7 Z.J / 0 f\l u.o / 1 90 1 .zu J.7J n a / 1 \RaHpr 1 1 f DaUCl RAD 1 0 J.OJ 0 67 u.o / A 98 H.ZO 7 01 / .U 1 U D \ z / vjrdpeiruu or '70 zu H.Z4 n Ai U.4 / j.Zo ^ 7 1 J.Z 1 a } fill *^ A \ z 1 urange UK ZU o.oz (\ AI u.4 / j.Oj 7 ^8 /.JO (5 1 1 Z D 1 til Ur J J.I 1 u.yj j.o4 /. /U 1*2 Bl OR 5 7.92 0.95 5.99 9.84 1*2 B24B GF 5 6.12 0.95 4.19 8.05 1*2 B24B OR 5 7.21 0.95 5.28 9.14 1*2 B22 GF 5 0.99 0.95 -0.94 2.91 1*2 B22 OR 5 4.15 0.95 2.22 6.08 1*2 BAD GF 5 4.10 0.95 2.18 6.03 1*2 BAD OR 5 7.19 0.95 5.26 9.12

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137 Table A.5. Transformed means, standard error of means (SEM), and ± 95% confidence interval limits for total numbers of Eutetranychus banksi motiles l" sample dates Factor Factor n mean SEM_ 95 % + 95% CnfLmt CnfLmt Total 40 2.82 0.32 2.16 3.47 {l}Blockl Bl 10 1.62 0.46 0.69 2.54 {l}Block24B B24B 10 4.02 0.46 3.09 4.95 {l}Block22 B22 10 0.71 0.64 -0.61 2.02 {l}Bader BAD 10 1.87 0.64 0.56 3.18 {2} Grapefruit GF 20 0.98 0.64 -0.33 2.29 {2} Orange OR 20 7.71 0.64 6.40 9.02 1*2 Bl GF 5 0.71 0.91 -1.15 2.56 a 1*2 Bl OR 5 0.81 0.91 -1.05 2.67 a 1*2 B24B GF 5 0.81 0.91 -1.05 2.67 a 1*2 B24B OR 5 4.13 0.91 2.28 5.99 a 1*2 B22 GF 5 0.71 0.91 -1.15 2.56 a 1*2 OR B-24B 5 2.93 0.91 1.07 4.79 a 1*2 OR B-22 5 1.15 0.91 -0.71 3.01 a 1*2 OR Bader 5 11.28 0.91 9.43 13.14 b 2"'^ sample dates Factor Factor n mean SEM_ 95 % + 95% CnfLmt CnfLmt Total 40 5.28 0.27 4.74 5.83 {l}Blockl Bl 10 5.78 0.54 4.69 6.87 {l}Block24B B24B 10 7.35 0.54 6.26 8.45 {l}Block22 B22 10 3.37 0.54 2.28 4.47 {IjBader BAD 10 4.62 0.54 3.53 5.71 {2} Grapefruit GF 20 4.00 0.38 3.22 4.77 a {2} Orange OR 20 6.57 0.38 5.79 7.34 b 1*2 Bl GF 5 5.20 0.76 3.65 6.74 1*2 Bl OR 5 6.37 0.76 4.82 7.91 1*2 B24B GF 5 5.63 0.76 4.08 7.18 1*2 B24B OR 5 9.08 0.76 7.53 10.62 1*2 B22 GF 5 1.35 0.76 -0.19 2.90 1*2 B22 OR 5 5.39 0.76 3.84 6.94 1*2 BAD GF 5 3.80 0.76 2.26 5.35 1*2 ^BAD OR 5 5.44 0.76 3.89 6.98

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-T'154 YOTHERS, W.W. 1914. The rust mite and its control. Proceedings of the Florida Horticultural Society 27: 1 15-1 19. YOTHERS, W.W. 1918. Spraying for the control of insects and mites attacking citrus trees in Florida. USDA Fanners Bulletin 933. 39 pp. YOTHERS, W.W., AND A. C. MASON. 1930. The citrus rust mite and its control. USDA Technical Bulletin. 176. YUE, B., AND C.C. CHILDERS. 1994. Effects of temperature on life table parameters of Agistemus exertus Gonzales (Acari: Stigmaeidae) and its attack rate on Panonychus citri eggs. International Journal of Acarology 20: 109-1 13. ZAR, J. H. 1984. Biostatistical analysis. 2nd Ed. Prentice Hall Inc. Englewood Cliffs, N.J. 718 pp ZEMEC, R., AND E. PRENEROVA. 1997. Powdery mildew an alternative food for the predatory mite Typhlodromus pyri Scheuten (Acari: Phytoseiidae). Experimental and Applied Acarology 21: 405-414.

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BIOGRAPHICAL SKETCH Raul T. Villanueva was bom in Recuay, Ancash, Peru, a small village in the Peruvian Andes, ca. 3500 m above sea level. He moved to Lima, and completed his entire primary and secondary studies there. He obtained his bachelor's degree in Agriculture (with honors) from the Universidad Nacional Agraria La Molina in Lima. Then he moved to Canada, and lived there for several years. Raul obtained his M.Sc. degree in Biology from Queen's University at Kingston, Ontario. Raul moved to the United States in July 1997 when he started his Ph.D. studies in the Department of Entomology and Nematology at the University of Florida. Raul was a teaching assistant of undergraduate courses at Queen's University and at the University of Florida. He likes to conduct research on the biology, and ecology of agricultural arthropods with emphasis on the biological control of pests. 155

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I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. Carl C. ChilHers, Chair Professor of Entomology and I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. Courtesy Assistant Professor Division of Plant hidustry. Florida I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. Maria Gallo-Meagher / (j Assistant Professor of Agronomy

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This dissertation was submitted to the Graduate Faculty of the College of Agricultural and Life Sciences and to the Graduate School and was accepted as partial fulfillment of the requirements for the degree of Doctor of Philosophy. May, 2002 /f^ • KJo^ Dean, College of A, Sciences Dean, Graduate School