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1 ASSOCIATIONS BETWEEN HEMIPTERAN HONEYDEW PRODUCERS AND NYLANDERIA FULVA (MAYR), WITH MANAGEMENT IMPLICATIONS FOR FLORIDA LANDSCAPES By SHWETA SHARMA 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 2013
2 2013 Shweta Sharma
3 To my husband, my parents and my brothers
4 ACKNOWLEDGMENTS I express my sincere gratitude to my major advisor Dr. Eileen A. Buss for her continuous encouragement, guidance and constructive criticism throughout my period as a Ph D student at the University of Florida. I am very grateful to her for believing in me and providing me with this opportunity when I needed it the most. Her friendly behavior made this whole time easy and comfortable. I would like to thank my Co advisor Dr. David Oi for his support and guidance on ant biology and for allowing me to work in h is laboratory I really appreciate hi m spending his valuable time in my writing and for improving my project with his deep knowledge in the subject. I am grateful to my other supervisory committee members Dr. Greg Hodges and Dr. Ed Gilman for their guidanc e and suggestions on how to improve my work. I would like to acknowledge the Entomology and Nematology Department and the USDA Tropical and Subtropical Agricultural R esearch (T STAR C ) program for providing financial assistance for the duration of my Ph D research. I am thankful to Department of plant protection (DPI), Gainesville for helping in species identification I appreciate the help of Mr. Lyle Buss identify ing some insects for my study and also providing important species information I am grate ful for the technical help Paul Ruppert provided I would like to thank all the wonderful lab assistants: Tamika Garrick, Manish Poudel, Karen Beaulieu, David Sekora, Jenna McDaniel and Chris Rachel all of who m helped me collect data and do lab tests. I d eeply appreciate Sarah Rachel for always being there. I would like to give her special thanks for supporting me as a lab assistant and as a friend. I would like to thank my colleagues Navneet Kaur, Julianna Xu and Dawn Calibeo for their support and encoura gement.
5 I appreciate the love and support that I received from my Nepalese friends in Gainesville. I would like to thank all of them for making Gainesville a home away from home. I am very grateful for the love and support of my parents (Mr. Madhu Sudan Sharma and Mrs. Ram Pyari Sharma) and my brothers (Shwadhin Sharma and Swapnil Sharma). They made me who I am and always encouraged me to achieve more. I would like to thank my sister s in law and family for their support and understanding. F inally, I cannot express in words how thankful I am to my wonderful husband for his love and support. None of this work would be possible without his encouragement.
6 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF FIGURES ................................ ................................ ................................ ........ 10 ABSTRACT ................................ ................................ ................................ ................... 12 CHAPTER 1 INTRODUCTION ................................ ................................ ................................ .... 14 Invasive Ants ................................ ................................ ................................ .......... 14 Social Structu re of Invasive Ants ................................ ................................ ............ 14 Taxonomy, Origin and Distribution of Nylanderia sp. ................................ .............. 15 Morphological Description ................................ ................................ ....................... 17 Nesting and Feeding Behaviors ................................ ................................ .............. 18 Damage ................................ ................................ ................................ .................. 19 Interactions with Non ant Species ................................ ................................ .......... 22 Reaction to Different Honeydew Sources and Concentrations ............................... 24 Non chemical Control of N. fulva ................................ ................................ ............ 24 Effect of Essential Oils on N. fulva ................................ ................................ .......... 27 Objectives ................................ ................................ ................................ ............... 27 Outline of Dissertation ................................ ................................ ............................. 28 2 HONEYDEW PRODUCING HEMIPTERANS IN FLORIDA ASSOCIATED WITH NYLANDERIA FULVA (HYMENOPTERA: FORMICIDAE), AN INVASIVE CRAZY ANT ................................ ................................ ................................ ........... 31 Background ................................ ................................ ................................ ............. 31 Materials and Methods ................................ ................................ ............................ 33 Hemipterans Associated with N. fulva ................................ .............................. 33 Seasonal Sampling ................................ ................................ .......................... 33 Results and Discussion ................................ ................................ ........................... 34 Hemipterans Associated with N. fulva ................................ .............................. 34 Seasonal Sampling ................................ ................................ .......................... 35 Conclusions ................................ ................................ ................................ ............ 39 3 EFFECT OF COTTONY CUSHION SCALE (HEMIPTERA: MONOPHLEBIDAE) CONTROL ON NYLANDERIA FULVA SURVIVAL AND TRAILING ACTIVITY ...... 49 Background ................................ ................................ ................................ ............. 49 Materials and Methods ................................ ................................ ............................ 51 Laboratory Test ................................ ................................ ................................ 51 Field Trial ................................ ................................ ................................ .......... 52 Results and Discussion ................................ ................................ ........................... 54
7 Laboratory Test ................................ ................................ ................................ 54 Field Trial ................................ ................................ ................................ .......... 55 Preliminary samp ling data ................................ ................................ .......... 55 Numbers of cottony cushion scale on branches ................................ ......... 56 Ants on sausage sample ................................ ................................ ............ 56 Twenty second count ................................ ................................ ................. 57 Ant trail count ................................ ................................ ............................. 57 Ant ne sting in the pots ................................ ................................ ............... 58 Correlation between I. purchasi and N. fulva ................................ ............. 58 Conclusions ................................ ................................ ................................ ............ 59 4 EFFECT OF VARIOUS MULCHES ON NESTING SUBSTRATE PREFERENCE, TOXICITY AND REPELLENCY OF AROMATIC CEDAR MULCH ON FORAGING BEHAVIOR OF NYLANDERIA FULVA ........................... 69 Background ................................ ................................ ................................ ............. 69 Materials and Methods ................................ ................................ ............................ 70 Mulches ................................ ................................ ................................ ............ 70 Nylanderia fulva Colonies ................................ ................................ ................. 71 Nesting Substrate Preference: Choice Test ................................ ..................... 71 Artificial Nest vs. Mulch Test ................................ ................................ ............ 72 Foraging Barrier Test ................................ ................................ ....................... 72 No choice Toxicity Test ................................ ................................ .................... 73 Toxicity of Mulch over Time ................................ ................................ .............. 73 Mulch Barrier Test ................................ ................................ ............................ 74 Results and Discussion ................................ ................................ ........................... 74 Nesting Substrate Preference: Choice Test ................................ ..................... 74 Artificial Nest Vs. Mulch: No choice Mulch Test ................................ ............... 75 Foraging Barrier Test ................................ ................................ ....................... 75 No choice Toxicity Test ................................ ................................ .................... 76 Toxicity of Mulch over Time ................................ ................................ .............. 76 Mulch Barrier Test ................................ ................................ ............................ 76 Twenty second count ................................ ................................ ................. 76 Ant trail count ................................ ................................ ............................. 77 Ants on sausage sample ................................ ................................ ............ 77 Number of nests in each treatment ................................ ............................ 78 Conclusions ................................ ................................ ................................ ............ 79 5 NYLANDERIA FULVA PREFERENCE FOR HONEYDEW FROM DIFFERENT HEMIPTERANS OR SUCROSE CONCENTRATIONS ................................ .......... 91 Background ................................ ................................ ................................ ............. 91 Materials and Methods ................................ ................................ ............................ 93 Hemipteran and Host Plant Preference ................................ ............................ 93 Different Sugar Concentrations Test ................................ ................................ 95 Results and Discussion ................................ ................................ ........................... 96 Hemipteran and Host Plant Preference ................................ ............................ 96
8 Different Sugar Concentrations Test ................................ ................................ 97 Conclusions ................................ ................................ ................................ ............ 98 6 SUMMARY AND CONCLUSIONS ................................ ................................ ........ 103 APPENDIX : EFFECTS OF ESSENTIAL OILS ON NYLANDERIA FULVA (MAYR) .... 105 LIST OF REFERENCES ................................ ................................ ............................. 111 BIOGRAPHICAL SKETCH ................................ ................................ .......................... 129
9 LIST OF TABLES Table page 2 1 Hemipterans tended by N. Fulva in Gainesville, Fl, from July 2010 to October 2010. ................................ ................................ ................................ .................. 40 2 2 List of trees, sampling locations, insects and number of leaves sampled. .......... 41 4 1 Presence or abse nce of N. fulva colony fragments (500 workers + 4 queens) among five types of landscape mulch. ................................ ................................ 85 4 2 Prevalence of N. fulva colony fragments nesting in pine bark or aromatic cedar mulch.. ................................ ................................ ................................ ...... 86 4 3 Percentage of N. fulva crossing the foraging barrier to ac cess food and water. ................................ ................................ ................................ .................. 87
10 LIST OF FIGURES Figure page 1 1 Distribution of Nylanderia fulva in the U.S. as of 2012. ................................ ....... 30 2 1 Intact (right) and broken (left) carton shelters showing tuliptree scale ( Toumeyella liriodendri (Gmelin)) on a magnolia ( Magnolia grandiflora L.) branch ................................ ................................ ................................ ................ 42 2 2 Carton shelters on the bark of a southern red cedar ( Juniperus silicicola (Small)) tree ................................ ................................ ................................ ........ 43 2 3 Number of N. fulva and hemipterans on holly ................................ ..................... 44 2 4 Number of N. fulva and hemipterans on live oak. ................................ .............. 45 2 5 Number of N. fulva and hemipterans on magnolia ................................ .............. 46 2 6 Number of N. fulva and hemipterans on sugarberry ................................ ........... 47 2 7 Mean number of ants crossing a specific point in 20 sec x the number of trails (trailing intensity) on different tree species in 2011 and 2012. .................. 48 3 1 Set up for the laboratory experiment. ................................ ................................ 60 3 2 Data from the lab experiment with Safari 20 SG and Merit 75 WP. ................... 61 3 3 Potted pittosporum plants placed next to a hedge in a northern Gainesville business complex. ................................ ................................ ............................. 62 3 4 Preliminary data collected from the pittosporum plants b efore the tr eatment application ................................ ................................ ................................ .......... 63 3 5 Number of I. purchasi counted on the branches of pittosporum plants. ............. 64 3 6 Number of N. fulva collected from sausage samples place d near the base of the trunk of pittosporum plant. ................................ ................................ ............ 65 3 7 Number of N. fulva crossing a specific point on the trunk of pittosporum plant in 20 sec ................................ ................................ ................................ ............. 66 3 8 Mean number of N. f ulva trails on the trunk of each plant. ................................ 67 3 9 Correlation between the mean number of I. purchasi and N. fulva ..................... 68 4 1 Aromatic cedar mulch and other mulches used for the nesting substrate pre ference test. ................................ ................................ ................................ ... 80
11 4 2 Laboratory set up for artificial nest vs. mulch test. ................................ .............. 81 4 3 Aromatic cedar mulch barrier with N. fulva on one side and food and water on the other side (foraging barrier test). ................................ ............................ 82 4 4 Nylanderia fulva placed on aromatic cedar mulch with food and water provided (Mulch toxicity test) ................................ ................................ .............. 83 4 5 Mulch placed around the live oak trees in the mulch barrier test ........................ 84 4 6 Average number of N. fulva crossing specific point in 20 sec (trailing intensity) ................................ ................................ ................................ ............. 88 4 7 Average number of N. fulva trails on the trunk of the trees surrounded by the mulches ................................ ................................ ................................ .............. 89 4 8 Average number of N. fulva on sausage samples that were place d on the mulches around the trees ................................ ................................ ................... 90 5 1 Uninfested and infested twigs of four different plant species arranged on a wire mesh in a lab. ................................ ................................ .............................. 99 5 2 Infested pittosporum twigs arranged on a wire mesh with different concentration of sucrose solutions in a lab ................................ ....................... 100 5 3 Mean number of N. fulva on the uninfested and infested t wigs of different plant species ................................ ................................ ................................ ..... 101 5 4 Compari son of mean number of N. fulva foraging on the twigs with N. fulva foraging on the su crose solutions and water tubes ................................ ........... 102 A 1 Different type of essential oils along with the N. fulva in the vials treated with these oi ls ................................ ................................ ................................ .......... 109 A 2 Time required in hours to achieve 100% mortality for 20 N. fulva ..................... 110
12 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 ASSOCIATIONS BETWEEN HEMIPTERAN HONEYDEW PRODUCERS AND Nylanderia F ulva (MAYR), WITH MANAGEMENT IMPLICATIONS FOR FLORIDA LANDSCAPES By Shweta Sharma May 2013 Chair: Eileen Buss Cochair: David Oi Major: Entomology and Nematology Nylanderia fulva (Mayr) (Hymenoptera: Formicidae) is an invasive pest ant in the southeastern U.S. Workers tend various honeydew producing hemipterans in managed landscapes and natural areas. I sought to understand the seasonal foraging activities of N. fulva its relationship with honey dew producing hemipterans and determine if N. ful va could be culturally suppressed. Nylanderia fulva hemipteran species different plant species in Gainesville, Florida from July 2010 to July 2012 Numbers of hemipterans from plant samples and N. fulva workers on or near four woody plan t s pecies were compared Nylanderia fulva and hemipteran densities were positively correlated over the s pring, summer and fall C arton shelters presumably constructed by N. fulva covered the juniper aphid, ( Cinara juniperivora (Wilson) ; Hemiptera: Aphididae) o n s outhern red cedar ( Juniperous silicicola (Small) ; Pinales: Cupressaceae ) and tuliptree scale ( Toumeyella liriodendr i (Gmelin) ; Hemiptera: Coccidae) o n magnolia ( Magnolia grandiflora L. ; Magnoliales: Magnoliaceae ).
13 Next, I determined if N. fulva populations would decline after treating their hemipteran honeydew producers in a laboratory test. Two imidacloprid formulations, CoreTect and Merit 2F were used to control cottony cushion scale ( Icerya purchasi Maskell ; Monophlebidae ) and to monitor changes in ant survival and foraging behavior. Fewer ants, number of trails, and less trailing occurred on plants in pots treated with either product than in untreated pots. Nylanderia fulva preference for different mulches was studied. A romatic cedar ( Juniperus virginiana L. ; Pinales: Cupressaceae ) mulch with known insecticidal properties r epel led N. fulva and killed colonies that were confined to that mulch. Colonies could survive and nest in several other mulches. When N. fulva was presented wi th four honeydew producer species to tend, the ants demonstrated no preference B ut when three sugar concentrations (0, 10, and 20% sucrose) were provided to N. fulva more ants visited vials with the highest sucrose concentration Several essential oil s with insecticidal properties were tested against N. fulva Camphor oil killed ants the fastest, whereas cinnamon and clove were least effective Th is new information will enhance our understand ing of how N. fulva interacts with hemipteran insects and to suppress N. fulva indirectly by controlling the hemipterans. Also, by us ing aromatic cedar mulch in landscapes, an ts could be deterred from tending honeydew producers or establishing nests within that mulch, and thus potentially reduces insecticide use in urban landscapes
14 CHAPTER 1 INTRODUCTION Invasive A nts Order 13112. Invasive species have become a worldwide ecological and conservation crisis as they are changing the terrestrial and aquatic ecosystem globally (Gurevitch and Padilla 2004). Pimental et al. (2005) mentioned that at least 50,000 non native species have been introduced in to the U.S. and their estimated damage and control costs are ap proximately $120 billion per year In troduced species cause about $13 billion just in crop losses each year. Of the ~ 4,500 invasive arthropod pest species in U S crops (Pimental et al. 2005), 949 species have invaded Florida (Frank and McCoy 1995). More than 150 species of ants (Hymenoptera: Formicidae) have been accidentally transported to new areas via global trade (McGlynn 1999) and among them nine are listed as invasive (invasive.org). Florida has the greatest number of introduced ant species (>50) wi thin the U.S. (Deyrup et al. 2000). Ants are important members of many ecosystems (Hlldobler and Wilson 1990) and they are successful due to their highly developed social structures, which allow thousands of individuals to function cooperatively (Wilson 1 971). Social Structure of Invasive Ants Eusocial species should have following characteristics: adults live in groups, cooperative care for brood s that is not their own, reproductive division of labor, and overlap of generations (Wilson 1971). All ant species are eusocial, which means their colonies have members that are behaviorally and morphologically specialized for
15 reproduction, foraging, tending larvae, and defense (Grimaldi and Agosti 2000). Ant colonies are typically divided into castes o f queens (i.e reproductive females), males, and workers (e.g., non reproductive females). A virgin queen takes the nuptial flight by leaving the nest, mating with one or more males, and gets inseminated. This is the only function of males. The q ueen cons tructs the first nest cell and rears the first brood of workers. Thereafter, the queen continuously lays eggs, and the non reproductive workers take over the task of foraging, nest enlargement and brood care. The workers are coordinated through the pheromo nes produced by queen and through body contact with each other ( Hlldobler and Wilson 1990) Unicoloniality is more common in invasive species (Passera 1994) which means that many invasive ants liv e in widespread colonies with many separate but interconne cted nests that contain many queens ( e.g., polygynous colonies ). Individual ants from separate nests within the same colony lack aggressi on towards each other and the colony extends over a large area (Abbott 2005). This allows the ants to share resources toward colony growth and interspecific competition ( Holway et al. 1998 ). Invasive ant species are also successful because of their ability to initiate new colonies by budding, or colony fragmentation which increases the opportunity for fra gments to be transported or disperse d to new areas (Wilson 1971). Taxonomy, Origin and Distribution of Nylanderia sp. The genus Nylanderia (Hymenoptera: Formicidae: Formicinae ) includes 134 species and subspecies (LaPolla et al. 2010). Nine native and six introduced species have been collected in the continental U S (Trager 1984, Bolton et al. 2006, Meyers 2008). Two species that have been of concern and which have been taxonomically confused are N pubens (Forel) and N. fulva (Mayr) Both species were recently in the
16 genus Paratrechina However, LaPolla et al. (2010) revised the Prenolepis group and with the exception of Paratrechina longicornis (Latreille) moved all Paratrechina species to the genus Nylanderia or Paraparatrechina. Nylanderia fulva is likely native to South America where the type locality is Brazil (Gotzek et al. 2012), while N pubens may have originated from the St. Vincent Island and the Lesser Antilles (Trager 1984 Forel 1893) Nylanderia pubens was first detected in the U .S. in 1953 in Coral Gables and Miami, Florida (Trager 1984) T he earliest record of this species being a pest was in 1990 after infestations were reported in Boca Raton, Homestead and Miami (Klotz et al. 1995, Deyrup et al. 2000 ), however Gotzek et al (2 012) hypothesize that pest outbreaks attributed to N. pubens may actually be N. fulva By 2012, N fulva is reported to be found in 23 counties of Florida (Fig. 1 1). N ylanderia pubens and N. fulva workers are morphologically similar, but the two species can be differentiated by examin ing the male parameres or by genetic comparisons. U sing molecular analyses in combination with specimen comparisons, it was determined that ants in Alachua County, FL were actually N. fulva (Gotzek et al. 2012). Nylanderia fulva c erratic and quick movements (Warner and Scheffrahn 2010). Th is species, when it was thought to be N. pubens has had several unofficial common names, including Caribbean crazy ants b rown crazy ants and hairy crazy ants (Wetterer 2007). After the species clarification by Gotzek et al. (2012) as an official common name for N. fulva (D. H. Oi, personal communication).
17 In 2002 a nts resembling and b ehaving like what at the time were thought to be Paratrechina pubens from Florida was found by a pest control operator (Tom Rasberry) in Pasadena Texas (Meyers 2008). The Texas ants were slightly larger than those in Florida, creating uncertainty about whether or not they were the same species. So, the Texas ants were called the Rasberry crazy ant, Paratrechina sp. near pubens (MacGown and Layton 2010 ) Mor phometric and DNA studies conducted at Texas A&M University to determine identity w ere in conclusive (MacGown and Layton 2010). However, recent molecular comparison s indicated that the crazy ants in Texas and Florida we re the same species (Zhao et al. 2012) and that all of these populations we re N fulva (Gotzek et al. 2012) N ylanderia fulva now occurs in 24 counties in Texas ( McDonald 2012 ). Nylanderia fulva was also co llected at a residence in Waveland, Hancock County, Mississippi by a pest control technician on 20 July 2009 (MacGown and Layton 2010). The technician reported that the ants were in the garage, on the patio, and around the house forming trails. T he ants were nesting in cars, motor homes, in a motorcycle, in electrical boxes and in house s (MacGown and Layton 2010) R epeated pesticide applications were in effect ive and new population s quickly replaced the old ones MacGown and Layton (2010) recommended careful monitoring and aggressive control measures. Morphological Description Nylanderia fulva are medium to small, reddish brown monomorphic ants (Warner and Schef frahn 2010). The body surface is shiny with dense pubescence. W orkers are 2.0 2.3 mm in length, males are slightly larger (2.4 2.7 mm), and queens are ~4.0 mm or longer (MacGown and Layton 2010) Workers lack ocelli, have dense
18 pubescence on the alitru nk (mesosoma) and gaster, and have long, somewhat flexuous (bending alternately from side to side), light brown macrochaetae (series of long hairs) on the body. The gaster looks striped after feeding due to the stretching of the inter segmental membranes. A ntenna e are 12 segmented Terminal segments do not form a club. Rather than a stinger, an acidopore is present at the tip of gaster (Zenner Polania 1990). Males have 13 segmented antennae and the eyes are much bigger than that of the workers (Zenner Polan ia 1990). Other than the size of the reproductive females, other features are similar to the workers. Nesting and Feeding B ehavior s Nylanderia fulva nests occur in leaf litter, soil, rotten logs, under potted plants, and along underground electrical condu its (Warner and Scheffrahn 2010), rather than as isolated mounds. As ambient temperature s increase foraging trails become several ants wide. Colonies consist of a large number of ants with more than one queen (polygamous) and are polydomous (nestin g in several locations) (Warner and Scheffrahn 2010). MacGown and Layton (2010) reported that N fulva often occur in huge numbers and display supercolony characteristics such as yellow crazy ants, Anoplolepis gracilipes Smith (Hymenoptera: Formicidae) Argentine ants Linepithema humile (Mayr) (Hymenoptera: Formicidae) and big headed ants Pheidole megacephala (F.) (Hymenoptera: Formicidae) where multiple interconnected nests could be hundreds of kilometers long ( Giraud et al. 2002 ). Nylanderia fulva is omnivorous (MacGown and Layton 2010) feeding both on live and dead arthropods or small vertebrates for protein (Meyers 2008), as well as tending honeydew producing hemipterans for carbohydrates Workers tend hemipteran insects
19 (e.g., aphids, scale inse cts) and other insects that excrete honeydew. They are also attracted to sweet parts of plants including nectaries and over ripe fruit Damage Ants are one of the most damaging invasive pest groups (Williams 1994 Moller 1996). For example, the costs asso ciated with the invasion of the red imported fire ant, Solenopsis invicta Buren (Hymenoptera: Formicidae) in the United States have been estimated at $1 billion per year (Pimentel et al. 2000). The control of infestations and repair of damage caused by in vasive ant species can be costly to home and business owners. The annual economic impact in the U S alone is estimated to exceed $6 5 bill ion in both urban and agriculture sector s (Oi and Drees 2009). When the fire ants were detected in the U. S in the an eradication program beg a n. It cost state and federal government s mor e than $200 million in 26 years (Myers et al. 1998). Jones et al. (1994) mentioned that even though ants may belong to different trophic level s (e.g., leaf cutter and har vester ants are primary consumers and predator y ants as secondary consumers ) they all can be classified as ecosystem engineers. Exotic ants often have negative impacts on native ant, invertebrate and vertebrate communities (Williams 1994). Thus, changes i n ant diversity or abundance as a result of the introduction of an invasive ant species can s ignificant ly a ffect the ecosystem. For example, the yellow crazy ant had a devastating effect on the ecosystem of Christmas Island where it killed land crabs resu lting in increased accumulated leaf litter and 003). The ants also promoted scale insect population s by tending them to obtain the carbohydrate rich honeydew and the scales in turn damaged the vegetation (Abbott 2005). The initial rapid response program for the
20 Christ mas Island National Park cost $ 1.5 million (AUD) in 2002 2003 (Reaser et al. 2007). Nylanderia fulva are a nuisance and the huge number of workers in infested areas can make human activities uncomfortable and di fficult (MacGown and Layton 2009). Nylanderia fulva has killed honey bee larvae and after driving the bees away even us ed the hives as their own nests (Harmon 2009). They have also been problematic in parts of South America (Meyers 2008) and the Caribbea n Islands because of their huge colonies. Hemipteran insects were tended by N. fulva increasing their population and d amag ing a variety of fruit crops like coconut (Wetterer and Keularts 2008). S tudies in both agricultural (Reimer et al. 1993, Gonzalez Hernandez et al. 1999) and non crop systems (Bach 1991) have reported that ants increase hemipteran numbers by removing predators and often by interfering with parasitoids I n the absence of ant tending, hemipteran densities tend to decrease (Krus helnycky et al. 2005). Nylanderia fulva can out compete and displace existing ant populations, thus reducing ecological diversity (Zenner Polania 1990) The i ntroduction of N. fulva into La Reserva Natural Laguna de Sonso in Colo mbia reduc ed native ant s pecies by 74% (Aldana et al. 1995). N ylanderia fulva has also been displacing the red imported fire ant, and homeowners have even said that they prefer red having fire ants over N. fulva (Wynalda 2008). Nylanderia fulva has caused the extinction of native v egetation and grassland habitats to dry out as a result of increased hemipteran pop ulations on plants in Colo mbia (Arcila et al. 2002, Meyers 2008). Many reports of infestation of sidewalks,
21 buildings and gardens have come from the pest control operators i n Florida (Warner and Scheffrahn 2010). Nylanderia fulva has caused electrical shortages of phone lines, air conditioning units, computers and sewage lift pump stations (Meyers 2008). Even a radiation scanner at the Port of Houston was reportedly due to N. fulva (Holden 2007) Wynalda (2008) mentioned a report of N. fulva invading the electrical wiring of a vehicle that caused it to ignite when the owner tried to start it. R eal estate values have decreased d ue to N. fulva in and around the structures and pe ople fil l trash cans with ant cadavers after pesticide applications (Wynalda 2008) Gaudin (2008) reported that due to the concerns of ants threatening critical systems of the National Aeronautics and Space Administration (NASA), Johnson Space Center contr acted a pest management professional T om Rasberry to monitor and treat for N. fulva The population of N. fulva can rebound within hours to days after pesticide use (Meyers 2008). Nylanderia fulva was considered a significant agricultural pest afte r its introduction to Colombia as a biological control agent aga inst snakes and leaf cutting ants (Campos Farinha and Zorzenon 2005). Nylanderia fulva caused blindness in large animals like cattle by attacking their eye s and caused asphyxia in poultry by c logging the nasal passage s (Zenner economy. Florida ranks second in the United States with $1.8 billion in vegetable production and first in the nation for citrus production (Gueder 2011). In ad dition to crop damage, the large numbers of ants were a nuisance to agricultural workers (Aldana et al. 1995). Severe infestations of N. fulva caused some farms in Columbia to be abandoned (Zenner Polania 1990).
22 Interactions with N on a nt S pecies species can transmit plant diseases (Buckley 1987). Ants tend hemipterans and protect them from predators and parasitoids resulting in greater hemipteran survival and population size (Fritz 1982, Bristow 1983, Fowler and MacGarvin 1985 Buckley 1987) and sometimes provide shelter (Sheppard et al. 1979, Stout 1979, Anderson and McShea 2001, Moya Raygoza and Larson 2008, Vanek and Potter 2010a). Ants can sometimes assume parental care of the nymphs, enabling adult female hemipteran s to produce more offspring (Bristow 1983). So me hemipterans live inside ant nests (e.g., root feeding aphids and coccids ) so they are protected from climatic variations (Buckley 1987). Ants, in turn, harvest th e carbohydrate rich honeydew which in turn, reduces the accumulation of sooty mold (Haines and Haines 1978 Fokkema et al. 1983). Sooty mold can suffocate hemipteran eggs (Moya Raygoza and Nault 2000), affect crawler settling (Bess 1958), and cause adult mortality (Way 1954, Das 1959). Hemipterans pu nctur e plant tissue with a stylet and withdraw large volume s of liquid excrete d by the insects as a complex mixture of water soluble carbohydrates, amino acids, amides, alcohol, auxins and salts from partially digested plant sap mixed with products of malpighian tubules (Hackman and Trikojus 1952, Way 1963, Delabie 2001). Hemipterans can excrete honey dew several times an hour and may be several times an insect body mass (Larsen et al. 1992). Ants may feed on the hemipterans they tend if the honeydew is not sufficient or protein is necessary for colony growth (Hlldobler and Wilson 1990, Sakata 1994). Many ants build protective shelters made of soil and plant debris (carton) over honeydew producing hemipterans (Wheeler 1910, Way 1963). These structures have
23 1898). Ants can a lso hide hemipterans in hollow stems or thorns, plant cavities or crevices (Hlldobler and Wilson 1990). These shelters prevent the hemipterans from escaping (Das 1959), protect hemipterans and ants from adverse environments (Way 1963, Helms and Vinson 200 2), increase the availability of honeydew to the ants due to the increased population of the hemipterans (Dejean et al. 1997), allow ants to cull the hemipterans when the population is too large (Brian 1983) and protect hemipterans from predators and paras itoids (Das 1959, Gibernau and Dejean 2001, Moya Raygoza and Larsen 2008). Ants also appear to regulate hemipteran populations by protecting just enough individuals to supply honeydew for the ants and using the remainder of the hemipterans to be used as p rotein supply for the colony (Way 1963, Addicott 1978 Hlldobler and Wilson 1990, Sakata 1994). Sakata (1994) found that Lasius niger L. (Hymenoptera: Formicidae) would prey more often on the aphids Lachnus tropicalis (Van der Goot) (Hemiptera: Aphididae) and Myzocallis kuricola (Matsumura) (Hemiptera: Aphididae) on chestnut trees when the aphid density per ant was high. When the density of aphids per ant decreased, L. niger tended those aphids Ants may also have a negative impact on the reproductive rate and offspring size of hemipterans (Stadler and Dixon 1998, Yao et al. 2000). S ome aphid species may alter their feeding behavior and the composition of their honeydew by increasing the concent rations of amino acids at the expense of
24 their own growth and fecundity in the presence of tendi ng ants (Stadler and Dixon 1998, Yao et al. 2000, Yao and Akimoto 2002). Reaction to Different Honeydew Sources and Concentrations When several hemipteran spec ies and ant colonies co occur ant workers focus their tending on those hemipterans that produce a greater volume or larger droplets of honeydew, or have honeydew with higher sugar conte nt ( Hlldobler and Wilson 1990; Detrain et al 1999; Mailleux et al 2 000, 2003 ; Romeis and Wckers 2000 ). A nts tend to be more attracted to honeydew containing high a mounts of melezitose (Kiss 1981, Vlkl et al. 1999) gut from two units of glucose and one unit of fru ctose (Bacon and Dickinson 1957, Ashford et al. 2000). The concentration of a mino acids in honeydew can change when individuals of the same species feed on different host plants (Hendrix et al. 1992, Douglas 1993, Fischer and Singleton 2001). H o neydew composition could als o vary among and within hemipteran species despite all of them sharing the same host plant (Sandstrm et al. 2000) possibly reflecting the different metabolic rates in the different species (Wool et al. 2005) For example, o nly 40% of aphid species are te nded by ants although all aphids produce honeydew ( Stadler 1997). The degree of ant tending is affected by an aphid s honeydew production rate (Banks and Nixon 1958, Takeda et al. 1982 Del Claro and Oliveira 1993, Fischer et al. 2001 ). Non chemical C ontrol of N. fulva Little information about the biology and behavior of N. fulva is available, which hinders the development of effective control strategies. Eradication of this species is nearly impossible after the ants have become established (Meyers 2 008). Multiple control techniques includ ing sanitation, baits, and insecticidal sprays will be necessary
25 to suppress populations to tolerable levels (Calibeo and Oi 2011). Many control methods that are used for other ants have been in effective on N. fulva which causes concern over wasted time and money, plus negative impacts to the environment. Some research suggested limiting honeydew sources to reduce Argentine ant L humile (Mayr), densities as an indirect method of controlling infestations of this in vasive pest (Rust et al. 2003, Silverman and Brightwell 2008, Brightwell and Silverman 2009). Huge densities of invasive ants achieve their superiority through the dominance of carbohydrate resources like honeydew (Davidson et al. 2003) So, b y limiting th e honeydew sources, ants may consume more of liquid carbohydrate based toxic bait (Silverman and Brightwell 2008). For example, Brightwell and Silverman (2009) excluded Argentine ant foragers from the native terrapin scale, Mesolecanium nigrofasciatum (Per gande) (Hemiptera: Coccidae) which led to a dramatic decline in the number of Argentine ant nests at the base of the host red maple, Acer rubrum L. (Sapindales: Aceracae). The decline was due to relocation of the nests after exclusion from honeydew resources within the host tree. Given that a colony of N. fulva may contain 16 000 individuals ( D Calibeo, personnel communication) baits and contact insecticides are rendered ineffective when, after the initial group of ants have died, subsequent nestmates walk over them and continue the infestation. Thus, identification of novel tools and/or strategies is essential to curbing the r apid spread of this pest. Since direct contro l of ant infestations is challenging, it is important to look for the indirect control of N. fulva It has not been previously evaluated how insecticidal control of hemipterans might affect the foraging and tending behavior of N. fulva and if this control will indirectly reduce ant densities.
26 Nylanderia fulva typically nests in rotting wood, soil, various types of debris and landscape objects and under landscape mulch (MacGown 2012). Mulch is typically used as a protective organic or inorganic covering ar ound the base of a plant. M ulched landscape s can better absorb water, conserve the soil, buffer the soil temperature, prevent water loss from evaporation and suppress weed growth (Duryea 2000). Organic mulch breaks down, improving the soil nutrient content and physical structure by increasing the soil organic matter content. Mulch also adds to the aesthetic value of the landscape. Various mulches are used in Florida, but the most common are cypress (60%), pine bark (20%), pine straw (1%), eucalyptus (1%), m elaleuca (1%) and red mulch which is composed of recycled woods (17%) (Stake 2000). Since mulch is so commonly used it may contribute to the presence of N. fulva around or even inside buildings Aromatic cedar mulch or e astern red c edar, Juniperus virginiana L. (Pinales: Cupressaceae) mulch contains cedrol, A cedrene and thujopsene as defensive compounds (Technical Resources International 2002) that appear to be toxic to some insects. Eastern red cedar wood has been thought to possess insecticidal properties (Back and Rabak 1922, Sweetman et al. 1953). Argentine ants and odorous house ants, Tapinoma sessile (Say) (Hym enoptera: F ormicidae) avoided aromatic cedar mulch and nest ed on other mulches and died when confined to the mulch (Meissner and Si lverman 2001). T he t oxic effect of this mulch was also demonstrated in studies of the clothes moth, Tineola bisselliella (Hum.) (Lepidoptera: Tineidae) (Scott et al. 1918) black carpet beetle, Attagenus piceus (Oliv.) (Coleoptera: Dermestidae) termites Reticu litermes flavipes Kollar (Blattodea:
27 Rhinotermitidae) (Adams et al. 1988), and house dust mite, Dermatophagoid es spp (Arachnida: Pyroglyphididae) (Enomoto et al. 1999). This mulch was repellent to German cockroach, Blattella germanica (Linn.) (Blattodea: Blattellidae) (Appel and Mack 1989), and S invicta Buren (Thorvilson and Rudd 2001). The effects of different types of mulches on N. fulva have not been determined and may be an important component of integrated control strategies Effect o f Essential Oils on N. fulva Research are focused on the s earch for natural, safe and non polluting insecticides and exploring bioactive chemical compounds from plants (Vogt et al. 2002, Appel et al. 2004, Cheng et al. 2004). Essential oils could decrease the quantity of toxic insecticide residues and contribute to a healthier environment (Regnault Roger 1997). Essential oils are naturally occurring volatile oils that give distinctive odor, flavor or taste to a plant (En an 2001 ). They are the by products of plant metabolism that are found in glandular hairs or cavities of plant cell wall and are present in the leaves, stems, bark, flowers, roots and fruits in plants (Koul et al. 2008). Many control methods that are used for oth er ants are not effective on N. fulva which causes concern over wasted time and money, plus negative impacts to the environment. Thus, the identification of novel tools and/or strategies is essential to curbing the rapid spread of N. fulva Objectives 1. To describe associations between N. fulva and several hemipteran populations and to identify the plant and hemipteran species involved ; 2. To study the effect of hemipteran control on N. fulva foraging, tending behavior, survival ;
28 3. To study the effect of differe nt aromatic cedar mulch on the foraging and nesting behavior of N. fulva ; 4. To determine the response of N. fulva to the honeydew from different hemipterans ; and 5. To study the effect of different type s of essential oils on N. fulva Outline of Dissertation This dissertation presents important aspects of the biology of the pest ant Nylanderia fulva in the Florida landscape Since this is a relatively new pest in the United States, the literature presents very little information on its biolog y physiolog y and ecolog y My research investigated the seasonal foraging activity of N. fulva and its interaction with several hemipteran species in the laboratory and field, and evaluated the potential for culturally suppressing ant nesting or survival in mulched areas or with the use of essential oils In Chapter 2, the seasonal activity of N. fulva workers from February 2011 to July 2012 was monitored along with the population dynamics of four abundant hemipteran species on their host plants. Given that the ants need a carbohydrate source for most of the year, Chapter 3 explored whether or not chemically suppressing a hemipteran infestation [e.g., cottony cushion scale ( I. purchasi Maskell) on pittosporum] that was tended by N. fulva would result in a similar suppressi on of N. fulva Because N. fulva tends to nest in loose debris and landscape mulch, I evaluated the potential for repelling or killing N. fulva through the use of mulch from different tree species in laboratory and field tests in Chapter 4.
29 Chapter 5 is a study of whether or not N. fulva preferred the honeydew produced by four different hemipterans (an aphid, whitefly, mealybug, and scale insect), or if the ant would prefer a solution with a higher sucrose content compared to scale honeydew. Finally, a synopsis of the major results and their implications for N. fulva management, as well as potential future studies, are presented in C hapter 6.
30 Figure 1 1. Distribution of Nylanderia fulva in the U.S. as of 2012. Photo Credits: http://mississippientomologicalmuseum.org.msstate.edu//Researchtaxapages /Formicidaepages/genericpages/Nylanderia_fulva.htm
31 CHA PTER 2 HONEYDEW PRODUCING HEMIPTERANS IN FLORIDA ASSOCIATED WITH NYLANDERIA FULVA (HYMENOPTERA: FORMICIDAE), AN INVASIVE CRAZY ANT B ackground Nylanderia fulva (Mayr) (Hymenoptera: Formicidae) is an invasive pest ant from South America that heavily infests buildings and landscapes in Florida, Louisiana, Mississippi and Texas (MacGown and Layton 2010, Warner and Scheffrahn 2010, Aguillard et al. 2011). The ants in Florida were originally identified as N. pubens (Forel) and have been called the brown crazy ant, the Caribbean crazy ant, and the hairy crazy ant (Wetterer and Keularts 2008, Warner and Scheffrahn 2010, Calibeo and Oi 2011). T he ants from Texas and Missi ssippi were called 1 the Rasberry crazy ant, and were designated as Nylanderia sp. near pubens (Meyers and Gold 2008, MacGown and Layton 2010 ). However, Zhao et al. (2012) determined that all the specimens of Nylanderia species in both Texas and Florida are identical and Gotzek et al. (2012) confirmed that the pestiferous ants identified as N. pubens were actually N. fulva Nylanderia fulva has caused electrical short circuits in Texas (Meyers 2008), destroyed and occupied bee hives in Texas (Harmon 2009), b itten people, and has displaced some native ant species in Colombia and Texas (Meyers 2008). Nylanderia fulva also has caused problems in parts of South America (Zenner Polania 1990) and the Caribbean Islands because of their abundance and mutualism with h emipteran populations that damage crops, such as coconut (Wetterer and Keularts 2008). 1 This chapter will be published as Sharma, S., D. H. Oi, and E. A. Buss. 2013. Honeydew producing hemipterans in Florida associated with Nylanderia fulva (Hymenoptera: Formicid ae), an invasive crazy ant. Fl a. Entomol. ( In Press )
32 Mutualisms between ants and honeydew producing hemipterans (e.g., aphids, scales, mealybugs, leafhoppers) are well documented. Ants benefit from their association with hemipterans because their honeydew is an important source of carbohydrates (Stout 1979, Anderson and McShea 2001, Moya Raygoza and Larsen 2008, Vanek and Potter 2010). Furthermore, hemipterans can serve as a protein source if they are directly consumed by ants (Rosengren and Sundstrm 1991, Sakata 1994, Gullan 1997). Conversely, hemipterans that produce honeydew benefit when they are tended by ants. Ants protect hemipterans from natural enemies and they sometimes provide them shelter (Sheppard et al. 1979 Stout 1979, Anderson and McShea 2001, Moya Raygoza and Larsen 200 8, Vanek and Potter 2010), allowing more hemipterans to survive and reproduce (Fritz 1982, Bristow 1983, Fowler and MacGarvin 1985, Buckley 1987). In addition, hemipterans that live inside ant nests (e.g., root feeding aphids and soft scales) are protected from extreme weather (Buckley 1987). Some ants have been reported to assume parental care of hemipteran nymphs, enabling adult female hemipterans to produce more offspring (Bristow 1983). The removal of honeydew by mold that grows on honeydew (Fokkema et al. 1983, Haines and Haines 1978). Sooty mold can suffocate hemipteran eggs (Moya Raygoza and Nault 2000) affect crawler settling (Bess 1958), and cause adult mortality (Way 1954, Das 1959). In this study, we identified hemipteran species that were associated with N. fulva and documented N. fulva foraging activity with seasonal hemipteran abundance.
33 Material s and Methods Hemipterans Associated with N. fulva From Jul to Oct 2010, colonies of N. fulva were observed in parks, natural areas and neighborhoods in Gainesville (Alachua Co.), Florida, for any tending behavior on natural infestations of hemipterans on trees, shrubs and other plants. Nylanderia fulva trails on plants were visually followed until ants were seen tending individual hemipterans. Leaves or stems with the hemipterans were collected and insects and plants were identified by specialists at the Florida Department of Agriculture and Consumer Services, Division of Plant Industry (FDACS/DPI) Seasonal Sampling Nylanderia fulva populations and honeydew producing hemipterans were monitored monthly from Feb 2011 to Jul 2012 at 3 sites in Gainesville, Florida. Four plant species [live oak ( Quercus virginiana Mill.; Fagales: Fagaceae ), holly ( Ile x cornuta Lindl.; Aquifoliales: Aquifoliaceae ), magnolia ( Magnolia grandiflora L.; Magnoliales: Magnoliaceae) and sugarberry ( Celtis laevigata Willd.; Urticales: Ulmaceae)] that were infested with at least one honeydew producing hemipteran species, and had trails of N. fulva going up the stem were selected for monitoring. Four plants of each species were sampled except for magnolia, for which only one plant was sampled in 2011 and three more plants were added in January 2012. The selected plant species were located in managed landscapes, usually along driveways paved with blacktop asphalt. Six to ten terminal shoots per plant with at least six to ten leaves (depending on the size of the plant and leaves) were arbitrarily chosen, cut with a pole pruner and tr ansported in plastic bags to the laboratory on ice. The total number of honeydew producing insects on terminal shoots was counted within 24 h of collection.
34 Nylanderia fulva activity was estimated in three ways for each plant. First, a slice of sausage (0 .5 2.5 cm Armour Original Vienna Sausage, Cherry Hill, New Jersey) was placed on the center of an index card (7.6 12.7 cm) that was positioned about 25 cm from the base of the plant on the northern and southern sides. After 15 min, each card with ants was individually bagged, frozen and the number of ants per sausage was determined. Second, the number of N. fulva foraging trails going up one major trunk per plant from the ground was counted. Finally, the number of workers walking past a specific point ( ~30.5 cm above the ground) on the most active foraging trail was counted for 20 sec. All sampling occurred between 0900 1500 h when the air temperature was > by placing a digital thermo/hygrometer (Acu Rite 00891) near the base of the plants. was used to examine the association between the total number of hemipterans on a shoot and the 20 sec ant count. Due to the increased number of trails per trunk in the summer, the 20 sec count was multiplied by the number of trails to provide an index of trailing intensity. Analysis of variance (ANOVA) was conducted to determine differences in 20 sec ant counts from the most active ant trail among plant species an among plant species (R Development Core Team 2012). Results and Discussion Hemipterans Associated with N. fulva A total of 17 species from seven families of honeydew producing hemipterans were tended by N. fulva on ten plant species (Table 2 1). Nylanderia fulva exhibited no preference for any particular hemipteran species. With six species of aphids and five specie s of mealybugs, these were the most prevalent hemipterans and had the greatest
35 diversity. Zenner Polania (1990) also reported N. fulva tending whiteflies and scale insects in orange trees and mealybugs in rangeland grass, sugarcane and coffee berries. They also observed ants transporting hemipterans from infested to uninfested plants and protected the hemipterans from predators by constructing protective shelters over the mealybugs. We observed carton shelters on magnolia branches, where N. fulva had cover ed tuliptree scales ( Toumeylla liriodendri (Gmelin); Hemiptera: Coccidae) using soil and plant debris (Fig. 2 1). Shelters were also observed on the trunk and near the base of the trunk where there were pruning wounds. Nylanderia fulva also built carton sh elters around juniper aphids ( Cinara juniperivora (Wilson); Hemiptera: Aphidida e) along a split on the trunk of southern red cedar ( Juniperous silicicola (Small); Pinales: Cupressaceae) (Fig. 2 2). The structures were built in such a way that ants had acce ss to enter and exit those shelters. When the carton was broken apart, many N. fulva workers scrambled out of the shelters and the juniper aphids or tuliptree scales that were previously covered were visible. Many ants like Acropyga Formica Lasius Odont omachus and Oecophylla build protective shelters of soil and plant debris, or carton, over honeydew producing hemipterans (Wheeler 1910, Way 1963, Evans and Leston 1971). These shelters are thought to protect the hemipterans from adverse environments (Way 1963, Helms and Vinson 2002), thereby increasing hemipteran populations and the availability of honeydew for the ant colony (Dejean et al. 1997). Seasonal Sampling The hemipterans collected from plants that were sampled monthly from Feb 2011 to Jun 2012 a re listed in Table 2 2. The predominant hemipteran species on each host plant was Florida wax scale, Ceroplastes floridensis Comstock ( Hemiptera: Coccidae),
36 on holly; the aphid, Myzocallis puncata (Monell) (Hemiptera: Aphididae), on live oak; the tuliptree scale, Toumeyella liriodendri (Gmelin) (Hemiptera: Coccidae) on magnolia; and the Asian wooly hackberry aphid, Shivaphis celti Das ( Hemiptera: Aphididae) on sugarberry. Nylanderia fulva were observed tending five hemipteran species on live oak and three species on sugarberry. Two hemipterans were sampled from holly, and only one was on magnolia. Among the sampled tree species, live oak and sugarberry were large mature trees while holly a nd magnolia were smaller, probably younger in age, and were pruned (Table 2 2). Thus, the difference in plant age and architecture could have affected the diversity of herbivorous hemipterans. Hemipteran populations were higher during the warmer months (M ay to Sep t ) and decreased as ambient temperatures dropped at the end of Oct (Fig. 2 3a to 2 6a). Similar temporal patterns of hemipteran distribution have been reported for Myzus persicae (Sulzer) (H emiptera: Aphididae) on potatoes, Macrosiphum euphorbiae (Thomas) (H emiptera: Aphididae) on tobacco, walnut aphid ( Chromaphis juglandicola (Kalt.) (H emiptera: Aphididae ) ), lime aphid ( Eucallipterus tiliae L. (H emiptera: Aphididae ) ), and black bean aphid ( Aphis fabae Scopoli (H emiptera: Aphididae ) ) (by Barlow 196 2, Dixon 1977). Stevens et al. (1998) found that the populations of several ant species that were tending the honeydew producing hemipterans decreased in cooler months when hemipteran numbers declined and increased in warmer months when hemipterans returne d. Similarly, N. fulva activity was greater during the warmer months (e.g., May to Aug) on all four plant species (holly, live oak, magnolia and sugarberry). Figures 2 3c to 2 6c show the number of ants foraging on the sausage provided during the sampling s.
37 The average number of ants crossing a specific point in 20 sec during the 16 mo period in 2011 2012 is presented in Figures 2 3d to 2 6d. As the daily average temperature decreased in Oct and Nov (< 21C), ant numbers declined slightly. When the tempera tures cooled further in Dec and Jan (< 19C), ant foraging decreased dramatically, suggesting that the N. fulva activity is largely dictated by temperature. Ants are poikilothermic, thus their foraging activity depends on different abiotic factors like rel ative humidity and soil temperature (Traniello 1989, Valenzuela et al. 1995). Porter & Tschinkel (1987) reported that Solenopsis invicta Buren (Hymenoptera: Formicidae) foraging activity was limited due to low temperatures in several locations of southeast ern United States. Shorter day length during cooler months might also have some impacts on the ant activities. An Australian arid Iridomyrmex purpureus form viridiaeneus Viehm e yer (Hymenoptera: Formicidae ) foraging activity temperature was suitable (Greenway 1981). Nylanderia fulva less in 2012 than in 2011 during similar months (Figs. 2 3acd to 2 6acd) This was possibly due to the lower average RH (< 30%) in 2012 than in 2011. In addition, the average precipitation for Dec to Apr was also lower in 2012 (3.6 cm) than in 2011 (7 cm). Hlldobler and Wilson (1990) suggested that warmer temperatures may not be enough for high ant activity. A relatively humid environment may be needed to avoid desiccation and to resume their activities in the open. Therefore, lower humidity and rainfall might have contributed to reduced N. fulva abundance in 2012 as compared to similar time periods in 2011. Hemipteran population size (Figs. 2 3a to 2 6a) was also lower in 2012 than in 2011. Our protocol was not designed to determine the cause of
38 hemipteran population reduction. More research will be required to evaluate this r elationship. Significant positive correlations were found between the total number of ants (trailing intensity) and the number of hemipterans in holly (r = 0.57, P < 0.0001), magnolia (r = 0.63, P < 0.0001), live oak (r = 0.80, P < 0.0001) and sugarberry (r = 0.44, P < 0.0001) (Figs. 2 3b to 2 6b). Trees with higher hemipteran populations had higher N. fulva trailing intensity (i.e., the number of trailing ants crossing a specific point in 20 sec number of trails). Trailing intensity over time differed significantly among the four plant species (Fig. 2 7). Magnolia had an average trailing intensity of 136 ants throughout the sampling period which was significantly higher than the 43 ants on live oak. The trailing intensity on sugarberry and holly were si milar at eight and seven ants, respectively (Fig. 2 7). Greater N. fulva trailing on magnolia and live oak could be attributed to greater hemipteran diversity and abundance. Tuliptree scale in magnolia is present in all stages of development during the win ter in southern states (Donley and Burns 1971) and even though most insect species found in live oak were present in warmer months, lace bugs were present throughout the year in areas with mild winters like Florida (Dreistadt and Perry 2006) providing a co ntinuous supply of honeydew to N. fulva. In contrast, on holly and sugarberry hemipterans were found only during the warmer months (Jul to Oct), and as a deciduous tree, sugarberry was defoliated from Oct to Feb. Brightwell and Silverman (2011) determined that there were fewer Linepithema humile (Mayr) ( Hymenoptera: Formicidae ) nests around deciduous trees in fall, but were present around evergreen species Pinus taeda L. (Pinales: Pinaceae) throughout winter and successfully foraged in these trees even when ambient
39 temperatures were below the minimum foraging threshold. This pattern of behavior may be similar for the deciduous trees in our study where N. fulva moved their nests away from sugarberry in the winter, thus reflecting the reduced number ants in su garberry after Oct 2011 (Fig. 2 6acd). Conclusions In this study, the activity periods of honeydew producing hemipterans and N. fulva corresponded, which reflected the s easonal changes in temperatures During the warmer months (May to Oct), the presence an d abundance of hemipterans increased, which was associated with greater ant foraging. Our paper also documents that some species of honeydew producing hemipterans were apparently protected by shelters created by N. fulva This provides evidence of mutualis m between N. fulva and honeydew producing hemipterans. Understanding the seasonal phenology between honeydew producing hemipteran species tended by N. fulva could be important in developing control strategies for this invasive ant. Controlling hemipteran s pecies would remove a food resource of N. fulva and could help decrease the density of ants in the landscape.
40 Table 2 1. Hemipterans tended by N. Fulva in Gainesville, Fl, from July 2010 to October 2010. Insect Species Family Scientific name Common name Host plant Aleyrodidae Dialeurodes citri (Ashmead) Citrus whitefly Ligustrum Japonicum Aphididae Cowpea aphid Sesbania exaltata Willow aphid Salix nigra Juniper aphid Juniperus silicicola Juniperus silicicola Asian wooly hackberry aphid Celtis laevigata Coccidae Florida wax scale Ilex cornuta Cottony maple scale Parthenocissus quinquefolia Kermesidae Kermes sp. Kermes scale Quercus virginiana Pseudococcidae Rhodes grass mealybug Stenotephrum secundatum Noxious bamboo mealybug Arundinaria tecta Pineapple mealybug Quercus virginiana Quercus virginiana Arundinaria tecta Psyllidae Pachypsylla venusta (Osten Sacken) Hackberry petiole gall psyllid Celtis laevigata Tingidae Corythucha floridana Heidemenn Florida oak lace bug Quercus virginiana
41 Table 2 2. List of trees, sampling locations, insects and number of leaves sampled. Tree Site Tree ht (m) Honeydew producers No. of leaves/branch Burford holly ( Ilex cornuta Lindl.) Site 1 0.76 Florida wax scale ( Ceroplastes floridensis (Comstock)) Mealybug ( Dysmicoccus texensis (Tinsley)) 6 Live oak ( Quercus virginiana Mill.) Site 1 4.27 Lace bug ( Corythucha floridana Heidemenn) Aphids ( Myzocallis puncata (Monell)) Kermes scale ( Allok ermes sp. ) Bullet gall ( Disholcaspis quercusvirens (Ashm.)) Pineapple mealybug ( Dysmicoccus brevipes (Cockerell)) 10 Magnolia ( Magnolia grandiflora L.) Site 2/3 2.29 Tuliptree scale ( Toumeyella liriodendri (Gmelin)) 3 Sugarberry ( Celtis laevigata Willd.) Site 2 6.4 Asian wooly hackberry aphid ( Shivaphis celti Das) Flatid plant hopper Psyllid gall ( Pachypsylla sp.) 10
42 Figure 2 1. Intact (right) and broken (left) carton shelters showing tuliptree scale ( Toumeyella liriodendri (Gmelin)) on a magnolia ( Magnolia grandiflora L.) branch. Photo courtesy of Eileen A. Buss.
43 Figure 2 2. Carton shelters on the bark of a southern red cedar ( Juniperus silicicola (Small)) tree. Intact (A) and broken carton shelters (B) showing juniper aphids ( Cinara juniperivora (Wilson) ). Photo courtesy of Shweta Sharma. B A
44 Figure 2 3. Num ber of N. fulva and hemipterans on h olly. A ) Mean number of hemipterans collected from the twigs every month from holly in Gainesville fr om February 2011 to June 2012. B ) Correlation between number of hemipterans and number of ants trailing intensity (i.e. number of ants crossing a specific point in 20 sec number of trails) on holly (r = 57; n = 72). C ) Mean number of ants collected on sausage samples every month at the base of holly in Gainesville fr om February 2011 to June 2012. D ) Mean number of N. fu lva crossing a specific point in 20 sec in holly from February 2011 to June 2012.
45 Figure 2 4. Number of N. fulva and hemipterans on live oak. A ) Mean number of hemipterans collected from the twigs every month from live oak in Gainesville fr om February 2011 to June 2012. B ) Correlation between number of hemipterans and number of ants trailing intensity (i.e., number of ants crossing a specific point in 20 sec number of trails) o n live oak (r = 0.80; n = 72). C ) Mean number of ants collected on sausage samples every month at the base of live oak in Gainesville fr om February 2011 to June 2012. D ) Mean number of N. fulva crossing a specific point in 20 sec in live oak from February 2011 to June 2012.
46 Figure 2 5. Number of N. fulva and hemipte rans on m agnolia. A ) Mean number of hemipterans collected from the twigs every month from magnolia in Gainesville fr om February 2011 to June 2012. B ) Correlation between number of hemipterans and number of ants trailing intensity (i.e., number of ants cros sing a specific point in 20 sec number of trails) o n magnolia (r = 0.63; n = 35). C ) Mean number of ants collected on sausage samples every month at the base of magnolia in Gainesville fr om February 2011 to June 2012. D ) Mean number of N. fulva crossing a specific point in 20 sec in magnolia from February 2011 to June 2012.
47 Figure 2 6. Number of N. fulva and hemipterans on sugarberry. A ) Mean number of hemipterans collected from the twigs every month from sugarberry in Gainesville from February 2011 to June 2012. B ) Correlation between number of hemipterans and number of ants trailing intensity (i.e., number of ants crossing a specific point in 20 sec number of trails) on sugarberry (r = 0.44; n = 72). C ) Mean number of ants collected on sausage samples every month at the base of sugarberry in Gainesville fr om February 2011 to June 2012. D ) Mean number of N. fulva crossing a specific point in 20 sec in sugarberry from February 2011 to June 2012.
48 Figure 2 7. Mean number of ants c rossing a specific point in 20 sec x the number of trails (trailing intensity) on different tree species in 2011 and 2012. Different letters indicates significantly different ( P < 0.05) means by analysis of variance and Tukey HSD test (R Development Core T eam 2012). 0 20 40 60 80 100 120 140 160 180 200 Magnolia Live oak Sugarberry Holly Mean number of N. fulva Tree species a bab bbb b cccccccc ccccc c c
49 CHAPTER 3 EFFECT OF CO TTONY CUSHION SCALE (HEMIPTERA: MONOPHLEBIDAE) CONTROL ON NYLANDERIA FULVA SURVIVAL AND TRAILING ACTIVITY Background The cottony cushion scale, Icerya purchasi Maskell (Hemiptera: Monophlebidae), is a large, polyphagous insect that primarily infests citrus and pittosporum in Florida ( Caltagirone and Doutt 1989 Thrarinsson 1990 ). Although native to Australia, I. purchasi is now dispersed throughout the world ( Ebeling 1959 ). In general, I. purchasi are kept under control by the vedalia beetle, Rodolia cardinalis (Mulsant), and other natural enemies ( Thrarinsson 1990) but its populations occasionally increase significantly enough to cause plant dieback and the sooty mold that accumulates on honeydew coated leaves and branches is aesthetically displeasing in urban environments ( Baker and Frank 1994, Hamon and Fasulo 1998 ). An invasive crazy ant, Nylanderia fulva (Mayr) (Hymenoptera: Formicidae) was observed tending I purchasi on pittosporum hedges and bushes in Gainesville, FL, which had not been previously documented for either species. Although not an obligate mutualism, N. fulva as that of several other hemipteran species (Sharma et al. 2013) By co nsuming the nutrient rich honeydew (Way 1963, Buckley 1987, Larsen et al. 1992 Delabie 2001), the ants presumably minimize d the buildup of sooty mold on the host plant s and kep t the hemipteran s environment cleaner (Haines and Haines 1978, Majer 1982, Fokkema et al. 1983). As with other ant hemipteran interactions ( Nixon 1951, El Ziady and Kennedy 1956) N. fulva could potentially protect I. purchasi from its natural enemies and allow it to become a primary citrus pest.
50 However, N. fulva is an urban pes t in its own right, and its distribution continues to expand. Established colonies of N. fulva in loose litter or mulched areas may harbor up to 16,000 workers (Calibeo 2013), and are difficult to manage solely by insecticides (Calibeo and Oi 2011) B aits are quickly consumed, and as the initial workers die, both baits and contact insecticides become covered with ant cadavers allowing other workers to esca pe insecticide exposure. T he identification of novel tools and/or strategies is essential to limiting the rapid spread of this pest. The effect of excluding ants from the hemipteran s that they tend for honeydew has been previously studied with other ant and hemipteran species (Davidson et al. 2003 Kenne et al. 2003, Altfeld and Stiling 2006, Piol et al. 2008, Vanek and Potter 2010b) but not with N. fulva or any of the hemipterans that its workers tend. For example, invasive Argentine ant [ Linepithema humile Mayr (Hymenoptera: Formicidae) ] foragers were excluded from the native terrapin scale, Mes olecanium nigrofasciatum (Pergande) (Hemiptera: Coccidae) which reduc ed the number of Argentine ant nests at the base of the host red maple trees, Acer rubrum L (Sapindales: Aceracae) ( Brightwell and Silverman 2009). The ants relocated their nests becaus e they lost access to their immediate carbohydrate resource. In addition, limiting the honeydew or nectar resources of invasive ants may increase the attractiveness of non repellent, liquid carbohydrate based baits (Silverman and Brightwell 2008). Thus, the objective of this study was to determine if certain neonicotinoid insecticides could adequately control plant feeding hemipterans in field tests, and if N. fulva densities would decrease because of the reduction of hemipterans or direct mortality from the insecticides.
51 Materials and Methods Laboratory T est A laboratory test was conducted to evaluate if two formulations of imidacloprid labeled to control hemipteran insects could suppress N. fulva without directly killing them. The test was conducted in a randomized comp lete block design, with four re plications assigned as blocks. Each replication consisted three plastic tray s (27.18 cm 19.56 cm 9.4 cm) with sides coated with Fluon and filled with untreated soil (50% autoclaved sand and 50% Fafard potting mix) to a depth of 2.5 cm. T wo 0.47 liter pots filled with the same soil were placed at the center of each tray (Fig. 3 1) The soil in one pot was treated with insecticide, while the other pot remained untreated A N. fulva colony of 500 work ers and two queens collected in Gainesville, FL (Alachua Co.) was placed on the soil in each tray. The ant colony was provided water and a 10% sucrose sol ution through cotton plugged 20 ml vials, and frozen house crickets were provided as a protein source. Preliminary trials were conducted with a variety of common insecticides used against hemipterans, such as Safari 20 SG (20% dinotefuran, Valent U.S.A. Corporation, Walnut Creek, CA) and Merit 75 WP (75% imidacloprid, Bayer Environmental Science, Research Triangle Park, NC) to see if they fit the major criteria for this study, which was to control the N. fulva without directly killing them. However, the ant mortality rates with these chemicals were high and therefore were not suitable for the use in our laboratory test (Fig. 3 2) CoreTect (20% imidacloprid, Bayer Environmental Science, Research Triangle Park, NC) and Merit 2F (21.4% imidacloprid, Bayer Environmental Science, Research Triangle Park, NC) were chosen
52 as the next potential pesticides for the trial since they had a lower percentage of active ingredient in them and were labeled as toxic for hemipte rans, but not for ants. One CoreTect tablet (dry formulation ) was placed 5.1 cm below the soil surface at the rate of one tablet per 3.75 liter pot Merit 2F (liquid formulation) was applied as a soil drench with a pipette at the rate of 146.2 l/m 2 Eac h pot received 25 ml of water post application, regardless of treatment. Control pots remained untreated. Ant mortality was assessed a fter 2 wk T he soil from each pot and tray was spread separately in larger tubs (45.5 cm 34.3 cm 7.6 cm) and allowed t o dry. Because ants preferred moist areas over dry soil, they were provided with artificial nest sites and allowed to move out of the drying soil. A dark container (4.1 cm 7.5 cm 12.5 cm) (to simulate the dark nest of ants in nature) with a hole just enough for the ants to get in and out, on each side was placed in the middle of each tub Two 20 ml test tubes with water retained half way with cotton and dental plaster (Castone) were placed inside each tub as nest sites (Banks et al. 1981). The number of live N. fulva in the test tubes were counted after 48 h Percentage of ant mortality was based on the number of dead ants determined by subtracting the number of live ants from the initial number of ants. D ata w ere square root transformed before analysis. D ifferences in N. fulva mortality among the two imidacloprid treatments and untreated control were then determined by a one way analysis of variance (ANOVA) using R (R Development Core Team 2012). Field Trial Twenty four pittosporum ( Pittosporu m tobira (Thunb) ) (Apiales: Pittosporaceae) plants (<1 m tall) in 11.34 liter pots were artificially infested with I purchasi in a greenhouse, then transferred to a bu siness complex in northern Gainesville that was
53 naturally infested with N. fulva In Aug ust 2012, p ots were placed on the soil surface 2 m apart in between a hedge and a road, in a completely randomized design with eight replications (Fig. 3 2). Potted plants became naturally infested with N. fulva After 1 wk, pretreatment data were collect ed, which included the mean number of I. purchasi per three branches, mean number of N. fulva on sausage samples, mean number of ants trailing at one spot for 20 sec, and the mean number of ant trails per plant stem, as described in Sharma et al. (2013) (C hapter 2). Scale and ant infested pittosporum pots were randomly assigned one of three treatments. In the first treatment, t hree CoreTect tablets were spaced equally apart, inserted ~5.1 cm down into the potting media (~1 5.2 cm from the base of the plant ) in each pot, and irrigated (28. 8 ml water per p ot. Merit 2F was applied a s a soil drench in the second treatment (6 ml mixed with 28. 8 ml wat er per 30.5 cm of shrub height). The untreated c ontrol pots received 28.8 ml of water. All pots were drip irrigat ed daily for 10 min. Icerya purchasi and N fulva survival and activity in the infested plants w ere monitored weekly between 0900 1500 h, when the ambient temperatures were for 2 mo Air temperature and relative humidity (RH) were recorded o n each sampling date by placing a digital thermo/hygrometer (Acu Rite 00891) near the base of the pot t hree branches w ere randomly flagged on each plant before the stud y began and the mean n umber of I. purchasi per three branch sample was determined Two months post treatment, the plants were removed from their pots placed in fluon coated tubs (45.5 cm 34.3 cm 7.6 cm) the r oots were removed, and the soil
54 was allo we d to air dry in the laboratory. The ants were supplied with water and a 10% sucrose solution through cotton plugged 20 ml vials, and frozen house crickets were provided as a protein source. All live ants that moved into the nest tubes were counted after 48 h. Mean number of N. fulva and standard error of mean are presented in result section. Repeated measure a nalysis was conducted to determine differences in the number of I. purchasi and the number of N. fulva collected on the sausage samples, on 20 sec count and trail count among treatments for the preliminary data as well as the data collected every week after the treatments were applied. If significant, means averaged over every week were separated by the Tu key HSD test (R Development Core Team 2012). was used to examine the association between the number of I. purchasi and number of N. fulva for each treatment. Results and Discussion Laboratory T est Nylanderia fulva survival when expos ed to soil treated with either imidacloprid formulation did not significantly differ from the untreated control ( F = 16.54; df = 2, 6 ; P > 0.003 ). The mean number of live ants (n = 500) averaged 432 (mean) 15 ( standard error, SE ) in untreated soil, 390 21.6 in soil treated with Merit 2F and 357 22.9 in soil treated with CoreTect. The ant mortality that occurred in this test may be attributed to the drier, less hu mid laboratory environment (~ than what occurs under outdoor conditions T he survival rates of different ant species is often correlated with relative humidity. Hood and Tschinkal (1990) reported that ants (e.g., Paratrechina spp., Solenopsis spp., Formica spp., Camponotus spp.) in a lab oratory in Tallahassee, FL, had greater su rvival when reared under 75% RH Hlldobler and Wilson (1990 )
55 suggested that low humidity was not suitable for ant activity and survival. Given that N. fulva prefers to nest in loose litter and mulched areas ( MacGown 2012 ), soil moisture may also have been a mortality factor. Alt hough Merit 2F and CoreTect were not specifically labeled to control ant s, other imidacloprid formulations are registered for use and are efficacious against nuisance ants ( e.g., Maxforce Quantum, Temprid etc.). It was observed that the workers avoided the imidacloprid treated potting media inside the pots, despite imidacloprid being considered a non repellent insecticide (Rust et al. 2004, Hu et al. 2005). Nylanderia fulva nested with in the potting media of the untreated pots, but nested beneath the pots treated with either imidacloprid product This suggested the treated soil was repellent and deterred N. fulva from nesting in the soil within the pots. Field Trial Preliminary sampling data The main purpose of collecting prelim inary data on the number of I. purchasi on pittosporum branches, number of ants on sausage and the ant trailing intensity (number of ants crossing a specific point) on the pittosporum trunk was to determine if the starting conditions of the treatments were generally similar among all the pots. This was crucial to ensure reliability of the inferences that would be drawn from the subsequent data collected after treatment applications. The analysis of variance of these data indicated that the number of scales did not differ significantly among the pittosporum branches before the pesticides were appli ed (Fig. 3 4). Similarly, the number of ants on the sausage samples, trailing intensity as well as number of ant trails also did not vary significantly in all the environments (Fig. 3 4). This indicated that the initial infestation levels as well as the ant densities in and around the plants were similar in all the pots.
56 Numbers of cottony cushion scale on branches T he number of I. purchasi was significantly higher on t he control compared to the imidacloprid treatments throughout the sampling dates ( F = 8.9 ; df = 2, 21 ; P > 0.001 ) (Fig. 3 5 ) However, n o statistical difference between the numbers of I. purchasi between the pesticide treated pots was observed. The nu mber of scales in treated plants began to decrease 1 wk post treatment and was almost zero at 7 wk post treatment. However the scale density decreased in September and October even in the control pots, possibly due to the falling ambient temperature s in S eptember (24C) and October (20C) compared to August (26C) (Florida Automated Weather Network) In 1962, Barlow reported a decrease in the survival and fecundity rate of M. persicae (Sulzer) (in potatoes) and Macrosiphum euphorbiae Thomas (in tobacco) du e to lower ambient temperatures. Smaller number of scale insects therefore might have resulted due to the reduced survival rate coupled with reduced reproduction thus potentially decreasing the density of ants associated with the hemipteran species. Ants on sausage sample The number of ants foraging on the sausage samples differed significantly between the control and the two imidacloprid treatments throughout the sampling dates ( F = 11.1 ; df = 2, 21 ; P > 0.0 00 5) (Fig. 3 6 ). However, the mean number of N. fulva workers in the pots treated with CoreTect and Merit 2F did not differ significantly suggesting a similar level of effectiveness of the two pesticides. It was observed that most of the ants on the sausages at the base of the control plants emerged f rom the potting soil within minutes of placing the sausages. In contrast, the ants found on sausages at the base of treated plants appeared to move in from the surrounding area but not from the insecticide treated soil It can be presumed that these ants followed the
57 odor trails and came to the sausage from around the pots, which would have increased searching and recruitment times. This difference in the time required to find the sausage and recruit the ants from the surroundings of the pot might have res ulted in the significant difference in the observed number of ants between the control and the treatments. It appeared that N. fulva did not nest in the pesticide treated pots because ants recruited to the sausages came from the surrounding area. This is c onsistent with the laboratory trial where the ants tended to nest below rather than in, the treated soil. Twenty second count In the plants that were treated with CoreTect or Merit 2F, the trailing intensity was significantly lower (two ants per 20 sec) than the trailing intensity on the untreated plants (15 ants per 20 sec) ( F = 20.6 ; df = 2, 21 ; P > 0.0 001 ) However, the t railing intensity did not differ significantly between the treatments (Fig. 3 7) thus further confirming the results from the ant number on the sausage sample that the two pesticides were of similar efficacy. The difference in the trailing intensity possibly resulted due to the lower number of I. purchasi on the pesticide treated plan ts (Fig. 3 5). Ant trail count S ignificantly fewer ant trails occurred on the main stems of treated plants than on the stems of control plants whereas the average number of trails on the stems of pesticide treated plants did not differ significantly ( F = 26.0 ; df = 2, 2 1 ; P > 0.0 001 ) (Fig. 3 8 ). T he lower number of ant trails on the treated plants could also be attributed to the lower abundance of I. purchasi on the branches (Fig. 3 5) Due to the reduction in the honeydew source ants might have divert ed their attention to other plants that still had honeydew producers on them resulting in less intensive ant activity on the plants.
58 Ant nesting in the pots After 2 mo of being in the ant infested area, all pots were examined for the sts. All untreated control pots either had nests or ants present in them. Half (n = 4 ) of the untreated pots had some tunnels and brood present in them averag ing 545 222 ants. The remaining control pots contained 250 86 adults and lacked brood These observations suggest that the a nts had create d a satellite nest in the containers of pittosporum plants in the absence of insecticides while none of the treated pots contained any nests or ant tunneling. It was found that t hree of the treated pots (two with Merit 2F and one with CoreTect) contained a small number of ants averaging 100 50 (n = 3) workers per pot. The data also indicated that only <19% of the treated pots contained some number of ants at the time of final sampling. This indicates that the ants avoided the treated soil Since imidacloprid is considered a non repellent insecticide (Rust et al. 2004, Hu et al. 2005) it is unclear why the ants avoided the treated soil. In the laboratory test when Merit 75WP with 75% im idacloprid as active ingredients was used, significant mortality of N. fulva was seen. CoreTect tablets had 20% imidacloprid and Merit 2F had 21.6% imidacloprid as the active ingredients in them. It is possible that at lower concentrations imidacloprid is not toxic enough to kill the N. fulva but can repel them from the soil. Correlation between I. purchasi and N. fulva I. purchasi and 20 sec N. fulva count s in all three treatments. A positive correlation between the scales and N. fulva was found in the pots treated with CoreTect (r = 0.88), Merit 2F (r = 0.88) and control (r = 0. 60) (Fig. 3 9 ). This is consistent with the results of the previous experiment (Chapter 2) where both ants and hemipteran
59 numbers were positively correlated throughout a sampling season. This suggests that when the scale population was higher, more ants tended the m bec ause of the abundance of the honeydew source. As the number of hemipterans decrease d honeydew bec a me scarce for N. fulva populations which may have trigger ed the ants to search food elsewhere. Conclusions This study showed that it is possible to s ignif icant ly reduc e N. fulva activity by controlling I. purchasi on pittosporum This study therefore has very important implications in the control of N. fulva in infested landscapes. The results also suggest that k eeping the plants free of honeydew producing insects is important to reduce the density of invasive N. fulva from the landscape. Although this study involved only one hemipteran species associated with N. fulva similar results can be expected with other hemipterans as well since their honeydew is an important re source for the ants. This study therefore has important implications on the effective control of N. fulva not only by controlling I. purchasi but also other honeydew producing hemipterans that are common pests.
60 Figure 3 1. Set up for the l ab oratory experiment. Photo courtesy of Shweta Sharma.
61 Figure 3 2. Data from the lab experiment with Safari 20 SG and Merit 75 WP. D ifferent letters indicates significantly different ( P < 0.05) means by analysis of variance and Tukey HSD test (R Development Core Team 2012). 0 100 200 300 400 500 600 Safari 20 SG Merit 75 WP Control Number of live N. fulva Treatments a b c
62 Figure 3 3 P otted p ittosporum plants placed next to a hedge in a northern Gainesville business complex. Photo courtesy of Shweta Sharma.
63 Figure 3 4 Preliminary data collected from the pittosporum plants bef ore the treatment application. A ) Number of cottony cushion scale ( I. purchasi ) on the pittosporum plants B ) number of N. fulva collected from the sausage samples C ) Number of N. fulva crossing a specific point on the trunk of the pittosporum plants and D ) Number of N. fulva trails on the trunk of pittosporum plants.
64 Figure 3 5 Number of I purchasi counted on the branches of pittosporum plants. D ifferent letters indicates signif icantly different ( P < 0.05) means by repeated measures analysis of variance and Tukey HSD test (R Development Core Team 2012). 0 10 20 30 40 50 60 70 22-Aug 29-Aug 5-Sep 12-Sep 19-Sep 26-Sep 3-Oct 10-Oct 17-Oct Number of I. purchasi Weeks after treatment CoreTect Merit 2F Control b b a
65 Figure 3 6. N umber of N. fulva collected from sausage samples placed near the base of the trunk of pittosporum plant D iffe rent letters indicates significantly different ( P < 0.05) means by repeated measures analysis of variance and Tukey HSD test (R Development Core Team 2012). 0 20 40 60 80 100 120 140 22-Aug 29-Aug 5-Sep 12-Sep 19-Sep 26-Sep 3-Oct 10-Oct 17-Oct Number of N. fulva Weeks after tretment CoreTect Merit 2F Control a b b
66 Figure 3 7 Number of N. fulva crossing a specific point on the trunk of pittosporum plant in 20 sec. D ifferent letters indicates significantly different ( P < 0.05) means by repeated measures analysis of variance and Tukey HSD test (R Development Core Team 2012). 0 5 10 15 20 25 22-Aug 29-Aug 5-Sep 12-Sep 19-Sep 26-Sep 3-Oct 10-Oct 17-Oct Number of N. fulva Weeks after treatment CoreTect Merit 2F Control b b a
67 Figure 3 8 Mean number of N. fulva trails on the trunk of each plant. Di fferent letters indicate significantly different ( P < 0.05) means by repeated measures analysis of variance and Tukey HSD test (R Development Core Team 2012). 0 0.5 1 1.5 2 2.5 3 22-Aug 29-Aug 5-Sep 12-Sep 19-Sep 26-Sep 3-Oct 10-Oct 17-Oct Number of N. fulva Weeks after treatment CoreTect Merit 2F Control b b a
68 Figure 3 9 Correlation between the mean number of I purchasi and N. fulva in A ) CoreTect (r = 0.88; n = 8) B ) Merit 2F (r = 0.88; n = 8) and C ) Control (r = 0.60; n = 8) treated pots.
69 CHAPTER 4 EFFECT OF VARIOUS MULCHES ON NESTING SUBSTRATE PREFERENCE, TOXICITY AND REPELLENCY OF AROMATIC CEDAR MULCH ON FORAGING BEHAVIOR OF NYLANDERIA FULVA B ackground Nylanderia fulva (Mayr) is a difficult to control nuisance pest in various landscapes and buildings because of its polygynous and polydomous colonies ( MacGown and Layton 2010 Warner and Scheffrahn 2010) Baits and contact insecticides provide minimal efficacy (Warner and Scheffrahn 2010) and biological control is not yet feasible (Valles et al. 2012) However, because N. fulva is known to nest in mulch (MacGown 2012), it may be possible to identify a repellent mulch type that c ould prevent ants from nesting in sensitive areas and/or serve as a barrier for ants searching for honeydew producing, plant infesting hemipterans (Shweta et al. 2013) One such m ulch is produced from e astern red cedar wood ( i.e., Juniperus virginiana L. (Pinales: Cupressaceae) contains cedrol, A cedrene and thujopsene, which are considered insecticidal in nature (Back and Rabak 1922, Sweetman et al. 1953 Technical Resources International 2002). When confined onto aromatic cedar mulch, Argentine ants, Linepithema humile (Mayr) (Hymenoptera: Formicidae) and odorous house ants, Tapinoma sessile (Say) (Hymenoptera: Formicidae) avoided it and nested on other mulches or died (Meissner and Silverman 2001). Aromatic cedar wood is also toxic to the clothes moth, Tineola bisselliella (Hum.) (Lepidoptera: Tineidae) (Scott et al. 1918) the black carpet beetle, Attagenus piceus (Oliv.) (Coleoptera: Dermestidae) termites (Isoptera) ( Adams et al. 1988 ) and house dust mite s Dermatophagoid es spp. (Arachnida: Pyroglyphididae) (Enomoto et al. 1999). It is also reported to be repellent to the German cockroach, Blattella
70 germanica (Linn.) ( Blattodea: Blattellidae) ( Appe l and Mack 1989 ) and red imported fire ant, Solenopsis invicta Buren (Hymenoptera: Formicidae) ( Thorvilson and Rudd 2001) The red imported fire ant also avoided the leachate from cedar shavings ( Anderson et al. 2002). The objective of this study was to evaluate different mulches (e.g., aromatic cedar, melaleuca, eucalyptus, pine bark, cypress and cedar mulches) for their suitability as nesting sites or for their potential repellency or toxicity to N. fulva as a cultural control option Materials and M ethods Mulches Six mulches were used in the study. Aromatic cedar mulch was chosen based on its insecticidal properties, and the other five mulches were chosen because of their common use in Florida (Stake 2000) Aromatic cedar mulch (Mid America Mulch, Inc. Bradenton, FL) was shredded reddish yellow colored mulch. Pine bark mulch (Margo Garden Products, Inc. Folkston, GA) was brown in color in the form of nuggets. Eucalyptus mulch (Scotts Company, LLC Palmdale, FL) was shredded and yellowish brown in color. Eucalyptus essential oil contains 1, 8 cineol and limonene, which are used as natural insecticides (Manzoor et al. 2012). Cedar mulch (Mid America Mulch, Inc. Bradenton, FL) was shredded and dark brown in color. Melale uca mulch (Forestry Resources, I nc. Ft. Myers, FL) was light brown shredded mulch. Major constituents of melaleuca essential oils are 1, 8 cineole terpineol which are used as an insect repellent (Sakasegawa et al. 2003). Cypress mulch (Margo Garden Products, Inc. Folkston, GA) was made of 100% wood, light brown shredded mulch. Pine bark and
71 cypress mulch are not known to possess any insecticidal properties A lthough live pine trees are resistant to certain pests ( Chorbadjian et al. 2011 ), pine bark mulch seem s to harbor more insect pests than any other mulch (Gill et al. 2011) Nylanderia fulva Colonies N ylanderia fulva colonies FL (Alachua County) and were reared at the United States Department of Agriculture Centre for Medical, Agricultural, and Veterinary Entomology ( USDA CMAVE) lab oratory for 3 5 mo. The colony was provided with frozen house crickets, water and 10% sucrose solutions. The N. fulva rearing methods are a modification of the fire an t rearing methods of Banks et al. (1981). Nesting Substrate Preference : Choice Test A series of laboratory comparisons between aromatic cedar mulch and five other mulches ( pine bark, eucalyptus, cedar, cypress, or melaleuca ) were conducted to determine whi ch mulches would be suitable as nesting substrates for N. fulva Methods were modified from Meissner and Silverman (2001) Each arena ( length width height = 45.5 cm 34.3 cm 7.6 cm) contained two mulch piles (aromatic cedar mulch vs. pine bark, eucalyptus, cedar, cypress, or melaleuca ) t hat were equal in volume and were 8.9 cm thick (Fig. 4 1). An artificial nest was placed on the arena between the two piles. It contained 500 workers four queens and some amount of brood that could not be separated during the counting process. Each nest was co vered by an opaque container (4.1 cm 7.5 cm 12.5 cm) F ood (frozen crickets and 10% sucrose solution) and water was placed near each nest. After 24 h each mulch pile was examined, and the number of ants and brood were counted
72 There were ten sets of five comparisons in a test and the whole test was repeated twice in different time period ( n = 100 replicates ) Artificial Nest vs Mulch Test This test was con ducted to determine if N. fulva laboratory colonies would choose to nest in an artificial nest, aromatic cedar mulch, or pine bark mulch. An artificial nest of N. fulva (500 workers and four queens and some brood) was placed in each arena (45.5 cm 34.3 cm 7.6 cm) F ood ( 7 10 frozen crickets and 10% sucrose solution) and water were placed near the nest. An 8.9 cm thick aromatic cedar mulch pile (Fig. 4 2) was placed in each arena for 24 h Presence and absence of the colony in the mulch was recorded after 24 h (i.e., workers or queens walking on/in the mulch or transferring its nests to or from the mulch) The aromatic cedar mulch was then replaced with an equal volume of pine bark mulch in the same arena s and with the same N. fulva colonies Pine bark mulch was used because it was readily available commonly used and was already being used a t the field trial site. Presence and absence of the colony in the mulch was recorded after 24 h (i.e., workers or queens walking on/in the mulch or tran sferring its nests to or from the mulch) T he whole test was conducted twice in different time period s (n = 20 replicates) Foraging Barrier Test This test was conducted to determine if aromatic cedar mulch could provide a barrier to N. fulva foraging A strip ( 8.9 cm wide ) of aromatic cedar mulch was placed across the middle of an arena (45.5 cm 34.3 cm 7.6 cm) to separate a group of 500 N fulva workers four queens and some brood from food ( frozen crickets, sucrose solutions) and water (Fig. 4 3 ). After 24 h any ants that crossed the mulch barrier to
73 access food and water were counted. Each arena represents one replication (n = 10) T he experiment was conducted twice with a n artificial nest (test tube with Castone and opaque covering) and twice without a nest to force N. fulva to go into the aromatic cedar mulch or cross the mulch for food and water. No other mulch was used for comparison. No choice T oxicity Test This no choice test was conducted to determine if aromatic cedar mulch was toxic to N. fulva A romatic cedar mulch was added evenly to a depth of 8.9 cm to each a rena (45.5 cm 34.3 cm 7.6 cm) (Fig. 4 4). Five hundred workers and four queens were placed on top of the mulch layer They were provided with food (frozen crickets and 10% sucrose solution) and water. After 24 h the number of dead N. fulva was count ed. There were ten replications (arenas) and the test was repeated twice (n = 20) No other mulch was used for comparison. Toxicity of Mulch over Time This test was conducted to evaluate how long aromatic cedar mulch might retain its toxicity against N. fulva compared with pine bark, after the products we re subjected to natural weather conditions. Fresh a romatic cedar mulch and pine bark mulch were placed outside a greenhouse in a N. fulva free area in Gainesville. Every 15 d for up to 75 d 2 g o f mulch were weighed and placed into a 50 ml vial. There were 20 vials for each type of mulch for each date (da y 0, 15, 30, 45, 60, and 75) Twenty N. fulva were placed in each vial v ials were plugged with cotton and held in the USDA CMAVE laboratory at A nt mortality was assessed after 2 4 (until day 45) or 48 h (until day 75)
74 Mulch Barrier Test This test was conducted to compare the effectiveness of pine bark and aromatic cedar mulches to repel N. fulva in outdoor landscaping. Twenty four live oak trees ( Quercus virginiana Mill Fagales: Fagaceae ) were selected in a N. fulva infested business complex in northern Gainesville, FL P ine bark mulch that surrounded the tree s w as either 1) left as a control, i.e. old pine bark mulch, 2) replaced with aromatic cedar mulch or 3) replaced with a fresh volume of pine bark mulch The experiment was conducted in a randomized complete block design with each treatment replicated eight times (= 8 trees) Existing p ine bark mulch was replaced with fresh pine bark mulch to test the impact of disturbance during mulch replacement o n N. fulva nesting beha vior. Either aromatic cedar or pine bark m ulch was placed 2.5 m around the trunk of each tree to a depth of 10.2 cm (Fig. 4 5). Sampling was conducted every 15 d for 4 mo starting from Aug to Nov of 2012. Nylanderia fulva activity was estimated using sausage counts, number of ant trails up the stem, and a 20 sec count on the main trail (see C hapter 2 ) T he mulch around each tree was searched at the end of the test to count the number of N. fulva nests present Analysis of variance was performed for th e trailing intensity, number of ant trails and number of ants on the sausage samples and Tukey HSD test on each variable was conducted for mean separation using the statistical computing software R (R Core Team 2012). Results and Discussion Nesting Substr ate Preference : Choice Test In all ten replications and in both times when the experiment was repeated, 100% of the N. fulva avoided the aromatic cedar mulch and nested in all other mulches that
75 were provided as an alternative (Table 4 1) This test demons trated that aromatic cedar mul ch was not preferred by N. fulva and was repelled by the mulch Artificial Nest Vs Mulch : No choice Mulch Test Since N. fulva avoided aromatic cedar in the presence of other suitable mulches the question arises if it will still avoid this mulch in absence of other mulches. The no choice test showed that N. fulva preferred to stay in the artificial nests rather than move their co lony to nests under the aromatic cedar mulch. After 24 h, when the aromatic cedar mulch was replaced by pine bark mulch, the N. fulva relocated their colony to the pine bark mulch in all ten replications and when the experiment was repeated (Table 4 2) Th is test further support s that N. fulva has some repellent properties against N. fulva Foraging Barrier Test T he mulch foraging barrier experiment was conducted to determine if N. fulva would cross the aromatic cedar mulch which has insecticidal properties to find food within 24 h O nly four ants crossed the mulch barrier to find food when the nests were provided (Table 4 3) Mortality on both sides was 17% when the nests were provided. Nylan deria fulva did not vacate the nest tubes to nest in the aromatic cedar mulch. Only three ants crossed the mulch barrier and went to the side where food was kept when the nests were not provided. Mortality was 100% without the nests most likely because of desiccation due to the aromatic cedar mulch preventing access to the water Barriers of non repellent mulches were not tested, thus how the cedar mulch prevented ant access to water and food was not determined.
76 No choice T oxicity Test When N. fulva was confined in aromatic cedar mulch, 100% mortality of the ants w as reported within 24 h, even with food and water present. The ants may have died from being exposed to the volatile compounds present in the mulches direct contact with the mulch or may have desiccated due to the lack of a nesting site. However, comparisons with other non repellent or non volatile mulches were not conducted, thus precluding evaluation of other potential causes of the observed mortality. Toxicity of Mulch over Time N ylanderia f ulva workers kept in vials with aromatic cedar mulch had 100% mortality within 24 h until the mulch was weathered for 45 d. No mortality was observed in vials with pine bark within that time frame. It took up to 48 h of exposure to aromatic ceda r mulch by day 60 to achieve 100% mortality. T here was no mortality in vials with pine bark mulch e ven after 48 h. By da y 75 aromatic cedar mulch had lost its toxicity However, ants stayed near the cotton plugs in the vials with cedar, while in the other vials they remained inside the pine bark mulch This suggested that aromatic cedar mulch still has repellent properties by day 75 Meissner and Silverman (2001) showed 100% mortality due to aromatic cedar mulch for up to 140 d but the mulch was not completely exposed to the natural conditions in their experiment. Mulch Barrier Test Twenty second count There was significantly less trailing intensity on trees surrounded with aromatic cedar mulch than on trees with both the replaced and or old (control) pine bark mulch ( F = 23.2; df = 2, 117 ; P < 0.0001 ) (Fig. 4 6). T railing intensity was similar for trees with replaced or intact pine bark mulch The average trailing intensity was 34 4. 2 ants on
77 trees surrounded by aromat ic cedar mulch but was 91 16.5 ants in the undisturbed pine bark and 116 11.3 ants in the disturbed pine bark mulch. Al though trailing intensity did not differ among the two pine bark treatments, perhaps the s lightly greater trailing intensity in replac ed pine bark could be related to the disturbance created during the mulch replacement. Tramp ants like Argentine ants prefer to nest in disturbed habitats (Bestelmeyer and Wiens 1996), so N. fulva might favor t he recently replaced pine bark over an undistu rbed mulched area Fewer N. fulva in the aromatic cedar mulch suggested that the ants were repelled or killed by the mulch Furthermore, few N. fulva cross ed the aromatic cedar mulch barrier to tend hemipterans despite the presence of honewdew producing hemipteran s in the trees [ e.g ., Allok ermes spp. (Hemiptera: Kermesidae), Corythucha floridana Heidemenn (Hemiptera: Tingidae) etc. ] throughout the business complex. Ant trail count Trees su rrounded by aromatic cedar mulch had significantly fewer trails going up their stems (1.5 0.1), compared to trees surrounded by the replaced pine bark mulch or control ( 3 .1 0.1 ) ( F = 16.3; df = 2, 117 ; P < 0.0001 ) (Fig. 4 7) Ants on sausage sample The number of ants foraging on the sausage samples was significantly less on aromatic cedar mulch when compared to the control and replaced pine bark mulch ( F = 8.6; df = 2, 116 ; P < 0.0003 ) (Fig. 4 8). Over all sampling dates, t he average number of ants on the sausage samples placed at the base of the trees surrounded by aromatic cedar mulch was 73 9. 8 and the average number of ants on the sausage samples of
78 the control and replaced pine bark mulch were 109 13.2 and 119 13.8, respectively. Even tho ugh there was minimal N. fulva activity on trees with the aromatic cedar mulch, ants from adjacent area s were observed crossing the aromatic cedar mulch barrier through fallen leaves, twigs and grass clippings that were blown over the mulch. Mulch in the f ield study might have also lost its toxici ty and insect repellent properties due to environmental exposure Number of nests in each treatment Destructive sampling was conducted at the end of the sampling period to count the number of N. fulva nests in ea ch treatment. There was a significant difference in the nest count between the aromatic cedar mulch and the old pine bark mulch (control) or the replaced pine bark mulch. T hree N. fulva nests occurred in aromatic cedar mulch, ten nests in the old pine bark mulch and 11 nests in replaced pine bark mulc h The difference in nest counts could be attributed to the toxic and /or repellent properties of the aromatic cedar mulch. This sampling could not be done immediately after the experiment was complet ed (November 20 th ) time destructive sampling was conducted (January 4 th ) the mulch was in place for 18 wk. The toxicity of mulch over the time test indicated that the mulch was not very effective by day 75. Thus, mu lch might have lost it s toxicity and repellent nature after 18 wk (126 d) and ants could have nested in the aromatic cedar mulch. Nevertheless, ant activity and nests were significantly lower on the trees surrounded by aromatic cedar mulch than the control and the trees surrounded by the fresher, replaced pine bark mulch.
79 C onclusions The lab oratory experiments showed that aromatic cedar mulch was repellent and even toxic to N. fulva populations as noted by the lack of nesting and dead ants after 24 h expos ure. However, it should be noted that comparisons with non repellent mulch were not conduc ted in the laboratory tests examining foraging barriers and no choice toxicity of the aromatic cedar mulch. Field results showed there was low ant trailing intensity, fewer ant trails and few ants on the sausage pieces near trees surrounded by aromatic cedar mulch than the trees surrounded with undisturbed or rep laced pine bark mulch. However, 100% mortality or repellency of N. fulva by the aromatic cedar mulch was not obtained in the field, unlike the laboratory tests. Aromatic cedar mulch could potentially be used as a part of an integrated pest management program for N. fulva Using repellent mulch in the landscape to interfere wit h ant access to honeydew producers could help reduce the availability of food resources and improve ant bait efficacy.
80 Figure 4 1. Aromatic cedar mulch and other mulches used for the nesting substrate preference test Photo courtesy of Shweta Sharm a.
81 A B Figure 4 2. Laboratory set up for artificial nest vs. mulch test. A) Aromatic cedar mulch replaced with pine bark mulch after 24 h. B) P ine bark mulch observed after 24 h to see the ant preference for the artificial nest against the mulch. Photo courtesy of Shweta Sharma.
82 Figure 4 3. Aromatic cedar mulch barrier with N. fulva on one side and food and water on the other side ( foraging barrier test) Photo courtesy of Shweta Sharma.
83 Figure 4 4. Nylanderia fulva placed on aromatic cedar mulch with food and water provided (Mulch toxicity test) Photo courtesy of Shweta Sharma.
84 Figure 4 5. Mulch placed around the live oak trees in the mulch barrier test. A ) Tre e with fresh pine bark mulch. B) Tr ee with aromatic cedar mulch. C) Tree with undisturbed pine bark mulch. Photo courtesy of Shweta Sharma. A B C
85 Table 4 1. Presence or absence of N. fulva colony fragments (500 workers + 4 queens) among five types o f landscape mulch that were individually paired with aromatic cedar mulch. Each pairing was replicated 10 times. Mulch type No. colony fragments in specified mulch type No. colony fragments in aromatic cedar mulch Cedar 10 0 Cypress 10 0 Eucalyptus 10 0 Melaleuca 10 0 Pine bark 10 0
86 Table 4 2 Pre valence of N. fulva colony fragments nesting in pine bark or aromatic cedar mulch. Fragments consisted of 500 worker + 4 queens. Mulch no. of colony fragments nesting in mulch Pine bark 10 Aromatic cedar 0
87 Table 4 3 Percentage of N. fulva crossing the foraging barrier to access food and water. Nest provided Nest not provided % of ants crossing for food % ant mortality % of ants crossing for food % ant mortality 0.89 16.95 0.56 100 Note: n = 10
88 Figure 4 6. Average number of N. fulva crossing specific point in 20 sec (trailing intensity). Statistical difference ( P < 0.05) among means are indicated by different letters (ANOVA, Tukey HSD test, R Development Core Team 2012). 0 50 100 150 200 250 Aug 22 Sept 05 Sept 19 Oct 05 Oct 19 2-Nov 20-Nov Average number of N. fulva Sampling dates (2012) Control Pine Bark Aromatic Cedar a a b a a b a b c a a b a a b a a b a a b
89 Figure 4 7. Average number of N fulva trails on the trunk of the trees surrounded by the mulches Statistical differences ( P < 0.05) among means are indicated by different letters. (ANOVA, Tukey HSD test R Development Core Team 2012). 0 0.5 1 1.5 2 2.5 3 3.5 4 Aug 22 Sept 05 Sept 19 Oct 05 Oct 19 2-Nov 20-Nov Average number of N. fulva trails Sampling dates Control Pine Bark Aromatic Cedar a a b a a b a a b a a b a a b a a b a b c
90 Figure 4 8. Average n umber of N. fulva on sausage samples that were placed on the mulches around the trees Statistical differences ( P < 0.05) among means are i ndicated by different letters. (ANOVA, Tukey HSD test R Development Core Team 2012). 0 20 40 60 80 100 120 140 160 180 Aug 22 Sept 05 Sept 19 Oct 05 Oct 19 2-Nov 20-Nov Average number of N. fulva Sampling dates Control Pine Bark Aromatic Cedar a a b a a b a a b a b a a a b a a b a a b
91 CHAPTER 5 NYLANDERIA FULVA PREFERENCE FOR HONEYDEW FROM DIFFERENT HEMIPTERANS OR SUCROSE CONCENTRATIONS Background Obligate mutualistic relationships between ants and honeydew producing hemipterans have been well documented, detailing the benefits of a stable food source for the an ts ( El Ziady and Kennedy 1956, Pontin 1959, El Ziady 1960, Way 1963, Fokkema et al. 1983, Samways 1983, Buckley 1987, Haines and Haines 1987, Hlldobler and Wilson 1990, Yao et al 2000, Van Emden and Harrington 2007) and the protection from natural enemi es, movement within or among host plants, or greater survival and fecundity for the hemipterans (Way 1963, Burns 1973, Sheppard et al. 1979, Stout 1979, Wood 1977, Fritz 1982, Bristow 1 983, Fowler and MacGarvin 1985, Buckley 1987, Anderson and McShea 2001, Moya Raygoza and Larson 2008, Vanek and Potter 2010 ). However, a nts can also regulate hemipteran populations by protecting just enough individuals to and preying on the remainder of the hemipterans as a protein source (Way 1963, Addicott 1979). For example, Lasius niger L. (Hymenoptera: Formicidae) killed more aphids Lachnus tropicalis (Van der Goot) (Hemiptera: Aphididae) and Myzocallis kuricola (Matsumura) (Hemiptera: Aphididae) when aphid density per ant was high but w hen aphid density per ant decreased L. niger switched to tend ing the aphids ( Sakata 1994) Similarly, if alternative carbohydrate resources are available, ants may increase their predation of hemipterans, rather than tending them, as occurred with L. niger and Aphis fabae Scopoli (H emiptera: Aphididae ) ( Offenberg ( 2001) S ome aphid species have alter ed t heir feeding behavior and honeydew composition by increasing the concentrations of
92 amino acids in the presence of tending ants, which reduces their ow n growth and fecundity (Stadler and Dixon 1998 Yao et al. 2000, Yao and Akimoto 2002). A nt preferences could depend on the sugar concentration and composition of existing honeydew sources ( Romeis and Wckers 2000 ). This is illustrated by honeydew producing hemipterans not all being ant tended. For example, only 40% of aphid species are tended by ants despite all aphids produc ing honeydew ( Stadler 1997). Aphids, such as Metopeurum fuscoviride Stroyan (Hemiptera: Aphididae) on tansy or Cin ara spp. on conifers, excrete honey dew contain ing between 30% and 70% melezitose and are mostly tended by ants. Melezitose is synthesized in gut from two units of glucose and one unit of fructose (Bacon and Dickins on 1957, Ashford et al. 20 00). Alternatively species like Macrosiphoniella tanacetaria Kaltenbach (H emiptera: Aphididae ) on tansy or Macrosiphum euphorbiae (Thomas) (H emiptera: Aphididae ) on tomato, produce a negligible amount of melezitose in their h oneydew and are rarely ant tend ed (Hendrix et al. 1992 Volkl et al. 1999). Ant s respond to the most profitable source of honeydew available (Davidson 1978, Krebs and Kacelnik 1991, Nonacs and Dill 1991). Honeydew produced by individuals of the same hemipteran species can vary i n amino acid (Douglas 1993) and sugar composition (Hendrix et al. 1992) depending on which plants individual hemipterans feed ( Sandstrm et al. 2000 Wool et al. 2005 ) It can also vary among hemipteran species (Fischer and Singleton 2001). H oneydew mainl y consists of monosaccharide (glucose and fructose) and d isaccharide (maltose, sucrose) sugars. It also may also contain trisaccharides (melezitose, raffinose, erlose), as well as small amounts of amino acids, proteins and lipids ( Baker and Baker 1983 ;
93 Vl kl et al 1999; Wckers 2000, 2001). Some studies indicate d that ants are more attracted to honeydew with high melezitose content (Kiss 1981, Vlkl et al. 1999). Nylandaria fulva is an invasive ant species spreading rapidly through Fl orida and the southern U.S. It has been observed tend ing multiple hemipteran species throughout the year on different herbaceous and woody plant s (Sharma et al. 2013 (In Press ) ; Chapter 2 ) and does not appear to have any obligate mutualistic relationships with any of t he hemipterans that it tends Limited information exists on the biology of N. fulva and its behavior i n landscape s such as food preferences T he objectives of this study were to determine if N fulva preferred the honeydew produced by species representing four hemipteran families and if differences in sucrose concentration could affect N. fulva feeding preferences. Materials and Methods Hemipteran and Host Plant Preference A laboratory test was conducted to evaluate whether or not N. fulva would prefer entially tend certain honeydew producing insects (whiteflies, mealybugs, scales or aphids) on different host plant species. All plants (hibiscus, holly, milkweed, and pittosporum) were naturally infested either in the landscape or in a gr eenhouse in Gainesville, FL. Hibiscus, Hibiscus rosa sinensis L. (Malvales: Malvaceae) was infested with silverleaf whiteflies ( Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae ), holly ( Ilex cornuta Lindl. (Aquifoliales: Aquifoliaceae) plants were infes ted with coffee root mealybugs ( Dysmicoccus texensis (Tinsley) (Hemiptera: Pseudococcidae) ) pittosporum ( Pittosporum tobira (Thunb) ) (Apiales: Pittosporaceae) was infested with cottony cushion scale ( Icerya purchas i Maskell (Hemiptera: Monophlebidae) ) and milkweed
94 ( Asclepias curassavica L. (Gentianales: Asclepiadaceae) was infested with oleander aphid ( Aphis nerii Boyer de Fonscolombe ( Hemiptera: Aphididae) ) Two twigs (~12 cm long) from each plant species were cut on the day of the test, and inserted in to 11 cm long plastic water pics (tubes with a hole on the cap). One twig was infested with one of the representative hemiptera species and the other twig was uninfested, for each species of plant. The number of hemipterans on the infested twigs was counte d before ants were introduced. Each pair of twigs was obtained from a different hibiscus, holly, milkweed, or pittosporum plant to avoid pseudoreplication. The infested and uninfested twigs per plant species were held upright and ~10 cm apart within separa te holes in one wire mesh station (6.4 cm 30.5 cm; 0.5 0.5 cm mesh ). F our wire mesh stations were placed in a Fluon coated plastic arena (45.5 cm 34.3 cm 7.6 cm ) ( Fig. 5 1) and held in the USDA CMAVE laboratory ( ambient temperature was 26C with 45% RH ) Five hundred N. fulva workers and four queens were placed in each arena with artificial nests covered with dark containers (4.1 cm 7.5 cm 12.5 cm) ) Food and water was withheld from the ants for the duration of the experiment (48 h) to elicit tending of the representative hemipteran species. The number of ants visiting each twig was counted once in late morning (1000 1100 h) and once in late aftern oon (1500 1600 h) for 48 h Water in the pics was refilled as needed. The test was discontinued after 48 h to prevent ant desiccation due to the lack of additional water sources. The test was replicated eight times (one arena = a replicate) in a complete ly randomized design, and the entire test was repeated four times. There were a total of 32 re plicates (8 replicates per 48 hours, which was repeated on four dates). The data was averaged
95 over all sampling dates and time and the data analysis was based on 8 replicates A 2 way analysis of variance was conducted on the average number of N. fulva on infested and uninfested twigs of different host plants for the 48 hour period using R software (R development Core 2012) and Tukey HSD was used for mean separatio n. Different Sugar Concentrations Test A laboratory test was used to evaluat e whether or not N. fulva preferred a certain percentage of sucrose solution. In this test there were three treatments 1) 10% sucrose solution, 2) 20% sucrose solution and 3) water as a control Each treatment received a pittosporum twig infested with I purchasi The sugar concentration of I. purchasi honeydew was measu r ed to be 15% using a Vee Gee Scientific BTX1 handheld refractometer (0.2% accuracy). The water, 10% and 20% sucrose solution were provided in 20 ml plastic tubes with a cotton plug. An I purchasi i nfested pittosporum twig was inserted in a water pic and held upright within the wire mesh stations, as previously described (Fig. 5 2). Each arena either re ceived 1) 10% sucrose solution tube, water tube and pittosporum twig, 2) 20% sucrose solution tube, water tube and pittosporum twig or 3) water tube and pittosporum twig. Five hundred workers and four queens which were not starved prior to the test, were placed in the center of each arena (45.5 cm 34.3 cm 7.6 cm ) The number of ants visiting each tube or pic was counted once in the late morning (1000 1100 h) and once in the late afternoon (1500 1600 h) for 48 h and water in the pic was re fille d as needed The treatments were organized in randomized complete block design with six replications (arenas ) The number of ants visiting the sucrose tube, water tube and the twig was compared by a one way analysis of variance for each of the three treat ments.
96 Data for the comparison of number of ants visiting the water tubes and twigs were analyzed using a t test (R development Core 2012). Results and Discussion Hemipteran and Host Plant Preference Preliminary count s of hemipterans from the infested shoots showed that the average ( 9.2) number of hemipteran s among the four plant species were: holly twigs (67 8.8), milkweed (59 12.7), pittosporum (40 14) and hibiscus (23 1.6). Fewer than two ants occurred on a ny uninfested twigs whereas most ants occurred on milkweed twigs t ending oleander aphids Despite higher numbers of mealybugs occurring on the holly twigs, there were fewer numbers of ants tending the mealybugs as compared to other infested twigs There w as a significant difference between number of N. fulva visiting the infested and uninfested twigs over the different species of host plants ( F = 132.7; df = 1, 49 ; P < 0.0001) (Fig. 5 3) as expected. Nylanderia fulva was visitin g and tending the twigs that contained honeyde w producing hemipterans on them while very few a nts were found in the uninfested twigs at the sampling dates. However, there was no significant difference between the numbers of N. fulva visiting different host plant species ( F = 1.5; df = 3, 49 ; P > 0.2) This result is consistent with field observations gathered in chapter 2, where N. fulva did not appear to favor any particular hemipteran species. This could be attributed to the high density of the N. fulva population which may result in less selective foraging behavior. S ugar and amino acid analysis on the honeydew was not conducted due to the lack of laboratory facility to conduct such tests, and the effects of these compounds in the honeydew of the hemipterans used in this test could not be determined. The sugar content and quality of the honeydew could be different among hemipterans species
97 despite feeding on the same hos t plant (Sandstrm et al. 2000), Also, hemipterans within the same species could have different amount s of amino a cids in their honeydew when feeding on different host plants (Douglas 1993). T here is no information about the sugar content and presence of any other compound in the honeydew and one can only speculate on the implications of the study T here is no difference in the honeydew quality and ant tending between the four hemipteran sp ecies used in these experiments as indicated by foraging activity. D ue to the high po pulation density and colony nutritional needs, N. fulva collect ed any food avai lable without exhibiting a preference. The samples were collected late in the morning and late in the afternoon to emulate the foraging behavior of N. fulva under field conditions. Nylanderia fulva were most active in the day time when the temperature is around 21C. In the laboratory, the average temperature was 26C; and the ants could be active when the sampling was not conducted. Different Sugar Concentrations Test The N. fulva worker count on the twigs (honeydew sugar concentration = 15%) was significantly higher than the 10% sucrose solution and the water ( F = 50.9; df = 2 10; P < 0.0001) (Fig. 5 4a). There also was significantly high er number s of N. fulva visiting the 20% sucrose solution a nd the twig than the water tube s ( F = 26.7; df = 2 10; P < 0.0001) (Fig. 5 4b). The comparison of N. fulva counts between only the water tubes and the twig s, revealed significantly higher number s of ants on the twigs than on the water tubes (Fig. 5 4c). This data indicates that N. fulva prefer liquids with a higher sugar concentration than those with a lower sugar concentration. This evidence of N. fulva preferring a higher sugar concentration indicates that this ant species i s likely to
98 tend the hemipteran species that produce honeydew with higher sugar concentrations. However, no difference in preference was seen between the 20% sucrose solution and I. purchasi honeydew that contained 15% sugar concentration which suggests th at even when an artificial, sugar source of higher concentration than the honeydew is provided, the ants may keep tending the hemipterans. C onclusions The results of this experiment showed that higher number s of N. fulva tended to visit the hemipteran inf ested twigs of each host plant. Nylanderia fulva showed no preference to any parti cular hemipteran insect species in the first lab test. Despite the different number of hemipterans on the twigs, N. fulva tend ed all infested twigs equally. Honeydew analysis was not conducted for this experiment so one can only speculate that there might have been no difference in the honeydew of different hemipteran species used in this test I n the second experiment, ant tending was greater when higher sugar concentration w as provided. This s uggests that N. fulva will likely tend hemipterans which produce higher honeydew sugar concentrations when presented a choice. More research is needed to make any recommendations from this experiment.
99 Figure 5 1 Uninfested and infested twigs of four different plant species arranged on a wire mesh in a lab. Photo courtesy of Shweta Sharma.
100 Figure 5 2. Infested pittosporum twigs arranged on a wire mesh with different concentration of sucrose solutions in a lab Photo courtesy of Shweta Sharma.
101 Figure 5 3 Mean number of N. fulva on t he uninfested and infested twigs of different plant species. D ifferent letters indicates significantly different ( P < 0.0001 ) means by analysis of variance and Tukey HSD test (R Development Core Team 2012). 0 2 4 6 8 10 12 14 Pittosporum Holly Milkweed Hibiscus Number of N. fulva Tpye of plants Uninfested Infested a b a b a b a b
102 Figure 5 4 Comparison of mean number of N. fulva foraging on the twigs with N. fulva foraging on the sucrose solutions and water tubes. A ) 10% sucrose solution and water tubes B ) 20% sucrose solution and water tubes, and C ) water tubes. Different letters indicates significantly different ( P < 0.05) means analyzed by one way analysis of variance in a) and b) and by t test in c) (R Development Core Team 2012).
103 CHAPTER 6 SUMMARY AND CONCLUSIONS In chapter 2, many hone ydew producing insects were found to be tended by N ylanderia fulva (Hymenoptera: Formicidae). A positive relationship between hemipterans and N. fulva activity which reflected the seasonal changes in temperature was observed. Nylanderia fulva was also fo und constructing carton shelter covering the honeydew producing hemipterans. It is essential to u nderstand the seasonal phenology between honeydew producing hemipteran species tended by N. fulva which could be important in developing control strategies for this invasive ant. Future research could be conducted in other N. fulva infested regions to further understand their relationship with other hemipteran insects and the potential implications this has to the development of IPM strategies. In chapter 3, neonicotinoid insecticide formulations containing the active ingredient i midacloprid were used to control Icerya purchasi o n pittosporum plants which in turn reduc ed N. fulva activity This experiment showed a potential approach of indirectly controlling N. fulva populations by suppressing hemipteran populations on plants. Controlling hemipteran insects in the landscape and maintaining low hemipteran populations by pruning periodically could reduce the ant density and formicide use in the land scape. This chapter however, did not address the observation of N. fulva avoiding soil treated with the supposedly non repellent pesticide i midacloprid In chapter 4, a series of lab and field experiment s was conducted with various types of mulch es which showed that aromatic cedar mulch was repellent and even toxic to N. fulva colonies. However, all N. fulva did not die and were not completely repelled by the aromatic cedar mulch in the field test, in contrast to the laboratory test results. It
104 is possible that natural weathering broke down the repellent compounds in the mulch However, significantly less ant activity occurred on the trees surrounded by aromatic cedar mulch than on the trees surrounded by pine bark mulch. This suggested the potential use of aromatic cedar as a mulch to deter N. fulva nesting in landscapes. Finally, c hapter 5 briefly investigated the preference of N. fulva to tend four different hemipteran species from four different taxonomic families. No apparent differences in tending occurred for any of the species A second lab experiment showed a difference in ant tending behavior when higher sugar concentration was provided to the ant More research and hon eydew analysis will be needed to make any recommendations from this experiment. The essential oil experiment in the appendix demonstrated that several oils could kill N. fulva under laboratory conditions. However, further research is needed to determine t heir potential for N. fulva control in the field
105 APPENDIX EFFECTS OF ESSENTIAL OILS ON NYLANDERIA FULVA (MAYR) B ackground T he identification of novel tools and/or strategies is essential to minimize the spread of the invasive ant Nylanderia fulva (Mayr) The search for natural, relatively safe and non polluting insecticides is important and research is focused on exploring bioactive chemical compounds from plants (Vogt et al. 2002 Appel et al. 2004). Naturally occurring insecticides have been used in pest control for centuries (Ebeling 1971, Coats 1994) with the goal of replacing or reducing the amount of conventional insecticide use (Regnault Roger 1997). Essential oils are naturally occurring volatile oils t hat give a distinctive odor, flavor or taste to a plant (Enan 2001). Koul et al. (2008) mentioned that essential oils we re the by products of plant metabolism. Essential oils occur in glandular hairs or cavities of plant cell wall s and are present in the l eaves, stems, bark, flowers, roots and fruits in plants (Koul et al. 2008). In this appendix the effect of five essential oils with insecticidal properties was examined on N. fulva C amphor [ Cinnamomum camphora (L.) J. Presl (Laurales: Lauraceae ) ] cinna mon [ Cinnamomum verum J. Presl (Laurales: Lauraceae ) ] citronella [ Cymbopogon winterianus Jowitt (Poales: Poaceae)] peppermint [ Mentha piperita L. (Lamiales: Lamiaceae)] and clove [ Eugenia caryophyllata (L.) Merrill and Perry (Myrtales: Myrtaceae)] essential oils were used as treatments with canola oil as a control. Canola oil was chosen as the control because it i s also plant based derived from rape seeds and was considered a better comparison than petroleum oil because it was not expected to ca use any insect mortality ( Stark and Walter 1995 )
106 Leaf essential oils from Cinnamomum spp have anti termite, antibacterial, anti mite, anti mosquito, anti pathogenic and antifungal properties (Chang et al. 2001, Chang and Cheng 2002, Wang et al. 2005, Ch eng et al. 2006). The active ingredient in cinnamon oil is cinnamaldehyde (Woolf 1999, Isman 2000). Cheng et al (2008) determined that cinnamon leaf essential oil is effective against Solenopsis invicta Buren (Hymenoptera: Formicidae) They reported that at 2.0% the oil killed all red imported fire ant s after 40 min of exposure. Camphor ( C camphora ) is used to control tracheal mites in beehives (Koul et al 2008). It also act s as a growth inhibitor and anti feedant in the variegated cutworm Peridro ma saucia Hubner (Lepidoptera: Noctuidae) (Dale and Saradamma 1981 Koul et al 1990). The compounds linalool, limonene and camphore in C. camphora oil were reported to be toxic, repellent or fumigant against insects ( Tripathi et al. 2000 Hummelbrunner and Isman 2001 Tripathi et al. 2003 ). Citronella ( C nardus ) essential oil has been used for over fifty years as an insect repellent (Koul et al 2008). The authors also mentioned that a few drops of citronella, lemon [ Citrus limon (L.) Burm. F. (Sapinda les: Rutaceae)] rose [ Rosa R damascene Mill. (Rosales: Rosaceae)] lavender and basil essential oils with one liter of distilled water is effective to ward off indoor insect pests. The larvicidal activity of citronella oil has been attributed to its monoterpenic constituent citronellal (Zaridah et al 2003). Citronellal is toxic to Spodoptera litura (F.) ( Lepidoptera: Noctuidae ) Musca domestica L. (Diptera: Muscidae) (Lee et al. 1997 Hummelbrunner and Isman 2001), cowpea weevil [ Callosobruchus maculatus (F ) (Coleoptera: Chrysomelidae)], and Drosophila
107 melanogaster Mei gen (Diptera: Drosophilidae) (Don Pedro 1996). Citronellal was also effective against Aedes aegypti (L.) (Diptera: Culicidae) (Coats et al. 1991). Eugenol, the major constituent of cloves was effective against several beetles including, red flour beetle [ Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae)], maize weevil [ Sitophilus zeamais (Motschulsky) (Coleoptera: Curculionidae)], S. granarius (Motschulsky) (Coleoptera: Curculionidae) and Prostephanus truncatus (Horn) (Coleoptera: Bostrichidae) ( Obeng Ofori and Reichmuth 1997 ). Clove essential oil has been used in a cleaning solution with pyrethroids and destroyed eggs and larvae of Blattaria and also prevented reinfestation (Heinmenberg 1992). Application of a solution containing clove essential oil on a woolen cloth is effective against cloth infesting moths and de rmestid or carpet beetles (Riedel et al 1989). Peppermint ( M piperita ) can repel ants, flies, lice and moths (Koul 2008). A flea collar for pet dogs was manufactured by adding essent ial oils c itronella and p eppermint to ethylene vinyl acetate (Seto 1987). Appel et al. ( 2004 ) tested the repellency and toxicity of mint oil granules and found that all Solenopsis invictus Buren mounds that were treated with mint oil granules were abandoned Peppermint o il is composed of menthol menthone (Edris and Farrag 2003) and pulegone (Clark and Menary 1980). Pulegone is effective against M domestica L. Diabrotica virgifera LeConte (Coleoptera: Ch rysomelidae) Peridroma saucia Hubner (Lepidoptera: Noctuidae) and Spodoptera litura (F.) ( Lepidoptera: Noctuidae ) (Harwood et al. 1990, Lee et al. 1997, Hummelbrunner and Isman 2001). Pulegone containing diet at 0.1% retarded development and inhibited rep roduction of last instar of southern armyworm [ Spodoptera eridania (Cramer) ( Lepidoptera: Noctuidae )] (Gunderson et al. 1985).
108 Materials and Methods Cotton balls were placed at the bottom of the 50 ml vials. Twenty N. fulva workers were placed in each vial and the vials were plugged with cotton balls (Fig. A 1). One hundred microliters of oil was gently injected in to the top cotton ball, but not in to the vials to avoid any contact toxicity The amount of time required for 100% morta lity to occur was recorded based on observations made at 15 min intervals for 8 h and then once every 16 h. There were five treatments ( camphor, cinnamon, clove, peppermint and citronella ) and a control ( canola oil ). Each treatment had five replicates. The experiment continued for 48 h and was repeated twice. Results and Discussions The amount of time needed to kill all N. fulva within the treatment vials is presented in Figure A 2. Camphor oil was the most effective against the N. fulva None of the 20 an ts used in this study survived after 15 min of exposure. Peppermint oil killed all N. fulva in 45 min and it took citronella oil 70 min to achieve 100% mortality. Cinnamon oil and clove oil kill ed 100% of N. fulva with in ~ 24 h (observations were taken ever y 16 h for cinnamon and clove) The ants were unharmed by the canola oil e ven after 48 h of exposure. Chen (2009) reported that camphor oil showed significant repellency to S invicta at the concentration of 100 mg/kg but not at the concentration of 1 mg/kg and 10 mg/kg. Essential oils were expensive and cost as much as $6 for 15 ml. Due to the cost and due to the lack of willingness of property owner s to spray oil in i nfested buildings; a field test could not be conducted. Thus, results are preliminary and further testing is needed to assess their efficacy under various environmental conditions.
109 Figure A 1 Different type of essential oils along with the N. fulva in the vials treated with these oils. Photo courtesy of Shweta Sharma.
110 Figure A 2 Time required in hours to achieve 100% mortality for 20 N. fulva No mortality was seen with canola oil (control) even after 48 h which is not shown in this graph. 0 5 10 15 20 25 30 Camphor Peppermint Citronella Cinnamon Clove Time required for 100% mortality Essential oils
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129 BIOGRAPHICAL SKETCH Shweta Sharma was born in Chitwan, Nepal. She completed her undergraduate degree at the Institute of Agriculture and Animal Sciences in Rampur, Nepal. She joined the University of Florida in spring 2007 and graduated with an M.S. degree in turfgrass scienc e from the Department of E nvironmental H orticulture in spring, 2009. Dr. Laurie Trenholm was her advisor. During her M.S., she earned a prestigious award through the Florida Turfgrass Association. S he continued on for a doctorate degree in Plant Medicine at the University of Florida. However, i n the summer of 2011, she joined Dr. Ph D candidate in the Department of Entomology and Nematology. Her area of interest is urban landscape entomology and her research was focused on the interactions of the ants Nylanderia fulva with honeydew producing hemipteran insects. This dissertation is the culmination of the studies she conducted during 2011 2012 as part of her Ph D research. Funding was provided by the USDA T STAR C program. In spring 2013, she successfully defended her dissertation and earned the doctorate degree in e ntomology and n ematology.