Manipulation, Rearing and Storage of Tamarixia Radiata (Hymenoptera

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Manipulation, Rearing and Storage of Tamarixia Radiata (Hymenoptera Eulophidae) Parasitoid of Diaphorina Citri (Hemiptera: Psyllidae)
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1 online resource (73 p.)
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english
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Chen, Xulin
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University of Florida
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Gainesville, Fla.
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Thesis/Dissertation Information

Degree:
Master's ( M.S.)
Degree Grantor:
University of Florida
Degree Disciplines:
Entomology and Nematology
Committee Chair:
Stansly, Philip Anzolut
Committee Members:
Frank, J H
Rohrig, Eric Andrew

Subjects

Subjects / Keywords:
citri -- diaphorina -- radiata -- tamarixia
Entomology and Nematology -- Dissertations, Academic -- UF
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Entomology and Nematology thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

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Abstract:
Tamarixia radiata (Waterston) (Hymenoptera: Eulophidae), is anarrhenotokous ectoparasite of the Asian citrus psyllid (ACP) Diaphorina citri (Kuwayama) (Hemiptera: Psyllidae),vector of citrus greening disease or huanglongbing (HLB). Tamarixia radiata is being tested as an augmentive biological control agent, since the number present in the field is low following winter when few hosts are available. Any program aimed at augmentation of a natural enemy would require an efficient system of mass production. Key components would include methods of manipulating, rearing and storing large populations of insects. This research investigated the use of carbon dioxide (CO2)to anesthetize T. radiata, determined optimal host densities for rearing the parasitoid, and evaluated diets to maintain fecundity during storage periods. Tamarixia radiata adults held in an atmosphere of100% CO2 for 5 min were immobilized for about 4 min. However,survivorship and fecundity were reduced significantly, although sex ratio of progeny from treated adults was not affected. Consequently, lighter doses of CO2or other methods of anesthesia are needed. One pair of three-day-old T. radiata was released in a centrifuge tube for five days with different 4th instar D. citri nymphs densities: 10, 20, 30, 40, 50, and 60. At a density 40, 4thinstar nymphs per female fecundity, incidence of parasitism and superparasitism were all optimal. The pattern of parasitism for the first five days conformed to a Type II functional response. Estimated searching efficiency was 0.442 ± 0.036 per day and estimated handing time was 0.045 ± 0.008 days. Therefore, 40 hosts per female T. radiata should be a target for mass rearing.Females feeding on eight different diets were dissected every 5, 10, 15 and 20 days to check the number of eggs in ovaries. The result showed that T. radiata formed more eggs feeding on mixed diets (Nu-Lure+ honey+nymphs or Nu-Lure+ nymphs) compared to nymphs alone. Thus, it is recommended that protein and carbohydrate supplements along with host nymphs be provided to T. radiata before release in the field.
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In the series University of Florida Digital Collections.
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Includes vita.
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Includes bibliographical references.
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Description based on online resource; title from PDF title page.
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This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility:
by Xulin Chen.
Thesis:
Thesis (M.S.)--University of Florida, 2013.
Local:
Adviser: Stansly, Philip Anzolut.
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RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2013-11-30

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UFE0045482:00001


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1 MANIPULATION, REARING AND STORAGE OF TAMARIXIA RADIATA (HYMENOPTERA: EULOPH IDAE) PARASITOID OF DIAPHORINA CITRI (HEMIPTERA: PSYLLIDA E) By XULIN CHEN A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2013

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2 2013 Xulin Chen

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3 To my parents

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4 ACKNOWLEDGMENTS Funding for this research was provided by Dr. Phi l Stansly from Citrus Research and Development Foundation. I thank my major professor Dr. Phil Stansly for his patient guidance, support and pertinent suggestions. I also thank my other two committee members, Dr. Howard Frank and Dr. Eric Rohrig, for their continuous support and advice. I thank Dr. Jawwad Qureshi for providing suggestions in my research

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ ............ 8 LIST OF FIGURES ................................ ................................ ................................ .......... 9 ABSTRACT ................................ ................................ ................................ ................... 10 CHAPTER 1 LITERATURE REVIEW ................................ ................................ .......................... 12 Huanglongbing ................................ ................................ ................................ ........ 12 Distribution ................................ ................................ ................................ ....... 12 Symptoms ................................ ................................ ................................ ........ 12 Pathogen ................................ ................................ ................................ .......... 13 Diaphorina Citri ................................ ................................ ................................ ....... 13 Distribution ................................ ................................ ................................ ....... 14 Transmission Mechanism between Pathogen and Vector ................................ 14 Host Plants ................................ ................................ ................................ ....... 15 Biology of D. Citri ................................ ................................ .............................. 16 Life cycle ................................ ................................ ................................ .... 16 Seasonal history ................................ ................................ ........................ 18 Dispersion of D. citri ................................ ................................ ................... 19 Damage by D.citri ................................ ................................ ...................... 19 Influence of temperature and humidity ................................ ....................... 20 Influence of light ................................ ................................ ......................... 21 Natural Enemies ................................ ................................ ............................... 21 Predators ................................ ................................ ................................ ... 21 Parasitoids ................................ ................................ ................................ 22 Tamarixia Radiata ................................ ................................ ................................ ... 22 Distribution ................................ ................................ ................................ ....... 22 Taxonomy and Identification ................................ ................................ ............. 23 Host Specificity ................................ ................................ ................................ 23 Biology of T. Radiata ................................ ................................ ........................ 24 Life cycle ................................ ................................ ................................ .... 24 Mating ................................ ................................ ................................ ........ 25 Oviposition ................................ ................................ ................................ 25 Host preference ................................ ................................ ......................... 26 Host feeding ................................ ................................ ............................... 26 Sex ratio ................................ ................................ ................................ ..... 27 Reproduction ................................ ................................ .............................. 27 Supe rparasitism ................................ ................................ ......................... 28

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6 Host density effects ................................ ................................ .................... 28 Influence of food ................................ ................................ ........................ 29 Deve lopment time ................................ ................................ ...................... 29 Temperature and humidity effects ................................ .............................. 30 2 CARBON DIOXIDE ANESTHESIA OF TAMARIXIA RADIATA (WATERSTON) (HYMENOPTERA: E ULOPHIDAE) PARASITOID OF DIAPHORINA CITRI (HEMIPTERA: PSYLLIDAE) ................................ ................................ ................... 33 Introduction ................................ ................................ ................................ ............. 33 Materials and Methods ................................ ................................ ............................ 34 Colonies ................................ ................................ ................................ ........... 34 Gas Chamber ................................ ................................ ................................ ... 34 Recovery Time ................................ ................................ ................................ 35 Survival Rate ................................ ................................ ................................ .... 35 Percent Parasitism ................................ ................................ ........................... 36 Results ................................ ................................ ................................ .................... 36 Discussion ................................ ................................ ................................ .............. 37 3 FUNCTIONAL RESPONSE OF TAMARIXIA RADIATA (HYMENOPTERA: EULOPHIDAE) TO DENSITIES OF ITS HOST, DIAPHORINA CITRI (HEMIPTERA: PSYLLIDAE) ................................ ................................ ................... 40 Introduction ................................ ................................ ................................ ............. 40 Materials and Methods ................................ ................................ ............................ 42 Colonies ................................ ................................ ................................ ........... 42 Arenas ................................ ................................ ................................ .............. 43 Statistical Analysis ................................ ................................ ................................ .. 43 Results ................................ ................................ ................................ .................... 44 Fecundity, Percent Parasitism and Percent Superparasitism ........................... 44 Functional Response ................................ ................................ ........................ 44 Discussion ................................ ................................ ................................ .............. 45 4 THE INFLUENCE OF DIET ON EGG FORMATION IN TAMARIXIA RADIATA (HYMENOPTERA: EULOPHIDAE), A PARASITOID OF DIAPHORINA CITRI (HEMIPTERA: PSYLLIDAE) ................................ ................................ ................... 50 Intro duction ................................ ................................ ................................ ............. 50 Materials and Methods ................................ ................................ ............................ 51 Colonies ................................ ................................ ................................ ........... 51 Diets ................................ ................................ ................................ ................. 52 Egg Load ................................ ................................ ................................ .......... 53 Statistical Analysis ................................ ................................ ................................ .. 53 Results ................................ ................................ ................................ .................... 54 Discussion ................................ ................................ ................................ .............. 54 5 DISCUSSION AND CONCLUSIONS ................................ ................................ ...... 61

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7 LIST OF REFERENCES ................................ ................................ ............................... 64 BIOGRAPHICAL SKETCH ................................ ................................ ............................ 73

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8 LIST OF TABLES Table page 1 1 Demographic parameters T (generation time) per day, R 0 (net reproductive rate) nymphs per female, and r (intrinsic rate of increase) per day. .................... 32 3 1 Number of parasitized hosts (Mean SEM) at different host densities .............. 47 3 2 Percent parasitism (Mean SEM) at different host densities ............................. 47 3 3 Percent superparasitism (Mean SEM) in different host densities, mean number ................................ ................................ ................................ ............... 47 4 1 Mean SEM number of eggs by treatment from dissections after 5, 10, 15, and 20 days at 17 C ................................ ................................ .......................... 56

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9 LIST OF FIGURES Figure page 2 1 Carbon dioxide gas chamber ................................ ................................ .............. 38 2 2 Tamarixia radiata female and male recovery time frequency (%) distribution .... 38 2 3 Mean (SEM) survivorship of CO 2 treated and untreated Tamarixia radiata adults. ................................ ................................ ................................ ................. 39 3 1 Number of parasitized host at different host densities, error ba r stands for SEM ................................ ................................ ................................ .................... 48 3 2 Percent parasitism at different host densities, error bar stands for SEM ............ 48 3 3 Percent superparasitism in different host densities ................................ ............ 49 3 4 Number of D. citri parasitized by T. radiata and the functional response curve (Type II) ................................ ................................ ................................ .............. 49 4 1 Newly emerged (unfed) T. radiata female digestive system ............................... 57 4 2 Newly emerged (unfed) T. radiata female ................................ .......................... 57 4 3 Paired T. radi ata ovaries after feeding on honey for 20 days (egg resorption) ... 58 4 4 T. radiata ovary after feeding on Nu Lure+honey for 10 days ............................ 58 4 5 Paired T. radiata ovaries after feeding on nymphs for 5 days ............................ 59 4 6 Paired T. radiata ovaries after feeding on honey +nymphs for 10 days .............. 59 4 7 Paired T. radiata ovaries after feeding on Nu Lure+ nymphs for 10 days ........... 60 4 8 Paired T. radiata ovaries after feeding on Nu Lure+honey+nymphs for 5 days .. 60

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10 Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Master of Science MANIPULATION, REA RING AND STORAGE OF TAMARIXIA RADIATA (HYME NOPTERA: EULOPHIDAE) PARASITOID OF DIAPHORINA CITRI (HEMIPTERA: PSYLLIDA E) By Xulin Chen May 2013 Chair: Phil. Stansly Major: Entomology and Nematology Tamarixia radiata (Waterston) (Hymenoptera: Eulophidae), is an arrhenotokous ectoparasite of the Asi an citrus psyllid (ACP) Diaphorina citri (Kuwayama) (Hemiptera: Psyllidae), vector of citrus greening disease or huanglongbing (HLB). Tamarixia radiata is being tested as an augmentive biological control agent, since the number present in the field is low following winter when few hosts are available. Any program aimed at augmentation of a natural enemy would require an efficient system of mass production. Key components would include methods of manipulating, rearing and storing large populations of insects This research investigated the use of carbon dioxide (CO 2 ) to anesthetize T. radiata determined optimal host densities for rearing the parasitoid, and evaluated diets to maintain fecundity during storage periods. Tamarixia radiata adults held in an atm osphere of 100% CO 2 for 5 min were immobilized for about 4 min. However, survivorship and fecundity were reduced significantly, although sex ratio of progeny from treated adults was not affected. Consequently, lighter doses of CO 2 or other methods of anest hesia are needed.

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11 One pair of three day old T. radiata was released in a centrifuge tube for five days with different 4 th instar D. citri nymphs densities: 10, 20, 30, 40, 50, and 60. At a density 40, 4 th instar nymphs per female fecundity, incidence of pa rasitism and superparasitism were all optimal. The pattern of parasitism for the first five days conformed to a Type II functional response. Estimated searching efficiency was 0.442 0.036 per day and estimated handing time was 0.045 0.008 days. Theref ore, 40 hosts per female T. radiata should be a target for mass rearing. Females feeding on eight different diets were dissected every 5, 10, 15 and 20 days to check the number of eggs in ovaries. The r esult showed that T. radiata formed more eggs feeding on mixed diets (Nu Lure+ honey+ nymphs or Nu Lure+ nymphs) compared to nymphs alone. Thus, it is recommended that protein and carbohydrate supplements along with host nymphs be provided to T. radiata before release in the field.

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12 CHAPTER 1 LITERATURE REVI EW This chapter reviews (1) distribution, symptoms and pathogen of Huanglongbing, (2) biology, distribution of Diaphorina citri Kuyawama, (Hemiptera: Psyllidae), vector of Huanglongbing, the transmission mechanism between pathogen and vector, and natural e nemies of D. citri (3), biology of Tamarixia radiata (Waterston) (Hymenoptera: Eulophidae), a major biological control agent for D. citri. Huanglongbing Distribution Huanglongbing (HLB) is one of the most destructive diseases of citrus in the world (Halb ert and Manjunath 2004, Teixeira et al. 2005, Bov 2006, Wang et al. 2006, Batool et al. 2007, Manjunath et al. 2008). It has been reported from Asia: eastern Japan, southern China, Indian subcontinent and Pakistan, and also in the Arabian peninsula for so me time. An African form is found in eastern, central, and southern Africa (Gottwald 2007). HLB was first discovered in the western hemisphere in Brazil in July 2004 (Teixeira et al. 2005). The first discovery of HLB in North America occurred in south Flor ida in August 2005 (Halbert 2005). By December 2007, it had spread through 30 counties which were all located south of Marion County (Hall 2008a). Symptoms Huanglongbing means "yellow dragon disease" in Chinese because of the yel low shoots which are early symptoms of the disease. In addition to yellow shoots, typical symptoms also include blotchy (asymmetric) mottling, chlorosis resembling zinc deficiency which is followed by leaf drop and twig dieback (Halbert 2004). Symptomatic fruit are small, lopsided with small, dark aborted seed and

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13 discolored vascular bundles in the fruit axis. The stylar end of symptomatic fruit remains green even as they mature (Gottwald 2007). Roistacher (1996) reported that HLB damaged at least 10 15% of trees each year in Thailand, which could lead many citrus areas to go out of business. Infected citrus trees do not die immediately but decline within 8 9 years (Roistacher 1996). Pathogen The causative agent is a vector proteobacterium (Jagouei x et al.1994) which has not yet been isolated in pure culture (Garnier and Bov 1993). Three etiologic agents of HLB have been implicated based on their 16S rRNA sequence: Candidatus Liberibacter asiaticus (Asia, North America, and Brazil), Ca Liberibacte r americanus (Brazil), and Ca Liberibacter africanus (Africa) (Garnier et al. 1984, Jagoueix e t al. 1996, Sagaram et al. 2009 ). Of the three agents, only Candidatus Liberibacter asiaticus (Las) was discovered in Florida in August 2005 (Halbert 2005), whic h led to an estimated 1.6% of orange trees in Florida being infected by 2008 (Morris et al. 2009a,b). Candidatus Liberibacter asiaticus is transmitted by psyllid vectors, and can also be transmitted by grafting, dodder, and possibly by seed, but not by con tamination of personnel and tools or by wind and rain (Halbert 2010). Diaphorina C itri To date, two vectors have been reported for HLB disease in the world, Asian citrus psyllid, Diaphorina citri (Kuwayama) (Hemiptera: Sternorrhyncha: Psyllidae), and Afri can psyllid, Trioza erytreae (del Guercio) (Hemiptera: Sternorrhyncha: Triozidae). D. citri is responsible for transmitting Ca. L asiaticus in North America, Brazil and Asia, and Ca. L americanus in Brazil, while T. erytreae transmits Ca. L africanus in th e Middle East, Reunion and Africa (Halbert and Manjunath 2004). Of the two species, D. citri is

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14 more heat tolerant, while T. erytreae is sensitive to high temperatures ( Massonie et al. 1976). D. citri was first detected in Florida in 1998 (Halbert 1998) an d later become established throughout the state ( Halbert 2010) and in other southeastern states Distribution Diaphorina citri is thought to be indigenous to tropical and subtropical Asia and has been reported in the following geographical areas: China, India, Burma, Taiwan, Philippine Islands, Malaysia, Indonesia, Ceylon, Pakistan, Thailand, Nepal, Sikkim, Hong Kong, Ryukyu Islands, Afghanistan, Saudi Arabia, Reunion and Mauritius ( Mead 1977, Halbert and Manjunath 2004). D. citri was also known to occur in South America in Brazil (Lima 1942, Mead 1977). During the 1990s, D. citri invaded the West Indies (Guadeloupe), Abaco Island, Grand Bahama Island, and Cayman Islands (Halbert and Nez 2004). In June 1998, D. citri was detected on the east coast of Flo rida, from Broward to St. Lucie counties (Halbert 1998), by September 2000, this pest had spread to 31 counties in Florida (Halbert et al. 2001). During 2001, it was found in the Dominican Republic, Cuba (Halbert and Nez 2004), Puerto Rico (Pluke et al. 2008) and Texas (French et al. 2001). Transmission Mechanism between Pathogen a nd Vector Information about interactions between D. citri and the pathogen Las is quite limited. Nymphal stages of D. citri have the ability to acquire HLB pathogen during the later instar development when growing on the pathogen infected trees. Adults emerging from these infected nymphs can transmit the pathogen immediately after emergence (Capoor et al. 1974, Xu et al. 1988). Uninfected adults feeding on infected trees can als o acquire the pathogen (Xu et al. 1988). However, compared with the adults, later instar nymph stages have a higher efficiency in acquiring the pathogen in nature (Xu et

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15 al. 1988). In more recent studies, nymphs reared on Las infected trees were also more likely to acquire the pathogen than adults (Pelz Stelinski et al. 2010). Acquisition access period (AAP), latent period, and inoculation access period (IAP) all varied a lot in the past studies. An AAP of 15min to 24h was reported for both D. citri and T. erytreae by Capoor et al. (1974), and Buitendag and von Broembsen (1993), whereas Xu et al. (1988) reported an AAP of between 30min to 5 h for D. citri The transmission mechanism between D. citri and the pathogen Las is persistent and presumably propagat ive transmission. The pathogen apparently multiplies in the vector, and the adult D. citri generally remain infective during their whole lifespan (Xu et al. 1988, Hung et al. 2004); but Pelz Stelinski et al. (2010) found that the ratio of Las infected adul ts declined over time when reared on healthy trees, even though the pathogen appears to multiply in the vector body. Following acquisition, adults of T. erytreae may require 21 days of latent period before they can transmit the pathogen (Moll and Van Vuur en 1977). Latent period for D. citri was also reported to be 8 to 12 days by Cappor et al. (1974). IAP was reported from 15min to 7h for both psyllid species (Capoor et al. 1974, Buitendag and von Broembsen 1993), but a study with D. citri concluded that t he disease could be transmitted after adults had fed on healthy trees for 5hr to 7hr (Xu et al. 1988). Transovarial transmission was not reported to occur between D. citri parents and offspring (Xu et al. 1988, Capoor et al. 1974, and Hung et al. 2004). Ho wever, Pelz Stelinski et al. (2010) reported that transovarial transmission does occur at a rate from 2% to 6%. Host Plants Diaphorina citri has a restricted range of host plants including citrus, orange jasmine ( Murraya paniculata) orange boxwood ( Seve rinia buxifolia ), and some other

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16 related species of Rutaceae (Halbert and Manjanath 2004). Murraya paniculata was reported to be more tolerant than citrus of direct feeding damage from D. citri (Skelley and Hoy 2004). Murraya paniculata has been widely use d to rear D. citri and it is also an ornamental plant and a host for both psyllids and bacteria. However, Walter et al. ( 2012) suggested that, M. paniculata is serving as a minor source of Candidatus Liberibacter asiaticus inoculum, because they found less than 1% of psyllids and 1.8% of plants were positive, using sensitive quantitative polymerase chain reaction (qPCR) tar geting at two prophage genes of Candidatus Liberibacter asiaticus. The development, longevity and fecundity of D. citri varied on different host plants (Fung and Chen 2006, Nava et al. 2007). Tsai and Liu (2000) reported that D. citri has a higher rate of development on grapefruit compared with rough lemon, sour orange and orange lime and M. paniculata (Nava et al. 2007). Fecundity was higher on M. paniculata Diaphorina ci tri developed better on Rangpur lime and orange jasmine compared to Sunki mandarin (Nava et al. 2007). Biology o f D C itri Life cycle The life cycle of D. citri is incomplete metamorphosis, so there are only three life stages: egg, nymph and adult. The eg g has been described as oval or almond shaped, elongated, slightly curved with ends in a blunt point (Husain and Nath 1927). A slender stalk like process appears from its rounded base which links the egg with the plant tissue (Husain and Nath 1927, Tsai an d Liu 2000). Egg length was reported to be 0.3 mm on average without the stalk, or about 0.038 mm with the stalk. The great est diameter was around

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17 0.13 mm (Husain and Nath 1927). The color of the eggs is light yellow when newly deposited, gradually turning bright orange with two distinct red spots, the eyes of the nymph, just before hatching (Husain and Nath 1927, Hall 2008a). D. citri development includes five nymphal instar stages. The general color of the nymph is light yellow or dull orange (Husain and Nath 1927, Hall 2008a). As nymphs mature, the abdominal color of some turn green while some others turn pale orange (Tsai and Liu 2000). The first nymphal instar is 0.3mm long and 0.17 mm wide with two visible red eyes (Tsai and Liu 2000). Wing pads are n ot yet visible. Some setae arise from the surface of the dorsum and two golf club shaped tarsal setae arise from of the middle and hind legs with a single seta on each of the fore legs (Husain and Nath 1927). All three features differentiate the first ins tar from the other stages (Husain and Nath 1927). The second instar is 0.45mm long and 0.25 wide (Tsai and Liu 2000), with small triangular wing pads distinct on the dorsum of the thorax (Tsai and Liu 2000, Husain and Nath 1927). The third instar nymphs ar e 0.74mm long and 0.43 mm wide on average, the wing pads are well developed and the antennal segmentation is visible (Tsai and Liu 2000). There is a single lanceolate seta on each antenna (Husain and Nath 1927). The fourth instar nymphs have two setae on e ach of the antennae (Husain and Nath 1927). The fifth instar nymphs averaged 1.6mm long and 1.02mm wide (Tsai and Liu 2000), with a distinctly marked thorax and well developed wing pads (Husain and Nath 1927). There is only one seta on each tarsus on the l eg, and three setae on each antenna (Husain and Nath 1927). Paiva and Parra ( 2012) found that, the egg to adult survivorship was really low with only 7 adults emerging from 406 eggs under field

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18 conditions Of all the generations, egg to first nymphal instar show ed the lowest survival of 38.8% in field D.citri adults are 2.7 to 3.3 mm in length, with mottled brown wings (Hall 2008a). When feeding or resting, they always align their heads against the surface of the lea ves, such that the axis of the body is at 45angle with the leaf surface (Hall 2008a). Adults can copulate soon after emergence, and eggs are laid if young shoots are available (Husain and Nath 1927). Both oviposition and development of immature D. citri a re confined to youn g, new growth (Hall and Albrigo 2007), and all the activities are restricted to the daylight (Wenninger and Hall 2007). Sex ratio of adult D. citri was reported around 1:1 by Aubert and Quilici (1988). New adults become physically mature in 2 to 3 days, and female adults begin to lay eggs 1 or 2 days after mating (Wenninger and Hall 2007). Females can lay eggs continuously throughout their lifetime if new and young shoots are available (Hall 2008a). Adult females live approximately 40 to 48 days, and lay 500 to 800 eggs on average during lifetime, and 1900 at most (Husain and Nath 1927, Nava et al. 2007, Tsai and Liu 2000). Adults usually lay eggs in the f olds of partially opened leaves, push them between the bud and stem or petioles of le aves and axillary buds, and other similar situations (Husain and Nath 1927). Eggs are attached to plant tissue by means of their stalks, which can protect them from bad climatic conditions, such as washing off by heavy rain (Husain and Nath 1927). Seasonal history All three stages of D. citri are found on citrus plants throughout the year, and no hibernation of a specific stage has been discovered (Husain and Nath 1927). Adults have been observed throughout winter, but usually lay no eggs until spring beca use of the lack of new growth. Large numbers of eggs and nymphs were found during March

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19 and April, but in the northern Punjab, the most frequent stage found in the dry May and June period is the adult stage (Husain and Nath 1927). If rains start in July, t here will be a second peak of the nymphal population coinciding with the burst of new growth. Population s begin to decline in the middl e of October, and always remain low until January the next year (Husain and Nath 1927). Dispersion of D. c itri Adults a re very active; they can move a short distance through jumping and flying (Husain and Nath 1927, Hall 2008a). Aubert and Hua (1990) reported that adults fly to disperse all day long, but are most apt to move during warm, sunny afternoons between 4 to 6 pm. Hall (2008a) speculated that flying psyllids could be carried by wind over a 0.5 to 1.0 km distance according to wind speed and the duration of flight. Adults congregate on young and fresh leaves, and move quickly when searching for a place to oviposit (H usain and Nath 1927). Husain and Nath (1927) stated that adult D. citri are positively phototropic and negatively geotropic (Husain and Nath 1927). Nymphs concentrate on the young leaves close to the emergence site. Nymphs do not move much, but will crawl down the stem to larger leaves when the population density is high (Husain and Nath 1927). Damage by D.citri Besides being the vector of the HLB pathogen, D. citri can also cause damage through feeding on plants. D. citri is a sucking insect that inserts its mouthparts into the plant tissue to suck the sap (Husain and Nath 1927, Hall 2008). High adult populations cause defoliation of shoots attacked and dieback of branches (Husain and Nath 1927). Nymphs suck the cell sap and exclude thick sugary honeydew f rom the anus encased in a waxy secretion secreted by the circumanal glands (Husain and Nath 1927). Nymphs

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20 move their abdomen to dislodge the wax covered honeydew, and some fungi will also grow on it, which makes the leaves look to be covered by a black soo ty deposit (Husain and Nath 1927). Influence of temperature and humidity Tsai and Liu (2000) recorded that eggs hatch in 4 days at 25 C At this temperature, the nymphal stage lasts over 13 d leading to a total 17 days from egg to adult. The life cycle varies significantly with different temperatures, 24 to 28 C being the optimal temperature range. Diaphorina citri totally fails to develop at temperatures above 33 C or below 10 C (Liu and Tsai 2000). Nava et al. (2007) reported that, with temperature varying from 18 to 32 the duration of the egg an d nymphal stages is 2.6 to 7.7 d and 9.4 to 35.8 d, respectively. Lower temperature development threshold (TT) and thermal constant (K) for egg, nymph and whole life cycle were 12.0 C 52.6 Degree Day (DD ), 13.9 C 156.9 DD and 13.5 C 210.9 DD respectively (Nava et al. 2007). Longevity differs between females and males even at the same temperature ; adult males live 21 to 25 d while females live 31 to 32 d on average (Nava et al. 2007). Different temp eratures also influence adult longevity which ranges from 117 days at 15 C to 51 days at 30 C (Liu and Tsai 2000). Skelley and Hoy (2004) showed that D. citri stopped laying eggs at 34 or above. However, fecundity returned if temperature was later reduced. Fecundity is reduced at RH below 40% (Hall 2008a). The survival of D. citri may increase with increasing relat ive humidity (McFarland and Hoy 2001). Yang (1989) reported that nympha l mortality is low from RH 43% to 75% but increases at RH 85% to 92%. The combined influence of temperature and humidity can reinforce the effect. Nymphal mortality was high at high temperature (34

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21 ) and a high humidity (82 92%) and low at moderate temper ature (20 30 ) and a low humidity level (43 75%) (Yang 1989). However, in general, temperature plays a more important role in influencing nymphal survival than humidity (Yang 1989). Influence of light Yang (1989) found that the number of eggs laid per female increased with increasing light intensity and duration under constant conditions below 11000lux and l ight duration less than 18 h per day. Both light duration and light intensity influenced preoviposition period, fecundity, and mortality of females. However, light duration ha d a slightly greater effect (Yang 1989). Natural Enemies Predators Diaphorina citri is commonly attacked by ladybeetles (Coleoptera: Coccinellidae), syrphid flies (Diptera: Syrphidae), lacewings (Neuroptera: Chrysopidae, Hemero biidae), and spiders (Araneae) (Michaud 2002, Michaud 2004, Gonzalez et al.2003, Qureshi and Stansly 2009). Adults are not very vulnerable to these natural enemies (Husain and Nath 1927). Coccinellid predators, such as Harmonia axyridis Pallas and Olla V n igrum Mulsant have been considered the most important predators in Florida (Michaud 2002, 2004). Exochomus childreni Mulsant, Cycloneda sanguinea L. (Michaud 2004), and Curinus coeruleus Mulsant (Michaud and Olsen 2004) have also been observed to attack D. citri in Florida. Two lacewings, Ceraeochrysa sp. and Chrysoperla rufilabris Burmeister, were reported to contribute to psyllid mortality in Florida (Michaud 2004). The spider Hibana velox (Becker) was reported to be of great importance in Florida (Michaud 2004). Ants can just prey on immature D. citri in Florida (Michaud 2002).

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22 (J uan Blasco et al. 2012) found that Amblyseius swirskii (Acari: Phytoseiidae) showed significant predation on D. citri eggs, and also suck body fluids of first instar nymphs. Parasitoids Tamarixia radiata (Waterston) (Hymenoptera: Eulophidae) and Diaphore ncyrtus aligarhensis (Shafee, Alam and Agarwal) (Hymenoptera: Encyrtidae) are two well know parasitoids of D. citri (Hall 2008b Grafton Cardwell et al.2013 ). The ectoparasitoid T. radiata and endoparasitoid D. aligarhensis were recorded as primary parasit es (Tang 1990) Marietta leopardina was found attacking immature T. radiata and D. aligarhensis (Hoddle 2012) D. aligarhensis was released in Florida but has not yet been reported to have established (Rohrig et al. 201 1) Tamarixia R adiata Distribution Tamarixia radiata the arrhenotokous ectoparasite of D. citri, was first recorded In Pakistan (Waterston 1922) In 1978, T. radiata was introduced to Reunion Island from the Punjab of India and was credited with achieving control of D. citri (Etienne and Aubert 1980). It was also reported to have b een successfully introduced in Mauritius in 1984 (Aubert 1987). It is well known to occur in Jiangxi and Fujian provinces in China (Tang 1998), but was not released there on purpose (Yang et al. 2006). It was introduced to Taiwan in 1983 for control of D. citri and is very effective there (Chien and Chu 1996) D istribution s of T. radiata in Asia include India (Husain and Nath 1927), Japan (Kohon et al. 2002), Nepal (Lama et al. 1988), Thailand (Markote and Nanta 1995), and Vietnam (Hoy et al. 1999). The pa rasitoid was imported from Taiwan and Vietnam and released in Florida in 1999 2001 (Hoy and Nguyen 2001). Tamarixia radiata has been detected in Texas (French et al. 2001), Brazil (Gomez et al. 2006),

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23 and Puerto Rico (Pluke at al.2008) although no releases were ever recorded for those locations. Tamarixia radiata is established in Florida now, although the parasitism rate is relatively low except for late in the growing season (Tsai at al. 2002, Qureshi et al. 2009). Taxonomy a nd Identification Tamarixia radiata was originally described as Tetrastichus radiatus by Waterston (1922) from specimens collected at Lyallpur, India (now Faisalabad Pakistan). The genus Tamarixia was split off as a separate genus from Tetrastichus by Mercet (1924). All are parasit oids of Psylloidea (LaSalle 1994). Tang and Aubert (1990) described some distinguishing characteristics of T. radiata : about 1 mm long including head; eyes red in fresh specimens; head and body blackish and shining but without a metallic sheen; underside o f gaster pale with a large whitish basal patch on the 5 th tergite of dorsum; legs totally pale white and wings transparent. Male and female are the same in color and body structure, except for antennae. The female antenna has eight segments, both funicle a nd club with three segments covered with fine, short setae. The funicle is slender with the 1 st segment longer than the 2 nd and the 2 nd segment longer than the 3 rd The length of the 3 rd segment is almost equal to the width. The male antenna is more slende r, and nine segmented. The four segmented funicle is covered with long, slightly curved hairs, and the ventral scapal sensorium is near to the base of the scape (Tang and Aubert 1990). Host Specificity T. radiata is not known to attack any psyllid other t han D. citri (Aubert and Quilici 1984). (Mann et al. 2010) reported that T. radiata can be attracted by volatiles emanating from D. citri nymphs.

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24 Biology o f T. R adiata Life cycle Structure of the 4 life stages of T radiata was described by Chen et al. (1991a). The egg is translucent, ivory, and reniform, with one end adhesive to the host. There are four larval instars, each distinguished by head length (Chen et al. 1991a). Development of the immature stages was rep orted by Xu and Tang (1993) and Chien et al. (1991a) The newly eclosed larva sucks fluids externally from the site where it is 1991, Hall 2008b). The third instar crawls to t he ventral side of the host thorax to feed ( Chien et al. 1991a). The parasitized nymph continues to live and secrete honeydew for some time (Husain and Nath 1923). By the time the parasitoid molts to the fourth instar, all contents of the nymph have been c onsumed and the nymph turns to a dark brown mummy (Husain and Nath 1923, Chien et al. 1991a ). The mature larva ceases to feed as it progresses to the prepupal stage which secures the mummy to the plant surface by means of silken threads ( Chien et al. 1991 a ). After expelling the meconium, it molts to the pupal stage which turns yellow, with red ommatidia and ocelli (Chen et al. 1991a). Emergence As soon as the adult hardens, it makes its way out of the mummy by chewing a round hole of approximately 0.5 mm diameter in the region of thorax (Husain and Nath 1923, Chien et al. 1991a, Aubert 1987 ). Over 80% of adults emerge between 5 a.m. to 10 a.m., with the peak between 7 a.m. to 8 a.m. Male emergence occurs 1.5 hours earlier than the female on average ( Chien et al. 1991a ).

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25 Mating Males use their antennae to locate receptive females. Once found, the male crawls onto the dorsum of the female to make contact for an average of 687 seconds before mating which lasts an average 333 seconds ( Chien et al. 1991a ). Ab out 93% of females mate once and only once during the first day of emergence. The remainder mate twice during the first two days following emergence. Fecundity and longevity of females were not affected by mating frequency. Males are capable of multiple ma tings over a lifetime ( Chien et al. 1991a ). Oviposition Eggs can be laid immediately after emergence by mated or unmated females ( Chien et al. 1991a) From 5 am to 10 am is the most active time of day for ovi position (Chu and Chien 1991). Host volatiles mediate host location (Mann et al. 2010). The female moves actively among D. citri nymphs using her antennae to search for a suitable host (Husain and Nath 1923). After an acceptable host is found, she deposits an egg or occasionally two on the underside of the nymph, usually next to the mid or posterior coxae (Husain and Nath 1923, Aubert 1987, Chien et al. 1991a Hall 2008b, Tang and Huang 1991). Oviposition took 3 to 4 min according to Husain and N ath (1923), but only 618 s according to Chien et al. (1 991a) Chien et al. (1991a) also reported that the female T. radiata injects venom into the host nymph through the ovipositor, im mobilizing it for 4 to 8 min If the egg was removed, the host nymph could not molt, and died 8 days later at a temperature of 25 C A first, second, or third stage parasitoid larva placed on an unparasitized 5 th instar nymph could not attach, and dropped off when the nymph began crawling ( Chien et al. 1991a)

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26 Host preference Studies on host selection of T. radiata females have given varying results. Chien et al. (1991a) and Chu and Chien (1991) reported that 5th instar nymphs w ere preferred for oviposition. Survival rate was 85%, compared with 33% and 71% from 3 rd and 4 th instars, resp ectively (Chien et al. 1991a). Body length was also greater among offspring from 5 th instar hosts compared to 4 th instar hosts: 1.12 mm compared with 0.91 mm (female), 1.03 mm compared with 0.86 mm (male). The pattern was repeated with fecundity: 215 compared to 120 eggs per female, and longevity: 18.0 d compared t o 14.4 d (females), or 11.6 d compared to 7.2 d (males) (Chien et al. 1991). However, 4 th instar nymphs are parasitized significantly more than 3 rd or 5 th instars according to Tang and Huang (1991). Host feeding Both sexes feed on honey dew from D. citri nymphs, and females can suck hemolymph after puncturing the nymph with the ovipositor (Chien et al.1991a, Skelley and Hoy 2004). The T. radiata population oviposited and host fed almost simultaneously (Chu and Chien 1991). Host feeding oc curs in daytime and takes an average of 212 s (Chien et al. 1991a). The host will die once fed upon and females avoid laying eggs and feeding on the same host (Chien et al. 1991a, Tang and Huang 1991). Ratio of host feeding to oviposition correlates with host density and paras itoid age (Chien et al. 1991a). One egg was laid for an average 0.18 hosts fed upon by fema les between 4 to 18 d old. Younger or older females laid one egg for an average 0.29 0.38 hosts fed upon respectively (Chien et al. 1991a). An average of 80% mortality resulted from parasitism with an additional 20% from host feedin g (Chien et al. 1991a, 1994b). In this way, a single female T. radiata could kill up to 500 nymphs during her lifetime (Chu and

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27 Chien 1991). However, Chien et al. (199 3) estimated host killing capacity at 16, 25, 245, 196 nymphs per female at 15 C 20 C 25 C and 30 C respectively. Sex ratio T. radiata is arrhenotokous, meaning virgin females deposit eggs which produce males, while eggs deposited by mated females c an develop into either sex. The average number of eggs deposited by virgin and mated females in one study was 209.2 and 215.4 respectively (Chu and Chien 1991). Female progeny ratio is highly correlated with parasitoid age (Tang and Huang 1991, Chu and Chi en 1991). The proportion of female progeny increased as the mother aged, from 0.5 from a 1 d old female to 0.77 from a 22 d old female (Chu and Chien 1991). Sex ratio is also correlated with host stage, although published results differ. Tang and Huang (19 91) reported female ratios of 0.88 from 5 th instar hosts, 0.75 from 4 th instars and 0.41 from 3 rd instars. However, Chu and Chien (1991) found female ratios of 0.67 from 5 th instar nymphs, compared with 0.16 from 4th instars. Skelley and Hoy (2004) reporte d female ratios of 0.64 and 0.67 with 4 th instar nymphs for their Taiwanese and Vietnamese colonies of T. radiata Reproduction Tamarixia radiata can be characterized as synovigenic autogenous, meaning that some eggs become mature in the newly emerged was p without feeding, but that host feeding is later required to mature more eg gs. Most synovigenic parasitoids, including T. radiata can resorb eggs when hosts are absent or scarce (Chien et al. 1994a), thereby maintaining reproductive resources and synchr ony with the host po pulation (Jervis et al. 2001). Egg resorption is thus a mechanism that aids adaption to host biology and ecology. It can occur in T. radiata at either 15 C or 25 C when only honey is provided and is positively related to host deprivat ion time (Chien et al. 1991a, 1994a).

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28 Once suitable hosts are fed upon, new mature eggs can be produced in the ovary and oviposition can proceed normally (Chien et al. 1994a). Little or no effect on total fecundity was observed after host deprivation for 1 0 days at 25 C with honey provided as food (204 eggs), but there was a difference af ter host deprivation for 20 d (1 56 eggs) (Chien et al. 1994a). Wasps thus stored for 10 to 20 d wasps at 25 C laid significantly more eggs (15 6 eggs) than wasps stored a t 15 C (98 eggs). Fecundity decreased greatly following ho st deprivation for 30 to 40 d at 15 C (25~59 eggs). Superparasitism Female T. radiata can discriminate between parasitized and unparasitized hosts to avoid superparasitism (Chien et al. 1991a). H usain and Nath (1923) observed superparasitism during December and January when hosts were scarce, but not when hosts were abundant. Chien et al. (1991a, 1991b) observed superparasitism rates of up to 5.6% when the host density was low and active space was limited. Host density effects Longevity, fecundity, sex ratio, and ratio of host feeding to oviposition all correlate with host density (Chu and Chien 1991, Chien et al. 1991a 1995). The relationship between host density and wasp longevity (both males and females) has been described as following a domed parabolic response, meaning that female longevity and fecundity ascend with host density to a peak and then decrease as host density increases (Chien et al. 1995). Average female longevity increased fro m 15.9 to 18.6 to 20.3 d when host densities of 10, 20 and 30 were provided daily. However, female longevity decreased from 23.6 to 17.2 to 11.2 over a range of 40, 60, 80 hosts per day, respectively (Chien et al. 1995). Chu and Chien (1991) reported that females lived an average 23.6 days and males 14.8 d when 40, 5 th instars were provided at 25

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29 C 14:10 (L: D) photoperiod and 100% RH. Both daily and lifetime fecundity showed similar parabolic responses to host density, with the peak at 40 hosts per day ( Chien et al. 1995). Influence of food Adults deprived of food or water survived 1.0 to 1.7 d (Chien et al. 1994b). These authors found no difference in longevity among female adults deprived of hosts and fed either honey alone (22.5 d), honey and pollen (2 3.0 d), or honey and yeast extract (23.4 d ). However, all these food supplements increased fecundity and progeny survivorship compared with adul ts held without food or water. Adults fed on a diet of honey and yeast extract significantly decreased host feed ing while maintaining or improving intrinsic rate of increase (0.2976 to 0.3014 per day) and the net reproductive rate (140 to 187 female eggs per female), respectively (Chien et al. 1994b). Development time Chien et al. (1991a) found the duration of on e generation for T. radiata on orange jasmine (egg to adult e mergence) to be around 11.4 d at 25 C 14:10 (L: D) photoperiod, and 100% RH. This would include 45 h for the egg, 24, 24, 22, 26 h for the 1 st through 4 th instars, respectively, 14.4 h for the prepupal, and a 117.6 h pupal stage. Xu and Tang (1993) reported a 12.6 d generation time at 25 1 14:10 (L:D) photoperiod, and 75% to 85% RH: 40 h for the egg, 119 h = 25, 28, 32 and 34 h for 1 st to 4 th larval instar respectively, 24 h for the prepupa and 120 h for the pupa. These results could indicate that humidity may play an important role in development rate especially in the prepupal and pupal stages

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30 Temperature and humidity effects Tamarixia radiata completes development at 15 C to 32 C with an optimum temperature of 25 (Chien et al. 1993). Gomez Torres et al. (2012) found parasitism rates to be highest at 25 and 30 C (85.5 and 72.8%, respectively) compared to 23.1 and 40.2 % at 15 and 35 C respectively. They also found emergence rates to be highest, 86.7 and 88.3% a t 25 and 30 C respectively, compared to about 50% in the 15 to 20 C range. At 70 10% RH, and L: D= 14:10 they estimated maximum parasitism rates of 77.2% at 26.3 whereas emergence was greatest (89.9%) at 30.8 Pre imaginal development was longer f or females, varying from 489.6 h at 15 C to 247.2 h at 35 C compared to males at 343.2 to 146.4 h over this same range. Longevity with access to pure honey negatively correlated with temperature between 8 C to 30 C (Quilici and Fauvergue 1990). These authors found that adult longevity decreased from 34 d at 20 C to 22 d at 22 C 10 d at 30 C and 8 d at 35 C Chien et al. (1993) found longevity to increase from 45.5 to 59.5 between 8 and 15 C but then decrease to 22.5 and 9.6 days at 25 and 30 C r espectively. Ten percent of T. radiata adults survived for 50 d when stored at 25 C with access to honey and yeast extract (Skelley and Hoy 2004). McFarland and Hoy (2001) reported that T. radiata adults from Vietnam survived longer without food and wate r compared to wasps from Taiwan over a range of RH from 7% to 9 7% at 25 C and especially 30 C. They attributed this difference to greater moisture requirements of wasps from Taiwan. Chien et al. (1993) estimated host killing capacity at 16, 25, 245, 196 nymphs per female at 15 C 20 C 25 C and 30 C Estimates of intrinsic rate of increase (r), net

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31 reproductive rate (R 0 ), and mean generation time (T) for pairs of T. radiata provided 20 or 30, 5 th instar nymphal hosts by Chien et al. (1993) and Gomez Torres (2012) respectively varied considerably, especially at low temperatures (Table 1 1 ). Different results may have been due to different condit ions during these two studies. Chien et al. (1993) did theirs at host density 20 and 100% RH for five replica tions whereas Gomez Torres et al. (2012) conducted their study at density 30, 70 10% RH for ten replications, which may have le d to the different results. Differences could also be inherent in the races of T. radiata tested from Taiwan and Brazil respect ively. Skelley and Hoy (2004) showed that T. radiata stored for up to 35 d at 17 C with honey and yeast s uffered less than 5% mortality. Chien et al. (1994a) reported that females stored for 20 d at 25 C were able to lay a total of 156 eggs, compared wit h 98 eggs when stored at 15 C W e may conclude that less mortality was experienced at low temperature but that production suffered Therefore, ideal storage temperature should be determined according to specific objectives (establishment or augmentation) and conditions (host availability).

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32 Table 1 1 Demographic parameters T (generation time) per day, R 0 (net reproductive rate) nymphs per female, and r (intrinsic rate of increase) per day as estimated by Chien et al. (1993) and Gomez Torres et al. (2012). Parameter C Chien et al. (1993) Gomez Torres et al. (2012) T 15 39.9 20.3 T 20 22.8 18.8 T 25 16.1 15.5 T 30 12.3 11.8 T 35 NA 10.4 R 15 2 9.9 R 20 6 23.6 R 25 140 126.8 R 30 90 58.6 R 35 NA 21.3 r 15 .0011 .18 r 20 .0081 .25 r 25 .31 .37 r 30 .37 .34 r 35 NA .25

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33 CHAPTER 2 CARBON DIOXIDE ANESTHESIA OF TAMARIXIA RADIATA (WATERSTON) (HYMENOPTERA: EULOPHIDAE) PARASITOID OF DIAPHORINA CITRI (HEMIPTERA: PSYLLIDAE) Introduction Tamarixia radiata (Waterston) (Hymenoptera: Eulophidae), is an arrheno tokous ectoparasite of the Asian citrus psyllid (ACP) Diaphorina citri (Kuwayama) (Hemiptera: Psyllidae), vector of citrus greening d isease or huanglongbing (HLB). The parasitoid is reported to have controlled ACP populations to low levels on the islands o f Runion, Guadeloupe and Puerto Rico (Aubert & Quilici 1984; Etienne et al. 2001, Pluke et al. 2008). Tamarixia radiata was first imported to Florida from Taiwan and Vietnam in 1998 and released in 1999 2001 (Hoy & Nguyen 2001). A survey conducted in 2006 07 determined that T. radiata was well distributed in citrus orchards throughout the state (Qureshi et al. 2009). However, incidence of parasitism was generally low, especially early in the growing season, suggesting a need for augmenting parasitoid popul ations at that critical time as component of an integrated management progra m (Qureshi et al. 2007, 2009). Studies of T. radiata biology and current efforts at mass rearing and release of this species might benefit from an ability to inactivate adults by C O 2 anesthetization, including separation of emergent wasps and psyllids. Carbon dioxide (CO 2 ) is widely used to anesthetize insects, but may also cause deleterious side effects on biology and behavior. (Brooks 1957) found that development rate of German cockroach, B lattella germanica L. (Blattodea: Blattellidae), nymphs decreased 53% when exposed weekly to high CO 2 concentrations for 3 min. (Crystal 1967) reported significantly decreased survival rates and fertility of New World screwworm, Cochliomyia hominivorax (Coquerel) (Diptera: Calliphoridae), exposed to

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34 100% CO 2 for 30 min. Sherman (1953) reported that CO 2 anesthesia of Mediterranean fruit fly, Ceratitis capitata (Wiedemann) (Diptera: Tephritidae), led to increased mortality. This study was undertaken as a first step toward possible use of anesthesia for mass rearing by evaluating the response of T. radiata to CO 2 exposure. The objective was to test the effectiveness of CO 2 anesthesia of T. radiata and to determine the incidence and severity of side effects of CO 2 anesthesia on longevity, parasitism rate and sex ratio. Materials a n d Methods Colonies A T. radiata colony was maintained at FDACS DPI in Gainesville on ACP nymphs using orange jasmine, Murraya paniculata (l.) Jack (Sapindales: Rutaceae), as plant host. Six newly trimmed plants with new growth were held in an acrylic 62 cm cubic cage and 600 D. citri adults were released for 72 h for oviposition in a g reenhouse under natural sunlight 25 5 C and 50% ~ 70% RH. Adults were removed and plants held for 10 d until 4th instar nymphs were available. Plants were moved into another clean cage of the same type for 20 days into which 100 T. radiata were released. Adult progeny were later collected daily until no more emerged. Gas Chamber A gas chamber was constructed consisting of a vial, 6.50 cm in diam and 12 cm in height (Fisher S cientific, Pittsburgh, Pennsylvania) provided with two 0.50 cm diam holes in the lid into each of which was fitted a 0.5 cm plastic tube inserted either 1 cm or 11 cm into the chamber for a gas outlet and inlet respectively (Fig 2 1) Plasticine modeling clay (Flair Leisure Products, Cheam Surrey, England) was molded around the openings of the lid to prevent leakage.

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35 Flow time of gas at 3.8 kpa (2 psi) needed to displace all air in the chamber was assessed by filling the vial with water and then replacin g with CO 2 through the inlet. All th e water was displaced in 10 s. A CO 2 sensor (K 33 ICB 30% CO 2 Sensor, CO 2 Meter Inc., Ormond Beach Florida, USA) was used to determine that 3 s of flow time were necessary to attain a 30% CO 2 concentration confirming th e earlier result. The CO 2 sensor was also used to test for leaks by confirming that a given concentration remained constant over several min. Recovery Time Five wasps having emerged within 24 h or less were placed in the chamber. The lower of the vial wa s covered with black cloth to induce the wasps to walk to the top and thus avoid injury from inrushing gas. Gas was introduced through an inlet from a CO 2 tank at 3.8 kpa for 15 s to exchange all the air, and then the 2 tubes closed with metal clamps. Wasp s were removed after a 5 min exposure and observed with the naked eye using a stop watch to record recovery time (normal movement). Males and females were treated separately each with eight replications so that a total of 80 wasps was used. Survival Rate To evaluate survival, 5 anesthetized wasps were collected into each of 6 small glass vials (1.5 cm in diam, 5.3 cm high) and provided pure honey on a tissue paper strip. On the same day control wasps not anesthetized were placed in 6 other vials. Vials wit h wasps were held in a growth chamber at 25 C 14:10 h L: D and 60 5% RH, and checked daily, noting sex of all cadavers until all had died.

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36 Percent Parasitism Six newly trimmed plants were held in a ventilated 62 cm acrylic cubic cage until there were a t least 3 new shoots 3 cm in length upon which to evaluate parasitism. Plants were infested by releasing 600 ACP adults for a 24 h oviposition period. ACP adults were removed and the plants were held for 9 days in a rearing room at 25 and 60 5% RH. A small brush was used to remove nymphs until exactly 120 fourth instars remained on each plant. Each plant was then placed individually into a clear acrylic cylinder (12.5 cm diam, 43 cm high) into which 3 T. radiata females and 2 males were released. Cages were randomly selected to receive either anesthetized or untreated wasps ( N = 8). Newly emerged T. radiata offspring were collected daily from day 7 until day 19 after which no more new progeny were found. Progeny were counted and sexe d and parasitism rate calculated based on 120 original hosts. Results Seventy percent of T. radiata females recovered from anesthesia with CO 2 within 4 min, males recovered about as quickly. Indeed, there was no significant difference between male and fe male recovery time ( 2 = 13.04, df = 7, P = 0.071, Fig 1). It was noted that a wasp often would recover immediately after being crawled over by another recovering individual. Survival rate for the treated wasps was consistently lower than the control over the entire study period (Fig. 2). Insect days, the area under the curve of insect numbers by time (Ruppel 1983), was significantly less for the CO 2 treatment (3445.3 348.6) than the control (5610.5 836.6) ( t = 2.39, df = 7, P 0.05). A mean of 59.0 3.8 wasps emerged over 12 days from 120 fourth instar hosts exposed to 3 female and 2 male T. radiata treated with CO 2 compared to a mean of

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37 85.2 4.5 in the control. This corresponded to a parasitism rate of 49.2 3.2% for trea ted wasps compared to 71.0 3.7% for untreated wasps ( t = 4.46, df = 14, P = 0.00054). There was no significant difference in progeny sex ratio ( t = 1.03, df = 14, P = 0.32) between the treated and the control. Discussion Carbon dioxide anesthesia is a co nvenient tool for manipulating insects, but can cause deleterious side effects. In this case, a 5 min exposure of Tamarixia radiata adults to 100% CO 2 concentration caused a knockdown of about 4 min, significantly reduced survivorship and fecundity, but di d not affect the sex ratio of progeny from treated adults. Future research will focus on using less concentrated doses or shorter exposure times to inactivate the wasps in order to improve survival and fecundity.

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38 Figure 2 1. Carbon dioxide gas cha mber (Photo courtesy of Xulin Chen) Figure 2 2 Tamarixia radiata female and male recovery time frequency (%) distribution

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39 Figure 2 3 Mean (SEM) survivorship of CO 2 treated and untreated Tamarixia radiata adults.

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40 CHAPTER 3 FUNCTIONAL RESPONSE OF TAMARIXIA RADIATA (HYMENOPTERA: EULOPHIDAE) TO DENSITIES OF ITS HOST, DIAPHORINA CITRI (HEMIPTERA: PSYLLIDAE) Introduction Tamarixia radiata (Waterston) (Hymenoptera: Eulophidae), is an arrhenotokous ectoparasite of the Asian citrus psyllid (ACP) Diaphori na citri (Kuwayama) (Hemiptera: Psyllidae), vector of citrus greening disease or huanglongbing (HLB). The parasitoid is reported to have controlled ACP populations to low levels on the islands of Runion, Guadeloupe and Puerto Rico (Aubert & Quilici 1984, Etienne et al. 2001, Pluke et al. 2008). Tamarixia radiata was first imported to Florida from Taiwan and Vietnam in 1998 and released in 1999 2001 (Hoy & Nguyen 2001). A survey conducted in 2006 07 determined that T. radiata was well distributed in citrus orchards throughout the state. However, incidence of parasitism was generally low, especially early in the growing season, suggesting a need for augmenting parasitoid populations at that critical time as component of an integrated management program (Qures hi et al. 2007, 2009). The success of such a program would depend, in part, on the efficiency of mass rearing the parasitoid. Thus optimization of the host: parasitoid ratio is of critical importance. Optimal host: parasitoid ratio may also be an important consideration for guiding field release strategies. It has been reported that the fecundity of T. radiata is highly correlated with host density (Chu and Chien 1991, Chien et al. 1991a 1995). The relationship between both daily and lifetime fecundity wit h host density showed similar parabolic responses, meaning that female fecundity ascends with host density to a peak of 40 hosts, and then decreases as host density increases (Chien et al. 1995) The decline in fecundity

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41 at high host density could be due t o host defense or excessive honey dew. In fact, he experiments were conducted at 100% RH which would increase honeydew and consequent sooty mold that would interfere with the T. radiata Another issue may be that late 5 th instar n ymphs are not suitable hosts especially when they are about to emerge as adults Similar behaviors have been observed in Eretmocerus mundus (Mercet) (Hymenoptera: Aphelinidae), parasitoid of whitefly Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae). The female E. mundus never attacked late 4 th instar white fly nymphs once adult development was initiated (Gelman et al. 2005) Therefore, it is necessary to repea t these experiments under more favorable conditions of relative humidity and host availability. The relationship between number of hosts parasitized per parasitoid and host density is known as functional response It has been us e d t o illustrate the compl ex interactions between parasitoids and their hosts, and is an important character influ encing biocontrol success (Jones et al. 200 3). F unctional response has been described as Holling (1959) Type I, II or III, characterized by linear, decelerating and sig moidal responses of parasitism rate to increasing host density Most laboratory studies show that parasitoids exhibit Type II functional response, Type III functional response s are also common (Holling 1959). The shape of the functiona l response curve vari es with parasitoid species, and also parasitoid age (Jones et al. 2003). My objective was to evaluate the influence of host density on percent parasitism and the number of hosts parasitized by T. radiata ( functional response ) to help optimize rearing con ditions in the laboratory as well as release conditions in the field.

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42 Materials a nd Methods Colonies Murraya paniculata (L.) Jack (Rutaceae) contained in 3.92 L pots with 40: 60 mix of Canadian sphagnum peat (Fafard 4P mix Professional Growing Mix soil, Conrad Fafard Inc.) was used to rear D. citri Each plant selected had 2 5 shoots of new growth, with each new shoot about 4 cm in length. Plants were maintained using M pede soap (Dow AgroSciences LLC) as a contact chemical to control unwanted psyllids an d other pests soap sprayed pla nts cannot be used within 3 d All plants used in this experiment were grown in an unheated screen houses (hoop style trussed greenhouse with insect screen me sh) with natural ventilation and sunlight. A wood framed cubic cage was used (62 cm in each dimension) 3 sides of which were covered with fine mesh for ventilation with the other 3 sides enclosed with clear acrylic Six flushing M. paniculata plants were placed in the cage and 600 D. citri released and held there for 24 h for oviposition P lants were then moved to a similar clean cage for about seven days until the eggs hatched and nymphs developed to the 4 th insta r. All cages were held in an air conditioned rearing room at 25 30 C 60 80 % RH (Extech RH10 humidity and tem perature datalogger, Grainger, Inc) and L: D=14:10. Murraya paniculata with 4 th nymphal instar psyllids were transferred into a clear acrylic cylinder (12.5 cm diam, 43 cm high) with mesh on top in a growth c hamber (Percival model I36LLC8 Perry Iowa) at 24 4 C RH 60 80 %, L: D=14:10 (Extech RH10 humidity and temperature datalogger, Grainger, Inc). Five pairs of newly emerged T. radiata were released into this cylinder for three days to allow the parasitoids to mature.

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43 Arenas Arenas were prepared using centrifuge tubes (11 cm long and 3 cm diam) Into each arena was placed a 7 cm young shoot of M. paniculata infested with 10, 20, 30, 40, 50, or 60, 4th instar psyllid nymphs ; the number of nymphs was controlled using a small paintbrush. O ptimal young shoo ts were chosen as those with more than the number of hosts needed A small paintbrush w as used to remove extra nymphs to achieve the desired density Placing additional nymphs onto the young growth was avoided, because the newly placed nymphs always crawle d off the new shoot. One randomly chosen 3 d old parasitoid pair was released into each centrifuge tube and sealed with Parafilm provided with small holes made by a No.1 insect pin for ventilation. A renas were placed in the growth chamber and shoots replac ed every 24 h. Exposed nymphs were inverted under a stereoscopic microscope to check for parasitoid eggs. Each pair of T. radiata was held in the arena for 5 d and six replications were completed for each host density. Statistical Analysis Data on number and proportion of parasitized and superparasitized hosts in each arena among six host densities over the 5 d period were transformed using square root for normality fitness and then subjected to Analysis of Repeated Measures using JMP software (SAS Insti polynomial regression of proportion of hosts consumed versus density was used to distinguish between Type II and Type III functional response (Juliano 2001). A significant positive linear term coefficient indicates Type III whereas a significant negative linear term coefficient implies a Type II functional response. Data were then fitted to the corresponding functional response equation using nonlinear regression in

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44 JMP and the functional respon se parameters: searching efficiency (a) and handing time (TH) were reported from the regression. Results Fecundity, Percent Parasitism a nd Percent Superparasitism Inspection of the box plot revealed no outliers among the data, and no significant differenc es were observed among replications (F = 0.6461 df = 5, P = 0. 6648). Fecundity was significantly different among the six host densities (F = 111.2432, df = 5, P < 0.0001), and it increased with host density to a maximum of 11 to 12 eggs per day with no si gnificant differences among host densities of 40, 50, and 60 (Table 3 1 and Fig 3 1). P ercent parasitism w as significantly different among six host densities (F = 49.6352, df = 5, P < 0.0001) : highest at 43% with 10 hosts and least at 18.7% with 60 hosts (Table 2, Fig. 2). Percent s uperparasitism (superparasitized hosts number over parasitized hosts) w as also significantly different among the six host densities (F = 140.7006, df = 5, P < 0.0001) ; highest (37.9%) at the lowest host density level of 10 hosts per female followed by 15. 4 % at 20 hosts per female Superparasitism was negligible at densities over the range of 30 to 60 hosts per female (Table 3) Functional Response The estimated linear term coefficient for the polynomial regression was 0.0099 0 .0015 and significant ( t = 4.01, df = 177, P < 0.001), indicating a type II functional response (Juliano 2001). When the data were fitted to the disc equation (Holling 1959), the estimated searching efficiency (a) was 0.442 0.036 per day (95% confidence interval was 0.378~0.520) and estimated handing time (T H ) was 0.045 0.008 d (95% confidence interval was 0.032 ~ 0.063), which were all significant (Fig 4).

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45 Discussion Percent p arasitism and percent superparasitism changed with host densit y The number o f hosts parasitized increased from density 10 to 40 per female after which it remained constant. Host availabl ility w as limited at low host densities, indicating that fecundity was constraine d by lack of host s The number of eggs laid did not increase bey ond 40 hosts per female, indicating that the upper limit had been reached. Parasitism rate was highest at density 10, but 37.9% of the parasitized hosts were superparasitized, which meant the hosts were not used effectively as only one par asitoid adult can emerge and supernumerary parasitoids are thus wasted ( Chien et al. 1991a) Therefore a host density of 10 per female would not be effi cient for parasitoid mass rearing. A maximum number of hosts were p arasitized a host density of 40, and percent parasiti sm was highest at densit ies of 20 and 40 hosts per female However, 15.4% superparasitism was observed at 20 hosts per female whereas superparasitism was negli gible at a host density of 40. Therefore a density of 40 host per female optimal because it maxim ized number of percentage of hosts parasitized with minimal superparasitism. When host density exceeded 40, the number of hosts parasitized did not increase but the percent parasitism declined. However, no decrease in number of hosts parasitized was observ ed at host densities above 40 per fem ale as reported by Chien et al. (1995). In conclusion, a host density of 40 was the optimal choice maximal progeny and the best for usage of D. citri. Superparasitism was highest at a host density of 10, and was not si gnificantly different when the density exceeded 30. Chien et al. (1991) reported that female T. radiata were able to discriminate between parasitized and non parasitized hosts. But

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46 gravid female s still laid eggs even though there were not enough suitable h osts available resulting in superparasiti sm Superparasitism is waste ful because only one egg can mature to the adult and any others will die. ( Chien et al. 1991a) To avoid superparasitism, host number should be controlled in excess of 20 per female. The p attern of host s parasitized by T radiata over the first five days conformed to a functional response Type II, which means the searching efficiency and the handing time were all constant in different host densities. These two parameters will be useful to c ompare T. radiata with other parasitoids, and they may help to make a better study of the behavior of T. radiata in the future.

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47 Table 3 1. Number of parasitized hosts (Mean SEM) at different host densities Host Density (4 th Instars/arena/day) Parasitize d Hosts (Mean SEM) 10 4.30 0.16 a 20 5.60 0.3 b 30 7.23 0.42 c 40 11.24 0.24 d 50 11.50 0.40 d 60 11.22 0.30 d Table 3 2. Percent parasitism (Mean SEM) at different host densities Host Density (4 th Instars/arena/day) Percent pa rasitism (Mean SEM) 10 43.0 0.016 a 20 28.0 0.015 b 30 24.1 0.006 c 40 28.1 0.006 b 50 23.0 0.008 c 60 18.7 0.005 d Table 3 3. Percent superparasitism (Mean SEM) in different host densities, mean number Host Density (4 th Instar s/arena/day) Percent superparasitism (%) (Mean SEM) 10 37.9 0.03a 20 15.36 0.03 b 30 1.24 0.004c 40 0.35 0.007c 50 0.43 0.004 c 60 0.27 0.002c

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48 Figure 3 1 Number of parasitized host at different host densities, error bar stands for SEM Figure 3 2 P ercent p arasitism at different host densities, error bar stands for SEM

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49 Figure 3 3. Percent s uperparasitism in different host densities Figure 3 4. Number of D. citri parasitized by T. radiata and the functional response curve (T ype II)

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50 CHAPTER 4 THE INFLUENCE OF DIET ON EGG FORMATION IN TAMARIXIA RADIATA (HYMENOPTERA: EULOPHIDAE), A PARASITOID OF DIAPHORINA CITRI (HEMIPTERA: PSYLLIDAE) Introduction Tamarixia radiata (Waterston) (Hymenoptera: Eulophidae), is an arrhenotokous ec toparasite of the Asian citrus psyllid (ACP), Diaphorina citri (Kuwayama) (Hemiptera: Psyllidae), vector of citrus greening disease or huanglongbing (HLB). The parasitoid is reported to have controlled ACP populations to low levels on the islands of Runi on, Guadeloupe, and Puerto Rico (Aubert & Quilici 1984, Etienne e t al. 2001, Pluke et al. 2008). Tamarixia radiata was first imported to Florida from Taiwan and Vietnam in 1998 and released in 1999 2001 (Hoy & Nguyen 2001). A survey conducted in 2006 07 de termined that T. radiata was well distributed in citrus orchards throughout the state. However, incidence of parasitism was generally low, especially early in the growing season, suggesting a need for augmenting parasitoid populations at that critical tim e as component of an integrated management program (Qureshi et al. 2009). It would thus be necessary to mass rear parasitoids to obtain adequate number s for release requiring temporary storage. During the holding period, food provided to females may affec t the number of eggs formed in ovaries, which may influence their efficiency as a biocontrol agent upon release Oogenesis is a nutrition limited process, and nutrition is obtained during the larval or adult stage; insufficient nutrients always affect egg production (Wheeler 1996) Carbohydrate is the major energy source for most insects; but it is not the main nutrition that triggers egg formation in female insects. Varley and Edwards (1957) reported that female Nasonia vitripennis (Wlkr.) (Pteromal idae) fed on carbohydrates only survived,

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51 but resorbed eggs resulting in gradual decreaseof eggs in the ovaries. (Bownes and Blair 1986) found that when Drosophila were feeding on sugar diet s, the number of eggs laid and the number of vitellogenic oocytes in ovaries were reduced significantly This was due to reduced transcription of y olk protein in the fat bodies resulting in reduced availability of yolk protein for oogenesis Host hemolym ph is rich in protein and an importan t source of nutrients to many hymenopterous female parasites for increasing fecundity (Howard 1910, Rockwood 1917) Varley a nd Edwards (1957 ) observed egg development by female N. vitr ipennis ,was initiated and/or accelerated shortly after host hemolymph was (Leius 1961) reported that female Itoplectis conquisitor (Say) (Hymenoptera: Ichneumonidae) which fed on host body fluid laid more eggs than those fed on carbohydrates alone Additionally, Leius (1961) f ound that p ollen in diets significantly increased female I. conquisitor fecundity, and females fed on a mixed diet (host body fluid and carbohydrates) deposited even more eggs. The objective of this experiment was to investigate wh ich food s promote egg production du ring storage presumable improv ing oviposition rate upon release. Materials a nd Methods Colonies Murraya paniculata (L. ) Jack (Rutaceae) w as used as a host plant to maintain a D. citri colony. Plants were grown in 3.9 L pots using 40: 60 mix of Canadian sphagnum peat potting soil (Fafard 4P Professional Growing Mix soil, Conrad Fafard Inc.) in a screen house (hoop style trussed, with insect screen mesh) under natural sunlight and passive ventillation Plants sprayed with 35 % M pede soap (Dow AgroSciences LLC) as needed to control psyllids insects and mites

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52 The D. citri colony was maintained in an air conditioned reari ng room at 28 2 C 60 80 % RH (Extech RH10 humidity and temperature datalogger, Grainger, Inc), and L: D= 14: 10. Six M. paniculata plants each with five to eight, 4 cm long new sprouts were placed a wood frame cage (60 cm 60 cm 120 cm) covered all three sides with fine mesh for ventillation Six hundred D. citri adults were released in each cage for 24 h for oviposition. All adult psylli ds were then c ollected and removed P lants were held for approximately 1 0 d (depending on temperature and humidit y until all nymphs reached 4 th instar. Six M. paniculata plants with 4 th instar nymphs were transferred into a clear acrylic cage (60 cm 80 c m 90 cm) with 3 sides of fine mesh in a n air conditioned greenhouse maintained at 28 2 C 75 5% RH. Fifty T. radiata females and 30 males were released in the cage for 48 hours to parasitize hosts, then c ollected and removed P l ants were held for anoth er 6 d until the parasitized host s d mumm ified Then, the young shoots with nymphs w ere excised and nymphs were examined under a microscope. When a nymph was found parasitized with its parasitoid wasp developed almost to the pupal stage, that part of the s hoot ( about 2 cm) was excised and placed in a glass tube (75 mm long, 12 mm in diameter, Fisher Scientific, Pyrex 9820) in an air c onditioned rearing room at 282 60 80 % RH (Extech RH10 humidity and temperature datalogger, Grainger, Inc). Tubes were che cked frequently, and once the wasp removed immediately after emerge Diets Each pair of newly emerged T. radiata was plac ed in a 50 ml centrifuge tube (Kendall Labware, Mansfield, MA) sealed with Parafilm ( Pechiney Plastic Packaging

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53 Company Chica go, Illinois ) and stored in a growth chamber (Percival Scientific model I36LLC8, Perry Iowa ) at 17 C 75~ 85 % RH and L: D = 14:10. Wasp were provided with 8 different diet treatments: water alone honey, Nu Lure (Miller Chemical & Fertilizer Corporatio n, Hanover, Pennsylvania), host nymphs, honey+ Nu Lure, honey+ host nymphs, Nu Lure+ host nymphs, and honey+ Nu Lure + host nymphs (water stripe was provided in every treatment) Nu Lure (Miller Chemical and Fertilizer, Hanover, Pa.) is a proteinaceous dar k brown liquid made from 44% hydrolyzed corn gluten meal, and 56% inert ingredients Water, honey and N Lure were all provided on 8 cm long, 1.5 cm wide cellulose strips (Wypall L30, Kimberly Clark Professional). Host nymphs were provided on a fresh cut s ho ut on which 2 nd 3 rd 4 th instar nymphs were mixed together. Water strips were changed every 24 h and honey, Nu Lure strips and host nymphs were changed every 48 hours to make sure they did not dry out and continued providing efficient food sources. W asps in each treatment were held separately for 5, 10, 15 and 20 d There were 10 replications of every treatment day combination. Egg Load T. radiata females under each different treatment we re dissected after 5, 10, 15 and 20 d as well as newly em erged T. radiata under a dissecting microscope (Olympus SZX 16, Olympus Corporation) Pi ctures were taken using an Olympus DP 21, and the number of eggs was recorded. Statistical Analysis Data from the eight treatments at each 5 day dissection interval we re compared test ( JMP SAS Institute Inc. 2012 ).

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54 Results Newly emerged T. radiata f emales averaged 4.6 eggs each. Egg loads were significantly different among the eight treatments at 5 days ( F = 11.1702, df = 55, P < 0.0001), 10 days ( F = 12.72, df = 40, P < 0.0001), 15 days ( F = 7.8989, df = 35, P < 0.0001), and 20 days ( F = 10.28, df = 34, P < 0.0001). Wasps fed on water and Nu Lure all died within 10 days. At each of the five day checks, females fed on honey had significantly fewer eggs compared with the other treatments except for water and Nu Lure. During the first five days, females fed on nymphs formed the same number of eggs as honey + Nu Lure, but with time females fed on honey+ Nu Lure formed fewer and fewer eggs until the 20 th day, when the difference was significant compared to those fed on nymphs. Females fed on Nu Lure + nymphs, and honey+ Nu Lure+ nymphs formed significantly more eggs than those provided with nymphs alone, and a little more than those on honey + ny mphs, although the dif ference was not significant (Table 4 1). Wasps fed on honey alone formed fewer and fewer eggs after 10 days ; wasps fed on honey +Nu Lure also formed gradually fewer eggs Discussion An average of 4.6 eggs were observed in ovaries of n ewly emerged female T. radiata despite not feeding on any diets (Fig 4 1, 4 2), so they may use reserved nutrition from larval stages to form the first clutch of eggs. Mating was not necessary for egg formation. This study showed that honey alone was enou gh to keep wasp females alive, but egg resorption took place within 5 days after emergence (Fig 4 3). This may mean that T. radiata females lack the ability to transfer carbohydrates into amino acids to form

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55 eggs, or oogenesis requires essential amino acid s which cannot be transferred from carbohydrates. Nu Lure alone was not sufficient to keep the females alive for more than 5 days which indicated that carbohydrate is a necessary energy resource needed for survival and they may lack the capability of tr ansforming amino acids into carbohydrate. The combination of honey + Nu Lure resulted in female survivorship similar to a diet of host nymphs, but egg formation was still less than if provided with nymphs ( Fig 4 4). Females with access to host nymphs (Fig 4 5) matured significantly more eggs than those fed on honey, Nu Lure, or honey+ Nu Lure, indicating that host body fluid contained essential nutrients not available in the food supplements. However, the combinations of honey+ Nu Lure+ nymphs (Fig 4 8) an d Nu Lure+ nymphs (Fig 4 7) resulted in significantly higher fecundity than a diet of nymphs alone honey + nymphs (Fig 4 6) was a little better, but not significant (Table 4 1). I body fluid is an irreplaceable nutrition source extra source of carbohydrates and amino acids provided by the supplements had a positive effect both in the absence and presence of nymphs In future studies, it will be interesting to investigate which amino acid s or other constituents of hemolymph are important for egg formation.

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56 Table 4 1. Mean SEM number of eggs by treatment from dissections after 5, 10, 15, and 20 days at 17 C Means in the same column followed by the same letter are not significantly di test HSD, P <0.05). 5 days 10 days 15 days 20 days Water 1.830.6 D ______ ______ ______ Nu Lure 2.140.7 D ______ ______ ______ Honey 3.5 0 0.4 C 3.670.6 C 2.4 0 0.4 D 1 .00 0.4 D Nymphs 5.5 0 0.6 B 6.3 0 0.8 AB 6.8 8 0.7 B C 6.5 0 0.8 B Honey+ Nu Lure 5.3 8 0.7 B 5.3 0 1.0 B 5 .00 0.7 C 4.6 0 0.8 C Honey+ Nymphs 6 .00 0.5 AB 7 .00 0.8 AB 7.7 0 0.6 AB 8 .00 1.5 A Nu Lure+ Nymphs 7.560.6 A 8 .00 0.9 A 8 .00 0.9 A 8.3 0 0.7 A Honey+ Nu Lure+ Nymphs 7.2 0 0.6 A 7.8 0 0.6 A 8 .00 0.8 A 8.2 0 0. 8 A

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57 Figure 4 1. Newly emerged (unfed) T. radiata female digestive system (Photo courtesy of Xulin Chen) Figure 4 2. Newly emerged (unfed) T. radiata female (Photo courtesy of Xulin Chen)

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58 Figure 4 3. Paired T. radiata ovaries after feeding on honey for 20 days (egg resorption) (Photo courtesy of Xulin Chen) Figure 4 4. T. radiata ovary after feeding on Nu Lure+honey for 10 days (Photo courtesy of Xulin Chen)

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59 Figure 4 5. Paired T. radiata ovaries after feeding on nymphs for 5 days (Photo courtesy of Xulin Chen) Figure 4 6. Paired T. radiata ovaries after feeding on honey +nymphs for 10 days (Photo courtesy of Xulin Chen)

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60 Figure 4 7. Paired T. radiata ovaries after feeding on Nu Lure+ nymphs for 10 days (Photo courtesy of Xulin Chen) Figure 4 8. Paired T. radiata ovaries after feeding on Nu Lure+honey+nymphs for 5 days (Photo courtesy of Xulin Chen)

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61 CHAPTER 5 DISCUSSION AND CONCLUSIONS Tamarixia radiata is an arrhenotokous ectoparasite of the Asian citrus psy llid (ACP) Diaphorina citri vector of citrus greening disease or huanglongbing (HLB). Tamarixia radiata is being tried as an augmentive biological control agent, since the number present in the field reduced in winter and by insecticide applications Any program aimed at augmentation of a natural enemy would require an efficient system of mass production. Key components would include methods of manipulating, rearing and storing large populations of insects. To make counting easily and separating D. citri and T. radiata after collection carbon dioxide was used as anesthesia. Carbon dioxide anesthesia can be a convenient tool for manipulating insects, but can also cause deleterious side effects. In this study, a 5 min exposure of Tamarixia radiata adults to 100% CO 2 concentration caused a knockdown of about 4 min, and significantly reduced survivorship and fecundity, but did not affect the sex ratio of progeny from treated adults. The h armful effects of CO 2 proscribe its routine under the tested exposure co nditions for rearing T radiate. Nevertheless the wasp showed s u r pris ing tolerance to high CO 2 concentration: the wasps acted normally when sealed in a gas chamber with 30 % CO 2 Perhaps lower concentrations and/or exposure times would provide more acceptab le alternatives. Also, the physiological mechanism of CO 2 catabolism could be an interesting research aspect. The effects of chilling to immobilize the insects should also be tested. Host density is an important factor influencing mass rearing efficiency Fourth and early 5 th instars are the preferred stage for oviposition by T. radiata M y study

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62 showed that a density 40, 4 th instar nymphs per female optimized fecundity, maximized incidence of parasitism and minimized incidence of superparasitism. This resu lt could be used to determine the idea number of parasitoids to be released into a rearing cage. The number of flushes in the rearing cage can be counte d, and the number of proper stage nymphs on one shoot can be estimated to calculate the number of nymphs in the whole rearing cage. Then, knowing the sex ratio and the ideal 4 th instar host to female ratio of 1:40, the best number of T. radiata females to release will be clear. The p attern of parasitism observed over the first five days conformed to a Type II functional response, estimated searching efficiency was 0.442 0.036 per day and estimated handing time was 0.045 0.008 days. These two parameters are important in comparing a T. radiata ate its parasitic capacity When T. radiata were stored for release, certain nutri tional food is necessary to maintain fecundity. My r esult s showed that, T. radiata formed more eggs feeding on mixed diets (Nu Lure+ honey+ nymphs or Nu Lure+ n ymphs) compar ed to nymphs alone. Until now, no diet has been found to completely substitute nymph al hemolymph. The present practice is to hold wasps in centrifuge tubes provided with honey on paper strips preparatory to release. My research would indicate that wasps should also be provided with a supplementary protein source such as Nu Lure, and also with hosts in order to maintain the fecundity Tamaraxia radiata is an efficient biological control agent of D. citri under optimal temperature, humidity, host densities and nutritional supplement in lab conditions More

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63 work need to be done to apply these lab results in fiel d to get a more optimal parasitism capacity of D. citri

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64 LIST OF REFERENCES A NONYMOUS 2004. Estudos indicam que a nova doena tem relao com o gr eening. Fundecitrus. http://www.fundecitrus.com A UBERT B., G RISONI M., V ILLEMIN M., R OSSOLIN G., D A G RACA J. V., M ORENO P., AND Y OKOMI R.K. 1996 A case study of Huanglongbing (greening) control in Reunion Proceeding s of the 13th Conference of the International Organization of Citrus Virologists, p. 276 278. A UBERT B., AND Q UILICI S. 1984 Biological control of the African and Asian citrus psyllids (Homoptera : Psylloidea) through eulophid and encyrtid parasites (Hymenoptera: Chalcidoidea) in Reunion Island Proceedings from the 9th Conference of the International Organization of Citrus Virologists, p.100 108. A UBERT B., AND H UA X. Y. 1990 Monitoring flight activity of Diaphorina citri on citrus and Murraya canopies, Rehabilitation of citrus industry in the Asia Pacific Region. Pr oc. 4th International Asia Pacific Conference on Citrus Rehabilitation, Chiang Mai, Thailand, pp. 181 187. A UBERT B., AND Q UILICI S. 1988 Monitoring adult psyllas on yellow traps in Reunion Island. Proceedings of the 10th Conference of International Org anization of Citrus Virologists. International Organization of Citrus Virologists, Riverside, CA, pp. 249 254. B ATOOL A., I FTIKHAR Y., M UGHAL S. M., K HAN M. M., J ASKANI M. J ., A BBAS M., AND K HAN I. A. 2007. Citrus greening disease a major cause of c itrus decline in the worlda review. Hort. Sci. 34: 159 166. B OV J. M. 2005. First report of a huanglongbing like disease of citrus in So Paulo Candidatus sease. Plant Dis. 89: 107. B OV J. M. 2006. Huanglongbing: a destructive, newly emerging, century old disease of citrus. J. Plant Pathol. 88: 7 37. B OWNES M., AND B LAIR M. 1986. The effects of a sugar diet and hormones on the expression of the drosophil a yolk protein genes. J.Insect Physiol. 32: 493 501. B ROOKS M. A. 1957 Growth retarding effect of carbon dioxide anaesthesia on the German cockroach. J. Insect Physiol. 1: 76 84. B ROOKS M. A. 1965. The effects of repeated anesthesia on the biology of Bl attella germanica (Linnaeus.). Entomol. Exp. Appl. 8: 39 48. B UITENDAG C. H., AND B ROEMBSEN L. A. 1993. Living with citrus greening in South Africa. Citrus J. 3: 29 32.

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73 BIOGRAPHICAL SKETCH Xul in Chen was born in Shandong Province, China. She began her undergra duate study in Shandong Agriculture University majoring in Plant Quarantine in 2007. After she graduated in July, 2011, she started her graduate study in University of Florida Entomology and Nematology Department under the supervision of Dr. Phil Stansly.