THE EFFECTS OF AN ARTIFICIAL HOLDING DIET ON THE
FECUNDITY OF THE ECTOPARASITIC WASP TAMARIXIA RADIATA
(WATERSTON) (HYMENOPTERA: EULOPHIDAE)
A Thesis
Presented to the
Faculty of
California State Polytechnic University, Pomona
In Partial Fulfillment
Of the Requirements for the Degree
Master of Science
In
Plant Science
By
Danielle Renay Ruais
2018
SIGNATURE PAGE
DISSERTATION: THE EFFECTS OF AN ARTIFICIAL HOLDING DIET ON THE FECUNDITY OF THE ECTOPARASISTIC WASPTAMARIXIA RADIATA (WATERSTON) (HYMENOPTERA: EULOPHIDAE)
AUTHOR: Danielle Renay Ruais
DATE SUBMITTED: Spring 2018
College of Agriculture
Dr. Valerie J. Mellano ______Department Chair Plant Sciences
Dr. Anna L. Soper ______Committee Chair Plant Sciences
Dr. Robert L. Green ______Adjunct Professor Plant Sciences
Dr. David J.W.Morgan ______CDFA Environmental Program Manager
ii
ACKNOWLEDGEMENTS
Thank you to the ARI for providing funding for this research under grant #15-04-
219. Thank you to the project director Valerie J. Mellano, PhD, Chair of the Plant
Sciences Department at California State Polytechnic University, Pomona. As well as the project collaborators Anna L. Soper, PhD, Assistant Professor of Plant Sciences at
California State Polytechnic University, Pomona, Brian Taylor of the California Citrus
Research Board, and David J. W. Morgan, PhD, Environmental Program Manager at the
California Department of Food and Agriculture at Mt. Rubidoux, Riverside, California.
Thank you to the California Citrus Research Board and California Citrus Mutual for your contributions.
Thank you to California State Polytechnic University, Pomona and my committee for your guidance in developing the process; Robert L. Green, PhD, Lecturer of Plant
Science at California State Polytechnic University, Pomona, David J. W. Morgan, PhD,
Environmental Program Manager at the California Department of Food and Agriculture at Mt. Rubidoux, Riverside, California, and my Committee Chair Anna L. Soper, PhD,
Assistant Professor of Plant Science at California State Polytechnic University, Pomona.
Thank you to the California Department of Food and Agriculture at Mt.
Rubidoux, Riverside, California for providing the Tamarixia for my experiments. I would like to especially thank David J. W. Morgan, PhD, Environmental Program Manager, and
Environmental Scientists Grace Radabaugh, Alex Muniz, and Judith Herreid.
Thank you Hoon Kim, PhD, Professor of Statistics and Co-Director of the
Consulting Center for Statistics and Applied Mathematics for your help with providing
iii
the statistical analysis for my experiments. Thank you also to the Spring 2017 Advanced
Statistics 559 course for your work with my data. Thank you David W. Still, PhD,
Executive Director of the California State University Agricultural Research Institute for your guidance and direction during the early part of the process.
I would like to thank Benjamin J. Lehan, MS, Lecturer at California State
Polytechnic University, Pomona, for managing the Cal Poly Pomona Entomology
Laboratory and making sure that I always had everything I needed to make my work go smoothly. I thank the following Plant Science undergraduate students for your help with the seemingly never-ending dissections and laboratory work. Thank you Christian
Buelna. You are a reliable man and intelligent problem solver. Thank you Kevin
Gonzalez for always making yourself available to me even when in the middle of your own important projects. Also thank you to University of California, Berkley undergraduate student Cindy E. Hsu for your help with the Tamarixia project.
Thank you to Robert R. Shortell, PhD for your unwavering belief in my abilities and for giving me that shove when I was too hesitant to take the leap. Thank you David
H. Headrick, PhD, Professor in the Horticulture and Crop Science Department at
California State Polytechnic University, San Luis Obispo, for your wise counsel and for making me realize that bugs have feelings too.
Thank you Paul Fletcher for introducing me to the love I feel for the trees, the bugs, and the soil. Thank you for cultivating my inquisitive spirit and connection to the earth. You have made a deep impact on how I view the world.
I thank my parents Lynn and Raymond Ruais for their continuous love and support along this journey. To my sister Brie Ruais, you have been my steadfast
iv
supporter who kept my mind on the big picture. And to Niles Ruais for causing me to realize my own strengths. Thank you to Enrique Wallace IV who supported my ambitions and encouraged me to persevere. To Haley E. McCown, MS, thank you for your positive attitude and love along the way. Thank you to Haley Chappell and Elliott Popel for your encouragement throughout the process. Thank you Heidi Arndt for your brilliance. And last but certainly not the least, thank you Rebecca A. Morris, MSG, MPH, for reminding me to believe in myself and for helping me to make my master’s degree a reality.
v
ABSTRACT
Since the detection of the invasive Asian Citrus Psyllid (ACP), Diaphorina citri
Kuwayama (Hemiptera: Psyllidae)—vector of the lethal citrus disease Huanglongbing
(HLB) Candidatus Liberibacter asiaticus—in LA County in 2008, millions of Tamarixia radiata (Hymenoptera: Eulophidae) have been reared and released as an augmentative biological control agent to mitigate the ACP threat to California’s massive commercial citrus production. T. radiata is an effective ectoparasitoid because it establishes quickly under optimal release conditions and the adult female wasp has dual impact on ACP mortality: death by host-feeding on all nymphal instars and through parasitisation of later instars.
As a synovigenic species, the female T. radiata wasp can emerge with a few eggs and after those eggs have been deposited onto ACP nymphs, she can produce new eggs.
However, the formation of these new eggs is contingent on the type of nutrients, or diet, available to her as an adult. Increasing her egg load through diet during storage periods prior to release has the potential to monstrously affect the threat of ACP.
Mass rearing these insects for release by the millions necessitates an efficient production system that includes manipulation of rearing conditions and storage for large populations of T. radiata. In this study, wasps received various diets containing various proteins, sugars, and amino acids for 24-hour increments prior to dissection to record EL.
This information will aid in optimizing T. radiata rearing and storage protocol, which allows for wasp peak performance in field releases throughout California.
vi TABLE OF CONTENTS
Signature Page ...... ii
Acknowledgments...... iii
Abstract ...... vi
List of Figures ...... ix
Literature Review. A Fatal Disease, Its Vector, and the Heroine ...... 1
Background on Biological Control ...... 1
The World Citrus Economy ...... 2
Huanglongbing (HLB) ...... 4
Asian Citrus Psyllid, Diaphorina citri (Kuwayama) (Hemiptera: Psyllidae) ...... 14
Tamarixia radiata (Waterston, 1922) (Hymenoptera: Eulophidae) ...... 22
Mass-Rearing the Star of Biological Control ...... 34
Experiment One. Regeneration of Egg Load ...... 37
Introduction. A Synovigenic Solitary Ectoparasitic Wasp ...... 37
Objective. Egg Regeneration through Diet ...... 38
Materials and Methods. Age, Exposure Lengths, and Diet Treatments ...... 42
Results. It’s All About the Egg Load ...... 52
Discussion. Age, Body Size, and Diet Treatment ...... 57
Conclusion. The Ultimate Factor ...... 61
Experiment Two. CPP Citrus Grove Trials ...... 63
Introduction. Exposed to the Elements...... 63
Objective. Observations of Wasp Activity ...... 66
vii Materials and Methods. The Quest for ACP ...... 68
Results. To Host-Feed or to Parasitize ...... 74
Discussion. In-field Diet Treatment Performance ...... 75
Conclusion. Limitations and Future Directions ...... 76
References ...... 80
Appendix A. Contacts for Thesis Work...... 97
Appendix B. Feeding Trials Data Collection ...... 99
Appendix C. Diet Treatment Formulations ...... 100
Appendix D. Tamarixia radiata Dissection Protocol ...... 101
Appendix E. Experiment One Figures ...... 106
Appendix F. Field Data Collection ...... 110
Appendix G. Experiment Two Figures ...... 111
viii LIST OF FIGURES
Figure 1. Correlation between Egg Load and Hind Tibia Length...... 106
Figure 2. Irregular Distribution of Tamarixia radiata Egg Load...... 106
Figure 3. Histogram of Wasp Age by Average Egg Load ...... 107
Figure 4. Histogram of Exposure Length to Diet by Average Wasp Egg Load ...... 107
Figure 5. Histogram of Average Egg Load by Diet Treatment...... 108
Figure 6. Histogram of Wasp Age by Diet Treatment ...... 108
Figure 7. Histogram of the Average Egg Load: Age by Exposure Length to Diet ...... 109
Figure 8. Histogram of Diet by Exposure Length ...... 109
Figure 9. Percent Parasitized and Percent Host-fed 6-day-old Tamarixia. radiata ...... 111
Figure 10. Percent Parasitized and Percent Host-fed 12-day-old Tamarixia radiata ...... 111
ix LITERATURE REVIEW. A FATAL DISEASE, ITS VECTOR, AND THE
HEROINE
Background on Biological Control
Invasive plant and animal species are crossing borders worldwide at increased rates made possible by our ever-expanding global human network (McNeely, J. A.,
Mooney, H.A., Neville, L. E., Schei, P., & Waage, J.K., 2001). According to the Center for Invasive Species Research, 50,000 or more nonnative species were introduced into the
United States in 2014, either accidentally or purposefully (Vogt & Koch, 2016).
Approximately 4,500 of these nonnative species were arthropod insect pests.
Furthermore, new establishments and expanded ranges of these nonnative species continue to spread rapidly, especially in tropical and temperate monocropping systems.
For example, an average of six new species are established per year in the state of
California, and an average of 15 exotic arthropod pests are established annually in both the states of Florida and Hawaii (Hoddle, 2004; Vogt & Koch, 2016).
It is therefore important to not only accurately describe these invasive arthropod pests, but to also study their native ranges, all possible plant hosts, the environmental conditions under which they have evolved, and any natural enemy or biological control agent that mitigates their damage in their native environments (Borror & White, 1970).
Thorough investigation is needed into whether these complex pest–natural enemy interactions can be recreated and successfully established in newly affected areas in order to protect economically important crops such as citrus (Aubert, 1987; Hoddle, 2004;
Qing, 1990; Qureshi, Hall, & Stansly, 2009).
1 The following is a review of biological control agents of the invading Asian citrus psyllid (ACP) and the disease it vectors (Huanglongbing; HLB) to citrus trees and closely-related plants worldwide, with special attention to southern California. This literature review specifically looks at (1) leading world citrus crop producers, the economics of world citrus production, and current citrus crop management practices in the United States; (2) distribution and symptoms of the pathogen of HLB and current control methods for bacterial management in other countries as well as the United States, with special attention to the states of California and Florida; (3) the biology and distribution of the HLB vector, Diaphorina citri (Kuwayama) (Hemiptera: Psyllidae) also known as Asian Citrus Psyllid (ACP); the transmission mechanism between the pathogen and vector; natural enemies of D. citri; and current chemical, biological, and cultural management strategies, (4) the biology of the current primary biological control agent for
D. citri, Tamarixia radiata (Waterston, 1922) (Hymenoptera: Eulophidae); the abiotic and biotic influences on T. radiata such as diet, host size, wasp age, wasp body size, temperature, and relative humidity (RH) effects on wasp reproductive mechanisms, the general behavior exhibited by the wasp during parasitism and host–feeding activities; coordinated biological control releases; and subsequent challenges with the establishment of T. radiata in California’s commercial, residential, and urban citrus; and (5) how this specific thesis aids in parasitoid mass-rearing procedures and fits into the grand scheme of sustainable and long-term protection of the citrus industry in southern California.
The World Citrus Economy
Global citrus production is concentrated around the Mediterranean basin and
2 North America; however, Brazil and China have both been increasing their citrus production in recent years (Ferris, 2015; U.S. Department of Agriculture [USDA]
Foreign Agricultural Service [FAS], 2016). When it comes to citrus production, there are two categories of fruit: whole fruit, which judges fruit aesthetics and taste, and fruit for processing that depends on high concentrations of sugars and amount of juice per unit of fruit (USDA FAS, 2016). Worldwide orange juice production is forecast to decline by
16% in 2017–2018 due to unfavorable weather in Brazil, the European Union, and the
United States as Brazil citriculture tries to recover from decades of record low yields
(USDA FAS, 2017; 2018). This is in contrast to China which is experiencing economic growth with their citrus production due to increases in acreage devoted to mandarin and tangerine production (USDA FAS, 2016). Conversely, production has decline overall in the United States as a result of rapidly decreasing yields of fruit available for processing in the state of Florida (USDA FAS, 2017).
Florida produces 60% of all citrus in the United States, with the bulk of fruit used for the juice market (USDA National Agricultural Statistics Service [NASS], 2016).
Although California has a $3.3 billion citrus industry and leads the country as the largest supplier of whole fruit—it grows 80% of fresh market oranges and 90% of lemons in the
United States—it still ranks second to Florida in terms of production (California
Department of Food and Agriculture, 2014; Civerolo, 2015; Ferris, 2015; Lee, 2015;
Milosavljević, Schall, Hoddle, Morgan & Hoddle, 2017). The Florida citrus industry contributes more than $9 billion annually to its state’s economy (Qureshi, Rohrig, &
Stansly, 2012). However, with the introduction and rapid spread of HLB, the entire
United States citrus industry could collapse in a matter of years (Halbert & Manjunath,
3 2004).
Historically, citrus trees do not have as many pests as other economically important crops and tend to be more resilient, which allows citrus to be grown across an expansive range of environmental conditions. Current citrus crop production practices in the United States rely on calculated preventative and curative chemical applications that are rotated over the course of the calendar year to avoid the buildup of pesticide resistance (University of California Integrated Pest Management [UC IPM], 2016). The
University of California Integrated Pest Management (UC IPM) website, a publication provided by the UC Division of Agriculture and Natural Resources, offers detailed information on how to manage citrus crops year-round (UC IPM, 2016). These suggestions include appropriate timing and application of relevant cultural and biological controls, target-specific and site-specific chemical use, and recommended fertilizer application rates. Furthermore, the website encourages consistent monitoring, thorough record keeping, and preventative strategies as the first lines of defense against citrus pests and diseases (UC IPM, 2016).
Huanglongbing (HLB)
HLB is one of the world’s most destructive citrus diseases and it presents an immediate threat to global citrus production (Bové, 2006; CDFA, 2014; Chen, 2013;
Halbert & Manjunath, 2004; Qureshi et al., 2009). Historically, HLB has been present in
China since the 1870s, but was of little importance until surveys in 1936 revealed that the infectious disease had spread to citrus growing provinces all over the country (Bové,
2006). Most citrus are susceptible to HLB, including hybrids, cultivars, and citrus
4 relatives (Wager-Page, 2010). Halbert and Manjunath (2004) included a comprehensive list of known plant hosts of the causal bacterial agent for HLB, Candidatus Liberibacter spp. Some of the included plant families were Atalantia, Balsamocitrus, Calodendrum,
Fortunella, Microcitrus, Poncirus, Severinia, Swinglea, Toddalia, and Triphasia (Halbert
& Manjunath, 2004).
“Huanglongbing” translated from Chinese means “yellow dragon disease.”
Southern Chinese rural citrus growers named the disease for the yellowing of the plant shoots that can be a symptom of the disease (Bové, 2006; Chen, 2013). Other symptoms include asymmetrical mottling of leaves with indistinct margins and leaf chlorosis that resembles zinc or other mineral deficiency, usually followed by heavy mature leaf drop and massive twig dieback (Garnier & Bové, 1993; Halbert & Manjunath, 2004).
Symptomatic fruit are small, lopsided, bitter-tasting, discolored, and mostly green, which is why the disease is also referred to as Citrus Greening disease (Bové, 2006;
Gottwald, 2010). Management controls recommend plant removal as soon as a certified laboratory confirms the disease so that the diseased crop will not become a reservoir for clean Asian Citrus Psyllid to contract the bacterium (Brlansky & Rogers, 2007; Moffis,
Burrow, Dewdney, & Rogers, 2016). However, the uneven distribution of the pathogen within infected plant tissues causes plants to unpredictably exhibit symptoms or to be completely asymptomatic. This means that once a tree is infected, not all parts of the tree will be an inoculum source for ACP to acquire the bacterium (Brlansky & Rogers, 2007;
Cen et al., 2012). Furthermore, there is a latency period of 6–12 months between the time that the citrus plant acquires the disease and when the citrus plant actually exhibits symptoms, if any (Warnert, 2017). This makes early detection based on visual scouting
5 methods difficult within a citrus grove, and subsequent management decisions based on symptomatic trees ineffective (Halbert & Manjunath, 2004; Meyer, 2007). Once a citrus plant contracts the disease it is estimated to decline and die within five to eight years
(Grafton-Cardwell & Daugherty, 2015; Moffis et al., 2016).
Recent studies in Florida reflect the devastation to citrus farmers statewide. Since its introduction into Florida in 1998, the presence of HLB has increased farmer production costs by 40%, there has been a loss of over 60,000 acres of prime citrus production, 8,000 jobs, and $7 billion in state revenue (CDFA, 2014; Emerson, 2014;
Milosavljević, Schall, Hoddle, Morgan, & Hoddle, 2017; Qureshi et al., 2012; Wager-
Page, 2010).
The causative agent of HLB is a gram-negative, vector-borne α-proteobacterium that cannot be maintained in pure laboratory cultures, which is designated by the term
Candidatus (Garnier & Bové, 1993; Meyer, 2007; Miles Stover, Ramadugu, Manjunath,
& Lee, 2017). Three etiological agents of HLB have been implicated based on 16S ribosomal RNA sequencing: Candidatus Liberibacter asiaticus (Asia and North
America), Candidatus Liberibacter africanus (Africa), and Candidatus Liberibacter americanus (Brazil) (Brlansky & Rogers, 2007; Chen, 2013; Garnier & Bové, 1993;
Halbert & Manjunath, 2004). Of the three agents, only Candidatus Liberibacter asiaticus
(CLas) was been detected in Florida in 2005, but it led to an estimated 1.6% of the state’s citrus trees to be infected by 2008 and more than 80% of its trees infected by 2015 (Miles et al., 2017; Morris, Erick, & Estes, 2009).
CLas can be transmitted by psyllid insect vectors, the grafting of infected plant material, the parasitic plant dodder (Cuscuta spp.), and possibly by seed (Halbert &
6 Manjunath, 2004; Meyer, 2007; Moffis et al., 2016). Though it is a bacterium, CLas has shown that it cannot be transmitted through contamination of personnel, tools, wind, or rain (Brlansky & Rogers, 2007; Chen, 2013; Halbert, 2010). The bacterium physically clogs the phloem, resulting in a blockage that prevents the movement of carbohydrates from the leaves to the plant’s roots. The roots, without necessary carbohydrates from the leaves to perform basic functions, cannot reallocate nutrients and water from the environment. The leaves do not receive the nutrients via the xylem that are integral to photosynthesis, and the tree essentially starves to death, which results in the yellowing symptom present in the leaf veins and shoots (Miles et al., 2007; Moffis et al., 2016).
In a 2015 CNBC interview with Joel Nelsen, president of California Citrus
Mutual (CCM) and farmer based in California’s San Joaquin Valley, said that the state of
California currently spends $25 million annually on HLB prevention. Nelsen said that
$15 million of that expense is covered by the citrus industry and the rest comes from federally funded programs to protect California’s citrus growers from the disease (Ferris,
2015). CCM is a non-profit advocacy organization that represents California’s citrus growers concerning economic issues and policy regulations (Lee, 2015). The California strategy, based off of Florida’s experiences, is focused on early detection of HLB- positive trees, strict quarantines of infected areas, inoculum destruction through removal of infected plants, eradication efforts for ACP where applicable, and biological and chemical treatments against psyllid vectors (Aubert, 1987; Bové, 2006; Flores &
Ciomperlik, 2017; Grafton-Cardwell, Daugherty, Jetter, & Johnson, 2018; Halbert &
Manjunath, 2004; Hall, Richardson, Ammar, & Halbert, 2012; Meyer, 2007; Paiva &
Parra, 2012).
7 Nelsen goes on to explain that state and local government employees scout trees in areas that are known to have high psyllid populations, which occur mostly in residential neighborhoods in Los Angeles and the surrounding southern California areas
(UC IPM, 2016). Generally, when clean ACP first appear in a region, numbers are low and the population can potentially be eradicated locally (Gentz, Murdoch, & King, 2010;
Grafton-Cardwell & Daugherty, 2015; UC IPM, 2016). The hope is to catch HLB- positive cases before the disease can spread to the 270,000 acres of commercial citrus crops (Ferris, 2015; Lee, 2015; Wager-Page, 2010).
New technologies are being developed in order to more accurately detect HLB and CLas early; even prior to a citrus tree displaying symptoms, which would allow for the removal of infected plants as quickly as possible (Civerolo, 2015). Early detection of
HLB is determined by advancements in polymerase chain reaction (PCR) techniques that allow for rapid and economical detection of the bacterium in both the infected citrus tree as well as in the individual psyllid vector (Garnier & Bové, 1993; Hoy, Jeyaprakash, &
Nguyen, 2001; Hung, Hung, Chen, Hsu, & Su, 2004; Meyer, 2007). Furthermore, since the presence of CLas can now be determined within individual psyllids, the percentage of infected ACP individuals in a population can be tracked throughout the season which creates new opportunities for vector epidemiology and disease detection (Garnier &
Bové, 1993).
Strict quarantines for the CLas pathogen and its psyllid vector are in place in affected citrus-growing regions of the United States and in residential areas of southern
California (Flores & Ciomperlik, 2017; Grafton-Cardwell & Daugherty, 2015). Because only psyllid vectors, grafting infected materials, Cuscuta spp., and infected seed can
8 transmit HLB, regulations have been focused on curbing the movement of infected citrus materials into uninfected areas (Grafton-Cardwell & Daugherty, 2015; Hall et al., 2012).
This includes the movement of whole citrus trees, citrus plant nursery stock, untreated and unprocessed fruit, and fruit waste (Halbert & Manjunath, 2004; Ke, Li, Ki, & Tsai,
1991; Meyer, 2007; Warnert, 2013a). Alternate host-plants for ACP such as the popular residential landscape plant, Orange Jessamine, Murraya paniculata, are also on the quarantine checklist (Grafton-Cardwell & Daugherty, 2015; Halbert & Manjunath, 2004;
Skelley & Hoy, 2004).
Inoculum destruction is an important part in the management of the HLB disease- vector complex. The commercial citrus tree inventory has reported on the abundance of abandoned citrus groves in the state of Florida (USDA NASS, 2014, 2016). These neglected trees can serve as a haven for populations of HLB-positive psyllids to build and become a potential threat to commercial citrus in the area (Hall et al., 2012). Some
Florida citrus groves previously identified as “abandoned” have remained vacant, have been destroyed, or have been returned to production (Brlansky & Rogers, 2007; USDA
NASS, 2014, 2016). In southern California things look grim with over 400 backyard citrus trees slated for removal and destruction since early 2018 (D. Morgan, personal communication, February 9, 2018).
Management of HLB and its spread depends largely on biological and chemical treatments for vector control to either reduce disease transmission to healthy trees, or to prevent the re-inoculation of already-infected trees (Brlansky & Rogers, 2007; Qureshi et al., 2012). Chosen biological and chemical controls must be specific to the pest, the plant host, and to the infected site. Most of these control techniques have limited success when
9 used individually, and are not appropriate under all circumstances (Aubert, 1987; Halbert
& Manjunath, 2004; Pilkington, 2010; Qureshi & Stansly, 2009). For instance, in Florida, where there is extensive establishment of both ACP populations and HLB, chemical eradication of the insect vector is no longer a feasible management goal. Instead, combination tactics that encompass the planting of healthy nursery stock, prompt culling of diseased trees, horticultural practices to combat stress in infected trees by fertilization, and the use of biological controls should all be practiced to reduce the need for insecticides, while concurrently providing vector suppression in residential, unmanaged, and abandoned groves (Hall & Albrigo, 2007; Meyer, 2007; Miles et al., 2017; Qureshi et al., 2012).
If both the insect pest and the pathogen are present, scientific literature generally agrees that chemical control of pest psyllids is necessary (Halbert & Manjunath, 2004;
Warnert, 2013b). The California Department of Food and Agriculture (CDFA), together with the Citrus Research Board (CRB), UC IPM, and Cooperative Extension, the county agricultural commissioner’s office, and the US Department of Agriculture (USDA), all encourage farmers and homeowners to be involved by self-reporting to the CDFA Pest
Hotline (800-491-1899) or through self-education with the CDFA public website (CDFA,
2014; Grafton-Cardwell & Daugherty, 2015; Teiken, Lemaux, Grafton-Cardwell, &
McRoberts, 2015; UC IPM, 2016).
In order to time critical pesticide applications in commercial citrus groves, scouting, heavy monitoring with yellow sticky panel traps, and branch tap samplings are important, especially during citrus flushing episodes (Brlansky & Rogers, 2007; Grafton-
Cardwell & Daugherty, 2015; Hall & Albrigo, 2007; Qureshi et al., 2009; UC IPM,
10 2016). Citrus tree flush refers to the tender new leaf tissues that are appropriate for ACP oviposition, nymphal development, and as a food source for young ACP (Qureshi et al.,
2009). It is the developing tissues themselves that attract the gravid, egg-laying adult female psyllids (Hall & Albrigo, 2007).
Some of the recommended chemical controls for California are systemic soil drenches or trunk injections of insecticides with the active ingredient imidacloprid— which remains within the plant tissue for an extended period of time—and are effective against a wide array of known phloem-sucking insects, including ACP (CDFA, 2014;
Gentz et al., 2010; Grafton-Cardwell & Daugherty, 2015; Hall et al., 2012; Monzo,
Qureshi, & Stansly, 2014). Plants with visible psyllids should be treated with a foliar application of the contact insecticide cyfluthrin in order to knock-down present populations (Warnert, 2013b). Soft-chemicals and botanicals like natural oils and soaps have had some success against select psyllid species (Halbert & Manjunath, 2004; Gentz et al., 2010; Meyer, 2007; Monzo et al., 2014).
For backyard citrus in urban and residential areas, chemical and biological controls can be used to prevent the establishment of ACP and HLB, as well as to mitigate direct feeding damage caused by the pest psyllid. Dr. E. Grafton-Cardwell, entomologist at UC Riverside (UCR) Kearny Agricultural Center, has developed a website designed to disseminate detailed management information to both farmers and residents who grow citrus in southern California (Grafton-Cardwell, 2017). The website (ucanr.edu) details
ACP and HLB distribution in California, ACP monitoring tips, and treatment options with relevant costs (Grafton-Cardwell, 2017). A recent survey reported that 60% of
California residents have at least one citrus tree in their landscape, which makes the
11 potential devastation from HLB a real and present issue within California (Citrus Matters,
2016). However, Grafton-Cardwell assures homeowners that there are “safe and reliable ways to reduce ACP presence, which can reduce the chance of losing a tree to HLB”
(Warnert, 2015). One such example would be the use of cultural methods to limit the amount of flush on a tree as well as seasonal flushing episodes which can be achieved through timely pruning techniques (Chien & Chu, 1996). The research supporting this management tactic is based on the positive correlation between the ratio of ACP-infested shoots and the flush density of the tree, which concludes that flush availability is a potential limiting factor on psyllid populations (Hall & Albrigo, 2007; Qureshi et al.,
2009).
There has been interest developing in recent years over the use of biological control methods to suppress psyllid populations. A study published in 1987 by B. Aubert showed that a certain pathogenic fungus could be the most important element of ACP mortality in the field. Aubert observed ACP mortality rates up to 60–70% when environmental humidity was in excess of 87%; however, the use of insect pathogenic fungal applications has yet to be researched in depth and exploited for the protection of citrus production (Aubert, 1987).
Currently, there are two insect biological control agents that have been researched, imported, and released in select citrus growing regions of the world (Flores &
Ciomperlik, 2017; Halbert & Manjunath, 2004). The insects are T. radiata and
Diaphorencyrtus aligarhensis (Shafee, Alam, & Argarwal, 1975) (Hymenoptera:
Encyrtidae), parasitic wasps, which have been behind the successful reduction of ACP populations in the countries of Guadeloupe, Réunion Island, and Taiwan (Aubert, 1987;
12 Chen, 2013; Chien & Chu, 1996; Étienne et al., 2001; Hall et al., 2012). Both biological control are parasitoids that are host-specific to ACP; however, after many trials, T. radiata has proven to be the more efficient and the more readily established biological agent than D. aligarhensis (Halbert & Manjunath, 2004; Hall, 2008b; McFarland & Hoy,
2001). The success of a biological control agent is dependent on the specific environmental context of the affected area (Hoddle, 2004). The mere application of biological control agents is not a “silver bullet” for ACP and HLB control, as it is recommended to be used in conjunction with other site-appropriate integrated pest management (IPM) tools (Garnier & Bové, 1993; Gentz et al., 2010; Hall et al., 2012;
Meyer, 2007; Qureshi et al., 2009).
Another option that is soon-to-be available is genetically engineered citrus plants.
There are several approaches to the genetic engineering of both CLas and HLB-resistant or tolerant citrus rootstocks. This technology could potentially save the citrus industry
(Hall et al., 2012; Warnert, 2013a). With these advances in genetic engineering come benefits such as decreased insecticide use, the reduction in the development of pesticide resistance, decreased rates of secondary pest outbreaks, fewer negative impacts on natural enemies and pollinators, and the overall reduced risk to the environment as well as to agricultural workers (Étienne et al., 2001; Teiken et al., 2015). Though many citrus industry professionals are interested in genetically modified rootstocks, it would take years before these plants could be commercially available, and then several more years before the citrus trees could bear mature fruit (Teiken et al., 2015). Citrus growers urgently need tools in order for current and future citrus production areas to survive
(Halbert & Manjunath, 2004; Miles et al., 2017; Monzo et al., 2014; Warnert, 2013a).
13 Currently, work is carried out to identify and understand in detail the ACP feeding mechanism—particularly regarding the molecular synthesis and formation of the salivary sheath—which is critical for regular psyllid feeding to occur (Ammar, Hall, & Shatters,
2013). Researchers report that the formation of the salivary sheath can be inhibited, and that this mechanism could be used to produce transgenic citrus plants that are mechanically resistant to psyllid feeding (Shatters, 2011).
Asian citrus psyllid, Diaphorina citri (Kuwayama) (Hemiptera: Psyllidae)
D. citri, has risen to the top of the charts as one of the world’s most economically important psyllid species. This is not just due to the direct damage caused by pest feeding, but due to the lethal citrus pathogen (HLB) it transmits to healthy citrus trees and closely-related plant species (Grafton-Cardwell & Daugherty, 2015; Meyer, 2007; Pluke et al., 2008; UC IPM, 2016).
Biologically speaking, D. citri goes through a hemimetabolous metamorphosis from egg, through five nymphal instars, and into its final adult stage in approximately 13 days at 25°C (Fung & Chen, 2006; Husain & Nath, 1927). At 15 to 17 days old, the females are reproductively mature and mate with several different males. A few days later, the females lay their eggs on new flush of citrus plants producing an average of 800 eggs over the course of their lives (Hall, 2008a; Husain & Nath, 1927; Mead & Fasulo,
1998; Meyer, 2007). The developmental periods of eggs and nymphs vary greatly with temperature, humidity, resource availability, and predator abundance (Fung & Chen,
2006; Husain & Nath, 1972). The amygdaliform eggs are approximately 0.3mm long, resemble pale yellow pearls stuffed between the young expanding leaves, and are
14 anchored to the tips of new citrus flush (Mead & Fasulo, 1998).
The nymphal instars resemble small yellow scabs with red eyes that measure 0.25
st th mm long at the 1 instar, and up to 1.7 mm long at the 5 instar (Grafton-Cardwell &
Daugherty, 2015; Mead & Fasulo, 1998). The ACP instars are much more sedentary than the adults because they lack wings and can travel only a few centimeters from the flushing foliage tips where they were laid as eggs, down the leaf petiole, and onto young stems and the undersides of mature leaves to continue to feed and molt into adulthood
(Mead & Fasulo, 1998).
The adult stage of the psyllid is approximately 2.0–5.0 mm, with mottled brown wings that are held like a peaked roof over its body and jump short distances when disturbed (Borror & White, 1970; Meyer, 2007). Adult D. citri actively feed and mate in the groves during windless, sunny, warm days and live an average of 20 to 30 days (Hall,
2008b; Nava et al., 2007). When searching for new food sources, even with relatively weak flight muscles, adult psyllids can jump and fly up to 1.5 meters into adjacent grove trees and into neighboring citrus farms and residential plantings (Bové, 2006; Halbert &
Manjunath, 2004; Hall et al., 2012; Meyer, 2007).
The D. citri adult has an opisthognathous head with piercing-sucking mouthparts that form a three-segmented beak, which causes the psyllid to raise its body to a 45° angle to gorge on the vascular contents of the plant (Cen et al., 2012; Grafton-Cardwell &
Daugherty, 2015; Hall et al., 2012; Meyer, 2007). During the initial feeding and probing stages, psyllids secrete a saliva that solidifies immediately upon exposure to air, and forms a straw-like tube (Shatters, 2011). This feeding tube allows the psyllid to remove large quantities of sugars and sap from the phloem of the citrus tree (Cen et al., 2012;
15 Wager-Page, 2010). As the psyllid feeds, it injects the host-citrus tree with a toxin that triggers phytotoxemia in the tree’s young leaves and shoots, causing the new foliage to distort, twist, and easily break from the tree (Grafton-Cardwell & Daugherty, 2015;
Husain & Nath, 1927; Meyer, 2007; UC IPM, 2016). As the tree’s defense mechanism to psyllid infestations, it sheds its affected foliage; however, when compounded with severe psyllid feeding damage, the citrus tree falls victim to extreme defoliation and suffers a considerable set-back to fruit development (Chen, 2013; Husain & Nath, 1927).
Not only does direct D. citri feeding damage negatively affect citrus yield, but the nymphs also excrete curly, waxy tubules called frass as a result of their continuous feeding on citrus tree sugars and sap (Grafton-Cardwell & Daugherty, 2015; Hall et al.,
2012; Husain & Nath, 1927). This frass is highly attractive to ant species (Herrera &
Pellmyr, 2002). Video-recorded observations made by UCR researchers depict Argentine ants tending to the honeydew-producing D. citri nymphs on the citrus leaves and stems.
The ants aggressively defend ACP nymphs against natural enemies and parasitoids such as T. radiata (UCR CISR, 2014). In order to increase the effectiveness of parasitoids and other predators in the area, it is recommended to use an adhesive chemical barrier such as
Tanglefoot aerosol spray in order to keep the ants from farming and protecting the ACP frass in the field. (Navarrete et al., 2013).
The accumulation of frass—especially in humid regions and on leaves in shady, dense canopies of citrus trees—leads to the growth of black sooty mold; technically the mycelium of a fungus (Chen, 2013; Chien & Chu, 1996; Grafton-Cardwell & Daugherty,
2015; Hall, 2008a; Perez et al., 2009). This fungus can then be spread throughout the orchard easily by wind, rain, and by psyllid movement (Perez et al., 2009; Tashiro et al.,
16 2013). These fungi do not infect citrus plants and cause more cosmetic damage rather than economic damage. However, extreme fungal growth can stunt plants, decrease photosynthetic rates, and interfere with parasitoids’ and natural enemies’ abilities to locate their hosts (Chien & Chu, 1996; Laemmlen, 2011; Perez et al., 2009; Tashiro et al.,
2013; Wager-Page, 2010).
Studies generally agree on the transmission mechanism of CLas: the pathway of
CLas begins with psyllid ingestion and continues into the psyllid’s hemolymph, fat, muscle tissues, and then into the salivary glands where the bacterium is transmitted via salivary secretions from the piercing-sucking mouthparts of the psyllid into the citrus
th th tree’s vascular tissues (Hall et al., 2012). Some reports suggest that 4 and 5 instar psyllids can acquire the pathogen after 30 minutes of feeding on an infected tree (Halbert
& Manjunath, 2004; Mead & Fasulo, 1998; Meyer, 2007). The psyllid retains the bacterium through successive molts and into adulthood, transmitting the bacterium as it feeds for the rest of its life (Brlansky & Rogers, 2007; Hall et al., 2012). Current evidence shows that the bacterium can be further transmitted sexually from infected adult males to uninfected adult females, and also transovarially from the adult female psyllid to her eggs
(Hall et al., 2012; Mann et al., 2011; Pelz-Stelinski et al., 2010).
Considering the ease with which this infectious bacterium spreads, distribution of
D. citri worldwide needs to be of concern to all citrus producers and consumers alike. D. citri was first detected in 1998 in the U.S. state of Florida in a backyard planting of the ornamental shrub Murraya paniculata (Mead & Fasulo, 1998). By 2001, D. citri had spread to 31 Florida counties, closely following the residential landscape plant distribution through various plant nurseries as well as following the movement of
17 infected plant material by individual people (Hall et al., 2012; Qureshi & Stansly, 2009;
Wager-Page, 2010). Several years later, D. citri had moved across the country to southern
California in 2008, followed closely by CLas in 2012 (Civerolo, 2015; Emerson, 2014).
Due to the presence of both the vector and bacterium, HLB eradication is no longer a realistic approach in southern California; therefore, other management strategies have to be employed to suppress the disease, as explained earlier. From a sustainability perspective, it is critical to acquire knowledge about the seasonal trends within a psyllid population, the psyllid physiology, and its CLas acquisition and transmission rates. This information is necessary to make chemical applications at the appropriate times to be the most effective against psyllids and have the least impact on natural enemies, pollinators, and non-target species (Brlansky & Rogers, 2007).
The U.S. Department of Agriculture's National Institute of Food and Agriculture
(USDA-NIFA) has funded $13.6 million for research grants to combat HLB (USDA
FAS, 2017). The financial support is sanctioned by Title VII of the 2014 United States farm bill which authorizes funding for research, extension, and education—including competitive grants for land grant institutions and state agricultural experimental stations, as well as intramural funding for USDA research agencies. One of the highlights of the bill requires mandatory funding for specialty crop research to increase to $80 million annually, which includes at least $25 million for emergency citrus disease research
(USDA NASS, 2016).
One of the projects currently funded by the USDA-NIFA, is the creation of
NuPsyllid (Teiken et al., 2015). It is an effort to genetically engineer D. citri that are incapable of transmitting CLas. A proponent of the project states that once NuPsyllid is
18 released into a wild ACP population, it will out-compete, and potentially eliminate the
“wild” type psyllid population capable of spreading the bacterium (Teiken et al., 2015;
Warnert, 2013a).
Classical biological control has afforded long-term and sustainable management of several invasive insect pests (Godfray, 1994; Malais & Ravensberg, 1992; Qureshi et al., 2009). This management tactic is designed to reconstruct specific elements of natural enemy complexes, particularly the role of parasitic wasps that may have coevolved with the invasive pest and with other natural enemies and predators, including hyperparasitoids (Aubert, 1987; Hoddle, 2004; Pilkington, 2010; Qureshi et al., 2009;
Qureshi et al., 2012).
Exclusion studies suggest that there are both native and established natural enemies already present in commercial perennial citrus orchards that are responsible for a broad portion of psyllid mortality (Aubert, 1987; Hall, 2008a; Monzo et al., 2014). These natural enemies include several generalist predators observed in southern California orchards; hover flies (Syrphidae), green lacewings (Chrysopidae), arboreal spiders
(Araneae), and predaceous lady-beetles (Coccinellidae) (Hall et al., 2012; Michaud,
2004; Qureshi & Stansly, 2009; Wager-Page, 2010). There was a study reviewed by J.P.
Michaud in 2004 that mentioned that the survival rate of ACP nymphs increased by a factor of 120 when the nymphs were in cages that excluded all predation activity. This indicated the importance of natural predator populations as a critical source of ACP nymphal mortality within commercial monocultures (Michaud, 2004). In the United
States, extensive studies continue to determine which selective insecticides, soaps and oils, and dormant spray combinations may be supplemented with the augmentation of
19 natural enemies and the release of pest-specific parasitoids (Gentz et al., 2010; Hall &
Nguyen, 2010; Monzo et al., 2014; Qureshi & Stansly, 2009).
The release of ACP-specific parasitoids, together with the culling of HLB- infected trees, has led to the elimination of HLB in Réunion Island in the Indian Ocean
(Aubert, 1987; Garnier & Bové, 1993; Hall, 2008a; Hall & Nguyen, 2010). The success of this case—which has since inspired other countries to do the same—was due to the unique nature of its tropical island ecology, which provided for an array of niches for introduced species to readily establish (Chien, Chiu, & Ku, 1989; Emerson, 2014;
Cooksey, 2017). Furthermore, the parasitoids imported to control the ACP populations were introduced to the island without their hyperparasitoids—the parasitoid’s natural enemies—as a natural population check (Étienne et al., 2001). Other Caribbean island nations such as Puerto Rico and Guadeloupe have also sufficiently suppressed ACP while maintaining their citrus production and keeping HLB at low levels (Qureshi et al., 2009).
Currently, T. radiata and D. aligarhensis are the two parasitoids that are reared and released in the United States as part of an IPM strategy to slow the spread of HLB into clean citrus production areas (Chen & Stansly, 2014b; Meyer, 2007). D. aligarhensis is an obligate endoparasitoid that is host-specific to D. citri (Aubert, 1987; Aubert &
Quilici, 1984; Chien et al., 1989; Cooksey, 2017; Milosavljević, Schall, & Hoddle, 2017).
D. aligarhensis development from egg to adult at 25°C occurs between 16 and 18 days (Rohrig et al., 2012; Chien, 1995). The larval to pupal stage are completed within the D. citri body which has since died upon parasitism and has hardened into a brown, protective mummy that envelops the wasp during its pupation. Once the endoparasitic wasp has gone through eclosion, it exits the mummy through a hole in the nymph’s
20 abdomen, not in the thorax like T. radiata (Grafton-Cardwell & Daugherty, 2015;
Milosavljević, Schall, & Hoddle, 2017). The location of the exit hole in an ACP mummy is telling, specifically as to which parasitoid successfully emerged from that ACP nymph.
It has been calculated that an adult female D. aligarhensis can kill up to 280 ACP
nd rd th nymphs through a combination of parasitization of 2 , 3 , and 4 nymphal instars, and
st nd through host-feeding on 1 and 2 nymphal instars (Chien, 1995; Chien & Chu, 1996;
Milosavljević, Schall, & Hoddle, 2017; Skelley & Hoy, 2004; Meyer, 2007). T. radiata
th th st nd rd parasitizes 4 and 5 ACP instars, and host-feeds on the 1 , 2 , and 3 instars, allowing one adult female T. radiata to kill approximately 500 ACP nymphs during her adult stage
(Chu & Chien, 1991; Chen & Stansly, 2014b; Hall, 2008a; Meyer, 2007; Qureshi et al.,
2012).
The T. radiata populations that were released between 1999 and 2001 in commercial citrus groves across the state of Florida were originally collected from a strain of Tamarixia found in Taiwan and South Vietnam (Chien & Chu, 1996; Hoddle,
2012). Though these wasps have established in the southern United States, they persist at low numbers with inconsistent parasitism rates that can dip to 1–3% throughout the
Floridian growing season (Chen & Stansly, 2014b). Conversely, the strain of wasp released in southern California was collected from Pakistan, an area that has a 70% climate match to southern California (Hoddle, 2011). Releases of this Pakistani strain of wasps in southern California first began in December 2011, after the USDA and the
Animal and Plant Health Inspection Service (APHIS) cleared the parasitoid for release from the UCR quarantine facility (Hoddle, 2011). Since 2011, over nine million T.
21 radiata have been released in a 40,000 square-mile area that covers 46 cities, including the Los Angeles urban sprawl, Orange County, Riverside County, and San Bernardino
County (D. Morgan, personal communication, May 18, 2018). The release of T. radiata was determined to not have “any negative cumulative impacts on the continental US” because it is host-specific to D. citri (Wager-Page, 2010). The UCR releases have proven to be successful as these parasitoids have been detected five to seven miles beyond the release sites (Milosavljević, Schall, & Hoddle, 2017).
On December 16, 2014, Dr. Mark Hoddle, Biological Control Specialist, Principal
Investigator, Director of the Center for Invasive Species Research (CISR), and professor at UCR, and his team of entomologists made the first release in California of the second
ACP parasitoid, D. aligarhensis. The release was made at the UCR Biological Control
Citrus Grove (Hoddle, 2015). It was suggested that the presence of this second parasitoid would synergistically enhance ACP suppression because with two host-specific parasitoids, a wider range of ACP nymphal instars would be susceptible to attack, which would reduce the rate of the spread of HLB into the commercial production orchards of southern California (Meyer, 2007; Milosavljević, Schall, & Hoddle, 2017).
Tamarixia radiata (Waterston, 1922) (Hymenoptera: Eulophidae)
The very nature of a parasitoid is that it lives within or on a larger host-insect for a portion of its life, ultimately killing its host. According to population dynamics, parasitoids are not that dissimilar from predatory insects and their “top-down” control exhibited on a given pest (Cooksey, 2017).
The tiny primary ACP parasitoid, T. radiata, was originally described in 1922 in
22 the Punjab region which spanned parts of India and Pakistan. It has since been recorded as an indigenous species to other areas such as Saudi Arabia, Nepal, Indonesia, Mainland
China, Japan, Vietnam, and Thailand (Qing, 1990; Wager-Page, 2010). In 1978, T. radiata was introduced into Réunion Island and achieved rapid control of ACP there. The parasitoid later again was successful when it was released in 1983 on the island of
Taiwan (Chien & Chu, 1996; Étienne & Aubert, 1980). Furthermore, the same T. radiata wasps from this established Taiwanese population have been imported into Florida between 1999 and 2001 for release (Hoy et al., 2001).
The wasp is now considered established in Florida and in other states where it was not purposefully introduced, like Texas (Hoddle, 2012). It is interesting that the wasp does not perform as well in certain release sites as others (Chen, 2013; Chien & Chu,
1996; Étienne et al., 2001). One compelling suggestion is that there are genetic differences between T. radiata populations due to geographic isolation from each other and the origins of the founding populations introduced (Hall & Nguyen, 2010; Paiva &
Para, 2012, Weiner, 1994). In response to this observation, entomology professor Dr. R.
Stouthamer of University of California, Riverside, has made it a personal goal to preserve the natural genetic variation within the collected populations of Tamarixia, which could be the key that allows this strain of parasitoid to adapt to citrus production regions of southern California and its inland valleys (Chen, 2013; Hoddle, 2012).
T. radiata adults have a black head and thick thoracic region, a black and yellow abdomen, pale yellow legs, and deep crimson eyes (Aubert & Quilici, 1984; Meyer,
2007). The clue to sexual dimorphism in T. radiata lies within the minor disparities between both antennal and abdominal features. Male antennae are covered in long,
23 greasy, stringy hairs, and have a rounder, tubby, terminal abdominal segment, whereas females have much shorter hairs on their antennae, and have a full but tapered terminal abdominal segment (Chen, 2013; Chu & Chien, 1991; Mann & Stelinski, 2014; Skelley
& Hoy, 2004).
Most hymenopteran insects are produced by haplodiploidy, which means that not all eggs are fertilized, but these eggs can still develop into viable adults (Jervis et al.,
2001). The sex determination of T. radiata is classified as arrhenotokous parthenogenesis, which means that unfertilized eggs will result in males and fertilized eggs will produce either males or females (Chen, 2013; Chien & Chu, 1996; Cooksey,
2017). Males usually have multiple matings, whereas 93% of females mate once after emergence (Chien, Chu, & Ku, 1991).
In most parasitoids, mating is immediate upon emergence and is promoted physiologically through males having a shorter developmental time than females
(Cooksey, 2017). This allows the males to be “ready and waiting” to mate with freshly- emerged females (Cooksey, 2017). Males use their antennae to locate responsive females
(Onagbola et al., 2009). Once the male has located a receptive female, the male makes contact with her dorsum for an average of 687 seconds. After which time, the male will penetrate the female for an additional 333 seconds (Chien et al., 1991). Mating and mating frequency are not significant factors when determining the potential fecundity of a female T. radiata (Chen, 2013).
Female T. radiata move actively among ACP nymphal colonies by using her clubbed antennae and host-plant volatiles to locate appropriate hosts (Gould et al., 2011;
Mann & Stelinski, 2010). Once a fitting host for parasitism is found, the female uses her
24 ovipositor to inject a temporary paralytic into the nymph, thereby immobilizing the ACP nymph for four to eight minutes while she oviposits an egg next to the nymph’s posterior coxae. This takes between one and four minutes (Chen, 2013; Chien et al., 1991). If the developing wasp egg were to be removed from the psyllid nymph, the nymph would not molt and would die. Additionally, the T. radiata egg would not mature in the absence of its ACP host—its provider of nutrients and protection. When a T. radiata larva is placed onto an un-parasitized nymph, the T. radiata larva will not attach and instead disconnects from the nymph as soon as the nymph mobilizes (Chen, 2013; Chien et al., 1991).
The T. radiata egg, translucent and reniform, is deposited onto and is attached to the ventral side of the nymph, usually behind the rear coxae (Aubert & Quilici, 1984;
Chu & Chien, 1991). Once the larva emerges it feeds on its host’s hemolymph at the point of attachment. Tamarixia radiata continues to develop through four larval stages before it fastens the drained ACP exoskeleton to the plant surface with long silk threads
(Aubert & Quilici, 1984; Chien et al., 1991). The ACP exoskeleton now serves as a shelter for the T. radiata to safely pupate beneath. The parasitized ACP nymph continues to feed and produce frass before it turns into a dark brown, hardened corpse—referred to as a mummy. The wasp discharges its meconium and molts into its pupal stage, and her body turns a honey blond with scarlet ommatidia and ocelli. Once the adult wasp hardens, it emerges from the mummified ACP host by penetrating the integument and making a
0.5mm diameter hole in the dorsal thorax (Chen, 2013). At the optimal temperature of
25°C, development of T. radiata from egg to adult averages 11.4 days. Average durations for the developmental stages of T. radiata are 1.9 days, 4.0 days, 0.6 days, and 4.9 days for the egg stage, larval stage, prepupal stage, and pupal stage, respectively (Chien et al.,
25 1991).
The adult female wasp can also kill ACP nymphs by host-feeding. This doubles her impact on psyllid populations (Étienne et al., 2001). Female T. radiata use their ovipositors to pierce and wound vulnerable ACP nymphs. Through host-mutilation the T. radiata female can feed on oozing ACP hemolymph and body fluids, which are a good source of protein needed to form and mature eggs (Chen, 2013; Cooksey, 2017; Wager-
Page, 2010). Male T. radiata have been observed to also feed on the gushing wounds of nymphs created by female wasps (Chen & Stansly, 2014b). Both male and female T. radiata have been observed to feed on the frass excreted by the feeding ACP nymphs which is a good source of carbohydrates (Skelley & Hoy, 2004). The female wasp has also been observed to simultaneously oviposit and host-feed, though on different nymphs
(Chu & Chien, 1991). Host-feeding time generally takes 20 seconds and once the host is fed upon it is no longer a suitable host for parasitism (Chien et al., 1991; Cooksey, 2017).
T. radiata is a synovigenic species, which means that the adult female has the capacity to continuously produce eggs throughout her life, which averages 28.6 days
(Aung, Takagi, & Ueno, 2010; Aung, Takasu, Ueno, & Takagi, 2012; Jervis & Kidd,
1986). Though she can mature her first clutch of eggs within her ovaries without first mating or host-feeding (Chen & Stansly, 2014b; Heimpel et al., 1997), after the deposition of these first eggs, further maturation of new ova and later ovipositionary activities are dependent on access to adequate nutrition (Chen, 2013; Headrick et al.,
1999; Heimpel & Collier, 1996; Heimpel et al., 1997; Jervis & Kidd, 1986), rather than on the metabolites and reserve nutrition that were acquired during her larval stages (Aung et al., 2010; Marchioro & Foerster, 2013).
26 The age of the adult female wasp has a significant effect on the amount of eggs she can produce from each host-fed meal (Aung et al., 2012). It is generally thought that as the female parasitoid ages, her physiological processes slow down, which include her reproductive processes and parasitism capacity (Aung et al., 2012; Sinatra, 1998). In a
1991 study performed by Drs. Chien, Chu, & Ku, the parasitic strategy, morphology, and life history of T. radiata were assessed. It was concluded that female T. radiata between the ages of 4 days old and 18 days old deposited one egg for every 0.18 ACP fed on
(Chien et al., 1991), and those T. radiata that were younger than 4 days old but older than
18 days old deposited one egg per every 0.29–0.38 ACP hosts fed upon (Chien et al.,
1991).
Most synovigenic parasitoids can absorb their eggs—a mechanism referred to as ovisorption—when unable to obtain proteinaceous food, such as when proper hosts are absent or at low levels. This maintains reproductive synchrony with insect host populations (Chien & Chu, 1996; Cooksey, 2017; Heimpel et al., 1997; Jervis, 2001).
Therefore, the number of eggs potentially laid by a female parasitoid and her consequent parasitism capacity can be largely determined by the available hosts and nutrients in her immediate environment (Chu & Chien, 1991; Gόmez-Torres et al., 2014). Generally, within the environment, the main nutrients available for adult parasitoids are sugars— found in the frass of ACP and in the frass of other soft-bodied homopterans—plant nectaries, honey, pollen, and nectar (Aung et al., 2010; Ball, 2007).
Typically, insects that feed on these sugar sources in the environment respond positively to a feeding solution of honey-water when in short-term storage under laboratory conditions (Grenier et al., 2005; Hall & Klein, 2014; Jervis & Kidd, 1986). It
27 has been shown that feeding female hymenopterous insects honey-water—a supersaturated sugar source that is usually 38.5% fructose and 31.0% glucose (da Silva et al., 2016)—increased both fecundity and longevity over nutrient deprivation or water alone (Aung et al., 2012; Ball, 2007; Chen, 2013; Étienne et al., 2001; Heimpel et al.,
1997). In the case of short-term storage of T. radiata, honey-water alone does not prevent ovisorption (Chen, 2013; Chien & Chu, 1996). Arrhenotokous female insects seem to either lack the ability to transfer the carbohydrates in honey-water into the essential amino acids required for egg formation, or that the oogenesis process requires other specific amino acids that cannot be obtained through honey-water feeding alone (Aung et al., 2012).
Due to the unique dual-killing capacity of T. radiata, it has been concluded that the proteins obtained specifically from ACP hemolymph are an invaluable nutrient source for further egg formation in adult females (Chien et al., 1991; Étienne et al., 2001; Meyer,
2007). Generally, sugars are thought to be fuel for energy and somatic maintenance, whereas proteins are thought to be assimilated to form mature eggs (Heimpel et al.,
1997). However, with other parasitoids that host-feed, when honey is a part of their diet, those wasps have increased rates of both fecundity and longevity (Aung et al., 2012;
Heimpel et al., 1997; Jervis & Kidd, 1986). If additional sugar sources are needed to complete the egg maturation process, there are several essential nutrients found in honey, such as carbohydrates at 65–80%; (da Silva et al., 2016), amino acids (Marchioro &
Foerster, 2013), vitamins, minerals, and proteins (Ball, 2007; Grenier et al., 2005).
Feeding trials with T. radiata confirm the earlier nutrition-requirement stereotype of this ectoparasitoid: adult female wasps need to both host-feed and sugar-feed in order
28 to survive and be reproductively successful. ACP body fluids are a tedious ingredient to acquire for a diet provided during the storage and transportation of wasps. However, ACP hemolymph is not necessarily a limiting factor for wasp longevity, as other trials record storing T. radiata for weeks with only honey-water as a food source (Chen, 2013; Hall &
Klein, 2014; Skelley & Hoy, 2004).
Drs. Chen and Stansly, who have previously researched T. radiata holding diets, found that by feeding T. radiata females mixed diets that included proteins, sugars, and carbohydrates, caused those female wasps to form more eggs than the other female wasps that were fed the individual ingredients (Chen & Stansly, 2014b; Grenier et al., 2005).
The most successful diet in those trials was honey-water and Nu-Lure (Chen & Stansly,
2014b). Nu-Lure is a corn gluten meal derived proteinaceous liquid marketed for agricultural use as an attractant and insect bait to be added to insecticidal sprays (Miller
Chemical & Fertilizer, LLC, 2011). Results from this study indicated that the additional amino acids provided by Nu-Lure satisfied the nutritional needs of the wasp, but were insufficient for oogenesis. Carbohydrates were found to not be a limiting factor for oogenesis; the addition of carbohydrates, as well as amino acids, was found to be beneficial (Chen & Stansly, 2014b).
There has yet to be a fully artificially-produced diet that can substitute for the essential proteins and amino-acids found in ACP hemolymph (Chen, 2013; Skelley &
Hoy, 2004). The best artificial diet results formulated thus far still have an insect-derived component to them, such as the inclusion of host hemolymph, host body fluids, or ova juices (Carpenter et al., 2001; Ferkovich et al., 2000, 1999; Grenier et al., 2005). If it were known how each essential nutritional element were assimilated by the wasp, the
29 resulting artificial diet would triumph over the limitations of large-scale production of biological controls for release (Grenier et al., 2005).
According to evolutionary theory, this relationship among host selection, progeny sex-ratio, and resultant progeny body size, exists because females stand to lose more by being physically smaller than males for reasons related to fitness and metabolic body maintenance (Reeve, 1987). When a small female wasp emerges, she has few eggs, approximately 4.6, and needs to host-feed immediately in order to survive (Chen, 2013;
Chen & Stansly, 2014b). If appropriately-sized ACP hosts are not in the vicinity, the small female wasp must absorb the eggs she emerged with and divert that metabolic energy to flight muscles for host-searching activities (Godfray, 1994). There is a trend among parasitoids addressing this very situation and is exemplified by the popular biological control agent, Aphytis melinus (Hymenoptera: Aphelinidae) (Flint & Dreistadt,
1998). Smaller hosts are selected for oviposition of male eggs, and larger hosts are chosen for deposition of female eggs. Consequently, smaller hosts yield more males that are smaller overall, and larger hosts yield more females that are larger overall (Reeve,
1987). With this strategy, it is advantageous for female eggs to be allocated to larger host nymphs for the reproductive success and greater fitness of future generations of parasitic wasps (Flint & Dreistadt, 1998; Reeve, 1987). This trend is noticeable with T. radiata as well; larger ACP instars have a higher percentage of female emergence: 88% female
th th progeny from 5 instars and 75% female progeny from 4 instars (Chen & Stansly,
2014b).
Various studies on solitary parasitoids like T. radiata have shown that wasps assess host nutritional quality by discrimination of host-nymph body size (Ellers et al.,
30 1998; Godfray, 1994). In general, the smaller nymphs are relegated to host-feeding activities, and the larger nymphs are used for parasitization (Chu & Chien, 1991; Skelley
& Hoy, 2004). Those eggs that were oviposited onto larger nymphs had greater survival
th th rd rates: 85%, 71%, and 33% emergence when 5 , 4 , and 3 instar nymphs were chosen, respectively (Chen & Stansly, 2014b; Chien et al., 1991; Étienne et al., 2001). Not only
th was survival rate measured, but progeny size was also measured: progeny wasps from 5
th instars measured 1.12 mm, while those that emerged from 4 instars were 0.91 mm
(Chien et al., 1991). Researchers also noted that progeny wasps that emerged from larger nymphs had a higher fitness level and greater longevity than those that emerged from smaller nymphs (Chen, 2013; Chu & Chien, 1991; Lopez & Hoddle, 2014; Tang &
Huang, 1991).
T. radiata uses host-volatiles to discriminate between available ACP hosts and already-parasitized ACP hosts in order to avoid super-parasitism (Mann et al., 2010; Sirot et al., 1996). Super-parasitism is the situation in which more than one T. radiata parasitizes the same individual host (Husain & Nath, 1927). Super-parasitism does not result in success for both deposited eggs, as, at he most, only one egg will complete its development; the other dies and is a waste of an expensive egg (Bruce & Wadhams,
2005). Super-parasitism is sporadically observed in citrus groves, usually during the cooler months when there are reduced ACP population levels (C. Buelna, personal communication, September 2016; Chien et al., 1991).
Since the potential success of an individual parasitoid in its environment is mostly size-dependent—which can be modified by genetics and environmental conditions—then
31 wasp body size is the criterion for enhanced fitness not only for an individual wasp, but for an entire generation of wasps (Ellers et al., 1998; Lopez & Hoddle, 2014). For a wasp, being physically larger means having greater fat reserves to fuel flight activity, preserve somatic maintenance, and increase egg formation (Ellers et al., 1998; Lopez & Hoddle,
2014). This means that a larger body size translates into having greater dispersal ability, which means that larger wasps can more rapidly colonize release sites. This also means that a larger female is less likely to absorb already-formed eggs because she has enough stored nutriment to sustain her while she searches for an appropriate host. And finally, larger females have increased egg formation which means that they will produce more eggs overall (Lopez & Hoddle, 2014). Furthermore, it has been posited that the smaller adult parasitoid should compensate for its small body size and fewer metabolic reserves by host-feeding more (Berger et al., 2012).
The success of a field-released biological control agent depends not only on the nuances of the direct interactions between the introduced parasitoid and its host-insect, but it also extends to biotic and abiotic factors that cause compounding selection pressures on the parasitoid’s physiological processes (Aung et al., 2010; Berger et al.,
2012; Gómez-Torres et al., 2014). Temperature and relative humidity are crucial abiotic elements that influence all facets of T. radiata development; in particular, her reproductive activity and subsequent parasitism rate (Aung et al., 2010; Chen, 2013;
Gómez-Torres et al., 2014). For instance, it has been recorded in the laboratory that the most favorable temperature for T. radiata reproduction is 25°C; which is when female fecundity reaches 300 eggs over 24 days (Étienne et al., 2001). It is therefore paramount that thermal hygrometric requirements be considered, not only during mass-rearing and
32 storage, but also when matching biological control agents to release sites (Gómez-Torres et al., 2014).
Host-density—the immediate amount of available ACP nymphs—influences the longevity of adult female wasps and has a strong effect on wasp behavior, such as choosing whether to parasitize or to host-feed (Chen, 2013; Chu & Chien, 1991; Reeve,
1987). The relationship between host-density and adult wasp longevity follows a domed parabolic response, which means that both adult female wasp longevity and adult female
th th wasp fecundity rise along with host-density to a crest—at 40 4 and 5 instars per T. radiata, 25°C, 14L:10D, and 100% RH, adult females lived 23 days and adult males lived 14 days—then both values decline as host-density continues to increase (Chien,
1995). In this way, T. radiata can maintain synchronicity with its ACP host (Chien &
Chu, 1996). Host-density plays a further role in influencing T. radiata mass-rearing
th th efficiency: 40 4 and 5 instar nymphs per female, at 25°C, 14L:10D, and 100% RH not only optimizes female wasp fecundity, but also further maximizes the incidence of parasitism (an average of 513–525 ACP nymphs [Chen, Wong, & Stansly, 2016; Chien et al., 1991; Ellers et al., 1998; Meyer, 2007]), and minimize incidence of super-parasitism
(Chen, 2013; Chu & Chien, 1991; Skelley & Hoy, 2004). This density correlation can be used to determine the ideal number of parasitoids to use in rearing cages during greenhouse mass-production or can be used to encourage T. radiata parasitism activity over host-feeding behavior once field-released (Chen et al., 2016).
Part of the reason that T. radiata releases have been successful in southern
California—and have had earlier fortunes in island ecosystems—is that T. radiata has
33 been imported, bred, and released, all without introducing its hyperparasitoids or natural enemies (Aubert, 1987; Pilkington, 2010; Qureshi et al., 2009; Qureshi et al., 2012). This is not to say that T. radiata is the top predator when released in citrus groves—many other insects hinder her impact on ACP suppression—such as ants which are the most disastrous by far (Hall & Nguyen, 2010; Qureshi et al., 2009; UC Riverside CISR, 2014).
In central Florida, certain coccinellid species exhibit intraguild predation, and cause 64% to 95% of T. radiata mortality (Michaud, 2004). Furthermore, T. radiata must compete with other insect predators for ACP nymphs. One Riverside County trial release-site reported a 66.3% T. radiata parasitism rate when walking insect predators were excluded, and only a 1.4% T. radiata parasitism rate when the ACP nymphs were exposed to all insect predators (Kister, 2014; Kister et al., 2016; Navarrete et al., 2013).
Taking into account the citrus landscape of southern California—most of which meanders through residential and private properties—T. radiata is the best option for biologically suppressing ACP (Grafton-Cardwell, 2017). These backyard niches are fragmented and inaccessible to large-scale chemical control of ACP. T. radiata can reach areas that are inappropriate for chemical or mechanical ACP control, especially in backyard plantings and urban citrus (Grafton-Cardwell & Daugherty, 2015).
Mass-Rearing the Star of Biological Control
For many experts, biological control is the only feasible sustainable method for controlling ACP on a regional scale (Hoddle, 2004). Compared to pesticide use, the augmentation of host-specific parasitoids is more likely to result in long-term, cost- effective control of ACP with fewer negative impacts on non-target species (Hoddle &
34 Hoddle, 2013; UC Riverside CISR, 2014). In order to inundate release sites with wasps, there need to be more efficient methods to mass-produce, store, transport, and release the wasps (Chen, 2013).
As of this publication, HLB has been confirmed in citrus trees in Riverside
County, southern California in July 2017 (Citrus Insider, 2017), and this discovery pressures insectaries to ramp up mass-production of T. radiata. Mass-rearing of ACP parasitoids for release in southern California—now firmly the joint responsibility of the
CDFA and CRB (Milosavljević, Schall, & Hoddle, 2017)—requires greenhouse production of clean curry leaf plants (Murraya koenigii), which are then inoculated with
ACP adults in a separate and secure location, and are finally exposed to the parasitic wasps for oviposition in a third discrete location (California Department of Food and
Agriculture [CDFA] Mt. Rubidoux, 2017; Flores & Ciomperlik, 2017; Qureshi et al.,
2012).
Once the wasps emerge, they are collected from the CDFA greenhouses and are stored under cooler conditions (12.8°C, RH 60−80%, in the dark) in the CDFA
Laboratory (CDFA Mt. Rubidoux, 2017). During this holding period, also known as short-term storage (Hall & Klein, 2014), nutritional foods provided to the adult female affect the number of eggs she forms, which influence her parasitism capacity, and thus, her efficiency as a biocontrol agent upon release (Chen, 2013; Marchioro & Foerster,
2013). Holding periods can last for days or weeks before the wasps are transported from the rearing insectary to the citrus producers, or to state and county agencies for release
(A. Muniz, personal communication, 2017; Hall & Klein, 2014). Furthermore, the longer the parasitoids are in storage, the fewer eggs they have and the lower their parasitism
35 rates become (Hall & Klein, 2014).
This demonstrates the need for better rearing and storage conditions (with a nutritious diet being paramount), and fitter strains of the wasp in order to maintain synchrony within the plant-pest-parasitoid complex: the timing and availability of plant flush, ACP oviposition and nymphal developmental rates, and T. radiata host-feeding and parasitism rates once released (Chien & Chu, 1996; Hall & Nguyen, 2010; Monzo et al., 2014; Qureshi et al., 2012; Skelley & Hoy, 2004; Wager-Page, 2010).
36 EXPERIMENT ONE. REGENERATION OF EGG LOAD
Introduction. A Synovigenic Solitary Ectoparasitic Wasp
With the introduction of the Asian citrus psyllid (ACP) (Diaphorina citri)
(Kuwayama) (Hemiptera: Psyllidae) and the lethal bacterium Candidatus Liberibacter asiaticus (CLas) to southern California, mass production of the natural enemy of ACP
Tamarixia radiata (Waterston) (Hymenoptera: Eulophidae) (T. radidata) is well underway. The mass production of T. radiata for biological control releases has created the need for optimal storage conditions that enhance the wasp’s egg load upon release
(Chen, 2013). The diet provided to female wasps during the storage period prior to release may be the foremost factor that determines the number of mature ova destined for oviposition, which greatly influences the wasp’s efficiency once released (Chen &
Stansly, 2014b; Gómez-Torres, Nava, & Para 2012).
Synovigenic species such as T. radiata emerge with few mature eggs but can produce new eggs when proper food resources are made available in the environment.
This means that the reproductive success of this parasitoid is partially determined by diet
(Aung et al., 2010). Furthermore, host-derived nutrients (ACP nymph hemolymph) contribute to overall fecundity of the wasp, whereas sugar and carbohydrate feeding are credited with contributing to the longevity of the insect (Aung, Takagi, & Ueno, 2010;
Jervis & Kidd, 1986).
This link between dietary nutrition intake and egg formation in certain synovigenic species of parasitic wasps has been well-detailed (see, for example: Chien &
Chu, 1996; Cooksey, 2017; Jervis, Heimpel, Ferns, Harvey, & Kidd, 2001). In this case,
37 the female T. radiata is able to deposit 4.6 mature eggs on average directly after eclosion
(Chen, 2013), but prior to host-feeding or mating (Jervis & Kidd, 1986). The metabolic compounds that she obtains from her larval and pupal stages of development are typically exhausted after this deposition of her first egg load (Cooksey, 2017; Étienne, Quilici,
Marival, & Franck, 2001; Heimpel & Collier, 1996; Wheeler, 1996). In order for a young female to then produce additional eggs later in life, she relies on the nutrition she can obtain from feeding during her adult stage (Aung et al., 2010). Necessary materials needed for egg development such as sugars, amino acids, inorganic salts, vitamins, and proteins can be environmentally available to the adult female T. radiata through host- feeding on ACP and its nymphal honeydew frass secretions (Chen & Stansly, 2014b), by receiving mating packages from male T. radiata, and through accessibility to plant-host nectaries (Aung et al., 2010; Cooksey, 2017; Halbert & Manjunath, 2004; Heimpel &
Collier, 1996; Stenberg, Heil, Ahman, & Björkman, 2015).
Objective. Egg Regeneration Through Diet
No artificial diets currently exist to rear T. radiata, and so the ACP host must also be reared (Skelley & Hoy, 2004). The main purpose of Experiment One was to determine the effects of an artificial diet on oogenesis with the ultimate goal of maintaining, increasing, and possibly regenerating reproductive potential during storage, and furthermore optimizing the wasp’s parasitism rate once released. The experiment measured egg load after feeding on a sugar solution (honey-water) and then switching to one of four diet treatments (A, C, G, or I) for a 24-hour, 48-hour, or 72-hour exposure length. The main concerns of Experiment One were the interacting factors of age, diet,
38 and exposure length to that diet. Since T. radiata is capable of continuously producing eggs when nutrients are abundant, she can likewise resorb her eggs in the interest of self- preservation when resources are scarce (Chen, 2013).
When T. radiata employs ovisorption, she does so in the interest of somatic maintenance. She does this by breaking down her already-formed egg material and redistributing those metabolic components for her corporal sustenance (Chen & Stansly,
2014b.; Chien & Chu, 1996; Heimpel, Rosenheim, & Kattari, 1997; Jervis et al., 2001;
Jervis & Kidd, 1986). This is common during the storage stage of mass production when she does not have access to ACP hosts on which to feed or deposit her eggs (Hall &
Klein, 2014). This ovisorption mechanism aids T. radiata with host synchronicity—a reaction to environmental triggers such as host-density, availability of appropriately-sized hosts, temperature, and relative humidity (Chen & Stansly, 2014b; Chu & Chien, 1991;
Jervis & Kidd, 1986).
Other factors besides diet that are known to influence T. radiata fecundity are her age (Chen, 2013; te Velde & Pearson, 2002), body size, and length of exposure to a nutritive diet (Heimpel & Collier, 1996; Honěk, 1994; Visser, 1994). These factors are recorded throughout this experiment with specific interest in the multi-way interactions among these factors and diet treatments.
Wasp Age
With attention to female T. radiata age, the general consensus is that the older she is, the smaller her egg load will be (Hall & Klein, 2014). This could be the result of her older ovaries not being as productive as they once were or that older females are deficient
39 in some nutrient that is responsible for stimulating egg formation (Acosta, Fonseca,
Desbats, Cerqua, Zordan, Trevisson, & Salviati, 2016; Ben-Meir, Burstein, Borrego-
Alvarez, Chong, Wong, Yavorska, Naranian, Chi, Wang, Bentov, Alexis, Meriano, Sung,
Gasser, Moley, Hekimi, Casper, & Jurisicova, 2015; te Velde & Pearson, 2002;
Yavorska, 2012). It has further been postulated that older females may be devoting more of their energy to somatic maintenance than reproduction, which also leads to a reduction in egg load (Jervis et al., 2001; Lang-Fen, Dong-Dong, Pan, Zhong-Shi, & Zai-Fu, 2015; te Velde & Pearson, 2002). In this experiment, I predicted that the experiment results will conclude similar findings: the older the female wasp, the fewer eggs she has. It was further proposed that the precise age of the female T. radiata at the time of feeding is of the utmost importance if those nutrients are supplied too late in life, or for too brief a period, her mature egg load could be significantly reduced (Aung et al., 2010; Jervis &
Kidd, 1986). However, if a nutritive meal given to older females under laboratory storage conditions could rejuvenate their egg-forming capacity, then even older females that were previously thought to have limited function in the field could potentially be reared to have a heavier egg load and a greater parasitism rate when field-released.
Wasp Body Size
Female T. radiata body size measured by a wasp’s right hind tibia length, can further influence her potential fecundity (Heimpel & Collier, 1996; Visser, 1994). Several studies have shown that body size correlates positively with oviposition rate and is generally a good indicator of potential fecundity (Berger, Olofsson, Friberg, Karlsson,
Wiklund, & Gotthard, 2012; Lopez & Hoddle, 2014). A larger female not only has
40 greater physiological reserves for egg production and longevity, but she has greater fitness in the field via enhanced searching efficiency, allowing her to hunt for her host over greater distances (Skelley & Hoy, 2004; Visser, 1994). As solitary adult female parasitoids can also attribute their greater mass to the size of the host from which they developed and emerged, genetics can also play a role in the body size of these types of insects (Visser, 1994). Although that information was not available for T. radiata explicitly it was still predicted that the resulting data would support the trend that the larger the female, the greater the number of eggs she would have.
Exposure Length to Diet
Exposure length to the diet treatments is another factor that was considered important in egg load recovery. Published reports indicate that at least 24 hours are necessary for other synovigenic wasp species to mature new eggs after a period of starvation (Aung et al., 2010; Heimpel et al., 1997; Jervis et al., 2001). For this experiment, it was expected that at least 24 hours would be needed for this metabolic assimilation and conversion process and that exposure to the diet for greater than 72 hours would be detrimental to her egg load, as the longer the period of time she was stored, the lower her egg load would be (Grenier, Gomes, Febvay, Bolland, & Parra,
2005; Hall & Klein, 2014). Exposure length to the diet treatments was measured with the intention of computing the assimilation time needed for ingested nutrients to be converted into mature, viable eggs without losing eggs to ovisorption (Chen & Stansly, 2014b;
Gόmez-Torres et al., 2012; Grenier et al., 2005; Heimpel & Collier, 1996; Jervis et al.,
2001).
41 Diet Treatments
The artificial diet treatments should be simple to acquire, store, and administer in laboratory situations where beneficial insects can be stored for up to one month (Skelley
& Hoy, 2004). The diet treatments also need to be rich in the nutrients that older females use for egg formation and maturation (Chen & Stansly, 2014b; Grenier et al., 2005;
Hervet, 2016; Jervis & Kidd, 1986; Wheeler, 1996). Maintaining or even increasing egg load within an individual female wasp through an artificial nutrient-rich diet could lead to greater parasitism rates and therefore quicker suppression of ACP populations at release sites (Chen, 2013; Chen & Stansly, 2014b; Hall & Klein, 2014).
Materials and Methods. Age, Exposure Lengths, and Diet Treatments
T. radiata were reared in tent-style bug dorms (BugDorm-2 400F, length 75 cm x width 75 cm x height 115 cm, mesh size 150 x 150) in glasshouses at the California
Department of Food and Agriculture (CDFA) rearing facilities at the Mt. Rubidoux Field
Station at 4500 Glenwood Drive, Riverside, CA 92501 (CDFA, 2017). Standard operating procedures were defined by the CDFA for the production of the two parasitic wasps: T. radiata and Diaphorencyrtus aligarhensis (Shafee, Alam, & Argarwal)
(Hymenoptera: Encyrtidae) (CDFA, 2017; Rohrig, 2014). Tamarixia. radiata populations were maintained in synchrony with a supply of D. citri nymphs and mature curry leaf plants (Murraya koenigii) (CDFA, 2017). CDFA staff collected T. radiata daily with automatic aspirators into plastic jars varying in size from 50–500 mL with approximately
200–500 T. radiata each (G. Radabaugh, personal communication, 2017; see Appendix A for thesis work contacts). Directly after collection from greenhouses, T. radiata (mated
42 males and females having mass-fed on feeding strips impregnated with a honey-water solution) were stored in dark laboratory coolers at 12.8ºC at the CDFA facility.
Tamarixia. radiata vials were carefully wrapped and packed with towels and small foam blocks next to an ice pack (Qureshi et al., 2009). Vials were transported to the CPP Plant
Sciences Laboratory in dark, insulated cooler bags.
As soon as T. radiata vials arrived at the CPP Plant Sciences Laboratory, they were stored in a dark wine cooler at 12.3°C, with access to a honey-water feeding strip that was replenished as needed (Hall & Klein, 2014). Tamarixia radiata were stored in this manner until old enough to participate in the feeding trials at 6 days-old (DO), 12DO, and 18DO (Heimpel & Collier, 1996; Marchioro & Foerster, 2013). Prior to the distribution of T. radiata in the CPP Plant Sciences Laboratory, comprehensive observations were made which included general T. radiata physical activity, overall body size, and an estimated sex ratio of the vial population.
Clear acrylic propagation cages with a mesh-side and tie-off sleeve (length 19 cm x width 19 cm x height 35.6 cm), a Craft Smart glue gun, Surebonder glue sticks for minor holes in the mesh sleeves, compound binocular light microscopes (American
Optical One-Fifty, American Optical Company), a three-prong extension cord, an upright freezer (-18°C; located in CPP Plant Sciences Laboratory), small wine coolers (Haier
Thermoelectric Wine Cellar, Model# HVTECO6ABS, Serial #15091110313), and the
Caron Insect Growth Chamber (Model #6025, 708L, W90 cm x H195 cm x 84.5 L) were used in this experiment.
When T. radiata were at the first designated test age—6DO, presumably mated, and satiated from the provided honey-water solution—they were randomly separated and
43 passively recollected into treatment vials in the CPP Plant Sciences Laboratory. Although all T. radiata were raised at the same CDFA facility, they were collected from different insect-rearing dorms and from different production glasshouses (CDFA, 2017). Even within an individual glasshouse there exist microclimates that can be the cause of extensive biological variation within the reared T. radiata populations (A. Muniz, personal communication, November 2015). The T. radiata populations fluctuated not only in body size, but also in sex ratio and egg load (C. Buelna, personal communication
December 2015; CDFA, 2017).
In order to maintain a representative sample for the experiment, the 6DO T. radiata were released into small, clear acrylic containers in the CPP Plant Sciences
Laboratory, and were then passively recollected through manipulating the light source into smaller, clean vials of 50–150 T. radiata each (CDFA, 2017; Skelley & Hoy, 2004).
[Author’s note: Manual aspirators were not used for T. radiata recollection as it caused unnecessary stress and physical damage to T. radiata. It was observed that females tended to be more active (flying and walking toward the light), whereas males were less mobile and remained near the bottom of the propagation cages (Skelley & Hoy, 2004)].
Four diet treatments were tested. Batches of each diet treatment were mixed in individual vials in the CPP Plant Sciences Laboratory. Diet treatment measuring tools included Eppendorf tubes (5.0 mL, clear polypropylene tubes with snap caps,
#0030119401), glass funnel, small silicone spatula, narrow honey spreader, small glass stirrer, a weight-measurement scale (Taylor digital glass-top food scale, #49178540,
UPC# 077784025468), plastic measuring boats for weighing solid ingredients, small measuring glass cylinders (Pyrex, 10−500 mL), all-purpose scissors, Scott blue super
44 absorbent shop towels, and small mortar and pestle. Access was provided to the following supplies: manual aspirator (BioQuip 6 mm, latex tubing, 1135A, 9-dram clear styrene vial with snap on cap, #1135Y HEPA filter) to remove males from vials, as well as collection vials with lids (9 and 15-dram; 55 mL, clear polystyrene vials with snap cap,
E&K Scientific), Ziploc bags (quart and gallon sizes), Sharpie pens, 1.0 inch binder clips,
3M colored masking tape, Parafilm (Sigma-Aldrich, #P7793), and plastic jars with screw- top lids (16 oz., clear BPA-free PET storage containers with screw-top sealing caps).
Recording materials included paper for collection data forms (see Appendix B), pens, calculator, clipboards, Microsoft Excel computer program, printer, printer ink, optical rulers for the American Optical compound microscope (WF10X, 18 mm, 0.1 mm scale), temperature and relative humidity monitoring devices (EXTECH Instruments
Hygro-Thermometer Clock #445702), and both AAA and AA batteries. In order to distinguish between female and male T. radiata, a personal iPhone 5S camera and a hand lens (magnifier-LED double-multiple jewelry identifying type, 30 x 22 mm, 60 x 12 mm) were used to observe their antennal and abdominal differences (Skelley & Hoy, 2004;
Waterston, 1922).
Diet treatment recipe ingredients included a large and accessible source of ACP nymphs, ACP adults, and ACP nymphal frass, honey (raw orange blossom virgin honey,
Winter Park, Winter Park, FL), deionized water, pre-made royal jelly and honey blend
(Y.S. Eco Bee Farms, royal jelly in honey), and a pure powder form of Coenzyme Q10
(Sigma-Aldrich Life Sciences, #C9538, 100 mg). Access to each assigned diet treatment was provided via a 2.0 cm x 3.0 cm feeding strip of Scott blue super-absorbent shop towel that was placed on the inside of each 50 mL vial cap. Each vial held a combined
45 total of 50−100 male and female T. radiata able to feed and mate ad lib for the designated exposure length.
Length of Exposure to Diet
Adult T. radiata were assigned to age groups (6DO, 12DO, or 18DO), treatment exposure lengths (24-hour, 48-hour, or 72-hour) (Chen & Stansly, 2014b; Gómez-Torres et al., 2012; Jervis & Kidd, 1986), and to diet treatment feeding groups (A, C, G, and I).
During the exposure times to diet treatments, T. radiata vials were placed inside a Caron insect growth chamber at 25ºC with 70–100% relative humidity (Caron, 2017; Chen &
Stansly, 2014b; Chu & Chien, 1991; Gómez-Torres et al., 2012; Hall & Klein, 2014;
McFarland & Hoy, 2001; Skelley & Hoy, 2004). After each exposure period had elapsed, participant T. radiata were terminated by means of the CPP Plant Sciences Laboratory upright freezer (-18°C). Vials remained in the freezer for at least 24 hours before dissection of the females. Dissections under the American Optical compound light microscope provided counts of mature egg load (A. Soper, personal communication,
September 2015; Aung et al., 2010).
Dissection tools included ultrafine forceps (BioQuip #4535 forceps #5, Swiss pattern, super fine-honed, high-quality stainless steel, 112 mm), plastic pipette dropper (4 mL), bent probe with sharp tip, needle-point scriber probe (full spear, stainless steel,
#4755), teasing needle (angled tip, #4752), dissecting scissors (straight, sharp points, stainless steel, 114 mm, #4713), glass microscope slides (Karter Scientific microscope slides, 206A2), and glass coverslips (Karter Scientific microscope cover glass, 22 x 22 mm, 0.13–0.16 mm, #141473, #211Z3).
46 Though hygrometric conditions were set to the optimal temperature and relative humidity for the T. radiata to feed and form eggs, according to current CDFA data and literature, there were vast irregularities with premature mortality between the T. radiata vials (CDFA, 2017; Gómez-Torres et al., 2014; McFarland & Hoy, 2001). This may have been the result of toxic gases emitted from the metals and plastics of the Caron insect growth chamber, or it may have been a defunct humidifier (C. Buelna, personal communication, September 2015). Furthermore, T. radiata are diurnal meaning that the majority of their life activities occur during daylight hours, and a fortiori, in the beginning hours of the morning (Chen, 2013; Chen & Stansly, 2014b). As the Caron insect growth chamber lacked an internal light source, the T. radiata feeding activity could have been thus impaired (Caron, 2017; Hall & Klein, 2014; Hervet, Laird, &
Floate, 2016; Skelley & Hoy, 2004).
Dr. David Morgan of the CDFA offered solutions to the issues with the Caron insect growth chamber. He suggested putting the vials of wasps into buckets of water inside the insect growth chamber (D. Morgan, personal communication, January 2016).
The idea was that these water reservoirs would cancel out noxious gases such as benzene, styrene, and phenol emitted from the growth chamber’s metal and plastic components (D.
Morgan, personal communication, January 2016; Destaillats, Maddalena, Singer,
Hodgson, & McKone, 2007). Additionally, wasp vials were sealed with parafilm and were double-bagged and clipped to the edge of the water containers in such a way that the vials would not be immersed in the reservoir water. It took several weeks to streamline practices so that every T. radiata vial placed inside the Caron insect growth chamber did not perish preceding the end of experiment. This was also the practice when storing T.
47 radiata vials inside the wine coolers in the CPP Plant Sciences Laboratory prior to feeding trials. This was done because of the irregular humidity and possible toxic gas emissions from the wine coolers that could have caused premature mortality of the wasps.
Diet Treatments
None of the diet treatments stored well between trials. In an effort to provide fresh diet treatments (see Appendix C for diet treatment formulations) to avoid further untimely deaths due to the possible lethality of the diets themselves, all diets were mixed and administered to wasps which were then placed in the growth chamber, all in under 15 minutes (Ball, 2007; da Silva, Gauche, Gonzaga, Costa, & Fett, 2016). Paper towel feeding strips measuring 2.0 cm x 3.0 cm were cut prior to mixing diets and were quickly dipped into the diet treatment solutions, blotted off, and placed on the inside of the plastic vials’ caps (Hall & Klein, 2014; Skelley & Hoy, 2004). Excess diet material was blotted off to minimize the incidence of T. radiata getting trapped in the diet treatment; wasps that were attached to feeding strips at time of dissection were not recorded (Hall & Klein,
2014).
Diet Treatment A consisted of 75% pre-made royal jelly and honey blend mixed with 25% deionized (DI) water. Royal jelly is a worker bee (Apis mellifera) glandular secretion (Grout, 1946). It is roughly comprised of 12−15% proteins, 10−12% sugars,
3−7% lipids, and varying amounts of amino acids, vitamins, and minerals (Miyashita,
Kizaki, Sekimizu, & Chikara, 2016). It is used exclusively by the worker bees to feed the queen bee in the hive (Grout, 1946; Morita, Ikeda, Kajita, Fujioka, Mori, Okada, Uno, &
Ishizuka, 2012; Wang, Ma, Zhang, Cui, Wang, & Xu, 2016). Royal jelly has a higher
48 percentage of protein, glucose, and sucrose than regular worker bee honey (Wang et al.,
2016). When the queen honeybee, a holometabolous hymenopteran insect, feeds on royal jelly, not only does it induce queen differentiation but it also uniformly enlarges her entire body. This effect, as well as increased longevity and higher fecundity, are also outcomes in non-holometabolous insects such as silkworms (Bombyx mori) and spotted crickets (Gryllus bimaculatus) when fed royal jelly during their immature stages
(Miyashita et al., 2016).
Diet Treatment C was a 75% honey and 25% DI water solution. This is what the
T. radiata fed on prior to being assigned to a diet treatment group (A, C, G, or I). Honey- water is the contemporary industry diet for stored hymenopteran parasitoids used for biological control releases (Aung et al., 2012; CDFA, 2017; Étienne et al., 2001;
Marchioro & Foerster, 2013; Skelley & Hoy, 2004). For this study, raw orange blossom honey was used and stored at room temperature (20–25°C) in the dark (Ball, 2007;
CDFA, 2017; da Silva et al., 2016; Hall & Klein, 2014; Jervis & Kidd, 1986).
Diet Treatment G was a mixture of 50% ACP, 25% honey, and 25% DI water
(Chen & Stansly, 2014b; Skelley & Hoy, 2004). Diaphorina citri eggs, all stages of nymphs, and adults were procured from colonies that were reared in the CPP Plant
Sciences Laboratory (C. Buelna, personal communication, September 2015) and at the
CDFA insect rearing facility at the Rubidoux Field Station (J. Herreid, personal communication, March 2016). ACP bodies were frozen at -18℃; their frosty bodies were crushed using a mortar and pestle (Grenier et al., 2005) and then mixed with a honey- water solution at the appropriate concentrations. ACP host tissues including hemolymph are rich in proteins, fats, sugars, essential vitamins, and salts that are needed both as fuel
49 for maintenance and for egg production (Heimpel & Collier, 1996; Jervis et al., 2001;
Jervis & Kidd, 1986).
The chief ingredient in Diet Treatment I was the nutritive supplement Coenzyme
Q10. Coenzyme Q10 is a human supplement that plays a key role in cell energy production, enhances egg quality and female fertility, and shows positive results in helping the ovaries of older mammals to perform better (Ben-Meir et al., 2015; Sinatra,
1998; te Velde & Pearson, 2002; Yavorska, 2012). In order to garner the benefits of
Coenzyme Q10, it must be dissolved in lipids, hence honey was added as an additional ingredient. Honey contains a number of lipids, although in small quantities. Such lipids include hydrocarbons, fatty acid esters, sterols, waxes, cholesterol esters, and fatty acids
(Kapoulas, Mastronicolis, & Galanos, 1977). In this treatment, pure Coenzyme Q10 powder was stored in the CPP Plant Sciences Laboratory upright freezer at -18°C in the dark. The mixture contained 50% Coenzyme Q10, 25% honey, and 25% DI water.
Studies on female mammals have concluded that a deficiency in Coenzyme Q10 in the body can impair important mitochondrial function and performance, such as lowered adenosine triphosphate (ATP) production which drives age-associated oocyte deficits and leads to infertility in older females (Acosta et al., 2016; Ben-Meir et al.,
2015; Sinatra, 1998). If the ovaries of older T. radiata were to be rejuvenated, not only would egg load be positively affected, but her energy stores would also be replenished and ultimately T. radiata would be able to be stored longer; she would be just as potent when released at an older age as when released at a younger age.
Because these trials focused on the female wasp’s ability to resorb her eggs and then produce more eggs at a later date, the T. radiata in Experiment One had previous
50 access to honey-water for the first five days of their lives until feeding trials took place at ages 6DO, 12DO, and 18DO. It was necessary to record if the female wasp was recycling egg components when diets were provided or if they were producing fewer eggs due to aging (Ben-Meir et al., 2015; te Velde & Pearson, 2002).
T. radiata populations were passively separated and randomly assigned to diet treatments and exposure lengths and placed into the insect growth chamber for their designated holding periods of 24 hours, 48 hours, or 72 hours (Fathipour, Hosseini,
Talebi, & Moharramipour, 2006). Tamarixia radiata that were to be used at the next data- collecting interval were returned to the CPP wine cooler until they were old enough to participate in later feeding trials (Destaillats et al., 2007).
Death of T. radiata
After the assigned exposure lengths had elapsed for each age parameter, the vials of T. radiata were placed in the CPP Plant Sciences Laboratory upright freezer. After 24 hours at -18°C, T. radiata were probed and dissected in accordance to the dissection protocol as recommended by A. Soper, professor of Plant Sciences at California State
Polytechnic University, Pomona (A. Soper, personal communication, September 2015).
For a detailed explanation of the dissection protocol, see Appendix D. The technique was complex and took a certain finesse. [Author’s note: CPP students helping with the project regularly had discrepancies in measurements. In order to maintain consistency and efficiency in the recording of data, only two people who conferred regularly performed the actual dissections on the female T. radiata under compound light microscopes (C.
Buelna, personal communication, February 2016; CDFA, 2017)].
51 The sample sizes of female T. radiata ranged between 100 and 300 individual female T. radiata for each age, diet treatment, and exposure length. Egg load and hind tibia length (HTL) were recorded for every single female T. radiata. It was necessary to overestimate the amount of test subject wasps needed from the CDFA in order to account for early mortality and unpredictable sex ratios. Also, T. radiata availability was variable due to mechanical difficulties with mass production (A. Muniz, personal communication,
2015) and with large quantities of wasps reserved for CDFA site releases in southern
California. Thus, weekly collection trips to the CDFA Mt. Rubidoux field station were made continuously from September 2015 through May 2016.
Results. It’s All About the Egg Load
It is because these trials dealt with a biological organism that the data from
Experiment One did not align with a typical bell-shaped-curve distribution. Additionally, it was not anticipated that there would be such a disproportionate level of zero egg load
(meaning the wasp had no eggs at the time of dissection) across each tested age (6DO,
12DO, 18DO), each tested exposure length (24 hours, 48 hours, 72 hours), and each tested diet treatment (A, C, G, I). The overwhelming amount of zero egg load data was a confounding issue but was ultimately determined to not be so dissonant as to manually remove all zero egg load counts from the analyzed data set, therefore zero egg loads were left in the data set (D. Morgan, personal communication, March 20, 2018).
Including Zero Egg Load
The entirety of the data including all those wasps with zero egg load (meaning all
52 4,556 wasps that were dissected) and the relationship between body size (HTL) and egg load are shown in Figures 1 and 2. The descriptive statistics used to evaluate the egg load of all recorded data points showed that the mean egg load was 2.681 and the standard deviation was 3.160. The mean and standard deviation for HTL were 3.110 and 0.396, respectively. To better evaluate the relationships between the recorded variables, a
Pearson correlation was used to analyze the numerical values of HTL and egg load, which showed a value of 0.142 and a significant p value < 10 -4. A Spearman rank correlation was used to evaluate the following relationships among the categorical data: egg load and age: -0.202, egg load and exposure length: -0.093, HTL and age: 0.111, and
HTL and exposure length: 0.056. All p values from the Spearman rank correlation were significant with a p value < 10 -4 for all categorical data.
The ANCOVA Model
Regression was used to evaluate the relationship between the numerical and categorical variables. Two-way Analysis of Covariance (ANCOVA) model is a general linear model which blends ANOVA and regression. ANCOVA evaluates whether the means of the dependent variables are equal across all levels of a categorical independent variable (treatment), while statistically controlling for the effects of other continuous variables that were not of primary interest—known as covariates (H. Kim, personal communication, June 2017). In this instance, ANCOVA used egg load as the dependent variable. The diet, exposure length, and age of wasp were dependent variables, and HTL was used as the covariate. ANCOVAs were performed using statistical software SAS
(SAS Institute, 2004). All significance testing was done at = .05, or 5% (H. Kim,
53 personal communication, March 22, 2018).
In the main analysis, since the F-value was sufficiently large (F-value = 189.37) and the p value was < 10 -4, the ANCOVA model was statistically significant at the 5% level. The r-squared value of 0.159 indicated that the model explains 15.9% of the total variability in the egg load.
Wasp Age and Egg Load
Data from Experiment One agrees with the literature supporting the theory that younger T. radiata have a greater egg load than older T. radiata (Hall & Klein, 2014).
The mean egg load for the wasp ages are as follows: 6DO wasps had 3.70 eggs on average, 12DO wasps had 2.53 eggs on average, and 18DO wasps had 1.73 eggs on average at the time of dissection (see Figure 3). The differences were all statistically significant (p value < 0.001).
Exposure Length and Egg Load
Exposure length to diet could be an important factor in preventing the loss of mature eggs to ovisorption or infertility due to reproductive aging (Sinatra, 1998, te
Velde & Pearson, 2002). The exposure lengths of both 24 hours and 48 hours yielded higher egg loads (2.84 eggs and 3.07 eggs, respectively) than the 72-hour exposure length
(2.05 eggs), which coincides with previous studies suggesting that longer storage and diet exposure lengths may prove detrimental to egg load (Hall & Klein, 2014; see Figure 4).
The difference between the average egg loads for the 24-hour and 48-hour exposure lengths was not statistically significant (P = 0.101 > 0.05). However, the differences
54 between the 24-hour and 72-hour exposure lengths and 48-hour and 72-hour exposure lengths are all statistically significant (p value < 0.001). The data shows that wasps should not be exposed to diet for longer than 48 hours, as egg load decreases with a longer exposure length.
Diet Treatment and Egg Load
Overall Diet G (ACP) was responsible for a greater egg load than the other three diets—Diet A (royal jelly), Diet C (honey), and Diet I (Coenzyme Q10)—all of which had a similar effect on egg load (see Figure 5). The average egg loads for the diet treatments, in order of greatest to least are Diet G (2.94 eggs), Diet I (2.61 eggs), Diet A
(2.54 eggs), and Diet C (2.53 eggs). The difference between Diet G (ACP) and all other diet treatments is statistically significant (p value < 0.05). However, the differences among diets A, C, and I, are not statistically significant (p value > 0.05). This means that although ACP is not feasible to use as an artificial storage diet, the other diet treatments
(royal jelly and Coenzyme Q10) increased egg load and could be added to the currently- used artificial diet of honey-water.
Two-Way Interaction: Diet Treatment and Age of Wasp
Different ages of wasps are affected by diet differently (F6, 4531 = 3.36, p = 0.001; see Figure 6). Overall, the 6DO T. radiata had a higher average egg load across all four diet treatments when compared to both the 12DO and 18DO T. radiata. In order, the greatest producing diet treatments for 6DO wasps were Diet Treatment G (ACP) with
3.84 eggs, Diet Treatment C (honey) with 3.77 eggs, Diet Treatment I (Coenzyme Q10)
55 with 3.66 eggs, and Diet Treatment A (royal jelly) with 3.52 eggs on average. The diet treatment and wasp age combination that produced the least amount of eggs on average was Diet Treatment C (honey) at 18DO (1.18 eggs).
Two-Way Interaction: Wasp Age and Exposure Length
If wasps of different ages are exposed to the diet treatments (A, C, G, or I) for different amounts of time (24 hours, 48 hours, or 72 hours), then the egg loads are different (F4, 4531 = 21.3, p < 0.001; see Figure 7). Among the results, wasps that were
6DO and exposed to diet treatments for 24 and 48 hours (4.26 and 4.22 eggs, respectively) produced the greatest egg loads while wasps that were 18DO and exposed to diet treatments for 72 hours produced the least amount of eggs (0.95 eggs). Overall, according to the data from Experiment One, the younger the wasp and the shorter the diet exposure length to diet treatment, the greater the wasp’s egg load.
Two-Way Interaction: Diet Treatment and Exposure Length
The two-way interaction between diet treatment and exposure length shows that wasps had different egg loads when fed different diets depending on how long they were
-4 exposed to those diets (F6, 4531 = 38.09, p value < 10 ; see Figure 8). Among them, the combination of Diet Treatment I (Coenzyme Q10) and the 24-hour exposure length produced the greatest egg load (4.25 eggs), followed by Diet G (ACP) at 48 hours (3.96 eggs), Diet C (honey) at 48 hours (3.56 eggs), and Diet A (royal jelly) at the 48-hour exposure length (3.02 eggs). The diet and exposure length combination that produced the least amount of eggs was Diet I at the 72-hour exposure length (1.77 eggs). Overall,
56 wasps that were held and exposed to diets for shorter periods of time, such as for 24 and
48 hours, had higher egg loads than those that were older and held for longer periods of time. This supports another study that finds shorter storage periods to be more beneficial for greater wasp egg loads (Hall & Klein, 2014).
Discussion. Age, Body Size, and Diet Treatment
The results of these trials showed a positive relationship among the factors of age, body size, diet treatment and the resulting egg loads.
Age of Wasp
Age is an important factor in wasp storage because wasps live longer under laboratory conditions (with younger and larger wasps tending to have more eggs than older wasps) than they do in their natural environment (Heimpel & Collier, 1996).
Moreover, there is a plasticity in the timing of reproductive activities that comes with being a synovigenic wasp species. It has previously been found that T. radiata has the flexibility to “shift” egg production toward an earlier age in order to take advantage of nutrients carried over from the larval stage (Jervis et al., 2001). If these eggs that she has upon eclosion are not removed immediately by oviposition (usually within five days when deprived of outside nutrients), these eggs must be eliminated in another fashion: through ovisorption (Chen & Stansly, 2014b). Ovisorption is dual purpose as it can be an economical way of providing space for newly-formed eggs as well as providing nutrients for adult maintenance and sustaining oogenesis until proper host-feeding and parasitisation can be resumed (Jervis & Kidd, 1986).
57 Wasp Age and Diet Treatment
Adult female T. radiata live an average of 23.6 days and at 12DO, the female T. radiata is halfway through her life cycle (Mann & Stelinski, 2010). Perhaps other physiological needs and limitations are activated at this point in her life cycle and a more complex diet is needed to satiate these chemical desires. Another possibility that may explain the differences in egg load between older and younger wasps is that as the adult female wasp ages, she needs different nutrients at different times in her life to produce eggs and restore her metabolic losses. Creating and administering these mixed diets positively contributes to producing more robust wasps as compared to providing them with only proteins or only sugars (as is the current practice) (Chen, 2013).
Age and Exposure Length
When considering the best age at which to release wasps, it is also imperative to consider the exposure length to diet of the wasps in storage. Wasps can live longer in controlled laboratory conditions, but the longer they are stored—even with access to a beneficial diet—the lower their parasitism rates are in the field (Hall & Klein, 2014).
Similar results have been recorded for wasps that were deprived of a diet altogether: the older the wasp, or the longer she was held, the lesser her parasitism rate (Chen, 2013;
Chen & Stansly, 2014a). The next question to answer is if the oldest wasps are capable of egg load recovery and which diet treatment best allows for that. This experiment did not show that egg load recovery was possible with these specific diet treatments and exposure lengths.
58 Diet Treatment
Oogenesis is a nutrient-limited process that is only initiated when sufficient nourishment is obtained for egg production (Carpenter, Ferkovich, & Greany, 2001;
Wheeler, 1996). More specifically, the types of nutrients obtained have a particular function on the oogenesis process. Feeding hymenopterous wasps sugar diets contribute positively to longevity whereas protein-rich meals increase fecundity (Heimpel,
Rosenheim, & Kattari, 1997). This is proven true in many instances in which honey, or a sugar diet alone, was not enough to prevent resorption of already-formed eggs (Chen &
Stansly, 2014b). Moreover, in rearing and storage conditions it is the goal that both sustained fecundity and increased longevity are obtained (D. Morgan, personal communication, March 2018).
ACP host hemolymph is rich in proteins and is a critical source of nutrients for female T. radiata as it increases wasp fecundity and egg maturation rates (Heimpel et al.,
1997; Skelley & Hoy, 2004). Results from Drs. Xulin Chen and Phil Stansly’s experiment on holding diet and egg formation in T. radiata (2014a) indicate that nutrients acquired through host-feeding are necessary for egg production. Still it was found in this study, as well as others, that additional carbohydrates, amino acids, and fatty acids found in Coenzyme Q10 and royal jelly, were beneficial to egg formation and egg maturation as well (Chen & Stansly, 2014a; Étienne et al., 2001; Hall & Klein, 2014; Wheeler, 1996).
In this current study of T. radiata diet and fecundity, it was found that nutrients other than those found in D. citri could produce similar or even better outcomes that result in an equal or greater egg load than previously-tested holding diets. Researchers involved in devising storage diets for T. radiata may wish to consider introducing
59 Coenzyme Q10 as found in Diet Treatment I, or royal jelly as found in Diet Treatment A, to their diet treatment programs, as its addition to honey-water produced significantly positive results in this experiment.
Body Size
Adult body size, measured by HTL, is genetically and physiologically determined but can be modified by environmental conditions during larval development with environmental factors such as temperature, relative humidity, and light to dark cycle influencing her fecundity in different ways (Honĕk, 1993). Having a larger HTL is a contributing factor to having a larger egg load, possibly having larger eggs, and having greater longevity (Heimpel & Collier, 1996; Honĕk, 1993; Lopez & Hoddle, 2014). More specifically, having a larger body size means having more resources that could be used to search for available hosts, as well as having a physically larger body cavity in which to form and carry eggs. Tamarixia radiata is extremely small and because of this, her egg load could be size-limited meaning that she can have all the nutrients she needs, be as young as she can be, and still she can only carry so many eggs without access to hosts on which to oviposit those eggs (D. Morgan, personal communication, March 2018; Honĕk,
1993). When she has all her eggs and cannot physically hold any more, she needs to find living ACP hosts on which to lay her eggs in order to have more room in her body to continue to produce eggs (D. Morgan, personal communication, March 2018). This could be the ideal circumstance under which she should be released for optimal dispersal ability and host suppression.
60 Conclusion. The Ultimate Factor
To truly maximize the use of this wasp, much still needs to be learned about how to improve the efficiency of mass rearing, storage, release, and the parasitoid’s ability to drive ACP to low numbers in California’s citrus niches. It is necessary to further develop rearing systems heavily based on artificial diets that can maintain or increase fecundity in order to economically produce the number of fit wasps needed to achieve ACP vector control (Chen & Stansly, 2014b; Ferkovich et al., 2000; Qureshi & Stansly, 2007).
In Response to Zero Egg Load
Previous research suggested that when female T. radiata are deprived of hosts and instead provided with an alternative diet, not only is oviposition suppressed (which leads to the resorption of her eggs) but that there is also a release of energy (Jervis & Kidd,
1986). This suggestion was confirmed by this experiment. The suppression of oviposition was overwhelming, with overall zero egg loads accounting for 33.9% of the data in
Experiment One. This additional release of energy that comes from ovisorption could also have contributed to the premature death of many T. radiata suffered prior to dissection. It was suggested by Dr. David Morgan from the CDFA that the wasps were
“tuckering themselves out,” and in such a way were speeding up their life cycles (D.
Morgan, personal communication, December 2016). The T. radiata simply had more energy after resorbing their eggs, either because they could not lay them or because they needed those metabolic nutrients and overexerted themselves within the vials.
The lack of access to healthy available hosts (and therefore her natural behavior as a parasitoid to extract needed nutrients through host-feeding) may have more of an
61 impact on ovigeny, or the production and retention of an egg load, than previously thought (Jervis et al., 2001; Heimpel & Collier, 1996). The T. radiata had been denied actual living host nymphs and kept in a vial under dark, humid conditions. It is thus not surprising that their egg loads were severely depressed overall, or that they had great amounts of zero egg loads.
This lack of energy may have caused the wasps to deplete all metabolic reserves and act as older wasps that devotes their energy to somatic maintenance instead of to egg formation. Taking this into consideration, while T. radiata are stored, the wasp’s age and her body size may be the best indicators for egg load, fitness, and longevity.
This study contributed to the goal of maintaining, increasing, and possibly regenerating reproductive potential during storage by showing that the age and body size of a wasp may be the foremost factors when determining the best circumstances under which to release this biological control agent for substantial control over ACP at releases sites in southern California.
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EXPERIMENT TWO. CPP CITRUS GROVE TRIALS
Introduction. Exposed to the Elements
The success of a biological control program depends on factors that affect the field efficiency of organisms raised under greenhouse and laboratory conditions (Gómez-
Torres, Nava, & Parra, 2012). The primary goal of citrus agencies, such as the Citrus
Research Board, has been to battle the citrus pest, Asian Citrus Psyllid, with integrated pest management practices which necessitate mass production and establishment of T. radiata as extensively as possible in California (Hoddle, Amrich, Stosic, & Kistner,
2016; Qureshi, Rogers, & Stansly, 2009). Factors such as nutrient-rich diet formulations can influence Tamarixia radiata (T. radiata) (Waterston) (Hymenoptera: Eulophidae) in various ways depending on the wasp’s age at the time of feeding and the length of time the wasp has access to the diet. Understanding how to manipulate female T. radiata egg load using diet and exposure length to that diet when storing and transporting T. radiata for release can positively contribute to expediting both wasp establishment and suppression of Asian citrus psyllid (ACP) Diaphorina citri (D. citri) (Kuwayama)
(Hemiptera: Psyllidae) at release sites (Gómez-Torres et al., 2012).
Inundating California’s diverse landscape with tiny ectoparasitoids is not a trivial exploit. Not only do T. radiata have to produce massive rates of ACP mortality on their end, but must also reproduce, fight off predators, compete for food, establish, and spread across release sites that cover marine, semi-arid, and arid citrus-growing regions (Hoddle et al., 2016; Michaud, 2004; Qureshi & Stansly, 2009; Pluke, Qureshi, & Stansly, 2008).
As of 2017, T. radiata has successfully been released and established as a classical
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biological control agent in both residential and urban citrus, as well as in organic citrus production sites (Chien, Chiu, & Ku, 1989; Hoddle et al., 2016; Milosavljević, Schall,
Hoddle, Morgan, & Hoddle, 2017). However, the spread of the lethal citrus disease,
Huanglongbing (HLB) and its ACP insect vector are out-pacing T. radiata production, releases, and establishments. With all other management tactics employed at full tilt— quarantined areas, identification of HLB-positive trees and their successive removal, and well-timed pesticide sprays in commercial groves—when it comes to this primary biological control tool, there is room for improvement (Hoddle et al., 2016).
Improvements need to mostly be made in the mass-production and storage phases of T. radiata (D. Morgan, personal communication, February 9, 2018).
Even with an average of several hundred thousand wasps released per year since
2013, not every wasp that is released is in prime shape (Hornbaker, 2016). For example, current mass-production of T. radiata, which is centered in Riverside County at the
California Department of Food and Agriculture’s (CDFA) Mt. Rubidoux facility, creates wasp cohorts that are heavily male-biased, have females with low egg load counts, and exhibit inactivity or premature aging and mortality (D. Morgan, personal communication,
February 2018). Moreover, there are always unpredictable circumstances and suboptimal conditions to contend with such as weather, the quality and state of the ACP hosts at the time of wasp release, and continual competition for ACP with other organisms at release sites (Chen, Triana, & Stansly, 2017; Wager-Page, 2010).
T. radiata’s behavior is such that a female will parasitize 4th and 5th ACP instars, and will host-feed on the 1st, 2nd, and 3rd (Chu & Chien, 1991; Meyer, 2007; Qureshi et al., 2012). Thus, allowing one adult female T. radiata to kill approximately 500 ACP
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nymphs during her adult stage (Chen & Stansly, 2014b; Hall, 2008a). In the laboratory, the optimal conditions for T. radiata activity are 25°C, 70−100% relative humidity, and
14-hour light/10-hour dark cycle (Chen, Wong, & Stansly, 2016; Chen & Stansly,
2014a). Under these conditions and when wasps are supplied with 40 4th and 5th ACP nymphal instars daily, T. radiata can kill an estimated 500 D. citri nymphs over the course of a month through both host-feeding and parasitisation (Chen & Stansly, 2014a;
Chu & Chien, 1991; Skelley & Hoy, 2004). Additionally, female T. radiata have been observed to simultaneously host-feed (21 ± 2 seconds [Chen & Stansly, 2014a]), and parasitize (4.8 minutes [Chen et al., 2017]) at a rate of 5.6 host-fed D. citri for every one parasitized D. citri (Chu & Chien, 1991; Hall, 2014; Paiva & Parra, 2012; Skelley &
Hoy, 2004). Furthermore, T. radiata has the ability to synchronize her life cycle with her
ACP hosts’ through ovisorption (the resorption of her egg load) which can be a complicating factor when held in storage prior to release and can complicate subsequent host-feeding and parasitisation behaviors after release (Michaud, 2004).
While host-feeding is considered necessary for the wasp to produce eggs (Varley
& Edwards, 1957; Wager-Page, 2010), and is an added benefit to the wasps’ killing potential, this behavior can complicate the initial establishment of wasps at a release site, especially if the released wasps do not have eggs or have low egg loads; particularly considering that a single female wasp can lay up to 300 eggs at 25–30°C (Chu & Chien,
1991; Étienne, Quilici, Marival, & Franck, 2001; Qureshi et al., 2009; Paiva & Parra,
2012). Not only will an ACP nymph be dead, but its death limits the amount of ACP that are available for a female to oviposit on (Paiva & Parra, 2012; Skelley & Hoy, 2004).
This means that potentially the female wasp will use smaller ACP nymphs on which to
65
oviposit, or that a single nymph will be super-parasitized (Chen & Stansly, 2014b).
Smaller ACP, which are of lesser quality, and ACP that have been parasitized more than once, tend to result in male wasps and smaller wasps (Chu & Chien, 1991; Reeve, 1987;
Tang & Huang, 1991). Male parasitoids are only valued for their reproductive activities as they are unable to host-feed or oviposit (Chen & Stansly, 2014b). An abundance of males at a release site could lead to a failure of wasps to find mates which would cause the released population to become extinct. Allowing wasps to host-feed and mate prior to field-release would encourage females to have more fertilized eggs, which would lead to a largely female-biased cohort that would establish and spread more quickly than their non- host-fed and unfertilized counterparts (A. Soper, personal communication, June
2016).
The field data produced by this study yields results to compare with wasp fitness studies that have been confined to the laboratory. The objective of these field studies is to aid in the reduction of the ACP threat and the transmission and spread of HLB through the identification of trends among tested diets, wasp egg loads, wasp parasitism rates, and wasp host-feeding rates in order to help guide current wasp production and releases
(Chen et al., 2017; Chen & Stansly, 2014a; Chen, Wong, & Stansly, 2016; Chu & Chien,
1991; Reeve, 1987).
Objective. Observations of Wasp Activity
This experiment was designed to satisfy the requirements of ARI Grant #15-04
219 and was proposed by Drs. Valerie Mellano and Anna Soper of the Plant Science
Department at California State Polytechnic University, Pomona (CPP), in participation
66
with Dr. David Morgan of the CDFA and Dr. Kris Godfrey of the University of
California, Davis. The goal of these field studies was to develop the most efficient system possible to provide the minimum numbers of T. radiata needed to establish in the ACP infested areas of southern California and to minimize the potential for HLB transmission.
In order to meet the goals of the proposed study, Experiment Two analyzed how the wasps performed in the field after feeding on one of four diet treatments in the CPP
Plant Sciences Laboratory. Providing wasps with a nutritious artificial diet to increase egg load would allow for higher parasitism rates and hence quicker establishment in the field (Chen & Stansly, 2014a; Chen & Stansly, 2014b; Hall & Klein, 2014). Field performance was assessed based on host-feeding and parasitism rates (Chu & Chen,
1991; Heimpel & Collier, 1996; Pluke et al., 2008).
Several factors that have been recorded as being highly correlated with wasp activity are wasp age, wasp diet, wasp body size, ACP host density, temperature, and relative humidity (Chen & Stansly, 2014a; Chen & Stansly, 2014b; Gόmez-Torres et al.,
2012; Mann & Stelinski, 2014; Skelley & Hoy, 2004). Ages of the wasps (6 and 12 days old) and diet treatment (Diets A, C, G, and I) were used from Experiment One (see
Appendix C). If diet affects egg load and in turn affects parasitism rates and can further regenerate the egg loads of older wasps, it would be expected that the parasitism rates among the ages are not significantly different so long as the same diet treatment was administered (Chen et al., 2016; Hall & Klein, 2014; Wright, Hoffmann, Chenus, &
Gardner, 2001). This data could potentially show that the egg load of older wasps can be rejuvenated by diet alone, and that the proper diet can outweigh all other factors that affect egg load. Providing the proper diet could increase parasitism rates and hence reveal
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the minimum number of individual wasps needed to establish at a release site.
Uncovering this relationship would have great influence on how diet is used during wasp storage prior to release.
Materials and Methods. The Quest for ACP
T. radiata were reared inside tent-style bug dorms (BugDorm-2 400F, insect rearing tent; length 75 cm x width 75 cm x height 115 cm, 150 x 150 mesh size) in glasshouses at the CDFA rearing facilities located at the Mt. Rubidoux Field Station
(California Department of Food and Agriculture, 2017). Tamarixia radiata were collected with automatic aspirators by the CDFA staff on the day of emergence in plastic jars varying in size from 50–500 mL with approximately 200–500 male and female T. radiata. The T. radiata were stored in a dark cooler at 12.8ºC with an impregnated paper towel strip of honey-water for 6 to 12 days (CDFA, 2017; Chen & Stansly, 2014b; Hall &
Klein, 2014).
Wasp vials were transported in an insulated cooler with ice packs, foam block and towels (Qureshi et al., 2009), from the CDFA to the CPP Plant Sciences Laboratory.
General observations were made which included T. radiata overall physical activity, body size, and sex ratio of the vial population.
In the Laboratory
Clear acrylic propagation cages with a mesh-side and tie-off sleeve (length 19 cm x width 19 cm x height 35.6 cm), Craft Smart glue gun, Surebonder glue sticks for minor holes in mesh sleeves, dissecting stereoscopic microscope (Olympus SZX16, Olympus
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Corporation) (Paiva & Parra, 2012; Qureshi et al., 2009), compound binocular light microscope (American Optical One-Fifty, American Optical Company), three-prong extension cord, upright freezer (-18°C), small wine cooler (12.8ºC, Haier, Thermoelectric
Wine Cellar, Model# HVTECO6ABS, Serial #15091110313), and Caron Insect Growth
Chamber (Model #6025, 708L, W90 cm x H195 cm x 84.5 L) were used. Tamarixia radiata were distributed in the clear acrylic cages. Wasps were passively recollected into labelled vials by manipulating the light source because the use of either a manual or automatic aspirator to recollect wasps caused unwanted physical stress to wasps.
The four diet treatments (see Appendix C) from Experiment One were tested.
Small batches of the diets were mixed in individual vials. The diet treatment measuring tools included Eppendorf tubes (5.0 mL, clear polypropylene tubes with snap caps,
#0030119401), glass funnel, small silicone spatula, small/narrow honey spreader, small stirrer, weight-measurement scale (Taylor digital glass-top food scale, #49178540, UPC#
077784025468), plastic measuring boats, small measuring glass cylinders (Pyrex, 10−500 mL), all-purpose scissors, Scott blue super-absorbent shop towels, and small mortar and pestle.
Diet treatment ingredients included a consistent and accessible source of D. citri including adults, all nymphal stages, and nymphal frass. Honey (raw orange blossom virgin honey, Winter Park Honey, Winter Park, FL), deionized (DI) water, pre-made royal jelly and honey blend (Y.S. Eco Bee Farms, royal jelly in honey), and a pure powder form of Coenzyme Q10 (Sigma-Aldrich Life Sciences, C9538-100 mg, 3050
Spruce St., St. Louis, MO) (see Appendix C) were also used in the diet treatments. Wasp access to each assigned diet was provided via a 2.0 cm x 3.0 cm feeding strip of Scott
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blue super-absorbent shop towel that was placed on the inside of the cap of the 50 mL clear plastic vials holding a total of 50–100 male and female T. radiata. Adult female and male T. radiata were allowed to mass-feed and mate ad nauseum for 24 hours before used in the field trials (Qureshi & Stansly, 2009).
Access to a manual aspirator was provided to remove males from vials after the mating period elapsed (BioQuip 6mm, latex tubing, 1135A, 9-dram clear styrene vial with snap on cap, #1135Y HEPA filter). Collection vials with lids (9 and 15-dram; 55 mL, clear polystyrene vials with snap cap, E&K Scientific), Ziploc bags (quart and gallon sizes), Sharpie pens, large binder clips (1inch), 3M colored masking tape, Parafilm
(Sigma-Aldrich, #P7793), and plastic jars with screw-top lids (16 oz, clear BPA-free PET storage containers with screw-top sealing caps) were used in the CPP Plant Sciences
Laboratory.
Recording materials included a calculator, clipboard, Microsoft Excel program, printer, printer ink, paper, pens, optic ruler for compound light microscope (WF10X, 18 mm, 0.1 mm scale), temperature and relative humidity monitoring device (EXTECH instruments Hygro-Thermometer Clock #445702), AAA and AA batteries. The personal camera on iPhone 5S, and hand lens (magnifier-LED double-multiple jewelry identifying type, 30 x 22 mm, 60 x 12 mm) were used to identify and distinguish between female and male T. radiata by deciphering antennal and abdominal details (Skelley & Hoy, 2004;
Waterston, 1922).
In the Field
The trial site grove—20 acres of outdoor, mixed, mature citrus located on the CPP
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campus—boasts 2,000 citrus trees that produce 180 tons of citrus per year from 23 varieties (Allen, 2008). No attention was paid as to which citrus varieties were used in these trials. Because of the low presence and unequal distribution of D. citri within the citrus grove, and because small tree branches were removed in order to record observations in the CPP Laboratory, some trees were hosts for the entire trial, and other trees were used only once during the trials (Chu & Chien, 1991).
Field materials included mesh sleeve-cages (conical organza mesh candy bags; double drawstring pull-tie; 10.8 cm opening; 20.0 cm long; PaperMart) (A. Soper, personal communication, July 2016), neon-colored flagging tape (ULINE), Tanglefoot aerosol ant trap (Tangle-trap Sticky Trap Coating aerosol spray, Tanglefoot,
#300000676) (Hoddle & Hoddle, 2013), FELCO spring-loaded bypass pruners, FELCO small snips, and antibacterial hand wipes.
Releasing the Treatment Wasp
T. radiata had been feeding on a honey-water diet up until they reached the designated ages of 6-day-old (DO) or 12DO. At 6DO and 12DO, wasps were switched to one of four diet treatments to feed on in the CPP Plant Sciences Laboratory for 24 hours with access to mates. The reason for mating the females was that she is an arrhenotokous species which means that her fertilized eggs will develop into females whereas her unfertilized eggs will be male (Chen, 2013; Chien & Chu, 1996; Cooksey, 2017). A single female treatment wasp—assumedly mated and from each of the four diet treatments (A, C, G, or I)—was then released into an ACP population enclosed within a drawstring sleeve-cage (Chen & Stansly, 2014; Qureshi & Stansly, 2009; Yu-qing & Zhi
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peng, 1991).
Estimations of enclosed ACP populations were made prior to releasing a treatment wasp. Low densities had between 10–40 4th and 5th instars, along with egg and adult stages of ACP (Chen & Stansly, 2014b). High densities included 50–100 4th and 5th instars, along with other stages of ACP (Chen & Stansly, 2014b). After releasing a treatment wasp into the sleeve cage, cages were tied and the base of the treatment branch and surrounding branches were sprayed with Tangle-foot sticky trap coating aerosol spray to dissuade ants from compromising the experiment (Hoddle & Hoddle, 2013;
Navarrete, McAuslane, Deyrup, & Pena, 2013).
Retrieval of Treatment Wasps from the Groves
There was a total of four trials performed in the CPP citrus groves in the summer of 2016 between the months of July and September. Each trial had 20 female T. radiata test wasps. In each group of twenty wasps there were 6 to 7 replicates for each tested diet.
Trial A was conducted with 12DO T. radiata, fed diets A, C, or G, for 24 hours prior to release in mesh sleeve-cages in the grove. Trial A mesh sleeve-cages were collected after
24 hours in the grove. Trial B was conducted with 12DO T. radiata, fed diets A, C, or I, for 24 hours prior to release in mesh sleeve-cages in the grove. Trial B mesh sleeve-cages were collected after 72 hours in the grove. Trial C was conducted with 6DO T. radiata, fed diets A, C, or G, for 24 hours prior to release in the field. Trial C mesh sleeve-cages were collected after 168 hours (seven days) in the grove. Trial D was conducted with
6DO T. radiata, fed diets A, C, or I, for 24 hours prior to release in mesh sleeve-cages in the grove. Trial D mesh sleeve-cages were collected after 72 hours in the grove.
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After the allotted grove exposure lengths (24 hrs, 72 hrs, 168 hrs, 72 hrs, respectively), the branches that contained all densities of ACP and T. radiata were removed from the trees. Each sleeve-cage and the branch sample within were placed into water beakers in the CPP Plant Sciences Laboratory and set in a wine cooler at 12.8ºC until they could be examined. All sleeve-cages were processed within 10 hours of collection from the field. The count of parasitized, un-parasitized, and host-fed D. citri nymphs (excluding adults) were recorded using a dissecting stereoscopic microscope and field data collection sheets (Paiva & Parra, 2012; See Appendix F).
Recordkeeping
Each individual wasp within her sleeve-cage was assigned an identification letter and number. All D. citri nymphs within the sleeve-cages were organized according to wasp ID. Each ACP nymph was examined to record life stage (1st–5th nymphal instar) and its current state: parasitized, host-fed, alive (no evidence of T. radiata activity), or dead due to natural causes (Paiva & Para, 2012). Percent parasitism rates were calculated based on the total count of nymphs that were parasitized divided by the count of nymphal stages that were available for parasitism (4th and 5th nymphal instars) (Paiva & Parra,
2012). To determine the percent host-feeding rates of each released female T. radiata, the number of nymphs that were host-fed was divided by the total amount of nymphs available for host-feeding, which included all five nymphal stages (Pluke et al., 2008;
Qureshi et al., 2009).
If the treatment female wasp could be recollected, she was dissected with a compound light microscope to determine remaining body size and egg load and by
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measuring right hind tibia length (HTL). Dissection tools included ultrafine forceps
(BioQuip #4535 forceps #5, Swiss pattern, super fine-honed, high-quality stainless steel,
112 mm), plastic pipette dropper (4 mL), bent probe with sharp tip, needle-point scriber probe (full spear, stainless steel, #4755), teasing needle (angled tip, #4752), dissecting scissors (straight, sharp points, stainless steel, 114 mm, #4713), glass microscope slides
(Karter Scientific microscope slides, 206A2), and glass coverslips (Karter Scientific microscope cover glass, 22 x 22 mm, 0.13–0.16 mm, #141473, #211Z3).
It was intended that the parasitized D. citri nymphs recovered from the field to be reared in the CPP Plant Sciences Laboratory until the preimaginal period had elapsed and an adult T. radiata emerged. Not only would egg viability be confirmed, but progeny sex and egg load of the emerged wasp could also be recorded (CDFA, 2017; Chu & Chien,
1991; Qureshi et al., 2009; Skelley & Hoy, 2004). Due to a variety of unfavorable circumstances in the CPP Plant Sciences Laboratory, all developing T. radiata within the parasitized D. citri nymphs suffered early mortality and could not be reared in order to collect such information (Qureshi et al., 2009).
Results. To Host-Feed or Parasitize
The following data was collected and recorded: individual treatment wasp ID, diet
푁௨푏푒 푓푃푎௦ℏ௧ℏ௭푒푑 푁௬ℎ௦ fed prior to release (A, C, G, or I), in-field parasitism rate ( ), 푁௨푏푒 푓 4௧ℎ 푎푑 5௧ℎ 퐼௦௧푎௦
푁௨푏푒 푓 퐻௦௧−퐹푒푑 푁௬ℎ௦ in-field host-feeding rate ( ), T. radiata age (6DO or 푁௨푏푒 푓 1௦௧ ௧ℎ௨푔ℎ 5௧ℎ 퐼௦௧푎௦
12DO), exposure length to CPP citrus groves (24 hrs, 72 hrs, 168 hrs, or 72 hrs), count of total nymphs (ACP host density), and life-stage of collected ACP (egg or 1st−5th instar)
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(Paiva & Parra, 2012). For the limited number of T. radiata that were re-collected, HTL and egg load were recorded (Lopez & Hoddle, 2014). Only Trials B (6DO wasps in the field for 72 hours) and D (12DO wasps in the field for 72 hours) are further explained due to the field exposure length parameters being similar.
The average parasitism and host-feeding rates from Trial D summarize 6DO wasps that were fed diet treatments A, C, or I, for 24 hours prior to release in the CPP orchard. Tamarixia radiata fed Diet Treatment A (royal jelly + honey-water) had an average 9.84% parasitism rate with an average 5.98% host-feeding rate (see Figure 9).
Tamarixia radiata fed Diet Treatment C (honey-water) had a 9.99% parasitism rate and a
1.25% host-feeding rate. Diet Treatment I (Coenzyme Q10 + honey-water) resulted in a parasitism rate of 2.16% and a host-feeding rate of 2.72%.
The average parasitism and host-feeding rates of the 12DO T. radiata that were fed diet treatments A, C, or I, for 24 hours (see Figure 10), were released into sleeve- cages in the field, and then re-collected after 72 hours. Wasps fed Diet Treatment A
(royal jelly + honey-water) had a 0.00% parasitism rate and a 10.85% host-feeding rate.
Tamarixia radiata provided with Diet Treatment C (honey-water) parasitized 0.00% and host-fed on 50.00% of D. citri nymphs within the sleeve-cage. Diet Treatment I
(Coenzyme Q10 + honey-water) resulted in a parasitism rate of 5.55% and a host-feeding rate of 36.35%.
Discussion. In-Field Diet Treatment Performance
With respect to differences in the host-feeding to parasitism ratio between the T. radiata ages, overall 12DO T. radiata tended to host-feed more on average and had lower
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parasitism rates than 6DO T. radiata. This concurs with a previous study by Hall & Klein which found that T. radiata held in storage conditions for a longer period of time had lower parasitism rates in the field than freshly-collected T. radiata that were not stored as long (Hall & Klein, 2014). This result also agrees with the Heimpel and Collier review that younger wasps have a larger egg load, or greater oviposition activity, than older wasps and can therefore parasitize more readily than older wasps can (Heimpel & Collier,
1996). Furthermore, egg load plays a crucial role in influencing host-feeding strategies.
Wasps with a lower egg load are predicted to host-feed more than wasps with a higher egg load (Heimpel & Collier, 1996).
Taking the age of wasp and her egg load into account when looking at the diet treatments, it seems that age may have more of an influence on host-feeding behavior and parasitism activity than diet does. Neither host-feeding nor parasitism rates correspond based on diet treatment between 6DO wasps and 12DO wasps.
During the beginning of the trials, the length of needed field exposure time for the treatment wasp was unknown. Treatment wasps were checked on at 24 hours (Trial A) and 48 hours, which yielded no visible activity from wasps. Treatment wasps were given a minimum of 72 hours exposure time in the field before re-collection to ensure that there was some sort of wasp activity (A. Soper, personal communication, July 2016). This was the reason that treatment wasps were left in the field for a high of 168 hours/seven days
(Trial C).
Conclusion. Limitations and Future Directions
The CPP research team, which was comprised of master’s student Dani Ruais and
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undergraduate student Christian Buelna (see Appendix A), began its search for D. citri on the trees along the perimeter, which were south-facing, and closest to dusty main roads.
Diaphorina citri colonization within the CPP citrus grove corresponded to light quality and availability, amount of flush (new growth) on the tree, temperature differences within the tree canopy, and grove-edge effects (Pluke et al., 2008; Qureshi et al., 2009). Once those hot-spots were identified, the CPP research team looked for flush on the sunny side of the tree. It was easy to find the tiny amber-colored D. citri eggs in the early sunlight, always perched at the newest and greenest flush points at the tips of tree limbs (Qureshi et al., 2009). Diaphorina citri adults were observed feeding on the new flush and on the underside of proximal leaves and stems. Occasionally both D. citri nymphs and adults were found feeding on the fruit, though no eggs were found on fruit (C. Buelna, personal communication, August 2016).
ACP Host Densities
Tamarixia radiata’s behavior in the field directly correlates with her ACP host’s density and this correlation could have a stronger impact on her activity than her diet does. But since the differentiation between the density of healthy available ACP nymphs and parasitized or unavailable ACP nymphs could not be determined in the field, results were unclear how heavily parasitism and host-feeding rates were influenced by ACP density in these studies (Heimpel & Collier, 1996).
Trial Limitations
There was difficulty at the beginning of the field trials in June 2016. During the
77
first few weeks, the research team found only D. citri eggs and adults present, which were tagged and revisited during the following weeks. It was necessary to wait for the D. citri eggs to develop to the 4th and 5th nymphal stages before populations could be estimated and sleeve-cages could be set up in which to release the treatment wasps
(Qureshi et al., 2009). The first sleeve-cage releases were not made until July 2016, when
D. citri densities were greater in the grove.
According to thermal hygrometric trials performed by Mariuxi Lorena Gómez-
Torres et al. (2012), temperature and relative humidity are dominant elements that act on
T. radiata’s and ACP’s development, survivability, activity, and fecundity. Even though the weather was superlative during the trials in the CPP grove for D. citri production
(25−28°C [Liu & Tsai, 2000; Pluke et al., 2008]) and T. radiata activity—with daytime temperatures at 31°C, nighttime temperatures at 13°C and an average 63% relative humidity (AccuWeather, 2017; Gómez-Torres et al., 2014)—inconsistencies with production at the CDFA rearing facilities (J. Herreid, personal communication, July
2016) and the CPP laboratory proved difficult and nigh impossible to discriminate from the data that reflected T. radiata inactivity (Hall & Nguyen, 2010).
These field trials had several limitations which could be responsible for the discrepancies in parasitism and host-feeding rates that have been collected by other states and countries such as Florida (Qureshi et al., 2009), Puerto Rico (Pluke et al., 2008), and
Réunion Island (Étienne et al., 2001). First, there was an unknown pre-existing presence of T. radiata within the CPP groves. More field trials were proposed; however, due to the presence of ACP mummies—parasitized remains with characteristic T. radiata emergence hole in the mummy’s thorax—throughout the grove, it could not be positively
78
concluded whether this ACP mortality was due to released treatment wasps or due to existing wasp populations whose origins were unknown.
Secondly, there was no attempt to account for background mortality of ACP—the placing of sleeve-cages over ACP populations throughout the orchard without introduction of a wasp (D. Morgan, personal communication, February 9, 2018). Doing so would have allowed for the comparison of ACP natural mortality that existed between treatment cages and non-treatment cages. It could have provided a baseline for the determination of how diet treatment, affected wasp activity. The observed wasp inactivity could have been due to the lack of healthy and available ACP nymphs which was possibly attributed to natural ACP mortality.
This trial was also limited by the availability of knowledgeable labor, low ACP density in the groves, ant interference within field sleeve-cages (Hoddle & Hoddle, 2013;
Navarrette et al., 2013), access to diet treatment ingredients (particularly fresh ACP for
Diet Treatment I: ACP + honey-water), and by access to appropriately aged, quality wasps. Other factors that were not isolated in the trial parameters—such as available D. citri overall density and available D. citri host sizes, which are linked to ACP host quality-based decisions—could have further influenced the parasitoid’s behavior in the field (Chen et al., 2016; Michaud, 2004). Due to these factors, the ultimate question of the most favorable circumstances, including diet, under which to ensure maximal parasitisation of D. citri in the field remains unanswered by this experiment.
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REFERENCES
1. AccuWeather, Inc.; Channel ABC7 (2017). AccuWeather Report. Retrieved from http://www. accuweather.com/en/us/pomona-ca/91766/july weather/331974?monyr=7/1/2016
2. Acosta, M. J., Fonseca, L. V., Desbats, M. A., Cerqua, C., Zordan, R., Trevisson, E., & Salviati, L. (2016). Coenzyme Q biosynthesis in health and disease. Biochimica et Biophysica Acta, 1857, 1079–1085.
3. Allen, D. (2008, January 27). Pomona A to Z: C is for citrus [blog post]. Retrieved from http://www.insidesocal.com/davidallen/2008/01/27/c/
4. Ammar, E. D., Hall, D. G., & Shatters, R. G. (2013). Stylet morphometrics and citrus leaf vein structure in relation to feeding behavior of the Asian citrus psyllid Diaphorina citri, vector of the citrus huanglongbing bacterium. PLoS ONE, 8(3). Retrieved from http://journals.plos.org/plosone/article?id=10.1371/journal. pone.0059914
5. Aubert, B. (1987). Trioza erytreae del Guercio and Diaphorina citri Kuwayama (Homoptera: Psylloidea), the two vectors of citrus greening disease: Biological aspects and possible control strategies. Fruits, 42(3), 149–162.
6. Aubert, B., & Quilici, S. (1984). Biological control of the African and Asian citrus psyllids (Homoptera: Psylloidea), through eulophid and encyrtid parasites (Hymenoptera: Challoidcidea) in Reunion Island. Riverside, CA. Proceedings of 9th Conference of the International Organization of Citrus Virologists, 100–108.
7. Aung, K. S. D., Takagi, M., & Ueno, T. (2010). Influence of food on the longevity and egg maturation of the egg parasitoid Ooencyrtus nezarae (Hymenoptera: Encyrtidae). Journal of the Faculty of Agriculture, Kyushu University, 55(1), 7981.
8. Aung, K. S. D., Takasu, K., Ueno, T., & Takagi, M. (2012). Effect of host-feeding on reproduction in Ooencyrtus nezarae (Ishii) (Hymenoptera: Encyrtidae), an egg parasitoid of the bean bug Riptortus clavatus. Journal of the Faculty of Agriculture, Kyushu University, 57(1), 115–120.
9. Ball, D. W. (2007). The chemical composition of honey. Journal of chemical education, 84(10), 1643–1646.
10. Bech, R. A. (2008). Federal domestic quarantine order: citrus greening disease (CG) and Asian citrus psyllid (ACP). Deputy administrator plant protection and quarantine federal order. 1–6, Retrieved from https://www.aphis.usda.gov/ plant_health/plant_pest_info/citrus.pdf
80
11. Ben-Meir, A., Burstein, E., Borrego-Alvarez, A., Chong, J., Wong, E., Yavorska, T., Naranian, T., Chi, M., Wang, Y., Bentov, Y., Alexis, J., Meriano, J., Sung, H. K., Gasser, D. L., Moley, K. H., Hekimi, S., Casper, R. F., & Jurisicova, A. (2015). Coenzyme Q10 restores oocyte mitochondrial function and fertility during reproductive aging. Aging Cell, 14(5), 887–895.
12. Berger, D., Olofsson, M., Friberg, M., Karlsson, B., Wiklund, C., & Gotthard, K. (2012). Intraspecific variation in body size and the rate of reproduction in female insects − adaptive allometry or biophysical constraint? Journal of Animal Ecology, 81, 1244–1258.
13. Bistline-East, A. (2016, January 11). UC Riverside Center for Invasive Species Research (CISR) Psyllaphycus diaphorinae: Another natural enemy from Pakistan for ACP biocontrol? [blog post]. Retrieved from http://cisr.ucr.edu/blog/ psyllids/ psyllaphycus-diaphorinae-another-natural-enemy-from-pakistan-for-acp biocontrol/
14. Borror, D. J., & White, R. E. (1970). The Peterson field guide: A field guide to the insects of America north of México. New York, New York: Houghton Mifflin Company.
15. Bové, J. M. (2006). Huanglongbing: A destructive, newly-emerging, century-old disease of citrus. Journal of Plant Pathology, 88(1), 7–37.
16. Bownes, M., & Blair, M. (1986). The effects of a sugar diet and hormones on the expression of the Drosophila yolk-protein genes. Journal of Insect Physiology, 32(5), 493–501.
17. Brlansky, R. H., & Rogers, M. E. (2007). Citrus Huanglongbing: understanding the vector-pathogen interaction for disease management. The American Pathological Society. Retrieved from http://www.apsnet.org/publications/ apsnetfeatures/Pages/Huanglongbing.aspx
18. Bruce, T. J. A., & Wadhams, L. J. (2005). Insect host location: a volatile situation. Trends in Plant Science, 10(6), 269–274.
19. Capinera, J. L. (2008). Semiochemical. Encyclopedia of entomology, 3343–3344.
20. Caron. (2017). Insect Growth Chambers: Model #6025. Retrieved from http://www.caron products.com/files/docs/Literature/LIT-InsectGrowth Chamber60256045.pdf
21. Carpenter, J. E., Ferkovich, S. M., & Greany, P. D. (2001). Fecundity and longevity of Diapetimorpha introita (Cresson) (Hymenoptera: Ichneumonidae)
81
reared on artificial diets: Effects of a lipid extract from host pupae and culture media conditioned with an insect cell line. The Florida Entomologist, 84(1), 43– 49.
22. California Department of Food and Agriculture. (2014, August 12). Asian Citrus Psyllid (ACP) Factsheet. Retrieved from https://www.cdfa.ca.gov/plant/factsheets/ACP_FactSheet.pdf
23. California Department of Food and Agriculture, Mt. Rubidoux. (2017, April 12). Riverside, CA. Standard operating procedure- production of Tamarixia radiata and Diaphorencyrtus aligarhensis, 1–62
24. Cen, Y., Yang, C., Holford, P., Andrew, G., Beattie, C., Spooner-Hart, R. N., Liang, G., & Deng, X. (2012). Feeding behavior of the Asiatic citrus psyllid, Diaphorina citri, on healthy and Huanglongbing-infected citrus. Entomologia Experimentalis et Applicata, 143, 13–22.
25. Chen, X. (2013). Manipulation, rearing, and storage of Tamarixia radiata (Hymenoptera: Eulophidae) parasitoid of Diaphorina citri (Hemiptera: Psyllidae). Master of Science Thesis, University of Florida.
26. Chen, X., & Stansly, P. A. (2014a). Effect of holding diet on egg formation of Tamarixia radiata (Hymenoptera: Eulophidae), parasitoid of Diaphorina citri (Hemiptera: Psyllidae). The Florida Entomologist, 97(2), 491–495.
27. Chen, X., & Stansly, P. A. (2014b). Biology of Tamarixia radiata (Hymenoptera: Eulophidae), parasitoid of the citrus greening disease vector Diaphorina citri (Hemiptera: Psylloidea): a mini review. The Florida Entomologist, 97(4), 1404– 1413.
28. Chen, X., Triana, M., & Stansly, P. A. (2017). Optimizing production of Tamarixia radiata (Hymenoptera: Eulophidae), a parasitoid of the citrus greening disease vector Diaphorina citri (Hemiptera: Psyllidae). Biological Control, 105, 13–18.
29. Chen, X., Wong, S. W. K., & Stansly, P. A. (2016). Functional response of Tamarixia radiata (Hymenoptera: Eulophidae) to densities of its host, Diaphorina citri (Hemiptera: Psylloidea). Annals of the Entomological Society of America, 109(3), 432–437.
30. Chien, C. (1995). The role of parasitoids in the pest management of citrus psyllid. Taichung, Taiwan. Proceedings of the Symposium on Research and Development of Citrus in Taiwan, 245–261.
31. Chien, C., Chiu, S. & Ku, S. (1989). Biological control of Diaphorina citri in
82
Taiwan. Fruits, 44(7), 401–407.
32. Chien, C. C., & Chu, Y. (1996). Biological control of citrus psyllid, Diaphorina citri in Taiwan. Taipei, Republic of China on Taiwan. Biological pest control in systems of integrated pest management, food and fertilizer technology center for the Asian and Pacific region, 93–105.
33. Chien, C. C., Chu, Y. I., & Ku, S. C. (1991). Parasitic strategy, morphology and life history of Tamarixia radiata (Hymenoptera: Eulophidae). (Abstract in English). Chinese Journal of Entomology, 11(4), 264–281. Retrieved from http://www.cabdirect.org/cabdirect/ abstract/19921166587
34. Chu, Y. I., & Chien, C. C. (1991). Utilization of natural enemies to control of psyllid vectors transmitting citrus greening. Taipei, Republic of China on Taiwan. Biological pest control in systems of integrated pest management, food and fertilizer technology center for the Asian and Pacific region, 135–145.
35. Citrus Insider. (2017, July 26). Citrus pest & disease prevention program- California agricultural commissioner’s office: agricultural officials taking steps to battle citrus disease recently detected in Riverside. Retrieved from http://citrusinsider.org/2017/07/agricultural-officials-taking-steps-to-battle-citrus disease- recently-detected-in-riverside/
36. Citrus Matters. (2016, August 25). Crop science, a division of Bayer. Retrieved from http://citrusmatters.bayercropscience.us/why-citrus-matters
37. Civerolo, E. (2015). ACP and HLB detection in California. Citrograph Magazine, 8–9.
38. Cooksey, D. (2017). Biological Control in the Western USA: Biological control in pest management systems of plants. University of California, Riverside (PowerPoint slides). Retrieved from https://nature.berkeley.edu/biocon/What%20is%20Biological%20control.htm
39. da Silva, P. M., Gauche, C., Gonzaga, L. V., Costa, A. C. O., & Fett, R. (2016). Honey: chemical composition, stability, and authenticity. Food Chemistry, 196, 309–323.
40. Destaillats, H., Maddalena, R. L., Singer, B. C., Hodgson, A. T., & McKone, T. E. (2007). Quantifying pollutant emissions from office equipment: indoor pollutants emitted by office equipment: a review of reported data and information. Atmospheric Environment. Retrieved from https://www.osti.gov/scitech/servlets/purl/924853/
41. Ellers, J., Van Alphen, J. J. M., & Sevenster, J. G. (1998). A field study of size
83
fitness relationships in the parasitoid Asobara tabida. Journal of Animal Ecology, 67, 318–324.
42. Emerson, S. (2014, December 18). Stingerless wasps released in Asian citrus psyllid fight. San Bernardino Sun. Retrieved from http://plantingseedsblog.cdfa.ca.gov/wordpress/?p=7394
43. Étienne, J., & Aubert, B. (1980). Biological control of psyllid vectors of greening disease on Reunion Island. Sidney, Australia. Proceeding of the 8th Conference of International Organization of Citrus Virologists. University of California Press, Riverside, CA.
44. Étienne, J., Quilici, S., Marival, D., & Franck, A. (2001). Biological control of Diaphorina citri (Hemiptera: Psyllidae) in Guadeloupe by imported Tamarixia radiata (Hymenoptera: Eulophidae). Fruits, 56(5), 307–315.
45. Fathipour, Y., Hosseini, A., Talebi, A. A., & Moharramipour, S. (2006). Functional response and mutual interference of Diaeretiella rapae (Hymenoptera: Aphidiidae) on Brevicoryne brassicae (Homoptera: Aphididae). Entomologica Fennica, 17, 90–97.
46. Ferkovich, S. M., Morales-Ramos, J. A., Rojas, M.G., Oberlander, H., Carpenter, J. E., & Greany, P. (1999). Rearing of ectoparasitoid Diapetimorpha introita on an artificial diet: supplementation with insect cell line-derived factors. BioControl, 44, 29–45.
47. Ferkovich, S. M., Shapiro, J., & Carpenter, J. (2000). Growth of a pupal ectoparasitoid, Diapetimorpha introita, on an artificial diet: stimulation of growth rate by a lipid extract from host pupae. BioControl, 45, 401–413.
48. Ferris, R. (2015, October 21). Fatal citrus disease HLB shows up in California. CNBC. Retrieved from http://www.cnbc.com/2015/07/20/fatal-citrus-disease-hlb shows-up-in-california.html
49. Flint, M. L. & Dreistadt, S. H. (1998). Natural enemies handbook: the illustrated guide to biological pest control. Oakland, California: Regents of the University of California Division of Agriculture and Natural Resources.
50. Flores, D. & Ciomperlik, M. (2017). Biological control using the ectoparasitoid, Tamarixia radiata, against the Asian citrus psyllid, Diaphorina citri, in the Lower Rio Grande Valley of Texas. Southwestern Entomologist, 42(1): 49–59.
51. Fung, Y. C., & Chen C. N. (2006). Effects of temperature and host plant on population parameters of the citrus psyllid (Diaphorina citri Kuwayama). Formosan Entomologist 26, 109–123.
84
52. Garnier, M., & Bové, J. M. (1993). Citrus greening disease and the greening bacterium. Proceedings of the 12th Conference of International Organization of Citrus Virologists. 212–219.
53. Gentz, M. C., Murdoch, G., & King, G. F. (2010). Tandem use of selective insecticides and natural enemies for effective, reduced-risk pest management. Biological Control, 52, 208–215.
54. Godfray, H. C. J. (1994). Parasitoids: behavioral and evolutionary ecology. Princeton, N. J.: Princeton University Press.
55. Gómez-Torres, M. L., Nava, D. E., & Parra, J. R. P. (2012). Life table of Tamarixia radiata (Hymenoptera: Eulophidae) on Diaphorina citri (Hemiptera: Psyllidae) at different temperatures. Journal of Economic Entomology, 105(2), 338–343.
56. Gómez-Torres, M. L., Nava, D. E., & Parra, J. R. P. (2014). Thermal hygrometric requirements for the rearing and release of Tamarixia radiata (Waterston) (Hymenoptera: Eulophidae). Revista Brasileira de Entomologia, 58(3), 291–295.
57. Gottwald, T. R. (2010). Current epidemiological understanding of citrus Huanglongbing. Annual Review of Phytopathology 48,119–139.
58. Gottwald, T. R., da Graca, J. V., & Bassanezi, R. B. (2007). Citrus Huanglongbing: the pathogen and its impact. Plant Health Progress. Retrieved from https://www.apsnet.org/publications/apsnetfeatures/Pages/Huanglongbing Impact.aspx
59. Gould, J. R., Ayer, T., & Fraser, I. (2011). Effects of rearing conditions on reproduction of Spathius agrili (Hymenoptera: Braconidae), a parasitoid of the emerald Ash borer (Coleoptera: Buprestidae). Journal of Economic Entomology, 104(2), 379–387.
60. Grafton-Cardwell, E. E. (2017). Asian citrus psyllid distribution and management. University of California Division of Agriculture and Natural Resources. Regents of the University of California. Retrieved from http://ucanr.edu/sites/ACP/Distribution_of_ACP_in_California/
61. Grafton-Cardwell, E. E., & Daugherty, M. P. (2015, July 27). Pest notes: Asian citrus psyllid and Huanglongbing disease (formerly titled Asian citrus psyllid). Agriculture and Natural Resources, University of California statewide Integrated Pest Management program. Retrieved from http://www.ipm.ucdavis.edu/pmg/pestnotes/pn 71455.html
85
62. Grafton-Cardwell, E. E., Daugherty, M., Jetter, K., & Johnson, R. (2018). Eradication strategy. University of California Division of Agriculture and Natural Resources. Regents of the University of California. Retrieved from http://ucanr.edu/sites/ACP/Grower_Options/Grower_Management/Eradication_St rategies/
63. Grenier, S., Gomes, S. M., Febvay, G., Bolland, P., & Parra, J. R. P. (2005). Artificial diet for rearing Trichogramma wasps (Hymenoptera: Trichogrammatidae) with emphasis on protein utilization. 2nd International Symposium on Biological Control of Arthropods, 480–486.
64. Grout, R. A. (1946). The Hive and the Honeybee; a New Book on Beekeeping to Succeed the Book “Langstroth on the Hive and the Honeybee.” Hamilton, IL: Dadant & Sons.
65. Halbert, S. E. (2010, November 1). Pest Alert -DPI-FDACS, Citrus greening/Huanglongbing. Retrieved from http://swfrec.ifas.ufl.edu/hlb/database/pdf/00002355.pdf
66. Halbert, S. E. & Manjunath, K. L. (2004). Asian citrus psyllids (Sternorrhyncha: Psyllidae) and greening disease of citrus: A literature review and assessment of risk in Florida. Florida Entomologist, 87(3), 330–353.
67. Hall, D. G. (2008a). Biological control of Diaphorina citri. Hermosillo, Sonora, México. North American Plant Protection Organization Workshop, 1–7.
68. Hall, D. G. (2008b). Biology, history and world status of Diaphorina citri. Hermosillo, Sonora, México. North American Plant Protection Organization Workshop, 1–11.
69. Hall D. G., & Abrigo, L.G. (2007). Estimating the relative abundance of flush shoots in citrus, with implications on monitoring insects associated with flush. HortScience 42, 364–368.
70. Hall, D. G., Hentz, M. G., & Ciomperlik, M. A. (2007). A comparison of traps and stem tap sampling for monitoring adult Asian citrus psyllid (Hemiptera: Psyllidae) in citrus. Florida Entomology, 90, 327–334.
71. Hall, D. G., & Klein, E.M. (2014). Short-term storage of adult Tamarixia radiata (Hymenoptera: Eulophidae) prior to field releases for biological control of Asian citrus psyllid. The Florida Entomologist, 97(1), 298–300.
72. Hall, D. G., & Nguyen, R. (2010). Toxicity of pesticides to Tamarixia radiata, a parasitoid of the Asian citrus psyllid. Biocontrol: Journal of the International Organization for Biological Control, 55(5), 601–611.
86
73. Hall, D. G., Richardson, M. L., Ammar, E., Halbert, S. E. (2012). Asian citrus psyllid, Diaphorina citri, vector of citrus Huanglongbing disease: A mini review. Entomologia Experimentalis et Applicata, 146, 207–223.
74. Headrick, D. H., Bellows, T. S., & Perring, T. M. (1999). Development and reproduction of a population of Eretmocerus eremicus (Hymenoptera: Aphelinidae) on Bemisia argentifolii (Homoptera: Aleyrodidae). Biological Control, 28(2), 300–306.
75. Heimpel, G. E., & Collier, T. R. (1996). The evolution of host-feeding behaviour in insect parasitoids. Biological Review, 71, 373–400.
76. Heimpel, G. E., Rosenheim, J. A., & Kattari, D. (1997). Adult feeding and lifetime reproductive success in the parasitoid Aphytis melinus. Entomologia Experimentalis Et Applicata, 83, 305–315.
77. Herrera, C. M., & Pellmyr, O. (2002). Plant-animal interactions: an evolutionary approach. Malden, MA: Blackwell Science Publishing Ltd.
78. Hervet, V. A. D., Laird, R. A., & Floate, K. D. (2016). A Review of the McMorran diet for rearing lepidoptera species with addition of a further 39 species. Journal of Insect Science, 16(1), 1–7.
79. Hoddle, M. (2011, December 20). UC Riverside Center for Invasive Species Research (CISR) First release of Tamarixia radiata in California for the biological control of Asian citrus psyllid. [blog post]. Retrieved from http://cisr.ucr.edu/blog/news/first-release-of-tamarixia-radiata-in-california-for the-biological-control-of-asian-citrus- psyllid/
80. Hoddle, M. (2012, July 19). UC Riverside Center for Invasive Species Research (CISR) Has the Asian citrus psyllid parasitoid, Tamarixia radiata, established in California? [blog post]. Retrieved from http://cisr.ucr.edu/blong/asian-ctirus psyllid-2/has-the-asian-citrus-psyllid-parasitoid-tamarixia-radiata-established-in california/
81. Hoddle, M. S. (2004). Restoring balance: using exotic species to control invasive exotic species. Conservation Biology, 18 (1), 38–49.
82. Hoddle, M. S. (2015). USDA-APHIS approves release of second ACP parasitoid from Pakistan. Citrograph Magazine, 16–17.
83. Hoddle, M. S., & Hoddle, C. D. (2013). Classical biological control of Asian citrus psyllid with Tamarixia radiata in urban Southern California. Citrograph Magazine, 52–58.
87
84. Hoddle, M., Amrich, R., Stosic, C. & Kistner, E. (2016). Where’s Tamarixia? Citrograph Magazine, 7, 14–16.
85. Honěk, A. (1993). Intraspecific variation in body size and fecundity in insects: a general relationship. Oikos, 66(3), 483–492.
86. Hornbaker, V. (2017). ACP and HLB: The California situation. Arizona Grower Meetings. ACP-HLB Update. (Microsoft PowerPoint). Retrieved from http://extension.arizona.edu/extension.arizona.edu/files/resources/acp-hlb california-situation-ctirus.pdf
87. Hoy, M. A., Jeyaprakash, A., & Nguyen, R. (2001). Long PCR is a sensitive method for detecting Liberobacter asiaticum in parasitoids undergoing risk assessment in quarantine. Biological Control, 22, 278–287.
88. Hung, T. H., Hung, S. C., Chen, C. N., Hsu, M. H., & Su, H. J. (2004). Detection by PCR of Candidatus Liberibacter asiaticus, the bacterium causing citrus Huanglongbing in vector psyllids: application to the study of vector-pathogen relationships. Plant Pathology, 53, 96–102.
89. Husain, M. A. & Nath, D. (1927). The citrus psylla (Diaphorina citri, Kuwayama) (Psyllidae: Homoptera). Memoirs of the Department of Agriculture, India, 10, 1–27.
90. Jervis, M. A., Heimpel, G. E., Ferns, P. N., Harvey, J. A., & Kidd, N. A. C. (2001). Life- history strategies in parasitoid wasps: a comparative analysis of ‘ovigeny’. Journal of Animal Ecology, 70(3), 442–458.
91. Jervis, M. A., & Kidd, N. A. C. (1986). Host-feeding strategies in hymenopteran parasitoids. Biological Reviews, 61, 395–434.
92. Jones, D. B., Giles, K. L., Berberet, R. C., Royer, T. A., Elliott, N. C., & Payton, M. E. (2003). Functional responses of an introduced parasitoid and an indigenous parasitoid on Greenbug at four temperatures. Environmental Entomology, 32(3), 425–432.
93. Kapoulas, V. M., Mastronicolis, S. K., & Galanos, D.S. (1977, Feb. 25). Identification of the lipid components of honey. Z Lebensm Unters Forsch, 163(2), 96–99.
94. Ke, S., Li, K. B., Ki, C., & Tsai, J. H. (1991). Transmission of the Huanglongbing agent from citrus to periwinkle by dodder. Proceedings of the 10th Conference of International Organization of Citrus Virologists, 258–264.
88
95. Kidd, N. A. C., & Jervis, M. A. (1989). The effects of host-feeding behaviour on the dynamics of parasitoid-host interactions, and the implications for biological control. Researchers on Popular Ecology, 31, 235–274.
96. Kister, E. (2014, October 31). UC Riverside Center for Invasive Species Research (CISR) Tamarixia radiata and natural enemy impacts on the invasive Asian citrus psyllid in southern California. [blog post]. Retrieved from http://cisr.ucr.edu/blog/uc-riverside/tamarixia-radiata-natural-enemy-impacts invasive-asian-citrus-psyllid-southern-california/
97. Kister, E., Amrich, R., Castillo, M., Strode, V., & Hoddle, M. S. (2016). Phenology of Asian citrus psyllid (Hemiptera: Liviidae), with special reference to biological control by Tamarixia radiata, in the residential landscape of southern California. Journal of Economic Ecology, 109(3), 1047−1057.
98. Kister, E.J. & Hoddle, M. S. (2015). The life of the ACP: field experiments to determine natural enemy impact on ACP in southern California. Citrograph, 6(2), 52–57.
99. Laemmlen, F. F. (2011). Integrated pest management for home gardeners and landscape professionals. Agriculture and Natural Resources, University of California statewide Integrated Pest Management program, 1–3. Retrieved from http://ipm.ucanr.edu/PMG/PESTNOTES/pn74108.html
100. Lang-Fen, H., Dong-Dong, F., Pan, L., Zhong-Shi, Z., & Zai-Fu, X. (2015). Reproductive modes and daily fecundity of Aenasius bambawalei (Hymenoptera: Encyrtidae), a parasitoid of Phenacoccus solenopsis (Hemiptera: Pseudococcidae). Florida Entomologist, 98(1), 358–360.
101. Lee, C. (2015, October 21). Citrus facts. California Citrus Mutual. Retrieved from http://www.cacitrusmutual.com
102. Leius, K. (1961). Influence of food on fecundity and longevity of adults of Itoplectis conquisitor (Say) (Hymenoptera: Ichneumonidae). Canadian Entomologist, 93, 771–780.
103. Liu, Y. H. & Tsai, J. H. (2000). Effects of temperature on biology and life table parameters of the Asian citrus psyllid, Diaphorina ctiri Kuwayama (Homoptera: Psyllidae). Annals of Applied Biology, 137, 201–206.
104. Lopez, V. M., & Hoddle, M. S. (2014). Effects of body size, diet, and mating on the fecundity and longevity of the goldspotted Oak borer (Coleoptera: Buprestidae). Annals of the Entomological Society of America, 107(2), 539–548.
105. Malais, M. H. & Ravensberg, W. J. (1992). Knowing and recognizing, the biology
89
of glasshouse pests and their natural enemies. The Netherlands: Koppert B.V.
106. Marchioro, C. A. & Foerster, L. A. (2013). Effects of adult-derived carbohydrates and amino acids on the reproduction of Plutella xylostella. Physiological Entomology, 38, 13–19.
107. Mann, R. S. & Stelinski, L. L. (2010). An Asian citrus psyllid parasitoid: Tamarixia radiata (Waterston) (Insecta: Hymenoptera: Eulophidae). University of Florida FDACS/DPI Entomology Circular. Retrieved from http://entnemdept.ufl.edu/creatures/beneficial/wasps /tamarixia_radiata.htm
108. Mann, R. S., Pelz-Stelinski, K., Herman, S. L., Tiwari, S., & Stelinski, L. L. (2011). Sexual transmission of a plant pathogenic bacterium, Candidatus Liberibacter asiaticus, between conspecific insect vectors during mating. PLoS ONE, 6(12): e29197.
109. McFarland, C. D. & Hoy, M. A. (2001). Survival of Diaphorina citri (Homoptera: Psyllidae) and its two parasitoids, Tamarixia radiata (Hymenoptera: Eulophidae) and Diaphorencyrtus aligarhensis (Hymenoptera: Encyrtidae), under different relative humidities and temperature regimes. Florida Entomologist, 84(2), 227– 233.
110. McNeely, J. A., Mooney, H.A., Neville, L. E., Schei, P., & Waage, J.K. (2001). Gland, Switzerland and Cambridge, United Kingdom. A global strategy on invasive alien species. In collaboration with the Global Invasive Species Programme. IUCN, 1–63. ISBN: 2-8317-0609-2.
111. Mead, F. W. & Fasulo, T. R. (1998). Asian citrus psyllid - Diaphorina citri Kuwayama. University of Florida FDACS/DPI Entomology Circular, 180. Retrieved from http://entnemdept.ufl.edu/creatures/citrus/acpsyllid.htm
112. Meyer, J. M. (2007). Microbial associates of the Asian citrus psyllid and its two parasitoids: symbionts and pathogens. Master of Science Thesis. University of Florida, Florida, USA.
113. Michaud, J. P. (2004). Natural mortality of Asian citrus psyllid (Homoptera: Psyllidae) in central Florida. Biological Control, 29, 260–269.
114. Miles, G. P., Stover, E., Ramadugu, C., Manjunath, K. L., & Lee, R. F. (2017). Apparent tolerance to Huanglongbing in citrus and citrus-related germplasm. HortScience, 52(1), 31–39.
115. Milosavljević, I., Schall, K. A., & Hoddle, M. S. (2017). Classical Biological Control of Asian Citrus Psyllid, Diaphorina citri (Hemiptera: Liviidae), in California. University of California, Riverside. Retrieved from
90
http:biocontrol.ucr.edu/asian_citrus_psyllid.html
116. Milosavljević, I., Schall, K., Hoddle, C., Morgan, D., & Hoddle, M. (2017). Biocontrol program targets Asian citrus psyllid in California’s urban areas. California Agriculture, 71 (3), 169–177.
117. Miller Chemical and Fertilizer, LLC. (2011). NU-LURE INSECT BAIT: Active and inert ingredients. Hanover, Pennsylvania. Retrieved from https://greenbook assets.s3.amazonaws.com/L118397.pdf
118. Miyashita, A., Kizaki, H., Sekimizu, K., & Chikara, K. (2016). Body-enlarging effect of royal jelly in a non-holometabolous insect species, Gryllus bimaculatus. Biology Open, 0, 1–7. doi:10.1242/bio.019190.
119. Moffis, B. L., Burrow, J. D., Dewdney, M. M., & Rogers, M. E. (2016). Frequently asked questions about Huanglongbing (HLB; citrus greening) for homeowners. UF/IFAS Extension, PP326. Retrieved from https://edis.ifas.ufl.edu/pdffiles/PP/PP32600.pdf
120. Monzo, C., Qureshi, J. A., & Stansly, P. A. (2014). Insecticide sprays, natural enemy assemblages and predation on Asian citrus psyllid, Diaphorina citri (Hemiptera: Psyllidae). Bulletin of Entomological Research, 1–10.
121. Morita, H., Ikeda, T., Kajita, K., Fujioka, K., Mori, I., Okada, H., Uno, Y., & Ishizuka, T. (2012). Effect of royal jelly ingestion for six months on healthy volunteers. Nutrition Journal, 11(77). DOI: 10.1186/14752891-11-77.
122. Morris, R. A., Erick, C., & Estes, M. (2009). Greening infection at 1.6%, survey to estimate the rate of greening and canker infection in Florida citrus groves. Citrus Industry, 90, 16–18.
123. Nava, D. E., Torres, M. L. G., Rodrigues, M. D. A., Bento, J. M. S., & Parra, J. R. P. (2007). Biology of Diaphorina citri (Hemiptera: Psyllidae) on different hosts and at different temperatures. Journal of Applied Entomology, 131: 709D715.
124. Navarrete, B., McAuslane, H., Deyrup, M., & Pena, J. E. (2013). Ants (Hymenoptera: Formicidae) associated with Diaphorina citri (Hemiptera: Lividae) and their role in its biological control. Florida Entomologist, 96(2), 590– 597.
125. Onagbola, E., Boina D., Hermann, S., & Stelinski, L. (2009). Antennal sensilla of Tamarixia radiata (Hymenoptera: Eulophidae), a parasitoid of Diaphorina citri (Hemiptera: Psyllidae). Annals of the Entomological Society of America, 102, 523–531.
91
126. Paiva, P. E. B., & Parra, J. R. P. (2012). Natural parasitism of Diaphorina citri (Kuwayama) (Hemiptera: Psyllidae) nymphs by Tamarixia radiata (Waterston) (Hymenoptera: Eulophidae) in Sao Paulo orange groves. Revista Brasileira de Entomologia, 56(4), 499–503.
127. Pelz-Stelinski, K. S., Brlansky, R. H., Ebert, T. A., & Rogers, M. E. (2010). Transmission parameters for Candidatus Liberibacter asiaticus by Asian citrus psyllid (Hemiptera: Psyllidae). Journal of Economic Entomology, 103(5), 1531– 1541.
128. Perez, J. T., French, J. V., Summy, K. R., Baines, A. D., & Little, C. R. (2009). Fungal phyllosphere communities are altered by indirect interactions among trophic levels. Microbial Ecology, 57(4), 766–774.
129. Pilkington, L. J. (2010). Protected biological control- biological pest management in the greenhouse industry. Biological Control, 52, 216–220.
130. Pluke, R. W. H., Qureshi, J. A., & Stansly, P. A. (2008). Citrus flushing patterns, Diaphorina citri (Hemiptera: Psyllidae) populations and parasitism by Tamarixia radiata (Hymenoptera: Eulophidae) in Puerto Rico. Florida Entomologist, 91(1), 36–42.
131. Qing, T. Y. (1990). On the parasite complex of Diaphorina citri (Kuwayama) (Homoptera: Psyllidae) in Asian-Pacific and other areas. Proceedings of the 4th International Asia Pacific Conference on Citrus Rehabilitation, 240–245.
132. Qureshi, J. A., Rogers, M. E., Hall, D. G., & Stansly, P. A. (2009). Incidence of invasive Diaphorina citri and its introduced parasitoid Tamarixia radiata in Florida citrus. Journal of Economic Entomology, 102(1), 247–56.
133. Qureshi, J. A., Rohrig, E., & Stansly, P. A. (2012). Introduction and augmentation of natural enemies for management of Asian citrus psyllid and HLB. Citrus Industry, 14–16. Retrieved from http://swfrec.ifas.ufl.edu/docs/pdf/entomology/research/pubs/ci_june_2012.pdf
134. Qureshi, J. A., & Stansly, P. A. (2009). Exclusion techniques reveal significant biotic mortality suffered by Asian citrus psyllid Diaphorina citri (Hemiptera: Psyllidae) populations in Florida citrus. Biological Control, 50, 129–136.
135. Reddy, G.V.P. & Guerrero, A. (2004). Interactions of insect pheromones and plant semiochemicals. Trends in Plant Science, 9(5), 253–261.
136. Reeve, J. D. (1987). Foraging behavior of Aphytis melinus: effects of patch density and host size. Ecology, 68(3), 530–538.
92
137. Rohrig, E. (2014). An Asian citrus psyllid parasitoid: Diaphorencyrtus aligarhensis (Shafee, Alam, and Argarwal) (Insecta: Hymenoptera: Encyrtidae). University of Florida FDACS/DPI Entomology Circular. Retrieved from http://entnemdept.ufl. edu/creatures/beneficial/wasps/Diaphorencyrtus_ aligarhensis.htm
138. Rohrig, E. A., Hall, D. G., Qureshi, J. A., & Stansly, P. A. (2012). Field release in Florida of Diaphorencyrtus aligarhensis (Hymenoptera: Encyrtidae), an endoparasitoid of Diaphorina citri (Homoptera: Psyllidae), from mainland China. The Florida Entomologist, 95(2), 479–481.
139. Sanchez, F. & Smitz, J. (2012). Molecular control of oogenesis. Biochimica et Biophysica Acta 1822, 1896–1912.
140. SAS Institute. 2004. SAS for Windows, version 9.1. SAS Institute, Cary, NC.
141. Shatters, R. G. (2011). The psyllid feeding process: composition and biosynthetic inhibition of the salivary sheath. Proceedings of the 2nd International Research Conference on Huanglongbing, 43. Retrieved from https://www.plantmanagemen tnetwork.org/ proceedings/irchlb/2011/presentations/IRCHLB_2011_2.4.pdf
142. Sinatra, S. T. (1998). The coenzyme Q10 phenomenon. Chicago, Illinois: Keats.
143. Sirot, E., Ploye, H., & Bernstein, C. (1996). State dependent superparasitism in a solitary parasitoid: egg load and survival. Behavioral Ecology, 8(2), 226–232.
144. Skelley, L. H., & Hoy, M. A. (2004). A synchronous rearing method for the Asian citrus psyllid and its parasitoids in quarantine. Biological Control, 29, 14–23.
145. Stenberg, J. A., Heil, M., Ahman, I., & Björkman, C. (2015). Optimizing crops for biocontrol of pests and disease. Trends in Plant Science, 20(11), 698–712.
146. Tang, Y. Q., & Huang, Z.P. (1991). Studies on the biology of two primary parasites of Diaphorina citri Kuwayama (Homoptera: Psyllidae). Proceedings of the 6th International Asia Pacific Workshop Integrated Citrus Health Management, 91–98.
147. Tashiro, N., Noguchi, M., Ide, Y., & Kuchiki, F. (2013). Sooty spot caused by Cladosporium cladosporioides in postharvest Satsuma mandarin grown in heated greenhouses. Journal of General Plant Pathology, 79, 158–161. DOI: 10.1007/s10327-013-0430-1
148. te Velde, E. R., & Pearson, P. L. (2002). The variability of female reproductive ageing. PubMed-NCBI, 8(2), 141–154.
93
149. Teiken, C., Lemaux, P., Grafton-Cardwell, E. E., & McRoberts, N. (2015). Genetic engineering to protect citrus from HLB. Citrograph Magazine, 24–31.
150. Uchida, K., Moribayashi, A., Matsuoka, H., & Oda, T. (2003). Effects of mating on oogenesis induced by amino acid infusion, amino acid feeding, or blood feeding in the mosquito Anopheles stephensi (Diptera: Culicidae). Journal of Medical Entomology, 40(4), 441–446.
151. University of California Integrated Pest Management: UC management guidelines for Asian citrus psyllid on citrus. (2015, September 15). Agriculture and Natural Resources, University of California statewide Integrated Pest Management program. Retrieved from http://www.ipm.ucdavis.edu/pmg/pestnotes /r107304411.html
152. University of California Riverside, Center for Invasive Species Research (CISR). (2014, October 31). Tamarixia and Argentine ant. (Video File). Retrieved from https://youtu.be/VGXay2RYDuI
153. U.S. Department of Agriculture, Foreign Agricultural Service. (2016). Citrus: world markets and trade. Retrieved from http://usda.mannlib.cornell.edu/usda/fas/ citruswm//2010s/2016/citruswm-01-20-2016.pdf
154. U.S. Department of Agriculture, Foreign Agricultural Service. (2017). Citrus: world markets and trade. Retrieved from https://apps.fas.usda.gov/psdonline/circulars/citrus.pdf
155. U.S. Department of Agriculture, Foreign Agricultural Service (2018 January). Citrus: world markets and trade. Retrieved from https://apps.fas.usda.gov/psdonline/circulars/citrus.pdf
156. U.S. Department of Agriculture, National Agricultural Statistics Service. (2014). Citrus: abandoned acres. Retrieved from https://www.nass.usda.gov/Statistics_by _State/Florida/Publications/ Citrus/Abandoned_Acreage/CitAA14.pdf
157. U.S. Department of Agriculture, National Agricultural Statistics Service. (2016). Citrus: abandoned acres. Retrieved from https://www.nass.usda.gov/Statistics _by_State/Florida/Publications/Citrus/Abandoned_Acreage/CitAA16.pdf
158. Varley, G. C., & Edwards, R. L. (1957). The bearing of parasite behaviour on the dynamics of insect host and parasite populations. Journal of Animal Ecology, 26(2), 471–477.
159. Visser, M. (1994). The importance of being large: the relationship between size and fitness in females of the parasitoid Aphaereta minuta (Hymenoptera: Braconidae). Journal of Animal Ecology, 63 (4), 963–978.
94
160. Vogt, J.T. & Koch, F. H. (2016). The evolving role of forest inventory and analysis data in invasive insect research. American Entomologist, 62(1), 46–58.
161. Wager-Page, S. (2010, June). Proposed release of a parasitoid (Tamarixia radiata) (Waterston) for the biological control of Asian citrus psyllid (Diaphorina citri) (Kuwayama) in the continental United States. USDA Environmental Assessment. 1–21.
162. Wang, N., Stelinski, L. L., Pelz-Stelinski, K. S., Graham, J. H., & Zhang, Y. (2017). Tale of Huanglongbing disease pyramid in the context of the citrus microbiome. Phytopathology Review, 107, 380–387.
163. Wang, Y., Ma, L., Zhang, W., Cui, X., Wang, H., & Xu, B. (2016). Comparison of the nutrient composition of royal jelly and worker jelly of honey bees (Apis mellifera). Apidologie, 47, 48–56.
164. Warnert, J. E. (2013a, August 2). Newly found Asian citrus psyllids prompt quick action in Tulare. [blog post]. Retrieved from http://ucanr.edu/blogs/ANRnewsreleases/
165. Warnert, J. E. (2013b, August 19). New resource for growers and homeowners waging Asian citrus psyllid battle. [blog post]. Retrieved from http://ucanr.edu/blogs/ANRnewsreleases/
166. Warnert, J. E. (2015, July 22). UC ANR scientists urge citrus farmers and backyard growers to be vigilant in face of new disease find. [blog post]. Retrieved from http://ucanr.edu/blogs/ANRnewsreleases/
167. Warnert, J. E. (2017, July 28). UC has boots on the ground in an unrelenting search for Asian citrus psyllid. [blog post]. Retrieved from http://ucanr.edu/blogs/blogcore/postdetail.cfm?postnum=24752
168. Waterston, J. (1922). On the chalcidoid parasites of psyllids (Hemiptera, Homoptera). Bulletin of Entomological Research, 13, 41–58.
169. Weiner, J. (1994). The beak of the finch. New York City, New York: Vintage Books.
170. Wenninger, E. J., & Hall, D. G. (2007). Mating age and time in Diaphorina citri: daily timing of mating and age at reproductive maturity in Diaphorina citri (Hemiptera: Psyllidae). The Florida Entomologist, 90(4), 715–722.
171. Wheeler, D. (1996). The role of nourishment in oogenesis. Annual Review of Entomology, 41, 407–431.
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172. Wright, M. G., Hoffmann, M. P., Chenus, S. A., & Gardner, J. (2001). Dispersal behavior of Trichogramma ostriniae (Hymenoptera: Trichogrammatidae) in sweet corn fields: implications for augmentative releases against Ostrinia nubilalis (Lepidoptera: Crambidae). Biological Control, 22, 29–37.
173. Yavorska, T. (2012). Role of TAp73 in female reproductive aging and fertility. University of Toronto (Canada), ProQuest (2012). Web.
174. Yu-qing, T., & Zhi-peng, H. (1991). Studies on the biology of two primary parasites of Diaphorina citri Kuwayama (Homoptera: Psyllidae). The 6th International Asia Pacific Workshop on Integrated Citrus Health Management, 91–98.
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APPENDIX A. CONTACTS FOR THESIS WORK
California State Polytechnic University, Pomona
Buelna, C. Plant Science Department undergraduate student (class of 2016)
CPP University Police Non-emergency/Business Line 909-869-3070; Anonymous Crime Tip Line 909-869-3399; Live Scan Information Line 909-869-6738; [email protected]; for notification of CPP grove work, especially when working early mornings and weekends
Green, R., Ph.D. Lecturer in the Plant Sciences Department; Thesis Advisor; 909- 869-5293; [email protected]
Harshberger, T. Administrative Support Coordinator; 909-869-2214; [email protected]
Lehan, B. J., MS Lecturer in the Plant Sciences Deparment, ACP Outreach Coordinator; [email protected]
Kim, H., Ph.D. Professor of Statistics; Co-Director of the Consulting Center for Statistics and Applied Mathematics; Advisor to students from Spring 2017 Advanced STATS 559 class; 909-869-3493; [email protected]
Matias, D. Farm Supervisor in the Plant Sciences Department; 909-869-2061; [email protected]
Mellano, V., Ph.D. Plant Sciences Department Chair, Professor of Agribusiness & Food Industry Management, Agricultural Sciences and Plant Sciences; 909-869- 2809; [email protected]
Soper, A., Ph.D. Assistant Professor in the Plant Sciences Department, Thesis Committee Chair; 909-869-4367; [email protected]
Still, D., Ph.D. Executive Director of the California State University Agricultural Research Institute; 909-869-2138; [email protected]
California Department of Food & Agriculture at Mt. Rubidoux Field Station
Herreid, J., MS Agricultural Specialist; [email protected]
Morgan, D., Ph.D. Environmental Program Manager; Thesis Advisor; [email protected]
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Muniz, A. Environmental Scientist; [email protected]
Radabaugh, G. Environmental Scientist; [email protected]
University of California, Riverside
Hoddle, M. S., Ph.D. Biological Control Specialist; Principal Investigator & Director of the Center for Invasive Species Research (CISR); 951-827-4714; [email protected]
Irvin, N., Ph.D. Control Specialist; Research Scholar; 951-827-4360, [email protected]
Stouthamer, R., Ph.D. Professor of Entomology; 951-827-2422; [email protected]
University of California, Cooperative Extension Division of Agriculture & Natural Resources, ANR Lindcove Research & Extension Center
Grafton-Cardwell, E., Ph.D. Director of Lindcove Research & Extension Center; Research Entomologist at UCR; 559-592-2408 ext. 152; [email protected]
University of Florida/IFAS, Research and Education Center, Immokalee, Florida
Stansly, P. A., Ph.D. Professor of Entomology; 239-658-3427; [email protected]
Chen, X., Ph.D. Graduate Scholar 2015-2017; [email protected]
United States Department of Agriculture, Agricultural Research Service, United States Horticultural Research Laboratory, Florida
Stover, E., Ph.D. Research Horticulturalist & Geneticist
Stoller International Enterprises, Stoller USA, International Marketing, Research, & Development for Growers and their Products, USA & International
Shortell, R., Ph.D. Vice President of Global Marketing, Stoller Group
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APPENDIX B. FEEDING TRIALS DATA COLLECTION
Egg Load # Tibia Length 1 Tibia Length 2 Notes 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 TODAY’S DATE: DIET: DATE EMERGED: DATE FED: #HOURS FED: #DAYS POST EMERGENCE: DATE KILLED: HOW KILLED: DISSECTION DATE:
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APPENDIX C. DIET TREATMENT FORMULATIONS
Diet Treatment ID A C G I Ingredients RJ and DI Honey and ACP, Honey CoQ10, water DI water and DI water Honey and DI water Ratios and 3 RJ:1 DI 3 Honey: 2 ACP: 2 CoQ10: Percent water 1 DI water 1 Honey: 1 Honey: Concentrations 1 DI water 1 DI water
75% RJ 75% Honey 50% ACP 50% CoQ10 25% DI 25% DI 25% Honey 25% Honey water water 25% DI water 25% DI water Notes on Blot extra Blot extra All ACP stages Pure, dry, Administration mixture off mixture off were used; powder form and Storage of feeding strip feeding strip manually must be stored Diet or wasps or wasps will crushed via at -18℃ in the Treatments will stick to stick to it mortar and upright it while while flying pestle; make freezer in the flying and and feeding fresh mixture CPP Plant feeding inside the for each diet Sciences inside the vial; store in treatment Laboratory, in vial; store in airtight event; mixed an airtight airtight container in diet treatment container to container in CPP Plant does not store prevent CPP Plant Sciences and will be oxidation and Sciences Laboratory contaminated spoilage Laboratory wine cooler by fungus wine cooler 20−25℃ 20−25℃ ACP, Asian citrus psyllid; CoQ10, Coenzyme Q10, DI, deionized water; RJ, Royal jelly
Diet Treatment A: Pre-made royal jelly and honey
Diet Treatment C: Honey
Diet Treatment G: D. citri (ACP) nymphs and adults, D. citri nymphal frass
Diet Treatment I: Pure powder form of Coenzyme Q10
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APPENDIX D. TAMARIXIA RADIATA DISSECTION PROTOCOL
1. Dead T. radiata that need to be dissected are in the CPP Plant Sciences Laboratory upright freezer at -18℃. They are in a labeled tub. Each vial should have approximately 50−150 wasps. Collection and recording data is on each vial with a piece of colored label tape.
Figure A. Bin of dead T. radiata vials that need to be dissected to record egg load and hind tibia length (HTL).
2. Data collection sheets are located in Dani’s T. radiata drawer in the CPP Plant Sciences Laboratory. Transfer the appropriate data label information from the vial to the data collection sheet. The following information must be recorded: diet treatment, date of emergence, date fed, date of dissection, diet exposure length, T. radiata age, egg load, and hind tibia length (HTL).
Figure B. Record sheet to record egg load and HTL for individual T. radiata.
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3. Use a binocular compound light microscope (American Optical One-Fifty) with the 10X lens located in the CPP Plant Sciences Laboratory. Ocular ruler (WF10X, 18 mm; 0.1 mm scale) for compound microscope should be attached to the microscope, or in the ACP secret stash cupboard. Please turn microscope lamp off, wrap cord, and cover with dust cover after every use.
Figure C. Compound Light Microscope (American Optical One-Fifty, American Optical Company) with optic micrometer ruler (WF10X, 18 mm; 0.1 mm scale).
4. The dissection tools that are needed include the following: ultrafine forceps (#4535 forceps #5, Swiss pattern, super fine-honed, high-quality stainless steel, 112 mm, imported), plastic pipette dropper (0.14 oz; 4 mL), bent probe with sharp tip, needle- point scriber probe (full-spear, stainless steel, #4755), teasing needle (angled tip, #4752), dissecting scissors (straight, sharp point, stainless steel (114 mm, #4713; DO NOT use for cutting tape), glass microscope slides (Karter Scientific microscope slides, 206A2), and glass coverslips (Karter Scientific microscope cover glass; 22 x 22 mm, 0.13−0.16 mm, 141473; 211Z3).
5. Use a plastic pipette dropper and small beaker with water to make wet mount slides of T. radiata. Place three drops of water on a single glass slide and put one wasp on each water droplet. Then put one coverslip over each wasp and droplet (x 3). Making three specimens per slide saves time and glass slides. [Author’s note: as the primary CPP researcher, Dani Ruais, gained speed and confidence, she dissected three wasps per water droplet (x 9 wasps per glass slide); therefore, less coverslips were used, less slides needed to be cleaned, and less time was wasted overall].
6. Once the slide is prepared, place it on the compound light microscope stage and use the metal stage clips to fasten. Start with the least powerful objective lens and continue to greater focus to see T. radiata abdomen and antennal characteristics. Adjust focus and lamp light strength as needed.
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Figure D. Female on slide. Looking through the ocular ruler; the female T. radiata is centered on a glass slide. She stays on the water droplet with a single coverslip on top of her.
7. Male wasps are not measured. They are identified by long, hairy segmented antennae whereas the females have shorter, less hairy antennae.
Figure E. Antennal differentiation between male and female. Male T. radiata has long hairy antennae. (A) Male T. radiata, (C) with close-up of antennae, (B) Female T. radiata, (D) with close-up of antennae.
8. Measure right leg HTL with ocular ruler to the closest micrometer (mm). Range of lengths can be between 1.3−4.3 mm. Record in appropriate space on data collection sheet.
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Figure F. Female wasp. Measure right HTL for every female. Depending on orientation, CPP researcher may need to adjust compound light microscope light to get a clear view of the beginning and end measurements of her HTL.
9. Use a probe to place pressure on the coverslip and slide around the abdomen of the wasp until it pops and the eggs spill out into the water under the slide. Sometimes it takes a lot of pressure with the probe (or whichever instrument is comfortable to work with). Be careful not to rupture the eggs. Also probe into the abdomen thoroughly to release all eggs. Mature eggs can be described as small gray kidney bean shapes. Record counts of mature eggs on the appropriate data collection sheet.
Figure G. T. radiata eggs. Eggs under compound microscope after CPP researcher ruptures her abdomen with probe and other dissection tools.
10. Do not record wasps that are stuck to feeding-strip on the inside of the vial lid. To prevent wasps from sticking, only dampen feeding-strip with diet treatment, blot feeding-strip thoroughly before adhering it to vial lid, do not shake vials, and do not put vials under direct light or intense heat during the administration of diet treatments. Furthermore, fungal growth occurs rapidly when there is excess of diet treatment and dead wasps stuck to it—which may ruin your samples prior to dissection.
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Figure H. Female and male T. radiata stuck to diet treatment feeding-strip on inside of vial lid.
11. Diet treatment trials are organized and entered (by wasp age, diet treatment, and exposure length to diet treatment) into the Microsoft Excel data collection sheet. Each wasp is assigned an individual identification number and letter and the following information is recorded digitally on the CPP Plant Sciences Laboratory computer.
Figure I. Microsoft Excel record sheet for Experiment One: Regeneration of Egg Load. All above information should be recorded correctly on raw data sheets in order to enter into the Microsoft Excel file.
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APPENDIX E. EXPERIMENT ONE FIGURES
Hind Tibia Length (HTL)
Egg Load
Figure 1. Correlation between egg load and hind tibia length (HTL). All collected data is included (N = 4,556). Pearson correlation between egg load and HTL was 0.142 with P = 0.001.
Frequency
Egg Load
Figure 2. Irregular distribution of Tamarixia radiata egg load. This is for all dissected wasps (N = 4,556). 33.9% of recorded wasps had zero egg load at time of dissection.
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4
3.5
3
2.5
2
Egg Load 1.5
1
0.5
0 6 12 18 Wasp Age (in days)
Figure 3. Histogram of wasp age (in days) by average egg load. The mean egg loads for each wasp age are: 6DO = 3.70 eggs, 12DO = 2.53 eggs, and 18DO = 1.73 eggs.
3.5
3
2.5
2
1.5 Egg Load 1
0.5
0 24 48 72
Exposure Length to Diet (in hours)
Figure 4. Histogram of exposure length to diet (in hours; 24 hours, 48 hours, and 72 hours) by average wasp egg load. The average egg loads are 2.84 eggs at the 24-hour exposure length, 3.07 eggs at the 48-hour exposure length, and 2.05 eggs at the 72-hour exposure length. The difference between 24-hour and 48-hour exposure lengths is not statistically significant (P = 0.101 > 0.05).
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3
2.9
2.8
2.7
2.6
LoadEgg 2.5
2.4
2.3
A C G I
Wasp Diet
Figure 5. Histogram of average egg load by diet treatment. Diet A (royal jelly), Diet C (honey), Diet G (ACP), and Diet I (Coenzyme Q10). The average egg loads for the diet treatments are 2.94 eggs (Diet G), 2.61 eggs (Diet I), 2.54 eggs (Diet A), and 2.53 eggs (Diet C).
4
3 I 2 G
Diet 1 C Egg Load A 0 6 12 18 Wasp Age (in days)
A C G I
Figure 6. Histogram of wasp age by diet treatment (F6, 4531 = 3.63, p = 0.001). The greatest egg load was produced by 6DO wasps fed Diet G (ACP) with an average of 3.84 eggs, followed by 6DO wasps fed Diet C (honey) with 3.77 eggs, 6DO wasps fed Diet I (Coenzyme Q10) with 3.66 eggs, and 6DO wasps fed Diet A (Royal jelly) with 3.52 eggs. The least amount of eggs was produced by 18DO wasps fed Diet C (honey) with 1.18 eggs.
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5 4
3
72 2 48 Egg Load 1 24 0 Exposure to Length Diet 6 12 18 Wasp Age
24 48 72
Figure 7. Histogram of the average egg load of wasp: age by exposure length to diet. The greatest egg-producing combinations were 6DO wasps exposed to diet treatments for 24 hours (4.26 eggs) and for 48 hours (4.22 eggs), while 18DO wasps exposed to diets for 72 hours produced the least amount of eggs (0.95 eggs).
5 4
3 I
2 C Egg Load G Diet 1 A 0 24 48 72 Exposure Length to Diet (in hours)
A G C I
Figure 8. Histogram of diet by exposure length (F6, 4531 = 38.09, p value < .001). Diet I (Coenzyme Q10) at 24-hour exposure length produced greatest egg load (4.25 eggs), then Diet G (ACP) at 48 hours (3.96 eggs), Diet C (honey) at 48 hours (3.56 eggs), and Diet A (royal jelly) at 48-hour exposure length (3.02 eggs). The diet and exposure length combination that produced the least amount of eggs was Diet I at 72-hour exposure length (1.77 eggs).
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APPENDIX F. FIELD DATA COLLECTION
Trial #: Wasp ID: Diet: Wasp Age: Date Released: Date Processed: # Hrs Exp. to Diet: Date Collected:
Nymphal Instar Parasitized Host Fed Notes 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
If Wasp Collected, Hind Tibia Length (HTL): Egg Load#:
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APPENDIX G. EXPERIMENT TWO FIGURES
Figure 9. Percent parasitized and percent host-fed 6-day-old Tamarixia radiata. Trial D wasps were fed diet treatments A, C, or I for 24 hours prior to release in CPP grove. Sleeve-cages were collected after 72 hours in the grove.
Figure 10. Percent parasitized and percent host-fed 12-day-old Tamarixia radiata. Trial B wasps were fed diet treatments A, C, or I for 24 hours prior to release in CPP grove. Sleeve-cages were collected after 72 hours in the grove.
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