**United States**

**Department of Agriculture**  

# Field **Release of the** 

**Parasitoid Diaphorencyrtus**  **aligarhensis for the**  **Biological Control of the Asian Citrus Psyllid**  **in the Conti**  **guous United**  **States Environmental Assessment,**  **June 2014**  **Marketing and Regulatory**  **Programs** Animal and Plant Health Inspection Service

# Field **Release of t he Parasitoid** 

**Diaphorencyrtus aligarhensis for the**  **f or the Biological**  **Control of the Asian Citrus**  **Psy llid**  **in the Conti guous**  **United States**

**Env ironmental**  **As sessment,**  **June 2014**  **Ag ency** **Contact** : Shirley W ager- Page, Ph.D. Pest Permitting Plant Protection a nd Quarantine Animal and Plant Health Inspection Service U.S. Department of Agriculture 4700 River Road, Unit 133 Riverdale, MD 20737

– 1236Non
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– 2600 (voice and TDD). M en tion o f c o m pan ies o r c o mm erc ial produ cts in t h i s repor t do e s no t i m p ly re c o m m enda t ion o r endors e m e n t b y USDA o v e r o t her s no t me n t i oned

. U S DA ne i t he r guaran t ee s no r w arran ts t h e st andard o f an y produ ct m en tio ned

. P rodu ct n a m e s are me n t i one d to repor t fa ct ua lly on a v a il ab le da ta an d to pro v ide s p e c i fic inf o rmatio n
. This pub l i c a tion repor

ts re s earch i n vo l v i n g pe sti ci d e s. All u s e s o f p e st i cid e s m u s t b e reg i st ere d b y appropr i a te St a te an d / o r F edera l agen ci e s be f ore t he y c a n b e re c o m m ended. C A U TIO N : P e s t i c id e s c a n b e inj u rio u s to h u m a n s, d o m e st i c an i m a l s , de sira b le p la n t s , an d fi sh and o t he r wil d l i fe— if t he y are no t hand led o r app li e d prope r l y . U se a ll pe s t i cid e s s e le ct i ve ly an d c are fu ll y . Foll o w re c o mme nde d label p r a ct i c e s fo r t h e use and d i s p o s a l o f pe s ti c i d e s an d pe s t i cide c o n t a in ers
.

## Table of Contents
I. Purpose and Need for the Proposed Action

................................ . 1 II. Alternatives

................................ ................................ ................. 2 III

. Affected Environment

................................ ................................ . 5 I V. Environmental Con sequences
................................ ................... 7 V. Other Issues

................................ ................................ ............. 1 2 VI

. Agencies, Organizations, and Individuals C onsulted

............... 1 3 VII. References

................................ ................................ ............. 1 4 **Ap pendix 1.**  National Quarantine Map for ACP and HLB. **Ap pendix 2.**  Host
- specificity testing from Hoddle and Bistline, 2013.1



## I. **Purpose and Need for the Proposed Ac**  **tion** The U.S. Department of Agriculture (USDA), Animal and Plant Health Inspection Service (APHIS), Plant Protection and Quarantine (PPQ), Pest Permitting Branch is proposing to issue permits for release of the insect parasitoid 1 species Diaphorencyrtus aligarhensis ( D. aligarhensis ) (H ym enoptera: Ency

rt idae)

. This organism would be used by the permit applicant for biological control of the nonindigenous Asian citrus psyllid ( ACP ) Diaphorina citri (Hemip tera: Liviidae) in the conti guous United States

. This environmental assessment2 (EA) has been prepared, consistent with USDA, APHI S' National Environmental Policy Act (NEPA) implementing procedures (Title 7 of the Code of Federal Regulations (CFR), part 372). It examines the potential effects on the quality of the human environment that may be associated with the release of the parasitoid wasp, D. aligarhensis to control ACP within the conti guous United States

. This EA considers a “no action” alternati ve and the potential effects of the proposed action. The applicant’s purpose for releasing D. aligarhensis is to reduce infestations of ACP in California, although this agent could spread or be released an ywh ere in the conti guous United States

. The ACP is an insect pest of citrus because it vectors a plant pathogenic bacterium, Candidatus Liberibacter asiaticus Jagoueix, Bove ́ and Garnier, that causes a lethal citrus disease, known as huanglongbing (HLB) or citrus greening ( Halbert and Manjunath, 2004 ; Bov é 2006 ; Grafton

- Cardwell et al., 2013 ). ACP and HLB are serious invasive species that cause immense economic loses in countries with important citrus industries. In Florida, ACP was first detected in 1998 and HLB was discovered in 2005 (Hall and Hentz, 20 11)

. Since that time, HLB has been found in commercial and residential sites in all counties of Florida with commercial citrus , and it is having an adverse economic impact on citrus production in that State (Hodges and Spreen, 2012)

. HLB has also been det ected in California, Georgia, Louisiana, South Carolina , and Texas. Other S tates, including Alabama, Arizona, Hawaii , and Mississippi, are at risk for HLB because populations of ACP have been found in those S tates. See Appendix 1 for a map of national quarantine boundaries for ACP and HLB.
1 In this case, small, stingless wasps that during their development, live in the body of a single host individual, eventually killing that individual. 2 Regulations implementing the National Environmental Policy Act of 1969 (42 United States Code 4321 et seq. ) provide that an environmental assessment “[shall include brief discussions of the need for the proposal, of alternatives as required by section 102(2)(E), of the environmental impacts of the proposed action and alternatives, and a listing of agencies and persons consulted.” 40 CFR § 1508.9.2 E conomic losses to growers in Florida attributable to HLB have b een estimated at greater than $1.7 billion which translates to approximately a 16 percent reduction in grower revenues (Hodges and Spreen, 2012). An effecti ve ACP management program is considered an important part of reducing the spread of HLB (USDA

–APHI S, 2012)

. All of the existing ACP management options (discussed below) are expensive, temporary , ineffective, and/or include non

- target impacts. For these r easons, there is a need to identify and release an effective, host

specific biological control organism against ACP in the conti guous United States , particularl y for control of ACP in backyard citrus

. 

## II. **Al ternati**  **ve s**  This sectio n will explain the two alternatives available to APHI S

–PPQ: no action (no issuance of permits) and issuance of permits for environmental release of D. aligarhensis in the conti guous United States. Although APHI S’ alternatives are limited to a decision of whether to issue permits for release of D. aligarhensis , other methods are described that are currentl y used to control ACP in the United States . Use of these control methods is not an APHI S decision, and their use is likely to continue whether or not APHI S

–PPQ issue s permits for environmental release of D. aligarhensis. APHI S

–PPQ considered a third alternative but will not analyze it further. Under this third alternative, APHI S

–PPQ would issue permits for the field release of D. aligarhensis . The permits , however, would contain special provisions or requirements concerning release procedures or mitigating measures , such as limited releases of D. aligarhensis in the United States. There are no issues raised i ndicat ing that special provisions or requirement s are necessary . 

### A. **No Ac**  **tion** Under the no action alternative, APHI S

–PPQ would not issue permits for the field release of D. aligarhensis for the control of ACP

— the release of this biological control agent would not occur, and current methods to control ACP in the United States will continue. Use of these methods is likely to continue even if APHI S

–PPQ issues permits for release of D. aligarhensis. Presently , control of ACP in the United States is limited to chemical and biological control methods. **1 . Chemical Control**  There are man y insecticides that may be applied to citrus as sy stemic and foliar spray s against ACP

. Systemic insecticides are applied and3 translocated throughout the tree. Sy stemic insecticides includ e imidacloprid , aldicarb, and thiamethoxam

. Foliar spray s are applied to the leaves and include insecticides such as fenpropathrin, zeta

- cy permthrin, chlorpy rifos, chlorantraniliprole , spirotetramat, abamectin , and carbaryl. Organic insecticide options are available to control ACP includi ng Neem oil, py rethrin, and narrow range oil. There are no chemical controls for HLB. **2 . Biological Control**  The parasitic wasp Tamarixia radiata has been released against ACP in Florida and California . It causes considerable mortality of ACP in some locations in Florida

. Efficacy of T. radiata is still being evaluated in California , but it is not widely established there . APHI S prepared an environmental assessment and finding of no significant impact for environmental release of T. radiata in the contiguous United States (USDA

−APHIS, 2010a)

. D. aligarhensis originating from Taiwan and from China w ere previously released into Florida (McFarland and Ho y, 2001) but with mixed results (Halbert and Manjunath, 2004). Ithas no t established in Florida , p erhaps due to competition from T. radiata , generalist predators, and pesticides (Rohrig et al., 2012). Native predators, such as spiders, co ccinellids (lady beetles), lace wings, sy rphids, and minute pirate bugs attack ACP (Quereshi and Stansly , 2009; UC

- D ANR , 200 6 ). Coccinellids are important predators of ACP in Florida (Michaud, 2002; 2004). 

### B **. Issue Permits for Environmental Release of**  **D. aligarhensis (Preferred**  **Al ternative)**  Under this alternative, APHI S

–P PQ would issue permits upon request and after evaluation of each application for the field release of D. aligarhensis for the control of ACP in the conti guous United States . These permits would contain no special provisions or requirements concerning relea se procedures or mitigating measures. **1. D. aligarhensis**  **Taxonomic Information** (From Hoddle and Bistline, 2013). Diaphorencyrtus aligarhensis is an endo parasitoid attacking second to fourth instar ACP ny

mphs

. O viposition (egg laying) by D. aligarhensis i n the ACP ny

mph , and feeding of the larval D

. aligarhensis inside of the ACP ny mph eventuall y kills the pest.4 **a. Taxonomy**  **.** Diaphorencyrtus aligarhensis (Shafee, Alam & Agarwal) (Hy menoptera: Ency rtidae: Ency

rtinae) was originally

described from a spec imen reared from “Psylla sp. on Citrus sp.” that was collected in September 1968 in Aligarh, Uttar Pradesh, India (Shafee et al., 1975) and has been subsequently described multiple times (Ruggiero et al., 2011). The Universal Chalcidoidea Database (No yes , 2013) lists the following sy nonomies: Aphidencyrtus aligarhensis Shafee, Alam & Agarwal, 1975 Aphidencyrtus diaphorinae Myartseva & Trjapitzin, 1978 Aphidencyrtus sacchari Kaul & Agarwal, 1986 Diaphorencyrtus aligarhensis (Shafee, Alam & Agarwal, 1975) Diaphorencyrtus diaphorinae (L in & Tao, 1979) Diaphorencyrtus diaphorinae (Myartseva & Trjapitzin, 1978) Psyllaephagus diaphorinae Li n & Tao, 1979 Syrphophagus aligarhensis (Shafee, Alam & Agarwal, 1975) **b. Natural geographic range, other areas of intro**  **duction of**  **D. aligarhensis**  **.** Noyes (2013) lists the following countries from which D. aligarhensis has been recorded: India (Andhra Pradesh, Maharashtra, Punjab, Rajasthan, Tamil Nadu, Uttar Pradesh), China (Fujian [Fukien], Guangdong [Kwangtung], Guangxi [Kwangsi]), Philippines, Réunion, South Africa, Taiwan, United States (Florida), and Vietnam. Diaphorencyrtus aligarhensis has been deliberately released for the biological control of ACP in Florida and Réunion (Rohrig , 2011). It established in Réunion (v an den Berg , 1985) but failed to establish in Florida despite multiple releases of this species that were sourced from Taiwan (released 2000

- 2002) and China (released 2007

- 2009) (Rohrig et al., 2012). This parasitoid was deliberatel y released in South Afri ca (imported from Réunion in 1983) for control of the citrus pest Trioza erytreae (Del Guercio) (Hemiptera: Triozidae) and failed to establish because it was too host specific (van den Berg , 1985). In Taiwan, where D. aligarhensis is native, it is not as e ffective as T. radiata (introduced into Taiwan over 1984

- 1988 from Réunion) possibly because D. aligarhensis suffers high levels of attack by hy perparasitoids (Chien et al., 1989). In Saudi Arabia, D. aligarhensis is the dominant parasitoid attacking ACP nymphs in Mexican lime orchards (Al

- Ghamdi and Faragalla , 2000a) where it achieves maximum parasitism rates of approximately 60

− 70 percent (Al

- Ghamdi and Faragalla , 2000b). It was likely self

- introduced5 with ACP into Saudi Arabia , or it may be native having moved onto ACP from an unknown ps yl lid host.
**c. Life history**  **of D. aligarhensis**  Diaphorencyrtus aligarhensis is a solitary endoparasitoid which can parasitize second through fourth instar ACP ny mphs (Rohrig et al., 2012). An endoparasitoid completes all development inside the host. Besides parasitizing ACP, adult f

emales also kill ACP by host feed ing on ny mphs (Skelley and H oy , 2004) ; females insert their egg laying organ (ovipositor) into the ACP ny

mph to make a hole and then suck up the liquid that leaks out (Hoy and Nguyen, 2001) . ACP has five n ymphal instars but the fifth instar is unsuitable for parasitization or host fe eding b y D. aligarhensis (Skelley and Hoy , 2004). Adult females are capable of killing up to 280 ACP ny mphs through host feeding and parasitism combined (Chien , 1995). Development f

rom when the D. aligarhensis egg is laid inside an ACP ny mph to when D. al igarhensis emerge s as an adult requires about 12

- 16 day s at 25 ° C, depending on the ny mphal instar (Rohrig et al., 2012). Diaphorencyrtus aligarhensis larvae attach the parasitized ACP ny mph to plant material by

chewing a hole through the top of the host; f luids ooze out through the hole and “glue” the parasitized ACP ny mph to the plant

. After development is completed inside the ACP host, an a dult D. aligarhensis wasp emerge s from the back end of the dead ACP host

. Populations of D. aligarhensis imported int o Florida from Taiwan and China for biological control of ACP were thel yt okous (only females are produced) , and the production of all female offspring in both of these populations is likely

the result of infection with Wolbachia , a sex ratio altering bacte ria, for which both populations tested positive (Rohrig et al., 2012; Jeyaprakash and Hoy , 2000). In comparison, D. aligarhensis collected from Punjab Pakistan are bi

- parental and the sex ratio is one male to one female. All host range experiments presente d in this document were performed with this bi

- parental population of D. aligarhensis that is maintained in quarantine at University of California Riverside, as described by Hoddle and Bistline (2013). 

## III **. Af**  **fected Env ironment**  **A. A** **sian Citrus Psy llid**  ACP is a pest of citrus and close relatives of citrus, and is the targe t of D. aligarhensis . P syllids resemble miniature cicadas and are sometimes called jumping plant

- lice. ACP damages plants through its feeding activities. New shoot growth that is heavil y infested b y ACP does not expand or develop normally , and is more susceptible to breaking off. While direct damage is6 serious, there is even greater concern as ACP is an efficient vector of the bacterium that causes HLB. ACP is found in tropical and sub tropical Asia, Afghanistan, Saudi Arabia, Réunion, Mauritius, parts of South and Central America, Mexico, and the Caribbean. In the United States and its territories, this species is present in all or parts of Alabama, California, Florida, Georgia, Guam, H awaii, Louisiana, Mississippi, Puerto Rico, South Carolina, and Texas. ACP is known to develop only on members of the plant family Rutaceae including the genera Aegle, Aelopsis, Afraegle, Atalantia, Balsamocitrus, Citropsis, Citrus, Clausena, Eretmocitrus, Fortunella, Limonia, Merrillia, Microcitrus, Murraya, Naringi, Pampurus, Poncirus, Severina, Swinglea, Toddalia, Vepris, and Zanthoxylum. Murraya paniculata (L .) Jack (orange jasmine) and Citrus spp. are the preferred hosts of ACP (Aubert, 1987; Halbert and Manjunath, 2004; Yang et al., 2006). **B. North A merican Psy**  **llid Species** Hundreds of ps yl lid species occur on both native and introduced landscape plants in the United States. Several ps yl lid species are pests of crops such as pear, potato, and tomato. Each kind of ps yl lid feeds on onl y one plant species or closely

related group of plants. Some psyllids have been released for biological control of weeds, such as Boreioglycaspis melaleucae , r eleased in 2002 in Florida against the invasive plant Melale uca quinquenervia
. California has the richest native psyllid fauna in North America, and the influence of climat e and plant diversity in this region is considered key

to the diversification of native psyllids. There are currentl y 165 psyllid species known from California

. Most psyllids native to the United States are relatively uncommon and rarely

become pests. Ps yl lid species in North America could possibly be attacked by D. aligarhensis

. **C. Citrus Resources in North Am**  **erica** Citrus resources in the Unit ed States are at risk from ACP and HLB. The major citrus producing states in the United States include Florida, California, Texas , and Arizona. Of these states, Florida produced 63 percent of the total U.S. citrus crop in 2012, California produced 34 perce nt, and Texas and Arizona combined pro duced the remaining 3 percent (NASS , 2013)

. Florida is the largest producer of oranges, accounting for about 70 percent of total U.S. production, and of grapefruit, producing nearly

65 percent of total production (NAS S, 2013) . California is the largest producer of lemons, producing more than 92 percent of production, and of tangerines, accounting for a bout 80 percent of production (NASS , 2013)

.7 In 2012, total U.S. citrus exports were valued at $1.0 billion, a 2 perce nt decrease from 2011. In terms of value, fresh oranges and orange juice were the major U.S. citrus export items, followed by fresh grapefruit, lemons and grapefruit juice (FAS, 2012) . The top three overseas markets for U.S. citrus were Japan, South Korea and Canada (FAS , 2012)

. There are man y nursery

operations in the United States that produce citrus trees, orange jasmine, curryleaf, and other ACP host plants and will be adversel y affected b y AC P and HLB. Man y of these operations are identified by

the Small Business Administration as small businesses. 

## I **V .**  **En vi**  **ronmental Consequences A.**  **No Ac**  **tion A lternative**  **1. Impact of ACP**  **and HLB on Hosts of ACP and Citrus Resources**  ACP damages citrus b y feeding on young foliage, depleting the sap, and causing galling or curling of leaves. High populations feeding on a citrus shoot can kill the growing tip. ACP also causes damage b y excreting honeydew that coats plant leaves, allowing the growth of sooty mold that reduces or inhibits sunlight penetration (Chien and Chu, 1996). However, the worst threat is that ACP is an efficient vector of the bacterium which causes HLB. This is the most serious disease of citrus in the world, causing reduced production of fruit and eventual death of the trees. HLB

- infected trees show a blotch y, mottled condition of the leaves which results in the development of yellow shoots, the characteristic symptom of the disease. Trees are stunted, decline, and bear very few lop

- sided, poor quality fruit. The fruit produced b y infected trees is not suitable for either the fresh market or juice processing due to the significant increase in acidity and bitter taste. The lack of effective control measures to prevent the spread of HLB from sites of infestation to other areas and counties could le ad to higher production costs and an increase in shortages of citrus fruits and plants to the general economy . This would potentially result in increased costs for survey , detection, and treatment for the control of ACP and HLB as they spread to other area s and counties. United States citrus production for 2013/2014 is forecast down 11 percent to 6.7 million tons (FAS, 2014) . In Florida, early dry weather and HL B have caused severe fruit drop (FAS, 2014) . In California, production is down 5 percent and as a result of the December freeze, damaged fruit will be sent to be juiced (FAS, 2014). B etween 2006 and 2012, HLB was estimated to have cost Florida’s econom y $4.5 billion and 8, 257 jobs (Hodges and Spreen, 2012)

.8 Insecti cide usage to control ACP has increa sed significantly in Florida and other citrus

- producing States since the introduction of HLB, with 6 or more applications per ye ar. Prior to this, ACP was considered a minor pest. However, growers must apply both s ys temic soil drench treatments along with multiple foliar sprays (especiall y to protect newly planted and young (4 to 8

- ye ar old) trees that are more vulnerable to ACP attack because they flush more frequently . This pattern is similar to what has occurred in Brazil, South Africa, and other countri es where HLB has been introduced. In Brazil, foliar applications of insecticides have steadily increased from 12 up to 24 per year, and soil treatments from 2 up to 4 per year. In an effort to reduce costs, pesticides with longer residual effects are being substituted. Pesticides also are being applied aerially over large areas to achieve greater control of ACP. ACP populations are normall y higher in areas where there are commercial citrus plantings. When coordinated insecticide applications are completed on a majority of commercial citrus plantings in an area during a relativel y short window of one or two weeks, ACP is easier to manage. In addition, many residential areas have citrus trees in private gardens that can harbor ACP. Homeowner resistance to the application of insecticides has prevented treatment of infested trees in some locations. For nurseries in the United States that produce citrus trees, orange jasmine, and curryleaf, the effects of ACP and HLB will be adverse. For areas quarantined for HLB, nursery operations will suffer a complete loss of domestic market access. For areas quarantined only for ACP but not for HLB, nursery

operations will incur costs associated with treatment prior to interstate movement of the articles. APHI S published regulations that allow the movement of regulated nursery stock , under specific conditions , from an area quarantined for ACP, but not for an area quarantined for HLB (USDA

−APHIS, 2011). **2. Impact from the Use of Other**  **ACP and HLB Control Methods**  The continued use of chemical and biological control at current levels would result if the “no action” alternative is chosen, and may continue even if permits are issued for environmental release of D

. aligarhensis.
**a . Chemical Control**  The environmental consequences of man y of the insecticides used for control of ACP were analyzed b y APHIS (USDA

–APHI S, 20 10b ) and will not be discussed further in this document. However, an additio nal environmental consequence of the use of insecticides for control of ACP is a reduction in occurrence of natural predators of ACP, such as lady beetles, sy rphids, and spiders. M any of the insecticides used to control ACP are9 broad

- spectrum insecticides which disrupt not only native predators of ACP but also of other citrus pests. **b . Biological Control**  Native predators and previously released biological control agents ( Tamarixia radiata ) will remain in the environment and exert some level of control o ver ACP. However, these are not adequate to control ACP populations in the United States. Impacts of T. radiata have been modest at best, with parasitism levels often less than 10 percent on average, in commercial citrus production areas (Michaud, 2004; Q ureshi and Stansly , 2009). 

### B. **Bio logical**  **control alternative (preferred alternative)**  **1. Environmental Impacts of the Proposed Release of**  **D. aligarhensis.** 
**a. Known impact on vertebrates including humans**  **.** Diaphorencyrtus aligarhensis is an obligate parasitoid of psyllids , specificall y ACP . As such, it will rarel y come into contact with humans or other vertebrates, and if it does, it is incapable of stinging or biting. Ency rt id wasps have no known adverse impacts on humans or other vertebrates.
**b .**  **Direct impact of D. aligarhensis**  **(e.g. ,**  **intended effects on AC**  **P , direct effects on non**  **- targets).**  To evaluate the effects of D. aligarhensis on non - target ps yl lid species, host specificity

tests were conducted in the United States to determine possible direct effects on non

- target ps yl lid species. Six non

- target psyllid species were tested for their suitability as hosts for D. aligarhensis reproduction. Host range was assessed u s ing seven experimental designs encompassing no

- choice sequential tests, pro longed no choice and choice tests. The non

- target test species were comprised of: (1) Bactericera cockerelli (the native pestiferous potato/tomato psyllid) ( n = 480 total ny

mphs exposed); (2) Heteropsylla sp. (the native Acacia ps yl lid) ( n = 390 ny mphs exposed); (3) Arytainilla spartiophylla (a self

- introduced natural enem y of the noxious weed scotch broom psyllid) ( n = 380 n ymphs exposed); (4) Euphyllura olivina (the invasive olive ps yl lid) ( n = 320 n ymphs exposed);10 (5) Heteropsylla texana (the nati ve honey mesquite psyllid) ( n = 395 ny mphs exposed); (6) Dichlidophlebia fremontiae (the native fremontia psyllid) ( n = 375 ny mphs exposed). Successful parasitism of a non

- target species b y D. aligarhensis was observed for only

one species tested: B. co ckerelli , the native and pestiferous potato ps yl lid, the vector of the zebra chip bacterium that causes significant economic los s es in potatoes (Butler and Trumble , 2012). Observed parasitism rates ranged from 11 −18 percent for the treatment ty pes. Two of the treatment ty pe s exhibited moderate levels of parasitism, at 17 −18 percent . When compared to D. aligarhensis ACP parasitism rates across all treatment ty

pes (27

− 45 pe r cent) , it is likely B. cockerelli is not a highl y preferred host for D. aligarhensis , but it may be attacked b y D. aligarhensis if released into the environment. In California, there is a guild of generalist predators and parasitoids that attack B. cockerelli (Butler and Trumble, 2012). See Appen dix 2 for a more detailed discussion of ACP host specificity

testing conducted by

Hoddle and Bistline (2013)

. In addition, studies have conclusively demonstrated that D. aligarhensis does not transmit Candidatus Li beribacter asiaticus, the pathogen that c auses HLB in citrus (Hoy et al. , 2001). **2 .**  **Uncertainties Regarding the Environmental R**  **elease of D.**  **aligarhensis .**  Once a biological control agent such as D. aligarhensis is released into the environment and become s established, there is a possibility that it could move from the target insect ( ACP ) to attack nontarget insects , such as native psyllid s . Based on host specificity testing conducted, D. aligarhensis only successfully parasitized one psyllid species other than ACP , B

. cockerelli

, the potato/to mato psyllid . The rate of parasitism was less on B

. cockerelli than on ACP

. Nativ e species that are closely related to the target species are the most likely to be attacked (Louda et al., 2003)

. If other insect species were to be attacked b y D. aligarhen sis , the resulting effects could be environmental impacts th at may not be easily

reversed. Biological control agents such as D. aligarhensis generall y spread without intervention by man. In principle, therefore, release of these parasitoids at even one sit e should be considered equivalent to release over the entire area in which potential hosts occur and in which the climate is suitable for reproduction and survival. In addition, D. aligarhensis may not be successful in reducing ACP populations in the cont iguous United States. Approximately 12 percent of all parasitoid introductions have led to significant sustained control of the target pests, but the majority of intro ductions have failed to provide11 control of the pest (Greathead and Greathead, 1992) eithe r because intro duction did not lead to establishment or establishment did not lead to control (Lane et al., 1999). Actual impacts on ACP populations by D. aligarhensis will not be known until after release occurs

. T he environmental consequences discussed under the “no action” alternative may

occur even with the implementation of the biological control alternative, depending on the efficacy of D. aligarhensis to reduce ACP populations in the contiguous United States. **3 . Cumulative Impacts**  “Cumulative imp acts are defined as the impact on the environment which results from the incremental impact of the action when added to other past, present and reasonably

foreseeable future actions regardless of what agencies or person undertakes such other actions” (40 C FR 1508.7). Three USDA agencies have primary roles in combating HLB: APHIS , Agricultural Research Service (ARS), and National Institute of Food and Agriculture (NIFA). ARS and NIFA focus on research. APHI S focuses on survey and detection, regulatory act ion, and the development of data and protocols the citrus industry can use to manage, suppress, and slow the spread of the disease. Specificall y, APHIS' Citrus Health Response Program (CHRP) funds the administration of domestic regulations, pest surveys, coordinated area

wide suppression of ACP , and several initiatives in cooperation with State regulatory

agencies and the citrus industry in all of the major citrus

producing States: Arizona, California, Florida, and Texas. APHI S also conducts a public outr each and education program to increase the urgency about the risk of moving citrus plants in States and Territories with Federal citrus quarantines. ARS conducts HLB

- related research. Currently , ARS is using a multi

- faceted approach aimed at the three comp onents of the disease: resistance in the citrus host, suppression of the ACP vector, and control of the HLB causal organism. NI FA supports research on HLB , including way s to limit the spread of the disease and innovative management techniques to address th e ACP vector

. States are heavily

involved in implementing a variety of response activities to ACP and HLB , such as conducting ACP and HLB survey s and ACP treatments, entering into compliance agreements with packing houses to ensure that fruit packing pro cesses meet certain standards to minimize disease spread, and certifying citrus products. The States use their regulatory

authority to establish intrastate HLB quarantine areas that parallel the Federal quarantine areas.12 The citrus industry

works closely with USDA and State departments of agriculture to combat HLB . The industry coordinates its HLB and ACP management efforts based on USDA

- and State

- provided data on ACP and HLB detections and population distribution. In dustry

- funded initiatives include op erational activities, research, and outreach and education. Growers in the citrus

- producing States conduct management program s for ACP in commercial citrus; remove HLB

- infected commercial and residential citrus; and conduct outreach to homeowners. Releas e of D. aligarhensis is not expected to have any negative cumulative impacts in the contiguous United States because of its host specificit y to ACP. Effective biological control of ACP would have beneficial effects for ACP and HLB management programs, and may result in a long

- term, non
- damaging method to assist in the control of ACP and HLB, and prevent their spread into other areas potentially at risk from invasion. Suppression of ACP in citrus located in residential areas will not be achieved as it is in orchards where insec ticides are routinel y used. The primary means of suppressing ACP in residential citrus will be through biological control (USDA

−APHIS, 2012). **4 .**  **Endangered Species A ct**  Section 7 of the Endangered Species Act (ESA) and ESA’s implement ing regulations require Federal agencies to ensure that their actions are not likely to jeopardize the continued existence of federall y listed threatened or endangered species or result in the destruction or adverse modification of critical habitat. APH IS has determined that, based on the host specificity of D. aligarhensis, there will be no effect on an y listed insect in the conti guous United States. In host specificity testing, the biological control organism preferred ACP and only

successfull y parasit ized one non

- target psyllid species, B

. cockerelli

, the potato/tomato psyllid

. No psyllids are federall y listed threatened or endangered insects (FWS , 20 14 ). In addition, no listed species is dependent on ACP as a food source. Although certain federally l isted plants occur in the family Rutaceae and may serve as hosts of ACP, release of D. aligarhensis will not benefit any of these species because all occur outside of the conti guous United States (Hawaii, Puerto Rico, or the Virgin Islands)

. D. aligarhensi s will only

be released within the conti guous United States. 

## V **. Other Issues**  Consistent with Executive Order (EO) 12898, “Federal Actions to Address Environmental Justice in Minority Populations and L ow - income13 Populations,” APHI S considered the potential for disproportionately high and adverse human health or environmental effects on any minorit y populations and low

- income populations. There are no adverse environmental or human health effects from the field release of D. aligarh ensis , and their release will not have disproportionate adverse effects to an y minority or low

- income populations. Consistent with EO 13045, “Protection of Children From Environmental Health Risks and Safet y Risks,” APHI S considered the potential for dis proportionately

high and adverse environmental health and safety risks to children. No circumstances that would trigger the need for special environmental reviews are involved in implement ing the preferred alternative. Therefore, it is expected that no di sproportionate effects on children are anticipated as a consequence of the field release of D. aligarhensis

. EO 1317 5 , “ Consultation and Coo rdination with Indian Tribal Governm ents ” , w a s issued to ensure th at there would be “ m eaningful consultation and co llaboration with tribal officials in the dev e lop m ent of Federal policies that have tribal i m pli c ations....” Consistent w ith EO 131 7 5, A P H I S s ent letters of notification an d requests fo r comm ent an d consultation on the pro p osed action to tribes in California, Arizona, Florida, and Texas

. A P HIS will continue to consult and c o llaborate with Indian trib al officials to ensure that they are wel l

- infor m ed and represent e d in policy a

nd program decisions th a t m ay i m pact their agricultural interests, in accordan ce with EO 1 3175
. 

## V **I .**  **Ag encies, Organizations, and**  **Indiv iduals Consulted**  This EA was prepared and reviewed b y APHI S. The addresses of participating APHI S units, cooperators, and consultants (as applicable) follow. University

of California, Riverside Department of Entomology 900 University

Avenue Riverside, CA 92521 U.S. Department of Agriculture Animal and Plant Health Inspection Service Policy and Program Development Environmental and Risk Analy sis Services 4700 River Road, Unit 149 Riverdale, MD 20737

– 123814 

## V **II . References**  Al

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menoptera: Ency

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. An Environment Assessment Report for USDA

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- 2010/11. EDIS document FE903, Food and Resource Economics Department, Florida Cooperative Extensi on Service, Institute of Food and Agricultural Sciences, Universit y of Florida, Gainesville, FL. Accessible from http://edis.ifas.ufl.edu

. Hoy , M.A., and R. Nguyen. 2001. Classical biological control of Asian citru s psylla. 2001 Citrus Guide. Pp. 48

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. 22: 278

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–405. Lane, S.D., Mills, N.J., and Getz, W.M., 1999. The effects of parasitoid fecundity and host taxon on the biological control of insect pests: the relationship between theory and data. Ecological Entomol. 24:181 −190. Louda, S.M., R.W. Pemberton, M.T. Johnson, and P.A. Follett. 2003. Nontarget effects −The Achilles’ Heel of bio logical control? Retrospective anal ys es to reduce risk associated with biocontrol introductions. Annual Review of Entomology

. 48: 365

– 396. McFarland, C.D., and Hoy, M.A., 2001. Survival of Diaphorina citri16 ( Homoptera: Ps yl lidae), and its two parasitoid s, Tamarixia radiata ( Hy menoptera: Eulophidae) and Diaphorencyrtus aligarhensis (Homoptera: Ency

rtidae), under different relative humidities and temperature regimes. Florida Entomologist. 84: 227 −233. Michaud, J.P., 2002. Biological control of Asian citrus psyllid, Diaphorina citri (Hemiptera: Psyllidae) in central Florida: a preliminary report. Entomological News. 113: 216 −222. Michaud, J.P., 2004. Natural mortality of Asian citrus psyllid (Homoptera: Psyllidae) in central Florida. Biological Control. 29: 260 −269. NASS

— see U.S. Department of Agriculture, National Agricultural Statistics Service. Noyes, J.S. , 2013. Universal Chalcidoidea Database. World Wide Web e lectronic publication. http://www.nhm.ac.uk/research

curation/research/projects/chalcidoids/

. ( last accessed March 11, 2014 ). Qureshi , J.A., and Stansly , P.A., 2009 . Exc lusion techniques reveal significant biotic mortality suffered by

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of Florida. http://entnemdept.ufl.edu/creatures/beneficial/wasps/diaphorency rtus_alig arhensis.htm ( last accessed March 11, 2014 ). Rohrig, E., D.G. Hall, J.A. Qureshi, and P.A. Stansly . 2012. Field release in Florida of Diaphorencyrtus aliga rhensis (Hy menoptera: Ency

rtidae), an endoparasitoid of Diaphornia citri (Homoptera: Psyllidae), from mainland China. Fla. Entomol. 95: 479 −481. Ruggiero M., D. Gordon, N. Baill y, P. Kirk, and D. Nicolson. 2011. The Catalogue of Life Taxonomic Classification, Edition 2, Part A. In Species 2000 & IT IS Catalogue of L ife: 2011 Annual Checklist. Bisby F.A., Y.R. Roskov, T.M. Orrell, D. Nicolson, L. E. Paglinawan, N. Bailly , P.M. Kirk, T. Bourgoin, G. Baillargeon, and D. Ouvrard (eds). DVD; Species 2000: Reading, U.K. Shafee S.A., S.M. Alam, and M.M. Agarwal. 1975. Taxonomic survey of ency rtid parasites (Hy

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— see United States Department of Agriculture, Animal and Plant Health Inspection Service. U.S. Department of Agriculture, Animal and Plant Health Inspection Service, 2010 a . Environmental assessment: Proposed release of a parasitoid ( Tamarixia radiata Waterston) for the biological control of Asian citrus psyllid (

Diaphorina citri Kuwayama) in the continental United States, June, 2010. [Online]. Available: http://www.aphis.usda.gov/plant_health/ea /downloads/tamarixia

- radiata

acp.pdf ( last accessed March 11, 2014)

. U.S. Department of Agriculture, Animal and Plant Health Inspection Service, 2010 b

. Environmental Assessment: Asian Citru s Psyllid Control program in the Continental United States and P uerto Rico, August 2010. U.S. Department of Agriculture, Animal and Plant Health Inspection Service, 201 1. Citrus Canker, Citrus Greening, and Asian Citrus Psyllid; Interstate Movement of Regulated Nursery Stock . Federal Register. 76(81): 23449 −23459. Ap ril 27, 2011. U.S. Department of Agriculture, Animal and Plant Health Inspection Service, 201 2
. Technical Working Group (TWG) Report: HLB in Texas. April 5, 2012, 21 pp. http://nvap.aphis.usda.gov/plant_health/plant_pest_info/citrus_greening/d ownloads/pdf_files/twg/HLB

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− 352.19

**Appendix 1.** National Quarantine Map for ACP and HLB.20

**Appendix 2.** Host
- specificity testing from Hoddle and Bistline, 2013. **MATERIALS AND METHODS FOR HOST SPECIFICITY TESTING A. REARING**  **Asian citrus psyllid, Diaphorina citri**  **(Hemiptera: Psyllidae):** Asian citrus psyllids (ACP) u s ed for host range assessment experiments w ere taken from colonies established at the University

of California, Riverside (UCR), in Insectary

- level secure laboratories at the Insectary and Quarantine Facility

(I QF) (Facility

Number 93). Colonies were maintained under California Department of Food and Agriculture (CDFA) Permit No. 2870, and in accordance with designated CDFA and USDA protocols. All ACP used were from colonies initiated from California collected material and confirmed to be huanglongbing (HLB) free. **Host plant production for ACP col onies:**  Seedlings of Citrus volkameriana used as host plants for rearing ACP were obtained either through Willits and Newcomb Inc. citrus nursery (Arvin, CA), or from the CDFA rearing facility at Mt. Rubidoux Field Station (Rubidoux, CA). ACP were maintaine d on C. volkameriana seedlings which ranged from less than one year to approximately

two years of age, potted in 4 in ch diameter pots. Success of ACP reproduction, regardless of plant age, is strongl y stimulated by the presence of young budding flush (“feather flush”) and leaf axils, on which ACP females lay eggs. To maintain constant numbers of ACP for experimental use, it is critical to have plants producing feather flush on a regular basis. All C. volkameriana seedlings were maintained in greenhouses at UCR Agricultural Operations (AgOps) outside of quarantine, and were grown in UCR III typ e soil and watered and fertilized as needed. **Developing new flush for ACP oviposition:** To promote numerous lateral branching points that would bear flush growth, C. v olkameriana seedlings were initially pruned to within 15

- 20 centimeters ( cm ) above soil level in pots. In addition, the lower trunks of these seedlings were stripped of all developing buds, leaving onl y three to five possible branching points concentrated within the upper third of the trunk. After this initial round of intensive pruning, plants had to be regularl y pruned in order to induce continuous flushing on developing branches. In order to ensure a continuous supply of plants with plentiful feather flu sh for ACP reproduction, a pruning schedule was developed and implemented. Seedlings were grouped into approximately ten batches, dependent upon the total number of plants in circulation at any one time. Plants were pruned twice weekl y, with one batch each being pruned either Monday or Friday

. Feather flush suitable for ACP egg

- laying was attained approximately 10

- 12 day s after plant pruning, under AgOps greenhouse conditions (27±2°C; 50% RH; L :D under natural day length). Seedlings with sufficient cluste rs of feather flush were moved from AgOps greenhouses into an Insectary
- level laboratory in the UCR IQF and inoculated with ACP adults. Feather flush on plants not utilized for ACP rearing was allowed to develop into mature leaves and grow for an21

additio nal four to five weeks, after which the plant was subsequentl y pruned back to encourage the development of additional flushing points. Such a cycle of plant re

- use reduced the number of waste plants, and ensured seedlings would fit into the acrylic ACP rearing cages once they were ready for use. **ACP rearing cages in Quarantine:** Colonies of ACP were reared on the Insectary level of UCR IQ F u s ing a double

- cage sy stem (i.e., a cage within a cage) in order to prevent escape of ps yl lids. Survivability

of ACP o utside of rearing cages was further reduced b y there being no other available plant material in the rearing rooms on which escapees could feed. The outer cage was comprised of a large Bugdorm 2400 (Megaview Science, Taiwan). Within the Bugdorm were the pri mary rearing cages constructed of acrylic risers (construction described below) containing a potted C. volkameriana and psyllids. This outer cage contained up to a maximum of 16 smaller rearing cages. ACP rearing cages were constructed b y modifying two transparent acrylic risers (SW Plastics, Riverside, CA) which each measured 15 cm x 15.3 cm x 15.3 cm (h x w x d). The open back face was covered with white no

- see - um mesh netting (Skeeta, Bradenton, FL), and the front face was covered with a sleeve approxi mately 30cm in length sewn from no

- see - um which provided access to the cage’s contents. **ACP inoculation schedule of host plants:** Approximately 10

- 12 day s after initial pruning, C. volkamariana seedlings displaying adequate amounts of feather flush were s elected from UCR AgOps greenhouses and brought to an IQF Insectary laboratory

for ACP inoculation. Prior to the introduction of ACP, seedling pots were prepared by placing a length of nylon stocking over the top of the pot (covering the soil), secured around the base of the plant with a plastic twist

- tie. This nylon barrier was put in place to reduce the number of soil

- dwelling fungus gnats emerging into rearing cages, but was porous enough to allow for the plant to be watered without first removing it. Plants were also stripped of a majority (> 75 percent) of mature leaves, so as to force ACP females to utilize feather flush for feeding and ovipositing. Plants were then placed into primary

rearing cages with a saucer to contain excess water draining after w atering. An average of 18 cages (six each every Monday , Wednesday

and Friday) of C. volkameriana bearing feather flush were set up ea ch week for ACP oviposition in quarantine. Approximately 15

− 20 ACP adults were released into each cage and allowed to oviposit for 2

− 4 day s, after which they

were removed and transferred into a new cage needing inoculation. Any ACP adult mortality was mitigated by

supplementing additional adults sourced from HLB

- f ree ACP California strain lab colonies. Plants were watered three times per week (Monday, Wednesday and Friday). **THE SELECTION OF NON -**  **TARGET PSYLLID SPECIES IN CALIFORNIA FOR HOST SPECIFICITY TESTING OF**  **DIAPHORENCYTRUS ALIGARHENSIS SOURCED**  **FROM PUNJAB PA KISTAN**  California has the richest native psyllid fauna in North America, and the influence of climatic and floristic diversity

in this region is considered key to the diversification of native ps yl lids. There are currently

165 psyllid species (~5

− 6% of the world’s fauna) known from California and22

a revision of the ps yl lid fauna of California was used to guide selection of psyllid species for host specificity

testing of D. aligarhensis sourced from Punjab Pakistan (Percy et al. , 2011). Obviously , it would be an almost impossible job to test all of California’s psyllid species for suitability as hosts to D. aligarhensis . A basic assumption in conducting host range tests is that native species most closely related to the target pest are likel y to be more at risk from biological control agents under consideration for use against the target pest. There are no native representatives in the genus Diaphorina or the tribe Diaphorininae in California to which ACP belongs which means the closest relatives that should be selected for host range testing should come from Family

level representation. However, in addition to taxonomic relatedness, other factors could influence host location and use b y parasitoids including cues released b y native plants related to citrus, or exploitation of sub

- optimal hosts under conditions when the preferred target, ACP, is not available. In consultation with Dr. Diana Percy , a world authority on ps yl lid taxonomy and ph yl ogenetics, the testing strategy shown in Table 1 was designe d to select pertinent California ps yl lid spe cies for host range testing in q uarantine. **Table 1.** Experimental selection criteria and representative species of non

- target California psyllids used in host specificity testing of Diaphorencyrtus aligarhensis at UCR I Q. **Criterion Selected Representative Species**  Target pest species Diaphornia citri (Asian citrus psyllid [invasive pest] [L iviidae: Euphyllurinae]) Phylogenetic proximity

to ACP Dichlidophlebia fremontiae (fremontia

psyllid [native] [L iviid ae: L iviinae]) Euphyllura olivina (olive psyllid [invasive pest] [L iviidae: Euphyllurinae]) High probability

for occurrence environmentally

in native vegetation Heteropsylla texana (honey mesquite psyllid : native [Psyllidae: Ciriacreminae]) Heterops ylla sp. (acacia psyllid [native] [Psyllidae: Ciriacreminae]) Native ps yl lid species (pest) Bactericera cockerelli (potato psyllid [native] [Triozidae: no sub

- family designations exist for Triozidae]) Biological control agent of invasive weed Arytai nilla spartiophylla (scotch broom psyllid [self

- introduced] [Psyllidae: Psyllinae]) **Non -**  **target psyllid sources used for initiating colonies:** Inaddition to the various native psyllids throughout California, there are also many known natural enemies asso ciated with each species (Percy

et al., 2011). To avoid contamination and potential influence of specificity trials, all psyllid species native to California were collected from field populations and brought to UCR IQ F. From these initial collections, colo nies were set up and reared in an IQF Insectary23

laboratory . Establishment and maintenance of colonies allowed positive confirmation that all colonies and subsequently individual ny

mphs used in host specificity trials were free of parasitoids. Broom ps yl lid ( A. spartiophylla ) and olive psyllid ( Euphyllura olivine ) were field collected for use in experiments. Both of these exotic psyllids lack a parasitoid fauna associated with the ny

mphs. **Non -**  **target psyllid rearing: Host plants:**  Successful rearing of non

- target psyllids was based primaril y on having a continuous supply

of host plants (specific to the species being reared) with adequate feather flush for oviposition. Since each non

- target ps yl lid species was highly specific to their own host plant, colonies needed to be maintained on species

- appropriate host plants (mainly California native plants). In host specificity trials, each non

- target species was maintained on seedlings of their choice host plant (with the exception of Bactericera cockerelli , the pes tiferous poly phagous potato psyllid, which was reared on eggplant seedlings but tested on Capsicum seedlings). Table 2 lists the host plant associated with each representative psyllid species. **Table 2**  **.** Host plants and associated ps yl lid species which were used for maintaining colonies of selected non

- target ps yl lid species for testing against D. aligarhensis in UCR I QF. **Non -**  **target Psyllid Species Host Plant**  1 Bactericera cockerelli Egg plant Solanum melongena 1 Heteropsylla sp. Sweet aca cia Acacia farnesiana 1 Heteropsylla texana Honey mesquite Prosopis glandulosa 1 Dichlidophlebia fremontiae Fremontia bush Fremontodentron sp. 2Euphyllura olivina Olive Olea europaea 2 Arytainilla spartiophylla Scotch broom Cytisus scoparious
1 Colony maintained in Quarantine, UCR IQF. 2 Colony not maintained in Quarantine, insects were collected from wild populations and transferred to potted host plants for experiments. Potted F. californicum seedlings were acquired from a local Californi a native plant nursery (Moosa Creek Nursery , Valley Center, CA). Eggplants used in colony maintenance were matured at AgOps from commercially

- available seedlings (“Long Purple” variety, Botanical Interests, Broomfield, CO). Seedlings of Acacia farnesiana , and P. glandulosa were provided b y CDFA (Mt. Rubidoux Field Station) once per week on an as

- needed basis for use in experiments. Scotch broom seedlings were harvested from wild populations in El Dorado Co., and transplanted into experimental pots in Quarantine. Potted olive seedlings were provided b y CDFA. Broom and olive plants were used to maintain reproducing populations of the associated psyllid. All host plants were maintained in greenhouses at UCR AgOps and provided necessary fertilization and water until use in colony cages. Plants were pruned whenever necessary to maintain a small size to fit within the colony Bugdorms, as well as to promote growth of feather flush for ps yl lid oviposition.24

**N on**  **- target psyllid rearing:**  Non
- target ps yl lid colonies were maintained in Insectary
- level laboratories of UCR IQF in Bugdorms (model 2120 MegaView Science, Taiwan). Based on the status of the psyllid species (i.e., pre

- testing; currently undergoing testing; post

- testing), colonies were expanded (usuall y encompassi ng three to five Bugdorms) or reduced (one Bugdorm) as needed. Mature potted seedlings of native host plants obtained from CDFA were maintained at UCR AgOps until time of use. Plants were selected for use based on amounts of feather flush present, and were subsequently moved from AgOps to I QF. Fresh plants were placed into Bugdorms already containing their particular non

- target ps yl lid on older plants, and old, dying, or infested (usually

by plant

- feeding mites) plants were removed and disposed of according to the corresponding species’ CDFA permit instructions. In this manner, native psyllid colonies were continually

“refreshed” and kept robust. All psyllids kept in colony , with the exception of B. cockerelli , utilized young feather flush for oviposition. N ymphs would emerge and mature through the first and second instars within the feather flush, then move down onto the plant trunk or mature leaf surfaces to feed as later instars. In contrast, B. cockerelli oviposits directly onto the leaf lamina and along the margin of the leaf, with ny mphs maturing and moving to feed along leaf veins. This ps yl lid doesn’t require flush growth to stimulate oviposition. **Developmental notes of non -**  **target psyllids:** Nymphs of B. cockerelli (potato psyllid) developed on mature leaf surfaces (as this is where eggs are oviposited by females) and ranged from nearl y transparent (earl y instars) to a very light yellow

- green color which was most obvious in fourth and fifth instars. Psyllids retained a round, dome

- like body shape until the final molt to adulthood. Heteropsylla sp. (acacia psyllid) and Heteropsylla texana (honey mesquite psyllid) had earl y instars which were light yellow in color, subsequently developing to green as they matured. Dichlidophlebia fremontia ny mphs were white after first emerging, and matured to a very light whitish

- green. They

formed small clusters which were often covered with dense, white cottony wax. Scotch broom psyllid, A. spartiophylla , and olive psyllid, E. olivina , were not reared in the laboratory . Because these exotic psyllids lack parasitoids in California, they were collected from wild populations and tested on their respective host plants at UCR I QF without concern that ny mphs had been previously parasitized by

species other than D. aligarh ensis to which they were exposed to in quarantine. A. spartiophylla ny mphs were a nearl y uniform light yellow in color, and were found feeding on both the leaves and stems of host plant cuttings. This species is very active and will move readil y with very little stimulus, making them easy to transfer from cuttings to experimental seedlings. E. olivine ny

mphs, much like D. fremontia , are white and produce a fluffy white wax, which shelters clusters of young instars. Once older, E. olivine ny mphs tend to be more solitary , however, they

remain protected under waxy exudates. This species is also very active and, once found by removing the wax around the bases of leaf axils or along young branches, n ymphs were readil y transferred onto experimental seedlings.25

**B. HOST RANGE TESTING** Young seedlings used as host plants in D. aligarhensis exposure trials ( Capsicum , Acacia farnesiana , Prosopis sp., and Olea europaea ) were grown b y CDFA at their Mt. Rubidoux Field Station in outdoor greenhouses. Capsicum seedlings were the California Wonder variety (Ferry Morse Seed Compan y, Felton KY). Fremontodendron seedlings were obtained from Moosa Creek Nursery

(Valley Center, CA). Scotch broom seedlings were harvested from wild populations in northern California, with assist ance from the CDFA Biocontrol Program, Sacramento (Dr. Kris Godfrey now at UC Davis), and transplanted into experimental cones in Quarantine at UCR IQF. A summary of host plants involved in D. aligarhensis testing is provided in Table 3
. **Table 3**  **.** Host pl ants used for testing target and non

- target ps yl lid species. All host plants u sed for testing were seedlings; however the size of plant varied. **Non -**  **target Psyllid (Testing)**  **Host Plant Seedling Cone**  **- tainer or D40?**  Diaphorina citri Volkamer lemon ( Citrus volkameriana ) Cone

- tainer Bactericera cockerelli Sweet pepper ( Capsicum annum ) Cone

- tainer Heteropsylla texana Honey mesquite (

Prosopis glandulosa ) Cone

- tainer Heteropsylla sp. Sweet acacia ( Acacia farnesiana ) Cone

- tainer **Test plant pre paration:**  Prosopis, Acacia, Olea , and Capsicum were obtained from the CDFA Mt. Rubidoux Field Station as seedlings already i n cone - tainers (SC7 Stubby , low densit y, 3.8 cm diameter, 114 mL capacity , Ray
- leach, Stewe and Sons Inc., OR). Mature leaves on th ese seedlings were removed, leaving onl y the youngest leaves at the apex of the plant. A piece of circular upholstery

foam lined with netting was placed within the cone over the surface of the soil to reduce fungus gnat emergence, as well as aiding in the recovery of test insects that could fall onto the soil (this was important for determining the ultimate outcome of target ps yl lid’s fate [i.e., alive vs. dead]). The bottom of the cone

- tainer was passed through a plastic lid with a hole cut out, which was secured near the top of the cone

- tainer with two thumb tacks. Finally , a ventilated plastic vial was inverted over the test plant and secured to the lid, confining the psyllids and D. aligarhensis on the test plant. Standard experimental setup of these con e - tainers is illustrated below in Fig. 1
.26

**Fig. 1**  **.** Preparation of experimental seedlings in cone

- tainers with test ps yl lid species for D. aligarhensis exposure trials. 1) original seedling in cone

- tainer; 2) a trimmed seedling, with mature foliage re moved; 3) netted upholstery foam disc placed over soil (top of foam is level with top of cone

- tainer); 4) lid fitted around the top of the cone

- tainer and secured with thumb tacks; 5) ventilated vial inverted and placed over the test plant, secured to the lid from step 4.

We were unable to maintain scotch broom seedlings, as they are classified as a quarantined noxious weed, so seedlings used for testing were harvested from wild populations in northern California (the same location the psyllids were col lected), shipped under CDFA permit (no. 2977), and transplanted into cone

- tainers in Quarantine at UCR I QF. All other experimental setup procedures for scotch broom seedlings were identical to the process described above. Fremontia seedlings purchased fro m local California native

- plant nurseries were larger than expected upon arrival. The above preparation process was modified to accommodate the larger growing cones (“D40”) the Fremontia seedlings arrived in (Fig. 2 ). Paper discs 7mm in diameter, cut from wax y paper plates, were fitted on top of the cone and around the plant stem, with the same purpose of the netted foam from the cone

- tainer setup, to prevent fungus gnat emergence. The D40 growing cones were fitted with an 11mm diameter clear plastic bevera ge lid, with a rubber band (size 32, 1⁄4 lb) fitted underneath for support against slipping and to the reinforce seal. A 16

- ounce capacit y clear plastic beverage cup, with the bottom removed and replaced with no

- see - um netting for ventilation, was then inverted and secured over the plant, to restrict the Fremontia psyllids ( Dichlidophlebia fremontiae ) and D. aligarhensis to a small area surrounding the test plant.27

**Fig. 2.** Adapted experimental setup for Dichlidophlebia fremontiae on Fremontodendron sp. (Fremontia) in D40 growing cones.

**Transferring psyllid nymphs to test plants:** Ittook an average of approximately 7

− 8 day s post
- oviposition for ACP ny mphs to reach the early

third instar (a preferred host stage of D. aligarhensis ) under experimental c onditions in the Insectary rearing room at UCR I QF (26.7 ± 1° C and 40 ± 5% RH). Native non

- target psyllid species that were reared in colonies were maintained in rearing rooms separate from ACP at a slightly lower temperature of 25±1°C and 40 ± 5% on the Insectary level of IQF, because this slightly lower temperature improved colony vigor. A total of five third

- instar psyllids on a single host cone

- tainer seedling comprised a treatment which was exposed to a single female D. aligarhensis . To achieve this precise number of ps yl lids on each testing plant, a method of transferring n ymphs from large colon y plants onto smaller cone - tainer seedlings was developed. Ny mphs of the appropriate age/life stage were located on large colon y plants and disturbed by gent le tapping with a fine

- haired paintbrush to encourage ny mphs to become mobile. Once ps yl lids ceased feeding and/or clinging to the host plant and began walking, they

were gently

lifted off of the colony

plant and individually

placed onto the yo ung flush of the test plant. All ny mphs were moved onto testing plants the morning before their exposure to D. aligarhensis , so they had no additional time to mature and were guaranteed to be in an appropriate life stage for D. aligarhensis to parasitize. Because psyl lids were very yo ung at the time of transfer (sometimes as young as late second instar) the trauma of plant transfer caused some mortality , but overall this transfer method achieved a success rate of over 80% survivorship. **Test cage setup:** Clear plastic vials of 148 mLcapacity (Thornton Plastic Co, Salt Lake City , Utah) with three ventilation holes 12 mm in diameter (two on opposite sides, one on the bottom)28

and covered with an ultra

- fine white organza mesh were inverted to fit into the vial lid that was firmly secured around the cone

- tainer. This ventilated inverted vial enclosed the test plant and psyllids, and confined D. aligarhensis with the psyllid species of interest. **Diaphorencyrtus aligarhensis:** Adult D. aligarhensis (this population is bi

- pare ntal) originall y collected from the Punjab of Pakistan, were sourced from colonies maintained in Quarantine greenhouses at UCR IQF. Mating pairs of 1 male and 1 female were collected in 200 μ L microcentrifuge tubes with honey added for feeding, and allowed to mate for no less than 24 hours. Immediatel y before day 1 testing commenced, female D. aligarhensis were separated into a 0.6 mLmicrocentrifuge tubes, from which they were introduced into the inverted ventilated cages described above. At the end of the prescribed duration of exposure to test psyllids, the vial was opened within an observation cage and the parasitoid was recaptured in the microcentrifuge tube. Each D. aligarhensis female was used only once for choice and prolonged no

- choice tests over th e indicated 2

- day

period. In sequential no

- choice tests, the same female was tested four consecutive times. **Cone -**  **tainer setup:** Experimental blocks were set up in shoebox

- sized plastic storage boxes, with three rows of five (15 in total per lid) holes cut into the plastic lid of the box with a diameter the same as that of the testing cone

- tainers, and cone

- tainer seedlings were placed through the holes which supported them in a vertical position. Approximately two to three inches of tap water was added to the box to allow for continuous watering of testing plants (through absorption through the exposed soil tip at the base of each cone

- tainer) without the need for removing the ventilated vials at the top of the cone

- tainer during the testing period (Fig. 3)

. **Fig. 3**  **.** One experimental block of cone

- tainer seedlings, showing constant plant access to water provided at the bottom of the testing tray

.

**Host specificity test methods:** Host specificity testing for D. aligarhensis was conducted under choice and no

- choice conditions involving sequential and prolonged exposure experiments over the course of two day s. D. aligarhensis used in short

- term no
- choice sequential treatments were allowed to rest overnight between day one (d1) and day

2 (d2) replicates (Table 4 ). D.29

aligarhensis used in prolonged exposure treatments were given a 24 hour exposure window on each d1 treatment before being immediatel y transferred to d2 treatments. **Sequential no choice tests:** Short

- term no
- choice sequential exposure treatments fol lowed one of two sequences. In the first treatment (T1), D. aligarhensis were exposed to ACP for four hours, then immediately

transferred to a plant with a non

- target psyllid (NTP). The second treatment (T2) was the reciprocal, introducing D. aligarhensis first to NTP for four hours, then being moved to ACP for an additional four hours. After the second four

- hour exposure (whether on ACP or NTP), D. aligarhensis were removed and transferred into a 200 μ L microcentrifuge tubes with honey , and stored overnight in a temperature control cabinet at 13.2

- 14.4°C. Day 2 replicates were set up identically to day 1 replicates (i.e.: the D. aligarhensis used for T1d1 was then used for T1 day

2). The second

- day

exp osure (day

2) was conducted to measure the effects of previous ps yl lid exposure and host

- feeding on parasitism rates on both ACP and NTP. **Prolonged exposure no choice tests:** This design consisted of four different treatment ty pes, which varied the order o f host psyllid species presented to D. aligarhensis under prolonged exposure. Each prolonged exposure treatment consisted of a day 1 24

- hour exposure to either ACP or NTP, before being moved immediatel y onto a secondary day 2 plant containing either ACP or NTP for another 24

- hour exposure. This experimental design was implemented to determine attack rates on NTP when D. aligarhensis repeatedl y encountered NTP, or when D. aligarhens is was able to host

- feed on or parasitize ACP before or after encountering NT P, and was intended to more closely mimic conditions in a natural California habitat should D. aligarhensis find itself in areas with only non - target psyllids to attack. **Choice tests:** Choice tests (T3) were conducted by simultaneously exposing D. aligarh ensis to ACP and NTP on their respective host plants and allowing the parasitoid to forage freel y and choose between ps yl lid species for attack. These treatments were conducted using the same acryl ic riser cages constructed for use in colony maintenance (s ee above), giving D. aligarhensis a greatl y expanded area of foraging and host encounters. Treatments were set up on day 1 and left to run for 24

- hour, after which D. aligarhensis was captured, the day 1 plants removed, and day 2 plants with fresh psyllids placed into the choice cage. D. aligarhensis was subsequentl y released and given access to day 2 plants for an additional 24

- hour before final removal. **En vironmental test conditions in q**  **uarantine:** The Insectary laboratories where NTP colonies were maint ained were regulated at 25oC, 40% RH and 14:10 L :D cycle. In order to promote increased ACP reproduction for testing, the specific ACP rearing laboratory was maintained at constant environmental conditions of 28.33 oC, 40% RH, and 14:10 L :D. The quarantine testing room where trials took place was maintained at a mid

- range temperature, with environmental conditions set at 26.7oC, 40% RH, and 14:10 L :D throughout all testing phases. **Experimental Execution:** A single mated adult female D. aligarhensis (approx. 2

− 7 day s of age) was introduced into a test cage for a 4 hour exposure period in sequential no choice (T1, T2) treatments, or for 24 hours for choice (T3) and prolonged sequential no

- choice (T4

- 7) treatments. Baseline mortality

rates of psyllid ny

mphs dying from natural causes were derived from Control treatments (T8), in which a set of ps yl lids was set up on test plants in an identical manner to those used in experiments and maintained under the same q uarantine conditions, but were not30

exposed to parasitoid s. Experiments were set up in blocks comprised of T1

- T8 treatments, which were run in eight two

- day replications for each test species and treatment ty pe. Sequential no

- choice tests (T1, T2). During initial setup, two D. aligarhensis females were released into one of two test vials, one containing ACP (T1) and the other containing NTP (T2). After a period of 4 hours, parasitoids were removed and transferred immediately to a second vial containing the reciprocal psyllid ty pe (NTP for T1, ACP for T2) for an additiona l 4 hours. At the end of this 8

- hour rotation, females were removed and transferred into a 200 μ L microcentrifuge tube with honey , and rested overnight at 13

− 14°C. The same test sequences were repeated for female D. aligarhensis on day 2. Choice tests (T3). Ventilated inverted vials on individual test plants were removed in order to expose test ps yl lids on their respective host plants. Two cone

- tainers, one containing ACP and the other NTP, were placed inside an acrylic riser cage (identical to those constructed for use in colony maintenance [see above]), giving D. aligarhensis a greatl y expanded area of foraging. Treatments were set up on day 1 and run for 24 hours, after which D. aligarhensis was captured, the day 1 plants removed, and day 2 plants with fresh psyllids placed into the choice cage. D. aligarhensis was subsequently released and given access to day 2 plants for an additional 24 hours. After removal, ventilated vials were replaced over each individual test plant and its psyllids, and the cone

- tainer was returned to its respective block’s watering tray

. Prolonged sequential exposure test (T4

- 7). These 24

- hour sequential no

- choice treatments aimed to evaluate whether D. aligarhensis females would attack NTP under an exposure period longer than 4 hours, and with varying host sequence exposure. It was theorized that D. aligarhensis attacks on NTP would be possible if the exposure time was lengthened, either because females would have more time to act on NTP or because there was an extensive period during which the parasitoid was restricted to a single non

- choice species and in the absence o f the preferred host, ACP, oviposition on less desirable hosts may result. This setup also evaluated whether the order of exposure to the preferred (ACP) or less desired (NTP) host had an y effect on attack rates on either species. The first treatment (T4 ) had D. aligarhensis exposed to ACP on day 1, then moved to ACP on day 2. The second treatment (T5) had D. aligarhensis exposed to ACP on day 1, then moved to NTP on day

2. The third treatment (T6) had D. aligarhensis exposed to NTP on day 1, then transfe rred to ACP for day 2. In the final treatment (T7), D. aligarhensis were exposed to NTP on day 1, and transferred to NTP for day 2. In this manner, all combinations of exposures were addressed. Control (T8). One set of five ACP or five NTP nymphs on thei r respective host plants in cone

tainers were set up and maintained in a manner identical to that for test psyllids exposed to D. aligarhensis for each treatment day (day 1, day 2). Control psyllids of each test species were not exposed to any

predator or parasitoid. These control cages were used to provide estimates of naturally

- occurring mortality due to the process of transferring ps yl lids onto seedlings used in testing, and subsequent maintenance in q uarantine testing room conditions to determine develo pmental fate (i.e., death from unknown causes or development to adult psyllids).31

**Table 4**  **.** Treatment summary for exposure tests of female D. aligarhensis to Asian citrus psyllid (ACP) and non

- target psyllid (NTP) species. Treatments Day 1 (d1) Night Day 2 (d2) T1 Sequential (

D. citri first) [D. citri] ►[ NTP] rest ►[D. citri] ►

[NTP] T2 Sequential (NTP first) [NTP] ►[D. citri ] rest ► [NTP] ►[D. citri ] T3 Choice test [D. citri+NTP] [D. citri+NTP] T4 Prolonged sequential (ACP/ACP) [D. citri] ► [D. citri] T5 Prolonged sequential (ACP/NTP) [D. citri ] ► [NTP] T6 Prolonged sequential (NTP/ACP) [NTP] ► [D. citri] T7 Prolonged sequential (NTP/NTP) [NTP] ► [NTP] T8 Control [D. citri ] / [NTP] No parasitoid exposure to measure natural nymph mortality under experimental conditions [D. citri ] / [NTP] No parasitoid exposure to measure natural nymph mortality under experimental conditions ► D. aligarhensis mo vement to new psyllid hosts rest = containment of female D. aligarhensis in a 200 μ L microcentrifuge tube with honey and no psyllid exposure for ~16 hours at 13.2

- 14.4oC. **Data Recording:** All treatments were observed at least twice to record ps yllid developmental outcomes. Observation 1 was made 7

− 9 day s post
- day 1 exposure to D. aligarhensis

. Observation 2 was completed 14

− 16 day s post
- day 1 exposure. If at this point there remained mummies or live psyllid ny mphs, a third observation was taken 21 day s post
- day 1 exposure to determine developmental fate. An y intact mummies (i.e., no emergence of parasitoids) observed at this point were deemed dead and s ubsequently discarded. Counts were recorded of the number of psyllids that successfully matured to adulthood, psyllid ny mphs found dead (phy sicall y accounted for), the number of successfull y parasitized and mummified nymphs (observation 1 only), and number and sex of D. aligarhensis that emerged from hosts for each treatment. **RESULTS AND DISCUSSION** Six non

- target psyllid species were tested for their suitability as hosts for D. aligarhensis reproduction. Host range was assessed u si ng seven experimental d esigns encompassing no

choice sequential tests, prolonged no

- choice and choice tests. The non

- target test species were comprised of: (1) Bactericera cockerelli (the native pestiferous potato/tomato psyllid) ( n = 480 total ny mphs exposed); (2) Heteropsylla sp. (the native Acacia ps yl lid) ( n = 390 ny mphs); (3) Arytainilla spartiophylla (a self

- introduced natural enem y of the noxious weed scotch broom psyllid) ( n = 380 n ymphs); (4) Euphyllura olivina (the invasive olive ps yl lid) ( n = 320 n ymphs); (5) Heteropsy lla texana (the native honey mesquite psyllid) ( n = 395 n ymphs); and (6) Dichlidophlebia fremontiae (the native F remontia psyllid) ( n = 375 n ymphs). A s ummary (mortality , parasitism, survival to adulthood, etc.) for each ps yl lid species is presented in Tab le 5
. Successful parasitism of a non - target species by D. aligarhensis was observed for onl y one32

species tested: B. cockerelli , the native and pestiferous potato ps yl lid, the vector of the zebra chip bacterium which causes significant economic loses in p otatoes (Butler and Trumble 2012). Observed parasitism rates ranged 11

- 18% for the four treatment ty pes. Two treatment ty

pes (T2, the short no choice sequential trial in which NTP was presented first and T3 choice test) which exhibited moderate levels of parasitism, at 17

- 18%. P arasitism of B. cockerelli was lower in ACP

- first sequential no - choice test and prolonged exposure tests (11

- 13%). When compared to D. aligarhensis parasitism rates of ACP across all treatment ty pes (27

- 45%), it is likel y B. cock erelli is not a highl y preferred host for D. aligarhensis . Because of these findings, as well as the fact that there is robust guild of generalist predators and parasitoids native to California attacking B. cockerelli (Butler and Trumble, 2012), it is unl ikely that D. aligarhensis will pose a significant threat to B. cockerelli populations in the wild once released from Quarantine. Elevated mortality

l evels classified as a result of “unknown causes” (dead ny mphs which were found and confidentl y accounted for) were observed across all ps yl lid species exposed to D. aligarhensis (refer to Table 6 for the complete breakdown of confirmed mortality f rom unknown causes by

p syllid species). T his mortality i ncrease was measured against control treatments consistin g of host plant seedlings with NTP ny mphs identical in setup to experimental seedlings and kept under identical conditions, but lacking exposure to D. aligarhensis

. It is likel y that elevated mortality

when compared to controls was a combination of natura l ny mph mortality , trauma of moving early instars between plants, possible host feeding by D. aligarhensis (not confirmed), unsuccessful parasitism attempts (not confirmed), and (in some cases as discussed for A. spartiophila ) unavoidable external conditio ns due to field collection, subsequent transportation, and set up in quarantine. Mortality experienced by B. cockerelli ny mphs (potato psyllid, tested on pepper [ Capsicum ]) undergoing D. aligarhensis exposure ranged from 3

- 27%, as compared to 4% control m ortality

. Mortality rates for ACP, excluding death caused by successful parasitism of D. aligarhensis , ranged from 4

- 11%. Control mortality for ACP was 6%. No parasitism by D. aligarhensis was recorded for Heteropsylla sp. In creased mortalit y was obser ved for Heteropsylla sp. (acadia psyllid, tested on Acacia sp. seedlings) in the presence of D. aligarhensis . Control mortality f or this species, was around 8%, but mortality r anged from 5

- 17% across treatments with D. aligarhensis (Table 5). T hese data suggest that D. aligarhensis could cause additional, albeit, minor increases in host

- death when confined in small cages with this psyllid species. Observed mortality f or ACP was between 20% and 23% (independent of parasitism), markedl y higher than the mort alit y rate for Heteropsylla sp. (Table 5). No parasitism by D. aligarhensis was recorded for A. spartiophila (Table 5). Of all non

- target species tested, A. spartiophila (scotch broom psyllid, tested on transplanted scotch broom seedlings) had the highes t overall ny mph mortality

rates (Table 6). All of the experimental treatment ty

pes, except T1 (sequential 4 hr exposure to ACP first) have lower mortality rates compared to the control (14

- 32% with D. aligarhensis exposure vs. 33% in control treatments lacking D. aligarhensis exposure [Table 5]). This elevated control mortality may be due to a number of external factors, including same day field

- harvesting and transporting (via ice chest with psyllids flown Economy class from Sacramento to Riverside) o f psyllids and low survivorship of field

- harvested and transplanted scotch broom seedlings. There appears to be no33

significant elevation in A. spartiophila mortality when D. aligarhensis is confined with this psyllid. This is very

relevant as this particul ar ps yl lid is considered a fortuitous self

- introduced biological control agent of invasive scotch broom in northern California. Further, collections of A. spartiophila were made from high elevation sites in northern California. These collecting areas are s ubject to snowfall, a climate t ype that is not conducive for ye ar round persistence of D. aligarhensis

. Observed ACP mortality rates in this experimental set ranged from 13

- 17%, and ACP control mortality

was 19%. No parasitism by D. aligarhensis was reco rded for E. olivina (Table 5 ). E. olivina mortality in the presence of D. aligarhensis ranged 16

- 23% which was similar or slightly higher than control mortality which was recorded at 15% for olive ps yllid (Table 6). In comparison, ACP mortality was similar , ranging 12

- 22%, and parasitism range 16

- 28%. ACP mortality for control treatments was 15%

. No parasitism by D. aligarhensis was recorded for H. texana (Table 5 ). H. texana experienced little or no increase in NTP mortality in the presence of D. aligarh ensis (Table 5 ). Control mortality for H. texana was 21%, mortality when exposed to female D. aligarhensis ranged 8

- 24% for H. texana (Table 6). These data sets suggest that D. aligarhensis may not be significantl y affecting mortality rates of this non

- tar get species. In comparison, to H. texana , ACP mortality

across exposure experiments ranged 16

- 23%, parasitism ranged 14

- 28%, and control mortality

was 26%. No parasitism by D. aligarhensis was recorded for D.fremontiae (Table 5 ). Control mortality for D. fremontiae was 3%, and D. fremontiae exposed to D. aligarhensis suffered mortality rates between 8% and 16%. In contrast, ACP mortality was 24% in control treatments and ranged 17

- 25% in exposure treatments and ACP parasitism ranged 8

- 24%. This only slight increase in non

target mortality

under Quarantine conditions suggests that D. aligarhensis may affect natural death rate of Fremontia psyllids, but not significantly

.34

**Table 5**  **.** C onsolidated summary of data for exposure and c o ntrol treatments pertaini ng to ps yl lid mortality and parasitism rates in the presence of a single mated D. aligarhensis female under each of seven different exposure treatments
. **Non -**  **Target Psyllid Species**  **Test Type % NTP**  **Mortality %NTP** 

**Parasitism % ACP** 

**Mortality %ACP** 

**Parasitism %**  **NTP Control**

**Mortality %ACP Control** 

**Mortality** Bactericera cockerelli (Potato/Tomato Psyllid) (PTP) T1 (4hr, ACP

first) 18 13 11 39
4 6 T2 (4 hr NTP first) 3 18 7 42 Choice Test (T3) 13 17 4 27 Prolonged Exposure (T4

- 7) 27 11 1 45 Heteropsylla sp. ( Acacia farnesiana Psyllid) (AFP) T1 (4hr, ACP

first) 8 0 20 28 8 1 1 T2 (4 hr NTP first) 5 0 2 3 2 1 Choice Test (T3) 1 7 0 2 1 21 Prolonged Exposure (T4

- 7) 10 0 2 3 3 2 Arytainilla spartiophila (Scotch Broom Psyllid) (SBP) T1 (4hr, ACP

fi rst) 41 0 16 18 33 19 T2 (4 hr NTP first) 32 0 1 7 1 6 Choice Test (T3) 14 0 1 3 29 Prolonged Exposure (T4

- 7) 25 0 1 6 21 Euphyllura olivina (Olive Psyllid) (OLV) T1 (4hr, ACP

first) 21 0 1 6 1 6 15 15 T2 (4 hr NTP first) 23 0 2 2 2 5 Choice T est (T3) 16 0 19 2 1 Prolonged Exposure (T4

- 7) 19 0 1 2 2 8 Heteropsylla texana (honey mesquite (Prosopis) T1 (4hr, ACP

first) 24 0 18 20 21 26 T2 (4 hr NTP first) 13 0 9 2035

psyllid) (PRO) Choice Test (T3) 8 0 2 3 2 8

Prolonged Exposure (T4

- 7 ) 19 0 1 6 14 Dichlidophlebia fremontidae (Fremontia psyllid) (FRE) T1 (4hr, ACP

first) 11 0 25 8 3 24 T2 (4 hr NTP first) 14 0 25 23 Choice Test (T3) 8 0 1 7 1 5 Prolonged Exposure (T4

- 7) 16 0 19 2 436

All species of non

- target psyllid ny mphs experienced a modest increase in mortality due to unknown causes under experimental conditions when compared to control data. Elevated levels of unknown mortality

are likely the result of a combination host feeding or unsuccessful parasitism by D. aligarh ensis . However, the ecological importance of this is likely to be very

low as D. aligarhensis was unable to reproduce on these hosts, and elevated levels of mortality (< 15% on average: Heteropsylla sp. = 14%; A. spartiophila = 3%; E. olivina = 6%; H. texa na = 0%; D. fremontidae = 8%) could have possibly been an artifact of containment in small artificial arenas that prevented females leaving unsuitable host patches. In the unlikely event that these attack rates that should manifest themselves in nature, it is highl y unlikely there would be an y significant impact on non

- target ps yl lid species. Additonally , these exposure trials in Quarantine do not take into account various other biotic and abiotic factors that would act as barriers to D. aligarhensis interf

ering with native populations (e.g., native natural enem y guilds, plant community

composition, and the potential rarity of non

- target species for most of the year). The only native ps yl lid species D. aligarhensis was able to reproduce on was the potato ps yl lid, B. cockerelli . This psyllid is a pest of tomatoes, egg plants, peppers (Capsicum), and potatoes (Butler and Trumble 2012). It is most destructive in potatoes because it spreads a phloem

- limited bacterium, possibly Candidatus Li beribacter solanacearum, that causes zebra chip in potatoes and related maladies in other solanaceous crops. D. aligarhensis both increased unknown n ymph mortality (b y 24%) and successfull y parasitized B. cockerelli ny mphs at an average rate of ~15%. It is possible that D. aligarhensis could encounter B. cockerelli frequently under certain circumstances, particularly in urban environments, where tomatoes/potatoes/eggplants/peppers are grown in gardens co

- inhabited with citrus. No adverse environmental impacts from attacks on B. cockerelli by D. aligarhensis , should they occur, are anticipated. Further, there is a guild of resident natural enemies already exploiting B. cockerelli in California (Casey and Trumble , 2012) and an exotic parasitoid that has relatively weak attack rates under optimal conditions is unlikely to compete effectively

under field conditions. **CONCLUSIONS** Under stringent testing conditions that evaluated the host range of the bi

- parental ency rtid parasitoid, Diaphorencyrtus aligarhensis , sourced from Punjab Pakistan under four different testing strategies against six non

- target ps yl lid species, D. aligarhensis was unable to parasitize on five of these six species. L ow rates of parasitism, averaging 15%, were observed on the native pestiferous potato ps yl lid B . cockerelli . In comparison, parasitism rates on the target, Asian citrus psyllid, ranged 8

− 50% (average 23% parasitism rate). Mortality rates due to unknown causes across all non

- target ps yl lid species showed a slight elevation in the presence of D. aligarhensis as compared to control psyllids not exposed to D. aligarhensis . An exception was H. texana which exhibited no measurable differences in n ymphal death rates between exposure and control treatments. It is concluded that it is highly unlikely that such low rates of increased mortality and a lack of parasitism would be sufficient to affect n ative populations of these non

- target psyllids in California. Permission to release D. aligarhensis has been granted for Florida and releases of two strains of uniparental populations sourced from Taiwan and China as part of a biological control program t argeting ACP have so far failed to establish (Rohrig et al., 2012). A different outcome from releasing D. aligarhensis may occur in California because the strain to be released is sexual (as opposed to being parthenogenic, whereb y females reproduce asexual ly as is the case37

in Florida) and a competing ACP parasitoid, T. radiata , is not yet widespread in California which affords D. aligarhensis the opportunity to establish and spread in areas lacking this competitor. **References:** Butler, C.D., and J.T. Trumb le. 2012. I dentification and impact of natural enemies of Bactericera cockerelli (Hemiptera: Triozidae) in southern Cailfornia. Journal of Economic Entomology . 105: 1509 −1519. Percy , D.M., A. Rung, and M.S. Hoddle. 2011. An annotated checklist of the psyllids of California (Hemiptera: Psylloidea). Zootaxa 3193: 1 −27. Rohrig, E., D.G. Hall, J.A. Qureshi, and P.A. Stansly . 2012. Field release in Florida of Diaphorencyrtus aligarhensis (H ym enoptera: Ency rtidae), an endoparasitoid of Diaphornia citri (Homopt era: Ps yl lidae), from mainland China. Fla. Entomol. 95: 479

− 481.