United States Department of Agri culture 

# Field Release of the
Parasitoid **Diaphorencyrtus aligarhensis**  for the Biological Control of the Asian Citrus Psyllid in
the Conti guous United
States

Environmental Assessment,
October 2014 Marketing and Regulatory Programs

Animal and
Plant Health Inspection
Service

# Field Release o f the Parasitoid **Diaphorencyrtus aligarhensis**
for the f or the Biological
Control of the Asian Citrus Psyllid in the Conti guous United States

Environmental Assessment,
October 2014

Agency Contact : Shirley Wager

- Page, Ph.D. Pest Permitting
Plant Protec tion and 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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M en tion o f c o m pan ies o r c o mm er c ial produ c ts 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 endor s 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 D A ne i t he r guaran t ee s no r w arra n ts t h e
st andar d o f an y produ ct m en tio ned

. P rodu c t n a m e s ar e me n t i one d to repor t f a 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 r matio n

.

This pub l i c a tion repor ts re s ear ch i n v o 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
appro pr i a te St a te an d / o r F edera l agen ci e s be f or e t he y c a n b e r e c o m m ended.

C A U TIO N : P e s t i c id e s c a n b e in j u r io u s to h u m a n s, d o m e st i c an i m a l s

, de sir a b le p la n t s

, an d f i sh and

o t he r w il d l i f e

— if t he y ar e 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 v e ly an d c are f u ll y

. Foll o w re c o mme nde d label p r a ct i c e s f o 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 er s

.

## Table of Contents

I. Purpose and Need for the Proposed Action

................................ . 1

II. Alternatives

................................ ................................ ................. 2

III

. Affected Environment

................................ ................................ . 5

I V. Environment al Consequences

................................ ................... 7

V. Other Issues

................................ ................................ ............. 1 2

VI

. Agencies, Organizations, and Individuals C onsulted

............... 1 3

VII. References

................................ ................................ ............. 1 4

Appendix 1. National Quarantine Map for ACP and HLB.

Appendix 2. Host

- specificity testing from Hoddle and Bistline, 2013.

Appendix 3

. Response to Comments on the Draft Environmental

Assessment.

## I. Purpose and Need for the Proposed
Action

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 ) (Hymenoptera: Encyrt idae)

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

States

.

This en vironmental assessment 2 (EA) has been prepared, consistent with USDA, APHIS' 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 cont rol ACP within the conti guous United States

. This EA considers a “no action” alternative 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 anywhere in the conti guous United States

. The ACP is a n insect pest of citrus because it vectors a plant pathogenic bacterium, Candidatus Liberibacter asiaticus Jagoueix, Bove ́ and Garnier, that causes a lethal

citrus dis ease, known as huanglongbing (HLB) or citrus greening ( Halbert and Manjunath, 2004 ; Bové 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 Flo rida, ACP was first detected in 1998 and HLB was discovered in 2005 (Hall and Hentz, 2011)

. 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 impa ct on citrus production in

that State (Hodges and Spreen, 2012)

. HLB has also been detected in California, Georgia, Louisiana, South Carolina

, and Texas. Other S tates, including Alabama, Arizona, Hawaii , and Mississippi, are at risk for HLB because popula tions 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 R egulations 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.

1E conomic losses to growers in Florida attributable to HLB have b een estimated at greater than $1.7 billion which translates to approx imately a

16 percent reduction in grower revenues (Hodges and Spreen, 2012). An effective ACP management program is considered an important part of reducing the spread of HLB (USDA

– APHIS, 2012)

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

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

specific biological control organism against ACP in the conti guous United

States , particularly for con trol of ACP in backyard citrus

.



## II. Alternatives

This section will explain the two alternatives available to APHIS

– PPQ: no action (no issuance of permits) and issuance of permits for environmental

release of D. aligarhensis in th e conti guous United States. Although APHIS’ alternatives are limited to a decision of whether to issue permits

for release of D. aligarhensis , other methods are described that are currently used to control ACP in the United States . Use of these control

met hods is not an APHIS decision, and their use is likely to continue

whether or not APHIS

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

APHIS

– PPQ considered a third alternative but will not analyze it further.

Under this third alternati ve, APHIS

– 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 Unit ed States.

There are no issues raised i ndicat ing that special provisions or requirements

are necessary.



### A. No Action

Under the no action alternative, APHIS

– PPQ would not issue permits for

the field release of D. aligarhensis for the control of ACP

— t h e 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 APHIS

– PPQ issues permits for release of D. aligarhensis. Presently, contro l of ACP in the United States is limited to

chemical and biological control methods.

1 . Chemical Control

There are many insecticides that may be applied to citrus as systemic and

foliar sprays against ACP

. Systemic insecticides are applied and

2transloca ted throughout the tree. Systemic insecticides includ e imidacloprid , aldicarb, and thiamethoxam

. Foliar sprays are applied to the

leaves and include insecticides such as fenpropathrin, zeta

- cypermthrin, chlorpyrifos, chlorantraniliprole , spirotetramat, ab amectin

, and carbaryl. Organic insecticide options are available to control ACP including Neem oil, pyrethrin, 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 . APHIS 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 Hoy, 2001) but with mixed results (Halbert and Manjunath, 2004). It has no t established in Florida , perhaps due to competition from T. radiata , generalist predators, and pesticides (Rohrig et al., 2012).

Native predators, such as spiders, co ccinellids (lady beetles), l ace wings, syrphids, and minute pirate bugs attack ACP (Quereshi and Stansly , 2009;

UC

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



### B

. Issue Permits for Environmenta l Release of **D. aligarhensis** (Preferred Alternative)

Under this alternative, APHIS

– PPQ 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 Sta tes . These permits would contain no special provisions or requirements concerning release procedures or mitigating measures.

1. **D. aligarhensis** Taxonomic Information (From Hoddle and Bistline, 2013).

Diaphorencyrtus aligarhensis is an endo parasitoid att acking second to
fourth instar ACP nymphs

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

, and feeding of the larval D

. aligarhensis inside of the

ACP nymph eventually kills the pest.

3a. Taxonomy

.

Diaphorencyrtus aligarhensis (Shafee, Ala m & Agarwal) (Hymenoptera: Encyrtidae: Encyrtinae) was originally described from a specimen 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 describ ed multiple times (Ruggiero et al., 2011). The Universal Chalcidoidea Database (Noyes

, 2013) lists the following synonomies:

Aphidencyrtus aligarhensis Shafee, Alam & Agarwal, 1975 Aphidencyrtus diaphorinae Myartseva & Trjapitzin, 1978 Aphidencyrtus sacc hari Kaul & Agarwal, 1986 Diaphorencyrtus aligarhensis (Shafee, Alam & Agarwal, 1975) Diaphorencyrtus diaphorinae (Lin & Tao, 1979) Diaphorencyrtus diaphorinae (Myartseva & Trjapitzin, 1978) Psyllaephagus diaphorinae Lin & Tao, 1979 Syrphophagus aligarh ensis (Shafee, Alam & Agarwal, 1975)

b. Natural geographic range, other areas of introduction of **D. aligarhensis** 

.

Noyes (2013) lists the following countries from which D. aligarhensis has been recorded: India (Andhra Pradesh, Maharashtra, Punjab, Rajast han, 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 biolog ical control of ACP in Florida and Réunion (Rohrig

, 2011). It established in Réunion (van 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 20 07

- 2009) (Rohrig et al., 2012). This parasitoid was deliberately released in South Africa (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 sp ecific (van den Berg

, 1985). In Taiwan, where D. aligarhensis is native, it is not as effective as T. radiata (introduced into

Taiwan over 1984

- 1988 from Réunion) possibly because D. aligarhensis suffers high levels of attack by hyperparasitoids (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

- introduced

4with ACP into Saudi Arabia , or it may be native having moved onto ACP from an unknown psyllid host.

c. Life history of **D. aligarhensis**

Diaphorencyrtus aligarhensis is a solitary endoparasitoid which can parasitize second through fourth instar ACP nymphs (Rohrig et al., 2012).

A n endoparasitoid completes all development inside the host. Besides parasitizing ACP, adult f emales also kill ACP by host feed ing on nymphs (Skelley and H oy
, 2004) ; females insert their egg laying organ (ovipositor)

into the ACP nymph to make a hole and then suck up the liquid that leaks out (Hoy and Nguyen, 2001) . ACP has five nymphal instars but the fifth

instar is unsuitable for parasitization or host fe eding by D. aligarhensis (Skelley and Hoy

, 2004). Adult females are capable of killing up to 280

ACP nymphs through host feeding and parasitism combined (Chien

, 1995).

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

- 16 days at 25 ° C, depending on the nymphal instar (Rohrig et al., 2012). Diaphorencyrtus aligarhensis larvae attach the parasitized ACP nymph 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 nymph 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 thelytokous (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 . Affected Environment

A. Asian Citrus Psyllid

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 by ACP does not expand or develop normally, and is more susceptible to breaking off. While direct damage is

5serious, 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, Eretmocitru s, 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 American Psyllid Species

Hundreds of psyllid species occur on both native and introduced landscape plants in the United States. Several psyllid species are pests of crops such as pear, potato, and toma to. Each kind of psyllid feeds on only 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 currently 165 psyllid species known from California

. Most psyllids native to the United States are relatively uncommon and rarely become pests. Psyllid species in North America could possibly be attacked by D. aligarhensis

.

C. Citrus Resources in North America

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)

.

6I n 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 many n ursery operations in the United States that produce citrus trees, orange jasmine, curryleaf, and other ACP host plants and will be adversely affected by ACP and HLB. Many of these operations are identified by the Small Business Administration as small businesses.



## I V

. Environmental Consequences

A. No Action Alternative

1. Impact of ACP and HLB on Hosts of ACP and Citrus

Resources

ACP damages citrus by 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 by 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 blotchy, 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 by 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 HLB

ha ve 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 economy $4.5 billion and 8, 257 jobs (Hodges and Spreen, 2012)

.

7Insecti 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 year. Prior to this, ACP was considered a minor pest. However, growers must apply both systemic soil drench treatments along

with multiple foliar sprays (especially to protect newly planted and young (4 to 8

- year 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 normally 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 relatively 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 t he 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. APHIS 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 envir onmental release of D

. aligarhensis.

a . Chemical Control

The environmental consequences of many of the insecticides used for control of ACP were analyzed by APHIS (USDA

– APHIS, 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, syrphids, and spiders. M any of the insecticides used to control ACP are

8broad

- 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 radiat a ) 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. radiat a 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

, specifically ACP . As such, it will rarely come into contact with humans or other vertebrates, and if it does, it is incapable of stinging or biting. Encyrt id wasps have no known adverse impacts on humans or other

vertebrates.

b

. Direct impact of **D. aligarhensis** (e.g.

, intended effects on
ACP , direct effects on non

- targets).

To evaluate the effects of D. aligarhensis on non

- target psyllid species, host specificity tests were conducted in the United States to determine possible direct effects on non

- target psyllid 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 nymphs exposed); (2) Heteropsylla sp. (the native Acacia psyllid) ( n = 390 nymphs exposed ); (3) Arytainilla spartiophylla (a self

- introduced natural enemy of the noxious weed scotch broom psyllid) ( n = 380 nymphs exposed ); (4) Euphyllura olivina (the invasive olive psyllid) ( n = 320 nymphs exposed );

9(5) Heteropsylla texana (the nati ve honey mesquite psyllid) ( n = 395 nymphs exposed ); (6) Dichlidophlebia fremontiae (the native fremontia psyllid) ( n = 375 nymphs exposed ).

Successful parasitism of a non

- target species by D. aligarhensis was observed for only one species tested: B. co ckerelli , the native and pestiferous potato psyllid, 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
types. Two of the treatment type s exhibited moderate levels of parasitism,

at 17−18 percent . When compared to D. aligarhensis ACP parasitism rates across all treatment types (27

− 45 pe r cent ) , it is likely B. cockerelli is not a highly preferred host for D. aligarhensis

, but it may be attacked by 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 Liberibacter 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 t hat 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 by D. aligarhen sis , the resulting effects could be environmental impacts th at may not be easily reversed. Biological control agents such as D. aligarhensis generally 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

re production 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 int ro ductions have failed to provide

10control of the pest (Greathead and Great head, 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. APHIS 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.

Specifically, 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. APHIS 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. NIFA supports

research on HLB

, including ways 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 surveys 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.

11The 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. Industry

- 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 specificity 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 routinely used. The primary means of suppressing ACP in residential citrus will be through biological control (USDA

− APHIS, 2012).

4

. Endangered Species Act

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 federally 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 any listed insect in the conti guous United States. In host specificity testing, the biological control organism preferred ACP and only successfully parasit ized one non

- target psyllid species, B

. cockerelli

, the potato/tomato psyllid

. No psyllids are federally 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 Low

- income

12Populations,” APHI S considered the potential for disproportionately high and adverse human health or environmental effects on any minority 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 any minority or low

- income populations.

Consistent with EO 13045, “Protection of Children From Environmental Health Risks and Safety Risks,” APHIS 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 r dination with Indian Tribal

Govern m ents ” , w a s issued to ensure t h 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 mpli c ations....” Consistent w ith EO

131 7 5, A P H I S s e nt letters of notification a n d requests fo r com m ent a n 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 tri b 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 t heir agricultural interests, in accorda n ce with EO 1 3175

.


## V I

. Agencies, Organizations, and
Individuals Consulted

This EA was prepared and reviewed by APHIS. The addresses of participating APHIS units, cooperators, and consultants (as applicable)

follow.

U niversity 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 Analysis Services

4700 River Road, Unit 149 Riverdale, MD 20737

– 1238

13

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18Appendix 1. National Quarantine Map for ACP and HLB.

19Appendix 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 (IQF) (Facility Number 93). Colonies were maintained under California Department of Food a nd 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 strongly stimulated by the presence of young budding flush (“fea ther 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 q uarantine, and were grown in UCR III type 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 only 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 regularly 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 weekly, with one batch each being pruned either Monday or Friday.

Feather flush suitable for ACP egg

- laying was attained approximately 10

- 12 days 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 an

20additio nal four to five weeks, after which the plant was subsequently 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 rea ring cages once they were ready for use.

ACP rearing cages in Quarantine: Colonies of ACP were reared on the Insectary level of UCR IQF u s ing a double

- cage system (i.e., a cage within a cage) in order to prevent escape of psyllids. Survivability of ACP o utside of rearing cages was further reduced by 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 by modifying two t ransparent 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 days 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 arou nd 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. Pla nts 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 q uarantine. Approximately

15

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

− 4 days, 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 psyllids. There are currently 165 psyllid species (~5

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

21a revision of the psyllid 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 likely 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 tha t 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 by parasitoids including cues released by 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 psyllid taxonomy and phylogenetics, the testing strategy shown in Table 1 was designe d to select pertinent California psyllid spe cies for host range testing in q uarantine.

Table 1. Experimental selection criteria a nd representative species of non

- target California psyllids used in host specificity testing of Diaphorencyrtus aligarhensis at UCR IQ.

Criterion Selected Representative Species

Target pest species Diaphornia citri (Asian citrus psyllid [invasive pest] [Liviidae: Euphyllurinae])

Phylogenetic proximity to ACP Dichlidophlebia fremontiae (fremontia psyllid [native] [Liviid ae: Liviinae]) Euphyllura olivina (olive psyllid [invasive pest] [Liviidae: 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 psyllid 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: In addition 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 IQF. From these initial collections, colo nies were set up and reared in an IQF Insectary

22laboratory. Establishment and maintenance of colonies allowed positive confirmation that all colonies and subsequently individual nymphs used in host specificity trials were free of parasitoids. Broom psyllid ( 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 nymphs.

Non

- target psyllid rearing:

Host plants: Successful rearing of non

- target psyllids was based primarily on having a continuous supply of host plants (specific to the species being reared) with adequate feather flush for oviposition. Since each non

- target psyllid 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 polyphagous 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 psyllid species which were used for maintaining colonies of

selected non

- target psyllid species for testing against D. aligarhensis in UCR IQF.

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.

2 Euphyllura 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 by 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 Quaran tine. Potted olive seedlings were provided by 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 u ntil 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 psyllid oviposition.

23N on

- target psyllid rearing: Non

- target psyllid 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 (usually 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 IQF. Fresh plants were placed into Bugdorms already containing their particular non

- target psyllid 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 nymphs maturing and moving to feed along leaf veins. This psyllid 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 nearly transparent (early 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 early instars which were light yellow in color, subsequently developing to green as they matured. Dichlidophlebia fremontia nymphs 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

labora tory. Because these exotic psyllids lack parasitoids in California, they were collected from wild populations and tested on their respective host plants at UCR IQF without concern that nymphs had been previously parasitized by species other than D. aligarh ensis to which they were exposed to in q uarantine. A. spartiophylla nymphs were a nearly 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 readily with very little stimulus, making them easy to transfer from cuttings to experimental seedlings.

E. olivine nymphs, much like D. fremontia , are white and produce a fluffy white wax, which shelters clusters of young instars. Once older, E. olivine nymphs 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, nymphs were readily transferred onto experimental seedlings.

24B. HOST RANGE TESTING

Young seedlings used as host plants in D. aligarhensis exposure trials ( Capsicum

, Acacia farnesiana

, Prosopis sp., and Olea europaea ) were grown by CDFA at their Mt. Rubidoux Field Station in outdoor greenhouses. Capsicum seedlings were the California Wonder variety (Ferry Morse Seed Company, 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 psyllid 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 ( Cit rus 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 in cone

- tainers (SC7 Stubby, low density, 3.8 cm diameter, 114 mL capacity, Ray

- leach, Stewe and Sons Inc., OR). Mature leaves on th ese seedlings were removed, leaving only 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 psyllid’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

.

25Fig. 1

. Preparation of experimental seedlings in cone

- tainers with test psyllid 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 IQF. 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 waxy 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 capacity clear plastic beverage cup, with the bottom removed and replaced with no

- see

- um netting for ventilation, was then inver ted and secured over the plant, to

restrict the Fremontia psyllids ( Dichlidophlebia fremontiae ) and D. aligarhensis to a small area surrounding the test plant.

26Fig. 2. Adapted experimental setup for Dichlidophlebia fremontiae on Fremontodendron sp. (Fremontia) in D40 growing cones.

Transferring psyllid nymphs to test plants: It took an average of approximately 7

− 8 days
post

- oviposition for ACP nymphs to reach the early third instar (a preferred host stage of D. aligarhensis ) under experimental c onditions in the Insectary rearing room at UCR IQF (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 psyllids on each testing plant, a method of transferring nymphs from large colony plants onto smaller

cone

- tainer seedlings was developed. Nymphs of the appropriate age/life stage were located on large colony plants and disturbed by gent le tapping with a fine

- haired paintbrush to encourage nymphs to become mobile. Once psyllids 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 young flush of the test plant. All nymphs 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 young 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 mL capacity (Thornton Plastic Co, Salt Lake City, Utah) with three ventilation holes 12 mm in diameter (two on opposite sides, one on the bottom)

27and 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) originally 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. Immediately before day 1 testing commenced, female D. aligarhensis were separated into a 0.6 mL microcentrifuge 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 days. 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.

28aligarhensis used in prolonged exposure treatments were given a 24 hour exposure window on each d1 treatment before being immediately 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 psyllid exposure and host

- feeding on parasitism rates on both ACP and NTP.

Prolonged exposure no choice tests: This design consisted of four different treatment types,

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 immediately 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 repeatedly 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 freely and choose between psyllid species for attack. These treatments were conducted using the same acrylic riser cages constructed for use in colony maintenance (s ee above), giving D. aligarhensis a greatly 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 subsequently 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 q uarantine 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 days 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 nymphs dying from natural causes were derived from Control treatments (T8), in which a set of psyllids 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 not

29exposed 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 type.

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 type (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 yllids 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 greatly expanded a rea 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 te st (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 any 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 psyllids 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).

30Table 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. citr i] ►[ 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 movement 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 psyllid developmental

outcomes. Observation 1 was made 7

− 9 days post

- day 1 exposure to D. aligarhensis

. Observation 2 was completed 14

− 16 days post

- day 1 exposure. If at this point there remained mummies or live psyllid nymphs, a third observation was taken 21 days post

- day 1 exposure to determine developmental fate. Any 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 nymphs found dead (physically accounted for), the number of successfully 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 nymphs exposed); (2) Heteropsylla sp. (the native Acacia psyllid) ( n = 390 nymphs); (3) Arytainilla spartiophylla (a self

- introduced natural enemy of the noxious weed scotch broom psyllid) ( n = 380 nymphs); (4) Euphyllura olivina (the invasive olive psyllid) ( n = 320 nymphs); (5) Heteropsy lla texana (the native honey mesquite psyllid) ( n = 395 nymphs); and (6) Dichlidophlebia fremontiae (the native F remontia psyllid) ( n = 375 nymphs). A s ummary (mortality, parasitism, survival to adulthood, etc.) for each psyllid species is presented in Tab le

5

.

Successful parasitism of a non

- target species by D. aligarhensis was observed for only one

31species tested: B. cockerelli , the native and pestiferous potato psyllid, 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 types. Two treatment types (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%. Parasitism 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 types (27

- 45%), it is likely B.

cock erelli is not a highly 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 levels classified as a result of “unknown causes” (dead nymphs which were found and confidently accounted for) were observed across all psyllid species exposed to D. aligarhensis (refer to Table 6 for the complete breakdown of confirmed mortality from unknown causes by psyllid species). This mortality increase was measured against control treatments

consistin g of host plant seedlings with NTP nymphs identical in setup to experimental seedlings and kept under identical conditions, but lacking exposure to D. aligarhensis . It is likely that elevated mortality when compared to controls was a combination of natura l nymph 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 nymphs (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. Increased mortality was

obser ved for Heteropsylla sp. (acadia psyllid, tested on Acacia sp. seedlings) in the presence of D. aligarhensis . Control mortality for this species, was around 8%, but mortality ranged from 5

- 17% across treatments with D. aligarhensis (Table 5). These 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 for ACP was between 20% and 23% (independent of parasitism), markedly higher than the mort ality 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 nymph mortality rates (Table 6). All of the experimental treatment types, 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

lac king 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) of ps yllids and low survivorship of field

- harvested and transplanted scotch broom seedlings. There appears to be no

32significant elevation in A. spartiophila mortality when D. aligarhensis is confined with this psyllid. This is very relevant as this particular p syllid 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 subje ct to snowfall, a climate type that is not conducive for year 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 psyllid (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 significantly 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 slig ht increase in non

target mortality under Quarantine conditions suggests that D. aligarhensis may affect natural

death rate of Fremontia psyllids, but not significantly.

33Table 5

. C onsolidated summary of data for exposure and c o ntrol treatments pertaini ng to psyllid mortality and parasitism rates in the presence of a single mated D. aligar hensis 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

Heteropsyl la 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 20

34psyllid) (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 4

35All species of non

- target psyllid nymphs 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 highly unlikely there would be any significant impact on non

- target psyllid 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 enemy guilds, plant community composition, and the potential rarity of non

- target species for most of the year). The only native psyllid species D. aligarhensis was able to reproduce on was the potato ps yllid,

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 Liberibacter solanacearu m, that causes zebra chip in potatoes and related maladies in other solanaceous crops. D. aligarhensis both increased unknown nymph mortality (by 24%) and successfully parasitized B. cockerelli nymphs at an average rate of ~15%. It is possible that D. alig arhensis 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 encyrtid parasitoid, Diaphorencyrtus aligarhensis , sourced from Punjab Pakistan under four different testing strategies against six non

- target psyllid species, D. aligarhensis was unable to parasitize on five of these six species. Low rates of parasitism, averaging 15%, were observed on the native pestiferous potato psyllid 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 psyllid 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 nymphal 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, whereby females reproduce asexual ly as is the case

36in 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. Identification 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 psy llids 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 (Hymenoptera: Encyrtidae), an endoparasitoid of Diaphornia citri (Homopt era: Psyllidae), from mainland China. Fla. Entomol. 95: 479

− 481.

37Appendix 3

. Response to Comments on the Draft Environmental Assessment.

Legal notice of t he EA was made available in the Federal Register on September 18, 2014 for a 30

- day public commen t period

. Fif teen comments were received on the EA. Thirteen were in favor and two were in opposition to the proposed release of D. aligarhensis

. The two negative comments are addressed in this Appendix. No substantive issues were raised in the comments.

One commenter was concerned that Diaphorencyrtus aligarhensis can sting humans.

As stated in the draft EA, Diaphorencyrtus aligarhensis is an obligate parasitoid of psyllids

, specifically Asian citrus psyllid (ACP) . As such, it will rarely come into co ntact with humans or other vertebrates, and if it does, it is incapable of stinging or biting. Encyrt id wasps have no known adverse impacts on humans or other vertebrates.

One commenter was concerned about what the wasp c ould destroy and if it will des troy necessary bacteria on plants.

Diaphorencyrtus aligarhensis will have no impact on plant bacteria. In addition , as another commenter pointed out, the EA did in fact contain an evaluation of the possible effects of the

release of D. aligharensis on the environment, including specificity data, and impact on non

target species and humans.

38