United States 

Department of 

Agriculture Field Release of the 

Parasitoid 

Diaphorencyrtus

aligarhensis for the 

Biological Control of the 

Asian Citrus Psyllid in 

the Contiguous United 

States

Environmental Assessment,

June 2014

Marketing and 

Regulatory

Programs

Animal and 

Plant Health 

Inspection 

Service
Field Release of the Parasitoid 

Diaphorencyrtus aligarhensis

for the for the Biological 

Control of the Asian Citrus 

Psyllid in the Contiguous

United States

Environmental Assessment,

June 2014

Agency Contact: 

Shirley Wager-Page, Ph.D.

Pest Permitting

Plant Protection and Quarantine

Animal and Plant Health Inspection Service

U.S. Department of Agriculture

4700 River Road, Unit 133

Riverdale, MD 20737–1236
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Table of Contents

I. Purpose and Need for the Proposed Action................................. 1

II. Alternatives ................................................................................. 2

III. Affected Environment................................................................. 5

IV. Environmental Consequences................................................... 7

V. Other Issues ............................................................................. 12

VI. Agencies, Organizations, and Individuals Consulted............... 13

VII. References ............................................................................. 14

Appendix 1. National Quarantine Map for ACP and HLB.

Appendix 2. Host-specificity testing from Hoddle and Bistline, 2013. 
1

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 

parasitoid1species Diaphorencyrtus aligarhensis (D. aligarhensis) 

(Hymenoptera: Encyrtidae). This organism would be used by the permit

applicant for biological control of the nonindigenous Asian citrus psyllid

(ACP) Diaphorina citri (Hemiptera: Liviidae) in the contiguous United 

States. 

This environmental assessment2(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 control ACP within the contiguous 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 contiguous 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; 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 Florida, 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 impact on citrus production in 

that State (Hodges and Spreen, 2012). HLB has also been detected in 

California, Georgia, Louisiana, South Carolina, and Texas. Other States, 

including Alabama, Arizona, Hawaii, and Mississippi, are at risk for HLB

because populations of ACP have been found in those States. 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

Economic losses to growers in Florida attributable to HLB have been 

estimated at greater than $1.7 billion which translates to approximately 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 include non-target impacts. For 

these reasons, there is a need to identify and release an effective, hostspecific biological control organism against ACP in the contiguous United 

States, particularly for control 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 the contiguous 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 

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

whether or not APHIS–PPQ issues permits for environmental release of D. 

aligarhensis. 

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

Under this third alternative, 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 United States. 

There are no issues raised indicating 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 — 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 APHIS–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 many insecticides that may be applied to citrus as systemic and 

foliar sprays against ACP. Systemic insecticides are applied and 
3

translocated throughout the tree. Systemic insecticides include

imidacloprid, aldicarb, and thiamethoxam. Foliar sprays are applied to the 

leaves and include insecticides such as fenpropathrin, zeta-cypermthrin, 

chlorpyrifos, chlorantraniliprole, spirotetramat, abamectin, 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 were previously 

released into Florida (McFarland and Hoy, 2001) but with mixed results 

(Halbert and Manjunath, 2004). It has not established in Florida, perhaps

due to competition from T. radiata, generalist predators, and pesticides 

(Rohrig et al., 2012).

Native predators, such as spiders, coccinellids (lady beetles), lacewings, 

syrphids, and minute pirate bugs attack ACP (Quereshi and Stansly, 2009; 

UC-DANR, 2006). Coccinellids are important predators of ACP in Florida 

(Michaud, 2002; 2004).

B. Issue Permits for Environmental 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 contiguous United States. 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 endoparasitoid attacking second to 

fourth instar ACP nymphs. Oviposition (egg laying) by D. aligarhensis in

the ACP nymph, and feeding of the larval D. aligarhensis inside of the

ACP nymph eventually kills the pest. 
4

a. Taxonomy.

Diaphorencyrtus aligarhensis (Shafee, Alam & 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 described 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 sacchari 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 aligarhensis (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, 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 (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 2007-2009) (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 specific (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 
5

with 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).

An endoparasitoid completes all development inside the host. Besides 

parasitizing ACP, adult females also kill ACP by host feeding on nymphs 

(Skelley and Hoy, 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 feeding 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 from when the D. aligarhensis egg is laid inside an ACP

nymph to when D. aligarhensis emerges 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; fluids ooze 

out through the hole and “glue” the parasitized ACP nymph to the plant. 

After development is completed inside the ACP host, an adult D. 

aligarhensis wasp emerges from the back end of the dead ACP host.

Populations of D. aligarhensis imported into 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 bacteria, 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 presented 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 target of D. 

aligarhensis. Psyllids resemble miniature cicadas and are sometimes called 

jumping plant-lice. ACP damages plants through its feeding activities. New 

shoot growth that is heavily infested by ACP does not expand or develop 

normally, and is more susceptible to breaking off. While direct damage is 
6

serious, there is even greater concern as ACP is an efficient vector of the 

bacterium that causes HLB. 

ACP is found in tropical and subtropical 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, Hawaii, 

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 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 tomato. 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, 

released in 2002 in Florida against the invasive plant Melaleuca 

quinquenervia. California has the richest native psyllid fauna in North 

America, and the influence of climate 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 United 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 

percent, and Texas and Arizona combined produced 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 (NASS, 2013). California 

is the largest producer of lemons, producing more than 92 percent of 

production, and of tangerines, accounting for about 80 percent of 

production (NASS, 2013). 
7

In 2012, total U.S. citrus exports were valued at $1.0 billion, a 2 percent 

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 nursery 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.

IV. 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 lead 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 areas 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 

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). Between 2006 and 2012, HLB was 

estimated to have cost Florida’s economy $4.5 billion and 8,257 jobs

(Hodges and Spreen, 2012).
8

Insecticide usage to control ACP has increased 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 countries 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 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. 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 environmental 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, 2010b) and will 

not be discussed further in this document. However, an additional 

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. Many of the insecticides used to control ACP are 
9

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 over 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; Qureshi and Stansly, 

2009).

B. Biological 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. 

Encyrtid 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 using seven experimental designs 

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 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); 
10

(5) Heteropsylla texana (the native 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. cockerelli, the native and 

pestiferous potato psyllid, the vector of the zebra chip bacterium that 

causes significant economic losses in potatoes (Butler and Trumble, 2012). 

Observed parasitism rates ranged from 11−18 percent for the treatment 

types. Two of the treatment types exhibited moderate levels of parasitism, 

at 17−18 percent. When compared to D. aligarhensis ACP parasitism rates 

across all treatment types (27−45 percent), 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 Appendix 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 

causes HLB in citrus (Hoy et al., 2001). 

2. Uncertainties Regarding the Environmental Release of D. 

aligarhensis.

Once a biological control agent such as D. aligarhensis is released into the 

environment and becomes established, there is a possibility that it could 

move from the target insect (ACP) to attack nontarget insects, such as 

native psyllids. Based on host specificity testing conducted, D. 

aligarhensis only successfully parasitized one psyllid species other than 

ACP, B. cockerelli, the potato/tomato psyllid. The rate of parasitism was

less on B. cockerelli than on ACP. Native 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. aligarhensis, the resulting 

effects could be environmental impacts that 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 site 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 contiguous United States. Approximately 12 percent of 

all parasitoid introductions have led to significant sustained control of the 

target pests, but the majority of introductions have failed to provide 
11

control of the pest (Greathead and Greathead, 1992) either because 

introduction 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.

The 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 impacts 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 CFR 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 action, 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 areawide suppression of ACP, and several initiatives in cooperation with State 

regulatory agencies and the citrus industry in all of the major citrusproducing States: Arizona, California, Florida, and Texas. APHIS also 

conducts a public outreach 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 

components 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 the 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 processes 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. Industry-funded initiatives 

include operational activities, research, and outreach and education. 

Growers in the citrus-producing States conduct management programs for 

ACP in commercial citrus; remove HLB-infected commercial and 

residential citrus; and conduct outreach to homeowners. 

Release 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 insecticides 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 implementing 

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. 

APHIS has determined that, based on the host specificity of D. 

aligarhensis, there will be no effect on any listed insect in the contiguous 

United States. In host specificity testing, the biological control organism 

preferred ACP and only successfully parasitized one non-target psyllid 

species, B. cockerelli, the potato/tomato psyllid. No psyllids are federally 

listed threatened or endangered insects (FWS, 2014). In addition, no listed 

species is dependent on ACP as a food source. Although certain federally

listed 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 contiguous United States (Hawaii, Puerto Rico, or the 

Virgin Islands). D. aligarhensis will only be released within the 

contiguous United States. 

V. Other Issues

Consistent with Executive Order (EO) 12898, “Federal Actions to Address 

Environmental Justice in Minority Populations and Low-income 
13

Populations,” APHIS 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. 

aligarhensis, 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 

disproportionately high and adverse environmental health and safety risks 

to children. No circumstances that would trigger the need for special 

environmental reviews are involved in implementing the preferred 

alternative. Therefore, it is expected that no disproportionate effects on 

children are anticipated as a consequence of the field release of D. 

aligarhensis.

EO 13175, “Consultation and Coordination with Indian Tribal 

Governments”, was issued to ensure that there would be “meaningful 

consultation and collaboration with tribal officials in the development of 

Federal policies that have tribal implications....” Consistent with EO 

13175, APHIS sent letters of notification and requests for comment and 

consultation on the proposed action to tribes in California, Arizona, 

Florida, and Texas. APHIS will continue to consult and collaborate with 

Indian tribal officials to ensure that they are well-informed and 

represented in policy and program decisions that may impact their 

agricultural interests, in accordance with EO 13175.

VI. 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.

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 Analysis Services

4700 River Road, Unit 149

Riverdale, MD 20737–1238
14

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Service, 2011. Citrus Canker, Citrus Greening, and Asian Citrus Psyllid; 

Interstate Movement of Regulated Nursery Stock. Federal Register. 

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March 11, 2014. 

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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) 

used for host range assessment experiments were 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 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 colonies: 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 maintained on C. volkameriana seedlings which ranged from less than one year to 

approximately two years of age, potted in 4 inch diameter pots. Success of ACP reproduction, 

regardless of plant age, is strongly 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 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. volkameriana 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 flush 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 clusters 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 
21

additional 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 rearing cages once they

were ready for use. 

ACP rearing cages in Quarantine: Colonies of ACP were reared on the Insectary level of UCR 

IQF using a double-cage system (i.e., a cage within a cage) in order to prevent escape of psyllids. 

Survivability of ACP outside 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 

primary 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 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 approximately 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 selected 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 watering. 

An average of 18 cages (six each every Monday, Wednesday and Friday) of C. volkameriana 

bearing feather flush were set up each week for ACP oviposition in quarantine. 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-free 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 PAKISTAN 

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 
22

a 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 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 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 designed to select 

pertinent California psyllid species for host range testing in quarantine. 

Table 1. Experimental selection criteria and 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] [Liviidae: 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]) 

Heteropsylla 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 Arytainilla 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 associated 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, colonies were set up and reared in an IQF Insectary
23

laboratory. 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 pestiferous 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

1Heteropsylla sp. Sweet acacia Acacia farnesiana

1Heteropsylla texana Honey mesquite Prosopis glandulosa

1Dichlidophlebia fremontiae Fremontia bush Fremontodentron sp.

2

Euphyllura olivina Olive Olea europaea

2

Arytainilla spartiophylla Scotch broom Cytisus scoparious

1Colony maintained in Quarantine, UCR IQF. 2Colony 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 California 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 Quarantine. 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 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 psyllid oviposition.
24

Non-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 encompassing 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. Nymphs 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 

laboratory. 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. aligarhensis to which they

were exposed to in quarantine. 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.
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 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 assistance 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 plants used for testing target and non-target psyllid species. All host plants used 

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 preparation: 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 these 

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 cone-tainers 

is illustrated below in Fig. 1.
26

Fig. 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 removed; 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 collected), 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 from 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 beverage 

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 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: 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 conditions 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 gentle 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 psyllids 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) 
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-parental) 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 μL 

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 the 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. 
29

aligarhensis 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 followed 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 μL 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 exposure (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 of 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. 

aligarhensis was able to host-feed on or parasitize ACP before or after encountering NTP, 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. aligarhensis 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 (see 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. 

Environmental test conditions in quarantine: The Insectary laboratories where NTP colonies 

were maintained 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 28.33oC, 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 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 quarantine conditions, but were not 
30

exposed to parasitoids. 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 additional 4 hours. At the 

end of this 8-hour rotation, females were removed and transferred into a 200 μL 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 psyllids 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 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 of 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 

transferred 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 their respective host plants in conetainers 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 quarantine testing room conditions to determine 

developmental 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 movement to new psyllid hosts rest = containment of female D. aligarhensis in a 200 μL 

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 subsequently 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 using seven experimental designs encompassing nochoice 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) Heteropsylla texana (the native honey mesquite psyllid) (n = 395 nymphs); and (6) 

Dichlidophlebia fremontiae (the native Fremontia psyllid) (n = 375 nymphs). A summary

(mortality, parasitism, survival to adulthood, etc.) for each psyllid species is presented in Table 

5.

Successful parasitism of a non-target species by D. aligarhensis was observed for only one 
32

species tested: B. cockerelli, the native and pestiferous potato psyllid, the vector of the zebra chip 

bacterium which causes significant economic loses in potatoes (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. 

cockerelli 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 unlikely 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 

consisting 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 natural 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 conditions 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 mortality. 

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 

observed 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 mortality 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 highest 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

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) of psyllids and low 

survivorship of field-harvested and transplanted scotch broom seedlings. There appears to be no 
33

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

psyllid. This is very relevant as this particular psyllid 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 subject 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 recorded 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. aligarhensis (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-target 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 nontarget mortality under Quarantine conditions suggests that D. aligarhensis may affect natural 

death rate of Fremontia psyllids, but not significantly.
34

Table 5. Consolidated summary of data for exposure and control treatments pertaining to psyllid 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 11

T2 (4 hr NTP 

first) 5 0 23 21

Choice Test (T3) 17 0 21 21

Prolonged 

Exposure (T4-7) 10 0 23 32

Arytainilla 

spartiophila 

(Scotch Broom 

Psyllid) 

(SBP) 

T1 (4hr, ACP 

first) 41 0 16 18

33 19

T2 (4 hr NTP 

first) 32 0 17 16

Choice Test (T3) 14 0 13 29

Prolonged 

Exposure (T4-7) 25 0 16 21

Euphyllura 

olivina (Olive 

Psyllid) 

(OLV) 

T1 (4hr, ACP 

first) 21 0 16 16

15 15

T2 (4 hr NTP 

first) 23 0 22 25

Choice Test (T3) 16 0 19 21

Prolonged 

Exposure (T4-7) 19 0 12 28

Heteropsylla 

texana (honey 

mesquite 

(Prosopis) 

T1 (4hr, ACP 

first) 24 0 18 20

21 26 T2 (4 hr NTP 

first) 13 0 9 20
35

psyllid) 

(PRO) 

Choice Test (T3) 8 0 23 28

Prolonged 

Exposure (T4-7)

19 0 16 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 17 15

Prolonged 

Exposure (T4-7) 16 0 19 24
36

All 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. aligarhensis. 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. texana = 

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 interfering 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 psyllid, 

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 solanacearum, 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. 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 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 native 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 targeting 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 asexually as is the case 
37

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. Trumble. 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 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 (Hymenoptera: Encyrtidae), an endoparasitoid of Diaphornia citri 

(Homoptera: Psyllidae), from mainland China. Fla. Entomol. 95: 479−481.