**United States**

**Department of Agriculture**  **Marketing and Regulatory**  **Programs Animal and**  **Plant Health Inspection**  **Service** 

# Field Release of **Aphelinus**

**rhamni (**  **Hy men**  **o ptera:**  **Aphelin idae**  **) for B**  **iological Control of**  **the Soybean Aphid,**  **Aphis glycines (**  **Hemiptera: A**  **p h**  **ididae )**  **, in**  **the Continental United States**  **Environmental Assessment ,**  **August 2013**

# Field Releas **e of Aphelinus**  **rhamni**

**(Hy menoptera: Aphelinidae) for**  **Biological Control of the Soybean Aphid,**  **Aphis glycines (Hemiptera:**  **Aphididae), in the Continental United State**  **s Env**  **ironmental Assessment, August 2013**  **Ag ency** **Contact** : Shirley W ager- 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

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## Table of Contents **I. Purpose and Need for the Proposed A** **ction ............................**  **1 II.**  **Al ternatives**  **................................ ................................**  **................ 2**  **III. Af**  **fected Environment ................................**  **.............................. 5**  **IV. En**  **vironmental Consequences ................................**  **............... 7**  **V. Other Issues**  **................................ ................................**  **............ 1**  **2 VI.**  **Ag encies, Organizations, and Individuals Consulted**  **........ 1**  **3 VII.**  **R eferences**  **................................ ................................**  **............ 1**  **5**
**Ap pendix**  **A ................................**  **................................ ..................**  **2 3**  **Ap pendix**  **B ................................**  **................................ ..................**  **2 5** **1** 

## I. **Purpose and Need for the Proposed Ac**  **tion** The U.S. Department of Agriculture (USDA), Animal and Plant Health Inspection Service (APHIS) , Plant Protection and Quarantine (PPQ) , P est Permitting Branch is proposing to iss ue permit s for release of the insect Aphelinus rhamni ( Hy

menoptera : Aphelinidae )
. This organism would be used by the appli cant for biological control of soybean aphid , Aphis glycines ( Hemiptera: Aphididae ) , in the c ontinental United States . Before issuing permit s for release of A phelinus rhamni , APHI S

–PPQ needs to anal yz e the potential impacts of the release of this organism into the continental United States
. This environmental assessment 1 (EA) was prepared to be c onsistent with USDA

–APHI S'National Environmental Policy Act of 1969 (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 associat ed with the release of the parasit oid wasp , A. rhamni , to control soy

bean aphid in the continental United States
. A parasitoid is an insect whose immature stages (larvae and pupae) live as parasites that eventuall y kill their hosts (typicall y other insects )
. This EA considers a “no action” alternative and the potential effects of the proposed action
. The applicant’s purpose for releasing A. rhamni is to reduce the severity of damage to soy

bean ( Glycine max) from infestations of soy bean aphid in the continental United State s . In 2000 , the so ybean aphid (native to Asia) , was found in North America , and is now a major soy bean pest infesting 42 million acres in 2003 which result ed in decreased soy bean yi elds and greatl y increased control costs (Ragsdale et al., 2011; L andis et al., 2008; Song and Swinton, 2009) . The soy bean aphid invaded most soy bean production regions in North America, including 21 states and three Canadian provinces since originall y reported in North America in 2000 (Ragsdale et al., 2004)
. The applicant identified several purposes for releasing this particular insect species for biological control of so ybean aph id. F irst, impacts to non - target species are expected to be low. Second, this parasitic wasp may exhibit utility

as a biological control agent on the soy bean aphid’s alternate buckthorn host, R hamnus species
. Lastl y , the distribution of A. rhamni in the United States may differ from the expected distribution of A. glycinis , a related biological control ag ent of so yb ean aphid recentl y 1 Regulations implementing the National Environmental Policy Act of 1969 (42 United States Code 4321 et seq. ) provide that an enviro nm ental assessment “[shall include brief discussions of the need for the proposal, of alternatives as required by section 102(2)(E), of the e nvironmental impacts of the proposed action and alternatives, and a listing of agencies and persons consulted” ( 40 CFR § 1508.9 )

.**2** approved for release in the United States . Deplo yment of both insect species has the potential to provide enhanced control of soy bean aphid over a broader area in the United States than A. glycinis alone
. These parasitic wasps are insect s whose immature stages (larvae and pupae) live as parasites that eventually kill their hosts (ty

pically other insects). Most of the e xisting soy

bean aphid management options (discussed below) are expensive, temporary , and/or include non - target impacts. For these reasons, there is a need to identify and release a complement of effective, host
- specific biological control organism s against soy bean aphid in the continental United States
. 

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

–PPQ : no action (no issuance of permits) and issuance of permit s for environmental release of A. rhamni in the continental United States
. Although APHIS ’ alternatives are limited to a decision o f whether to issue permit s for rele ase of A. rhamni , we describe other methods currently used to control soy bean aphid by soy bean producers in the United States
. Use of these control methods is not an APHI S decision , and their use is li kely to continue whether or not APHI S
- PPQ issued permit s for environmental release of A. rhamni
. APHI S

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

–PPQ would issue permits for the field release of A. rhamni . The permits, however, would contain speci al provisions or requirements concerning release procedures or mitigating measures , such as limited release s of A. rhamni in the United States
. There are no issues raised indicating that special provisions or requirements are necessary . 

### A. **No A** **ction** Un der the no action alternative, APHI S

–PPQ would not issue permit s for the field release of A. rhamni for the control of soy bean aphid

— the release of this biological control agent would not occur , and current methods t o control soy

bean aphid in the United States will continue
. Use of these methods is likely to continue even if APHIS

–PPQ issues permit s for release of A. rhamni
. Presently , control of soy bean aphid in the United States is limited to chemical control , plant resistance , and biological control methods
. Insecticide treatments are the most effective method to manage so ybean aphids in North America. Foliar sprays of insecticides provide temporary suppression (7 to 14 day s) of soy bean aphid populations. Insecticides **1. Chemical C**  **ontrol 3**  including esfenvalerate , dimetho ate, chlorp yrifos , zeta - cy permethrin , methyl parathion , permethrin , and lambda cy halothrin are or have been labeled for foliar application against soy bean aphid (Ostlie, 2002 ). Soy bean s eed treatments using the neonicotinoid insecticide s imidacloprid and thiamethoxam for systemic control of so ybean aphids also can be used (Pedersen and Lang, 2006 ; Magalhaes et al., 2009 )
. Aphid
- resistant so ybean plants were bred to support reduce d aphid populations. Host plant resistance works by inhibiting aphid growth and development and increasing plant tolerance to aphid feeding. Aphelinus certus is a parasitoid native to Asia , accidentally introduced into the United States when the soy bean aphid invaded (Heimpel et al., 2010)
. A
. certus has a broad host range (Heimpel et al., 2010; Frewin et al., 2010) , and was not considered for purposeful introduction against soy bean aphid
. Inthe long term, introduction of another parasitoid is unlikely to affect the abundance of A. certus because these insects coexist in Asia. Another Asian parasitoid, Binodoxys communis (Hy menoptera: Braconidae) , was released in the United States beginning in 2007; unfortunatel y, it did not establish in the environment (Ragsdale et al., 2011). In 2 002, a previously introduced strain of the European parasitoid Aphelinus atriplicis was released against so ybean aphid
. This strain was first released in the w estern United States against the Russian wheat aphid (Hopper et al., 1998; Prokry m, 1998; Heraty et al., 2007) , but it also attack s the so ybean aphid (Wu et al., 2004) . Although this strain of Aphelinus atriplicis was released in nine Minnesota sites in 2002 (Heimpel et al., 2004) , it was not recovered since th at time. Most recentl y , APHIS

–PPQ approved the parasitoid Aphelinus glycinis for environmental release into the United States ( Hopper, 2010; USDA
APHI S, 2012). This related species originated from X iy uan, Liaoning Province, Peoples Republic of China . The climate in this area closel y matches the climate in the northern Midwest United States where so ybean aphid is a major pest. It is expect ed that A. glycinis will be come limited to this region , but may extend its range into Ontario and Quebec provinces in Canada
. E stablishment is expected to occur primarily in soy bean fields infested with soy

bean aphid during the growing season
. 

### B **. Issue**  **P ermit**  **s for**  **E nvironmental**  **R elease**  **of A.**  **r hamni**  **.** Under this alternative, APHI S

–PPQ would issue permit s upon request and after evaluation of each application for the field release of A. rhamni for the control of soy

bean aphid in the continental United States . Th ese permit s would contain no special provisions or requirements concerning release procedures or mitigating measures
. **3. Biologica l**  **Control 2.**  **Host Plant Resistance** **4** Insect Taxonomy Order: Hy

menoptera Family : Aphelini dae Genus: Aphelinus Species: rhamni Woolley and Hopper Common name : none The species description of A. rh amni was published in 2012 (Hopper et al., 2012)
. Voucher specimens of A. rhamni are held at three locations. These include (1) the Beneficial Insect Introductions Research Unit, Agricultural Research Service (ARS) , United States Department o f Agriculture, Newark, Delaware , (2) Texas A & M University, Department of Entomology , and (3) the Smithsonian in the Systematic Entomology Laboratory , USDA

−ARS. Aphelinus sp ecies are tiny (about 1 millimeter (mm) in length) , stingless parasitic wasps. The immature st ages develop as internal parasit oids o f arthropods where , in this case, feeding of the wasp larva inside the host aphid eventuall y kills the host aphid
. Many species of Aphelinus have demonstrated their importance in the biological control of aphids ( Hemip tera: Aphididae). Aphelinus rhamni females prefer second to fourth instar (an immature developmental stage) aphid hosts for oviposition (egg laying) , but will lay eggs in all stages, including winged adults. Females use an egg laying organ called an ovip ositor to insert an egg into the aphid. The inserted egg hatches and the wasp larva feeds on the internal organs of the aphid host. At 20 o C, the wasp s develop from egg to pupa in about 14 day s; third instar wasps kill their hosts leaving intact the aphid h ost external skeleton ( exoskeleton) hardened and black, in a process called mummification. Adult A. rhamni wasps emerge from the mummified aphid about one week after pupation by chewing a hole through the host exoskeleton to escape. Adult wasps eat plant n ectar and honeydew (a sugary liquid secreted by aphids and other sap
- feeding insects) . A dult females also feed on aphids to obtain nutrients for egg production by piercing or “stinging” the aphids with their ovipositor and drinking the hemol ymph (insect bl ood) from the wound , killing about two aphids per day by such feeding. Females emerge as adults with five to 15 mature eggs, but produce more eggs throughout their lives, maturing about 10 eggs per day
. In the laboratory , adult wasps live for two to three weeks, but they are unlikel y t o survive this long in the field (Hopper, 2011)
. Aphelinus species are weak fliers, searching primarily by walking (Fauvergue et al. , 1995; Mason and Hopper, 1997)
. Another related parasitoid , Aphelinus asychis, dispersed an average of 13 feet in one generation and 26 feet in three generations in fields with abundant hosts **1. Aphelinus**  **rhamni Taxonomic**  **Information**

**2. Description**  **and Biology**  **of Aphelinis**  **rhamni 5**  (Fauvergue and Hopper, 2009)
. Insects would have to disperse much further at the end of the growing season to track their hosts. Aphelinus rhamni was collected from parasitized soy bean aphid s on Rhamnus species in 2005 near Daxing ( Beijing, Hebei Province ) Peoples Republic of China , and is not known to occur elsewhere (Hopper et al., 2012)
. The present distribution of soy bean aphid in the United States matched fairly

well with a climate
- based expected distribution of A. rhamni (Dy

me x Simulator Application software, version 2.01.025, (c) CSIRO, 2004). It is expect ed that A. rhamni may not establish in the full southern range of the so ybean aphid because this pest is found further south than the climate match for Beijing. A phelinus rha mni is expected to establish primarily in the northern half of the United States east of the Rocky Mountains (Hopper, 2011)
. In the laboratory , an individual female A. rhamni wasp parasitiz es or kills by host feeding over 20 0 aphids during a lifetime o f three weeks, but long lifetimes in the field are unlikely
. If females lived one week in the field , each is expected to p arasitize and host feed on 70 aphids, if aphids are sufficiently

abundant (Hopper, 2011)
. 

## III **. Af**  **fected Env ironment**  **A. Soy**  **bean Ap**  **hid** The soy bean aphid is a host alternating species, which means it must disperse between two different types of host plant s between summer and winter. Females reproduce asexually on soy bean from June to September, producing winged and wingless forms . In September, winged mal es and parthenogenetic (a form of reproduction in which an unfertilized egg develops into a new individual) females migrate to their primary host plants, Rhamnus sp ecies , and the migratory females bear sexual, flightless females, which mate with the migratory males and lay

overwintering eggs. In the spring, parthenogenetic females hatch from these eggs, and several generations of females reproduce asexually on Rhamnus species before winged aphids move to soy bean plants
. Although soy bean aphids are weak fliers, the rapid rate of spread of so yb ean aphid indicates a high rate of windborn dispersal. In the laboratory , soy bean aphid has a high rate of population increase at 25 o C, with a steep decline to a negative rate when temperatures are higher than 30 o C (McCornack et al., 2004) . This can explain why

it has not moved further south in the United S tates where temperatures are warmer
. At 25 o C, females can produce 73 offspring during their lifetime, with a generation time of 10 day s. The combination of high dispersal rate and high reproductive rate mean that soy bean aphid can rapidl y reach damaging levels over wide regions, if not properl y managed (Hopper, 2011)
. **3 .**  **Geographic R**  **ange of**  **Aphelinis rhamni** 

**4 . Impact**  **of Aphelinus**  **rhamni on**  **Soy bean**  **Ap hid** **6** 

### B **. A reas**  **A**

**ffected by Soy**  **bean Ap**  **hid** The soy bean aphid is native to E ast Asia, found in eastern Russia, China, Japan, and Southeast Asia (Vietnam, Thailand, Indonesia, Malay sia, and the Philippines). There are reports in Kenya, and it invaded Australia in 2000. In North America, it is cur rentl y known from Ontario and Quebec in Canada, and from the following 2 9 U.S. states: Arkansas, Connecticut, Delaware, Georgia, In diana, Iowa, Il linois, Kansas, Kentucky , Louisiana, Maine, Maryland, Massachusetts, Michigan, Minnesota, Mississippi, Missouri, Nebraska, New Jersey , New York, North Dakota, Ohio, Oklahoma, Penns ylvania, South Dakota, Tennessee, Virginia, West Virginia, and Wisconsin (Ragsdale et al. , 20 11 ). Soy bean ( Glycine max) is the summer host of soybean aphid while various buckthorn speci es (

Rhamnus sp ecies ) are the winter hosts. S everal native and a few introduced species of Rhamnus occur in the United States
. 

### C **. Insects**  **R elated to**  **Soy bean A**  **phid and**  **A. rhamni**  **in the United States** In formation regarding insects taxonomically related to soy

bean aphid is included because closel y related insect species have the greatest potential for attack b y A. rhamni
. Of the 497 species of Aphis w ith distributions listed in Blackman and Eastop (2006), 119 are r eported from North America. Of these sp ecies found in North America, 33 are widel y distributed throughout the world, and many of these are invasive exotic pests. Only 86 species of Aphis were described from collections in North America , and consequentl y may be considered native species. Fortunatel y, most native Aphis species are not closely related to soy

bean aphid (

Aphis glycines )
. The soy

bean aphid is closely related to the cotton aphid, Aphis gossypii , which is also native to Asia and a major agricultural pest, and to the buckthorn
- potato aphid, Aphis nasturtii , which shares its overwintering host ( Rhamnus sp ecies ). Aphis nasturtii is a pest of potatoes in North America , and believed to be native to Asia (Hopper, 2011)
. Thirty
- one species of Aphelinus are reported from North America , howev er , onl y about half of these species (14) were described from North America (listed in Appendix A with their authors and species complex membership, as well as reported distributions and hosts)
. These 14 Aphelinus species are reported exclusively or almost exclusively

from North America, and likely are native. The remaining Aphelinus species reported from North America were **2. Present**  **D istribution in**  **North Am**  **erica 1.**  **Native and**  **Worldwide Distribution** 

**3 .**  **Soy bean**  **Ap hid**  **H osts** 

**1 .**  **Insects Related**  **to Soy**  **bean Ap**  **hid**

**2 .**  **I nsects**  **Related to Aphelinus**  **rhamni 7**  described elsewhere, are reported from one to several other continents, and therefore are unlikel y to be native to North America. Several have been introduced for biological control of pest aphids (Hopper et al., 1998; Jackson et al., 197 1 ; van den Bosch et al., 1959)
. Three native Aphelinus species were reported from Aphis species, A. marlatti , A. nigritis , and A. semiflavus , but only A. semiflavus parasitizes a species closel y related to the soy bean aphid, being reported from the cotton aphid in the M idwestern United States
. The native parasitoid species a ppear unlikely

to switch to attack the soy

bean aphid, because these types of switches have not been observed (Lin and Ives, 2003; Rutledge et al., 2004; Kaiser et al., 2007; Costamagna and Landis, 2007; Noma and Brewer, 2008; Noma et al., 2010)
. Five n ative Aphelinus species ( A. lapisligni , A. mali , A. prociphili , A. sanborniae , and A. siphonophorae) are in the mali complex to which A. rhamni belongs . These species are more closel y related to A. rhamni than other native Aphelinus species , yet their hos ts are different from those parasitized by A. rhamni (see Appendix A)

. Therefore, A. rhamni is not expected to compete with native species of Aphelinus
. Climate projections suggest both A. rhamni and A. glycinis are likel y to establish in the northern Mi dwest of the United States (Hopper, 2010; Hopper, 2011; Hopper, 2012). Establishment of A
. glycinis may preferentiall y occur on the soybean host, while A. rhamni is expected to establish on the buckthorn hosts. 

## I **V. Env ironmental Consequences**  **A. No**  **A ction**  **a . Effect of**  **soy bean aphid**  **on host plants** Soy bean aphids suck sap from plants. When aphid infestations are large, infested leaves are wilted or curled. D irect feeding damage from aphids may include plant stunting, reduced pod and seed counts, and yellowing of leaves. Soy

bean aphids also transmit plant viruses such as alfalfa mosaic, soy bean mosaic , and bean yellow mosaic ; these diseases cause yi eld reduction in soy

bean s . For so ybean growers in the north central United States, the soy bean aphid is the first insect pest to consistently cause important losses over wide areas (Ragsdale et al., 2004) , with yield decreases of up to 40 percent (Ragsdale et al., 2007)
. Invasion of the s oy bean aphid led to dramatic changes in the pest management practices of soy bean producers. In 2000, less than one - tenth of one percent of so ybeans in the north central states were treated with insecticides . B y 2006, over 13 percent of soy beans in these states were treated with insecticides . This indicates soy bean aphid was re sponsible for a 130
- fold increase in the use of insecticides (Ragsdale et al., 2011) . Control of the soy bean aphid currentl y relies on **1 .**  **Impact of Soy**  **bean Ap**  **hid on the Environment** **8** insecticide use, and millions of acres of so ybeans have been sprayed sin ce the invasion of this pest . This is a major disruption in the 70 million acres of soy bean production with in the country
. The continued use of chemical control, plant resistance, 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 A. rhamni

. **a. Chemical**  **C ontrol**  Foliar applications may

not sufficiently

control soy

bean aphids and prevent yi eld loss, especially if large quantities of so ybean aphids are surviving on lower leaves. Foliar spray s may pose a threat to bees and aphid natural enemies , such as lady beetle larvae and predatory bugs. In a screening assay , both λ
- cy halothrin and dimethoate were harmful to A. certus (Frewin et al., 2012). This suggests chemical pest controls may limit the effectiveness of various parasitoid wasps released as potential biocontrol organisms. Seed treatments o f s ys temic insecticide s such as imidacloprid do not last the entire growing season. Inadditio n, seed treatment insecticides are applied at the beginning of the growing season before it is known if treatment for so yb ean aphi d is even necessary
. This creates a financial loss to growers if aphid populations would not have been high enough to trigger economic damage requiring pest treatments
. **b. Plant Resistance**  Although plant resistance inhibit s aphid growth and development and increas es plant tolerance to aphid feeding , p lant resistance in certain cases has been overcome by

soybean aphids. **c. Biological Control**  Two biological control organisms , Aphelinus atriplicis and Binodoxys communis were released against soy bean aphid , h owever, neither organism e stablished in the environment and provide s control of the soy bean aphid. The related A. glyc inis recentl y was approved for environmental release in the continental United States ( Hopper, 2010; USDA
- APHIS, 2012) . T his organism is expected to establish primarily on the soy bean host. These impacts from the use of other control methods may have environmental consequences even with the implementation of the biological control alternative , d epending on the efficacy of A. rhamni to reduce soy

bean aphid infestations in the continental United States
. **2. Impact from the U**  **se of O**  **ther C**  **ontrol Methods** **9** 

### B. **Issue P ermit**  **s for**  **E nvironmental**  **R elease**  **of A.**  **rhamni**
**a. Scientific Literature**  Published literature on the host range of A

. rhamni includes only the information in the species description. In the field, Aphis glycines is the only known host , but in labo ratory experiments, Aphelinus rhamni parasitizes A

phis glycines, closely

related species in th is genus , and rarel y R hopalosiphum padi L. and Schizaphis graminum (Hopper et al., 2012)
. Other species in the mali complex tend to have narrow host ranges; a not able exception is A. mali itself which reportedly has a broad host range. On the other hand, literature reports of broad host ranges may reflect confounding effect s from cry ptic species within this genus (Heraty et al., 2007). **b. Host**  **S pecificity**  **T estin**  **g** Host specificity

testing of A. rhamni in containment examined three ways that aphids were killed: oviposition leading to mummification, stinging the aphid to death without mummification, and host feeding (Hopper, 2011). Parasitism o f 13 aphid species in five genera in two tribes on 10 plant species in nine families was measured (Table 1) . The se aphids covered a range of closel y related aphids reported as hosts of the A. mali complex ( see Appendix A), of which A. rhamni is a member. Furthermore, the ap hid
- plant combinations provided contrasts of aphids in the same versus different genera , on the same versus different plants ( see Appendix B). Based on the aphid distributions in Blackman and Eastop (2006), most of these aphid species occur within the nati ve geographic range of A. rhamni , so these aphids and this parasitoid are likel y to have been in contact for at least 10,000 years. The exceptions are D iuraphis noxia , which is native to central to western Asia and Europe, and Aphis oestlundi and Aphis mon ardae , which are native to North America. **1. Impact of**  **A. rhamni**  **on Non**  **- target**  **Insects 10**  **Table 1**  **. Aphid and host plant species used to test**  **Aphelinus rhamni**  **host specificity. Aphid species**  **Plant species Plant family**  Aphis glycines Glycine max Fabaceae Aphis gossypii Gossypium hirsutum Malvaceae Aphis gossypii Asclepias syriaca Apocy naceae Aphis monardae Monarda fistulosa Laminacea Aphis oestlundi Oenothera biennis Onagraceae Aphis nerii Asclepias syriaca Apocy naceae Aphis asclepiadis Asclepias syriaca Apocy naceae Aphis craccivora Vigna unguiculata Fabaceae Aphis rumicis Rumex altissimus Polygonaceae Rhopalosiphum maidis Hordeum vulgare Poaceae Rhopalosiphum padi Hordeum vulgare Poaceae Schizaphis graminum Hordeum vulgare Poaceae Diuraphis noxia Hordeum vulgare Poaceae Myzus persicae Raphanus sativus Brassicaceae Tests of parasitism in 24
- hour exposures of aphids to A. rhamni consisted of A. rhamni females being placed in cages with 100 aphids of a single species on plant foliage. In th is study , A. rhamni rarel y or never parasitized aphids outside of the genus Aphis. Within the genus Aphis, A

. rhamni produced mummified aphids on species closely related to the target aphid, A. glycines (e.g., A. gossypii, A. monardae, and A. oestlundi) , b ut not on more distantly related Aphis species. Direct observation of the b ehavior of A. rhamni and aphids determine d host use patterns . Continuous microscopic observation of each female parasitoid (and the aphids to which she was exposed) for 25 minute s under magnification showed A. glycines is the most suitable species for A. rhamni development, by producing an adult parasitoid per oviposition . In most other species in the genus Aphis , there were less than half as many A. rhamni adults per oviposition produced
. In summary , laboratory

experiments showed oviposition and host feeding by A. rhamni was restricted almost completely to species in the genus Aphis closely

related to the target. Furthermore, parasitoid stinging that did not lead to mummificatio n or host feeding did not increase mortality of any aphid species. Handling times of aphids b y A. rhamni were long, so ant - fended native aphids, such as A. monardae and A. oestlundi , should not be at risk in the field. The target pest, soy bean aphid, was the most suitable aphid species for A. rhamni development (Hopper, 2011)
. See Appendix B for detail s on the host specificity study
. Additional studies determined if A. rhamni might be likely to shift to non
target aphid hosts under certain conditions. In these studies, it was found**11** that stresses decreas ing the length of life of A. rhamni did not increase its acceptance of low quality aphid host species. Specificity appears to be stable , and low
- quality, non - target species should not have increased risk of attack when A. rhamni is stressed from starvation or age. Once a biological control agent such as A. rhamni is released into the environment and become s established, there is a poss ibility it could move from the target insect ( soy bean aphid) to attack nontarget insects, such as native aphid species. Native species that are closely related to the target species are the most likely to be attacked ( Louda et al., 2003 ). If other aphid sp ecies were to be attacked b y A. rhamni , the resulting effects could be environmental impacts that may not be easil y reversed. B iological control agents such as A. rhamni generall y spread without intervention by man. Inprinciple, therefore, release of thes e 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, these agents may not be successful in reducin g soy bean aphid populations in the continental 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 control of the pe st (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 soy

bean aphid populations by A. rhamni will not be known until after release and establishment occurs
. Actual impacts on the establishment of A. glycinis , including potential competitive interactions will not be known until after release and establishment of A. rhamni occurs. The environmental consequences discussed under the no action alternative may

occur even with the implementation of the action alternative, depending on the efficacy of A. rhamni to reduce soy bean aphid in the continental United States
. “Cumulative impacts are defined as the impact s on the env ironment 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 o ther actions” (40 CFR 1508.7). To date, o ther so ybean aphid biological control organisms released in the United States have not successfull y established. Therefore, competitive interactions would not occur among any organisms previously released. The release of A. glycinis is not expected to produce competitive interactions with A. rhamni because habitat partitioning of the two parasitoids between so yb ean aphid s on the two alternate plant hosts (soy bean and Rhamnus species) is anticipated ( K. Hopper, pers. comm. )
. **2. Uncertainties**  **Regarding the Environmental**  **R elease of**  **A phelinus**  **rhamni.**

**Cumula tive**  **I mpacts**  **3. Cumulative Impacts** **12** The two species also have different predicted geographical distributions based on climate matching, with the distribution of A. glycinis extending further north and A. rhamni further south (Hopper, 2010). A s discussed previously , A. rhamni is not expected to compete with native species of Aphelinus
. The non - native parasitoid Aphelinus certus Yasnosh accidentall y established in North America (Heimpel et al., 2010, Frewin et al., 2010) . It was found over a wide geographic range in the eastern United States and Canada since 2005
. In laboratory experiment s, i t has a host range of at least four genera in two subfamilies of aphids, and on at least four host plant species ( Heimpel et al., 2010 ). The abundance of A. certus in the United States is unlikely

to be affecte d by

introduction of A. rhamni because th is parasitoid’s host range is narrower than for A. certus . Aphelinus certus might interfere with establishment of A. rhamni , but the coexistence of these parasitoid species in Asia suggests any interference is likel y to be short
- lived
. Release of A. rhamni is not expected to have any negative cumulative impacts in the continental United States because of its host specificity to soy bean aphids
. Effective biological control of soy bean aphid will have beneficial effec ts for soy

bean growers in the United States , may result in a long
- term, non - damaging method to assist in the control of soy bean aphid , and may reduce the use of insecticides on soy bean. Section 7 of the Endangered Species Act (E SA) and ESA’s implementing regulations require Federal agencies to ensure that their actions are not likely to jeopardize the continued existence of federall y listed threatened and endangered species , or result in the destruction or adverse modification of critical habitat. APHI S has determined that , based on the host specificity of A. rhamni

, there will be no effect on any listed insect species or designated critical habitat in the continental United States
. Inhost specificity testing, A. rhamni only att ack ed species of aphid s . There are n o federally listed threatened or endangered insects belong to the aphid family , Aph ididae (USFWS, 20 1 2 ). There are n o federall y listed species are known to depend on or utilize soy

bean aphid
. 

## V. Other Issues Consistent with Executive Order (EO) 12898, “Federal Actions to Address Environmental Justice in Minority Populations and L ow - income Populations,” 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 anticipated from the field release of **4. Endangered Species A** **ct 13**  A. rhamni and its 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,” APHI S considered the potential for disproportionately

high and adverse environmental health and safety risks to children. Th ere are no circumstances that would trigger the need for special environmental reviews involved in implementing the preferred alternative. Therefore, there are no disproportionate effects on children anticipated because of the field release of A. rhamni
. EO 1317 5 , “ Consultation and Coo rdination with Indian Tribal Governments ” , w a s issued to ensure th at there would be “meaningful consultation and collaboration with tribal officials in the dev e lop ment of Federal policies that have tribal i m pli c ations....” Cons istent w ith EO 131 7 5, AP H I S s ent letters of notification an d requests fo r comment an d consultation on the prop osed action to tribes in areas where the soy bean aphid occurs. AP HI S will continue to consult and c o llaborate with Indian trib al officials to ensure that they are wel l
- infor med and represent e d in policy a nd program decisions th a t may i m pact their agricultural interests, in accordan ce with EO 1 3175
. 

## VI. **Ag encies, Organizations, and**  **Indiv iduals Consulted**  This EA was prepared and reviewed b y APHI S and ARS
. The addresses of p articipating APHIS units and any applicable cooperators are provided. U.S. Department of Agriculture Animal and Plant Health Inspection Service Policy and Program Development Environmental and Risk Analy sis Services 4700 River Road, Unit 149 Riverdale, MD 20737 U. S. Department of Agriculture Animal and Plant Health Inspection Service Plant Protection and Quarantine Registrations, Identification, Permits, and Plant Safeguarding 4700 Ri ver Road, Unit 133 Riverdale, MD 20737 U
. S
. Department of Agriculture Agricultural Research Service Beneficial Insect Introductions Research Unit 501 South Chapel Street**14** Newark, DE 19713**15** 

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− 737.**23** 

### Appendix A. Aphelinus species described from North America and their species complexes, distributions, and reported hosts **Species Author**  **Species com** **plex Reported distribution**  **Rep orted hosts**  howardii Dalla Torre,

1898 abdominalis California (Howard, 1895) Acyrthosiphon pisum (Herting, 1972) ; Macrosiphum euphorbiae (Sullivan and van den Bosch, 1971) ; Illinoia liriodendri (Zuparko and Dahlsten, 1993) jucundus Gahan, 1924 abdominalis California (Gahan, 1924) ; New York (Griswold, 1926) Acyrthosiphon malvae (Griswold, 1926) ; Acyrthosiphon pseudodirhodum (Griswold, 1927) ; Aulacorthum solani (Herting, 1972) ; Macrosiphum euphorbiae (Gahan, 1924) ; Myzus persicae (Griswold, 1927) semiflavus Howard, 1908 asychis Colorado (Howard, 1908) ; Indiana (Hartley, 1922) ; Kansas, Oklahoma (Kelly, 1917) ; Minnesota, New Mexico (Webster and Phillips, 1912) ; New York (Griswold, 1927) ; Ohio (Girault, 1917) Acyrthosiphon pisum , Aphis gossypii , Cerusaphis viburnicola, Rhopalosiphum maidis, Macr osiphoniella sanborni

, Macrosiphum avenae (Hartley, 1922) ; Aphis rumicis, Brevicoryne brassicae, Lipaphis pseudobrassicae, Sitobion avenae (Peck, 1963) ; Chaitophorus vim inalis, Schizaphis graminum (Webster and Phillips, 1912) ; Myzus persicae (Howard, 1908) ; Myzaphis rosarum (Peck, 1963) marlatti Ashmead,

1888 asychis Kansas (Ashmead, 1888b) ; Ontario ; Quebec (Peck, 1963) Aphis sp. (Ashmead, 1888b) ; Myzus persicae (Herting, 1972) lapisligni Howard, 1917 mali Oregon (Howard, 1917) ; Idaho (Smith, 1923) ; Washington (Smith, 1923) Brachycaudus helichrysi (Peck, 1963) ; Nearctaphis bakeri (Howard, 1917) mali Haldeman,

1851 mali Arkansas (Becker, 1918) ; Colorado (Gillette and Taylor, 19 08) ; District of Columbia, Illinois, Missouri (Howard, 1881) ; Kansas (Lohrenz, 1911) ; Nebraska (Bruner, 1894) ; Pennsylvania (Cresson, 1862) Eriosoma americanum (Girault, 1909) ; Eriosoma crataegi (Howard, 1881) ; Eriosoma lanigerum (Haldeman, 1859)
prociphili Carver, 1980 mali Iowa (Carver, 1980) Prociphilus fraxinifolii (Carver, 1980) sanborniae Gahan, 1924 mali Penns ylvania (Gahan, 1924) ; Ohio (Delong and Jones, 1926) Sanbornia juniperi (Gahan, 1924) ; Nasonovia houghtonensis (Delong and Jones, 1926) siphonophorae A shmead,

1888 mali Florida (Ashmead, 1888a) ; Ne w Jersey (Peck, 1963) Macrosiphum sp. (Ashmead, 1888a) nigritus Howard, 1908 varipes South Carolina (Howard, 1908) ; Kansas, Minnesota (Webster and Phillips, 1912) ; Ne w Mexico (Webster, 1909) ; Oklahoma (Jackson et al., 1970) ; Texas (Archer et al., 1974) Schizaphis graminum (Howard, 1908) ; Hysteroneura setariae (Webster and Phillips, 1912) ; Aphis helianthi (Rogers et al., 1972) ; Rhopalosiphum maidi (Jackson et al., 1970)
aureus Gahan, 1924 subgenus Mesidia California (Gahan, 1924) Chaitophorus nigrae (Gahan, 1924) automatus Girault, 1911 subgenus Mesidia Illinois (Girault, 1911) ; Virginia (Girault, 1916) ;

California (Zuparko and Dahlsten, 1995) Chaitophorus sp. (Girault, 1911) ; Hysteroneura setariae (Girault, 1916) ; Eucalli

pterus tiliae (Zuparko and Dahlsten, 1995)**24**

gillettei Howard, 1914 subgenus Mesidia Colorado (Howard, 1914) Diuraphis tritici (Howard, 1914) perpallidus Gahan, 1924 subgenus Mesidia Iowa (Gahan, 1924) ; Alabama (Edelson and Estes,

1987) ; California (Peck, 1963) ; Florida (Mizell and Sc hiffhauer, 1990) ; Texas (Watterson and Stone, 1982) Chromaphis juglandicola (Pe ck, 1963) ; Melanocallis fumipennellus, Monellia costalis (Peck, 1963) ; Monellia caryella (Bueno and Stone, 1983) ; Monelliopsis pecanis (Edelson and Estes, 1987)**25** 

## Ap **pendix B**  **. Host**  **specificity tests for Aphelin**  **u s**  **rhamni (Hopper 2011; 2012)**  **.**
**Parasitism in 24 -** **hour exposure of aphids on plant.** Individ ual female parasitoids were exposed to one of 12 aphid species. One to five day old females were used that had been with males and aphids since emergence and thus had the opportunity to mate, host
- feed, and oviposit. F emales were isolated from males and ap hids for 24 hours before using them in experiments to insure that the females had a full egg load. E ach female parasitoid was placed in a cage (10 centimeters (cm) diameter b y 22 cm tall) enclosing the foliage of a potted plant of the appropriate species with 100 aphids of mixed instars of a single species. Female parasitoids were removed after 24 hours and were used only

once. Because these parasitoids can parasitize a maximum of 20 aphids in 24 hours, this abundance of aphids and period of exposure allow ed parasitoids to use their full egg complement. Furthermore, the density of aphids, amount of plant material, and cage size meant that parasitoids were not limited by search rate. Paras i tism scoring occurred after larval parasitoids killed and mummified their hosts. Therefore, a combination of acceptance of hosts for oviposition and suitability of hosts for parasitoid development were measured
. Female parasitoids had the choice to oviposit or not to oviposit in a particular host species. This is frequently

the choice parasitoids make in the field. The purpose of this study was to measure parasitism in an environment that appears to harbor only one aphid species on only one plant species and where female parasitoids re
- encounter this combination repeatedly starting with a full egg complement. Ten day s after exposure of aphids to parasitoids, any mummified aphids were collected and held for adult parasitoid emergence. After the adults emerged, the number of mummified aphids and the number and sex of adult parasitoids were recorded
. Eight to 32 females on each of 12 aphid species were tested
. After 24 hours exposure, the number of aphids mummified by A. rhamni varied with aphid species ( F = 13.1; df = 12, 213; P < 0.0001; Fig. 3). Female parasitoids rarely or never parasitized aphids outside the genus Aphis ; within the genus Aphis , female parasitoids produced mummified aphids on species phyl ogenetically close to A. glycines , e.g. A. gossypii , A. monardae , and A. oestlundi , but not on aphids that are more phyl ogenetically distant, e.g. A. nerii and A. rumicis . The low numbers of mummified aphids for A. nerii may have resulted from their host plant, A. syriaca, which is known to produce toxic secondary chemicals which aphids could sequester for their defense (Mooney et al., 2008, Warashina and Noro, 2000). This effect is supported by many fewer mummies for A. gossypii on A. syriaca than on G. hirsutum (Fig. 3). The proportion of mummies from which parasitoid adults emerged was generall y high (0.93±0.01) but varied among species ( F = 4.6; df = 8, 67; P = 0.0002) because A. oestlundi had a lower adult emergence rate (0.64±0.01) than the other aphids. Sex ratio of parasitoid progeny**26**

did not differ from equality (0.52±0.02) and did not differ among aphid species


### .

Direct observations of behavior in experiments on the stresses of starvation and age, egg load, and experience affecting acceptance of low versus high quality host species for oviposition by Aphelinus rhamni found f emale parasitoids may

behave differently

after encountering low versus high quality hosts for longer periods. Starved Aphelinus rhamni females persisted in ovipositing more in high
- quality

aphid species than in low quality aphid species
. Females oviposited about twice as often in Aphis glycines than in A. craccivora or R. padi ( F = 5.8; df = 2,128; P = 0.004). Starved and fed females laid about the same numbers of eggs (1.0 ± 0.2 versus 1.1 ± 0.2, respectivel y), and there was no interaction between nutrition and aphid species in their effect on oviposition. Aphid species and egg load did interact in their effect on oviposition ( F = 5.9; df = 3,124; P = 0.0009). Honey
- fed parasitoids exposed to A. glycines oviposited more when they carried more eggs, but oviposition in the other aphid species did not increase with egg load, and increased onl y slightly in water
- fed females exposed to A. glycines . This pattern is the opposite of what one would expect if female parasitoids became choosier as eggs became more limiting. The higher oviposition in A. glycines did not result from stinging this species more often. Indeed, female parasitoids stung A. craccivora more often than A. glycines , which was stung at the about the same rate as R. padi (6.3±0.4, 4.1±0.3, and 3.7±0.3 aphids, respectivel y; F**27**

= 17.3; d f = 2,156; P < 0.0001), although A. glycines was oviposited in twice as often as the other aphid species. The lack of correlation between stinging and oviposition, probabl y results because Aphelinus females usually do not directly contact aphids before ovipositor insertion, and thus are may not assess host quality until aphids are stung. On encountering a potential host, Aphelinus rhamni females stop at about half their body length away from the aphid. Without touching the aphid, females sway from side to s ide several times with antennae extended (an approach). Stinging did not vary with nutrition, egg load, nor did these factors interact with aphid species in its effect on numbers of aphids stung. **Effect of age on oviposition in low versus high quality hos ts.**  Ten
- day old Aphelinus rhamni females persisted in ovipositing more in a high quality aphid species than in a low quality aphid species. Females oviposited over twice as often in A. glycines than in R. padi ( F = 13.0; df = 1,76; P = 0.0006). Young and o ld females laid about the same number of eggs (1.1 ± 0.2 versus 0.9 ± 0.2 eggs, respectivel y), and there was no interaction between parasitoid age and aphid species. As in the nutrition experiment, there was a strong interaction between aphid species and egg load in their effect on oviposition ( F = 6.6; df = 3,125; P = 0.0002). Young parasitoids exposed to A. glycines oviposited more when they carried more eggs, but oviposition did not increase significantl y with egg load in parasitoids exposed to R. padi or in older parasitoids exposed to A. glycines . As in the nutrition experiment, the preference for high quality hosts persisted in the face of low versus high egg load. At the start of observations, young females carried about 3 more eggs than older female s (9.5 ± 0.9 versus 6.2 ± 0.6, respectivel y; F = 453.3; df = 2,77; P < 0.0001), and b y the end of observations, both young and old females still had eggs they had not oviposited (8.0 ± 0.8 and 5.3±0.6, respectivel y). The higher oviposition in A. glycines was partl y a result of stinging this aphid species more often. Unlike the starvation experiment, where A. glycines and R. padi where stung equall y often, female parasitoids in this experiment stung A. glycines slightly more often than R. padi

. **Effect of age on oviposition in low versus high quality hosts.**  Ten
- day old Aphelinus rhamni females persisted in ovipositing more in a high quality aphid species than in a low quality aphid species. Females oviposited over more than twice as often in A. glycines than in R. padi ( F = 13.0; df = 1,76; P = 0.0006). Young and old females laid about the same number of eggs (1.1± 0.2 versus 0.9 ± 0.2 eggs, respectively), and there was no interaction between parasitoid age and aphid species. As in the nutrition experiment, there was a strong interaction between aphid species and egg load in their effect on oviposition ( F = 6.6; df = 3,125; P = 0.0002). Young parasitoids exposed to A. glycines oviposited more when they carried more eggs, but oviposition did not increase signi ficantl y with egg load in parasitoids exposed to R. padi or in older parasitoids exposed to A. glycines . As in the nutrition experiment, the preference for high quality hosts persisted in the face of low versus high egg load. At the start of observations, young females carried about 3 more eggs than older females (9.5 ± 0.9 versus 6.2 ± 0.6, respectivel y; F = 453.3; df = 2,77; P < 0.0001), and b y the end of observations, both young and old females still**28**

had eggs they had not oviposited (8.0 ± 0.8 and 5.3±0. 6, respectivel y). The higher oviposition in A. glycines was partl y a result of stinging this aphid species more often. Unlike the starvation experiment, where A. glycines and R. padi where stung equally often, female parasitoids in this experiment stung A. glycines slightly more often than R. padi (5.6±0.4 versus 4.4±0.3, respectively ; F = 5.3; df = 1,77; P = 0.02).However, this difference was onl y 1.3 fold more stings in A. glycines than R. padi , whereas oviposition was 2.5 fold greater in A. glycines th an in R. padi . Stinging did not vary with age, egg load, nor did these factors interact with aphid species in its effect on numbers of aphids stung. The frequency of host feeding did not vary with age, but parasitoids fed on A. glycines more frequentl y than on R. padi (32 percent versus 6 percent of parasitoids fed on each species; likelihood ratio χ 2 = 8.2; n = 74; P = 0.003), and this difference was similar to that for honey
- fed parasitoids in the nutrition experiment of the same age. Host feeding did not vary with egg load for either aphid species, age, or their c ombinations. Dif ferences in aphid defense cannot explain differences in oviposition between species because A. glycines defended itself as much as R. padi (2.3 ± 0.3 versus 2.1±0.3 defensive behaviors per approach), but received over twice as many eggs. Amount of aphid de fense behavior did not vary with aphid species, age, or their interaction. Both young and old Aphelinus rhamni females produced more progeny on high quality host species than on low quality host species when exposed to the aphids for 24 hours
. **Conclusion s.**  Stresses that decreased life expectancy did not increase acceptance of low quality host species b y Aphelinus rhamni . Egg load was also lower in stressed females, which resorbed eggs in response to starvation and aging, so changes in life expectancy and egg load may

have counterbalanced one another. However, the decrease in egg load was small relative to the decrease in life expectancy , particularly for starved females, which carried about three
- quarters of the egg load of honey
- fed females, but would die the day following the assay s. Furthermore, although oviposition increased with egg load for females exposed to A. glycines , oviposition did not vary

with egg load for females exposed to low quality aphid species. The outcome of this pattern was that females became less choosy as egg load decreased, rather than becoming choosier , as theory predicts. Besides having lower life expectancy , older females were kept without aphids as they aged so they

experienced a more aphid
- poor environment than did yo ung para sitoids. It was expected this would make parasitism of low quality aphids by

older females more frequent, yet it did not. These results demonstrate that A. rhamni specificity is behaviorall y stable rather than dy namic, and low
- quality

non - target species sh ould not have increased risk of attack when parasitoids are stressed from starvation or age or when they accumulate eggs.