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I. Regulatory Authority

Applications for importation, interstate movement, and/or release into the environment of genetically engineered (=transgenic) arthropods (and other invertebrates) are submitted to the Animal and Plant Health Inspection Service (APHIS), U.S. Department of Agriculture (USDA) under the authority of the Federal Plant Pest Act (FPPA) and the Plant Quarantine Act (PQA). These statutes confer very broad authority to the Secretary of Agriculture to prevent the introduction or dissemination of plant pests into the United States or between states. The Federal regulations pursuant to the FPPA and the PQA that govern the introduction of transgenic organisms (7 CFR 340) authorize APHIS to regulate the “introduction of organisms and products altered or produced through genetic engineering which are plant pests or which there is reason to believe are plant pests.” An organism is considered to be regulated if it is genetically engineered (as defined in 7 CFR 340.1) from a donor organism, recipient organism, or vector or vector agent that is listed in 7 CFR 340.2 and/or meets the criteria of plant pest; i.e., if the organism “can directly or indirectly injure, or cause disease, or damage in any plants or parts thereof, or any processed, manufactured, or other products of plants.”

Applications for a permit are first reviewed to determine the regulatory status of the proposed organism; that is, whether the organism is included in the definition of a “regulated article” (see above). Secondly, the application is reviewed for possible invoking of regulations under the National Environmental Policy Act (NEPA). The APHIS NEPA Implementing Procedures (7 CFR 372) categorically exclude from NEPA consideration both the permitting of confined field releases of genetically engineered organisms and the permitting of interstate movement of nonindigenous (i.e., genetically engineered) species between containment facilities. For the particular application under consideration, this regulatory assessment (RA) has been prepared, which outlines APHIS regulatory authority, the biology of the specific organism, and the possible risk to the environment.

II. Proposed Importation and Interstate Movement

APHIS has received an application from the USDA-APHIS-Plant Protection and Quarantine (PPQ)-Phoenix Plant Protection Center (Dr. Robert T. Staten, Principal Investigator) requesting a permit authorizing interstate movement from Riverside, California to Phoenix, Arizona, of a transgenic pink bollworm, Pectinophora gossypiella (Lepidoptera: Gelechiidae). This application (98–244–02m) is viewable on the APHIS web page at http://www.aphis.usda.gov/biotech/arthropod/permits/9824402m/9824402m.html . The review and evaluation of the application, and any subsequent permit issuance, will be performed by Scientific Services (SS), PPQ.

The proposed interstate movement involves multiple shipments of transgenic moths during 1999–2000 from a research facility in California (University of California at Riverside) to a research facility in Arizona (APHIS Phoenix Plant Protection Center). The transgenic P. gossypiella would contain genetic material from a jellyfish, Aequoria victoria, and additional gene components from a pomace fly, Drosophila melanogaster. The gfp (green fluorescence protein) gene obtained from A. victoria is a marker encoding for fluorescence; portions of the transgenic moth with the gfp gene would produce a green fluorescence when excited by blue light (485 nm) and viewed at 510 nm. Such a marker would allow quick and simple screening to identify transformed individuals. The gene fragments added from D. melanogaster consist of the hsp70 promoter that controls the encoding of the vector (piggyBac) transposase.

The molecular biology and methodology associated with the development of the transgenic form of P. gossypiella, and associated reference citations, are extensively detailed in the application (98–244–02m). The application is incorporated by reference into this RA and is attached as Appendix A. The reader is referred to Appendix A for presentation and discussion of the biological characteristics of the transgenic form of P. gossypiella, as well as descriptions of the shipment and containment conditions.

III. Assessment

An initial review of the application was conducted to determine jurisdiction under the FPPA and the PQA; that is, whether this particular transgenic organism presented a risk of plant pest introduction. APHIS regulates similar nontransgenic organisms under regulations at 7 CFR 330.200 and transgenic organisms under regulations at 7 CFR 340.

Under 7 CFR 340, there are four criteria to determine regulatory status that are applied to each transgenic organism proposed for import/movement/release: (1) is the donor (the organism contributing genetic material) a plant pest, (2) is the vector entity (the vehicle for transfering genes from donor to recipient) derived from a plant pest, (3) is the recipient (the organism that will receive the genetic material) a plant pest, or (4) is there reason to believe that the final genetically engineered recipient organism has been altered such that it will pose a plant pest risk. A positive response to any of the four criteria would invoke regulatory oversight.

The donor organisms A. victoria and D. melanogaster are not considered plant pests and have not been previously regulated by APHIS under 7 CFR 330. The piggyBac vector is found in a number of arthropod species and in this case was acquired from a plant pest, Trichoplusia ni (Lepidoptera: Noctuidae), the cabbage looper. The recipient organism, P. gossypiella, is an economically important pest of cotton in the southwestern United States. Under current APHIS procedures relating to the release of nontransgenic moths, the nontransgenic (recipient) P. gossypiella would be considered a plant pest and thus would be a “regulated article” under 7 CFR 330. The transgenic form, under 7 CFR 340, would also be considered a “regulated article” due to the pest status of the recipient organism and the pest status of the organism from which the vector entity was derived.

A review under 7 CFR 340 of the biological characteristics of the transgenic moth P. gossypiella (see Appendix A) indicates that this organism poses no greater risk of becoming a plant pest than the nontransgenic form for the following reasons: (1) the green fluorescence marker gene in transformed moths confers no known advantage or disadvantage to the individual; (2) the piggyBac vector has been demonstrated to not be capable of transposition in the absence of exogenous piggyBac transposase; i.e., transgenes are expected to be stable; and (3) under conditions of shipment and subsequent laboratory containment, escape of the transformed moths is unlikely. Termination of laboratory experiments will be accompanied by freezing at -20º C, ensuring nonsurvival of the transformed moths. If the transgenic form of P. gossypiella did somehow escape from containment, additional plant pest risk is unlikely, given the presence of abundant populations of P. gossypiella in the immediate vicinity. This is also true if an escaped transgenic form somehow reverted to its original nontransgenic form.

On the basis of this assessment, APHIS concludes that the proposed interstate movement of a transgenic P. gossypiella between containment facilities presents no risk of the organism becoming a plant pest additional to that of the nontransgenic form. Because the organism is a regulated article under the FPPA, APHIS will issue a standard movement permit with associated conditions designed to ensure that the organism is safely moved from Riverside to Phoenix and that the Phoenix facility adequately ensures containment.

IV. Preparation and Review

This regulatory assessment (RA) was prepared by Dr. Orrey P. Young, Team Leader–Arthropods; Biotechnology Evaluation, Scientific Services, Plant Protection and Quarantine, Animal and Plant Health Inspection Service. He was assisted in the preparation by staff at the applicant facility. The RA was reviewed and modified by several members of the APHIS Transgenic Arthropod Team (see Appendix B for composition of the team). Unsolicited communications concerning the application were also received from the general public after the text of the application was placed on the APHIS-SS-BE web page [8 September 1998], and these comments were considered in the preparation of the RA.

Appendix A

Text of Application No. 98–244–02m Dr. Robert T. Staten, USDA-APHIS-PPQ, Phoenix, AZ, on APHIS-BBEP Form 2000, Application for Permit or Courtesy Permit under 7 CFR 340 (Genetically Engineered Organisms or Products).

Appendix B

APHIS Transgenic Arthropod Team
    (http://www.aphis.usda.gov/biotech/arthropod)

Orrey P. Young, Ph.D.
Environmental Protection Officer; Ecologist
Biotechnology Evaluation–PPQ–SS

Ved S. Malik, Ph.D.
Biotechnologist; Molecular Biologist
Biotechnology Evaluation–PPQ–SS

L. Joseph Vorgetts, Ph.D.
Biological Scientist; Monitoring Specialist
Technical and Scientific Services–PPD

Kenneth R. Lakin, Ph.D.
Entomologist; Risk Analyst
Center for Plant Health–PPQ

Michael J. Firko, Ph.D
Entomologist; Ecological Geneticist
Biological Assessment and Taxonomic Support–PPQ

Ralph D. Stoaks, Ph.D.
Biological Control/Biotechnology Operations Officer
Western Region–PPQ

Don C. Vacek, Ph.D.
Biological Scientist; Population Geneticist
Methods Development–PPQ

Glen Garris, Ph.D.
Medical/Veterinary Entomologist/Acarologist
Veterinary Services

Michael J. Oraze, Ph.D.
Biological Scientist; Biological Control
National Biological Control Institute–PPQ

Douglas C. Prasher, Ph. D.
Molecular Biologist
Methods Development PPQ

Lauren Jones, B.S.
Writer/Editor; Data Management Specialist
PPQ–SS

Interstate Movement of a Transgenic Pink Bollworm [98–244–02m]
[1 September 1998]

1. NAME AND ADDRESS OF APPLICANT

   Dr. Robert T. Staten
   U.S. Department of Agriculture
   Phoenix Plant Protection Center
   4125 East Broadway Road
   Phoenix, AZ 85040
   

2. PERMIT REQUESTED

   Limited—Interstate Movement, NOT FOR RELEASE

3. THIS REQUEST IS

   NEW

4. TELEPHONE

   602–379–6014, x222 VOX

   MEANS OF MOVEMENT

   Mail and or Common Carrier

5. ORGANISM INFORMATION

   Trade Name	Scientific Name	Common Name
   a. Donor	Aequora victoria	jellyfish
   b. Recipient	Pectinophora gossypiella	pink bollworm
   c. Vector	piggyBac	—
   d. Regulated organism or product	Pectinophora gossypiella	pink bollworm
   e. If product, list names of constituents	—	—

6. QUANTITY OF REGULATED ARTICLE TO BE INTRODUCED AND PROPOSED SCHEDULE AND NUMBER OF INTRODUCTIONS

   Shipments as necessary (no more than weekly) of up to 200 transgenic eggs, larvae, and pupae starting immediately upon receipt of permit.

7. DATE (OR INCLUSIVE DATES OF PERIOD) OF RELEASE

   NO RELEASE INTENDED. First shipment is planned for November 1, 1998, (or upon receipt of permit) and will continue until permit expires in 1 year.

8. COUNTRY OR POINT OF ORIGIN OF THE REGULATED ARTICLE

   United States. The original stocks of the pink bollworm (P. gossypiella) were derived from the Pink Bollworm Rearing Facility, USDA-APHIS-PPQ, 3645 East Chipman, Phoenix, AZ 85040. The insects shipped to the Phoenix Plant Protection Center will be transformed at the University of California in Riverside from this original stock.

9. SPECIFIC LOCATION OF RELEASE

   NO RELEASE INTENDED. Transgenic pink bollworms will be shipped to the USDA, Phoenix Plant Protection Center at 4125 East Broadway Road, Phoenix, AZ 85040. These insects will be reared in Phoenix and used to form a colony for laboratory research purposes, not for release.

10. ANY BIOLOGICAL MATERIAL ACCOMPANYING THE REGULATED ARTICLE DURING MOVEMENT

    An agar and wheat germ based pink bollworm diet will accompany the transgenic larvae. No material will accompany the transgenic eggs or pupae fragments.

11. APPLICANTS FOR A COURTESY PERMIT

    NO

12. ENCLOSURES

    See below.

13. SIGNATURE OF RESPONSIBLE PERSON

    /s/ Robert T. Staten

14. PRINTED NAME AND TITLE

    Dr. Robert T. Staten, Center Director

15. DATE

    August 20, 1998

ENCLOSURE A

Dr. Thomas A. Miller
Professor of Entomology
Department of Entomology
University of California
Riverside, CA 92521
909–787–3886

Dr. John J. Peloquin
Assistant Research Entomologist IV
Department of Entomology
University of California
Riverside, CA 92521
909–787–4680

Dr. Robert T. Staten
Center Director
Phoenix Plant Protection Center
4125 East Broadway Road
Phoenix, AZ 85040
602–379–6014, x222

Dr. Fred D. Stewart
Facilities Director
Pink Bollworm Rearing Facility
3645 East Chipman
Phoenix, AZ 85040
602–379–4828

ENCLOSURE B

The additional genetic material in the pink bollworm allows the expression of a modified version of the GFP (Green Fluorescent Protein) derived from the jellyfish Aequora victoria. The transgenic pink bollworm with the GFP gene fluorescences strongly green when viewed at 510 nm with a 460 to 500 nm illumination. GFP confers no known competitive advantage or disadvantage to the recipient, and no ecological or other consequences resulting from incorporation of this marker into the transgenic pink bollworm can be envisioned. The nonmodified pink bollworm has no GFP gene; therefore, it does not fluorescence strongly green when illuminated under the same light frequency. No piggyBac transposase activity nor any antibiotic resistance is conferred to the transgenic pink bollworm by the introduced genetic material.

ENCLOSURE C

PiggyBac is a DNA (deoxyribonucleic acid) transposable element that, only when its ITR (Inverted Terminal Repeats) are intact, is capable of integrating DNA flanked by element-specific DNA into other DNA through mediation of a transposase encoded by an ORF (Open Reading Frame) within the element. In the construct used for transformation of the pink bollworm, the transposase gene of the piggyBac element was irreversably destroyed by insertion of the GFP gene. Transformation was effected by introducing with the transforming construct a helper plasmid that supplied transposase activity but was itself unable to transpose into other DNA. This transposition-defective helper plasmid has an ORF (Open Reading Frame) encoding piggyBac transposase under the control of the Drosophila melanogaster hsp70 promoter. One of the inverted terminal repeats that flank the wild-type piggyBac transposase in piggyBac has been removed in the helper plasmid so that the helper plasmid cannot, itself, integrate even though it encodes for active piggyBac transposase.

The potential for instability and unwanted mobilization of piggyBac-derived transforming constructs must be addressed as follows. It could be argued that if there were endogenous, piggyBac-like elements in pink bollworm, they might provide a source of transposase that could mobilize transgenes flanked by piggyBac-derived ITRs. Demonstration of elements homologous to piggyBac in the recipient organism, pink bollworm, might then suggest caution regarding stability of the transgene. However, the DNA-mediated element, hermes, has been used to successfully transform Aedes aegypti with little or no evidence of instability of the transgenes over at least 10 generations, even though there are in Aedes aegypti endogenous elements (presumably hAt-like as is hermes) with close enough homology to hermes so that these endogenous hAt and hermeslike elements are detected even in higher stringency Southern blots with a hermes probe (Jasinskiene et al 1998, PNAS 95:3743–3747).

In the case of pink bollworm, low stringency Southern blot experiments on pink bollworm DNA with radiolabled DNA probes derived from piggyBac, which would be even more likely to detect elements with low homology to piggyBac than the higher stringency methods used in Jasinskiene, et al., 1998, were unable to detect any endogenous piggyBac-like elements. This suggests that there are no elements in pink bollworm that might reasonably be expected to mobilize a piggyBac-derived transgene. In addition, excission and transposition assays were performed in pink bollworm embryos with piggyBac. This was primarily to determine if piggyBac could integrate into the pink bollworm genome. However, our results showed no transposition of piggyBac in the absence of exogenous piggyBac transposase in these transposition assays, strongly suggesting there were no unknown elements in the pink bollworm genome. We can thus be reasonably certain there would not be unexpected interactions between the components of the pink bollworm genome and the transforming construct that would result in instability of the transgenes. In any event, experiments to be performed in Phoenix after transfer of the transformed pink bollworm strains will further demonstrate the stability of the transgenes.

ENCLOSURE D

United States, Riverside County, Riverside, California, University of California at Riverside is where all final engineering of the transforming constructs were performed. The genes used from the donor organism and the piggyBac-derived portions of the vectors used to build the transforming construct were cloned off site. Specifically, E. coli was the immediate host for the plasmids carrying the cloned genes used to make the transforming constructs.

The recipient organism—the pink bollworm, Pectinophora gossypiella—is an old world species whose origin is uncertain. It is not a native species of the Western Hemisphere though it is now endemic to the southwestern United States and Mexico, associated with commercial cotton production. Introduction of the pink bollworm into the United States appears to have been via infected cottonseed. The pink bollworm appeared in Hearn, TX in 1917, and within a decade it had spread across western Texas, New Mexico, and into Arizona by 1929. The colony transformed at University of California in Riverside originated from the Pink Bollworm Rearing Facility in Phoenix, Arizona.

ENCLOSURE E

The goal is to create a marked strain of pink bollworm with the GFP gene and to introduce this strain into the Pink Bollworm Rearing Facility (PBWRF) in Phoenix, AZ for onsite experimentation only and not for release. A genetically marked insect can be distinguished from a native pink bollworm by screening with a fluorescent microscope and/or with PCR (Polymerase Chain Reaction).

ENCLOSURE F

Shipment of transgenic pink bollworms will be in shatter-resistant plastic tubes containing diet, which are capped and sealed with tape. The tubes will be contained further in a cardboard box lined with styrofoam, filled with packing material, and the box closed and sealed with tape. The box will be labeled as to its contents, origin, destination, and contact telephone numbers. All shipments will be by overnight mail using the following protocol.

ENCLOSURE G

Upon arrival in Phoenix, the shipment of insects will be opened inside the Pink Bollworm Genetic Rearing Facility. The facility consists of a 12' X 40' trailer with 3 self-contained rearing rooms. Each room has a separate self-closing door with no windows in any room. All rooms are individually environmentally controlled with separate Sanyo ductless air conditioning and heat pump units. Access to this Facility is limited to authorized personnel only, with keys issued to authorized staff members only.

All transgenic insects are reared in Precision Instruments Growth Chambers Model 818. Each unit features a 17.8-cubic-foot rearing capacity with top mounted microprocessor-based controls, fully gasketed door with key lock. The growth chambers are locked at all times with access to keys limited. At this time there are no plans for release, dissemination, distribution, field trials, or sale of these insects. The received insects will be used to populate a laboratory colony that will be used for research purposes within the laboratory in cages under restrictive access conditions.

ENCLOSURE H

The only destination for the shipments of the transgenic pink bollworm insects will be to the Pink Bollworm Genetic Facility. As mentioned in item 13 G above, the Facility can be accessed by authorized personnel only, and access to keys is strictly limited. As a further safeguard, all insects are reared in locked Precision Instrument Growth Chambers, with access to keys limited.

ENCLOSURE I

Any insects that are no longer needed will be disposed of by freezing at -20 C for 24 hours. This will destroy any life stage of this insect.

REFERENCE

Jasinskiene, N.; Coates, C.J.; Benedict, M.Q.; Conrel, A.J.; Rafferty, C.S.; James, A.A.; Collins, F.H.: Stable transformation of the Yellow Fever Mosquito, Aedes aegypti, with the hermes element from the housefly. Proceedings of the National Academy of Sciences of the United States of America, 1998 March 31, 95(7):3743–7.