2005 Annual Report 1.What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? What does it matter? Researchers, insectary managers, and those involved in the mass production of insects to be used in control programs, have been looking for assistance for extending the shelf life of insects and devising methodology for storage. One of the expenses ancillary to the USDA APHIS programs involved in rearing insects for pest control purposes, as with the SIT (sterile insect technique), is the development and continued maintenance of back-up strains. Back up strains are typically reared from 1 3 years before they are use d as a production strain. Methods for the long term storage of insect germplasm would ensure the availability of a new strain for introduction into the production line that had not experienced a reduction in field efficiency caused by inadvertent selection through extended laboratory rearing. The current means of preserving most insects is by continuous culture. Not only can continuous culture be a costly activity, it can effect genetic drift and is subject to accidental loss of colonies, genetic strains and transformants. Two approaches, cryopreservation and dormancy, are being investigated to enable insects to survive a cold storage period. Cryopreservation involves combined chemical and physical manipulations of cells and organisms to enable long term storage at liquid nitrogen temperatures. Using dormancy as a cold storage method necessitates that the organisms be prepared physiologically and also developmentally competent to survive extended low temperature exposure. Successful dormancy induction often requires that precise manipulation of environmental conditions be made during the mass rearing regime. 2.List the milestones (indicators of progress) from your Project Plan. Objective 1. Develop and/or improve cold storage protocols for insects. FY 2005-2009 Sub-objective 1.1 Long-term cryogenic storage of parasitic wasps. FY 2006-2008 Assessing parasitoid development. FY 2006 Timing parasitism of the host. FY 2007 Employ & optimize cryopreservation protocol. FY 2008 Sub-objective 1.2 Develop a cryopreservation protocol for Heliothine moth embryos that will allow long-term storage. FY 2005-2007 Permeabilize egg membranes. FY 2005 Synchronize embryo development. FY 2005 Determine CPA embryo tolerance. FY 2006 Optimize protocol & recovery procedure. FY 2007 Sub-objective 1.3 Investigate the use of cryopreserved pre-blastoderm nuclei blastomeres, and/or primordial germ cells as useful germplasm storage alternatives. FY 2005-2007 Assess incidence of chimerism. FY 2005 In situ germplasm cryopreservation. FY 2006 Germline reconstitution and clone production. FY 2007 Sub-objective 1.4: Develop stockpiling procedures at subambient temperatures for three hymenopterous species of GWSS egg parasitoids. FY 2005-2007 Determine parasitoid cold tolerance. FY 2005-2007 Sub-objective 1.5: Increase cold tolerance of the GWSS parasitoids by bio-fortification of the diet fed to the adult wasps. FY 2008-2009 Feed and evaluate antioxidant compounds. FY 2008-2009 Analyze enzyme levels. FY 2009 4a.What was the single most significant accomplishment this past year? Storage of host eggs for parasitoid propagation. The USDA/ARS Biosciences Research Laboratory and the Department of Entomology at North Dakota State University collaborated on a project which demonstrated that eggs of the glassy-winged sharpshooter killed by a 5 day low temperature exposure were utilized by the parasitoid, Gonatocerus ashmeadi, for production of their progeny after refrigerated storage for more than 60 days. These findings aid the mass-propagation of this egg parasitoid by enabling insectary managers to accumulate large numbers of insects for use in innudative releases as a control strategy. G. ashmeadi is one of the egg parasitoids being tested for use as a biological agent to support the control program for the glassy-winged sharpshooter which vectors Pierce’s disease to a number of agriculturally-important crops in the western U.S. 4b.List other significant accomplishments, if any. 25,000 screwworm embryos cryopreserved and archived at NCGRP. Personnel from the USDA/ARS Biosciences Research and the Midwest Livestock Insects Laboratories collaborated on a project where nearly 25,000 screwworm embryos were cryopreserved using the technique developed by ARS and ND State University scientists. Closing of the ARS quarantine facility for screwworm research in Lincoln, NB precipitated this effort and it resulted in saving 10 of 15 screwworm strains from being otherwise unavoidably discarded. The vitrified embryos were transported in liquid nitrogen to the USDA/ARS National Center for Genetic Resources Preservation in Ft. Collins, CO where they are currently stored. The strains included mutant marker, transgenic and progenitors of current factory-reared strains that can be transported to, and revived at ARS laboratories equipped to handle quarantined insects for research purposes. Functional response of egg parasitoid to its host characterized. USDA/ARS Biosciences Research Laboratory and the Department of Entomology at North Dakota State University collaborated on a project which involved characterizing the response and superparasitism by the egg parasitoid, Gonatocerus ashmeadi, when offered various ages and densities of glassy-winged sharpshooter eggs. The attack rate is higher on 1-day-old hosts than other ages and the parasitoid has a greater tendency toward superparasitism at parasite-to-host ratios of 4c.List any significant activities that support special target populations. None. 4d.Progress report. (1) This report serves to document the research conducted under a specific cooperative agreement between ARS and the North Dakota State University Department of Entomology (5442-22000-040-04S). The title of the project is "Long-term cold storage of caribfly and medfly germplasm." Additional details of this research area can be found in the report for the parent CRIS 5442-22000-040-00D, "Development of Cold Storage Technology for Mass-reared and Laboratory-colonized Insects". Insect embryonic stem cell cryropreservation. As an alternative to embryo cryopreservation and in vitro fertilization, primordial germ cell and blastoderm nuclei transplantation studies were conducted with Caribbean fruit fly embryos. The effects of removal and transplantation of blastoderm nuclei and primordial germ cells between embryos were assessed. These studies were conducted with normal wild-type embryos and embryos bearing a GFP or RFP (green and red florescent protein) genetic markers to distinguish the success of the procedure. Chimeras bearing the genetic background of FP-labeled flies and also wild-type genes were constructed by reciprocal transplantation of blastoderm nuclei. Cryopreservation of the blastoderm nuclei or primordial germ cells by in situ freezing within the injection micropipettes is being attempted. We are also determining how many totipotent blastoderm nuclei or cells can be removed from individual embryos and still have the embryo proceed to hatching and subsequent development to a normal adulthood. Cryopreservation of biopsied blastoderm nuclei or cells will allow preservation of an insect’s germplasm without sacrificing the insect in the process. Cryopreservation quality assurance using differential gene display. Primary screening for differentially expressed genes was carried out using a modified differential display technique. The majority of the genes showed similar expression in both the cryopreserved and untreated flies. Thirteen PCR amplicons of possible differentially regulated genes were isolated and cloned. The BlastX software was used to screen the GeneBank sequence repository for homology. Putative genes displaying down-regulation were identified as ATP synthase, ribosylation factor, ribosomal protein L-4 and L-18, unspecified ras, growth inhibitor and imaginal disc growth factor 4 genes. Conversely, a gene involved in flight muscle development showed up-regulation in the cryopreserved flies. We are affirming these results by Northern blot analysis and are continuing to screen for other differentially expressed genes in cryopreserved insects. We also intend to examine whether differential expression of these and other genes persist in the subsequent generations. It is hoped that this new method of analyzing post cryopreservation quality will alleviate various doubts about the competence of cryopreserved organisms and/or lead to the improvement of the cryopreservation process. (2) This report represents a reimbursable agreement between ARS and APHIS (5442-22000-040-05R). The details of the research progress of the project funded by this agreement can be found, in part, in the report for the parent CRIS 5442-22000-040-00D entitled "Development of Cold Storage Technology for Mass-reared and Laboratory-colonized Insects" and more specifically in the subordinate CRIS project 5442-22000-0040-07S entitled "Cold storage of parasitized eggs of the glassy-winged sharpshooter, Homalodisca coagulata." (3) This report serves to document the research conducted under a specific cooperative agreement between ARS and the North Dakota State University Department of Entomology (5442-22000-040-07S). The title of the project is "Cold storage of parasitized eggs of the glassy-winged sharpshooter, Homalodisca coagulata." Additional details of this research area can be found in the report for the parent CRIS 5442-22000-040-00D entitled "Development of Cold Storage Technology for Mass-reared and Laboratory-colonized Insects." Cold storage of an egg parasitoid and its host. We are developing cold storage methods to facilitate the mass-rearing of an egg parasitoid that attacks the glassy-winged sharpshooter (GWSS), an insect vector of Pierce’s Disease of plants. The parasitoid shelf-life can be increased by cycling the in-storage temperature for the parasitized GWSS eggs as opposed to holding it constant. When the parasitoids are stored within their hosts under the cycled regime of 4.5, 6.0, and 7.5ºC changing at 8 hr intervals for 10, 20 and 25 days, the emergence of wasps was 66%, 59% and 59%, respectively. However, the chilling sensitivity of the unparasitized GWSS eggs is significantly greater than that of the parasitoids. Holding GWSS eggs at 10ºC for 8 days is lethal, yet hatching occurs within 30 days if storage is at 13ºC. This study shows that killing eggs by placing at 2ºC for 5 days before storage at 10ºC is an acceptable means for preserving hosts for parasitism by the wasps. We determined parasitism rates and progeny emergence from GWSS eggs that developed 1, 3, 5, 7 or 9 days before storage up to 65 days and exposure to wasps. Parasitism and progeny emergence was highest for eggs stored as either 1- or 3-days-old. For these two groups, we also observed that there was a linear negative correlation between parasitism and progeny emergence vs. storage time. Parasitism and progeny emergence remained at acceptable levels for 1-day-old eggs up to 20 days in storage (90 and 76%) but after 65 days they fall to 45 and 28%, respectively. Preliminary quality assessment testing of wasps reared from 3-day-old eggs stored for 55 days showed that the lifespan and fecundity did not differ from that of wasps reared from untreated eggs. Complicating these studies is the situation that plants acceptable as egg-laying sites to the GWSS must be provided and that cuttings from these plants that hold the egg masses must remain viable throughout the cold storage period. Studies designed to determine whether the source of the reduction in host suitability occurring over time in storage is caused by plant or egg host deterioration are continuing. 5.Describe the major accomplishments over the life of the project, including their predicted or actual impact. This is a new CRIS project – (See expired CRIS 5442-22000-038-00D report for FY 2005.) The primary thrust of this research is relevant to the National Program 304 Biology of Pests and Natural Enemies (Microbes), Action Plan Component II, Problem Statement A: Basic Biology and Problem Statement B: Rearing of Insects and Mites. Accomplishments of this project relate to Strategic Plan Goal 1, Objective 1.2, performance measures 1.2.4 and 1.2.5. 6.What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end-user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? The technology relating to cryopreservation of dipteran embryos is available to the end-users (scientists and insectary managers). We have traveled to scientist’s laboratories (ARS- Lincoln, NB; Gainesville, FL and APHIS- Edinburg, TX) instructing personnel on how to cryopreserve screwworms and Caribbean and Mexican fruit flies. Contacts have been made by scientists from the United Kingdom, Brazil and Australia to come to our laboratory next summer to learn our techniques. Adoption of the cryopreservation protocol has been positive when the scientist or technician travels to our laboratory to get “hands-on” instruction or a representative from our laboratory goes to the scientist’s laboratory and gives on site training. The durability of the technology is evidenced by reviving live Mexican fruit flies after they have been stored in liquid nitrogen for more than 2 ½ years. 7.List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: List your peer reviewed publications below). Leopold, R.A. Colony maintenance and mass-rearing: Using cold storage technology for extending the shelf-life of insects. FAO/IAEA International Conference on Area-wide Control of Insect Pests: Integrating the sterile insect and related nuclear and other techniques. May 9-13, 2005, Vienna, Austria. (Oral presentation to scientists and science administrators from 76 countries). Suszkiw, J. Frozen flies safeguard research, screwworm eradication efforts. In: Agricultural Research. [Ed. R. Sowers]. Feb. 2005, pp. 14-15. USDA-ARS, Beltsville, MD. (ARS’ publication on research of potential public interest occurring within the agency). Anonymous. Storing frozen maggots. In: LiveScience. Feb. 9, 2005. www.livescience.com/imageoftheday/siod_050209.html. (Internet web-site giving daily items of scientific interest). Leopold, R.A. Cryopreservation of dipteran insects: Development and evaluation. International Conference on Preservation of Genetic Resources. Oct. 19-22, 2004, St. Petersburg, Russia. (Oral presentation to mainly Russian scientists). Review Publications Leopold, R.A. Establishing a national insect germplasm storage program for the U.S.A.. Meeting Abstract. Leopold, R.A. 2004. Cryopreservation of dipteran insects: development and evaluation. Meeting Abstract.October 18-23, 2004. St. Petersburg, Russia. In: Materials of the International Conference "Preservation of Genetic Resources" 46(9):760. Leopold, R.A. 2004. Cryopreservation of dipteran insects: development and evaluation. Meeting Abstract. October 18-23, 2004. St. Petersburg, Russia. In: Materials of the International Conference "Preservation of Genetic Resources" 46(9):760-761. Leopold, R.A., Chen, W., Yocum, G.D. 2004. Effects of using constant and cyclical stepwise-increasing temperatures on parasitized and unparasitzed eggs of the glassy-winged sharpshooter during cold storage. In: Proceedings of the CDFA Pierce's Disease Control Program Research Symposium, December 7-10, 2004, Coronado, California. p. 124-127. Leopold, R.A., Chen, W. 2004. Parasitism of the glassy-winged sharpshooter: Functional responses and super-parasitism by the egg parasitoid, Gonatocerus ashmeadi. In: CDFA Pierce's Disease Control Program Research Symposium, December 7-10, 2004, Coronado, California. p. 128-131. Freeman, T.P., Leopold, R.A., Nelson, D.R., Buckner, J.S., Henneberry, T.J. 2004. Ultrastructural contributions to the study of the glassy-winged sharpshooter and Pierce's Disease. Proceedings of CDFA Pierce's Disease Control Program Research Symposium. p. 100-102. Leopold, R.A. 2005. Colony maintenance and mass-rearing: Using cold storage technology for extending the shelf-life of insects. FAO/IAEA International Conference on Area-wide Control of Insect Pests: Integrating the sterile insect and related nuclear and other techniques. May 9-13, 2005, Vienna, Austria. In: Book of Extended Synopses. pp. 100-103.