Progress 06/06/04 to 05/24/05
Outputs
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 use 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. Year 1 (FY 2000) Objective 1. Investigate formulation of a method for cryopreservation of screwworm
embryos. Year 2 (FY 2001) Objective 1. Examine whether the Drosophila method for insect embryo vitrification is applicable for cryopreservation of Caribbean and Mediterannean fruit flies. Year 3 (FY 2002) Objective 1. Optimize cryopreservation methods for tephritid flies, namely, the Caribbean, Mexican, and Mediterranean fruit flies. Objective 2. Initiate studies on chilling tolerance of parasitized and unparasitized glassy-winged sharpshooter eggs. Year 4 (FY 2003) Objective 1. Continue examination of the chilling tolerance of parasitized and unparasitized glassy winged sharpshooter eggs. Objective 2. Identify plant host(s) that will support cold storage of sharpshooter eggs. Year 5 (FY 2004) Objective 1. Studies will be conducted to determine whether the process of cryopreservation alters the genetic diversity of a Mexican fruit fly colony recovered from liquid nitrogen storage. Objective 2. Methods for insect sperm cryopreservation will be developed for use with dipteran in vitro
fertilization. Objective 3. Continue work on improving survival of GWSS and their egg parasitoids after cold storage. 3a List the milestones that were scheduled to be addressed in FY 2005. For each milestone, indicate the status: fully met, substantially met, or not met. If not met, why. 1. Permeabilize egg membranes.- Develop a cryopreservation protocol for Heliothine moth embryos that will allow long-term storage. FY 2005-2007 Milestone Not Met Critical SY Vacancy 2. Synchronize embryo development. - Develop a cryopreservation protocol for Heliothine moth embryos that will allow long-term storage. FY 2005- 2007 Milestone Not Met Critical SY Vacancy 3. Assess incidence of chimerism. - Investigate the use of cryopreserved pre- blastoderm nuclei, blastomeres, and/or primordial germ cells as useful germplasm storage alternatives. FY 2005-2007 Milestone Substantially Met 4. Determine parasitoid cold tolerance. - Develop stockpiling procedures at subambient temperatures for three
hymenopterous species of GWSS egg parasitoids. FY 2005-2007 Milestone Substantially Met 5. Protocol testing. - Develop quality assurance technology and test the fitness and genetic stability of insects that have experienced cold storage either as mass-reared insects or as research colonies. FY 2005-09. Milestone Substantially Met 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 Pierces 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 (NCGRP) 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 less than or equal to 1:10. It is expected that this information will be helpful in assessing the efficiency of the parasitoid as bio-control agent against the glassy- winged sharpshooter, for devising mass-rearing
protocols, and in the implementation of release programs for control of its host. 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. Since inception of this project, there have been four principal accomplishments for insect cryopreservation. First, the major barriers that dipteran embryos present that exclude the use of conventional cryopreservation protocols were identified. This led to the next accomplishment which was solving of the problems dealing with membrane permeabilities, chilling intolerance, ultra fast cooling modifications, cryoprotectant toxicity, and post storage recovery procedures. Thus, the third accomplishment was development of individualized embryo cryopreservation protocols resulting in adult survival and recolonization for five species of dipterans. The fourth accomplishment was showing that embryo cryopreservation has no effect on the quality factors required for successful implementation of the SIT.
Demonstrating the feasibility of the cryopreservation technique for long term storage of these insects confirms that this technology can be employed to cut costs involved with maintenance of the various stains of flies used in mass production and research. An example of the actual impact of this technology is that it will allow saving of 15 strains of the screwworm, including those with visible and transgenic markers. As mentioned in the above paragraph, these strains will be stored in Ft. Collins, CO. Thus, this is the beginning of an insect germplasm repository that can be used to store valuable insect strains and also maintain genetic diversity for insects important to agricultural research programs. Calorimetric and cryomicroscopic studies on modifying ethylene glycol- based vitrification solutions permitted the development of a method to store the samples of dipteran embryos at the higher temperature of -130 deg. C. These results increase the potential for the mass processing and
storage of large numbers of embryos in a mechanical freezer rather than at -196 deg. C in liquid nitrogen dewars. Cold tolerance studies show that unparasitized eggs of the glassy-winged sharpshooter (GWSS) withstand up to 25 days at 14 deg. C with little affect on hatching rates but continued development of the leafhoppers is affected by chilling when the egg massess are stored longer than 3 weeks. GWSS eggs stored up to 25 days at 14 deg. C were successfully parasitized by the parasitoid, Gonatocerus triguttatus, and wasp progeny were subsequently observed emerge from these eggs. These results suggest that chilling damage to the GWSS eggs is not inhibitory to successful parasitization and that live GWSS eggs may not be necessary for propagation of the egg parasitoids of the GWSS. Short-term chilling tolerance of house fly embryos was shown to be age- specific. Cold storage at 5 degrees C was best tolerated by 3-hr-old embryos as opposed to the younger and older ages. Storage
periods of longer than 3 days resulted in the expression of chilling injury. Three patterns of chilling injury expressed by the embryos after experiencing short-term cold storage were identified. They were characterized as immediate, accumulative and latent. Expression of immediate and accumulative was linked to the age of the embryos at the time of chilling injury was linked placement into cold storage, while the latent type of injury was expressed during the post-embryonic stages of development and related to the length of cold exposure. Thus, a protocol was developed for short-term storage of dipteran embryos which can be used to allow interruptions in daily harvest of eggs (weekends and holidays) and also to minimize the size of parent colonies needed to maintain production at a mass-rearing facility. Long-term survival of Mexican fruit fly embryos cryopreserved by using our protocol and stored in liquid nitrogen was assessed. Hatching of embryos immediately after experiencing the
cryopreservation protocol was 36.8 +/- 6.8 % while the collective hatching of embryos stored from 2 months up to 3 years and 4 months was 35.3 +/- 4.2%. Thus, there is no indication that deterioration of the hatching of embryos occurs stored while vitrified under liquid nitrogen. 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? Insect embryo cryopreservation technology developed during the life of
this project has been transferred to 2 APHIS mass-rearing facilities, one that rears Mediterranean fruit flies in Waimanalo, HI and the other that rears Mexican fruit flies in Edinburg, TX. In addition, this technology is being used to cryopreserve screwworm strains at the ARS Midwest Livestock Insect Research Laboratory in Lincoln, NE for eventual storage at the National Center for Genetic Resources Preservation. This technology has also been transferred to the FAO/IAEA Division of Entomology at Sibersdorf, Austria and to the Department of Forensic Entomology at the University of Alabama Birmingham. To date, this technology transfer has been limited to cryopreservation of flies in four families of dipterans. Time will tell whether it can be used to cryopreserve other insects outside of the Order Diptera.
Impacts
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Publications
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Progress 10/01/03 to 09/30/04
Outputs
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 use 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. Year 1 (FY 2000) Objective 1. Investigate formulation of a method for cryopreservation of
screwworm embryos. Year 2 (FY 2001) Objective 1. Examine whether the Drosophila method for insect embryo vitrification is applicable for cryopreservation of Caribbean and Mediterannean fruit flies. Year 3 (FY 2002) Objective 1. Optimize cryopreservation methods for tephritid flies, namely, the Caribbean, Mexican, and Mediterranean fruit flies. Objective 2. Initiate studies on chilling tolerance of parasitized and unparasitized glassy-winged sharpshooter eggs. Year 4 (FY 2003) Objective 1. Continue examination of the chilling tolerance of parasitized and unparasitized glassy winged sharpshooter eggs. Objective 2. Identify plant host(s) that will support cold storage of sharpshooter eggs. Year 5 (FY 2004) Objective 1. Studies will be conducted to determine whether the process of cryopreservation alters the genetic diversity of a Mexican fruit fly colony recovered from liquid nitrogen storage. Objective 2. Methods for insect sperm cryopreservation will be developed for use with
dipteran in vitro fertilization. Objective 3. Continue work on improving survival of GWSS and their egg parasitoids after cold storage. 3. Milestones: A. (No milestones were completed during the approximately one month reporting period of this bridging CRIS project.) B. Anticipated milestones for FY 2005. (No formal CRIS project plan has been written for this project. The following is a list of activities taken from the project prospectus to be undertaken during the life of this proposed project.) Objective 1- Extend existing knowledge about insect cold hardiness and use that knowledge to develop cold storage protocols for use in insect release control programs and for insect colonies reared for research purposes. Sub-objective 1.1 - Optimize stockpiling procedures at subambient temperatures for three species of egg parasitoids, Gonatocerus ashmeadi, G. triguttatus, and G. fasciatus by manipulation of pre- and within storage conditions. Sub-objective 1.2 - Increase cold tolerance of
egg parasitoids during storage by bio-fortification of the diet fed to the adult wasps. Sub-objective 1.3 - Investigate the use of cryogenic techniques for long- term storage of parasitic insects, such as Fopius arisanus, while they are within an egg host. Sub-objective 1.4 - Cryopreserve blastoderm nuclei and primodial germ cells and evaluate the production of chimeras and/or clones after transplantation into recipient embryos. (continuation of substituted objective 2 in FY 2004 for the expired CRIS project). Sub-objective 1.5 - Develop quality assurance technology for testing the fitness and genetic stability of insects that have experienced cold storage either as mass-reared insects or as research colonies. 4. What were the most significant accomplishments this past year? A. (See expired CRIS 5442-22000-034-00D report for FY 2004.) B. Other Significant Accomplishment(s). (See expired CRIS 5442-22000-034-00D report for FY 2004.) C. Significant Accomplishments That Support Special
Target Populations. None D. Progress Report of Subordinate Projects. None 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. None - (See expired CRIS 5442-22000-034-00D report for FY 2004.) 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? None - (See expired CRIS 5442-22000-034-00D report for FY 2004.) 7. List your most important publications in the popular press and presentations to organizations and articles written about your work. None - (See expired CRIS 5442-22000-034-00D report for FY 2004.)
Impacts
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Publications
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