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United States Department of Agriculture

Agricultural Research Service

Research Project: Insect Cryopreservation, Dormancy, Genetics and Biochemistry

Location: Insect Genetics and Biochemistry Research

2012 Annual Report


1a.Objectives (from AD-416):
Objective 1: Develop technology to preserve insect germplasm and increase the shelf-life of insects by devising cryogenic procedures for long-term storage in liquid nitrogen of embryos for lepidopterans and tephritid fruit flies, by developing mass-cryopreservation systems for insects used in control programs employing sterile insects, by designing short-term storage protocols using stage-specific cold tolerance techniques and by manipulating dormancy of pest and beneficial insects. Objective 2: Provide molecular genetic data defining biosystematics and population diversity of pest and beneficial insects such as Diabrotica, Lygus and Osmia species. Determine the molecular mechanism(s) of diapause physiology for beneficial insects. Objective 3: Provide a better understanding of the roles that lipids and other natural products play in overwintering/cold-tolerance processes, communication, and better management of beneficial insects.


1b.Approach (from AD-416):
1) We will develop an industrial scale cryogenic storage protocol for the screwworm (Cochlyomyia hominivorax) and work towards developing large-scale storage protocols for other mass-reared insects. A majority of our efforts will be to develop a simplified and automated process by which embryos can be prepared for vitrification. Fluctuating thermal regime (FTR) studies will be conducted on dormant and developing bees. All thermal regimes will be conducted in programmable environmental chambers, concentrating on optimizing the high temperature pulse of the FTR. Survival, defined as a bee’s ability to successfully emerge from the cocoon, will be assessed weekly. The effects of photoperiod and atmospheric conditions on storage survival will also be assessed. Photoperiodic manipulation will be achieved via boxes fitted with programmable shutters. Atmospheric manipulation will be achieved in air tight chambers, focusing on differential oxygen and carbon dioxide levels. Survival will be assessed as detailed above. 2) We will use mitochondrial DNA (mtDNA) and genomic sequences to measure diversity and mine for heritable differences to establish an evolutionary-based phylogeny of recognized insect species. The mtDNA barcode region will be used to establish an initial molecular phylogeny of Lygus plant bug species. Additional markers will be used as needed to clarify potential ambiguities. Because the barcode region lacks sufficient polymorphism other markers need to be identified in the blue orchard bee. To determine the distribution of the different strains of Wolbachia in northern corn rootworm we will use PCR primers that are specific for each of the five strains. To isolate diapause regulated genes and DNA-methylation during diapause. The bees will be removed from the field by mid-August and transferred to 6ºC. RNA will be collected monthly from November to July and screened using microarrays to determine developmental expression profiles. DNA-methylation levels will be will be determined by EZ DNA Methylation-Direct Kit. After incubation the samples concentrations will be determined with a spectrophotometer. 3) Lipids involved in the processes of insect cold storage, dormancy and cryopreservation will be extracted from insect tissues with appropriate organic solvents. Lipid class fractions will be obtained using silica gel adsorption chromatography or HPLC techniques. Individual lipid components will be characterized by capillary GC and GC-MS. For bees reared under different temperature regimes, recovered internal lipids (e.g., triacylglycerols) will be analyzed and quantified by HPLC and GC-MS. Potentially-active chemicals extracted from bee tissues and bee nesting materials will be obtained from either, GC-FID and GC-MS analyses or HPLC-UV-VIS, HPLC-ELS, HPLC-MS and silica gel chromatography. Active chemicals (fractions) will be tested by using Y-tube olfactory response bioassays. Identified bio-active chemicals and chemical blends will be used in field bioassays at specific bloom times in almond and apple orchards to further explore nest cavity preference due to specific nest components.


3.Progress Report:
This project, implemented on April, 21, 2011, is focused on the cold storage (including cryopreservation), population genetics, and lipid biochemistry of economically important pest and beneficial insects. Progress was made on all three objectives, which fall under NP304 Component II – Protection of Agricultural and Horticultural Crops. Under Objectives 1-A&B, we expanded research on the effects of heating and cooling on the survival of the alfalfa leafcutting bee, Megachile rotundata, during storage and we developed a lab-based flight metabolism assay that is sensitive to subtle non-lethal effects of low temperature storage. We also utilized second generation sequencing technology to document physiological changes occurring during the transition from diapause to post-diapause development. Our results will contribute to optimizing storage protocols for commercial-scale management of M. rotundata, the primary pollinator for the U.S. Alfalfa Seed Industry. We also demonstrated that the prepupal period in the blue orchard bee, Osmia lignaria, is likely a true summer diapause which suggests that this species exhibits both prepupal and adult diapause. These results in addition to being of scientific interest (two diapause intervals are rare in insects) will contribute to improving commercial-scale overwintering protocols for populations destined for almond pollination. Under Objective 1-C, we developed a cryopreservation protocol for the pink bollworm, Pectinophora gossypiela, which represents a significant advancement in this ARS-led technology and is the first report of effective cryopreservation of a non-fly species. Our results will contribute to improving the sterile male release portion of the pink bollworm eradication program by allowing managers to preserve germplasm essential to success. Under Objectives 2-A&B, we expanded research on population-level molecular markers for O. lignaria, a solitary bee seeing increasing use as commercial-scale pollinator of tree fruit crops – including CA almonds, and collected mitochondrial DNA sequences from a range of populations. Although the general expectation is that the greater the geographical separation of populations, the greater the mitochondrial DNA divergence, our results with general molecular markers show that the greatest source of genetic diversity seems to reside in northern Utah, not in California where these bees have the most short term potential. Ultimately, molecular markers associated with populations and specific traits will be important in developing latitude-specific commercial-scale pollinator populations for crops like almonds. Under Objectives 3-A&B, we verified that nesting M. rotundata females apply olfactory cues to nests for individual recognition, and chemical analyses of the deposits revealed the presence of hydrocarbons, fatty aldehydes, fatty alcohol, acetate esters, and wax esters, similar to the cuticular lipids of nesting bees, but with consistent differences in the percent composition of certain components. These results will contribute to the development of generalized nesting/establishment attractants for commercial-scale pollination providers.


4.Accomplishments
1. Cryopreservation of pink bollworm germplasm. The pink bollworm is a major pest in cotton growing regions of the United States. Since 2002, cotton growers and APHIS have partnered to eradicate this invasive specialty crop pest However, a lack of storage protocols for the pink bollworm leaves the eradication effort vulnerable to loss of germplasm critical to the success of this program. ARS researchers in collaboration with colleagues at North Dakota State University in Fargo have developed a cryopreservation protocol for this species, representing the first effective application of this nascent technology to a moth species. This advancement not only offers protection to valuable APHIS resources, but signifies a substantial expansion of insect cryopreservation technology.


Review Publications
Yocum, G.D., Rinehart, J.P., Kemp, W.P. 2012. Duration and frequency of the high temperature pulse affect survival of emergence-ready Megachile rotundata (Hymenoptera: Megachilidae) during low-temperature incubation. Journal of Economic Entomology. 105(1):14-19.

Yocum, G.D., Greenlee, K.J., Rinehart, J.P., Bennett, M.E., Kemp, W.P. 2011. Cyclic CO2 emissions during the high temperature pulse of fluctuating thermal regime in eye-pigmented pupae of Megachile rotundata. Comparative Biochemistry and Physiology. 160(4):480-485. doi:10.1016/j.cbpa.2011.08.004.

Roehrdanz, R.L., Bourchier, R., Cortilet, A., Olson, D.L., Sears, S. 2011. Phylogeny and genetic diversity of flea beetles (Aphthona sp.) introduced to North America as biological control agents for leafy spurge. Annals of the Entomological Society of America. 104(5):966-975. DOI: 10.1603/AN10145.

Sgolastra, F., Kemp, W.P., Buckner, J.S., Pitts Singer, T., Maini, S., Bosch, J. 2011. The long summer: Pre-wintering temperatures affect metabolic expenditure and winter survival in a solitary bee. Journal of Insect Physiology. 57(12):1651-1659.

Rinehart, J.P., Yocum, G.D., West, M.S., Kemp, W.P. 2011. A fluctuating thermal regime improves survival of cold-mediated delayed emergence in developing Megachile rotundata (Hymenoptera: Megachilidae). Journal of Economic Entomology. 104(4):1162-1166.

Sgolastra, F., Kemp, W.P., Maini, S., Bosch, J. 2012. Duration of prepupal summer dormancy regulates synchronization of adult diapause with winter temperatures in bees of the genus Osmia. Journal of Insect Physiology. 58(7):924-933.

Last Modified: 12/22/2014
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