INSECT CRYOPRESERVATION, DORMANCY, GENETICS AND BIOCHEMISTRY
Location: Insect Genetics and Biochemistry Research
Project Number: 5442-21220-027-00
Start Date: Apr 21, 2011
End Date: Mar 31, 2016
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.
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.