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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

2011 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
Implemented on April, 21, 2011, this project is focused on the diapause, cold storage, and cryopreservation of economically important pest and beneficial insects. Progress was made on all three objectives, which fall under National Program 304, Component II – Protection of Agricultural and Horticultural Crops. Under Objectives 1A&B, we examined the effect of the rate of heating and cooling on survival of Megachile rotundata during storage by focusing on diapause physiology and low-temperature storage. We also initiated research to determine the physiological changes occurring during the transition between diapause and post-diapause development via second-generation sequencing technology. Our results will contribute to the improvement of commercial-scale management of M. rotundata, the primary pollinator for the U.S. Alfalfa Seed Industry. Under Objective 1-C, we developed a prototype robotic system for high-throughput insect cryopreservation that is currently being tested to replace an automatic system built to develop a cryobank of screwworm embryos to be located in the mass-production facility in Pacora, Panama (USDA-APHIS-IS, Screwworm Eradication Program). The robotic system has the potential to cryopreserve over 10,000 screwworm embryos per 8 hr period, and the cryobank repository is expected to function as insurance against plant shutdown due to insect disease, mechanical and electrical failure, work stoppage and genetic drift caused by long-term continuous rearing. Under Objectives 2-A&B, we initiated research on population-level molecular markers for Osmia lignaria, a solitary bee seeing increasing use as commercial-scale pollinator of tree fruit crops – including CA almonds. We collected mitochondrial DNA sequences from a range of O. lignaria populations, and the general expectation is that the greater the geographical separation of populations, the greater is the mitochondrial DNA divergence. Preliminary results revealed, however, that O. lignaria populations separated by 4000 km displayed little genetic variation while, in contrast, individuals from certain montane valleys comprised two distinct groups. Ultimately, molecular markers associated with populations and specific traits will be important in the development latitude-specific commercial-scale pollinator populations for crops like almonds. Under Objectives 3-A&B, we compared the total surface lipid (TSL – thought to be critical to water retention and survival in commercial-scale pollinator populations) composition of M. rotundata 2nd – 5th instar larvae with the prepupae and adult forms (both previously published). In expanded experiments, involving a large number of immature larvae that were synchronized as they emerged, we obtained multiple samples of TSL, at 13 different points across the 5th instar interval, and found increasing amounts of 27-47 carbon chain length mono-, di- and tri-methyl branched hydrocarbons concurrent with decreasing amounts of 22-40 carbon chain lengths wax esters throughout the 5th instar. These results will also contribute to Objective 1 and the improvement of commercial-scale (mass storage) management of M. rotundata.


4.Accomplishments
1. Improving short-term storage of a critical alfalfa seed industry pollinator. Alfalfa flower pollination by leaf cutter bees is critical for high seed yield. The alfalfa leafcutting bee may be exposed to low temperatures during interrupted spring incubation as alfalfa seed producers throughout the western U.S. attempt to synchronize adult bee emergence with peak alfalfa bloom. However, static low temperature exposure can be stressful depending on the temperature, duration of exposure, and the developmental stage exposed, frequently resulting in reduced survival and poor pollinator performance following emergence. ARS researchers at Fargo, ND have demonstrated that short high-temperature pulses can alleviate the harmful effects of prolonged low-temperature exposure, and their results provide a foundation for the next-generation storage protocols that will increase the shelf life of alfalfa leafcutting bees and improve post-storage quality. The technology is currently being adopted by commercial-scale alfalfa seed producers and pollination professionals.


Review Publications
Yocum, G.D., Toutges, M.J., Roehrdanz, R.L., Dihle, P.J. 2011. Insertion of miniature subterminal inverted repeat-like elements in diapause-regulated genes in the Colorado potato beetle, Leptinotarsa decemlineata (Coleoptera: Chrysomelidae). European Journal of Entomology. 108(2):197-203.

Yocum, G.D., Rinehart, J.P., Larson, M.L. 2011. Monitoring diapause development in the Colorado potato beetle, Leptinotarsa decemlineata, under field conditions using molecular biomarkers. Journal of Insect Physiology. 57(5):645-652.

Bosch, J., Sgolastra, F., Kemp, W.P. 2010. Timing of eclosion affects diapause development, fat body consumption and longevity in Osmia lignaria, a univoltine, adult-wintering solitary bee. Journal of Insect Physiology. 56(12):1949-1957.

Chen, W., Leopold, R.A., Boetel, M.A. 2010. Host plant effects on development and reproduction of the glassy-winged sharpshooter, Homalodisca vitripennis (Homoptera: Cicadellidae). Environmental Entomology. 39(5): 1545-1553.

Rinehart, J.P., Yocum, G.D., Leopold, R.A., Robich, R.M. 2010. Cold storage of Culex pipiens in the absence of diapause. Journal of Medical Entomology. 47(6):1071-1076.

Yocum, G.D., Buckner, J.S., Fatland, C.L. 2011. A comparison of internal and external lipids of nondiapausing and diapause initiation phase adult Colorado potato beetles, Leptinotarsa decemlineata. Comparative Biochemistry and Physiology, Part B. 159(3):163-170.

Last Modified: 4/16/2014
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