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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

2013 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 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 sub-objectives 1A&B, we further refined storage protocols for both developing and overwintering alfalfa leafcutting bees and demonstrated a dramatic increase in shelf-life when fluctuating thermal regimes are employed when storing overwintering bees. 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. Under sub-objective 1C, we developed an automated system for the cryopreservation of dipteran species, increasingly reproducibility of this ARS-led technology. These 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 sub-objectives 2A&B, we have:.
1)discovered two molecular markers that appear to distinguish eastern and western populations of Osmia lignaria, a solitary bee seeing increasing use as commercial-scale pollinator. Ultimately, molecular markers associated with populations and specific traits will be important in developing latitude-specific pollinator populations for crops like almonds;.
2)confirmed, using DNA sequences of six different genes, that northern corn rootworms from the eastern corn belt are infected with multiple strains of an inherited bacteria, Wolbachia. These bacteria are known for disrupting their host's normal reproductive cycle and it may be possible to manage the bacteria to reduce northern corn rootworm populations;.
3)used DNA barcoding and other molecular markers to define tarnished plant bugs and related species (Lygus sp.) - pests of a wide variety of non-grassy plants. The most common species in eastern North America is readily identifiable both visually and molecularly. Because Lygus species do not all have the same host preferences, it is important to clearly define them in order to estimate pest potential accurately; and,.
4)used high-throughput RNA-seq methods to conduct a global assessment of gene expression changes during diapause in the alfalfa leafcutting bee. These results will not only make the diapause of this bee one of the most thoroughly studied at the molecular level, but will also assist in the development of biomarkers for improved management. Under sub-objectives 3-A&B, we verified that nesting O. lignaria 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. Improved shelf-life of an alternative pollinator. The alfalfa leafcutting bee is an important alternative pollinator for alfalfa seed production, and can be used in many other crops. One significant impediment to its more widespread use is yearly fluctuations in the price farmers pay for this species. ARS researchers in Fargo, North Dakota have demonstrated that by employing fluctuating thermal regimes during long-term storage of the overwintering stage, the shelf-life can be greatly enhanced, effectively making stored bees available for a second field season. Adoption of this technology by bee producers will greatly ameliorate yearly supply fluctuations, leading to price stability for this important alternative pollinator.


Review Publications
Burange, P.S., Roehrdanz, R.L., Boetel, M.A. 2012. Geographically based diversity in mitochondrial DNA of North American Lygus lineolaris (Hemiptera: Miridae). Annals of the Entomological Society of America. 105(6):917-929.

Yocum, G.D., Rinehart, J.P., Boetel, M.A. 2012. Water balance in the sugarbeet root maggot, Tetanops myopaeformis, during long-term low-temperature storage and after freezing. Physiological Entomology. 37(4):340-344.

Rinehart, J.P., Yocum, G.D., Robich, R.M. 2012. Sugar feeding improves survival of nondiapausing cold-stored Culex pipiens. Journal of Medical Entomology. 49(6):1347-1354.

Kuester, A.P., Jones, R.W., Sappington, T.W., Kim, K., Barr, N.B., Roehrdanz, R.L., Senechal, P., Nason, J.D. 2012. Population structure and genetic diversity of the boll weevil, Anthonomus grandis (Coleoptera: Curculionidae), on Gossypium in North America. Annals of the Entomological Society of America. 105(6):902-916.

Roehrdanz, R.L., Sears Wichmann, S.G. 2013. Wolbachia wsp gene clones detect the distribution of Wolbachia variants and wsp hypervariable regions among individuals of a multistrain infected population of Diabrotica barberi (Coleoptera: Chrysomelidae). Annals of the Entomological Society of America. 106(3):329-338.

Rinehart, J.P., Yocum, G.D., Kemp, W.P., Greenlee, K.J. 2013. A fluctuating thermal regime improves long-term survival of quiescent prepupal Megachile rotundata (Hymenoptera: Megachilidae). Journal of Economic Entomology. 106(3):1081-1088.

Bennett, M.M., Petersen, K., Yocum, G.D., Rinehart, J.P., Kemp, W.P., Greenlee, K.J. 2013. Effects of extended prepupal storage duration on adult flight physiology of the alfalfa leafcutting bee (Hymenoptera: Megachilidae). Journal of Economic Entomology. 106(3):1089-1097.

Pitts Singer, T., Buckner, J.S., Freeman, T.P., Guedot, C.N. 2012. Structural examination of the Dufour's gland of the cavity-nesting bees Osmia lignaria say and Megachile rotundata (Fabricius) (Hymenoptera: Megachilidae). Annals of the Entomological Society of America. 105(1): 103-110.

Last Modified: 8/27/2014
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