2007 Annual Report 1a.Objectives (from AD-416) Provide molecular genetic data defining biosystematics and population diversity (including Wolbachia dispersal) of pest and beneficial insects such as Diabrotica, Aphthona, Lygus et al. Determine the molecular mechanisms of diapause initiation and termination in insects especially leaf beetles and pollinators. Identify conserved sequence motifs associated with diapause specific genes, determine if products (e.g. noncoding RNAs) are produced, and examine the distribution and diversity of the sequences across insect species. 1b.Approach (from AD-416) Measure nuclear genetic diversity using microsatellite and AFLP markers. Use mtDNA PCR-RFLP and sequencing to quantitate additional diversity and identify species. Monitor the movement of Wolbachia strain boundaries over several seasons. Define the level of competition between insects infected with different Wolbachia strains and correlate Wolbachia infection, mtDNA genotype, and sex ratio. Identify and characterize genes differentially expressed in diapause and non-diapause insects. Use interfering RNA (iRNA) experiments to determine the significance of key genes in diapause physiology. Measure respiration quotients at different temperatures and stages of diapause to determine the transition from a diapause respiration pattern to a nondiapause pattern. Conduct comparative study of diapausespecific genes and their regulatory elements in other insects based on primers derived from the original Colorado potato beetle diapause regulated genes. 3.Progress Report SCA 58-5442-2-338 - In this reporting period we examined the supercooling points (SCP) of first, second and third instar field-collected larvae as well as diapausing larvae and larvae stored for 1, 2 and 3 years at 5ºC. Four distinct observations were made. 1)The field-collected larvae had high SCPs and were not freeze tolerant.. 2)Once the larvae entered diapause their average SCPs started decreasing from -5ºC in October diapausing larvae to -13ºC in two-year stored larvae.. 3)After four month in storage SCPs fall into two distinct groupings (High and low SCP) and the difference between the two groups increased over time. This observation of two distinct populations within the stored larvae agrees with our previous results.. 4)Both populations (high and low SCP) of larvae are freeze tolerant. The larvae ability to survive freezing with a SCP of -13ºC was unexpected in light of current literature, and raises a number of important questions as to the nature of cold tolerance mechanism(s). These results along with our previous work clearly demonstrate that the sugarbeet root maggot is a unique model system for studying diapause and cold physiology which may draw more researchers to study this important pest. SCA 58-5442-2-329 L. lineolaris, the tarnished plant bug, has been widely sampled in the eastern US and southern Canada. The mitochondrial cox1 barcode region and a cox1-cox2 overlap region were sequenced from the mitochondrial genome. Both the cox1-cox2 boundary sequence and the cox1 barcode region divide L. lineolaris into two major clades along with a small third group of very divergent individuals. These results raise the possibility of cryptic species within L. lineolaris. Well-defined geographic based population structure has not been uncovered in L. lineolaris. Strict monophyly of the various Lygus species has been difficult to demonstrate. With cox1-cox2 the majority of sequences from individuals identified as L. lineolaris, L. elisus, L. borealis, L. hesperus, and L. keltoni cluster together as species. The L. hesperus and L. keltoni clusters are intermixed. A few individuals from all of these species are scattered among other branches of the trees but these are believed to be derived from failures of the morphological characters to fully discriminate the species. SCA 58-5442-5-342 - Within this reporting period we hired a postdoctoral researcher, constructed two lambda libraries, and began primary screening. Homologues of genes of interest have also been isolated from a number of leaf beetle species. These results lay the groundwork for identifying key diapause regulating transcription factors and contributing to our understanding of the evolutionary development of diapause within insects. Understanding the mechanism regulating diapause is important in predicting the possible geographic range of introduced insect species, modeling winter survival, developing storage strategies for agriculturally important insects. Monitoring of these projects was done primarily through daily interaction with the Post Doctoral research scientist and graduate students along with information exchange between the PI and ADODR. 4.Accomplishments Sugarbeet root maggot survival of multiple years in cold storage We are interested in understanding how the sugarbeet root maggot, Tetanops myopaeformis, can survive up to six years at 5ºC. To characterize the physiological status of larvae achieving multi-year survival at 5ºC, respiration, lipids, and gene expression patterns of field-collected diapausing larvae were compared with those of one-, two-, and five-year laboratory stored larvae. Additional assessments were made on post-storage survival, emergence, and reproductive fitness of stored larvae. There is currently a pressing need to be able to store large numbers of agriculturally important insects for both research needs and for pest control programs. Understanding how the sugarbeet root maggot survives prolonged cold exposure may inform storage strategies for other insect species. Wolbachia infection and genetics of northern corn rootworm. We have been studying the effects of Wolbachia infection on the genetic diversity of northern corn rootworm (Diabrotica barberi)(NCR). NCR populations in the USA are infected with Wolbachia bacteria that has resulted in a genetically distinct population of NCR. Three geographically contiguous populations of NCR have been identified that are. 1)uninfected;. 2)infected with a single strain of Wolbachia; or. 3)infected with multiple strains of Wolbachia. Because infection with different Wolbachia strains can establish breeding barriers between the NCR populations, the insect bacteria interaction could lead to phenotypic differences, such as extended diapause or resistance to transgenic corn, cause the populations to react differently to control measures, or lead to the creation of rootworm subspecies. Isolation of diapause regulating transcription factors from the leaf beetles Understanding the mechanism regulating diapause is important in predicting the possible geographic range of introduced insect species, modeling winter survival, developing storage strategies for agriculturally important insects. We have constructed two lambda libraries, and began primary screening. Homologues of genes of interest have also been isolated from a number of leaf beetle species. These results lay the groundwork for identifying key diapause regulating transcription factors and contributing to our understanding of the evolutionary development of diapause within Insecta. Sequence the mitochondrial genome of the tarnished plant bug (Lygus) We have been examining the phylogenetics and molecular systematics of pest plant feeding bugs from the genus Lygus. The nearly complete mitochondrial genome comprising 14454 base pairs has been sequenced from the tarnished plant bug, L. lineolaris. The sequence allows direct mitochondrial genomic comparisons between the tarnished plant bug which is most common in eastern North America with its close relative the western tarnished plant bug, L. hesperus. The mitochondrial genome of the latter has been sequenced by a different group. These research accomplishments fall under national program 304: Component I: Identification and Classification of Insects and Mites, Problem C. Systematic Studies of High Priority Pest and Beneficial Insects and Mites; Component II: Biology of Pests and Natural Enemies (Microbes), Problem A. Basic Biology; Component IX: Biological Control of Weeds, Problem A. Agent Discovery and Selection and Risk Assessment. It also relates to Strategic Plan Goal 3: Objective 3.2, performance measure 3.2.5. 5.Significant Activities that Support Special Target Populations Participated in Native American Intern Program. 6.Technology Transfer Number of non-peer reviewed presentations and proceedings 9 Number of newspaper articles and other presentations for non-science audiences 5 Review Publications Coudron, T.A., Yocum, G.D., Brandt, S.L. 2006. Nutrigenomics: a case study in the measurement of insect response to nutritional quality. Entomologia Experimentalis et Applicata. 121:1-14. Rinehart, J.P., Aiqing, L., Yocum, G.D., Robich, R.M., Hayward, S.A., Denlinger, D.L. 2007. Upregulation of Heat Shock Proteins is Essential for Cold Survival during Insect Diapause. Proceedings of the National Academy of Sciences. 104(27):11130-11137. Roehrdanz, R.L., Levine, E. 2007. Wolbachia bacterial infections linked to mitochondrial DNA reproductive isolation among populations of Northern Corn Rootworm (Coleoptera: Chrysomelidae). Annals of the Entomological Society of America. 100(4):522-531.