Progress 04/28/10 to 04/20/11
Outputs
Progress Report 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. 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 diapause specific genes and their regulatory elements in other insects based on primers derived from the original Colorado potato beetle diapause regulated genes. This is the final report for the bridging project 5442-22000-044-00D, initiated 04/28/2010 and terminated 04/20/2011. Please refer to the new in-house project 5442-21220-027-00D. The alfalfa leafcutting bee, Megachile rotundata is commonly used to pollinate 2.8 million acres of alfalfa for seed production. M. rotundata is held at low temperatures for overwintering the prepupae or for interrupting spring incubation to synchronize adult emergence with the peak alfalfa bloom. However, static low temperature exposures can be stressful depending on the temperature, duration of exposure, and the developmental stage exposed. In order to develop the next generation of storage protocols it is essential to be able to measure subtle nonlethal effects of low-temperature exposure that can affect the bees post emergence quality. We have developed a sensor and computer program to measure the timing of M. rotundata adult emergence. Changes in the timing of emergence would indicate that the brain was affected negatively by the storage protocol. The group of Lygus bug pest species feeds on a large number of crops. The species can be difficult to distinguish because of similarities in their appearance. Ribosomal RNA spacer sequences were used as molecular markers to help define species differences. Most insects from the common species groups have DNA sequences that fall into species-specific clusters. However, one taxonomic species does not appear to have its own species recognizable sequence. Its DNA sequences fall into the sequence groups associated with another other well defined species. This raises legitimate concerns that the taxonomic group in question may not be a valid species. Efforts are continuing to clarify this further. Combination of proposed taxonomic groups into a single species could simplify the identification process. Accomplishments 01 Food reserves role in insect dormancy. Insect diapause (dormancy) is th chief physiological mechanism that enables insects to survive in a given geographical region by timing their development and reproduction to food resources. One of the key physiological characteristics of the diapause the accumulation of food reserves prior to entering diapause. This raise the question if food reserves are involved in regulating an insects� ability to enter diapause. The Colorado potato beetle is endemic in most of the potato growing regions in the northern hemisphere and is the majo defoliator of potato in those regions. ARS researchers in Fargo, ND determined the timing of accumulation and the chemical makeup of Colorad potato beetle lipid reserves. They demonstrated that there are no major quantitative or qualitative differences between diapause or nondiapause adult beetles. This suggests that as it relates to lipid metabolism, the decision to enter diapause or not only controls the final allocation of lipids either into metabolic reserves for diapause or into reproduction for nondiapausing beetles. Understanding how food reserves regulate diapause and how the reserves are used during diapause will enable the development more accurate model predicting overwintering survival and future range expansion of this pest. 02 Genetic diversity in a biological control insect. Leafy spurge is a noxious weed in temperate regions of the USA. Biological control of this weed has had some success due to release of five species of flea beetles in the USA and Canada. ARS researchers in Fargo, ND, used DNA markers t examine the genetic diversity of the flea beetles captured at various sites in the USA and Canada. Low genetic diversity could hinder the ability of a biological control insect to adapt to various environmental conditions and thus limit their effectiveness. They found that about 3 of the population of the most common brown flea beetle species has potentially reduced genetic variability due to a bacterial infection. If an uninfected strain could be obtained, it might provide new opportuniti for biological control. Additionally, it was discovered that the common black species has very little diversity in the USA, but shows extensive diversity in Canada. One Canadian population is so divergent that it cou be another species. This population might be effective in controlling leafy spurge at some sites where the other populations and species have not done well. In-depth research on biological control agents for leafy spurge could improve the effectiveness of these agents, which would economically benefit farmers, ranchers and land managers who, by law, mu control noxious weeds.
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Progress 10/01/09 to 09/30/10
Outputs
Progress Report 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. 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 diapause specific genes and their regulatory elements in other insects based on primers derived from the original Colorado potato beetle diapause regulated genes. This bridging project was initiated in April 2010 after the previous project 5442-22000-039-00D expired. A new 5-year project is currently in peer review and will be implemented early in FY 2011. Refer to project 5442-22000-039-00D for additional information. Initiated genetic studies of blue orchard bee. Specimens were obtained from eastern and western populations. DNA barcode sequences have been examined and they are being further tested for markers that will reveal intraspecific diversity. In Wolbachia infections of northern corn rootworm it has been confirmed that the newly discovered 4th genotype of Hypervariable region 1 is linked normally to genotypes #4 of Hypervariable regions 2-4. Thus the 4 main strains of Wolbachia each have a complete set of distinctive Hypervariable regions.
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