2012 Annual Report
1a.Objectives (from AD-416):
Develop Tools for Future Symbiosis-Targeted Biocontrol Efforts. The first comprehensive genetic dataset for Megacopta and its symbiont through next generation transcript sequencing will be generated. Afterwards attempt will be made to identify genes that may be involved in host-symbiont interactions by assessing gene expression differences in symbiotic and aposymbiotic hosts and in tissues where the symbionts are stored.
1b.Approach (from AD-416):
Four pools of long-transcript ribonucleic acid (RNAs) will be generated and sequenced, focusing on two development stages and targeting identification of genes involved in host-symbiont interactions (RNA from whole symbiotic nymphs, RNA from whole abosymbiotic nymphs, RNA from symbiont-housing organs of adults, RNA from whole adults). Samples will be prepared such that reads will include both insect and symbiont transcripts. Libraries will be prepared such that both host and symbiont transcripts will be represented (i.e., useing random primers rather than polyA tails to generate cDNA).
Sequencing will be carried at Emory University's Genomics Core facility, and supplemented by additional sequencing, if necessary, at the USDA Stoneville Core Genomics facility. Both facilities have carried out 454 sequencing of non-model insects. Sequences will be compared to established reference mRNA sequences from Tribolium (beetle), Apis (honey bee), and Acyrthosiphon (pea aphid) sequences using tBLASTx queries. expressed sequence tag (EST) and genome reference sequences from Rhodnius prolixus (Heteroptera: Reduviidae) and Cimex lectularius (Heteroptera: Cimicidae) will be used for reference as well, as they become available. It is also our hope that plant-feeding heteropterans that have been targets of mitochondrial genome projects such as Halyomorpha halys (Heteroptera: Pentatomidae) and Nezara viridula (Heteroptera: Pentatomidae) will be targets for transcriptome sequencing in the near future, providing other reference sequences.
Specimens were collected from Griffith Campus of University of Georgia and other locations and transported into Stoneville Research Quarantine Facility (SRQF) on several occasions. A variety of host plants and cages were used to feed and house insects. Eggs rapidly became covered with fungal growth and nymphs that hatched died apparently without molting. Rearing in quarantine became problematic, as adults and nymphs were dying. When possible, egg masses were dissected and separated into treatment groups: (1: with symbiont) and (2: symbiont-free). We then set up separate cages with plant material to allow nymphs to hatch and have access to host plants. No survivors from either treatment group (n=84 for each group) for first experiment. Additional treatment groups suffered the same fate. Final live specimen died 27 June. Total eggs used in experiments 278 (139/treatment group).
Additional adult specimens were donated and additional eggs were collected, separated, housed in a new rearing strategy directly on growing plants. Experiments yielded failed attemps. Total eggs used in experiments 527 (265/262 without/with packets).
Repeated experiment varying plant tissue preparations to try to achieve nymph survival. Experiment using 150 eggs (75/treatment group) was harvested 24 hours after hatch resulting in 26 nymphs collected per treatment group. A second experiment using 140 eggs resulted in 32 nymphs per treatment group harvested at 7 days post hatch.
A standardized procedure for collecting and preparing kudzu shoots that support insect growth was prepared in September for technician(s). Growth chamber problems resulted in colony loss and delayed experimentation. Additional adult samples were donated. Problems with survival of both adult and nymph specimens recurred. Egg samples were collected and processed. Insects not used for experimentation were nurtured through winter and did not produce viable eggs. Kudzu plants were maintained throughout winter in Quarantine Greenhouse 116 but did not thrive.
Nine laboratory raised Megacopa (M.) cribraria survives (as adults) over the winter on a potted kudzu plant. These were G2 laboratory insects. Insects were observed mating, and one egg mass was collected in April 2012 (below). Arrangements were made for collection of additional experimental insects and experiment was repeated, treated insects harvested for ribonucleic acid (RNA) extraction. Attempts were made to root some stems, bare crowns, and shooting kudzu crowns in the Quarantine Greenhouse 116 with very poor results.
Those specimens collected are in an ultralow temperature freezer in RNA extraction buffer. Numerous problems have prevented further processing of those specimens.
With the lessons and techniques learned it is hopeful that the insect specimens will yield usable samples of RNA and thence complementary 2-deoxyribonucleic acid (cDNA) from paired samples of nymphs raised with and without symbiont on kudzu.