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.
Maintaining a continuous rearing of Megacopta cribraria has proven challenging. Kudzu is difficult to grow in greenhouse. Adult bugs will oviposit in kudzu plants, but mass death of immature stages occurred within weeks of receiving shipment into quarantine. The few adults that survived reproduced for many months. A method for keeping adults in a small cage was designed for extended collection of egg samples for experiments.