1a. Objectives (from AD-416):
The objectives of this project are: 1) produce a transcriptome of codling moth heads; 2) identify and characterize neuropeptides expressed by codling moth; and 3) Clone and characterize neuropeptides and receptors from spotted winged Drosophila.
1b. Approach (from AD-416):
1) Messenger RNA will be extracted from heads of codling moth larvae, pupae and adults, and then converted to complementary DNA (cDNA). 2) The sequences of cDNAs representing head mRNA transcripts will be determined using 454 sequencing technology. 3) The sequences of assembled head cDNAs will be used to identify those encoding neuropeptides, peptide hormones, and other potential protein targets for codling moth control. 4) cDNAs from SWD that encode neuropeptides and receptors involved in regulation of feeding and reproduction will be cloned and characterized.
3. Progress Report:
The work summarized in this progress report relates to objective number 2 in the Project Plan for 001-00D: 2. Identify genes, receptor proteins, and respective ligands that are critical to codling moth development and reproduction. A codling moth neuropeptide F receptor was identified and cloned. Neuropeptide F regulates feeding and digestion and is expressed in codling moth neonates. Interestingly, we detected the neuropeptide F receptor transcript expressed in antennae of codling moth adult male and females. Because neuropeptide F is involved in regulation of feeding, it is hypothesized that activation of this receptor in antennae may turn on expression of gene transcripts encoding for receptors involved in host plant seeking or feeding behaviors. The transcriptome generated from RNA extracted from codling moth heads from all codling moth life stages (larvae through adults) was “data mined”, with results exceeding our expectations. The initial search for gene transcripts encoding for neuropeptides and peptide hormones yielded identification of transcripts encoding for 44 neuropeptide and peptide hormone families with the potential to produce over 100 biologically active peptides. We identified transcripts encoding for 45 putative neuropeptide receptors as well as the codling moth insulin receptor. We identified transcripts encoding for the protein targets of all major classes of insecticides used for codling moth control in the orchard, as well as transcripts encoding for over 150 potential insecticide detoxification enzymes. These results are already being used to help identify potential mechanisms that codling moth may use to develop resistance to current control agents, in hopes to delay or prevent the failure of neonicotinoids (e.g. Calypso®), spinosyns (e.g. Delegate®) and rynaxypyr (e.g. Altacor®). This transcriptome will allow us to develop future research projects to gain a greater understanding of the roles of key proteins in codling moth physiology and to identify novel targets for the potential development of new codling moth control agents or strategies. We focused our codling moth transcriptome search to transcripts encodling protein members of the endocrine system (neuropeptide, peptide hormones, and their receptors) because it controls almost every physiological function in the insect including reproduction, regulation of feeding, and development. For spotted wing drosophila, we cloned gene transcripts encoding for sex peptide and its putative receptor, as well as transcripts that encode for neuropeptide F, short neuropeptide F, and their putative receptors. These targets were chosen based on their roles in regulating reproduction (sex peptide) and feeding (neuropeptide F and short neuropeptide F). The nucleotide sequences of these clones are freely available to the research community in efforts to further characterize the neuropeptide/receptor interactions with the hope of developing control agents that disrupt their physiological function.