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Putative nicotinic acetylcholine receptor subunits express dif.erentially through the lif. cycle of.codling moth, Cydia pomonella （Lepidoptera： Tortricidae）

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Insect Science 2016年第2期23卷 277-287页

作者： Jessica A. Martin stephen f. garczynski USDA-ARS Yakima Agricultural Research Laboratory Wapato WA 98951 USA

Nicotinic acetylcholine receptors （nAChRs） are the targets of.neonicotinoids and spinosads, two insecticides used in orchards to ef.ectively control codling moth, Cydia pomonella （L.）（Lepidoptera： Tortricidae）. Orchardists in Washington State are concerned about the possibility of.codling moth f.eld populations developing resistance to these two insecticides. In an ef.ort to help mitigate this issue, we initiated a project to identif. and characterize codling moth nAChR subunits expressed in heads. This study had two main goals; （i） identif. transcripts f.om a codling moth head transcriptome that encode f.r nAChR subunits, and （ii） determine nAChR subunit expression prof.les in various lif. stages of.codling moth. f.om a codling moth head transcriptome, 24 transcripts encoding f.r 12 putative nAChR subunit classes were identif.ed and verif.ed by PCR amplif.cation, cloning, and sequence determination. Characterization of.the deduced protein sequences encoded by putative nAChR transcripts revealed that they share the distinguishing f.atures of.the cys-loop ligand-gated ion channel superf.mily with 9 α-type subunits and 3 β- type subunits identif.ed. Phylogenetic analysis comparing these protein sequences to those of.other insect nAChR subunits supports the identif.cation of.these proteins as nAChR subunits. Stage expression studies determined that there is clear dif.erential expression of.many of.these subunits throughout the codling moth lif. cycle. The inf.rmation f.om this study will be used in the f.ture to monitor f.r potential target-site resistance mechanisms to neonicotinoids and spinosads in tolerant codling moth populations.

关键词： codling moth nAChR expression nicotinic acetylcholine receptor subunits

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Identif.cation and initial characterization of.the 3＇ end of.gene transcripts encoding putative members of.the pheromone receptor subf.mily in Lepidoptera

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Insect Science 2012年第1期19卷 64-74页

作者： stephen f. garczynski Kevin W. Wanner Thomas R. Unruh USDA-ARS Yakima Agricultural Research Laboratory Wapato Washington Department of.Plant Sciences and Plant Pathology Montana State University Bozeman Montana USA

Semiochemicals, including pheromones and kairomones, used in pest man- agement programs reduce the need f.r chemical insecticides, and understanding their interactions with their membrane receptors may help make them more ef.ective in the f.eld. Identif.cation of.odorant receptors in the Lepidoptera has mainly been achieved us- ing bioinf.rmatics to search DNA sequences generated by genome or expressed sequence tag （EST） sequencing projects. This study reports a rapid method to identif. members of.the pheromone receptor subf.mily in Lepidoptera. Degenerate oligonucleotide primers were designed against a conserved amino acid sequence in the carboxyl terminus of.known lepidopteran pheromone receptors, and the primers were used in a 3＇ rapid amplif.ca- tion of.complementary DNA （cDNA） ends procedure. Polymerase chain reaction products generated f.om seven dif.erent lepidopteran species were TA cloned and sequenced. The cDNA sequences of.25 transcripts were determined to encode potential members of.the pheromone receptor subf.mily. These cDNAs ranged f.om 238 to 642 bp and encoded 49-54 amino acids of.the carboxyl terminus. Analysis of.the 3＇ untranslated region reveals that most of.the transcripts contain multiple polyadenylation signal sequences, and in the case of.anduca sexta, an alternate polyadenylation signal appears to be used in transcript processing. The 3＇ untranslated region was also usef.l in determining unique receptors en- coded by transcripts having highly similar nucleotide and amino acid sequences. Overall, this technique provides a complementary method of.pheromone receptor identif.cation in EST sequencing projects, or can be used as a stand-alone method in conjunction with 5＇ rapid amplif.cation of.cDNA ends procedures.

关键词： degenerate primer PCR odorant receptor pheromone receptor polyadeny- lation signals 3＇ untranslated region

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Application of.Cydia pomonella expressed sequence tags： Identif.cation and expression of.three general odorant binding proteins in codling moth

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Insect Science 2013年第5期20卷 559-574页

作者： stephen f. garczynski Brad S. Coates Thomas R. Unruh Scott Schaef.er Derick Jiwan Tyson Koepke Amit Dhingra USDA-ARS Yakima Agricultural Research Laboratory Wapato WA 98951 USDA-ARS Corn Insect and Crop Genetics Research Unit Genetics Laboratory Iowa State University Ames IA 50011 Department of.Horticulture and Landscape Architecture Washington State University Pullman WA 99164 USA

The codling moth, Cydia pomonella, is one of.the most important pests of.pome f.uits in the world, yet the molecular genetics and the physiology of.this insect remain poorly understood. A combined assembly of.8？341 expressed sequence tags was generated f.om Roche 454 GS-f.X sequencing of.eight tissue-specif.c cDNA libraries. Putative chemosensory proteins （12） and odorant binding proteins （OBPs）（18） were annotated, which included three putative general OBP （GOBP）, one more than typically reported f.r other Lepidoptera. To f.rther characterize CpomGOBPs, we cloned cDNA copies of.their transcripts and determined their expression patterns in various tissues. Cloning and sequencing of.the 698？nt transcript f.r CpomGOBP1 resulted in the prediction of.a 163 amino acid coding region, and subsequent RT-PCR indicated that the transcripts were mainly expressed in antennae and mouthparts. The 1？289 nt （160 amino acid） CpomGOBP2 and the novel 702 nt （169 amino acid） CpomGOBP3 transcripts are mainly expressed in antennae, mouthparts, and f.male abdomen tips. These results indicate that next generation sequencing is usef.l f.r the identif.cation of.novel transcripts of.interest, and that codling moth expresses a transcript encoding f.r a new member of.the GOBP subf.mily.

关键词： codling moth EST odorant binding proteins

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Characterization of.three transcripts encoding small heat shock proteins expressed in the codling moth, Cydia pomonella （Lepidoptera： Tortricidae）

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Insect Science 2011年第5期18卷 473-483页

作者： stephen f. garczynski Thomas R. Unruh Christelle Guedot Lisa G. Neven USDA-ARS Yakima Agricultural Research Laboratory Wapato Washington USA

Codling moth is a major pest of.apples and pears worldwide. Increasing knowledge of.how this insect responds to environmental stress will improve f.eld and postharvest control measures used against it. The small heat shock proteins （sHsps） play a maj or role in cellular responses to environmental stressors. A degenerate oligonucleotide primer, designed against the conserved α-crystallin domain, was used in 3＇ rapid amplif.cation of.complementary DNA （cDNA） ends reactions to amplif. transcripts encoding sHsps expressed in the codling moth cell line, Cp169, subjected to heat shock. Three f.ll-length cDNAs were cloned f.om Cp169 cells that contained open reading f.ames encoding sHsps. The cDNA f.r CpHsp 19.8 was 795 bp encoding 177 amino acids. The cDNA f.r CpHsp 19.9 was 749 bp encoding 175 amino acids. The cDNA f.r CpHsp22.2 was 737 bp encoding 192 amino acids. Analysis of.the protein sequences of.the three CpHsps indicated the presence of.83 amino acids with homology to the α-crystallin domain. f.r each of.the CpHsps, the α-crystallin domain was surrounded by divergent N- and C-terminal regions, consistent with the conserved structural f.atures of.sHsps. Real-time polymerase chain reaction, used to determine the expression patterns of.each of.the sHsps in dif.erent developmental stages of.codling moth revealed the presence of.transcripts in all stages tested. Consistent with characteristics of.other sHsps, expression of.CpHsp transcripts were greatly enhanced when insects were subjected to heat shock. The results of.this research can be used as a guide to study the roles of.sHsps in codling moth control using various post-harvest treatments.

关键词： α-crystallin domain heat shock response qPCR

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