ABSTRACT
A cDNA encoding allatostatin Bs (ASTBs) containing the W(X)6W motif was identified using a database generated by a next generation sequencer (NGS) in the two-spotted cricket, Gryllus bimaculatus. The contig sequence revealed the presence of five novel putative ASTBs (GbASTBs) in addition to GbASTBs previously identified in G. bimaculatus. MALDI-TOF MS analyses revealed the presence of these novel and previously identified GbASTBs with three missing GbASTBs. We also identified a cDNA encoding G. bimaculatus GbASTB receptor (GbASTBR) in the NGS data. Phylogenetic analysis demonstrated that this receptor was highly conserved with other insect ASTBRs, including the sex peptide receptor of Drosophila melanogaster. Calcium imaging analyses indicated that the GbASTBR heterologously expressed in HEK293 cells exhibited responses to all identified GbASTBs at a concentration range of 10(-10)-10(-5)M.
Subject(s)
Gryllidae/genetics , Insect Proteins/metabolism , Neuropeptides/metabolism , Receptors, Neuropeptide/metabolism , Animals , Cloning, Molecular , DNA, Complementary , Female , Gene Expression , Gryllidae/chemistry , HEK293 Cells/metabolism , Humans , Insect Proteins/genetics , Neuropeptides/chemistry , Neuropeptides/genetics , Receptors, Neuropeptide/genetics , Spectrometry, Mass, Matrix-Assisted Laser Desorption-IonizationABSTRACT
Noninvasive genetic sampling enables biomonitoring without the need to directly observe or disturb target organisms. This paper describes a novel and promising source of noninvasive spider and insect DNA from spider webs. Using black widow spiders (Latrodectus spp.) fed with house crickets (Acheta domesticus), we successfully extracted, amplified, and sequenced mitochondrial DNA from spider web samples that identified both spider and prey to species. Detectability of spider DNA did not differ between assays with amplicon sizes from 135 to 497 base pairs. Spider and prey DNA remained detectable at least 88 days after living organisms were no longer present on the web. Spider web DNA as a proof-of-concept may open doors to other practical applications in conservation research, pest management, biogeography studies, and biodiversity assessments.
Subject(s)
Black Widow Spider/genetics , DNA/genetics , Fibroins/genetics , Gryllidae/genetics , Polymerase Chain Reaction/methods , Animals , Conservation of Natural Resources , DNA/isolation & purification , DNA Barcoding, Taxonomic/methods , DNA Primers/chemical synthesis , Female , Fibroins/isolation & purification , Predatory Behavior/physiologyABSTRACT
To search for an insect homologue of antioxidant protein 1 (ATX1), a mole cricket, Gryllotalpa orientalis, cDNA library was screened and a cDNA clone, which encodes a 73 amino acid polypeptide with a predicted molecular mass of 8.0 kDa and pI of 5.68, was isolated. The G. orientalis ATX1 (GoATX1) cDNA features both a MTCXXC copper-binding site in the N-terminus and a KTGK lysine-rich region in the C-terminus. The deduced amino acid sequence of the GoATX1 cDNA showed 63% identity to Drosophila melanogaster ATX1 and 55% to Ixodes pacificus ATX1. Northern blot analysis revealed the presence of GoATX1 transcripts in midgut, fat body, and epidermis. When H2O2 was injected into the body cavity of G. orientalis adult, GoATX1 mRNA expression was up-regulated in the fat body tissue. Fat body expression level of GoATX1 mRNA in the fat body was increased following exposure to low (4 degrees C) and high (37 degrees C) temperatures, suggesting that GoATX1 plays a protective role against oxidative stress caused by temperature shock. This is the first report about a functional role of insect ATX1 in antioxidant defense.
Subject(s)
Antioxidants/metabolism , Gryllidae/genetics , Gryllidae/metabolism , Insect Proteins/genetics , Insect Proteins/metabolism , Amino Acid Sequence , Animals , Base Sequence , Blotting, Northern , Cloning, Molecular , Copper/metabolism , DNA, Complementary/genetics , Female , Hydrogen Peroxide/metabolism , Insect Proteins/biosynthesis , Male , Molecular Sequence Data , Phylogeny , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Reverse Transcriptase Polymerase Chain Reaction , Sequence Alignment , Up-RegulationABSTRACT
A Cu,Zn superoxide dismutase (SOD1) cDNA was cloned from the mole cricket, Gryllotalpa orientalis. The G. orientalis SOD1 (GoSOD1) cDNA contains an open reading frame of 462 bp encoding 154 amino acid polypeptide with a predicted molecular mass of 15.8 kDa and pI of 6.1, and possesses the typical metal binding ligands of six histidines and one aspartic acid common to SOD1s. The deduced amino acid sequence of the GoSOD1 cDNA showed 75% identity to Lasius niger SOD1, 73% to Apis mellifera SOD1, and 70-68% to SOD1 sequences from other insects. Northern blot analysis revealed the presence of GoSOD1 transcripts in all tissues examined. The expression level of GoATX1 mRNA in the fat body was induced when G. orientalis adult was exposed at low (4 degrees C) and high (37 degrees C) temperatures, suggesting that the GoSOD1 seems to play a protective role against oxidative stress caused by temperature shock.