摘要: MicroRNAs (miRNAs) are regulatory RNA molecules that bind to target messenger RNAs (mRNAs) and affect the stability or translational efficiency of the bound mRNAs. Single or dual-luciferase reporter systems have been successfully used to identify miRNA target genes in mammalian cells. These reporter systems, however, are not sensitive enough to verify miRNA-target gene relationships in insect cell lines because the promoters of the target luciferase (usually Renilla) used in these reporter systems are too weak to drive sufficient expression of the target luciferase in insect cells. In this study, we replaced the SV40 promoter in the psiCHECK-2 reporter vector, which is widely used with mammalian cell lines, with the HSV-TK or AC5.1 promoter to yield two new dual-luciferase reporter vectors, designated psiCHECK-2-TK and psiCHECK-2-AC5.1, respectively. Only psiCHECK-2 and psiCHECK-2-AC5.1 had suitable target (Renilla)/reference (firefly) luciferase activity ratios in mammalian (HeLa and HEK293) and insect (Sf9, S2, Helicoverpa zea fat body and ovary) cell lines, while psiCHECK-2-TK had suitable Renilla/firefly luciferase activity ratios regardless of the cell line. Moreover, psiCHECK-2-TK successfully detected the interaction between Helicoverpa armigera miRNA9a and its target, the 3 '-untranslated region of heat shock protein 90, in both mammalian and H. zea cell lines, but psiCHECK-2 failed to do so in H. zea cell lines. Furthermore, psiCHECK-2-TK with the target sequence, HzMasc (H. zea Masculinizer), accurately differentiated between H. zea cell lines with or without the negative regulation factor (miRNA or piRNA) of HzMasc. These data demonstrate that psiCHECK-2-TK can be used to functionally characterize small RNA target genes in both mammalian and insect cells.

摘要: The wood-feeding termite Coptotermes formosanus represents a unique and impressive system for lignocellulose degradation. The highly efficient digestion of lignocellulose is achieved through symbiosis with gut symbionts like bacteria. Despite extensive research during the last three decades, diversity of bacterial symbionts residing in individual gut regions of the termite and their associated functions is still lacking. To this end, cellulose, xylan, and dye-decolorization bacteria residing in foregut, midgut, and hindgut regions of C. formosanus were enlisted by using enrichment and culture-dependent molecular methods. A total of 87 bacterial strains were successfully isolated from different gut regions of C. formosanus which belonged to 27 different species of 10 genera, majorly affiliated with Proteobacteria (80%) and Firmicutes (18.3%). Among the gut regions, 37.9% of the total bacterial isolates were observed in the hindgut that demonstrated predominance of cellulolytic bacteria (47.6%). The majority of the xylanolytic and dye-decolorization bacteria (50%) were obtained from the foregut and midgut, respectively. Actinobacteria represented by Dietza sp. was observed in the hindgut only. Based on species richness, the highest diversity was observed in midgut and hindgut regions each of which harbored seven unique bacterial species. The members of Enterobacter, Klebsiella, and Pseudomonas were common among the gut regions. The lignocellulolytic activities of the selected potential bacteria signpost their assistance to the host for lignocellulose digestion. The overall results indicate that C. formosanus harbors diverse communities of lignocellulolytic bacteria in different regions of the gut system. These observations will significantly advance our understanding of the termite-bacteria symbiosis and their microbial ecology uniquely existed in different gut regions of C. formosanus, which may further shed a light on its potential values at termite-modeled biotechnology.

摘要: The E2F family of transcription factors is crucial for cell cycle progression and cell fate decisions. Although E2Fs have been widely studied in mammals, there have been few studies performed in insects. Here, we determined the function of E2F4 in the silkworm, Bombyx mori. We demonstrate that E2F proteins are highly conserved among species from lower animals to higher mammals. Overexpression of the BmE2F4 gene led to cell cycle arrest in the G1 phase, whereas interfering with the BmE2F4 mRNA led to accumulation of cells in the S phase. These results indicate that BmE2F4 is important in cell cycle regulation. We also demonstrate that the BmE2F4 gene is involved in DNA replication of BmN-SWU1 cells and DNA synthesis in the silk gland. Furthermore, we identified a protein called Bm14-3-3 zeta that can interact with BmE2F4 and allow it to localize in the nucleus. Overexpression of the Bm14-3-3 zeta gene led to cell cycle arrest in the G1 phase, while knocking down the gene increased the proportion of cells in S phase. These findings provide important insights into the function of E2F transcription factors and increase our understanding of their involvement in cell cycle regulation.

摘要: Infections by mosquito-borne diseases represent one of the leading causes of death in third world countries. The rapid progression of resistance to conventional insecticide causes a significant threat to the highly efficient preventive methods currently in place. Insect neuropeptidergic system offers potential targets to control the insect vectors. The essential roles of the neuropeptide ecdysis triggering hormone (ETH) in insect development and reproduction led us to attempt understanding of the fundamentals of the biochemical interaction between ETH and its receptor in the African malaria mosquito Anopheles gambiae. One of two ETH peptides of the African malaria mosquito (AgETH1), a small peptide hormone with 17 amino acid residues (SESPGFFIKLSKSVPRI-NH2), was studied to elucidate its molecular structure. N-termini deletions and mutations of conserved amino acids in the ligand revealed the critical residues for the receptor activation. The solution structure of AgETH1 using 2D H-1-H-1 nuclear magnetic resonance (NMR) spectroscopy and nuclear overhauser effect (NOE) derived constraints revealed a short alpha helix between residues 3S and 11S. The NMR solution structure of AgETH1 will be of significant assistance for designing a new class of insecticidal compounds that acts on the AgETH receptor aiming for in silico docking studies.

摘要: Insect CAPA neuropeptidesare considered to affect water and ion balance by mediating the physiological metabolism activities of the Malpighian tubules. In previous studies, the CAPA-PK analogue 1895 (2Abf-Suc-FGPRLamide) was reported to decrease aphid fitness when administered through microinjection or via topical application. However, a further statistically significant decrease in the fitness of aphids and an increased mortality could not be established with pairwise combinations of 1895 with other CAPA analogue. In this study, we assessed the topical application of new combinations of 1895 with five CAPA-PVK analogues on the fitness of aphids. We found that 1895 and CAPA-PVK analogue 2315 (ASG-[beta L-3]-VAFPRVamide) was statistically the most effective combination to control the peach potato aphid Myzus persicae nymphs via topical application, leading to 72% mortality. Additionally, the combination (1895+2315) was evaluated against a selection of beneficial insects, that is, a pollinator (Bombus terrestris) and three natural enemies (Chrysoperla carnea, Nasonia vitripennis, and Adalia bipunctata). We found no significant influence on food intake, weight increase, and survival for the pollinator and the three representative natural enemies. These results could facilitate to further establish and generate CAPA analogues as alternatives to broad spectrum and less friendly insecticides.