Shisa reduces the sensitivity of homomeric RDL channel to GABA in the two-spotted spider mite, Tetranychus urticae Koch.

The γ-aminobutyric acid receptors (GABARs) mediate fast inhibitory transmission in central nervous system of insects and are important targets of insecticides. An auxiliary subunit, Shisa7, was identified in mammals as a single-passing transmembrane protein. However, the homology gene(s) of Shisa in invertebrates has not been reported to date. In the present study, a homolog Shisa gene was identified from the two-spotted spider mite, Tetranychus urticae Koch. Its open reading frame had 927 base pairs and encoded 308 amino acid residues, which has a typical Shisa domain at 13th-181st amino acid residues. According to the phylogenetic tree, the invertebrate Shisa was categorized apart with those of vertebrate, and TuShisa showed closest relationship with the Shisa9 of velvet mite, Dinothrombium tinctorium (L.). In the electrophysiological assay with two-electrode voltage clamp, the GABA-activated TuRDL channel was functionally formed in the Africa clawed frog Xenopus laevis (Daudin) oocytes (EC50 = 53.34 µM). No GABA-activated current could be observed in TuShisa-expressed oocytes, whereas TuShisa could reduce the sensitivity of TuRDL/TuShisa (mass ratio of 1: 4) channel to GABA. The homology structural models of TuRDL and TuShisa were built by the SWISS-MODEL server, their interaction was predicted using Z-DOCK and three predicted hydrogen bonds and interface residues were analysed by PyMOL. Meanwhile, the key interface residues of TuShisa affected the stability of complex were calculated by Discovery Studio 2019. In conclusion, the TuShisa, as the first reported invertebrate Shisa, was explored and functionally examined as the GABARs auxiliary subunit. Our findings provide a basis for research of invertebrate Shisa.

Molecular identification of four novel cytochrome P450 genes related to the development of resistance of Spodoptera exigua (Lepidoptera: Noctuidae) to chlorantraniliprole.

BACKGROUND: The beet armyworm, Spodoptera exigua, is an omnivorous insect that damages a variety of crops worldwide. Chlorantraniliprole is a new diamide insecticide that acts on the ryanodine receptors in insects. The aim of this study was to explore key genes related to the development of chlorantraniliprole resistance in S. exigua. RESULTS: Transcriptomes were compared between beet armyworms from a susceptible laboratory strain (Sus-Lab) and Sus-Lab screened with LC25 sublethal doses of chlorantraniliprole for six generations (SE-Sel). Ten of 11 cytochrome P450 genes with upregulated expression verified by quantitative reverse transcription polymerase chain reaction (qRT-PCR) in SE-Sel strains were detected in SE-PZ16 and SE-ZY16, two extremely resistant field populations. In addition, expression of four new cytochrome P450 genes, CYP9A21v1, CYP9A21v2, CYP9A21v3 and CYP9A21v4, was shown in the two field populations and was significantly higher in the SE-Sel strain than in the Sus-Lab strain (P < 0.05). Their full-length and protein tertiary structures were also cloned and predicted. The function of CYP9A21v3 was analysed by RNA interference, and the relative expression of CYP9A21v3 in the SE-ZY16 population after feeding on dsRNA was lower than in the control group. Moreover, mortality rates in insects treated at the LC50 of chlorantraniliprole after dsRNA feeding were significantly higher than in the control group 24 h after treatment (P < 0.05). CONCLUSION: Overexpression of CYP9A21v3 may be a primary factor in the development of chlorantraniliprole resistance in beet armyworms. © 2018 Society of Chemical Industry.

The Effect of Chemical Composition and Bioactivity of Several Essential Oils on Tenebrio molitor (Coleoptera: Tenebrionidae).

The major chemical components of four essential oils (EOs) extracted from dry leaves of Citrus limonum, Cymbopogon citratus, Litsea cubeba, and Muristica fragrans were analyzed with gas chromatograph-mass spectrometer and their fumigant, contact, and repellent activities against 10th instar and adults of Tenebrio molitor were also assayed. The results indicated that the major constituents of C. limonum and Cy. citrates were D-limonene (38.22%) and 3,7-dimethyl-6-octenal (26.21%), while which of L. cubeba and M. fragrans were (E)-3, 7-dimethyl-2, 6-octadienal (49.78%) and (E)-cinnamaldehyde (79.31%), respectively. Contact activities of L. cubeba and C. limonum with LC50 values of 21.2 and 13.9 µg/cm(2) at 48 h and repellence activities (>89.0% repellence indexes) (P < 0.05) at 12 h on 10th instar were better than those of the other two EOs. Nevertheless, the fumigation activities of L. cubeba on 10th instar and adults (LC50 = 2.7, 3.7 µl/liter) were stronger than those of C. limonum (LC50 = 10.9, 12.0 µl/liter) at 96 h and significant (not overlapping confidence intervals). The EOs of L. cubeba and C. limonum have clearly elongated the growth and development of larvae, egg, and slightly shorten pupae and adults of T. molitor compared with the control. The mainly active ingredients of L. cubeba and C. limonum, including D-limonene and ß-pinene, were demonstrated to coinhibit the actives of AChE and enhance the toxicities on 10th instar of T. molitor. These results indicate that the EOs of L. cubeba and C. limonum could have great potential as botanical insecticides against T. molitor.