Functional characterization of carboxylesterase gene mutations involved in Aphis gossypii resistance to organophosphate insecticides.

Carboxylesterases (CarEs) play an important role in detoxifying insecticides in insects. Over-expression and structural modification of CarEs have been implicated in the development of organophosphate (OP) insecticide resistance in insects. A previous study identified four nonsynonymous mutations (resulting in four amino acid residue substitutions) in the open reading frame of the carboxylesterase gene of resistant cotton aphids compared to the omethoate susceptible strain, which has possibly influenced the development of resistance to omethoate (a systemic OP insecticide). The current study further characterized the function of these mutations, both alone and in combination, in the hydrolysis of OP insecticides. The metabolism results suggest that the combination of four mutations, mainly existing in the laboratory-selected OP-resistant cotton aphid population, increased the OP hydrolase activity (approximately twofold) at the cost of detectable carboxylesterase activity. The functional studies of single or multiple mutations suggest the positive effect of H104R, A128V and T333P on the acquisition of OP hydrolase activity, especially the combination of H104R with A128V or T333P. K484R substitution decreased both the OP hydrolase activity and the CarE activity, indicating that this mutation primarily drives the negative effect on the acquisition of OP hydrolase activity amongst these four mutations in the resistant strain. The modelling and docking results are basically consistent with the metabolic results, which strongly suggest that the structural gene modification is the molecular basis for the OP resistance in this laboratory-selected cotton aphid strain.

Sublethal effects of imidacloprid on the fecundity, longevity, and enzyme activity of Sitobion avenae (Fabricius) and Rhopalosiphum padi (Linnaeus).

The aphid species Sitobion avenae and Rhopalosiphum padi are the most important pests in wheat growing regions of many countries. In this study, we investigated the sublethal effects of imidacloprid on fecundity, longevity, and enzyme activity in both aphid species by comparing 3-h exposure for one or three generations. Our results indicated that 3-h exposure to sublethal doses of imidacloprid for one generation had no discernible effect on the survival, fecundity, longevity, or enzyme activity levels of aphids. However, when pulse exposures to imidacloprid were sustained over three generations, both fecundity and longevity were significantly decreased in both S. avenae and R. padi. Interestingly, the fecundity of R. padi had almost recovered by the F5 generation, but its longevity was still deleteriously affected. These results indicated that R. padi laid eggs in shorter time lags and has a more fast resilience. The change in reproduction behavior may be a phenomenon of R. padi to compensate its early death. If this is stable for the next generation, it means that the next generation is more competitive than unexposed populations, which could be the reason underlying population outbreaks that occur after longer-term exposure to an insecticide. This laboratory-based study highlights the sublethal effects of imidacloprid on the longevity and fecundity of descendants and provides an empirical basis from which to consider management decisions for chemical control in the field.

Yeast one-hybrid screening the potential regulator of CYP6B6 overexpression of Helicoverpa armigera under 2-tridecanone stress.

In insect, the cytochrome P450 plays a pivotal role in detoxification to toxic allelochemicals. Helicoverpa armigera can tolerate and survive in 2-tridecanone treatment owing to the CYP6B6 responsive expression, which is controlled by some regulatory DNA sequences and transcription regulators. Therefore, the 2-tridecanone responsive region and transcription regulators of the CYP6B6 are responsible for detoxification of cotton bollworm. In this study, we used yeast one-hybrid to screen two potential transcription regulators of the CYP6B6 from H. armigera that respond to the plant secondary toxicant 2-tridecanone, which were named Prey1 and Prey2, respectively. According to the NCBI database blast, Prey1 is the homology with FK506 binding protein (FKBP) of Manduca sexta and Bombyx mori that belongs to the FKBP-C superfamily, while Prey2 may be a homology of an unknown protein of Papilio or the fcaL24 protein homology of B. mori. The electrophoretic mobility shift assays revealed that the FKBP of prokaryotic expression could specifically bind to the active region of the CYP6B6 promoter. After the 6th instar larvae of H. armigera reared on 2-tridecanone artificial diet, we found there were similar patterns of CYP6B6 and FKBP expression of the cotton bollworm treated with 10 mg g-1 2-tridecanone for 48 h, which correlation coefficient was the highest (0.923). Thus, the FKBP is identified as a strong candidate for regulation of the CYP6B6 expression, when the cotton bollworm is treated with 2-tridecanone. This may lead us to a better understanding of transcriptional mechanism of CYP6B6 and provide very useful information for the pest control.

Comparative studies on acetylcholinesterase characteristics between the aphids, Sitobion avenae and Rhopalosiphum padi.

The aphids Sitobion avenae (Fabricius) and Rhopalosiphum padi (Linnaeus) (Hemiptera: Aphidiae) are serious pests on grain crops and usually coexist on late period of wheat growth in China. Bioassays showed that R. padi was more susceptible than S. avenae to pirimicarb that is used for wheat aphid control, and the determination of acetylcholinesterase (AChE, EC 3.1.1.7) sensitivity showed that the sensitivity of AChE to pirimicarb was significantly higher in R. padi than in S. avenae ( Lu and Gao 2009 ). AChE is the target enzyme of the carbamates, including pirimicarb, hence, to understand the mechanism responsible for the tolerance difference to carbamate insecticides of S. avenae and R. padi, we purified AChE from both aphid species using procainamide affinity column and characterized the AChE. The purification factor and yield from S. avenae (234.7-fold and 92.9%) were far higher than that from R. padi 17.3-fold and 13.9%. The results of substrate and inhibitor specificities of purified enzyme from both S. avenae and R. padi indicated that the purified enzyme was a typical AChE. The crude AChE extract from S. avenae was 5.4-, 4.3- and 8.1-fold less sensitive to inhibition by pirimicarb, methomyl and thiodicarb, respectively, than that from R. padi, whereas for the purified AChE, S. avenae was only 1.6-, 1.3- and 1.7-fold less sensitive to inhibition by pirimicarb, methomyl and thiodicarb, respectively, than R. padi. This suggests that eserine and BW284C51 may bind with other proteins, such as carboxylesterase, in the crude extract to reduce their inhibition against AChE. These results are useful for planning the chemical control of aphids on wheat.

[Initial exploration of choice of the donor site of flap and its repair strategy].

Objective: To explore the choice of the donor site of flap and the repair method of secondary wound of flap donor site in tissue repair and reconstruction operation. Methods: From January 2014 to September 2018, 62 cases of scar contracture deformity, 15 cases of skin tumor, 20 cases of skin and soft tissue injury, and 25 cases of chronic wound were admitted to the Burn Center of People's Liberation Army of First Affiliated Hospital of Air Force Medical University, with 84 males and 38 females, aged from 3 to 89 years. Four repair strategies adopted for tissue repair and reconstruction and good repair of the donor site of flap were as follows: designing the flap rationally according to the condition around the wound or the size and shape of wound, choosing pre-expanded technique of the donor site of flap for repair of scar deformity optimally, making full use of the surrounding condition of flap donor site, and repaired with the distal flap, i. e. replacing the important site with secondary site. The donor site of flap was repaired by direct suture or peripheral flap and distal flap. The wound size of patients ranged from 3.0 cm×2.0 cm to 20.0 cm×18.0 cm, and the flap area ranged from 3.5 cm×2.0 cm to 25.0 cm×22.0 cm. The survival condition of flap, healing condition of donor site and recipient site, and follow-up condition of donor site and recipient site were recorded. Results: Wounds of 122 patients were repaired with a total of 148 flaps designed by the above four repair strategies. All the flaps survived well, and the wound and flap donor site healed well. Follow-up for 3 to 36 months showed that the shape and function of recipient site and flap donor site were satisfactory. Conclusions: According to the specific condition of the wound and anatomical structure of the surrounding tissue of flap donor site, overall surgical design with flexibility and personalization can achieve effects of good repair of the wound and reduce the secondary damage of flap donor site.

[Effects of ultrathin abdomen flap on repairing deep electric burn wounds in finger of pediatric patients].

Objective: To investigate the effects of ultrathin abdomen flap in repairing deep electric burn wounds in finger of pediatric patients. Methods: A total of 14 pediatric patients with simple electric burns in finger were admitted to our unit from March 2013 to October 2017. Six patients had electric burns in one finger, 5 patients had electric burns in two fingers, and 3 patients had electric burns in three fingers. The size of wounds in single finger ranged from 2.0 cm×1.0 cm to 3.5 cm×2.0 cm. After complete preoperative examination, wounds debridement and ultrathin abdomen flap repair operation were performed on 3 to 6 days post injury. Six pediatric patients were treated with abdominal random flap, 4 patients were treated with inferior epigastric artery paraumbilical perforator bilobed flap, and the other 4 patients were treated with superficial circumflex iliac artery bilobed flap. The size of flaps ranged from 4.0 cm×2.0 cm to 8.0 cm×4.0 cm. The donor sites were sutured directly. Results: The flaps of 14 pediatric patients survived well after operation, and no flap showed blood supplying disorder. During follow-up of 3 to 24 months, the appearance and function of fingers were good, and the donor sites recovered well, with no cicatrix contracture deformity. Conclusions: The ultrathin abdomen flap is one of the good choices for repairing deep electric burn wounds in finger of pediatric patients.

Induction of Cytochrome P450 Activity by the Interaction of Chlorantraniliprole and Sinigrin in the Spodoptera exigua (Lepidoptera: Noctuidae).

The joint toxicity of chlorantraniliprole, a novel insecticide that acts on ryanodine receptors, and sinigrin, a natural plant defense compound from brassicaceous vegetables, to the larvae of Spodoptera exigua (Hübner) was determined in this paper. Additionally, the joint effects of the two compounds on cytochrome P450 enzyme activity and on the expression levels of mRNA of three P450 genes (including CYP9A9, CYP6B, and CYP4G37) and an NADPH cytochrome P450 reductase gene (HQ852049) were investigated. The toxicity of the mixture of chlorantraniliprole and sinigrin to fourth-instar S. exigua larvae was 1.60-fold higher than the toxicity of the chlorantraniliprole-only treatment after 24 h. Induced by chlorantraniliprole and sinigrin, the specific activity of the P450 O-deethylase was affected in a time-, dose-, and organ-specific manner in fifth-instar S. exigua larvae. The effects were more pronounced in the midgut than in the fat body. The specific activity of the P450 O-deethylase in almost all treatments increased at 12, 24, and 36 h posttreatment compared with that in the control. Based on real-time PCR analyses, the expression levels of the P450 genes CYP9A9, CYP6B, and CYP4G37 and the NADPH cytochrome P450 reductase gene HQ852049 in fifth-instar S. exigua larvae were induced by chlorantraniliprole and sinigrin, and the trends were similar to the specific activity of the P450 O-deethylase. Therefore, the CYP9A9, CYP6B, and HQ852049 in the tested genes were the most inducible genes that were expressed when the S. exigua larvae were exposed to chlorantraniliprole and sinigrin.

Quantification and characterisation of arteries in retinal images.

A computerised system is presented for the automatic quantification of blood vessel topography in retinal images. This system utilises digital image processing techniques to provide more reliable and comprehensive information for the retinal vascular network. It applies strategies and algorithms for measuring vascular trees and includes methods for locating the centre of a bifurcation, detecting vessel branches, estimating vessel diameter, and calculating angular geometry at a bifurcation. The performance of the system is studied by comparison with manual measurements and by comparing measurements between red-free images and fluorescein images. In general an acceptable degree of accuracy and precision was seen for all measurements, although the system had difficulty dealing with very noisy images and small or especially tortuous blood vessels.

Comparison of the acute toxicity for gamma-cyhalothrin and lambda-cyhalothrin to zebra fish and shrimp.

Gamma-cyhalothrin 15CS (GCH) contains only the active stereoisomer of the two isomers found in lambda-cyhalothrin 25EW (LCH). GCH (0.5 x rate) provides equivalent overall insect control as LCH (1 x rate). Both formulations showed high acute toxicity to zebra fish (Brachydanio rerio H.B.) and shrimp (Macrobrachium nippoensis de Haan). The 96-h LC(50(zebra fish,GCH)) is 1.93 microg a.i/L and LC(50(zebra fish,LCH)) is 1.94 microg a.i/L. LC(50(shrimp,GCH)) is 0.28 microg a.i./L and LC(50(shrimp,LCH)) 0.04 microg a.i./L. This indicates that the toxicity to shrimp is likely stereochemistry-dependent. The fates of GCH and LCH are similar in laboratory simulated rice paddy water and their concentrations decrease rapidly, with no GCH or LCH detected after 3 or 4 days. Both are toxic to shrimp in a simulated paddy irrigation reservoir even though treated return water is diluted 5 times. No shrimp fatality is shown in the GCH-treated paddy water after a 4-day holding period, and longer than 5 days is necessary to reach a zero fatality rate for LCH. This is compatible with the 7-day water holding period considered reasonable in agricultural practice.

Inhibitory effects of four carbamate insecticides on acetylcholinesterase of male and female Carassius auratus in vitro.

The inhibitory effects of four carbamate insecticides, methomyl, thiodicarb, carbofuran and carbosulfan, on acetylcholinesterase of male and female Carassius auratus were investigated in the laboratory. Kinetic constants, biomolecular rate constant (k(i)), carbamylation rate constant (k2) and decarbamylation rate constant (k3) were determined in vitro. The ratios of bimolecular rate constant (female/male) for AChE reacting with methomyl, thiodicarb, carbofuran and carbosulfan were 1.03, 2.44, 1.03 and 1.106, respectively. The k(i) of AChE for thiodicarb was significantly higher in female fish than in male fish (P<0.05). The ratios of carbamylation rate constant (female/male) for methomyl, thiodicarb, carbofuran and carbosulfan were 1.18, 4.29, 3.53, and 2.07, respectively. The k2 values of AChE for the above four carbamates were significantly higher in females than in males. The ratios of the decarbamylation rate constant (female/male) for methomyl, thiodicarb, carbofuran and carbosulfan were 1.02, 1.39, 1.06, and 1.98, respectively. Only for carbosulfan, the decarbamylation rate of AChE was significantly higher in the female than in the male, indicating that AChE of females inhibited by carbosulfan recovered more quickly than that of males. The above results suggest that the female in the sensitivity of AChE to carbamates was different from the male fish.