Effects of Methoxyfenozide-Loaded Fluorescent Mesoporous Silica Nanoparticles on Plutella xylostella (L.) (Lepidoptera: Plutellidae) Mortality and Detoxification Enzyme Levels Activities.

The diamond back moth, Plutella xylostella, causes severe damage at all crop stages, beside its rising resistance to all insecticides. The objective of this study was to look for a new control strategy such as application of insecticide-loaded carbon dot-embedded fluorescent mesoporous silica nanoparticles (FL-SiO2 NPs). Two different-sized methoxyfenozide-loaded nanoparticles (Me@FL-SiO2 NPs-70 nm, Me@FL-SiO2 NPs-150 nm) were prepared, with loading content 15% and 16%. Methoxyfenozide was released constantly from Me@FL-SiO2 NPs only at specific optimum pH 7.5. The release of methoxyfenozide from Me@FL-SiO2 NPs was not observed other than this optimum pH, and therefore, we checked and controlled a single release condition to look out for the different particle sizes of insecticide-loaded NPs. This pH-responsive release pattern can find potential application in sustainable plant protection. Moreover, the lethal concentration of the LC50 value was 24 mg/L for methoxyfenozide (TC), 14 mg/L for Me@FL-SiO2 NPs-70 nm, and 15 mg/L for Me@FL-SiO2 NPs-150 nm after 72 h exposure, respectively. After calculating the LC50, the results predicted that Me@FL-SiO2 NPs-70 nm and Me@FL-SiO2 NPs-150 nm exhibited better insecticidal activity against P. xylostella than methoxyfenozide under the same concentrations of active ingredient applied. Moreover, the activities of detoxification enzymes of P. xylostella were suppressed by treatment with insecticide-loaded NPs, which showed that NPs could also be involved in reduction of enzymes. Furthermore, the entering of FL-SiO2 NPs into the midgut of P. xylostella was confirmed by confocal laser scanning microscope (CLSM). For comparison, P. xylostella under treatment with water as control was also observed under CLSM. The control exhibited no fluorescent signal, while the larvae treated with FL-SiO2 NPs showed strong fluorescence under a laser excitation wavelength of 448 nm. The reduced enzyme activities as well as higher cuticular penetration in insects indicate that the nano-based delivery system of insecticide could be potentially applied in insecticide resistance management.

Two-step method for rapid isolation of genomic DNA and validation of R81T insecticide resistance mutation in Myzus persicae.

Isolation and amplification of nucleic acid (DNA) is considered a vital and potent instrument in molecular biological research. However, its functioning outside of a laboratory setting is difficult because of complex procedures that demand expert personnel and expensive equipment in addition to the fulfillment of several additional requirements. DNA isolation from minute insects is sometimes difficult, making diagnostic and genotyping procedures problematic. Thus, the current work offers a high-throughput, cost-effective, straightforward, and faster approach for isolating DNA from the aphid Myzus persicae. Intriguingly, two-step DNA extraction process yielded a high yield of extremely pure genomic DNA and required only 10 s to complete. PCR investigation aiming at amplifying the non-synonymous R81T region on the loop D site of the nAChR gene of M. persicae was subsequently utilized to successfully validate the recovered DNA. Moreover, the proposed method was compared in terms of yield and purity with conventionally used DNA isolation methods including, phenol:chloroform, salt out, and commercially available kits. In conclusion, this newly developed method would enable researchers to quickly process many biological samples used to analyze genetic diversity, mutant screening, and large spectrum diagnosis both in laboratory and field conditions.

Neonicotinoid's resistance monitoring, diagnostic mechanisms and cytochrome P450 expression in green peach aphid [Myzus persicae (Sulzer) (Hemiptera: Aphididae)].

Green peach aphid [Myzus persicae (Sulzer) (Hemiptera: Aphididae)] is a significant pest with a known history of insecticide resistance. Neonicotinoids could manage this pest; however, their frequent use led to the evolution of resistance in field populations of M. persicae. Toxicity data for neonicotinoid insecticides synergized with pipernyl butoxide (PBO) in a field population (FP) were collected and compared to a laboratory susceptible clone (SC) of aphids. The enhanced expression of metabolic resistance-related cytochrome P450 gene CYP6CY3 and an arginine-threonine substitution were detected in FP, causing a single point mutation (R81T) at ß1 subunit of nicotinic acetylcholine receptor (nAChR) within D loop. High level of resistance to imidacloprid was developed in FP with 101-fold resistance ratio and moderate resistance level (10.9-fold) to acetamiprid. The results of PBO synergized bioassay suggested that cytochrome P450 enzymes were involved in the resistance to neonicotinoids. The mRNA transcriptional level of CYP6CY3 gene was significantly higher (3.74 fold) in FP compared to SC. The R81T mutation associated with neonicotinoid resistance had 26% resistant allele frequency in FP. Both P450 enzymes and R81T mutation of nAChR were found in field-evolved neonicotinoid resistance. It is concluded that field-evolved resistance in green peach aphid could be managed by using appropriate synergists such as PBO.

Embryonic development and oxidative stress effects in the larvae and adult fish livers of zebrafish (Danio rerio) exposed to the strobilurin fungicides, kresoxim-methyl and pyraclostrobin.

Two important strobilurin fungicides, kresoxim-methyl and pyraclostrobin, are widely used globally. Their effects on embryonic development and oxidative stress effects in the larvae and adult fish livers of zebrafish (Danio rerio) were assessed in our study. The hatching, mortality, and teratogenic rates were determined when the eggs of fish were exposed to kresoxim-methyl and pyraclostrobin for 24-144 h postfertilization (hpf). For further study, the effects of kresoxim-methyl and pyraclostrobin on antioxidant enzymes [catalase (CAT), superoxide dismutase (SOD) and peroxidase (POD)], detoxification enzymes [carboxylesterase (CarE) and glutathione S-transferase (GST)] and the malondialdehyde (MDA) content of larval zebrafish (96 h) and male or female adult zebrafish livers (up to 28 d) were evaluated for potential toxicity mechanisms. The study of embryonic development revealed that both kresoxim-methyl and pyraclostrobin caused developmental toxicity (hatching inhibition, mortality, and teratogenic rates) increase with significant concentration- and time-dependent responses, and the 144-h median lethal values (LC50) of kresoxim-methyl and pyraclostrobin were 195.0 and 81.3 µg L-1, respectively. In the larval zebrafish study, both kresoxim-methyl and pyraclostrobin at the highest concentrations (100 µg L-1 and 15 µg L-1, respectively) significantly increased the CAT, POD and CarE activities and MDA content compared with those of the control group (P < 0.05). We further found that oxidative stress effects in adult zebrafish livers caused by long-term kresoxim-methyl and pyraclostrobin exposure differed with time and sex. Regarding the residues in natural waters, the potential adverse effects of kresoxim-methyl and pyraclostrobin would be relatively low for adult zebrafish but must not be overlooked for zebrafish embryos/larvae (hatching impairment). Our results from the detoxification enzyme study also initially indicated that adult zebrafish had a greater detoxification ability than larvae and that males had a greater detoxification ability than females.

Effects of temperature on baseline susceptibility and stability of insecticide resistance against Plutella xylostella (Lepidoptera: Plutellidae) in the absence of selection pressure.

Plutella xylostella L. (Lepidoptera: Plutellidae) is an important pest causing significant losses to vegetables worldwide. Insecticides resistance in P. xylostella is a serious issue for scientists since last 30 years. However, deltamethrin and Bt Cry1Ac are commonly used insecticides against P. xylostella but studies involving development of resistance in P. xylostella against these two insecticides at different temperatures are lacking. The current study was aimed to find out the toxicity of deltamethrin and Bt Cry1Ac, and resistance development in P. xylostella. Results showed that the positive correlation between the temperature and toxicities of deltamethrin and Bt Cry1Ac. The results indicated -0.051, -0.049, -0.047, and -0.046 folds of deltamethrin resistance at 15 °C, 20 °C, 25 °C, and 30 °C temperatures, respectively from 1st to 12th generations. The toxicity of Bt Cry1Ac after 24 h was 2.2 and 4.8 folds on 1st generation at 20 °C and 25 °C temperatures, respectively compared to the toxicity recorded at 15 °C (non-overlapping of 95% confidence limits). Based on the results of this study, it is concluded that the temperature has a positive correlation with the toxicity of deltamethrin and Bt Cry1Ac against the larvae of P. xylostella. This study suggests that deltamethrin and Bt Cry1Ac can be included in the management program of P. xylostella on many vegetable crops. The baseline susceptibility data might be helpful to understand the resistance mechanisms in P. xylostella.

Assessment of the potential of a reduced dose of dimethyl disulfide plus metham sodium on soilborne pests and cucumber growth.

Methyl bromide (MB), a dominant ozone-depleting substance, is scheduled to be completely phased out for soil fumigation by December 30th 2018, in China. The combined effects of dimethyl disulfide (DMDS) plus metham sodium (MNa) were assessed in controlling soilborne pests for soil fumigation. A study was designed in laboratory for the evaluation of the efficacy of DMDS + MNa to control major soilborne pests. At the same time, two trials were conducted in cucumber field located in Tongzhou (in 2012) and Shunyi (in 2013), respectively, in order to assess the potential of DMDS + MNa in controlling soilborne pests. Laboratory studies disclosed positive synergistic effects of almost all four used combinations on Meloidogyne spp., Fusarium spp., Phytophthora spp., Abutilon theophrasti and Digitaria sanguinalis. Field trials found that DMDS + MNa (30 + 21 g a. i. m-2), both at a 50% reduced dose, effectively suppressed Meloidogyne spp. with a low root galling index (2.1% and 11.7%), significantly reduced the levels of Phytophthora and Fusarium spp. with a low root disease index (7.5% and 15.8%), gave very high cucumber yields (6.75 kg m-2 and 10.03 kg m-2), and increased income for cucumber growers with the highest economic benefits (20.91 ¥ m-2 and 23.58 ¥ m-2). The combination treatment provided similar results as MB standard dose treatment (40 g a. i. m-2) or DMDS standard dose treatment (60 g a. i. m-2) in pest control and yield, but was more effective than MNa standard dose treatment (42 g a. i. m-2). Usage of all chemical treatments gave better significant results than the untreated group of control. Considering the economic benefits, the DMDS plus MNa combination (30 + 21 g a. i. m-2) could be used for soil fumigation in cucumber production in China.

Fitness parameters of Plutella xylostella (L.) (Lepidoptera; Plutellidae) at four constant temperatures by using age-stage, two-sex life tables.

Different temperature zones have significant impact on the population dynamics of Plutella xylostella. Effective management of P. xylostella requires the knowledge of temperature tolerance by different life stages. In the current study, fitness parameters of diamondback moth were reported by using age-stage, two-sex life table traits at four constant temperatures (15, 20, 25 and 30 °C). The life cycle of P. xylostella was significantly longer at 15 °C. The 20 °C level of temperature was found optimal for fecundity, gross reproductive rate (51.74 offspring) and net reproductive rate (44.35 offspring per individual). The adult pre-oviposition period was statistically at par at all four level of temperatures. However, the survival was maximum at 20 °C as compared to other three temperature ranges. Based on the current study, it was concluded that temperature has a great role in population build-up of P. xylostella and effective management tactics should be applied to prevent significant damage to cabbage and other cruciferous crops when the temperature in the field is near 20 °C.

Evaluation of Insecticides induced hormesis on the demographic parameters of Myzus persicae and expression changes of metabolic resistance detoxification genes.

Insecticide induced-hormesis is a bi-phasic phenomenon generally characterized by low-dose induction and high-dose inhibition. It has been linked to insect pest outbreaks and insecticide resistance, which have importance in the integrated pest management (IPM). In this paper, hormesis effects of four insecticides on demographic parameters and expression of genes associated with metabolic resistance were evaluated in a field collected population of the green peach aphid, Myzus persicae Sulzer. The bioassay results showed that imidacloprid was more toxic than acetamiprid, deltamethrin and lambda-cyhalothrin. After exposure to sublethal doses of acetamiprid and imidacloprid for four generations, significant prolonged nymphal duration and increased fecundity were observed. Subsequently, mean generation time (T) and gross reproductive rate (GRR) was significantly increased. Moreover, expression of CYP6CY3 gene associated with resistance to neonicotinoids was increased significantly compared to the control. For pyrethriods, across generation exposure to sublethal doses of lambda cyhalothrin and deltamethrin prolonged the immature development duration. However, the expression of E4 gene in M. persicase was decreased by deltamethrin exposure but increased by lambda cyhalothrin. Based on results, demographic fitness parameters were effected by hormetic dose and accompanied with detoxifying genes alteration, hence, which would be evaluated in developing optimized insect pest management strategies.

Computational modeling and functional characterization of a GgChi: A class III chitinase from corms of Gladiolus grandiflorus.

The present study describes the predicted model and functional characterization of an endochitinase (30 kDa) from corms of Gladiolus grandiflorus. ESI-QTOF-MS generated peptide showed 96% sequence homology with family 18, Class III acidic endochitinase of Gladiolus gandavensis. Purified G. grandiflorus chitinase (GgChi) hydrolyzed 4-methylumbelliferyl ß-d-N,N',N''-triacetylchitotriose substrate showing specific endochitinase activity. Since no structural details of GgChi were available in the Protein Data Bank (PDB), a homology model was predicted using the coordinate information of Crocus vernus chitinase (PDB ID: 3SIM). Ramachandran plot indicated 84.5% in most favored region, 14.8% in additional and 0.6% in generously allowed region while no residue in disallowed region. The predicted structure indicated a highly conserved (ß/α)8 (TIM barrel) structure similar to the family 18, class III chitinases. The GgChi also showed sequence and structural homologies with other active chitinases. The GgChi (50 µg/disc) showed no antibacterial activity, but did provide mild growth inhibition of phytopathogenic fungus Fusarium oxysporum at a concentration of 500 µg/well Similarly, insect toxicity bioassays of GgChi (50 µg) against nymphs of Bemisia tabaci showed 14% reduction in adult emergence and 14% increase in mortality rate in comparison to control values. The GgChi (1.5 mg) protein showed significant reduction in a population of flour beetle (Tribolium castaneum) after 35 days, but lower reactivity against rice weevil (Sitophilus oryzae). The results of this study provide detai.led insight on functional characterization of a family 18 class III acidic plant endochitinase.

Replacing methyl bromide with a combination of 1,3-dichloropropene and metam sodium for cucumber production in China.

The combination of 1,3-dichloropropene (1,3-D) and metam sodium (MNa) is a potential resource to replace methyl bromide (MB) as a soil fumigant. The efficacy of 1,3-D+MNa as a crucial factor to limit soil-borne pests was evaluated in one laboratory experiment and two cucumber greenhouse studies conducted in commercial operations. Laboratory results revealed that 1,3-D and MNa (10+20 mg a.i. kg-1 soil) provided the best complementary control of the root-knot nematode, Fusarium oxysporum and two species of weed seeds. Greenhouse trials revealed that the blend of 1,3-D and MNa (10+20 g a.i. m-2) greatly inhibited the ability of Meloidogyne incognita to form root galls. In addition, the number of colony-forming units of F. oxysporum declined substantially after growth on media, resulting in higher fruit yields and greater economic benefits. The combined use of 1,3-D and MNa exhibited a higher control efficacy than when 1,3-D or MNa was utilized alone. The ability of this chemical combination to control soil-borne organisms did not differ significantly from the MB treatment and maintained high cucumber yields, enhancing the income of the farmers. Compared to the untreated control group, all the chemical treatments prominently improved the control of the pests. These results show that applying a combination of 1,3-D and MNa provides a promising alternative to MB that enables the sustained growth of cucumber production in China.