Resistance to Beta-cypermethrin, Azadirachtin, and Matrine, and Biochemical Characterization of Field Populations of Oedaleus asiaticus (Bey-Bienko) in Inner Mongolia, Northern China.

Oedaleus asiaticus (Bey-Bienko) is an economically devastating locust species found in grassland and pastoral areas of the Inner Mongolia region of northern China. In this study, resistance to three frequently used insecticides (beta-cypermethrin, matrine, and azadirachtin) was investigated in six field populations of O. asiaticus using the leaf-dip bioassay method. The inhibitory effects of synergists and the activities of detoxification enzyme activities in the different populations were determined to explore potential biochemical resistance mechanisms. The results showed that the field populations SB (resistance ratio [RR] = 7.85), ZB (RR = 5.64), and DB (RR = 6.75) had developed low levels of resistance to beta-cypermethrin compared with a susceptible control strain. Both the SB (RR = 5.92) and XC (RR = 6.38) populations had also developed low levels of resistance against matrine, with the other populations remaining susceptible to both beta-cypermethrin and matrine. All field populations were susceptible to azadirachtin. Synergism analysis showed that triphenyl phosphate (TPP) and diethyl-maleate (DEM) increased the toxicity of beta-cypermethrin significantly in the SB population, while the synergistic effects of TPP, piperonyl butoxide (PBO), and DEM on the toxicity of matrine were higher in SB (SR 3.86, 4.18, and 3.07, respectively) than in SS (SR 2.24, 2.86, and 2.29, respectively), but no synergistic effects of TPP, PBO, and DEM on azadirachtin were found. Biochemical assays showed that the activities of carboxylesterases (CarEs) and glutathione-S-transferases (GSTs) were significantly raised in all field populations of O. asiaticus, with a significant positive correlation observed between beta-cypermethrin resistance and CarE activity. The activities of cytochrome P450 monooxygenases (P450) and multi-function oxidases (MFO) were elevated in all six field populations, and P450 activity displayed strong positive correlations with the three insecticides. Our findings suggest that resistance to beta-cypermethrin in O. asiaticus may be mainly attributed to elevated CarE and GST activities, while P450 plays an important role in metabolizing matrine and azadirachtin. Our study provides insights that will help improve insecticide resistance management strategies.

Phenotypic and Differential Gene Expression Analyses of Phase Transition in Oedaleus asiaticus under High-Density Population Stress.

The high-density-dependent phase change from solitary to gregarious individuals in locusts is a typical example of phenotypic plasticity. However, the underlying molecular mechanism is not clear. In this study, first, Oedaleus asiaticus were treated with high-density population stress and then analyzed by Illumina sequencing on days 1, 3, 5, and 7 of the body color change to identify the stage-specific differentially expressed genes (DEGs). The KEGG pathway enrichment analysis of the identified DEGs revealed their role in metabolic pathways. Furthermore, the expression patterns of the nine key DEGs were studied in detail; this showed that the material change in locusts began on the third day of the high-density treatment, with the number of DEGs being the largest, indicating the importance of this period in the phase transition. In addition, the phenotypic change involved several key genes of important regulatory pathways, possibly working in a complex network. Phenotypic plasticity in locusts is multifactorial, involving multilevel material network interactions. This study improves the mechanistic understanding of phenotypic variation in insects at the genetic level.

Synergy in Efficacy of Artemisia sieversiana Crude Extract and Metarhizium anisopliae on Resistant Oedaleus asiaticus.

In order to explore the synergistic control effect of crude extracts of Artemisia sieversiana and Metarhizium anisopliae on Oedaleus asiaticus, we used different doses of M. anisopliae and crude extracts of A. sieversiana singly and in combination, to determine their toxicities to fourth instar O. asiaticus. The results showed that the combination of 10% crude extract of A. sieversiana with 107 and 108 spores/g M. anisopliae concentrations and the combination of 20% crude extract of A. sieversiana with 107 and 108 spores/g M. anisopliae concentrations had significant effects on the mortality, body weight gain, body length gain, growth rate, and overall performance of O. asiaticus than those of the crude extract of A. sieversiana and M. anisopliae alone. Among them, the 20% A. sieversiana crude extract mixed with 108 spores/g M. anisopliae and 10% A. sieversiana crude extract combined with 107 spores/g M. anisopliae, had the best control efficacy. In order to clarify the biochemical mechanism underlying the immune responses of O. asiaticus to the pesticide treatments, we monitored the activities of four enzymes: superoxidase dismutase (SOD), peroxidase (POD), catalase (CAT), and polyphenol oxidase (PPO). The results showed that the activities of three enzymes (SOD, CAT, and PPO) were significantly increased from the treatment with the combination of M. anisopliae mixed with crude extract of A. sieversiana. Interestingly, compared to the crude extract, the combination treatment did not significantly induce the expression of POD enzyme activity, which may be a biochemical factor for increasing the control effect of the combination treatment. Our results showed that the combination treatment had synergistic and antagonistic effects on host mortality, growth, development, and enzyme activities in O. asiaticus.

Migratory Take-Off Behaviour of the Mongolian Grasshopper Oedaleus asiaticus.

Oedaleus asiaticus is one of the dominant species of grasshoppers in the rangeland on the Mongolian plateau, and a serious pest, but its migratory behavior is poorly known. We investigated the take-off behavior of migratory O. asiaticus in field cages in the inner Mongolia region of northern China. The species shows a degree of density-dependent phase polyphenism, with high-density swarming populations characterized by a brown morph, while low-density populations are more likely to comprise a green morph. We found that only 12.4% of brown morphs engaged in migratory take-off, and 2.0% of green morphs. Migratory grasshoppers took off at dusk, especially in the half hour after sunset (20:00-20:30 h). Most emigrating individuals did not have any food in their digestive tract, and the females were mated but with immature ovaries. In contrast, non-emigrating individuals rarely had empty digestive tracts, and most females were mated and sexually mature. Therefore, it seems clear that individuals prepare for migration in the afternoon by eliminating food residue from the body, and migration is largely restricted to sexually immature stages (at least in females). Furthermore, it was found that weather conditions (particularly temperature and wind speed at 15:00 h) in the afternoon had a significant effect on take-off that evening, with O. asiaticus preferring to take off in warm, dry and calm weather. The findings of this study will contribute to a reliable basis for forecasting migratory movements of this pest.

Three Chemosensory Proteins Involved in Chemoreception of Oedaleus asiaticus (Orthopera: Acridoidea).

Chemosensory proteins (CSPs) are thought to play roles in the insect olfactory system by binding and carrying hydrophobic odorants across the aqueous sensillar lymph. The band-winged grasshopper, Oedaleus asiaticus Bei-Bienko, is one of the most important grasshopper pests in northern China, but there is little information about its olfactory system. In order to investigate the olfactory functions of CSPs in this pest, three CSP genes (OasiCSP4, OasiCSP11 and OasiCSP12) were expressed in Escherichia coli, and the binding affinities of the three recombinant CSP proteins were measured for 16 volatiles from the host plant (Stipa krylovii), fecal material and body of live adult O. asiaticus using fluorescence competitive binding assays. To further verify their olfactory functions, RNA interference (RNAi) and electrophysiological recording were conducted. The three recombinant proteins displayed different degrees of binding to various volatiles in ligand-binding assays, with OasiCSP12 having higher binding affinities for more volatiles than OasiCSP4 and OasiCSP11. OasiCSP12 exhibited strong binding affinities (Ki < 20 µΜ) for five host plant volatiles and one volatile from the live body of adult O. asiaticus. The transcript levels of the three OasiCSP genes were significantly lower after silencing the individual genes by RNAi, which in turn reduced the EAG responses in adults of both sexes to most tested compounds. Our study indicates that these three OasiCSPs are involved in the detection of volatile semiochemicals, and may play important roles in finding host plants and in aggregation in O. asiaticus.

Role of PTP/PTK trans activated insulin-like signalling pathway in regulation of grasshopper (Oedaleus asiaticus) development.

Protein tyrosine phosphatase (PTPs) and protein tyrosine kinase (PTKs) genes are responsible for the regulation of insect insulin-like pathway (ILP), cells growth, metabolism initiation, gene transcription and observing immune response. Signal transduction in insect cell is also associated with PTPs and PTKs. The grasshopper (Oedaleus asiaticus) 'Bey-Bienko' were treated with dsRNA of protein tyrosine non-receptor type 4 (PTPN4) and protein tyrosine kinase 5 (PTK5) along with control (water). Applying dsPTK5 treatments in 5th instar of Oedaleus asiaticus, significant reduction was recorded in body dry mass, growth rate and overall performance except survival rate. Whereas with PTPN4, no such significant impact on all of these growth parameters was recorded. Expression of genes in ILP 5th instar of Oedaleus asiaticus by the application of dsPTPN4 and dsPTK5 revealed that PTK, INSR (insulin receptor), IRS (insulin receptor substrate), PI3K (phosphoinositide 3-kinase), PDK (3-phosphoinositide-dependent protein kinase), Akt (protein kinase B) and FOXO (forkhead transcription factor) significantly expressed with downregulation except PTPN4, which remained non-significant. On the other hand, the phosphorylation level of ILP four proteins in O. asiaticus with the treatment of dsPTPN4 and dsPTK5 significantly affected P-IRS and P-FOXO, while P-INSR and P-AKT remained stable at the probability level of 5%. This indicated that the stress response in the O. asiaticus insulin-like signalling pathway (ILP) reduced. Regarding association of protective enzymatic activities, ROS (relative oxygen species), CAT (catalase) and PO (phenol oxidase) increased significantly with exposure to dsPTK5 as compared to dsPTPN4 and control, while exposure of 5th instar of O. asiaticus to dsPTPN4 treatment slightly raised CAT and PO activities with but significant contribution. No such significant effect on MFO and POD was seen using dsPTPN4 and dsPTK5. This showed that in the ILP of O. asiaticus, PTK5 was detrimental to growth, body mass and overall performance, which ultimately benefited insect detoxification with high-energy cost.

Identification and expression profiling of candidate chemosensory membrane proteins in the band-winged grasshopper, Oedaleus asiaticus.

The band-winged grasshopper Oedaleus asiaticus (Orthoptera: Acridoidea) is an important insect pest in north China. Chemosensory membrane proteins are believed to be crucial factors in direct interactions with odorants in the chemosensory process. However, there is much limited information on the chemosensory system in this pest. In this study, a total of 69 candidate chemosensory membrane protein genes, including 60 olfactory receptors (ORs), 6 ionotropic receptors (IRs) and 3 sensory neuron membrane proteins (SNMPs), were identified for the first time from the antennal transcriptomes of O. asiaticus. Blastp match and phylogenetic analysis demonstrated that these chemosensory membrane proteins were the closest to their orthologous species, Locusta migratoria. The qRT-PCR analysis revealed that all tested 14 OR and two SNMP genes (OasiSNMP1 and OasiSNMP2a) were strongly expressed in adult antennae, and nearly all tested genes (15/16) displayed significant differences in the expression levels between both sexes. Moreover, two IR genes (OasiIR25a and OasiIR76b) had uniquely high expression levels in the antennae, labial palps and maxillary palps, while three IR genes (OasiIR1, OasiIR2 and OasiIR3) were highly expressed in most tested tissues (heads without antennae and mouthparts, labial palps, maxillary palps, labia without labial palps, thoraxes, tarsi, and abdomens) except for antennae, labra, and wings but OasiIR5a was just faintly expressed in the antennae, labia without labial palps, labial palps, maxillary palps and abdomen. Our results provide important molecular information for further investigation on the chemoreception mechanisms in O. asiaticus.

Molecular Ecological Basis of Grasshopper (Oedaleus asiaticus) Phenotypic Plasticity under Environmental Selection.

While ecological adaptation in insects can be reflected by plasticity of phenotype, determining the causes and molecular mechanisms for phenotypic plasticity (PP) remains a crucial and still difficult question in ecology, especially where control of insect pests is involved. Oedaleus asiaticus is one of the most dominant pests in the Inner Mongolia steppe and represents an excellent system to study phenotypic plasticity. To better understand ecological factors affecting grasshopper phenotypic plasticity and its molecular control, we conducted a full transcriptional screening of O. asiaticus grasshoppers reared in four different grassland patches in Inner Mongolia. Grasshoppers showed different degrees of PP associated with unique gene expressions and different habitat plant community compositions. Grasshopper performance variables were susceptible to habitat environment conditions and closely associated with plant architectures. Intriguingly, eco-transcriptome analysis revealed five potential candidate genes playing important roles in grasshopper performance, with gene expression closely relating to PP and plant community factors. By linking the grasshopper performances to gene profiles and ecological factors using canonical regression, we first demonstrated the eco-transcriptomic architecture (ETA) of grasshopper phenotypic traits (ETAGPTs). ETAGPTs revealed plant food type, plant density, coverage, and height were the main ecological factors influencing PP, while insect cuticle protein (ICP), negative elongation factor A (NELFA), and lactase-phlorizin hydrolase (LCT) were the key genes associated with PP. Our study gives a clear picture of gene-environment interaction in the formation and maintenance of PP and enriches our understanding of the transcriptional events underlying molecular control of rapid phenotypic plasticity associated with environmental variability. The findings of this study may also provide new targets for pest control and highlight the significance of ecological management practice on grassland conservation.

Gut Transcriptome Analysis Shows Different Food Utilization Efficiency by the Grasshopper Oedaleous asiaticus (Orthoptera: Acrididae).

Oedaleus asiaticus B. Bienko is a persistent pest occurring in north Asian grasslands. We found that O. asiaticus feeding on Stipa krylovii Roshev. had higher approximate digestibility (AD), efficiency of conversion of ingested food (ECI), and efficiency of conversion of digested food (ECD), compared with cohorts feeding on Leymus chinensis (Trin.) Tzvel, Artemisia frigida Willd., or Cleistogenes squarrosa (Trin.) Keng. Although this indicated high food utilization efficiency for S. krylovii, the physiological processes and molecular mechanisms underlying these biological observations are not well understood. Transcriptome analysis was used to examine how gene expression levels in O. asiaticus gut are altered by feeding on the four plant species. Nymphs (fifth-instar female) that fed on S. krylovii had the largest variation in gene expression profiles, with a total of 88 genes significantly upregulated compared with those feeding on the other three plants, mainly including nutrition digestive genes of protein, carbohydrate, and lipid digestion. GO and KEGG enrichment also showed that feeding S. krylovii could upregulate the nutrition digestion-related molecular function, biological process, and pathways. These changes in transcripts levels indicate that the physiological processes of activating nutrition digestive enzymes and metabolism pathways can well explain the high food utilization of S. krylovii by O. asiaticus.

Biological and ecological evidences suggest Stipa krylovii (Pooideae), contributes to optimal growth performance and population distribution of the grasshopper Oedaleus asiaticus.

Oedaleus asiaticus Bey. Bienko is a significant grasshopper pest species occurring in north Asian grasslands. Outbreaks often result in significant loss in grasses and economic losses. Interestingly, we found this grasshopper was mainly restricted to Stipa-dominated grassland. We suspected this may be related to the dominant grasses species, Stipa krylovii Roshev, and hypothesized that S. krylovii contributes to optimal growth performance and population distribution of O. asiaticus. A 4 year investigation showed that O. asiaticus density was positively correlated to the above-ground biomass of S. krylovii and O. asiaticus growth performance variables (survival rate, size, growth rate) were significantly higher in Stipa-dominated grassland. A feeding trial also showed that O. asiaticus had a higher growth performance when feeding exclusively on S. krylovii. In addition, the choice, consumption and the efficiency of conversion of ingested food (ECI) by O. asiaticus was highest for S. krylovii compared with other plant species found in the Asian grasslands. These ecological and biological traits revealed why O. asiaticus is strongly associated with Stipa-dominated grasslands. We concluded that the existence of S. krylovii benefited the growth performance and explained the distribution of O. asiaticus. These results are useful for improved pest management strategies and developing guidelines for the monitoring of grasshopper population dynamics against the background of vegetation succession and changing plant communities in response to activities such as grazing, fire and climate change.

Susceptibility and potential biochemical mechanism of Oedaleus asiaticus to beta-cypermethrin and deltamethrin in the Inner Mongolia, China.

Oedaleus asiaticus is a highly destructive grass pest in Inner Mongolia, China, and likely developed resistance to pyrethroid insecticides due to their frequent application for control of this locust. In this study, the susceptibility of five field populations of O. asiaticus to two pyrethroid insecticides was investigated. The Wulate Middle Banner (WB) population was the least susceptible, whereas the Ewenki Banner (EB) population appeared to be the most sensitive. The WB population was 3.16 and 5.15-fold less sensitive to beta-cypermethrin and deltamethrin than EB population, respectively. Further, the enzyme activities and mRNA expression levels of carboxylesterase (CarE) and glutathione-S-transferase (GST) were determined and we found that their activities in the WB population were 5.15 and 2.8-fold higher than those in the EB population, respectively. Quantitative real-time PCR (qRT-PCR) analysis demonstrated that the mRNA expression levels of CarE and GST genes were positively correlated with the LD50 in the WB, Siziwang Banner (SB) and EB populations. Our findings suggest that differences in susceptibility to pyrethroids in O. asiaticus might be attributed to the elevated activities and mRNA expression levels of CarE and GST genes.

Novel odorant-binding proteins and their expression patterns in grasshopper, Oedaleus asiaticus.

Insects use olfaction to detect exogenous odors and adapt to environments. In their olfaction systems, odorant-binding proteins (OBPs) are believed to be a key component. The unique OBP system of each species reflects the evolution of chemosensation of insects with habits. Here, we for the first time identified 15 OBPs, OasiOBP1-15, of a grasshopper, Oedaleus asiaticus, that lives in the grasslands of Northern China and is closely related to the locust, Locusta migratoria. OasiOBP9 and OasiOBP10 are specifically expressed in the antennae. Other OBPs are expressed in the antennae as well as other chemosensory organs, such as the mouthparts and wings. Significantly more OasiOBP7 was detected in male than female antennae, but there are 9 OBPs that were more expressed in female than male antennae by quantitative real-time PCR. Phylogenetic analysis indicated that most of the O. asiaticus OBPs are similar to those of L. migratoria, but some are substantially different. This indicates that the OBPs originally evolved in a common ancestor, but their unique chemosensory systems are adapted to different ecosystems.