The Odorant-Binding Protein 1 Mediates the Foraging Behavior of Grapholita molesta Larvae.

Since eggs are laid directly on fruit skin, it is typically believed that food odor has little impact on the foraging of Grapholita molesta larvae. It is crucial to note that larvae that hatch on twigs and leaves could need some sort of identification system when foraging. Here, 22 GmolOBP genes were identified from the G. molesta larval transcriptome via the comparison of conserved domain and homology in the protein level. GmolOBP1 had strong affinities for important pear-fruit volatiles, which caused larvae strong behavioral responses. However, after GmolOBP1 silencing, the larvae lost their attraction to methyl salicylate, α-farnesene, butyl acetate, ethyl butanoate, and ethyl hexanoate, and the effects of larvae seeking various pears were significantly reduced. Consequently, GmolOBP1 was required for the reception of pear volatiles and was involved in mediating how G. molesta larvae foraged. Our research revealed the GmolOBP1 foraging signal recognition mechanism as well as potential molecular targets for field pest management.

Effect of ultraviolet radiation on Beauveria bassiana virulence and development of protective formulations.

Locusta migratoria is a serious agricultural pest in China. Beauveria bassiana is one of the most important pathogens of grasshoppers and locusts. The effects of ultraviolet light were evaluated on the B. bassiana strain BbZJ1. The results showed that 253.7 and 360 nm wavelength UV (Ultra Violet) did not affect the germination of B. bassiana after its recovery from UV treatments. Nevertheless, the virulence of B. bassiana BbZJ1 after its recovery from radiation of UV (253.7 nm) increased. The mortality rates were 85.00% for the BbZJ1 control, was 96.67% for BbZJ1 recovered from radiation of UV (253.7 nm) for 60 min. After treatment with 253.7 nm UV radiation for 60 min, the expression levels of stress-resistant genes BbAlg9 and Bbadh2 in BbZJ1 strain were 2.68 and 2.29 times higher than those in the control group, respectively. Meanwhile, the B. bassiana prepared in 5% groundnut oil showed highest tolerance levels to the ultraviolet radiation. The 5% groundnut oil was the most suitable potential UV-protectant for B. bassiana in terms of cost and availability.

Coronatine-Based Gene Expression Changes Impart Partial Resistance to Fall Armyworm (Spodoptera frugiperda) in Seedling Maize.

In recent years, Spodoptera frugiperda (S. frugiperda, Smith) has invaded China, seriously threatening maize production. To explore an effective method to curb the further expansion of the harm of the S. frugiperda, this experiment used maize seedlings of the Zhengdan 958 three-leaf stage (V3) of maize as the material to study the effect of coronatine (COR) on the ability of maize to resist insects (S. frugiperda) at the seedling stage. The results showed that when maize was sprayed with 0.05 µM COR, the newly incubated larvae of S. frugiperda had the least leaf feeding. It was found that 0.05 µM COR significantly increased the contents of abscisic acid (ABA) and jasmonate (JA) in maize leaves through the determination of hormone content. Moreover, transcriptome sequencing revealed that the expression of six genes (ZmBX1, ZmBX2, ZmBX3, ZmBX4, ZmBX5 and ZmBX6), which are associated with the synthesis of benzoxazinoid, were upregulated. Nine genes (ZmZIM3, ZmZIM4, ZmZIM10, ZmZIM13, ZmZIM18, ZmZIM23, ZmZIM27, ZmZIM28 and ZmZIM38) of JA-Zim Domain (JAZ) protein in the JA signal pathway, and seven genes (ZmPRH19, ZmPRH18, Zm00001d024732, Zm00001d034109, Zm00001d026269, Zm00001d028574 and Zm00001d013220) of ABA downstream response protein Group A Type 2C Protein Phosphatase (PP2C) were downregulated. These results demonstrated that COR could induce anti-insect factors and significantly improve insect resistance in seedling maize, which laid a theoretical foundation for further study of the mechanism of COR improving insect resistance in seedling maize, and provided data references for the use of COR as an environmentally friendly pest control method.

Symbiotic Bacteria System of Locusta migratoria Showed Antifungal Capabilities against Beauveria bassiana.

The stability of symbiotic flora is an important indicator of the health of an organism. Symbiotic bacteria have been proven to be closely involved in the immune process of organisms. The pathogenicity of Beauveria bassiana was studied in relation to symbiotic bacteria on the surface and inside of the migratory locust (Locusta migratoria). The results showed that the surface disinfection of test locusts contributed to the pathogenicity of B. bassiana to locusts. Most of the surface bacteria of L. migratoria caused some inhibition of B. bassiana growth, and LM5-4 (Raoultella ornithinolytica), LM5-2 (Enterobacter aerogenes), and LM5-13 (Citrobacter freundii) showed the highest inhibitory effect on the growth of B. bassiana. The inoculation of locusts with additional surface symbiotic bacteria reduced the virulence of B. bassiana to L. migratoria. Infection by different strains of B. bassiana caused similar changes in the symbiotic flora of migratory locusts. The inoculation of locusts with additional intestinal symbiotic bacteria (Enterobacter sp.) reduced the virulence of B. bassiana to L. migratoria. These findings illustrate the effect of bacterial communities on fungal infections in L. migratoria when seen from the perspective of ecology in a microenvironment. The active antifungal substances of such bacteria and their mechanisms of action need further study.

Interference with orco gene expression affects host recognition in Diorhabda tarsalis.

Introduction: Diorhabda tarsalis Weise is an important insect pest of the Chinese licorice Glycyrrhiza uralensis Fisch. Behavior of the beetle, including host location, oviposition site selection, self-defense, and aggregation, were regulated by plant volatiles or insect pheromones. Aim: In this study, Identification of ORs and function research on orco were carried out, these could lead to the development of understand for olfaction mechanism in D. tarsalis. Methods: ORs were identified by PacBio RS II platform to sequence the antennas of adult D. tarsalis, the function of orco was explored by dsRNA interference. Results: 29 odorant receptor candidate genes of D. tarsalis were obtained, which code for 130-479 amino acids. Phylogenetic trees of olfactory receptors were constructed with 243 ORs from eight Coleoptera species. DtarORco, DtarOR7 and DtarOR26 are specifically expressed in the antenna, and the expression levels were significantly higher than other DtarORs in antenna, there were no differential expression between male and female beetles. An odorant coreceptor gene (DtarORco) has characteristics of an odorant receptor family member, the encoded mature protein has a predicted molecular weight of 53.898 kDa, dsRNA L4440 expression vectors were constructed and successfully transformed into ribonuclease III-deficient Escherichia coli strain HT115 DE3. After interference treatment, the relative expression level of DtarORco in D. tarsalis antennae significantly decreased and electrophysiological responses to host localization odor signals significantly decreased. At the same time, beetles lost the ability to locate hosts. Discussion: The research on its mechanism of olfaction may lead to the development of new control measures that are environmentally friendly.

Odorant degrading carboxylesterases modulate foraging and mating behaviors of Grapholita molesta.

Odorant degrading carboxylesterases (CXEs) play key roles in the process of odor signal reception via degrading ester odorants. But the functional mechanisms of CXEs in modulating insect behaviors are unclear. Herein, we studied the roles that CXEs played in mating, foraging, and signal receptions of sex pheromones and host volatiles in Grapholita molesta. As a result, 23 candidate CXEs were identified by transcriptome analysis of G. molesta. The GmolCXE1 and 5 highly expressed in the antennae of male moths and GmolCXE14 and 21 abundantly expressed in larval heads, were significantly upregulated after exposure with odors from female adults or fresh ripe fruits respectively. After knockdown of GmolCXE1 and 5, or GmolCXE14 and 21 by RNA interference, the behavioral responses of G. molesta to ester sex pheromones or host volatiles were decreased, by exhibiting an inhibited searching behavior of G. molesta for females or fruits, respectively. Then evidence form GC-MS analysis, showed that the protein GmolCXE1 and GmolCXE5 could metabolize the sex pheromone components (Z/E)-8-dodecenyl acetate to their metabolites products (Z/E)-8-dodecenol, and that GmolCXE14 and GmolCXE21 could metabolize ethyl butanoate and ethyl hexanoate of ripe pears. In addition, fluorescent binding assays verified that GmolCXEs could degrade the free ester odor molecules, but not degrade the odor molecules protected by odorant-binding proteins. Our study not only demonstrated CXEs modulated the mating and foraging behaviors of G. molesta through inactivation of ester sex pheromone and host volatiles, but also discovered great potential molecular targets to develop behavioral inhibitors for pest management.

Effects of a combined infection with Paranosema locustae and Beauveria bassiana on Locusta migratoria and its gut microflora.

Even though Paranosema locustae is widely used in China as a biological agent for controlling grasshoppers, the mortality rate is initially quite low. This study sought to determine whether the simultaneous use of P. locustae and Beauveria bassiana would be a more effective control strategy. Additionally, changes in the intestinal microbial communities of migratory locusts infected with the two pathogens were analyzed to investigate the roles of gut microbes in pathogen-host interactions. The mortality rate of locusts inoculated with B. bassiana and P. locustae simultaneously was not significantly higher than expected, but the mortality rates of locusts inoculated with B. bassiana 3, 6, and 9 days after inoculation with P. locustae were significantly higher than if their effects were additive, indicating synergism. A MiSeq analysis found that Weissella was the most common bacterium, representing 41.48% and 51.62% of the total bacteria in the mid- and hindguts, respectively, and the bacterial declines were greatest during dual infections with B. bassiana and P. locustae. The appropriately timed combined application of P. locustae and B. bassiana was more effective against locusts than either treatment alone. Moreover, the combined inoculation of the two pathogens changed the gut microflora of locusts, indicating the potential relevancy of their synergistic effects on locust control.

Dynamics of Aboveground Natural Enemies of Grasshoppers, and Biodiversity after Application of Paranosema locustae in Rangeland.

Substantial harm to ecosystems from the use of chemical pesticides has led to an increasing interest in the use of biopesticides to control grasshoppers in rangelands, including China. One such potential biopesticide for control of grasshoppers is the fungus Paranosema locustae. In this study, the dynamics of aboveground natural enemies of grasshoppers and arthropod diversity 0-9 years after application of P. locustae were investigated in rangeland in Qinghai Plateau, China. We found that the number of species and of individuals of aboveground natural enemies increased by 17-250% and 40-126%, respectively, after spraying P. locustae, and that the main natural enemies showed three peaks after treatment. The conventional indices of species diversity (H') and evenness (J') increased by 11-267% and 13-171%, respectively, after treatment with P. locustae. The results showed the positive effects of P. locustae on aboveground natural enemies and biodiversity in an arthropod community in Chinese rangeland. Paranosema locustae is thought to be a safe biological control agent for grasshopper management in Northwestern China.

Different predation capacities and mechanisms of Harmonia axyridis (Coleoptera: Coccinellidae) on two morphotypes of pear psylla Cacopsylla chinensis (Hemiptera: Psyllidae).

Pear psylla, Cacopsylla chinensis (Yang & Li) are present as two seasonal morphotypes in pear orchards where they, suck phloem sap, defoliate pear trees, and cause fruit russet. Despite the importance of natural enemies in psyllid control, the interactions between predators and the two seasonal morphotypes of psyllids remain poorly documented. Here we determined the predation efficiencies of the Asian lady beetle, Harmonia axyridis (Pallas) on the two psyllid morphotypes. Predation of H. axyridis on both morphotypes conformed to a Type II functional response: the proportion of consumed psyllids decreased with increasing prey densities. Predation efficiency of H. axyridis against the winterforms increased with temperature when measured from 8 to 25°C. Predation rate on the summerforms was significantly higher than that of the winterforms. This was linked to smaller body size, higher soluble protein level, thinner cuticle, and lower chitin content of summerform psyllids compared to winterform psyllids. Predation capacities of H. axyridis on both morphotypes indicated its potential as a biocontrol agent in psyllids management. Predation efficiency was higher on summerforms, likely due to the difference in body size, exoskeleton fragility, and nutritional value between the two morphotypes. Due to the Type II functional response of H. axyridis to both morphotypes of pear psylla, application of H. axyridis in pear orchards under suitable temperatures could be taken into consideration for suppression of C. chinensis, although further experiments conducted in field conditions are needed to validate our findings.

The impact of Bt maize expressing the Cry1Ac protein on non-target arthropods.

From 2014 to 2016, individuals of the principal non-target arthropod (NTA) species in a field of Bt maize expressing the Cry1Ac protein (Bt38) were compared to those in a control field of the corresponding non-transformed near isoline (Z58). For all 3 years, the population dynamics and biodiversity of NTAs were analyzed to determine if any differences might be attributable to the genetically modified (GM) maize being evaluated. The main NTAs in these fields were Aphidoidea, Pentatomidae,Araneae, and Coccinellidae. Temporal variation in NTA diversity across sample dates within a year showed no significant differences between the Bt maize and the non-Bt maize field in the total number of individuals of the dominant arthropod species per 25 plants, the Shannon index (H), Pielou index (J), Simpson index (D), and Bray-Curtis index. The cultivation of Bt corn failed to show any detrimental evidence on individuals, H, J, D, and Bray-Curtis index of NTAs, and these parameters were identical in Bt and non-Bt corn plots. These results provide further evidence of the lack of ecological impact of GM maize.

Role of the neuropeptide F 1 in regulating the appetite for food in Locusta migratoria.

BACKGROUND: Neuropeptide F (NPF) is an intercellular signaling molecule that mediates many physiological and behavioral processes. However, the function of neuropeptide F in mediating the feeding behavior of Locusta migratoria has been unclear. RESULTS: The neuropeptide F 1 precursor cDNA from L. migratoria was obtained and analyzed, and its amino acid sequence deduced. Mature LmiNPF1 was composed of 36 amino acids and was similar to that of Schistocerca gregaria. The spatial and temporal expression profiles of LmiNPF1 were investigated. LmiNPF1 was primarily expressed in the central nervous system, especially in the brain, and the expression levels were higher during the day than during the night. However, starvation activated LmiNPF1 expression increases, and downregulation of LmiNPF1 inhibited locust feeding behavior. CONCLUSION: LmiNPF1 promotes the feeding behavior of the locust and is a potential molecular target to control locust feeding. © 2018 Society of Chemical Industry.

Behavioral thermoregulation in Locusta migratoria manilensis (Orthoptera: Acrididae) in response to the entomopathogenic fungus, Beauveria bassiana.

Insects such as locusts and grasshoppers can reduce the effectiveness of pathogens and parasites by adopting different defense strategies. We investigated the behavioral thermopreference of Locusta migratoria manilensis (Meyen) (Orthoptera: Acrididae) induced by the fungus Beauveria bassiana, and the impact this behavior had on the fungal mycosis under laboratory conditions. By basking in higher temperature locations, infected nymphs elevated their thoracic temperature to 30-32.6 °C, which is higher than the optimum temperature (25°C) for B. bassiana conidial germination and hyphal development. A minimum thermoregulation period of 3 h/day increased survival of infected locusts by 43.34%. The therapeutic effect decreased when thermoregulation was delayed after initial infection. The fungus grew and overcame the locusts as soon as the thermoregulation was interrupted, indicating that thermoregulation helped the insects to cope with infection but did not completely rid them of the fungus. A significant enhancement in the number of haemocytes was observed in infected thermoregulating locusts, reaching levels that were even higher than those observed in the controls. In contrast, haemocyte concentration was severely reduced in infected insects that did not thermoregulate. In infected non-thermoregulating locusts, the reduction in haemocyte number was accompanied by an increase in fungal blastospore concentration that was obvious in the haemolymph by day four. In contrast, no circulating blastospores were found in the haemolymph of infected thermoregulating locusts three days post-inoculation. We also examined the phagocytic activity of infected insects in vivo by using fluorescein isothiocyanate (FITC)-labelled silica beads. The proportion of beads that was engulfed by haemocytes in infected, thermoregulating insects was similar to that in the controls throughout the experiment, whereas the rate of phagocytosis in infected, non-thermoregulating insects progressively decreased after infection. These findings demonstrated that behavioural thermoregulation can adversely affect B. bassiana mycosis in infected L. migratoria manilensis, thereby limiting the development of lethal entomopathogenic fungi in locusts. This is apparently accomplished through an increase in the levels of haemocytes, leading to greater phagocytic activity under certain environmental conditions.

Increase of Albinistic Hosts Caused by Gut Parasites Promotes Self-Transmission.

Paranosema locustae is a gut parasite that has been applied widely in the control of grasshoppers in many parts of the world. Usually, P. locustae is transmitted horizontally via passive modes under natural conditions but in the current study, a positive transmission strategy of P. locustae was demonstrated. First, infection by P. locustae resulted in the cuticula of infected Locusta migratoria nymphs to become lighter in color: normally only a small proportion of locusts are pale with most either being partly or mostly black; but locusts infected with P. locustae became pale. And it was found that the change to pale occurred even among uninfected black and partly black nymphs reared with infected locusts. The eumelanin of the thorax and abdomen of infected individuals decreased significantly, as did the level of dopamine. In addition, there was a decrease in phenol oxidase activity and the expression of henna and pale, which are involved in the synthesis of cuticle melanin, decreased. What is the ecological significance of this increase in light-colored hosts caused by P. locustae? We discovered that light-colored locusts were more susceptible to the microsporidian pathogen than dark-colored individuals were, because of their weaker melanization. Phenol oxidase activity in pale locusts was lower than that of black locusts, but the serpin expression level of pale locusts was higher than that of black individuals. When examined for infection, it was found that initially uninfected nymphs had picked up P. locustae infections indicating that infections are readily passed from one pale locust to another. The infection rate of healthy locusts reared with light-colored locusts infected with P. locustae was 100% which was more than with black-colored ones. The increase in albinistic locusts clearly promoted the prevalence of P. locustae in the total population. In conclusion, these results elucidated a new strategy of positive self-transmission in P. locustae. Importance: Mother Nature always grants wisdom to her creatures and feeds them carefully. This wisdom is particularly apparent in the relationships between two interacting species. In this study, our team focused on the interaction between L. migratoria and P. locustae. In a previous study, it was found that L. migratoria isolate infected individuals, reducing avoiding the spread of P. locustae, in a previous study. The solitary, pale individuals infected by P. locustae were left behind as locust groups marched ahead, leading to a kind of behavioral immunity in the insects. Here, we reported that P. locustae promotes pigmentation loss in L. migratoria, causing a larger proportion of light-colored individuals, and these lighter individuals which possessed weaker immunity against pathogens. This strategy is advantageous to P. locustae, as it promotes its propagation and spread. These extraordinary abilities of L. migratoria and P. locustae have accumulated over millennia of years of interaction.

Horizontal transmission of Paranosema locustae (Microsporidia) in grasshopper populations via predatory natural enemies.

BACKGROUND: Paranosema locustae Canning, 1953 (Microsporidia) provides effective control of grasshoppers. Horizontal transmission of P. locustae is known to occur, and evidence for the mechanism of this transmission via predatory natural enemies has been found. We conducted a 3-year laboratory and field study to assess the potential impact of feces from both grasshoppers Locusta migratoria L. and their natural enemies on the persistence of P. locustae. RESULTS: We found that P. locustae persisted among grasshopper populations in treated areas and in adjacent untreated areas for up to 2 years, and the density of grasshoppers decreased in both areas. We showed that healthy grasshoppers could be infected by eating food contaminated by feces from their natural enemies. Grasshopper predators retained a large number of spores acquired from eating grasshoppers infected with P. locustae. Spores in the feces of the main natural enemy, the beetle Pterostichus gebleri Dejean 1828, in the treated area showed clear viability. CONCLUSION: These results demonstrate that predatory natural enemies are important vectors for this microsporidian disease, and suggest that sustainable transmission and continuing population suppression might be achieved by horizontal transmission via natural enemies, which should be maximized to increase the effectiveness of P. locustae. © 2018 Society of Chemical Industry.

The mechanism for microsporidian parasite suppression of the hindgut bacteria of the migratory locust Locusta migratoria manilensis.

Locusts aggregate into bands of nymphs and swarms of adults that can pose a major threat to crop. Previous studies have shown that infection by the microsporidian parasite Paranosema locustae prevents locust aggregation behavior and we show that gut bacteria, which produce components of locust aggregation pheromones, are substantially reduced in locusts infected with P. locustae. We found that P. locustae could reduce the diversity, abundance and community composition of Locusta migratoria's gut bacteria. The parasite infection was also shown to interrupt the peroxidase activity of locust hindgut. Genome-wide expression analysis showed that the parasite infection suppressed peroxidase mRNA relative expression of locust hindgut, but had no effects on attacin expression and superoxide dismutase at 16 d post-inoculation with 20,000 P. locustae spores. Our findings reveal the mechanisms by which P. locustae impairs bacterial diversity and community structure of Locusta migratoria's gut bacteria.

Assembling of Holotrichia parallela (dark black chafer) midgut tissue transcriptome and identification of midgut proteins that bind to Cry8Ea toxin from Bacillus thuringiensis.

Holotrichia parallela is one of the most severe crop pests in China, affecting peanut, soybean, and sweet potato crops. Previous work showed that Cry8Ea toxin is highly effective against this insect. In order to identify Cry8Ea-binding proteins in the midgut cells of H. parallela larvae, we assembled a midgut tissue transcriptome by high-throughput sequencing and used this assembled transcriptome to identify Cry8Ea-binding proteins by liquid chromatography-tandem mass spectrometry (LC-MS/MS). First, we obtained de novo sequences of cDNAs from midgut tissue of H. parallela larvae and used available cDNA data in the GenBank. In a parallel assay, we obtained 11 Cry8Ea-binding proteins by pull-down assays performed with midgut brush border membrane vesicles. Peptide sequences from these proteins were matched to the H. parallela newly assembled midgut transcriptome, and 10 proteins were identified. Some of the proteins were shown to be intracellular proteins forming part of the cell cytoskeleton and/or vesicle transport such as actin, myosin, clathrin, dynein, and tubulin among others. In addition, an apolipophorin, which is a protein involved in lipid metabolism, and a novel membrane-bound alanyl aminopeptidase were identified. Our results suggest that Cry8Ea-binding proteins could be different from those characterized for Cry1A toxins in lepidopteran insects.

Unveiling the mechanism by which microsporidian parasites prevent locust swarm behavior.

Locusts are infamous for their ability to aggregate into gregarious migratory swarms that pose a major threat to food security. Aggregation is elicited by an interplay of visual, tactile, and chemical stimuli, but the aggregation pheromone in feces is particularly important. Infection by the microsporidian parasite Paranosema (Nosema) locustae is known to inhibit aggregation of solitary Locusta migratoria manilensis and to induce gregarious locusts to shift back to solitary behavior. Here we suggest that P. locustae achieves this effect by acidifying the hindgut and modulating the locust immune response, which suppresses the growth of the hindgut bacteria that produce aggregation pheromones. This in turn reduces production of the neurotransmitter serotonin that initiates gregarious behavior. Healthy L. migratoria manilensis exposed to olfactory stimuli from parasite-infected locusts also produced significantly less serotonin, reducing gregarization. P. locustae also suppresses biosynthesis of the neurotransmitter dopamine that maintains gregarization. Our findings reveal the mechanisms by which P. locustae reduces production of aggregation pheromone and blocks the initiation and maintainence of gregarious behavior.