Virulence of the fungal pathogen, Aspergillus oryzae XJ-1 to adult locusts (Orthoptera: Acrididae) in both laboratory and field trials.

BACKGROUND: Locusts and grasshoppers are pests of many agricultural crops, and their frequent outbreaks worldwide threaten food security. Microbial control agents are currently used to suppress the early (nymphal) stages of pests, but they are often less effective against adults, which are primarily responsible for locust plagues. The fungal pathogen Aspergillus oryzae XJ-1 has high pathogenicity in locust nymphs. To assess its potential for controlling locust adults, we evaluated the virulence of A. oryzae XJ-1 (i.e., locust Aspergillus, LAsp) in locust adults using laboratory, field-cage experiments, and a field trial. RESULTS: The lethal concentration of LAsp in adult Locusta migratoria was 3.58 ± 0.09 × 105 conidia mL-1 15 days after inoculation in the laboratory. A field-cage experiment showed that the mortalities of adult L. migratoria were 92.0 ± 4.6% and 90.1 ± 3.2% 15 days after inoculation with 3 × 105 and 3 × 103 conidia m-2 of LAsp, respectively. A large-scale field trial of 666.6 ha was conducted, in which a LAsp water suspension was applied at a concentration of 2 × 108 conidia mL-1 in 15 L ha-1 by aerial spraying via drones. The densities of mixed populations of L. migratoria and Epacromius spp. were significantly reduced by 85.4 ± 7.9%-94.9 ± 5.1%. In addition, the infection rates of surviving locusts collected from the treated plots were 79.6% and 78.3% on the 17th and 31st day after treatment, respectively. CONCLUSION: These results indicate that A. oryzae XJ-1 is highly virulent in adult locusts and that it has high potential for the control of locusts. © 2023 Society of Chemical Industry.

Suppression of yolk formation, oviposition and egg quality of locust (Locusta migratoria manilensis) infected by Paranosema locustae.

Locusta migratoria manilensis is one of the most important agricultural pests in China. The locust has high fecundity and consumes large quantities of food, causing severe damage to diverse crops such as corn, sorghum, and rice. Immunity against pathogens and reproductive success are two important components of individual fitness, and many insects have a trade-off between reproduction and immunity when resources are limited, which may be an important target for pest control. In this study, adult females L. migratoria manilensis were treated with different concentrations (5 × 106 spores/mL or 2 × 107 spores/mL) of the entomopathogenic fungus Paranosema locustae. Effects of input to immunity on reproduction were studied by measuring feeding amount, enzyme activity, vitellogenin (Vg) and vitellogenin receptor (VgR) production, ovary development, and oviposition amount. When infected by P. locustae, feeding rate and phenol oxidase and lysozyme activities increased, mRNA expression of Vg and VgR genes decreased, and yolk deposition was blocked. Weight of ovaries decreased, with significant decreases in egg, length and weight.Thus, locusts used nutritive input required for reproduction to resist invasion by microsporidia. This leads to a decrease in expression of Vg and VgR genes inhibited ovarian development, and greatly decreased total fecundity. P. locustae at 2 × 107 spores/mL had a more obvious inhibitory effect on the ovarian development in migratory locusts. This study provides a detailed trade-off between reproduction and immune input of the female, which provides a reliable basis to find pest targets for biological control from those trade-off processes.

The carbon catabolite repressor CreA is an essential virulence factor of Metarhizium acridum against Locusta migratoria.

BACKGROUND: CreA has been proved to be a core gene in asexual conidiation in Metarhizium acridum, which regulates the shift of normal conidiation and microcycle conidiation. At present, research on CreA in fungi has focused on carbon source metabolism. There is a lack of research on the effect of CreA in virulence of pathogenic fungi. RESULTS: The virulence of the MaCreA disrupted strain (ΔMaCreA) for Locusta migratoria was lost by topical inoculation bioassay. The formation rate and turgor pressure of the appressoria decreased. Growth of ΔMaCreA in host hemolymph was delayed, and the number of hyphal bodies was significantly reduced. The conidial cell wall of ΔMaCreA became thicker, the mannan content decreased, and the chitin content increased significantly, and it was more sensitive to calcofluor white and Congo Red. α-1,3-Glucan and ß-1,3-glucan are more exposed on the surface of ΔMaCreA conidia than on the wild type. Lmspätzle and Lmcactus, the immune response genes in the host Toll pathway, showed stronger transcriptional activities at the early stage of ΔMaCreA invasion. The phenoloxidase activity assay also showed stronger immunostimulation by ΔMaCreA in vitro. CONCLUSION: The main reasons for the loss of virulence of ΔMaCreA in the topical inoculation were the reduced penetration ability of appressoria, limited growth in hemolymph and stronger insect immunostimulation of ΔMaCreA. © 2022 Society of Chemical Industry.

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.

Differential responses of the antennal proteome of male and female migratory locusts to infection by a fungal pathogen.

The narrow host range entomopathogenic fungus, Metarhizium acridum, is an environmentally friendly acridid specific pathogen used for locust control. The locust is capable of responding within hours of infection, however, little is known concerning how the locust detects the pathogen. Here, we have identified 3213 proteins in the infected antennal proteome of the migratory locust, Locusta migratoria. iTRAQ comparative analyses of antennal proteomes identified 194 differentially abundant proteins (DAPs) between uninfected and infected males, 218 DAPs between uninfected and infected females, and 240 DAPs between infected males and infected females. In relation to olfaction, a total of 29 chemosensory proteins (CSPs), 9 odorant binding proteins (OBPs), 31 odorant receptors (ORs), and 8 ionotropic receptors (IRs) were differentially abundant after M. acridum infection, with a subset of 12 proteins found in both infected male and female antennae not present in uninfected individuals. The time course of the gene expression profiles of olfaction related DAPs were investigated by quantitative real-time PCR (qRT-PCR). Our data indicate significant changes in the antennal proteomes of male and female locusts in response to a microbial pathogen, highlighting the potential participation of olfactory processes in pathogen detection and response. BIOLOGICAL SIGNIFICANCE: The ability of an organism to detect microbial pathogens is essential for mounting a response to mitigate the spread of the infection. Using iTRAQ-based proteomic analyses changes in the protein repertoire of the antennae of male and female locusts in response to infection by a host-specific pathogen were determined. These data show proteomic alterations that are also sex-specific, identifying members of olfactory pathways that are modified in response to infection. Our data identify antennal and related olfactory proteins that are candidates for mediating host detection of pathogens, and that may contribute to subsequent behavioral and/or immune responses of the host to the infection challenge.

Transcriptome analysis of the molecular mechanism underlying immunity- and reproduction trade-off in Locusta migratoria infected by Micrococcus luteus.

Immune response and reproductive success are two vital energy-consuming processes in living organisms. However, it is still unclear which process is prioritized when both are required. Therefore, the present study was designed to examine this question arising for one of the world's most destructive agricultural pests, the migratory locust, Locusta migratoria. Transcripts from the ovaries and fat bodies of newly emerged locusts were analyzed, using RNA-seq based transcriptome and qualitative real-time PCR, at 4 h and 6 d after being infected with the gram-positive bacteria Micrococcus luteus. Changes in the main biological pathways involved in reproduction and immunization were analyzed using bioinformatics. After 4 h of infection, 348 and 133 transcripts were up- and down-regulated, respectively, whereas 5699 and 44 transcripts were up- and down-regulated, respectively, at 6 d after infection. Moreover, KEGG analysis indicated that vital pathways related with immunity and reproduction, such as Insulin resistance, FoxO signaling, Lysosome, mTOR signaling, and Toll-like receptor signaling pathways were up-regulated. Among the differentially expressed genes, 22 and 17 were related to immunity and reproduction, respectively. The expression levels of PPO1 and antimicrobial peptide defensin 3 were increased (log2FC = 5.93 and 6.75, respectively), whereas those of VgA and VgB were reduced (log2FC = -17.82 and -18.13, respectively). These results indicated that locust allocate energy and resources to maintain their own survival by increasing immune response when dealing with both immune and reproductive processes. The present study provides the first report of expression levels for genes related with reproduction and immunity in locusts, thereby providing a reference for future studies, as well as theoretical guidance for investigations of locust control.

Spatiotemporal expression of the putative MdtABC efflux pump of Phtotorhabdus luminescens occurs in a protease-dependent manner during insect infection.

Photorhabdus luminescens is an enterobacterium establishing a mutualistic symbiosis with nematodes, that also kills insects after septicaemia and connective tissue colonization. The role of the bacterial mdtABC genes encoding a putative multidrug efflux system from the resistance/nodulation/cell division family was investigated. We showed that a mdtA mutant and the wild type had similar levels of resistance to antibiotics, antimicrobial peptides, metals, detergents and bile salts. The mdtA mutant was also as pathogenic as the wild-type following intrahaemocoel injection in Locusta migratoria, but had a slightly attenuated phenotype in Spodoptera littoralis. A transcriptional fusion of the mdtA promoter (PmdtA) and the green fluorescent protein (gfp) encoding gene was induced by copper in bacteria cultured in vitro. The PmdtA-gfp fusion was strongly induced within bacterial aggregates in the haematopoietic organ during late stages of infection in L. migratoria, whereas it was only weakly expressed in insect plasma throughout infection. A medium supplemented with haematopoietic organ extracts induced the PmdtA-gfp fusion ex vivo, suggesting that site-specific mdtABC expression resulted from insect signals from the haematopoietic organ. Finally, we showed that protease inhibitors abolished ex vivo activity of the PmdtA-gfp fusion in the presence of haematopoietic organ extracts, suggesting that proteolysis by-products play a key role in upregulating the putative MdtABC efflux pump during insect infection with P. luminescens.

RNAi-knockdown of the Locusta migratoria nuclear export factor protein results in insect mortality and alterations in gut microbiome.

BACKGROUND: The migratory locust Locusta migratoria is one of the most important agricultural pests worldwide. The nuclear export factor 1 (NXF1) protein plays a crucial role in mediating mRNA transport from the nucleus to the cytoplasm. This study evaluates whether NXF1 could be a potential target for RNAi-mediated pest control of L. migratoria. RESULTS: We cloned and characterized the nuclear export factor lm-nxf1 of L. migratoria. Lm-nxf1 was expressed in all tissues examined, including head, fat body, hemolymph, trunk, leg and midgut, with high expression observed in the hemolymph and fat body. Injection of lm-nxf1 dsRNA into hemolymph resulted in inhibition of mRNA export in hemocytes, which were used as a target for observing mRNA export. Total hemocyte levels were reduced by ca. 97% in lm-nxf1-dsRNA-treated locusts, and high insect mortality occurred with LT50 = 7.75 day as compared with 18.15 day for gfp-dsRNA-treated controls. Further, the locust intestine became atrophy, and the opportunistic pathogens Enterobacter aerogenes, Klebsiella pneumoniae and Enterobacter asburiae were specifically detected in midgut after lm-nxf1 dsRNA treatment. CONCLUSIONS: The results reveal that knockdown of the lm-nxf1 gene affects the survival of L. migratoria, indicating that lm-nxf1 is a potential target for RNAi-mediated pest control. © 2018 Society of Chemical Industry.

Increased virulence in the locust-specific fungal pathogen Metarhizium acridum expressing dsRNAs targeting the host F1 F0 -ATPase subunit genes.

BACKGROUND: Metarhizium acridum is a host-specific fungal pathogen with great potential for locust control. However, the slow killing action of M. acridum has impeded its widespread application. To enhance fungal virulence, we constructed transgenic M. acridum strains that express double-stranded (ds)RNAs targeting the genes of the F1 F0 -ATP synthase α and ß subunits in Locusta migratoria. RESULTS: The two host genes were transcriptionally suppressed in L. migratoria nymphs (instar V) infected by RNA interference (RNAi) strains targeting one or two subunit genes of the host ATP synthase, followed by reduced ATPase activity and ATP synthesis. Consequently, the RNAi strain targeting both subunit genes displayed high virulence that was 3.7-fold that in the wild-type strain. CONCLUSION: Our results demonstrate that dsRNA expression in M. acridum can cause host RNA silencing during infection and greatly enhances the fungal virulence through interference with critical host genes, highlighting a new strategy for augmentation of fungal virulence against insect pests. © 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.

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.

Central Nervous System Responses of the Oriental migratory, Locusta migratoria manilensis, to Fungal Infection.

Responses of the central nervous system (CNS) to microbial challenge and the interplay between the CNS and the immune system are important for defending against pathogen attack. We have examined the CNS transcriptional response of Locusta migratoria manilensis to infection by the locust-specific fungal pathogen, Metarhizium acridum. CNS responses were examined during spore attachment, fungal germination and pre-penetration of the cuticle, and cuticle penetration/hemocoel ingress and proliferation. Effects were seen at the earliest time points (4 h post-infection) and the number of differentially expressed genes (DEGs) was highest during late mycosis (72 h post-infection). Significantly affected neurological pathways included genes involved in serotonergic, cholinergic, dopaminergic, GABAergic, and glutamergic synapse responses, as well as pathways responsible for synaptic vesicle cycle, long-term potentiation and depression, and neurotrophin and retrograde endocannabinoid signaling. In addition, a significant number of immune related DEGs were identified. These included components of the Toll, Imd and JAK/STAT pathways, consistent with interactions between the CNS and immune systems. The activation of immune response related CNS genes during early stage infection highlights the rapid detection of microbial pathogens and suggests an important role for the CNS in modulating immunity potentially via initiating behavioral adaptations along with innate immune responses.

Transferable Antibiotic Resistances in Marketed Edible Grasshoppers (Locusta migratoria migratorioides).

Grasshoppers are the most commonly eaten insects by humans worldwide, as they are rich in proteins and micronutrients. This study aimed to assess the occurrence of transferable antibiotic resistance genes in commercialized edible grasshoppers. To this end, the prevalence of 12 selected genes [aac(6')-Ie aph(2″)-Ia, blaZ, erm(A), erm(B), erm(C), mecA, tet(M), tet(O), tet(S), tet(K), vanA, vanB] coding for resistance to antibiotics conventionally used in clinical practice was determined. The majority of samples were positive for tet(M) (70.0%), tet(K) (83.3%) and blaZ (83.3%). A low percentage of samples were positive for erm(B) (16.7%), erm(C) (26.7%), and aac(6')-Ie aph(2″)-Ia (13.3%), whereas no samples were positive for erm(A), vanA, vanB, tet(O), and mecA. Cluster analysis identified 4 main clusters, allowing a separation of samples on the basis of their country of origin.

[Phenotypic and genetic changes of entomopathogenic ascomycete Beauveria Bassiana under passaging through various hosts].

Phenotypic and genetic estimations of entomopathogenic ascomycete B.bassiana (strain Sar-31) after 6-passaging through four hosts were shown. Increasing of virulence, changes in morpho-cultural characteristics and variations in Inter Simple Sequence Repeats (ISSR) assay between initial and reisolated cultures were registered. Six passages of entomopathogenic ascomycete Beauveria bassiana (strain Sar-31) through four hosts (Galleria mellonella, Tenebrio molitor, Leptinotarsa decemlineata, Locusta migratoria) and following estimation of phenotypic and genetic differences of the initial strain and reisolated cultures were conducted. The passaging of strain through certain host led to increasing of virulence for both this host and other test-insects. Unidirectional changes of morpho-cultural characteristics: colonies pigmentation and relief strengthening, increasing of conidia production and lipolytic activity were registered in all passaged cultures. Genetic analysis with 6 ISSR markers revealed variations between initial and reisolated cultures in 3 markers. Taken together, the results of this study help us understand potential ways of fungi strains changes during epizootic process and possibilities of ISSR assay applying for investigation of pathogen transmission.

A Family of CSαß Defensins and Defensin-Like Peptides from the Migratory Locust, Locusta migratoria, and Their Expression Dynamics during Mycosis and Nosemosis.

Insect defensins are effector components of the innate defense system. During infection, these peptides may play a role in the control of pathogens by providing protective antimicrobial barriers between epithelial cells and the hemocoel. The cDNAs encoding four defensins of the migratory locust, Locusta migratoria, designated LmDEF 1, 3-5, were identified for the first time by transcriptome-targeted analysis. Three of the members of this CSαß defensin family, LmDEF 1, 3, and 5, were detected in locust tissues. The pro regions of their sequences have little-shared identities with other insect defensins, though the predicted mature peptides align well with other insect defensins. Phylogenetic analysis indicates a completely novel position of both LmDEF 1 and 3, compared to defensins from hymenopterans. The expression patterns of the genes encoding LmDEFs in the fat body and salivary glands were studied in response to immune-challenge by the microsporidian pathogen Nosema locustae and the fungus Metarhizium anisopliae after feeding or topical application, respectively. Focusing on Nosema-induced immunity, qRT-PCR was employed to quantify the transcript levels of LmDEFs. A higher transcript abundance of LmDEF5 was distributed more or less uniformly throughout the fat body along time. A very low baseline transcription of both LmDEFs 1 and 3 in naïve insects was indicated, and that transcription increases with time or is latent in the fat body or salivary glands of infected nymphs. In the salivary glands, expression of LmDEF3 was 20-40-times higher than in the fat body post-microbial infection. A very low expression of LmDEF3 could be detected in the fat body, but eventually increased with time up to a maximum at day 15. Delayed induction of transcription of these peptides in the fat body and salivary glands 5-15 days post-activation and the differential expression patterns suggest that the fat body/salivary glands of this species are active in the immune response against pathogens. The ability of N. locustae to induce salivary glands as well as fat body expression of defensins raises the possibility that these AMPs might play a key role in the development and/or tolerance of parasitic infections.

Biochemical basis of synergism between pathogenic fungus Metarhizium anisopliae and insecticide chlorantraniliprole in Locusta migratoria (Meyen).

We challenged Locusta migratoria (Meyen) grasshoppers with simultaneous doses of both the insecticide chlorantraniliprole and the fungal pathogen, Metarhizium anisopliae. Our results showed synergistic and antagonistic effects on host mortality and enzyme activities. To elucidate the biochemical mechanisms that underlie detoxification and pathogen-immune responses in insects, we monitored the activities of 10 enzymes. After administration of insecticide and fungus, activities of glutathione-S-transferase (GST), general esterases (ESTs) and phenol oxidase (PO) decreased in the insect during the initial time period, whereas those of aryl acylamidase (AA) and chitinase (CHI) increased during the initial period and that of acetylcholinesterase (AChE) increased during a later time period. Activities of superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD) decreased at a later time period post treatment. Interestingly, treatment with chlorantraniliprole and M. anisopliae relieved the convulsions that normally accompany M. anisopliae infection. We speculate that locust mortality increased as a result of synergism via a mechanism related to Ca(2+) disruption in the host. Our study illuminates the biochemical mechanisms involved in insect immunity to xenobiotics and pathogens as well as the mechanisms by which these factors disrupt host homeostasis and induce death. We expect this knowledge to lead to more effective pest control.

Different Effects of Metarhizium anisopliae Strains IMI330189 and IBC200614 on Enzymes Activities and Hemocytes of Locusta migratoria L.

BACKGROUND: Metarhizium is an important class of entomopathogenic fungi in the biocontrol of insects, but its virulence is affected by insect immunity. To clarify the mechanism in virulence of Metarhizium, we compared the immunological differences in Locusta migratoria L. when exposed to two strains of Metarhizium anisopliae (Ma). RESULTS: The virulence of Ma IMI330189 was significantly higher than that of Ma IBC200614 to locust, and IMI330189 overcame the hemocytes and began destroying the hemocytes of locust at 72 h after spray, while locust is immune to IBC200614. IMI330189 could overcome the humoral immunity of locust by inhibiting the activities of phenol oxidase (PO), esterases, multi-function oxidases (MFOs) and acetylcholinesterases in locust while increasing the activities of glutathione-S-transferases (GSTs), catalase and aryl-acylamidase (AA). However IBC200614 inhibit the activities of GSTs and AA in locust and increase the activities of MFOs, PO, superoxide dismutase, peroxidase and chitinase in locust. The changes of enzymes activities in period of infection showed that the time period between the 2nd and the 5th day after spray is critical in the pathogenic process. CONCLUSION: These results found the phenomenon that Ma initiatively broke host hemocytes, revealed the correlation between the virulence of Ma and the changes of enzymes activities in host induced by Ma, and clarified the critical period in the infection of Ma. So, these results should provide guidance for the construction of efficient biocontrol Ma strains.

Microbial community assessment of mealworm larvae (Tenebrio molitor) and grasshoppers (Locusta migratoria migratorioides) sold for human consumption.

In Western countries, the popularity of edible insects as an alternative animal protein source is increasing. Nevertheless, there is a lack of profound insight into the microbial safety and shelf life of living insects sold for human consumption. The purpose of this study was to characterise the microflora of fresh edible mealworm larvae and grasshoppers in a quantitative and qualitative way. Therefore, culture-dependent analyses (the total viable aerobic count, Enterobacteriaceae, lactic acid bacteria, yeasts and moulds, and bacterial endospores) and next-generation sequencing (454amplicon pyrosequencing) were performed. High microbial counts were obtained for both insect species. Different insect batches resulted in quite similar microbial numbers, except for bacterial endospores. However, the bacterial community composition differed between both insect species. The most abundant operational taxonomic unit in mealworm larvae was Propionibacterium. Also members of the genera Haemophilus, Staphylococcus and Clostridium were found. Grasshoppers were mainly dominated by Weissella, Lactococcus and Yersinia/Rahnella. Overall, a variety of potential spoilage bacteria and food pathogens were characterised. The results of this study suggest that a processing step with a microbiocidal effect is required to avoid or minimize risks involved with the consumption of edible insects.

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.

Comparative transcriptomic analysis of immune responses of the migratory locust, Locusta migratoria, to challenge by the fungal insect pathogen, Metarhizium acridum.

BACKGROUND: The migratory locust, Locusta migratoria manilensis, is an immensely destructive agricultural pest that forms a devastating and voracious gregarious phase. The fungal insect pathogen, Metarhizium acridum, is a specialized locust pathogen that has been used as a potent mycoinsecticide for locust control. Little, however, is known about locust immune tissue, i.e. fat body and hemocyte, responses to challenge by this fungus. METHODS: RNA-seq (RNA sequencing) technology were applied to comparatively examine the different roles of locust fat body and hemocytes, the two major contributors to the insect immune response, in defense against M. acridum. According to the sequence identity to homologies of other species explored immune response genes, immune related unigenes were screened in all transcriptome wide range from locust and the differential expressed genes were identified in these two tissues, respectively. RESULTS: Analysis of differentially expressed locust genes revealed 4660 and 138 up-regulated, and 1647 and 23 down-regulated transcripts in the fat body and hemocytes, respectively after inoculation with M. acridum spores. GO (Gene Ontology) enrichment analysis showed membrane biogenesis related proteins and effector proteins significantly differentially expressed in hemocytes, while the expression of energy metabolism and development related transcripts were enriched in the fat body after fungal infection. A total of 470 immune related unigenes were identified, including members of the three major insect immune pathways, i.e. Toll, Imd (immune deficiency) and JAK/STAT (janus kinase/signal transduction and activator of transcription). Of these, 58 and three were differentially expressed in the insect fat body or hemocytes after infection, respectively. Of differential expressed transcripts post challenge, 43 were found in both the fat body and hemocytes, including the LmLys4 lysozyme, representing a microbial cell wall targeting enzyme. CONCLUSIONS: These data indicate that locust fat body and hemocytes adopt different strategies in response to M. acridum infection. Fat body gene expression after M. acridum challenge appears to function mainly through activation of innate immune related genes, energy metabolism and development related genes. Hemocyte responses attempt to limit fungal infection primarily through regulation of membrane related genes and activation of cellular immune responses and release of humoral immune factors.