Screening and Molecular Identification of Bacteria from the Midgut of Amphimallon solstitiale Larvae Exhibiting Antagonistic Activity against Bacterial Symbionts of Entomopathogenic Nematodes.

Entomopathogenic nematodes (Rhabditida: Steinernematidae and Heterorhabditidae) are a group of organisms capable of infecting larvae of insects living in soil, including representatives of the family Scarabaeidae. Their insecticidal activity is related to the presence of symbiotic bacteria Xenorhabdus spp. or Photorhabdus spp. in the alimentary tract, which are released into the insect body, leading to its death caused by bacterial toxins and septicemia. Although the antibacterial activities of symbionts of entomopathogenic nematodes have been well described, there is insufficient knowledge of the interactions between these bacteria and microorganisms that naturally inhabit the alimentary tract of insects infested by nematodes. In this study, 900 bacterial strains isolated from midgut samples of Amphimallon solstitiale larvae were tested for their antagonistic activity against the selected five Xenorhabdus and Photorhabdus species. Cross-streak tests showed significant antibacterial activity of 20 isolates. These bacteria were identified as Bacillus [Brevibacterium] frigoritolerans, Bacillus toyonensis, Bacillus wiedmannii, Chryseobacterium lathyri, Chryseobacterium sp., Citrobacter murliniae, Enterococcus malodoratus, Paenibacillus sp., Serratia marcescens and Serratia sp. Since some representatives of the intestinal microbiota of A. solstitiale are able to inhibit the growth of Xenorhabdus and Photorhrhabdus bacteria in vitro, it can be assumed that this type of bacterial interaction may occur at certain stages of insect infection by Steinernema or Heterorhabditis nematodes.

Bacteria from the Midgut of Common Cockchafer (Melolontha melolontha L.) Larvae Exhibiting Antagonistic Activity Against Bacterial Symbionts of Entomopathogenic Nematodes: Isolation and Molecular Identification.

The mechanisms of action of the complex including entomopathogenic nematodes of the genera Steinernema and Heterorhabditis and their mutualistic partners, i.e., bacteria Xenorhabdus and Photorhabdus, have been well explained, and the nematodes have been commercialized as biological control agents against many soil insect pests. However, little is known regarding the nature of the relationships between these bacteria and the gut microbiota of infected insects. In the present study, 900 bacterial isolates that were obtained from the midgut samples of Melolontha melolontha larvae were screened for their antagonistic activity against the selected species of the genera Xenorhabdus and Photorhabdus. Twelve strains exhibited significant antibacterial activity in the applied tests. They were identified based on 16S rRNA and rpoB, rpoD, or recA gene sequences as Pseudomonas chlororaphis, Citrobacter murliniae, Acinetobacter calcoaceticus, Chryseobacterium lathyri, Chryseobacterium sp., Serratia liquefaciens, and Serratia sp. The culture filtrate of the isolate P. chlororaphis MMC3 L3 04 exerted the strongest inhibitory effect on the tested bacteria. The results of the preliminary study that are presented here, which focused on interactions between the insect gut microbiota and mutualistic bacteria of entomopathogenic nematodes, show that bacteria inhabiting the gut of insects might play a key role in insect resistance to entomopathogenic nematode pressure.

Bacterial Communities and Midgut Microbiota Associated with Mosquito Populations from Waste Tires in East-Central Illinois.

Mosquito-microbe interactions tend to influence larval nutrition, immunity, and development, as well as fitness and vectorial capacity of adults. Understanding the role of different bacterial species not only improves our knowledge of the physiological and ecological consequences of these interactions, but also provides the basis for developing novel strategies for controlling mosquito-borne diseases. We used culture-dependent and culture-independent techniques to characterize the bacterial composition and abundance in water and midgut samples of larval and adult females of Aedes japonicus (Theobald), Aedes triseriatus (Say), and Culex restuans (Theobald) collected from waste tires at two wooded study sites in Urbana, IL. The phylum-specific real-time quantitative polymerase chain reaction assay revealed a higher proportion of Actinobacteria and a lower proportion of gamma-Proteobacteria and Bacteroidetes in water samples and larval midguts compared to adult female midguts. Only 15 of the 57 bacterial species isolated in this study occurred in both study sites. The number of bacterial species was highest in water samples (28 species from Trelease Woods; 25 species from South Farms), intermediate in larval midguts (13 species from Ae. japonicus; 12 species from Ae. triseriatus; 8 species from Cx. restuans), and lowest in adult female midguts (2 species from Ae. japonicus; 3 species from Ae. triseriatus). These findings suggest that the composition and richness of bacterial communities varies both between habitats and among mosquito species and that the reduction in bacteria diversity during metamorphosis is more evident among bacteria detected using the culture-dependent method.

Bio-products from Serratia marcescens isolated from Ghanaian Anopheles gambiae reduce Plasmodium falciparum burden in vector mosquitoes.

Novel ideas for control of mosquito-borne disease include the use of bacterial symbionts to reduce transmission. Bacteria belonging to the family Enterobacteriaceae isolated from mosquito midgut have shown promise in limiting Plasmodium intensity in the Anopheles vector. However, the mechanism of interaction between bacteria and parasite remains unclear. This study aimed at screening bio-products of two bacteria candidates for their anti-Plasmodial effects on mosquito stages of P. falciparum. Enterobacter cloacae and Serratia marcescens were isolated from field-caught Anopheles gambiae s.l. Spent media from liquid cultures of these bacteria were filtered, lyophilized and dissolved in sterile phosphate buffered saline (PBS). The re-dissolved bacterial products were added to gametocytaemic blood meals and fed to An. gambiae mosquitoes via membrane feeders. Control groups were fed on infected blood with or without lyophilized LB medium. The effect of the products on the infection prevalence and intensity of P. falciparum in mosquitoes was assessed by dissecting mosquito midguts and counting oocysts 10-11 days post-infection. S. marcescens bio-products elicited significant reduction in the number of mosquitoes infected (P=4.02 x10-5) with P. falciparum and the oocyst intensity (P<2 x 10-16) than E. cloacae products (P>0.05 for both prevalence and intensity) compared to the control (lyophilized LB medium). These data support the use of bioproducts released by S. marcescens for malaria control based on transmission blocking in the vector.

Guadipyr, a new insecticide, induces microbiota dysbiosis and immune disorders in the midgut of silkworms (Bombyx mori).

Guadipyr, which combines neonicotinoid and semicarbazone functional groups in one molecule, exhibits good activity on several pests and high acute and chronic toxicity to silkworms (Bombyx mori). In this report, the effects of low-dose guadipyr on the midgut microbiota and immune system of silkworms were studied. Results showed that the structure and richness of the midgut microbiota of silkworms were altered after being treated with 5.25 mg/L (1/10 of LC50) of guadipyr. The abundance of Pseudomonas was evidently increased, whereas Curvibacter was substantially reduced, which might be related to the growth and immunity of silkworms. The expression of key genes in the Toll, IMD, and JAK/STAT pathways, which ultimately led to the downregulation of antimicrobial peptide genes (AMPs), such as CecA, Defensin1, Leb, and glv2, was reduced upon guadipyr exposure. Simultaneously, the suppression of steroid hormone 20-hydroxyecdysone receptor and response genes, such as BR-C Z4, was detected in the exposed groups. The decreased expression of these immune regulatory pathway-related and 20-hydroxyecdysone signal pathway-related genes indicated that the immune system of silkworms was affected by low-dose guadipyr. Our results revealed the negative effects of guadipyr on silkworms and highlighted the unneglectable toxicity of low-dose guadipyr to this economic insect. Given the risk, it is necessary to control the application of guadipyr in or around the mulberry fields.

Microbiota modulates gut immunity and promotes baculovirus infection in Helicoverpa armigera.

Baculoviruses are natural enemies of agricultural and forest insect pests and play an important role in biological pest control. Oral infection by baculovirus in the insect midgut is necessary for establishing systemic infection and eventually killing the insect. Since the insect midgut continuously encounters microbiota, the gut microbiota could affect baculovirus infection. Here, we demonstrated that gut microbiota modulates immune responses and promotes baculovirus infection in the cotton bollworm, Helicoverpa armigera. After oral infection, numerous host immunity-related genes including genes encoding Toll and immune deficiency (IMD) pathway components were upregulated in the midgut. Elimination of the gut microbiota significantly increased the resistance to viral infection in H. armigera. Quantitative real-time reverse transcription polymerase chain reaction and proteomic analysis showed that downregulation of the antiviral factor prophenoloxidase (PPO) could be mediated by microbiota during infection. It implied that midgut microbiota diminishes the expression of PPO to facilitate viral infection in H. armigera. Our findings revealed that the microbiota plays an important role in modulating the resistance of H. armigera to baculovirus infection, providing new insights in applying biopesticide.

Longitudinal variations in the gastrointestinal microbiome of the white shrimp, Litopenaeus vannamei.

The shrimp gut is a long digestive structure that includes the Foregut (stomach), Midgut (hepatopancreas) and Hindgut (intestine). Each component has different structural, immunity and digestion roles. Given these three gut digestive tract components' significance, we examined the bacterial compositions of the Foregut, Hindgut, and Midgut digestive fractions. Those bacterial communities' structures were evaluated by sequencing the V3 hypervariable region of the 16S rRNA gene, while the functions were predicted by PICRUSt2 bioinformatics workflow. Also, to avoid contamination with environmental bacteria, shrimp were maintained under strictly controlled conditions. The pairwise differential abundance analysis revealed differences among digestive tract fractions. The families Rhodobacteraceae and Rubritalaceae registered higher abundances in the Foregut fraction, while in the Midgut, the families with a higher proportion were Aeromonadaceae, Beijerinckiaceae and Propionibacteriaceae. Finally, the Cellulomonadaceae family resulted in a higher proportion in the Hindgut. Regarding the predicted functions, amino acid and carbohydrate metabolism pathways were the primary functions registered for Foregut microbiota; conversely, pathways associated with the metabolism of lipids, terpenoids and polyketides, were detected in the Midgut fraction. In the Hindgut, pathways like the metabolism of cofactors and vitamins along with energy metabolism were enriched. Structural changes were followed by significant alterations in functional capabilities, suggesting that each fraction's bacteria communities may carry out specific metabolic functions. Results indicate that white shrimp's gut microbiota is widely related to the fraction analyzed across the digestive tract. Overall, our results suggest a role for the dominant bacteria in each digestive tract fraction, contributing with a novel insight into the bacterial community.

Interfaces between microbes and membranes of host epithelial cells in hemipteran midguts.

Certain families of plant-feeding insects in the order Hemiptera (infraorder Pentatomomorpha) have established symbiotic relationships with microbes that inhabit specific pouches (caeca) of their midgut epithelium. The placement of these caeca in a well-delineated region at the most posterior end of the midgut bordering the hindgut is conserved in these families; in situ the convoluted midgut is predictably folded so that this caecal region lies adjacent to the anterior-most region of the midgut. Depending on the hemipteran family, caeca vary in their number and configuration at a given anterior-posterior location. At the host-microbe interface, epithelial plasma membranes of midgut epithelial cells interact with nonself antigens of microbial surfaces. In the different hemipteran species examined, a continuum of interactions is observed between microbes and host membranes. Bacteria can exist as free living cells within the midgut lumen without contacting host membranes while other host cells physically interact extensively with microbial surfaces by extending numerous processes that interdigitate with microbes; and, in many instances, processes completely envelope the microbes. The host cells can embrace the foreign microbes, completely enveloping each with a single host membrane or sometimes enveloping each with the two additional host membranes of a phagosome.

Generational conservation of composition and diversity of field-acquired midgut microbiota in Anopheles gambiae (sensu lato) during colonization in the laboratory.

BACKGROUND: The gut microbiota is known to play a role in a mosquito vector's life history, a subject of increasing research. Laboratory experiments are essential for such studies and require laboratory colonies. In this study, the conservation of field-obtained midgut microbiota was evaluated in laboratory-reared Anopheles gambiae (s.l.) mosquitoes continuously hatched in water from field breeding habitats. METHODS: Pupae and late instars were obtained from the field and reared, and the emerged adults were blood-fed. The eggs obtained from them were hatched in either water from the field or in dechlorinated tap water. The mosquito colonies were maintained for 10 generations. Midguts of female adults from unfed F0 (emerging from field-caught pupae and larvae), F5 and F10 were dissected out and genomic DNA was extracted for 16S metagenomic sequencing. The sequences were compared to investigate the diversity and bacterial compositional differences using ANCOM and correlation clustering methods. RESULTS: Less than 10% of the bacterial families identified had differential relative abundances between generational groups and accounted for 46% of the variation observed. Although diversity reduced in F10 mosquitoes during laboratory colonization (Shannon-Weaver; P-value < 0.05), 50% of bacterial genera were conserved in those bred continuously in field-water compared to 38% in those bred in dechlorinated tap water. CONCLUSIONS: To our knowledge, this study is the first report on the assessment of gut bacterial community of mosquitoes during laboratory colonization and recommends the use of water from the natural breeding habitats if they are intended for microbiota research.