Microbial Gut Diversity of Africanized and European Honey Bee Larval Instars

The first step in understanding gut microbial ecology is determining the presence and potential niche breadth of associated microbes. While the core gut bacteria of adult honey bees is becoming increasingly apparent, there is very little and inconsistent information concerning symbiotic bacterial communities in honey bee larvae. The larval gut is the target of highly pathogenic bacteria and fungi, highlighting the need to understand interactions between typical larval gut flora, nutrition and disease progression. Here we show that the larval gut is colonized by a handful of bacterial groups previously described from guts of adult honey bees or other pollinators. First and second larval instars contained almost exclusively Alpha 2.2, a core Acetobacteraceae, while later instars were dominated by one of two very different Lactobacillus spp., depending on the sampled site. Royal jelly inhibition assays revealed that of seven bacteria occurring in larvae, only one Neisseriaceae and one Lactobacillus sp. were inhibited. We found both core and environmentally vectored bacteria with putatively beneficial functions. Our results suggest that early inoculation by Acetobacteraceae may be important for microbial succession in larvae. This assay is a starting point for more sophisticated in vitro models of nutrition and disease resistance in honey bee larvae.     

Evaluation of five antibiotics on larval gut bacterial diversity of Plutella xylostella (Lepidoptera: Plutellidae)

Larvae of the diamondback moth, Plutella xylostella L. (Lepidoptera: Plutellidae), have rich microbial communities inhabiting the gut, and these bacteria contribute to the fitness of the pest. In this study we evaluated the effects of five antibiotics (rifampicin, ampicillin, tetracycline, streptomycin sulfate and chloramphenicol) on the gut bacterial diversity of P. xylostella larvae. We screened five different concentrations for each antibiotic in a leaf disc assay, and found that rifampicin and streptomycin sulfate at 3 mg/mL significantly reduced the diversity of the bacterial community, and some bacterial species could be rapidly eliminated. The number of gut bacteria in the rifampicin group and streptomycin sulfate group decreased more rapidly than the others. With the increase of antibiotic concentration, the removal efficiency was improved, whereas toxic effects became more apparent. All antibiotics reduced larval growth and development, and eventually caused high mortality, malformation of the prepupae, and hindered pupation and adult emergence. Among the five antibiotics, tetracycline was the most toxic and streptomycin sulfate was a relatively mild one. Some dominant bacteria were not affected by feeding antibiotics alone. Denaturing gradient gel electrophoresis graph showed that the most abundant and diverse bacteria in P. xylostella larval gut appeared in the cabbage feeding group, and diet change and antibiotics intake influenced gut flora abundance. Species diversity was significantly reduced in the artificial diet and antibiotics treatment groups. After feeding on the artificial diet with rifampicin, streptomycin sulfate and their mixture for 10 days, larval gut bacteria could not be completely removed as detected with the agarose gel electrophoresis method.     

No evidence that gut microbiota impose a net cost on their butterfly host

Gut microbes are believed to play a critical role in most animal life, yet fitness effects and cost–benefit trade‐offs incurred by the host are poorly understood. Unlike most hosts studied to date, butterflies largely acquire their nutrients from larval feeding, leaving relatively little opportunity for nutritive contributions by the adult's microbiota. This provides an opportunity to measure whether hosting gut microbiota comes at a net nutritional price. Because host and bacteria may compete for sugars, we hypothesized that gut flora would be nutritionally neutral to adult butterflies with plentiful food, but detrimental to semistarved hosts, especially when at high density. We held field‐caught adult Speyeria mormonia under abundant or restricted food conditions. Because antibiotic treatments did not generate consistent variation in their gut microbiota, we used interindividual variability in bacterial loads and operational taxonomic unit abundances to examine correlations between host fitness and the abdominal microbiota present upon natural death. We detected strikingly few relationships between microbial flora and host fitness. Neither total bacterial load nor the abundances of dominant bacterial taxa were related to butterfly fecundity, egg mass or egg chemical content. Increased abundance of a Commensalibacter species did correlate with longer host life span, while increased abundance of a Rhodococcus species correlated with shorter life span. Contrary to our expectations, these relationships were unchanged by food availability to the host and were unrelated to reproductive output. Our results suggest the butterfly microbiota comprises parasitic, commensal and beneficial taxa that together do not impose a net reproductive cost, even under caloric stress.     

Microbial Community Profiling to Investigate Transmission of Bacteria Between Life Stages of the Wood-Boring Beetle, Anoplophora glabripennis

Many insects harbor specific bacteria in their digestive tract, and these gut microbiota often play important roles in digestion and nutrient provisioning. While it is common for a given insect species to harbor a representative gut microbial community as a population, how this community is acquired and maintained from generation to generation is not known for most xylophagous insects, except termites. In this study, we examined acquisition of gut microbiota by the wood-feeding beetle, Anoplophora glabripennis, by identifying and comparing microbial community members among different life stages of the insect and with microbes it encounters in the environment. Automated ribosomal intergenic spacer analysis was employed to compare bacterial communities present in the egg and larval stages of A. glabripennis as well as with microbes found in the oviposition site and the surrounding woody tissue. Multivariate analyses were used to identify relationships between sample type and specific bacterial types (operational taxonomic units). From this analysis, bacteria that were derived from the environment, the oviposition site, and/or the egg were identified and compared with taxa found in larvae. Results showed that while some larval microbes were derived from environmental sources, other members of the larval microbial community appear to be vertically transmitted. These findings could lead to a better understanding of which microbial species are critical for the survival of this insect and to development of techniques that could be used to alter this community to disrupt the digestive physiology of the host insect as a biological control measure.     

饲料类型对茶尺蠖幼虫肠道细菌多样性及幼虫存活率的影响

【目的】茶尺蠖是茶园中的重要害虫。研究茶尺蠖寄主食物-肠道菌群-茶尺蠖生长发育三者之间的关系对于茶尺蠖的防治具有重要的理论指导价值。【方法】分析不添加茶叶因子的纯人工饲料和茶树鲜叶对茶尺蠖幼虫的存活影响;用高通量测序技术分析不同饲料饲喂的茶尺蠖幼虫的肠道菌群异同。【结果】取食人工饲料的幼虫死亡率远远高于取食茶树鲜叶的幼虫;取食人工饲料的幼虫肠道细菌多样性和丰富度高于取食茶树鲜叶的幼虫;茶尺蠖幼虫肠道中存在很多促进宿主生长的细菌。【结论】饲料类型影响茶尺蠖幼虫的存活;饲料类型影响茶尺蠖幼虫肠道菌群结构。     

Changes in the Gut Microbiome of the Sea Lamprey during Metamorphosis

Vertebrate metamorphosis is often marked by dramatic morphological and physiological changes of the alimentary tract, along with major shifts in diet following development from larva to adult. Little is known about how these developmental changes impact the gut microbiome of the host organism. The metamorphosis of the sea lamprey (Petromyzon marinus) from a sedentary filter-feeding larva to a free-swimming sanguivorous parasite is characterized by major physiological and morphological changes to all organ systems. The transformation of the alimentary canal includes closure of the larval esophagus and the physical isolation of the pharynx from the remainder of the gut, which results in a nonfeeding period that can last up to 8 months. To determine how the gut microbiome is affected by metamorphosis, the microbial communities of feeding and nonfeeding larval and parasitic sea lamprey were surveyed using both culture-dependent and -independent methods. Our results show that the gut of the filter-feeding larva contains a greater diversity of bacteria than that of the blood-feeding parasite, with the parasite gut being dominated by Aeromonas and, to a lesser extent, Citrobacter and Shewanella. Phylogenetic analysis of the culturable Aeromonas from both the larval and parasitic gut revealed that at least five distinct species were represented. Phenotypic characterization of these isolates revealed that over half were capable of sheep red blood cell hemolysis, but all were capable of trout red blood cell hemolysis. This suggests that the enrichment of Aeromonas that accompanies metamorphosis is likely related to the sanguivorous lifestyle of the parasitic sea lamprey.     

Aquatic animals promote antibiotic resistance gene dissemination in water via conjugation: Role of different regions within the zebra fish intestinal tract,and impact on fish intestinal microbiota

The aqueous environment is one of many reservoirs of antibiotic resistance genes (ARGs). Fish, as important aquatic animals which possess ideal intestinal niches for bacteria to grow and multiply, may ingest antibiotic resistance bacteria from aqueous environment. The fish gut would be a suitable environment for conjugal gene transfer including those encoding antibiotic resistance. However, little is known in relation to the impact of ingested ARGs or antibiotic resistance bacteria (ARB) on gut microbiota. Here, we applied the cultivation method, qPCR, nuclear molecular genetic marker and 16S rDNA amplicon sequencing technologies to develop a plasmid‐mediated ARG transfer model of zebrafish. Furthermore, we aimed to investigate the dissemination of ARGs in microbial communities of zebrafish guts after donors carrying self‐transferring plasmids that encode ARGs were introduced in aquaria. On average, 15% of faecal bacteria obtained ARGs through RP4‐mediated conjugal transfer. The hindgut was the most important intestinal region supporting ARG dissemination, with concentrations of donor and transconjugant cells almost 25 times higher than those of other intestinal segments. Furthermore, in the hindgut where conjugal transfer occurred most actively, there was remarkable upregulation of the mRNA expression of the RP4 plasmid regulatory genes, trbBp and trfAp. Exogenous bacteria seem to alter bacterial communities by increasing Escherichia and Bacteroides species, while decreasing Aeromonas compared with control groups. We identified the composition of transconjugants and abundance of both cultivable and uncultivable bacteria (the latter accounted for 90.4%–97.2% of total transconjugants). Our study suggests that aquatic animal guts contribute to the spread of ARGs in water environments.     

Molecular Changes in Entamoeba histolytica in Response to Bacteria1

Entamoeba histolytica, the protozoan parasite, is the causative agent of amoebiasis. The degree of virulence, as inferred from invasiveness, of potentially pathogenic strains may be regulated by both host and parasite factors that determine the gut environment. One such factor that plays an important role is the bacterial flora in the gut. Previous studies have clearly shown that bacterial flora is an important determinant of virulence in E. histolytica. However, the exact nature of changes induced in E. histolytica in response to bacteria and their role in virulence is not clear. In this study the levels of a number of molecules potentially important in virulence mechanisms were determined in E. histolytica cells grown with and without normal human bacterial flora, using enzyme-linked immunosorbent assay. Significant changes were observed only after the E. histolytica cells had been adapted to grow with bacterial flora for a number of generations, and not in short term culture.     

Bacteria abundance and diversity of different life stages of Plutella xylostella (Lepidoptera: Plutellidae), revealed by bacteria culture‐dependent and PCR‐DGGE methods

Microbial abundance and diversity of different life stages (fourth instar larvae, pupae and adults) of the diamondback moth, Plutella xylostella L., collected from field and reared in laboratory, were investigated using bacteria culture‐dependent method and PCR‐DGGE analysis based on the sequence of bacteria 16S rRNA V3 region gene. A large quantity of bacteria was found in all life stages of P. xylostella. Field population had higher quantity of bacteria than laboratory population, and larval gut had higher quantity than pupae and adults. Culturable bacteria differed in different life stages of P. xylostella. Twenty‐five different bacterial strains were identified in total, among them 20 strains were presented in larval gut, only 8 strains in pupae and 14 strains in adults were detected. Firmicutes bacteria, Bacillus sp., were the most dominant species in every life stage. 15 distinct bands were obtained from DGGE electrophoresis gel. The sequences blasted in GenBank database showed these bacteria belonged to six different genera. Phylogenetic analysis showed the sequences of the bacteria belonged to the Actinobacteri, Proteobacteria and Firmicutes. Serratia sp. in Proteobacteria was the most abundant species in larval gut. In pupae, unculturable bacteria were the most dominant species, and unculturable bacteria and Serratia sp. were the most dominant species in adults. Our study suggested that a combination of molecular and traditional culturing methods can be effectively used to analyze and to determine the diversity of gut microflora. These known bacteria may play important roles in development of P. xylostella.     

Gut bacteria of cockroaches are a potential source of antibacterial compound(s)

Here, we hypothesized that the microbial gut flora of animals/pests living in polluted environments, produce substances to thwart bacterial infections. The overall aim of this study was to source microbes inhabiting unusual environmental niches for potential antimicrobial activity. Two cockroach species, Gromphadorhina portentosa (Madagascar) and Blaptica dubia (Dubia) were selected. The gut bacteria from these species were isolated and grown in RPMI 1640 and conditioned media were prepared. Conditioned media were tested against a panel of Gram‐positive (Methicillin‐resistant Staphylococcus aureus, Streptococcus pyogenes, Bacillus cereus) and Gram‐negative (Escherichia coli K1, Salmonella enterica, Serratia marcescens, Pseudomonas aeruginosa, Klebsiella pneumoniae) bacteria, as well as the protist pathogen, Acanthamoeba castellanii. The results revealed that the gut bacteria of cockroaches produce active molecule(s) with potent antibacterial properties, as well as exhibit antiamoebic effects. However, heat‐inactivation at 95°C for 10 min had no effect on conditioned media‐mediated antibacterial and antiamoebic properties. These results suggest that bacteria from novel sources i.e. from the cockroach's gut produce molecules with bactericidal as well as amoebicidal properties that can ultimately lead to the development of therapeutic drugs.

Significance and Impact of the Study

The bacteria isolated from unusual dwellings such as the cockroaches' gut are a useful source of antibacterial and antiamoebal molecules. These are remarkable findings that will open several avenues in our search for novel antimicrobials from unique sources. Furthermore studies will lead to the identification of molecules to develop future antibacterials from insects.     

Gut microbiomes of free‐ranging and captive Namibian cheetahs: Diversity,putative functions and occurrence of potential pathogens

Although the significance of the gut microbiome for host health is well acknowledged, the impact of host traits and environmental factors on the interindividual variation of gut microbiomes of wildlife species is not well understood. Such information is essential; however, as changes in the composition of these microbial communities beyond the natural range might cause dysbiosis leading to increased susceptibility to infections. We examined the potential influence of sex, age, genetic relatedness, spatial tactics and the environment on the natural range of the gut microbiome diversity in free‐ranging Namibian cheetahs (Acinonyx jubatus). We further explored the impact of an altered diet and frequent contact with roaming dogs and cats on the occurrence of potential bacterial pathogens by comparing free‐ranging and captive individuals living under the same climatic conditions. Abundance patterns of particular bacterial genera differed between the sexes, and bacterial diversity and richness were higher in older (>3.5 years) than in younger individuals. In contrast, male spatial tactics, which probably influence host exposure to environmental bacteria, had no discernible effect on the gut microbiome. The profound resemblance of the gut microbiome of kin in contrast to nonkin suggests a predominant role of genetics in shaping bacterial community characteristics and functional similarities. We also detected various Operational Taxonomic Units (OTUs) assigned to potential pathogenic bacteria known to cause diseases in humans and wildlife species, such as Helicobacter spp., and Clostridium perfringens. Captive individuals did not differ in their microbial alpha diversity but exhibited higher abundances of OTUs related to potential pathogenic bacteria and shifts in disease‐associated functional pathways. Our study emphasizes the need to integrate ecological, genetic and pathogenic aspects to improve our comprehension of the main drivers of natural variation and shifts in gut microbial communities possibly affecting host health. This knowledge is essential for in situ and ex situ conservation management.     

Microbial communities within field‐collected Culiseta melanura and Coquillettidia perturbans

Although mosquitoes are well‐known vectors of human and animal diseases, pathogens are only minor components of their total endogenous microbial communities. The midguts of many insects, including mosquitoes, contain diverse microbial communities. In this study, we used denaturing gradient gel electrophoresis to identify the diversity of bacteria in field‐collected adult female Culiseta melanura (Diptera: Culicidae) (Coquillett) and Coquillettidia perturbans (Diptera: Culicidae) (Walker). Few significant differences in bacterial fauna between the two mosquito species were found, but the results suggest that host life history may be a determinant of the endogenous bacterial communities in mosquitoes. In the present study, the dominant bacteria are frequently identified as major components of other mosquito species' microbial flora, suggesting the establishment of a stable association between the mosquitoes and the microbes after initial acquisition from the environment.     

Wild bacterial probiotics fed to larvae of mass‐reared Queensland fruit fly [Bactrocera tryoni (Froggatt)] do not impact long‐term survival,mate selection,or locomotor activity

Queensland fruit fly [Bactrocera tryoni (Froggatt), Diptera, Tephritidae] is the most devastating insect pest impacting Australian horticulture. The Sterile Insect Technique (SIT) is an important component of tephritid pest management programs. However, mass‐rearing and irradiation (to render insects sterile) may reduce the fitness and performance of the insect, including the ability of sterile males to successfully compete for wild females. Manipulation of the gut microbiome, including the supplementation with bacterial probiotics shows promise for enhancing the quality of mass‐reared sterile flies, however there are fewer published studies targeting the larval stage. In this study, we supplemented the larval stage of mass‐reared B. tryoni with bacterial probiotics. We tested several individual bacteria that had been previously isolated and characterized from the gut of wild B. tryoni larvae including Asaia sp., Enterobacter sp., Lactobacillus sp., Leuconostoc sp. We also tested a consortium of all four of these bacterial isolates. The fitness parameters tested included adult survival in field cages, laboratory mate selection of bacteria supplemented males by bacteria nonsupplemented females, and laboratory locomotor activity of adult flies. None of the bacterial probiotic treatments in the current study was significantly different to the control for field survival, mate selection or locomotor activity of adult B. tryoni, which agree with some of the other studies regarding bacterial probiotics fed to the larval stage of tephritids. Future work is needed to determine if feeding the same, and/or other probiotics to adults, as opposed to larvae can positively impact survival, mating performance, mating competitiveness and locomotor activity of B. tryoni. The bacterial group(s) and function of bacterial species that increase fitness and competitiveness is also of interest to tephritid mass‐rearing programs.     

An integrated overview of the bacterial flora composition of Hyalomma anatolicum,the main vector of CCHF

The microbial flora associated with Hyalomma anatolicum ticks was investigated using culture-dependent (CD) and independent (next generation sequencing, NGS) methods. The bacterial profiles of different organs, development stages, sexes, and of host cattle skins were analyzed using the CD method. The egg and female gut microbiota were investigated using NGS. Fourteen distinct bacterial strains were identified using the CD method, of which Bacillus subtilis predominated in eggs, larval guts and in adult female and male guts, suggesting probable transovarial transmission. Bacillus velezensis and B. subtilis were identified in cattle skin and tick samples, suggesting that skin is the origin of tick bacteria. H.anatolicum males harbour lower bacterial diversity and composition than females. The NGS analysis revealed five different bacterial phyla across all samples, Proteobacteria contributing to >95% of the bacteria. In all, 56611sequences were generated representing 6,023 OTUs per female gut and 421 OTUs per egg. Francisellaceae family and Francisella make up the vast majority of the OTUs. Our findings are consistent with interference between Francisella and Rickettsia. The CD method identified bacteria, such B. subtilis that are candidates for vector control intervention approaches such paratransgenesis whereas NGS revealed high Francisella spp. prevalence, indicating that integrated methods are more accurate to characterize microbial community and diversity.     

Characterization of bacterial communities from activated sludge: Culture-dependent numerical identification versus in situ identification using group- and genus-specific rRNA-targeted oligonucleotide probes

The structures of bacterial communities were studied in activated sludge samples obtained from the aerobic and anaerobic zones of a wastewater treatment plant showing enhanced phosphorous removal. Samples were analyzed by in situ hybridization with oligonucleotide probes complementary to selected regions of the 16S and 23S ribosomal RNA (rRNA) characteristic for defined phylogenetic entities (genera and larger groups). The microbial community structures revealed by molecular techniques were compared with the compositions of culturable bacterial communities, obtained from the characterization of 255 isolates from tryptone-soy (TS) agar and R2A agar. These isolates were characterized by 89 physiological tests and their cellular fatty acid patterns, and identified. Culture-dependent techniques indicated the following distribution: different Aeromonas spp. (2.7–8.3% on R2A agar; 45.0–63.7% on TS agar), Acinetobacter spp. (5.4–9.0% on R2A agar; 5.0–9.1% on TS agar), Pseudomonas spp. (up to 10% on R2A agar) and Shewanella putrefaciens (up to 3.0% on R2A agar), all members of the gamma subclass of Proteobacteria, were isolated most frequently. The relatively rare isolates of the beta subclass were identified as Acidovorax spp., Alcaligenes spp., and Comamonas spp., The Gram-positive bacteria (high DNA G+C) were assigned mainly to Arthrobacter spp., Microbacterium spp., and Mycobacterium phlei. In order to assess the in situ abundance of the most frequently isolated genus, Aeromonas, two rRNA-targeted oligonucleotide probes were developed. The two gamma proteobacterial genera Aeromonas and Acinetobacter constituted less than 5% of all bacteria. In situ, Proteobacteria belonging to the beta subclass and high G+C Gram-positive bacteria were dominant. From filamentous bacteria, Sphaerotilus spp. and Leptothrix spp. could be detected occasionally. In addition, one sample contained a high proportion of the morphologically distinct filaments of Microthrix parvicella.As for the genus Acinetobacter, the relative abundance of the most frequently gamma-proteobacterial genus Aeromonas was overestimated by the intrinsic selectivity of cultivation. Cultivation on nutrient-rich medium (TS-agar) especially supported an enhanced isolation of bacteria belonging to these two genera. Correspondence to: P. Kämpfer.     

SEASONALITY OF MICROBIAL FOULING ON ASCOPHYLLUM NODOSUM (L.) LEJOL., FUCUS VESICULOSUS L., POLYSIPHONIA LANOSA (L.) TANDY AND CHONDRUS CRISPUS STACKH1

The nature and seasonal extent of microbial fouling on Ascophyllum nodosum (L.) LeJol., Fucus vesiculosus L., Polysiphonia lanosa (L.) Tandy and Chondrus crispus Stackh. were observed by scanning electron microscopy. Bacterial filaments and smaller rod and coccoid forms dominated the fouling communities on all species, with pennate diatoms constituting a minor component in contrast to results with plastic substrates on which pennate diatoms dominated and preceded bacterial colonization. The total percent microbial coverage on the surfaces of all four seaweed species was determined by monthly stereological analyses of representative composite micrographs. These showed a simultaneous decline between April and May which could represent the die-off of the cold water bacterial flora when water temperature increased past the threshold for obligate psychrophiles. Microbial colonization patterns were directly correlated (P = 0.005) with maximum coverage in April and November–December and reduced levels from May to October. Significant inverse (P < 0.041) correlations between total percent coverage and water temperature indicate distinct seasonal cycles, however, the patterns of dominance by filamentous bacteria and rod and coccoid forms were markedly different. Total coverage patterns of both rhodophytes showed no apparent seasonal cycle and were not related to water temperature. Rod and coccoid bacteria were apparently suppressed year round on P. lanosa relative to the other species. These interspecific differences in seasonal fouling patterns are discussed in light of possible modes of regulation, especially algal antibiosis.     

Free-living culturable bacteria and protozoa from the rhizoplanes of three floating aquatic plant species

Terrestrial plant roots exude compounds that promote the proliferation of microorganisms, a phenomenon called the rhizosphere effect. However, little is known about the influence of roots of aquatic plants on microbial populations. We compared cultivable bacteria and protozoa from the rhizoplanes of Azolla filiculoides Lam., Lemna gibba L., and Ricciocarpus natans L., collected at the Tecocomulco Lagoon (Hidalgo, Mexico). The functional bacterial groups isolated from A. filiculoides, L. gibba and R. natans, were macro- and microscopically characterized, and phylogenetically identified using the 16S rDNA gen. About 96% of isolates corresponded to Gram-negative bacteria, and potential N-fixing free-living bacteria (diazotrophic bacteria) were the most abundant. Molecular analysis detected 15 bacterial genera in the rhizoplane of R. natans, whereas A. filiculoides and L. gibba only yielded five genera, among which Aeromonas was predominant. Twenty-five genera of flagellated and 20 genera of ciliated protozoa were identified. Bodo was the more abundant flagellated, whereas Halteria was the most frequent ciliate. All three aquatic plants showed the rhizospheric effects. The most abundant and diverse community of protozoa was found in A. filiculoides, which also had the most abundant bacterial community, but the highest bacterial diversity was found in R. natans.     

Autochthonous bacterial flora indicated by PCR-DGGE of 16S rRNA gene fragments from the alimentary tract of Costelytra zealandica (Coleoptera: Scarabaeidae)

Aims: To locate and identify putative autochthonous bacteria within the grass grub gut that may have a role in symbiosis. Methods and Results: Polymerase chain reaction-denaturing gradient gel electrophoresis (DGGE) fingerprinting was used to investigate bacterial diversity in the grass grub larval gut. The microbial community profiles from five geographically distinct populations were compared and the influence of feeding was analysed. Bacterial community in the midgut was highly variable between locations and was affected by feeding. The hindgut contained a more diverse but stable bacterial community that was less affected by external conditions. Forty-seven distinct DGGE bands, representing different bacterial genotypes, could be distinguished from all samples, with 34 different bands occurring in the hindgut. The 22 most common bands were isolated and DNA was sequenced. Sequence analysis revealed that most bacteria (16/22) were affiliated to the Clostridiales with the predominant bacteria affiliated to the genus Clostridium. The remaining bacteria were aligned to the Betaproteobacteria, Deltaproteobacteria and Bacteroidetes. Conclusions: The grass grub larva has an autochthonous microflora with predominance of Clostridium spp. in the hindgut. Significance and Impact of the Study: Occurrence of an autocthonous microflora in the grass grub hindgut suggests a symbiotic relationship which could help explain the ability of larval scarabs to feed on recalcitrant organic matter.