Bacterial Infection and Immune Responses in Lutzomyia longipalpis Sand Fly Larvae Midgut

The midgut microbial community in insect vectors of disease is crucial for an effective immune response against infection with various human and animal pathogens. Depending on the aspects of their development, insects can acquire microbes present in soil, water, and plants. Sand flies are major vectors of leishmaniasis, and shown to harbor a wide variety of Gram-negative and Gram-positive bacteria. Sand fly larval stages acquire microorganisms from the soil, and the abundance and distribution of these microorganisms may vary depending on the sand fly species or the breeding site. Here, we assess the distribution of two bacteria commonly found within the gut of sand flies, Pantoea agglomerans and Bacillus subtilis. We demonstrate that these bacteria are able to differentially infect the larval digestive tract, and regulate the immune response in sand fly larvae. Moreover, bacterial distribution, and likely the ability to colonize the gut, is driven, at least in part, by a gradient of pH present in the gut.     

肠道微生物与昆虫的共生关系

昆虫肠道栖息着大量的微生物。随着近年来研究肠道微生物的方法不断进步,尤其是基于16S rDNA的分子生物学方法的应用,人们对肠道微生物的了解逐渐加深。昆虫肠道对于微生物的拓殖存在一定的选择作用。肠道微生物对昆虫寄主的作用包括提供营养、利用拓殖抗性抵抗外来微生物侵袭、参与多重营养关系、引起昆虫免疫反应。长期进化过程中肠道微生物与昆虫发展出紧密的共生关系,微生物发展出一系列手段适应昆虫肠道环境。文章从以上几个方面对近年来的研究进展进行总结,并对昆虫肠道微生态学的实践意义和将来可能的研究热点进行展望。     

Composition and Stability of the Microbial Community inside the Digestive Tract of the Aquatic Crustacean <Emphasis Type="Italic">Daphnia magna</Emphasis>

Small filter-feeding zooplankton organisms like the cladoceran Daphnia spp. are key members of freshwater food webs. Although several interactions between Daphnia and bacteria have been investigated, the importance of the microbial communities inside Daphnia guts has been studied only poorly so far. In the present study, we characterised the bacterial community composition inside the digestive tract of a laboratory-reared clonal culture of Daphnia magna using 16S rRNA gene libraries and terminal-restriction length polymorphism fingerprint analyses. In addition, the diversity and stability of the intestinal microbial community were investigated over time, with different food sources as well as under starvation stress and death, and were compared to the community in the cultivation water. The diversity of the Daphnia gut microbiota was low. The bacterial community consisted mainly of Betaproteobacteria (e.g. Limnohabitans sp.), few Gammaproteobacteria (e.g. Pseudomonas sp.) and Bacteroidetes that were related to facultatively anaerobic bacteria, but did not contain typical fermentative or obligately anaerobic gut bacteria. Rather, the microbiota was constantly dominated by Limnohabitans sp. which belongs to the Lhab-A1 tribe (previously called R-BT065 cluster) that is abundant in various freshwaters. Other bacterial groups varied distinctly even under constant cultivation conditions. Overall, the intestinal microbial community did not reflect the community in the surrounding cultivation water and clustered separately when analysed via the Additive Main Effects and Multiplicative Interaction model. In addition, the microbiota proved to be stable also when Daphnia were exposed to bacteria associated with a different food alga. After starvation, the community in the digestive tract was reduced to stable members. After death of the host animals, the community composition in the gut changed distinctly, and formerly undetected bacteria were activated. Our results suggest that the Daphnia microbiota consists mainly of an aerobic resident bacterial community which is indigenous to this habitat.     

Honeybees and bumblebees share similar bacterial symbionts

Associations with symbiotic microorganisms are a major source for evolutionary innovation in eukaryotes. Arthropods have long served as model systems to study such associations, especially since Paul Buchner’s (1965) seminal work that beautifully illustrated the enormous diversity of microorganisms associated with insects. Particularly high taxonomic and functional diversities of microbial symbionts have been found in the guts and gut‐associated organs of insects. These microorganisms play important roles in the digestion, nutrition and defence of the host. However, most studies of gut microorganisms have focused on single host taxa, limiting the ability to draw general conclusions on composition and functional roles of the insect gut microbiota. This is especially true for the diverse and important insect order Hymenoptera that comprises the bees, wasps and ants. Recently, Russell et al. (2009) analysed the bacterial community associated with diverse ant species and found evidence for changes in the microbial gut community coinciding with the evolution of herbivory. In this issue of Molecular Ecology, Martinson et al. (2011) provide the first broad‐scale bacterial survey for bees. Their findings substantiate earlier evidence for a surprisingly simple gut microbiota in honeybees (Apis mellifera) that is composed of only six to ten major phylotypes. Importantly, Martinson et al. demonstrate for the first time that the same bacterial phylotypes are major constituents of other Apis as well as Bombus species, but not of any other bees and wasps outside of the corbiculate bees, a clade of four tribes within the subfamily Apinae. These results indicate that corbiculate bees harbour a specific and possibly co‐evolved bacterial community in their digestive tract. Furthermore, the comparison with other bees and wasps suggests that changes in social lifestyle may have had a stronger effect on the evolution of the gut microbiota than the dietary shift from predatory ancestors to pollen‐feeding (i.e. herbivorous) species. These findings have far‐reaching implications for research on the microbial symbionts of insects as well as on the nutritional physiology of the ecologically and economically important group of corbiculate bees.     

Green fluorescent protein (GFP)-labeling of enterobacteria associated with fruit flies (Diptera: Tephritidae) and persistence in their natural host Rhagoletis completa Cresson

Fruit flies (Diptera: Tephritidae) are a highly successful, widespread group of insects that cause economic damage in agriculture. Data available so far on the composition of the bacterial community associated with their digestive tract indicate that members of Enterobacteriaceae are the species most often isolated. Bacteria naturally occurring in insect guts may be engineered and used to study the spatial and functional interactions of microbes within the insect system and offer one route to meet the demand for novel insect pest management strategies. With this aim we introduced by conjugation the gfp gene carried by the suicide plasmid pTn5gfpmut1 into Klebsiella oxytoca and Raoultella (formerly Klebsiella ) spp. strains isolated from the oesophageal bulb of the fruit flies Ceratitis capitata (Wiedemann) and Rhagoletis completa Cresson, respectively. The GFP-encoding gene was stably maintained in two tested transgenic strains, both originally isolated from R. completa. In one case, GFP-labeled bacterial cells were used to feed larvae and adults of the original host. Genetically modified bacteria were able to colonize the gut of larvae and persisted through all larval instars to pupal stage.     

We and herbivores eat endophytes

Health depends on the diet and a vegetal diet promotes health by providing fibres, vitamins and diverse metabolites. Remarkably, plants may also provide microbes. Fungi and bacteria that reside inside plant tissues (endophytes) seem better protected to survive digestion; thus, we investigated the reported evidence on the endophytic origin of some members of the gut microbiota in animals such as panda, koala, rabbits and tortoises and several herbivore insects. Data examined here showed that some members of the herbivore gut microbiota are common plant microbes, which derived to become stable microbiota in some cases. Endophytes may contribute to plant fibre or antimetabolite degradation and synthesis of metabolites with the plethora of enzymatic activities that they display; some may have practical applications, for example, Lactobacillus plantarum found in the intestinal tract, plants and in fermented food is used as a probiotic that may defend animals against bacterial and viral infections as other endophytic-enteric bacteria do. Clostridium that is an endophyte and a gut bacterium has remarkable capabilities to degrade cellulose by having cellulosomes that may be considered the most efficient nanomachines. Cellulose degradation is a challenge in animal digestion and for biofuel production. Other endophytic-enteric bacteria may have cellulases, pectinases, xylanases, tannases, proteases, nitrogenases and other enzymatic capabilities that may be attractive for biotechnological developments, indeed many endophytes are used to promote plant growth. Here, a cycle of endophytic-enteric-soil-endophytic microbes is proposed which has relevance for health and comprises the fate of animal faeces as natural microbial inoculants for plants that constitute bacterial sources for animal guts.     

Influence of dietary aconitine and nicotine on the gut microbiota of two lepidopteran herbivores

The gut microbiota plays an important role in pheromone production, pesticide degradation, vitamin synthesis, and pathogen prevention in the host animal. Therefore, similar to gut morphology and digestive enzyme activity, the gut microbiota may also get altered under plant defensive compound-induced stress. To test this hypothesis, Dendrolimus superans larvae were fed either aconitine- or nicotine-treated fresh leaves of Larix gmelinii, and Lymantria dispar larvae were fed either aconitine- or nicotine-treated fresh leaves of Salix matsudana. Subsequently, the larvae were sampled 72hr after diet administration and DNA extracted from larval enteric canals were employed for gut microbial 16S ribosomal RNA gene sequencing (338 F and 806 R primers). The sequence analysis revealed that dietary nicotine and aconitine influenced the dominant bacteria in the larval gut and determined their abundance. Moreover, the effect of either aconitine or nicotine on D. superans and L. dispar larvae had a greater dependence on insect species than on secondary plant metabolites. These findings further our understanding of the interaction between herbivores and host plants and the coevolution of plants and insects.     

Complexity and variability of gut commensal microbiota in polyphagous lepidopteran larvae

Background

The gut of most insects harbours nonpathogenic microorganisms. Recent work suggests that gut microbiota not only provide nutrients, but also involve in the development and maintenance of the host immune system. However, the complexity, dynamics and types of interactions between the insect hosts and their gut microbiota are far from being well understood.

Methods/Principal Findings

To determine the composition of the gut microbiota of two lepidopteran pests, Spodoptera littoralis and Helicoverpa armigera, we applied cultivation-independent techniques based on 16S rRNA gene sequencing and microarray. The two insect species were very similar regarding high abundant bacterial families. Different bacteria colonize different niches within the gut. A core community, consisting of Enterococci, Lactobacilli, Clostridia, etc. was revealed in the insect larvae. These bacteria are constantly present in the digestion tract at relatively high frequency despite that developmental stage and diet had a great impact on shaping the bacterial communities. Some low-abundant species might become dominant upon loading external disturbances; the core community, however, did not change significantly. Clearly the insect gut selects for particular bacterial phylotypes.

Conclusions

Because of their importance as agricultural pests, phytophagous Lepidopterans are widely used as experimental models in ecological and physiological studies. Our results demonstrated that a core microbial community exists in the insect gut, which may contribute to the host physiology. Host physiology and food, nevertheless, significantly influence some fringe bacterial species in the gut. The gut microbiota might also serve as a reservoir of microorganisms for ever-changing environments. Understanding these interactions might pave the way for developing novel pest control strategies.     

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.     

Are you what you eat? A highly transient and prey‐influenced gut microbiome in the grey house spider Badumna longinqua

Stable core microbial communities have been described in numerous animal species and are commonly associated with fitness benefits for their hosts. Recent research, however, highlights examples of species whose microbiota are transient and environmentally derived. Here, we test the effect of diet on gut microbial community assembly in the spider Badumna longinqua. Using 16S rRNA gene amplicon sequencing combined with quantitative PCR, we analyzed diversity and abundance of the spider's gut microbes, and simultaneously characterized its prey communities using nuclear rRNA markers. We found a clear correlation between community similarity of the spider's insect prey and gut microbial DNA, suggesting that microbiome assembly is primarily diet‐driven. This assumption is supported by a feeding experiment, in which two types of prey—crickets and fruit flies—both substantially altered microbial diversity and community similarity between spiders, but did so in different ways. After cricket consumption, numerous cricket‐derived microbes appeared in the spider's gut, resulting in a rapid homogenization of microbial communities among spiders. In contrast, few prey‐associated bacteria were detected after consumption of fruit flies; instead, the microbial community was remodelled by environmentally sourced microbes, or abundance shifts of rare taxa in the spider's gut. The reshaping of the microbiota by both prey taxa mimicked a stable core microbiome in the spiders for several weeks post feeding. Our results suggest that the spider's gut microbiome undergoes pronounced temporal fluctuations, that its assembly is dictated by the consumed prey, and that different prey taxa may remodel the microbiota in drastically different ways.     

Geographical and ecological stability of the symbiotic mid‐gut microbiota in European firebugs,Pyrrhocoris apterus (Hemiptera,Pyrrhocoridae)

Symbiotic bacteria often play an essential nutritional role for insects, thereby allowing them to exploit novel food sources and expand into otherwise inaccessible ecological niches. Although many insects are inhabited by complex microbial communities, most studies on insect mutualists so far have focused on single endosymbionts and their interactions with the host. Here, we provide a comprehensive characterization of the gut microbiota of the red firebug (Pyrrhocoris apterus, Hemiptera, Pyrrhocoridae), a model organism for physiological and endocrinological research. A combination of several culture‐independent techniques (454 pyrosequencing, quantitative PCR and cloning/sequencing) revealed a diverse community of likely transient bacterial taxa in the mid‐gut regions M1, M2 and M4. However, the completely anoxic M3 region harboured a distinct microbiota consisting of facultative and obligate anaerobes including Actinobacteria (Coriobacterium glomerans and Gordonibacter sp.), Firmicutes (Clostri‐dium sp. and Lactococcus lactis) and Proteobacteria (Klebsiella sp. and a previously undescribed Rickettsiales bacterium). Characterization of the M3 microbiota in different life stages of P. apterus indicated that the symbiotic bacterial community is vertically transmitted and becomes well defined between the second and third nymphal instar, which coincides with the initiation of feeding. Comparing the mid‐gut M3 microbial communities of P. apterus individuals from five different populations and after feeding on three different diets revealed that the community composition is qualitatively and quantitatively very stable, with the six predominant taxa being consistently abundant. Our findings suggest that the firebug mid‐gut microbiota constitutes a functionally important and possibly coevolved symbiotic community.     

蜜蜂肠道微生物与其社会性的关系

蜜蜂是一种典型的营群居生活的社会性昆虫,相比独居生活的昆虫,其肠道微生物具有独特的区系结构。这种独特肠道微生物与其社会性之间的关系是一个重要的科学问题。现有研究显示,蜜蜂肠道的优势菌包括9大类群。消化道不同区段的优势菌种类和丰度存在差异。主要表现为前肠种类少、丰度低、后肠种类多、数量大,占了全消化道微生物的99%以上。不同社会分工的蜜蜂肠道微生物区系结构存在差异,肠道微生物会通过影响胰岛素信号的传导、保幼激素和卵黄原蛋白的合成以及蜜蜂抗氧化应激的能力等对蜜蜂的级型分化、社会分工、摄食行为及寿命长短产生调节作用。除此之外,蜜蜂肠道微生物还具有激活免疫、抑制病原菌生长、降解食物、促进养分吸收、解毒、发酵蜂蜜和蜂粮等作用。主要针对蜜蜂肠道微生物的基本特征及其与蜜蜂社会性的关系作一简要综述。     

Mining diversity of the natural biorefinery housed within Tipula abdominalis larvae for use in an industrial biorefinery for production of lignocellulosic ethanol

Abstract Although they are the largest taxonomic group of animals, relatively few insects have been examined for symbiotic relationships with micro-organisms. However, this is rapidly changing because of the potential for examination of the natural insect–microbe–lignocellulose interactions to provide insights for biofuel technology. Micro-organisms associated with lignocellulose-consuming insects often facilitate the digestion of the recalcitrant plant diet; therefore these microbial communities may be mined for novel lignocellulose-degrading microbes, or for robust and inexpensive biocatalysts necessary for economically feasible biofuel production from lignocellulose. These insect–microbe interactions are influenced by the ecosystem and specific lignocellulose diet, and appreciating the whole ecosystem–insect–microbiota–lignocellulose as a natural biorefinery provides a rich and diverse framework from which to design novel industrial processes. One such natural biorefinery, the Tipula abdominalis larvae in riparian ecosystems, is reviewed herein with applications for biochemical processes and overcoming challenges involved in conversion of lignocellulosic biomass to fuel ethanol. From the dense and diverse T. abdominalis larval hindgut microbial community, a cellulolytic bacterial isolate, 27C64, demonstrated enzymatic activity toward many model plant polymers and also produced a bacterial antibiotic. 27C64 was co-cultured with yeast in fermentation of pine to ethanol, which allowed for a 20% reduction of commercial enzyme. In this study, a micro-organism from a lignocellulose-consuming insect was successfully applied for improvement of biomass-to-biofuel technology.     

Comparison of gut microbiota of healthy and diseased walking sticks,Phasmotaenia lanyuhensis

Research on gut microbiota of phytophagous insects has shown to be important for the physiological functions of insect hosts; however, little is known about the changes in gut microbiota when they are suffering from environmental stress or pathogen infections. During rearing of Phasmotaenia lanyuhensis (Phasmatodea: Phasmatidae), sluggish locomotion was usually followed by the death of the insect with a symptom of melanization in the front part of the abdomen. Therefore, the abnormal individuals were initially classified into moribund, light- and serious-symptom based on the level of abnormal physiological circumstances and melanization. The gut microbiota of these samples were further investigated by 16S metagenomic sequencing and the differences in bacterial abundance and structure of bacterial community were analyzed. A decrease in microbiota diversity was observed in the diseased P. lanyuhensis, with the abundance of phyla Proteobacteria and Firmicute relatively higher compared to those without symptom. Interestingly, principal component analysis based on the bacterial richness was correlated to the level of melanization symptom in the diseased P. lanyuhensis, suggested the change in bacterial microbiota involved in this abnormal circumstance. However, the factor that caused the initial alternation of microbiota remains to be identified. Additionally, the lack of bacterial diversity (i.e., absence of Meiothermus and Nubsella spp.) in P. lanyuhensis might reduce the fitness for surviving. This report provided the comprehensive microbiota analysis for P. lanyuhensis and concluded that either the relative abundance or the bacterial diversity of microbiota in the insect digestive system may influence the physiological functions of phytophagous insects.     

Do diet and taxonomy influence insect gut bacterial communities?

Many insects contain diverse gut microbial communities. While several studies have focused on a single or small group of species, comparative studies of phylogenetically diverse hosts can illuminate general patterns of host–microbiota associations. In this study, we tested the hypotheses that (i) host diet and (ii) host taxonomy structure intestinal bacterial community composition among insects. We used published 16S rRNA gene sequence data for 58 insect species in addition to four beetle species sampled from the Sevilleta National Wildlife Refuge to test these hypotheses. Overall, gut bacterial species richness in these insects was low. Decaying wood xylophagous insects harboured the richest bacterial gut flora (102.8 species level operational taxonomic units (OTUs)/sample ± 71.7, 11.8 ± 5.9 phylogenetic diversity (PD)/sample), while bees and wasps harboured the least rich bacterial communities (11.0 species level OTUs/sample ± 5.4, 2.6 ± 0.8 PD/sample). We found evidence to support our hypotheses that host diet and taxonomy structure insect gut bacterial communities (P < 0.001 for both). However, while host taxonomy was important in hymenopteran and termite gut community structure, diet was an important community structuring factor particularly for insect hosts that ingest lignocellulose‐derived substances. Our analysis provides a baseline comparison of insect gut bacterial communities from which to test further hypotheses concerning proximate and ultimate causes of these associations.     

The effects of geographic origin and antibiotic treatment on the gut symbiotic communities of Bactrocera oleae populations

The olive fruit fly, Bactrocera oleae (Rossi) (Diptera: Tephritidae), is the major insect pest of olive orchards (Olea europaea L.), causing extensive damages on cultivated olive crops worldwide. Due to its economic importance, it has been the target species for a variety of population control approaches including the sterile insect technique (SIT). However, the inefficiency of the current mass‐rearing techniques impedes the successful application of area‐wide integrated pest management programs with an SIT component. It has been shown that insect mass rearing and quality of sterile insects can be improved by the manipulation of the insect gut microbiota and probiotic applications. In order to exploit the gut bacteria, it is important to investigate the structure of the gut microbial community. In the current study, we characterized the gut bacterial profile of two wild olive fruit fly populations introduced in laboratory conditions using next generation sequencing of two regions of the 16S rRNA gene. We compared the microbiota profiles regarding the geographic origin of the samples. Additionally, we investigated potential changes in the gut bacteria community before and after the first exposure of the wild adult flies to artificial adult diet with and without antibiotics. Various genera – such as Erwinia, Providencia, Enterobacter, and Klebsiella – were detected for the first time in B. oleae. The most dominant species was Candidatus Erwinia dacicola Capuzzo et al. and it was not affected by the antibiotics in the artificial adult diet used in the first generation of laboratory rearing. Geographic origin affected the overall structure of the gut community of the olive fruit fly, but antibiotic treatment in the first generation did not significantly alter the gut microbiota community.     

昆虫肠道防御及微生物稳态维持机制

在长期的进化过程中,昆虫形成了独特的肠道防御系统,主要由物理屏障和免疫系统共同作用来抵御外来微生物的入侵。如大部分后生动物一样,昆虫肠道上皮细胞无时无刻不与微生物接触,其种类从有益的共生菌、随食物进入的微生物到影响宿主生命的病原菌。在这样一种复杂的环境中,为了实现防御肠道病原微生物的同时又能维持共生微生物稳定的目的,宿主肠道上皮细胞必须在免疫应激和免疫耐受之间保持一种稳态平衡。Duox-ROS免疫系统和免疫缺陷(immune deficiency,Imd)信号通路作为肠道免疫反应的基本途径,必然参与调节此过程。本文从昆虫肠道防御组成、肠道免疫信号通路作用分子机制以及肠道免疫系统在肠道微生物群落稳态维持中的作用的最新研究进展进行综述。     

Angiosperm to Gymnosperm host‐plant switch entails shifts in microbiota of the Welwitschia bug,Probergrothius angolensis (Distant, 1902)

The adaptation of herbivorous insects to new host plants is key to their evolutionary success in diverse environments. Many insects are associated with mutualistic gut bacteria that contribute to the host's nutrition and can thereby facilitate dietary switching in polyphagous insects. However, how gut microbial communities differ between populations of the same species that feed on different host plants remains poorly understood. Most species of Pyrrhocoridae (Hemiptera: Heteroptera) are specialist seed‐feeders on plants in the family Malvaceae, although populations of one species, Probergrothius angolensis, have switched to the very distantly related Welwitschia mirabilis plant in the Namib Desert. We first compared the development and survival of laboratory populations of Pr. angolensis with two other pyrrhocorids on seeds of Welwitschia and found only Pr. angolensis was capable of successfully completing its development. We then collected Pr. angolensis in Namibia from Malvaceae and Welwitschia host plants, respectively, to assess their bacterial and fungal community profiles using high‐throughput amplicon sequencing. Comparison with long‐term laboratory‐reared insects indicated stable associations of Pr. angolensis with core bacteria (Commensalibacter, Enterococcus, Bartonella and Klebsiella), but not with fungi or yeasts. Phylogenetic analyses of core bacteria revealed relationships to other insect‐associated bacteria, but also found new taxa indicating potential host‐specialized nutritional roles. Importantly, the microbial community profiles of bugs feeding on Welwitschia versus Malvaceae revealed stark and consistent differences in the relative abundance of core bacterial taxa that correlate with the host‐plant switch; we were able to reproduce this result through feeding experiments. Thus, a dynamic gut microbiota may provide a means for insect adaptation to new host plants in new environments when food plants are extremely divergent.