Developmental stages and gut microenvironments influence gut microbiota dynamics in the invasive beetle Popillia japonica Newman (Coleoptera: Scarabaeidae).

Popillia japonica Newman (Coleoptera: Scarabaeidae) is a highly polyphagous invasive beetle originating from Japan. This insect is highly resilient and able to rapidly adapt to new vegetation. Insect-associated microorganisms can play important roles in insect physiology, helping their hosts to adapt to changing conditions and potentially contributing to an insect's invasive potential. Such symbiotic bacteria can be part of a core microbiota that is stably transmitted throughout the host's life cycle or selectively recruited from the environment at each developmental stage. The aim of this study was to investigate the origin, stability and turnover of the bacterial communities associated with an invasive population of P. japonica from Italy. Our results demonstrate that soil microbes represent an important source of gut bacteria for P. japonica larvae, but as the insect develops, its gut microbiota richness and diversity decreased substantially, paralleled by changes in community composition. Notably, only 16.75% of the soil bacteria present in larvae are maintained until the adult stage. We further identified the micro-environments of different gut sections as an important factor shaping microbiota composition in this species, likely due to differences in pH, oxygen availability and redox potential. In addition, P. japonica also harboured a stable bacterial community across all developmental stages, consisting of taxa well known for the degradation of plant material, namely the families Ruminococcacae, Christensenellaceae and Lachnospiraceae. Interestingly, the family Christensenallaceae had so far been observed exclusively in humans. However, the Christensenellaceae operational taxonomic units found in P. japonica belong to different taxonomic clades within this family.

A genomic investigation of ecological differentiation between free-living and Drosophila-associated bacteria.

Various bacterial taxa have been identified both in association with animals and in the external environment, but the extent to which related bacteria from the two habitat types are ecologically and evolutionarily distinct is largely unknown. This study investigated the scale and pattern of genetic differentiation between bacteria of the family Acetobacteraceae isolated from the guts of Drosophila fruit flies, plant material and industrial fermentations. Genome-scale analysis of the phylogenetic relationships and predicted functions was conducted on 44 Acetobacteraceae isolates, including newly sequenced genomes from 18 isolates from wild and laboratory Drosophila. Isolates from the external environment and Drosophila could not be assigned to distinct phylogenetic groups, nor are their genomes enriched for any different sets of genes or category of predicted gene functions. In contrast, analysis of bacteria from laboratory Drosophila showed they were genetically distinct in their universal capacity to degrade uric acid (a major nitrogenous waste product of Drosophila) and absence of flagellar motility, while these traits vary among wild Drosophila isolates. Analysis of the competitive fitness of Acetobacter discordant for these traits revealed a significant fitness deficit for bacteria that cannot degrade uric acid in culture with Drosophila. We propose that, for wild populations, frequent cycling of Acetobacter between Drosophila and the external environment prevents genetic differentiation by maintaining selection for traits adaptive in both the gut and external habitats. However, laboratory isolates bear the signs of adaptation to persistent association with the Drosophila host under tightly defined environmental conditions.

New Insights into the Microbiota of Moth Pests.

In recent years, next generation sequencing (NGS) technologies have helped to improve our understanding of the bacterial communities associated with insects, shedding light on their wide taxonomic and functional diversity. To date, little is known about the microbiota of lepidopterans, which includes some of the most damaging agricultural and forest pests worldwide. Studying their microbiota could help us better understand their ecology and offer insights into developing new pest control strategies. In this paper, we review the literature pertaining to the microbiota of lepidopterans with a focus on pests, and highlight potential recurrent patterns regarding microbiota structure and composition.

Modulation of fecal Clostridiales bacteria and butyrate by probiotic intervention with Lactobacillus paracasei DG varies among healthy adults.

BACKGROUND: The modulation of gut microbiota is considered to be the first target to establish probiotic efficacy in a healthy population. OBJECTIVE: This study was conducted to determine the impact of a probiotic on the intestinal microbial ecology of healthy volunteers. METHODS: High-throughput 16S ribosomal RNA gene sequencing was used to characterize the fecal microbiota in healthy adults (23-55 y old) of both sexes, before and after 4 wk of daily consumption of a capsule containing at least 24 billion viable Lactobacillus paracasei DG cells, according to a randomized, double-blind, crossover placebo-controlled design. RESULTS: Probiotic intake induced an increase in Proteobacteria (P = 0.006) and in the Clostridiales genus Coprococcus (P = 0.009), whereas the Clostridiales genus Blautia (P = 0.036) was decreased; a trend of reduction was also observed for Anaerostipes (P = 0.05) and Clostridium (P = 0.06). We also found that the probiotic effect depended on the initial butyrate concentration. In fact, participants with butyrate >100 mmol/kg of wet feces had a mean butyrate reduction of 49 ± 21% and a concomitant decrease in the sum of 6 Clostridiales genera, namely Faecalibacterium, Blautia, Anaerostipes, Pseudobutyrivibrio, Clostridium, and Butyrivibrio (P = 0.021), after the probiotic intervention. In contrast, in participants with initial butyrate concentrations <25 mmol/kg of wet feces, the probiotic contributed to a 329 ± 255% (mean ± SD) increment in butyrate concomitantly with an ∼55% decrease in Ruminococcus (P = 0.016) and a 150% increase in an abundantly represented unclassified Bacteroidales genus (P = 0.05). CONCLUSIONS: The intake of L. paracasei DG increased the Blautia:Coprococcus ratio, which, according to the literature, can potentially confer a health benefit on the host. The probiotic impact on the microbiota and on short-chain fatty acids, however, seems to strictly depend on the initial characteristics of the intestinal microbial ecosystem. In particular, fecal butyrate concentrations could represent an important biomarker for identifying subjects who may benefit from probiotic treatment. This trial was registered at www.controlled-trials.com/isrctn as ISRCTN56945491.

A 2000-Year-Old Bacillus stercoris Strain Sheds Light on the Evolution of Cyclic Antimicrobial Lipopeptide Synthesis.

Some bacteria (notably the genera Bacillus and Clostridium) have the capacity to form endospores that can survive for millions of years in isolated habitats. The genomes of such ancient bacteria provide unique opportunities to understand bacterial evolution and metabolic capabilities over longer time scales. Herein, we sequenced the genome of a 2000-year-old bacterial strain (Mal05) isolated from intact apple seeds recovered during archaeological excavations of a Roman villa in Italy. Phylogenomic analyses revealed that this strain belongs to the species Bacillus stercoris and that it is placed in an early-branching position compared to most other strains of this species. Similar to other Bacillus species, B. stercoris Mal05 had been previously shown to possess antifungal activity. Its genome encodes all the genes necessary for the biosynthesis of fengycin and surfactin, two cyclic lipopeptides known to play a role in the competition of Bacilli with other microorganisms due to their antimicrobial activity. Comparative genomics and analyses of selective pressure demonstrate that these genes are present in all sequenced B. stercoris strains, despite the fact that they are not under strong purifying selection. Hence, these genes may not be essential for the fitness of these bacteria, but they can still provide a competitive advantage against other microorganisms present in the same environment.

Exploring the expressiveness of abstract metabolic networks.

Metabolism is characterised by chemical reactions linked to each other, creating a complex network structure. The whole metabolic network is divided into pathways of chemical reactions, such that every pathway is a metabolic function. A simplified representation of metabolism, which we call an abstract metabolic network, is a graph in which metabolic pathways are nodes and there is an edge between two nodes if their corresponding pathways share one or more compounds. The abstract metabolic network of a given organism results in a small network that requires low computational power to be analysed and makes it a suitable model to perform a large-scale comparison of organisms' metabolism. To explore the potentials and limits of such a basic representation, we considered a comprehensive set of KEGG organisms, represented through their abstract metabolic network. We performed pairwise comparisons using graph kernel methods and analyse the results through exploratory data analysis and machine learning techniques. The results show that abstract metabolic networks discriminate macro evolutionary events, indicating that they are expressive enough to capture key steps in metabolism evolution.

Genome sequence of Blastococcus saxobsidens DD2, a stone-inhabiting bacterium.

Members of the genus Blastococcus have been isolated from sandstone monuments, as well as from sea, soil, plant, and snow samples. We report here the genome sequence of a member of this genus, Blastococcus saxobsidens strain DD2, isolated from below the surface of a Sardinian wall calcarenite stone sample.

Genome sequence of radiation-resistant Modestobacter marinus strain BC501, a representative actinobacterium that thrives on calcareous stone surfaces.

Here we report the full genome sequence of Modestobacter marinus strain BC501, an actinobacterial isolate that thrives on stone surfaces. The generated chromosome is circular, with a length of 5.57 Mb and a G+C content of 74.13%, containing 5,445 protein-coding genes, 48 tRNAs, and 3 ribosomal operons.

Delayed larval development in Anopheles mosquitoes deprived of Asaia bacterial symbionts.

BACKGROUND: In recent years, acetic acid bacteria have been shown to be frequently associated with insects, but knowledge on their biological role in the arthropod host is limited. The discovery that acetic acid bacteria of the genus Asaia are a main component of the microbiota of Anopheles stephensi makes this mosquito a useful model for studies on this novel group of symbionts. Here we present experimental results that provide a first evidence for a beneficial role of Asaia in An. stephensi. RESULTS: Larvae of An. stephensi at different stages were treated with rifampicin, an antibiotic effective on wild-type Asaia spp., and the effects on the larval development were evaluated. Larvae treated with the antibiotic showed a delay in the development and an asynchrony in the appearance of later instars. In larvae treated with rifampicin, but supplemented with a rifampicin-resistant mutant strain of Asaia, larval development was comparable to that of control larvae not exposed to the antibiotic. Analysis of the bacterial diversity of the three mosquito populations confirmed that the level of Asaia was strongly decreased in the antibiotic-treated larvae, since the symbiont was not detectable by PCR-DGGE (denaturing gradient gel electrophoresis), while Asaia was consistently found in insects supplemented with rifampicin plus the antibiotic-resistant mutant in the diet, and in those not exposed to the antibiotic. CONCLUSIONS: The results here reported indicate that Asaia symbionts play a beneficial role in the normal development of An. stephensi larvae.

The yeast Wickerhamomyces anomalus (Pichia anomala) inhabits the midgut and reproductive system of the Asian malaria vector Anopheles stephensi.

While symbiosis between bacteria and insects has been thoroughly investigated in the last two decades, investments on the study of yeasts associated with insects have been limited. Insect-associated yeasts are placed on different branches of the phylogenetic tree of fungi, indicating that these associations evolved independently on several occasions. Isolation of yeasts is frequently reported from insect habitats, and in some cases yeasts have been detected in the insect gut and in other organs/tissues. Here we show that the yeast Wickerhamomyces anomalus, previously known as Pichia anomala, is stably associated with the mosquito Anopheles stephensi, a main vector of malaria in Asia. Wickerhamomyces anomalus colonized pre-adult stages (larvae L(1)-L(4) and pupae) and adults of different sex and age and could be isolated in pure culture. By a combination of transmission electron microscopy and fluorescent in situ hybridization techniques, W. anomalus was shown to localize in the midgut and in both the male and female reproductive systems, suggesting multiple transmission patterns.

Complete Genome Sequence of Rhynchophorus ferrugineus Endocytobiont "Candidatus Nardonella dryophthoridicola" Strain NardRF.

We report the complete genome sequence and annotation of "Candidatus Nardonella dryophthoridicola" strain NardRF, obtained by sequencing its host bacteriome, Rhynchophorus ferrugineus, using Oxford Nanopore technology.

Molecular evidence for multiple infections as revealed by typing of Asaia bacterial symbionts of four mosquito species.

The recent increased detection of acetic acid bacteria (AAB) of the genus Asaia as symbionts of mosquitoes, such as Anopheles spp. and Aedes spp., prompted us to investigate the diversity of these symbionts and their relationships in different mosquito species and populations. Following cultivation-dependent and -independent techniques, we investigated the microbiota associated with four mosquito species, Anopheles stephensi, Anopheles gambiae, Aedes aegypti, and Aedes albopictus, which are important vectors of human and/or animal pathogens. Denaturing gradient gel electrophoresis (DGGE) analysis based on the 16S rRNA gene revealed the presence of several bacterial taxa, among which Asaia sequences were among the dominant in most of the samples. A collection of 281 Asaia isolates in cell-free media was established from individuals belonging to the four species. The isolates were typed by internal transcribed spacer (ITS)-PCR, tRNA-PCR, BOX-PCR, and randomly amplified polymorphic DNA (RAPD)-PCR, revealing that different Asaia strains are present in different mosquito populations, and even in single individuals.

Acetic acid bacteria, newly emerging symbionts of insects.

Recent research in microbe-insect symbiosis has shown that acetic acid bacteria (AAB) establish symbiotic relationships with several insects of the orders Diptera, Hymenoptera, Hemiptera, and Homoptera, all relying on sugar-based diets, such as nectars, fruit sugars, or phloem sap. To date, the fruit flies Drosophila melanogaster and Bactrocera oleae, mosquitoes of the genera Anopheles and Aedes, the honey bee Apis mellifera, the leafhopper Scaphoideus titanus, and the mealybug Saccharicoccus sacchari have been found to be associated with the bacterial genera Acetobacter, Gluconacetobacter, Gluconobacter, Asaia, and Saccharibacter and the novel genus Commensalibacter. AAB establish symbiotic associations with the insect midgut, a niche characterized by the availability of diet-derived carbohydrates and oxygen and by an acidic pH, selective factors that support AAB growth. AAB have been shown to actively colonize different insect tissues and organs, such as the epithelia of male and female reproductive organs, the Malpighian tubules, and the salivary glands. This complex topology of the symbiosis indicates that AAB possess the keys for passing through body barriers, allowing them to migrate to different organs of the host. Recently, AAB involvement in the regulation of innate immune system homeostasis of Drosophila has been shown, indicating a functional role in host survival. All of these lines of evidence indicate that AAB can play different roles in insect biology, not being restricted to the feeding habit of the host. The close association of AAB and their insect hosts has been confirmed by the demonstration of multiple modes of transmission between individuals and to their progeny that include vertical and horizontal transmission routes, comprising a venereal one. Taken together, the data indicate that AAB represent novel secondary symbionts of insects.

Mosquito-bacteria symbiosis: the case of Anopheles gambiae and Asaia.

The symbiotic relationship between Asaia, an α-proteobacterium belonging to the family Acetobacteriaceae, and mosquitoes has been studied mainly in the Asian malaria vector Anopheles stephensi. Thus, we have investigated the nature of the association between Asaia and the major Afro-tropical malaria vector Anopheles gambiae. We have isolated Asaia from different wild and laboratory reared colonies of A. gambiae, and it was detected by PCR in all the developmental stages of the mosquito and in all the specimens analyzed. Additionally, we have shown that it localizes in the midgut, salivary glands and reproductive organs. Using recombinant strains of Asaia expressing fluorescent proteins, we have demonstrated the ability of the bacterium to colonize A. gambiae mosquitoes with a pattern similar to that described for A. stephensi. Finally, fluorescent in situ hybridization on the reproductive tract of females of A. gambiae showed a concentration of Asaia at the very periphery of the eggs, suggesting that transmission of Asaia from mother to offspring is likely mediated by a mechanism of egg-smearing. We suggest that Asaia has potential for use in the paratransgenic control of malaria transmitted by A. gambiae.

Endosymbiosis morphological reorganization during metamorphosis diverges in weevils.

Virtually all animals associate with beneficial symbiotic bacteria. Whether and how these associations are modulated across a host's lifecycle is an important question in disentangling animal-bacteria interactions. We recently reported a case of complete morphological reorganization of symbiosis during metamorphosis of the cereal weevil, Sitophilus oryzae. In this model, the bacteriome, a specialized organ that houses the intracellular bacterium Sodalis pierantonius, undergoes a two-phase remodeling program synchronously driven by host and endosymbiont, resulting in a localization shift and the formation of multiple new bacteriomes. Here, we provide comparative data in a closely-related coleopteran, the red palm weevil Rhynchophorus ferrugineus, which is associated with the ancestral endosymbiont Nardonella. Using cell imaging experiments, we show that the red pal weevil bacteriome remains unchanged during metamorphosis, hence contrasting with what we reported in the cereal weevil S. oryzae. These findings highlight the complexity and divergence of host-symbiont interactions and their intertwining with host development, even in closely-related species. Abbreviations: DAPI: 4',6-diamidino-2-phenylindole; FISH: Fluorescence in situ hybridization; T3SS: Type III secretion system.

Acetobacter tropicalis is a major symbiont of the olive fruit fly (Bactrocera oleae).

Following cultivation-dependent and -independent techniques, we investigated the microbiota associated with Bactrocera oleae, one of the major agricultural pests in olive-producing countries. Bacterial 16S rRNA gene libraries and ultrastructural analyses revealed the presence of several bacterial taxa associated with this insect, among which Acetobacter tropicalis was predominant. The recent increased detection of acetic acid bacteria as symbionts of other insect model organisms, such as Anopheles stephensi (G. Favia et al., Proc. Natl. Acad. Sci. USA 104:9047-9051, 2007) or Drosophila melanogaster (C. R. Cox and M. S. Gilmore, Infect. Immun. 75:1565-1576, 2007), prompted us to investigate the association established between A. tropicalis and B. oleae. Using an A. tropicalis-specific PCR assay, the symbiont was detected in all insects tested originating from laboratory stocks or field-collected from different locations in Greece. This acetic acid bacterium was successfully established in cell-free medium, and typing analyses, carried out on a collection of isolates, revealed that different A. tropicalis strains are present in fly populations. The capability to colonize and lodge in the digestive system of both larvae and adults and in Malpighian tubules of adults was demonstrated by using a strain labeled with a green fluorescent protein.

Evidence for a conserved microbiota across the different developmental stages of Plodia interpunctella.

Diversity and composition of lepidopteran microbiotas are poorly investigated, especially across the different developmental stages. To improve this knowledge, we characterize the microbiota among different developmental stages of the Indian meal moth, Plodia interpunctella, which is considered one of the major pest of commodities worldwide. Using culture-independent approach based on Illumina 16S rRNA gene sequencing we characterized the microbiota of four developmental stages: eggs, first-, and last-instar larvae, and adult. A total of 1022 bacterial OTUs were obtained, showing a quite diversified microbiota associated to all the analyzed stages. The microbiotas associated with P. interpunctella resulted almost constant throughout the developmental stages, with approximately 77% of bacterial OTUs belonging to the phylum of Proteobacteria. The dominant bacterial genus is represented by Burkholderia (∼64%), followed by Propionibacterium, Delftia, Pseudomonas, and Stenotrophomonas. A core bacterial community, composed of 139 OTUs, was detected in all the developmental stages, among which 112 OTUs were assigned to the genus Burkholderia. A phylogenetic reconstruction, based on the 16S rRNA, revealed that our Burkholderia OTUs clustered with Burkholderia cepacia complex, in the same group of those isolated from the hemipterans Gossyparia spuria and Acanthococcus aceris. The functional profiling, predicted on the base of the bacterial 16S rRNA, indicates differences in the metabolic pathways related to metabolism of amino acids between preimaginal and adult stages. We can hypothesize that bacteria may support the insect host during preimaginal stages.

The Cost of Metabolic Interactions in Symbioses between Insects and Bacteria with Reduced Genomes.

Various intracellular bacterial symbionts that provide their host with essential nutrients have much-reduced genomes, attributed largely to genomic decay and relaxed selection. To obtain quantitative estimates of the metabolic function of these bacteria, we reconstructed genome- and transcriptome-informed metabolic models of three xylem-feeding insects that bear two bacterial symbionts with complementary metabolic functions: a primary symbiont, Sulcia, that has codiversified with the insects, and a coprimary symbiont of distinct taxonomic origin and with different degrees of genome reduction in each insect species (Hodgkinia in a cicada, Baumannia in a sharpshooter, and Sodalis in a spittlebug). Our simulations reveal extensive bidirectional flux of multiple metabolites between each symbiont and the host, but near-complete metabolic segregation (i.e., near absence of metabolic cross-feeding) between the two symbionts, a likely mode of host control over symbiont metabolism. Genome reduction of the symbionts is associated with an increased number of host metabolic inputs to the symbiont and also reduced metabolic cost to the host. In particular, Sulcia and Hodgkinia with genomes of ≤0.3 Mb are calculated to recycle ∼30 to 80% of host-derived nitrogen to essential amino acids returned to the host, while Baumannia and Sodalis with genomes of ≥0.6 Mb recycle 10 to 15% of host nitrogen. We hypothesize that genome reduction of symbionts may be driven by selection for increased host control and reduced host costs, as well as by the stochastic process of genomic decay and relaxed selection.IMPORTANCE Current understanding of many animal-microbial symbioses involving unculturable bacterial symbionts with much-reduced genomes derives almost entirely from nonquantitative inferences from genome data. To overcome this limitation, we reconstructed multipartner metabolic models that quantify both the metabolic fluxes within and between three xylem-feeding insects and their bacterial symbionts. This revealed near-complete metabolic segregation between cooccurring bacterial symbionts, despite extensive metabolite exchange between each symbiont and the host, suggestive of strict host controls over the metabolism of its symbionts. We extended the model analysis to investigate metabolic costs. The positive relationship between symbiont genome size and the metabolic cost incurred by the host points to fitness benefits to the host of bearing symbionts with small genomes. The multicompartment metabolic models developed here can be applied to other symbioses that are not readily tractable to experimental approaches.

Bacterial diversity shift determined by different diets in the gut of the spotted wing fly Drosophila suzukii is primarily reflected on acetic acid bacteria.

The pivotal role of diet in shaping gut microbiota has been evaluated in different animal models, including insects. Drosophila flies harbour an inconstant microbiota among which acetic acid bacteria (AAB) are important components. Here, we investigated the bacterial and AAB components of the invasive pest Drosophila suzukii microbiota, by studying the same insect population separately grown on fruit-based or non-fruit artificial diet. AAB were highly prevalent in the gut under both diets (90 and 92% infection rates with fruits and artificial diet respectively). Fluorescent in situ hybridization and recolonization experiments with green fluorescent protein (Gfp)-labelled strains showed AAB capability to massively colonize insect gut. High-throughput sequencing on 16S rRNA gene indicated that the bacterial microbiota of guts fed with the two diets clustered separately. By excluding AAB-related OTUs from the analysis, insect bacterial communities did not cluster separately according to the diet, suggesting that diet-based diversification of the community is primarily reflected on the AAB component of the community. Diet influenced also AAB alpha-diversity, with separate OTU distributions based on diets. High prevalence, localization and massive recolonization, together with AAB clustering behaviour in relation to diet, suggest an AAB role in the D. suzukii gut response to diet modification.

Differentiating current and past PCB and PCDD/F sources: The role of a large contaminated soil site in an industrialized city area.

Cities and contaminated areas can be primary or secondary sources of polychlorinated biphenyls (PCBs), dibenzo-p-dioxins (PCDDs), dibenzofurans (PCDFs), and other chemicals, into air and soil and can influence the regional level of some of these pollutants. In a contaminated site, the evaluation of such emissions can be crucial in the choice of the remediation technology to be adopted. In the city of Brescia (Northern Italy), more than 100 ha of agricultural areas were contaminated with PCBs, PCDD/Fs and heavy metals, originating from the activities of a former PCB factory. In order to evaluate the current emissions of PCBs and PCDD/Fs from the contaminated site, in a location where other current sources are present, we compared measured and predicted air concentrations, resulting from chemical volatilization from soils as well as fingerprints of Brescia soils and of soils contaminated by specific sources. The results confirm that the contaminated area is still a current and important secondary source of PCBs to the air, and to a lesser extent of PCDFs (especially the more volatile), but not for PCDDs. PCBs in soils have fingerprints similar to highly chlorinated mixtures, indicating contamination by these mixtures and/or a long weathering process. PCB 209 is also present at important levels. PCDD fingerprints in soil cannot be related to current emission sources, while PCDFs are compatible to industrial and municipal waste incineration, although weathering and/or natural attenuation may have played a role in modifying such soil fingerprints. Finally, we combined chemical and microbiological analyses to provide an integrated approach to evaluate soil fingerprints and their variation in a wider perspective, which accounts for the mutual effects between contamination and soil microbiota, a pivotal hint for addressing in situ bioremediation activities.