Species Composition and Barotolerance of Gut Microflora of Deep-Sea Benthic Macrofauna Collected at Various Depths in the Atlantic Ocean

The bacterial flora of marine animals collected at depths of 570 to 2,446 m was examined for population size and generic composition, and the barotolerant characteristics of selected bacterial isolates were determined. Total numbers of culturable, aerobic, heterotrophic bacteria were found to be low in animals collected at the greatest ocean depths sampled in this study. Vibrio spp. were predominant in 10 of 15 samples examined, and Photobacterium spp. and yeasts were the major components of the remainder. Pseudomonas, Achromobacter, and Flavobacterium spp. comprised minor components of the gut flora of deep-sea fish. Forty-six pure cultures isolated from samples of seven animals were tested for growth or viability after incubation for 1 week under pressures ranging from 100 to 750 atm. Strains of bacteria isolated from samples of fish intestine were more barotolerant than those from the stomach (P<0.01). When incubated at a pressure of 600 atm, viability of bacterial cultures originally isolated from fish caught at a depth of 570 m was significantly decreased in comparison with viability of cultures from animals caught at depths of 1,393 and 2,446 m (P<0.01). From results of this study, it is concluded that the gut microflora of animals that dwell in the deeper regions of the ocean are adapted to an increased hydrostatic pressure environment, that is, the gut microflora is less inhibited by elevated hydrostatic pressure with increasing depth from which the host animal was collected.     

Fluorescence analysis of zooplankton gut contents and an investigation of diel feeding patterns

A method is described for measurement of the in situ feeding rate of planktonic herbivores. Freshly caught Zooplankton are washed with filtered sea water and frozen. The gut contents are later analysed using acetone extracts of the entire sample. Fluorescence peaks due to ingested chlorophyll are easy to quantify for sample sizes of 20–200 animals. This method obviates some of the problems, such as confinement and previous feeding history, associated with extrapolation from laboratory experiments to field conditions. The method is illustrated by an investigation of diel feeding patterns of marine copepods. Initial results indicate the complexity of these patterns, with either a single night peak or dawn and dusk peaks for adult copepods. Diel variations in feeding activity appear to occur in addition to, but closely interacting with, the diel vertical migrations of the animals.     

External Microflora of a Marine Wood-Boring Isopod

Bacteria associated with the marine wood-boring isopod Limnoria lignorum were enumerated by acridine orange epifluorescence microscopy and by plate counts on several media; the plate-viable bacteria were isolated and identified. Similar procedures were followed to enumerate and identify bacteria associated with the wood substrate from which the isopods were collected and with the surrounding water from the isopod habitat. Approximately 1.4 × 10⁷ bacterial cells were associated with each individual L. lignorum. Aeromonas hydrophila, Pseudomonas, and Vibrio were the most common genera in the isopod microflora. Wood from L. lignorum burrows had an associated bacterial flora of 1.6 × 10⁷ cells per mg (damp weight). A. hydrophila also predominated in the wood microflora. The water from which the isopod population was collected contained 2.3 × 10⁶ bacteria per ml. Pseudomonas and Vibrio species were very common in the water microflora, but A. hydrophila was not detected. Interactions between the isopod, its associated microorganisms, and the microorganisms within the wood substrate are discussed in the light of the known absence of a resident digestive tract microflora in these animals.     

The relationships between faecal egg counts and gut microbial composition in UK Thoroughbreds infected by cyathostomins

A growing body of evidence, particularly in humans and rodents, supports the existence of a complex network of interactions occurring between gastrointestinal (GI) helminth parasites and the gut commensal bacteria, with substantial effects on both host immunity and metabolic potential. However, little is known of the fundamental biology of such interactions in other animal species; nonetheless, given the considerable economic losses associated with GI parasites, particularly in livestock and equines, as well as the global threat of emerging anthelmintic resistance, further explorations of the complexities of host-helminth-microbiota interactions in these species are needed. This study characterises the composition of the equine gut commensal flora associated with the presence, in faecal samples, of low (Clow) and high (Chigh) numbers of eggs of an important group of GI parasites (i.e. the cyathostomins), prior to and following anthelmintic treatment. High-throughput sequencing of bacterial 16S rRNA amplicons and associated bioinformatics and statistical analyses of sequence data revealed strong clustering according to faecal egg counts (P?=?0.003). A trend towards increased populations of Methanomicrobia (class) and Dehalobacterium (genus) was observed in Clow in comparison with Chigh. Anthelmintic treatment in Chigh was associated with a significant reduction of the bacterial Phylum TM7 14?days post-ivermectin administration, as well as a transient expansion of Adlercreutzia spp. at 2?days post-treatment. This study provides a first known insight into the discovery of the intimate mechanisms governing host-parasite-microbiota interactions in equines, and sets a basis for the development of novel, biology-based intervention strategies against equine GI helminths based on the manipulation of the commensal gut flora.     

The inconstant gut microbiota of Drosophila species revealed by 16S rRNA gene analysis

The gut microorganisms in some animals are reported to include a core microbiota of consistently associated bacteria that is ecologically distinctive and may have coevolved with the host. The core microbiota is promoted by positive interactions among bacteria, favoring shared persistence; its retention over evolutionary timescales is evident as congruence between host phylogeny and bacterial community composition. This study applied multiple analyses to investigate variation in the composition of gut microbiota in drosophilid flies. First, the prevalence of five previously described gut bacteria (Acetobacter and Lactobacillus species) in individual flies of 21 strains (10 Drosophila species) were determined. Most bacteria were not present in all individuals of most strains, and bacterial species pairs co-occurred in individual flies less frequently than predicted by chance, contrary to expectations of a core microbiota. A complementary pyrosequencing analysis of 16S rRNA gene amplicons from the gut microbiota of 11 Drosophila species identified 209 bacterial operational taxonomic units (OTUs), with near-saturating sampling of sequences, but none of the OTUs was common to all host species. Furthermore, in both of two independent sets of Drosophila species, the gut bacterial community composition was not congruent with host phylogeny. The final analysis identified no common OTUs across three wild and four laboratory samples of D. melanogaster. Our results yielded no consistent evidence for a core microbiota in Drosophila. We conclude that the taxonomic composition of gut microbiota varies widely within and among Drosophila populations and species. This is reminiscent of the patterns of bacterial composition in guts of some other animals, including humans.     

Diversity of the free-living marine and freshwater Copepoda (Crustacea) in Costa Rica: a review

The studies on marine copepods of Costa Rica started in the 1990’s and focused on the largest coastal-estuarine systems in the country, particularly along the Pacific coast. Diversity is widely variable among these systems: 40 species have been recorded in the Culebra Bay influenced by upwelling, northern Pacific coast, only 12 in the Gulf of Nicoya estuarine system, and 38 in Golfo Dulce, an anoxic basin in the southern Pacific coast of the country. Freshwater environments of Costa Rica are known to harbor a moderate diversity of continental copepods (25 species), which includes 6 calanoids, 17 cyclopoids and only two harpacticoids. Of the +100 freshwater species recorded in Central America, six are known only from Costa Rica, and one appears to be endemic to this country. The freshwater copepod fauna of Costa Rica is clearly the best known in Central America. Overall, six of the 10 orders of Copepoda are reported from Costa Rica. A previous summary by 2001 of the free-living copepod diversity in the country included 80 marine species (67 pelagic, 13 benthic). By 2009, the number of marine species increased to 209: 164 from the Pacific (49% of the copepod fauna from the Eastern Tropical Pacific) and 45 from the Caribbean coast (8% of species known from the Caribbean Basin). Both the Caribbean and Pacific species lists are growing. Additional collections of copepods at Cocos Island, an oceanic island 530 km away of the Pacific coast, have revealed many new records, including five new marine species from Costa Rica. Currently, the known diversity of marine copepods of Costa Rica is still in development and represents up to 52.6% of the total marine microcrustaceans recorded in the country. Future sampling and taxonomic efforts in the marine habitats should emphasize oceanic environments including deep waters but also littoral communities. Several Costa Rican records of freshwater copepods are likely to represent undescribed species. Also, the biogeographic relevance of the inland copepod fauna of Costa Rica requires more detailed surveys.     

Comparison of different DNA-extraction techniques to investigate the bacterial community of marine copepods

Marine zooplanktic organisms, such as copepods, are usually associated with large numbers of bacteria. Some of these bacteria live attached to copepods’ exoskeleton, while others prevail in their intestine and faecal pellets. Until now, general conclusions concerning the identity of these bacteria are problematic since the majority of previous studies focused on cultivable bacteria only. Hence, to date little is known on whether copepod genera or species harbour distinct bacterial populations and about the nature of this association. To shed more light on these copepod/bacteria consortia, the focus of this study was the development and evaluation of a suitable approach to extract bacterial DNA from different North Sea copepod genera. Furthermore, the bacterial DNA was analysed by PCR-DGGE and subsequent sequencing of excised bands. The result of this work was an appropriate extraction method for batches of ten to one copepod specimens and offered first insights as to which bacteria are attached to the copepods Acartia sp. and Temora sp. from Helgoland Roads (German Bight) and a laboratory-grown Acartia tonsa culture. It revealed the prevalence of Alphaproteobacteria.     

Analysis of gut microbiota in three species belonging to different genera (Hemitragus,Pseudois, and Ovis) from the subfamily Caprinae in the absence of environmental variance

This study aimed to identify the effects of host species on the gut microbial flora in three species (Hemitragus jemlahicus, Pseudois nayaur, and Ovis orientalis) from the subfamily Caprinae, by excluding the impact of environment factors. We investigated the differences in intestinal flora of three species belonging to Caprinae, which were raised in identical conditions. Fecal samples were collected from tahr, mouflon, and bharal, and the V3–V4 region of the 16S ribosomal RNA gene was analyzed by high‐throughput sequencing. The analysis of 16S rRNA gene sequences reveals that fecal samples were mainly composed of four phyla: Firmicutes, Bacteroidetes, Spirochaetes, and Proteobacteria. The most abundant phyla included Firmicutes and Bacteroidetes accounting for >90% of the bacteria, and a higher Firmicutes/Bacteroidetes ratio was observed in tahrs. Moreover, significant differences existed at multiple levels of classifications in the relative abundance of intestinal flora, differing greatly between species. Phylogenetic analyses based on 16S rRNA gene indicated that mouflon is closely related to bharal, and it is inconsistent with previous reports in the species evolutionary relationships. In this study, we demonstrated that the gut microbiota in tahr had a stronger ability to absorb and store energy from the diet compared with mouflon and bharal, and the characteristics of host–microbiome interactions were not significant.     

Variation in gut microbial communities and its association with pathogen infection in wild bumble bees (Bombus)

Bacterial gut symbiont communities are critical for the health of many insect species. However, little is known about how microbial communities vary among host species or how they respond to anthropogenic disturbances. Bacterial communities that differ in richness or composition may vary in their ability to provide nutrients or defenses. We used deep sequencing to investigate gut microbiota of three species in the genus Bombus (bumble bees). Bombus are among the most economically and ecologically important non-managed pollinators. Some species have experienced dramatic declines, probably due to pathogens and land-use change. We examined variation within and across bee species and between semi-natural and conventional agricultural habitats. We categorized as ‘core bacteria'' any operational taxonomic units (OTUs) with closest hits to sequences previously found exclusively or primarily in the guts of honey bees and bumble bees (genera Apis and Bombus). Microbial community composition differed among bee species. Richness, defined as number of bacterial OTUs, was highest for B. bimaculatus and B. impatiens. For B. bimaculatus, this was due to high richness of non-core bacteria. We found little effect of habitat on microbial communities. Richness of non-core bacteria was negatively associated with bacterial abundance in individual bees, possibly due to deeper sampling of non-core bacteria in bees with low populations of core bacteria. Infection by the gut parasite Crithidia was negatively associated with abundance of the core bacterium Gilliamella and positively associated with richness of non-core bacteria. Our results indicate that Bombus species have distinctive gut communities, and community-level variation is associated with pathogen infection.     

Bacterial genera isolated from field-collected (Diapausing) and laboratory-reared cotton boll weevils, Anthonomus grandis (Coleoptera: Curculionidae)

Little data exist on the bacterial flora of the cotton boll weevil, Anthonomus grandis, which is a common insect pest to cotton farmers in the United States. This investigation determined the total numbers of aerobic and anaerobic bacteria in diapausing field-collected boll weevils and active adult laboratory-reared boll weevils. Identifications were made of aerobic genera isolated from field-collected and laboratory-reared boll weevils that had previously been surface sterilized. The genera found, in order of decreasing frequency, in the field-collected boll weevils were Lactobacillus, Erwinia, Flavobacterium, Enterobacter, and Pseudomonas. The genera found, in order of decreasing frequency, in the laboratory-reared boll weevils were Lactobacillus, Pseudomonas, Streptococcus, Erwinia, Enterobacter, and Flavobacterium, Lactobacillus was the most frequently found genus in both groups.     

Marine microalgae attack and feed on metazoans

Free-living microalgae from the dinoflagellate genus Karlodinium are known to form massive blooms in eutrophic coastal waters worldwide and are often associated with fish kills. Natural bloom populations, recently shown to consist of the two mixotrophic and toxic species Karlodinium armiger and Karlodinium veneficum have caused fast paralysis and mortality of finfish and copepods in the laboratory, and have been associated with reduced metazooplankton biomass in-situ. Here we show that a strain of K. armiger (K-0688) immobilises the common marine copepod Acartia tonsa in a density-dependent manner and collectively ingests the grazer to promote its own growth rate. In contrast, four strains of K. veneficum did not attack or affect the motility and survival of the copepods. Copepod immobilisation by the K. armiger strain was fast (within 15 min) and caused by attacks of swarming cells, likely through the transfer and action of a highly potent but uncharacterised neurotoxin. The copepods grazed and reproduced on a diet of K. armiger at densities below 1000, cells ml⁻¹, but above 3500 cells ml⁻¹ the mixotrophic dinoflagellates immobilised, fed on and killed the copepods. Switching the trophic role of the microalgae from prey to predator of copepods couples population growth to reduced grazing pressure, promoting the persistence of blooms at high densities. K. armiger also fed on three other metazoan organisms offered, suggesting that active predation by mixotrophic dinoflagellates may be directly involved in causing mortalities at several trophic levels in the marine food web.     

The Gut Bacterial Communities Associated with Lab-Raised and Field-Collected Ants of Camponotus fragilis (Formicidae: Formicinae)

Camponotus is the second largest ant genus and known to harbor the primary endosymbiotic bacteria of the genus Blochmannia. However, little is known about the effect of diet and environment changes on the gut bacterial communities of these ants. We investigated the intestinal bacterial communities in the lab-raised and field-collected ants of Camponotus fragilis which is found in the southwestern United States and northern reaches of Mexico. We determined the difference of gut bacterial composition and distribution among the crop, midgut, and hindgut of the two types of colonies. Number of bacterial species varied with the methods of detection and the source of the ants. Lab-raised ants yielded 12 and 11 species using classical microbial culture methods and small-subunit rRNA genes (16S rRNAs) polymerase chain reaction-restriction fragment-length polymorphism analysis, respectively. Field-collected ants yielded just 4 and 1–3 species using the same methods. Most gut bacterial species from the lab-raised ants were unevenly distributed among the crop, midgut, and hindgut, and each section had its own dominant bacterial species. Acetobacter was the prominent bacteria group in crop, accounting for about 55 % of the crop clone library. Blochmannia was the dominant species in midgut, nearly reaching 90 % of the midgut clone library. Pseudomonas aeruginosa dominated the hindgut, accounting for over 98 % of the hindgut clone library. P. aeruginosa was the only species common to all three sections. A comparison between lab-raised and field-collected ants, and comparison with other species, shows that gut bacterial communities vary with local environment and diet. The bacterial species identified here were most likely commensals with little effect on their hosts or mild pathogens deleterious to colony health.     

Prevalent Ciliate Symbiosis on Copepods: High Genetic Diversity and Wide Distribution Detected Using Small Subunit Ribosomal RNA Gene

Toward understanding the genetic diversity and distribution of copepod-associated symbiotic ciliates and the evolutionary relationships with their hosts in the marine environment, we developed a small subunit ribosomal RNA gene (18S rDNA)-based molecular method and investigated the genetic diversity and genotype distribution of the symbiotic ciliates on copepods. Of the 10 copepod species representing six families collected from six locations of Pacific and Atlantic Oceans, 9 were found to harbor ciliate symbionts. Phylogenetic analysis of the 391 ciliate 18S rDNA sequences obtained revealed seven groups (ribogroups), six (containing 99% of all the sequences) belonging to subclass Apostomatida, the other clustered with peritrich ciliate Vorticella gracilis. Among the Apostomatida groups, Group III were essentially identical to Vampyrophrya pelagica, and the other five groups represented the undocumented ciliates that were close to Vampyrophrya/Gymnodinioides/Hyalophysa. Group VI ciliates were found in all copepod species but one (Calanus sinicus), and were most abundant among all ciliate sequences obtained, indicating that they are the dominant symbiotic ciliates universally associated with copepods. In contrast, some ciliate sequences were found only in some of the copepods examined, suggesting the host selectivity and geographic differentiation of ciliates, which requires further verification by more extensive sampling. Our results reveal the wide occurrence and high genetic diversity of symbiotic ciliates on marine copepods and highlight the need to systematically investigate the host- and geography-based genetic differentiation and ecological roles of these ciliates globally.     

Midgut Microbiota of the Malaria Mosquito Vector Anopheles gambiae and Interactions with Plasmodium falciparum Infection

The susceptibility of Anopheles mosquitoes to Plasmodium infections relies on complex interactions between the insect vector and the malaria parasite. A number of studies have shown that the mosquito innate immune responses play an important role in controlling the malaria infection and that the strength of parasite clearance is under genetic control, but little is known about the influence of environmental factors on the transmission success. We present here evidence that the composition of the vector gut microbiota is one of the major components that determine the outcome of mosquito infections. A. gambiae mosquitoes collected in natural breeding sites from Cameroon were experimentally challenged with a wild P. falciparum isolate, and their gut bacterial content was submitted for pyrosequencing analysis. The meta-taxogenomic approach revealed a broader richness of the midgut bacterial flora than previously described. Unexpectedly, the majority of bacterial species were found in only a small proportion of mosquitoes, and only 20 genera were shared by 80% of individuals. We show that observed differences in gut bacterial flora of adult mosquitoes is a result of breeding in distinct sites, suggesting that the native aquatic source where larvae were grown determines the composition of the midgut microbiota. Importantly, the abundance of Enterobacteriaceae in the mosquito midgut correlates significantly with the Plasmodium infection status. This striking relationship highlights the role of natural gut environment in parasite transmission. Deciphering microbe-pathogen interactions offers new perspectives to control disease transmission.     

Lactobacillus gasseri SF1183 Affects Intestinal Epithelial Cell Survival and Growth

It is now commonly accepted that the intestinal microbiota plays a crucial role in the gut physiology and homeostasis, and that both qualitative and quantitative alterations in the compositions of the gut flora exert profound effects on the host’s intestinal cells. In spite of this, the details of the interaction between commensal bacteria and intestinal cells are still largely unknown and only in few cases the molecular mechanisms have been elucidated. Here we analyze the effects of molecules produced and secreted by Lactobacillus gasseri SF1183 on human intestinal HCT116 cells. L. gasseri is a well known species of lactic acid bacteria, commonly associated to the human intestine and SF1183 is a human strain previously isolated from an ileal biopsy of an healthy volunteer. SF1183 produces and secretes, in a growth phase-dependent way, molecule(s) able to drastically interfere with HCT116 cell proliferation. Although several attempts to purify and identify the bioactive molecule(s) have been so far unsuccessful, a partial characterization has indicated that it is smaller than 3 kDa, thermostable and of proteinaceous nature. L. gasseri molecule(s) stimulate a G1-phase arrest of the cell cycle by up-regulation of p21WAF1 rendering cells protected from intrinsic and extrinsic apoptosis. A L. gasseri-mediated reduction of apoptosis and of cell proliferation could be relevant in protecting epithelial barrier integrity and helping in reconstituting tissutal homeostasis.     

Mesozooplankton Grazing on Picocyanobacteria in the Baltic Sea as Inferred from Molecular Diet Analysis

Our current knowledge on the microbial component of zooplankton diet is limited, and it is generally assumed that bacteria-sized prey is not directly consumed by most mesozooplankton grazers in the marine food webs. We questioned this assumption and conducted field and laboratory studies to examine picocyanobacteria contribution to the diets of Baltic Sea zooplankton, including copepods. First, qPCR targeting ITS-1 rDNA sequence of the picocyanobacteria Synechococcus spp. was used to examine picocyanobacterial DNA occurrence in the guts of Baltic zooplankton (copepods, cladocerans and rotifers). All field-collected zooplankton were found to consume picocyanobacteria in substantial quantities. In terms of Synechococcus quantity, the individual gut content was highest in cladocerans, whereas biomass-specific gut content was highest in rotifers and copepod nauplii. Moreover, the gut content in copepods was positively related to the picocyanobacteria abundance and negatively to the total phytoplankton abundance in the water column at the time of sampling. This indicates that increased availability of picocyanobacteria resulted in the increased intake of this prey and that copepods may rely more on picoplankton when food in the preferred size range declines. Second, a feeding experiments with a laboratory reared copepod Acartia tonsa fed a mixture of the picocyanobacterium Synechococcus bacillaris and microalga Rhodomonas salina confirmed that copepods ingested Synechococcus, even when the alternative food was plentiful. Finally, palatability of the picocyanobacteria for A. tonsa was demonstrated using uptake of ¹³C by the copepods as a proxy for carbon uptake in feeding experiment with ¹³C-labeled S. bacillaris. These findings suggest that, if abundant, picoplankton may become an important component of mesozooplankton diet, which needs to be accounted for in food web models and productivity assessments.     

Do viruses affect fecundity and survival of the copepod Acartia tonsa Dana?

Naturally occurring viruses are extremely abundant in aquaticsystems, and they infect bacteria, cyanobacteria, prokaryoticand eukaryotic phytoplankton, heterotrophic nanoflagellates,fish and mammals. Viral infections of single-celled organismshave been studied intensively in the past decade, but littleis known about the effects of viruses on aquatic metazoans,other than for some economically important species. Becausezooplankton assemblages are often dominated in number and biomassby copepods, we used them as model organisms to study the effectsof naturally occurring viruses on higher trophic levels. Weattempted to induce viral infection in laboratory-reared culturesof the estuarine copepod Acartia tonsa Dana by exposing themto elevated concentrations of natural viruses in seawater. Wefound no negative effects of such exposure on copepod fecundity,larval survival or adult survival.     

Understanding alternative fluxes/effluxes through comparative metabolic pathway analysis of phylum actinobacteria using a simplified approach

Actinobacteria are known for their diverse metabolism and physiology. Some are dreadful human pathogens whereas some constitute the natural flora for human gut. Therefore, the understanding of metabolic pathways is a key feature for targeting the pathogenic bacteria without disturbing the symbiotic ones. A big challenge faced today is multiple drug resistance by Mycobacterium and other pathogens that utilize alternative fluxes/effluxes. With the availability of genome sequence, it is now feasible to conduct the comparative in silico analysis. Here we present a simplified approach to compare metabolic pathways so that the species specific enzyme may be traced and engineered for future therapeutics.     

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.