Microbial artists: the role of parasite microbiomes in explaining colour polymorphism among amphipods and potential link to host manipulation.

Parasite infections are increasingly reported to change the microbiome of the parasitized hosts, while parasites bring their own microbes to what can be a multi-dimensional interaction. For instance, a recent hypothesis suggests that the microbial communities harboured by parasites may play a role in the well-documented ability of many parasites to manipulate host phenotype, and explain why the degree to which host phenotype is altered varies among conspecific parasites. Here, we explored whether the microbiomes of both hosts and parasites are associated with variation in host manipulation by parasites. Using colour quantification methods applied to digital images, we investigated colour variation among uninfected Transorchestia serrulata amphipods, as well as amphipods infected with Plagiorhynchus allisonae acanthocephalans and with a dilepidid cestode. We then characterized the bacteriota of amphipod hosts and of their parasites, looking for correlations between host phenotype and the bacterial taxa associated with hosts and parasites. We found large variation in amphipod colours, and weak support for a direct impact of parasites on the colour of their hosts. Conversely, and most interestingly, the parasite's bacteriota was more strongly correlated with colour variation among their amphipod hosts, with potential impact of amphipod-associated bacteria as well. Some bacterial taxa found associated with amphipods and parasites may have the ability to synthesize pigments, and we propose they may interact with colour determination in the amphipods. This study provides correlational support for an association between the parasite's microbiome and the evolution of host manipulation by parasites and host-parasite interactions more generally.

Exophiala amphawaensis sp. nov., a novel black yeast isolated from the gut of amphipods in Thailand.

The genus Exophiala is polymorphic, able to transition between yeast, hyphal and pseudohyphal forms. Species of the genus Exophiala are ubiquitous fungi that are distributed in various environments around the world. During a survey of fungal diversity in the gut of amphipods (Floresorchestia amphawaensis and undescribed Dogielinotid amphipods) from the Amphawa estuary, Samut Songkhram province, Thailand, five black yeast strains (DMKU-MG01, DMKU-MG07, DMKU-MG08, DMKU-HG10 and DMKU-FG04) were identified as representing a novel taxon on the basis of a combination of morphological and molecular phylogenetic features. The five strains did not produce filamentous hyphae or pseudohyphae. Only budding yeast cells were observed. On the basis of the phenotypic characteristics and the results of molecular analyses of the D1/D2 region of the large subunit (LSU) rRNA gene and the internal transcribed spacer (ITS) region, the five strains were identified as representing a novel species via applied nucleotide pairwise analysis. They differed from the most closely related species Exophiala alcalophiala by 3.54 % nucleotide substitutions (20 nucleotide substitutions in 572 bp) in the D1/D2 domains of the LSU rRNA gene. Moreover, the sequences of the ITS region of the five strains differed from those of the most closely related species E. alcalophiala, by 7.44-9.62 % nucleotide substitutions, and Exophiala halophiala, by 7.2-7.53 % nucleotide substitutions. The results of phylogenetic analyses based on the concatenated sequences of the ITS regions and the D1/D2 domains of the LSU rRNA gene confirmed that the five black yeast strains represented a single novel species of the genus Exophiala. In this study, Exophiala amphawaensis sp. nov. is proposed to accommodate these strains. The holotype is TBRC 15626T and the isotype is PYCC9020. The MycoBank accession number of the novel species is MB 851477.

Histopathological screening of Pontogammarus robustoides (Amphipoda), an invader on route to the United Kingdom.

Biological invasions may act as conduits for pathogen introduction. To determine which invasive non-native species pose the biggest threat, we must first determine the symbionts (pathogens, parasites, commensals, mutualists) they carry, via pathological surveys that can be conducted in multiple ways (i.e., molecular, pathological, and histological). Whole animal histopathology allows for the observation of pathogenic agents (virus to Metazoa), based on their pathological effect upon host tissue. Where the technique cannot accurately predict pathogen taxonomy, it does highlight pathogen groups of importance. This study provides a histopathological survey of Pontogammarus robustoides (invasive amphipod in Europe) as a baseline for symbiont groups that may translocate to other areas/hosts in future invasions. Pontogammarus robustoides (n = 1,141) collected throughout Poland (seven sites), were noted to include a total of 13 symbiotic groups: a putative gut epithelia virus (overall prevalence = 0.6%), a putative hepatopancreatic cytoplasmic virus (1.4%), a hepatopancreatic bacilliform virus (15.7%), systemic bacteria (0.7%), fouling ciliates (62.0%), gut gregarines (39.5%), hepatopancreatic gregarines (0.4%), haplosporidians (0.4%), muscle infecting microsporidians (6.4%), digeneans (3.5%), external rotifers (3.0%), an endoparasitic arthropod (putatively: Isopoda) (0.1%), and Gregarines with putative microsporidian infections (1.4%). Parasite assemblages partially differed across collection sites. Co-infection patterns revealed strong positive and negative associations between five parasites. Microsporidians were common across sites and could easily spread to other areas following the invasion of P. robustoides. By providing this initial histopathological survey, we hope to provide a concise list of symbiont groups for risk-assessment in the case of a novel invasion by this highly invasive amphipod.

Ultrastructure, molecular phylogeny, and prevalence rates of Alternosema bostrichidis gen. nov. sp. nov. (Microsporidia, Terresporidia), a parasite of Prostephanus truncatus and Dinoderus spp. (Coleoptera, Bostrichidae).

A new species and a new genus of a microsporidium Alternosema bostrichidis isolated from an adult Prostephanus truncatus in Mexico and from three species of the genus Dinoderus in Nigeria are described. The microsporidium is monomorphic, monoxenic, and develops in direct contact with host cell cytoplasm. The infection first appears with thoracic muscles, followed by a generalized invasion of the host. All developmental stages are diplokaryotic. Sporogony is disporoblastic. Mature spores are ovoid. Unfixed spores measure 3.7-4.2 × 2.0-2.6 µm, fixed and stained spores 3.5-5.0 × 2.4-2.8 µm. The polaroplast consists of dense lamellae and rare lamellae. The polar tube is slightly anisofilar, consisting of 11-17 coils, with 9-14 proximal (130 nm in diameter) and 2-3 distal coils (120 nm in diameter) arranged in one layer. Molecular phylogenetic analysis based upon a short portion of small-subunit ribosomal RNA gene (Genbank accession # KP455651) placed the new microsporidium within Liebermannia-Orthosomella lineage, which contains multiple undescribed parasites. In particular, A. bostrichidis showed maximal sequence similarity of 95% to Microsporidium sp. BBRE2 (# FJ755987) from Baikalian Diplacanthus brevispinus (Amphipoda: Acanthogammaridae) and Microsporidium sp. Comp CD Van 2 (# KC111784) from compost and soil in Canada. Frequent, devastating epizootics of laboratory cultures of A. bostrichidis support its potential as a biological control agent of grain borers.

Gut Microbial Divergence between Two Populations of the Hadal Amphipod Hirondellea gigas.

Hadal environments sustain diverse microorganisms. A few studies have investigated hadal microbial communities consisting of free-living or particle-associated bacteria and archaea. However, animal-associated microbial communities in hadal environments remain largely unexplored, and comparative analyses of animal gut microbiota between two isolated hadal environments have never been done so far. In the present study, 228 Gb of gut metagenomes of the giant amphipod Hirondellea gigas from two hadal trenches, the Mariana Trench and Japan Trench, were sequenced and analyzed. Taxonomic analysis identified 49 microbial genera commonly shared by the gut microbiota of the two H. gigas populations. However, the results of statistical analysis, in congruency with the alpha and beta diversity analyses, revealed significant differences in gut microbial composition across the two trenches. Abundance variation of Psychromonas, Propionibacterium, and Pseudoalteromonas species was observed. Microbial cooccurrence was demonstrated for microbes that were overrepresented in the Mariana trench. Comparison of functional potential showed that the percentage of carbohydrate metabolic genes among the total microbial genes was significantly higher in the guts of H. gigas specimens from the Mariana Trench. Integrating carbon input information and geological characters of the two hadal trenches, we propose that the differences in the community structure might be due to several selective factors, such as environmental variations and microbial interactions.IMPORTANCE The taxonomic composition and functional potential of animal gut microbiota in deep-sea environments remain largely unknown. Here, by performing comparative metagenomics, we suggest that the gut microbial compositions of two Hirondellea gigas populations from the Mariana Trench and the Japan Trench have undergone significant divergence. Through analyses of functional potentials and microbe-microbe correlations, our findings shed light on the contributions of animal gut microbiota to host adaptation to hadal environments.

Pathogens and other symbionts of the Amphipoda: taxonomic diversity and pathological significance.

With over 10000 species of Amphipoda currently described, this order is one of the most diverse groups of freshwater and marine Crustacea. Members of this group are globally distributed, and many are keystone species and ecosystem engineers within their respective ecologies. As with most organisms, disease is a key factor that can alter population size, behaviour, survival, invasion potential and physiology of amphipod hosts. This review explores symbiont diversity and pathology in amphipods by coalescing a range of current and historical literature to provide the first full review of our understanding of amphipod disease. The review is broken into 2 parts. The first half explores amphipod microparasites, which include data pertaining to viruses, bacteria, fungi, oomycetes, microsporidians, dinoflagellates, myxozoans, ascetosporeans, mesomycetozoeans, apicomplexans and ciliophorans. The second half reports the metazoan macroparasites of Amphipoda, including rotifers, trematodes, acanthocephalans, nematodes, cestodes and parasitic Crustacea. In all cases we have endeavoured to provide a complete list of known species that cause disease in amphipods, while also exploring the effects of parasitism. Although our understanding of disease in amphipods requires greater research efforts to better define taxonomic diversity and host effects of amphipod symbionts, research to date has made huge progress in cataloguing and experimentally determining the effects of disease upon amphipods. For the future, we suggest a greater focus on developing model systems that use readily available amphipods and diseases, which can be comparable to the diseases in other Crustacea that are endangered, economically important or difficult to house.

Molecular and Morphological Characterization of Anncaliia azovica sp. n. (Microsporidia) Infecting Niphargogammarus intermedius (Crustacea, Amphipoda) from the Azov Sea.

Five out of one hundred adults of Niphargogammarus intermedius caught at the Azov sea shore were found to be infected with microsporidia. The infection was found in the subcuticular fat body and myocytes. Parasites developed in direct contact with host cells, displayed a disporoblastic sporogony and diplokaryotic arrangement of nuclei at all stages. Spores were oval, 4.6-5.8 × 2.6-3.0 µm. Exospore appendages, vesicular-tubular secretions, and the anisofilar polar filament indicated a similarity to Anncaliia species. Sporont surfaces displayed ridges of amorphous material. Meronts and sporonts formed protoplastic extensions, similar to A. vesicularum and A. meligheti. Mature spores possessed a bipartite polaroplast. The polar tube was arranged in one row of 13-18 coils including 0-3 distal coils of lesser diameter. Partial sequencing of SSU, ITS, and LSU regions of rRNA gene (GenBank accessions: KY288064-KY288065) confirmed this new species to be congeneric with A. algerae (#AF069063) and A. meligheti (#AY894423). The SSU gene of this novel microsporidium shared 99.4% sequence similarity to A. algerae and 98.9% to A. meligheti. Genes for HSP70 and RPB1 amplified with primers designed for A. algerae orthologs displayed 99.7% and 97.4% similarity, respectively, between A. algerae and the novel microsporidium. A new species, Anncaliia azovica, is described based on morphological and molecular characterization.

'Candidatus Aquirickettsiella gammari' (Gammaproteobacteria: Legionellales: Coxiellaceae): A bacterial pathogen of the freshwater crustacean Gammarus fossarum (Malacostraca: Amphipoda).

Invasive and non-native species can pose risks to vulnerable ecosystems by co-introducing bacterial pathogens. Alternatively, co-introduced bacterial pathogens may regulate invasive population size and invasive traits. We describe a novel candidate genus and species of bacteria ('Candidatus Aquirickettsiella gammari') found to infect Gammarus fossarum, from its native range in Poland. The bacterium develops intracellularly within the haemocytes and cells of the musculature, hepatopancreas, connective tissues, nervous system and gonad of the host. The developmental cycle of 'Candidatus Aquirickettsiella gammari' includes an elementary body (496.73 nm ±â€¯37.56 nm in length, and 176.89 nm ±â€¯36.29 nm in width), an elliptical, condensed spherical stage (737.61 nm ±â€¯44.51 nm in length and 300.07 nm ±â€¯44.02 nm in width), a divisional stage, and a spherical initial body (1397.59 nm ±â€¯21.26 nm in diameter). We provide a partial genome for 'Candidatus Aquirickettsiella gammari', which clades phylogenetically alongside environmental 16S rRNA sequences from aquatic habitats, and bacterial symbionts from aquatic isopods (Asellus aquaticus), grouping separately from the Rickettsiella, a genus that includes bacterial pathogens of terrestrial insects and isopods. Increased understanding of the diversity of symbionts carried by G. fossarum identifies those that might regulate host population size, or those that could pose a risk to native species in the invasive range. Identification of 'Candidatus Aquirickettsiella gammari' and its potential for adaptation as a biological control agent is explored.

Colwellia marinimaniae sp. nov., a hyperpiezophilic species isolated from an amphipod within the Challenger Deep, Mariana Trench.

An obligately piezophilic strain was isolated from an amphipod crustacean obtained in the Challenger Deep region of the Mariana Trench during the DEEPSEA CHALLENGE expedition. The strain, MTCD1T, grew at extremely high hydrostatic pressures, with a growth range of 80-140 MPa (optimum, 120 MPa) at 6 °C. Phylogenetic analyses based on the 16S rRNA gene sequence indicate that it is closely affiliated with the genus Colwellia. Comparative 16S rRNA gene sequence analyses revealed 95.7, 95.5 and 95.2 % similarity to Colwellia maris ABE-1T, Colwellia piezophila Y233GT and Colwellia psychrerythraea ATCC 27364T, respectively. The major cellular fatty acids were C16 : 1, C16 : 0 and C22 : 6 (docosahexaenoic acid), and the sole isoprenoid quinone produced was ubiqinone-8. DNA G+C content was 48.6 mol%. The strain was positive for oxidase and catalase activities. Based on the results from this study, strain MTCD1T is a novel Gram-negative species of the genus Colwellia, and the name Colwellia marinimaniae sp. nov. (type strain MTCD1T=ATCC TSD-5T=JCM 30270T) is proposed. It is the most piezophilic organism yet described.

The diversity and antibiotic properties of actinobacteria associated with endemic deepwater amphipods of Lake Baikal.

The emergence of pathogenic bacteria resistant to antibiotics increases the need for discovery of new effective antimicrobials. Unique habitats such as marine deposits, wetlands and caves or unexplored biological communities are promising sources for the isolation of actinobacteria, which are among the major antibiotic producers. The present study aimed at examining cultivated actinobacteria strains associated with endemic Lake Baikal deepwater amphipods and estimating their antibiotic activity. We isolated 42 actinobacterial strains from crustaceans belonging to Ommatogammarus albinus and Ommatogammarus flavus. To our knowledge, this is the first report describing the isolation and initial characterization of representatives of Micromonospora and Pseudonocardia genera from Baikal deepwater invertebrates. Also, as expected, representatives of the genus Streptomyces were the dominant group among the isolated species. Some correlations could be observed between the number of actinobacterial isolates, the depth of sampling and the source of the strains. Nevertheless, >70% of isolated strains demonstrated antifungal activity. The dereplication analysis of extract of one of the isolated strains resulted in annotation of several known compounds that can help to explain the observed biological activities. The characteristics of ecological niche and lifestyle of deepwater amphipods suggests that the observed associations between crustaceans and isolated actinobacteria are not random and might represent long-term symbiotic interactions.

Bioaccumulation of Toxoplasma and Cryptosporidium by the freshwater crustacean Gammarus fossarum: Involvement in biomonitoring surveys and trophic transfer.

The protozoa Toxoplasma gondii and Cryptosporidium parvum are public health priorities because their oocysts can persist in recreational, surface, drinking, river, and sea water sources for a long time. To evaluate the capacity of the freshwater crustacean Gammarus fossarum to accumulate T. gondii and C. parvum oocysts, gammarids were exposed to 200, 2000 or 20,000 oocysts per gammarid and per day for 21 days followed by 5 days of depuration. C. parvum DNA was detected by qPCR in G. fossarum in only one out of four pools for the highest concentration and after 14 days of exposure, and T. gondii DNA was detected after 7 days of exposure to the two highest concentrations. Our results document the capacity of G. fossarum to accumulate T. gondii in its tissues proportionally to the ambient concentration; the maximum number of oocysts was detected in gammarid tissues after exposure to 20,000 oocysts per day. Mean values of 3.26 (±3), 21.71 (±15.18), and 17.41 (±10.89) oocysts were detected in gammarids after 7, 14, and 21 days, respectively, and after 5 days of depuration, T. gondii oocysts were still present in gammarid tissues. These results show for the first time that a freshwater crustacean can bioaccumulate T. gondii oocysts, suggesting that G. fossarum is a potential effective bioindicator of protozoan contamination in biomonitoring studies. Moreover, due to its key position in freshwater food webs, G. fossarum could also play a role in the trophic transfer of protozoa.

Predicting invasive species impacts: a community module functional response approach reveals context dependencies.

Predatory functional responses play integral roles in predator-prey dynamics, and their assessment promises greater understanding and prediction of the predatory impacts of invasive species. Other interspecific interactions, however, such as parasitism and higher-order predation, have the potential to modify predator-prey interactions and thus the predictive capability of the comparative functional response approach. We used a four-species community module (higher-order predator; focal native or invasive predators; parasites of focal predators; native prey) to compare the predatory functional responses of native Gammarus duebeni celticus and invasive Gammarus pulex amphipods towards three invertebrate prey species (Asellus aquaticus, Simulium spp., Baetis rhodani), thus, quantifying the context dependencies of parasitism and a higher-order fish predator on these functional responses. Our functional response experiments demonstrated that the invasive amphipod had a higher predatory impact (lower handling time) on two of three prey species, which reflects patterns of impact observed in the field. The community module also revealed that parasitism had context-dependent influences, for one prey species, with the potential to further reduce the predatory impact of the invasive amphipod or increase the predatory impact of the native amphipod in the presence of a higher-order fish predator. Partial consumption of prey was similar for both predators and occurred increasingly in the order A. aquaticus, Simulium spp. and B. rhodani. This was associated with increasing prey densities, but showed no context dependencies with parasitism or higher-order fish predator. This study supports the applicability of comparative functional responses as a tool to predict and assess invasive species impacts incorporating multiple context dependencies.

Analysis of bacterial communities associated with the benthic amphipod Diporeia in the Laurentian Great Lakes Basin.

Bacterial communities play important roles in the biological functioning of crustaceans, yet little is known about their diversity, structure, and dynamics. This study was conducted to investigate the bacterial communities associated with the benthic amphipod Diporeia, an important component in the Great Lakes foodweb that has been declining over the past 3 decades. In this study, the combination of 16S rRNA gene sequencing and terminal restriction fragment length polymorphism revealed a total of 175 and 138 terminal restriction fragments (T-RFs) in Diporeia samples following treatment with the endonucleases HhaI and MspI, respectively. Relatively abundant and prevalent T-RFs were affiliated with the genera Flavobacterium and Pseudomonas and the class Betaproteobacteria. T-RFs affiliated with the order Rickettsiales were also detected. A significant difference in T-RF presence and abundance (P = 0.035) was detected among profiles generated for Diporeia collected from 4 sites in Lake Michigan. Comparison of profiles generated for Diporeia samples collected in 2 years from lakes Superior and Michigan showed a significant change in diversity for Lake Superior Diporeia but not Lake Michigan Diporeia. Profiles from one Lake Michigan site contained multiple unique T-RFs compared with other Lake Michigan Diporeia profiles, most notably one that represents the genus Methylotenera. This study generated the most extensive list of bacteria associated with Diporeia and sheds useful insights on the microbiome of Great Lakes Diporeia that may help to reveal potential causes of the decline of Diporeia populations.

Niphargus-Thiothrix associations may be widespread in sulphidic groundwater ecosystems: evidence from southeastern Romania.

Niphargus is a speciose amphipod genus found in groundwater habitats across Europe. Three Niphargus species living in the sulphidic Frasassi caves in Italy harbour sulphur-oxidizing Thiothrix bacterial ectosymbionts. These three species are distantly related, implying that the ability to form ectosymbioses with Thiothrix may be common among Niphargus. Therefore, Niphargus-Thiothrix associations may also be found in sulphidic aquifers other than Frasassi. In this study, we examined this possibility by analysing niphargids of the genera Niphargus and Pontoniphargus collected from the partly sulphidic aquifers of the Southern Dobrogea region of Romania, which are accessible through springs, wells and Movile Cave. Molecular and morphological analyses revealed seven niphargid species in this region. Five of these species occurred occasionally or exclusively in sulphidic locations, whereas the remaining two were restricted to nonsulphidic areas. Thiothrix were detected by PCR on all seven Dobrogean niphargid species and observed using microscopy to be predominantly attached to their hosts' appendages. 16S rRNA gene sequences of the Thiothrix epibionts fell into two main clades, one of which (herein named T4) occurred solely on niphargids collected in sulphidic locations. The other Thiothrix clade was present on niphargids from both sulphidic and nonsulphidic areas and indistinguishable from the T3 ectosymbiont clade previously identified on Frasassi-dwelling Niphargus. Although niphargids from Frasassi and Southern Dobrogea are not closely related, the patterns of their association with Thiothrix are remarkably alike. The finding of similar Niphargus-Thiothrix associations in aquifers located 1200 km apart suggests that they may be widespread in European groundwater ecosystems.

Specific detection and localization of microsporidian parasites in invertebrate hosts by using in situ hybridization.

We designed fluorescence in situ hybridization probes for two distinct microsporidian clades and demonstrated their application in detecting, respectively, Nosema/Vairimorpha and Dictyoceola species. We used them to study the vertical transmission of two microsporidia infecting the amphipod Gammarus duebeni.

Effects of feminizing microsporidia on the masculinizing function of the androgenic gland in Gammarus duebeni.

Feminizing parasites enhance their vertical transmission to the host offspring by converting genetic male hosts into phenotypic females. Crustacea are the only invertebrates where sexual differentiation is controlled by a specialised endocrine organ, the androgenic gland, rather than by the gonads. We showed that a feminizing microsporidian Microsporidium sp. inhibits androgenic gland differentiation. We investigated the effect of Microsporidium sp. and a second feminizing microsporidium, Nosema granulosis, on the masculinizing function of the androgenic gland in Gammarus duebeni. Androgenic gland implants had a masculinizing effect on the sexual characteristics and sexual behaviour of recipient female hosts, reflecting the masculinizing function of the androgenic gland. Individuals that had received androgenic glands showed changed morphology in comparison with controls; they were bigger overall, they lost their oostegite marginal setae, developed calceoli and acquired a male-like behaviour. This effect was observed in uninfected females, as well as in females infected with the Microsporidium sp. The masculinizing effect of androgenic gland implants was smaller in N. granulosis infected individuals. N. granulosis and Microsporidium sp. fall into distinct clades of the Microspora. It appears that these divergent parasites both act by inhibiting the development of the androgenic gland. However, they differ in their ability to inhibit the host's response to the hormone that controls male sexual differentiation.

Influence of changing plant food sources on the gut microbiota of saltmarsh detritivores.

Changes in agricultural land-use of saltmarshes along the German North Sea coast have favoured the succession of the marsh grass Elytrigia atherica over the long-established Spartina anglica. Consequently, E. atherica represents a potential food source of increasing importance for plant-feeding soil detritivores. Considering the importance of this ecological guild for decomposition processes and nutrient cycling, we focussed on two sympatric saltmarsh soil macrodetritivores and their associated gut microbiota to investigate how the digestive processes of these species may be affected by changing plant food sources. Using genetic fingerprints of partial 16S rRNA gene sequences, we analysed composition and diversity of the bacterial gut community in a diplopod and an amphipod crustacean in relation to different feeding regimes representing the natural vegetation changes. Effects of syntopy on the host-specific gut microbiota were also taken into account by feeding the two detritivore species either independently or on the same plant sample. Bacterial community composition was influenced by both the host species and the available plant food sources, but the latter had a stronger effect on microbial community structure. Furthermore, bacterial diversity was highest after feeding on a mixture of both plant species, regardless of the host species. The gut microbiota of these two detritivores can thus be expected to change along with the on-going succession at the plant community level in this environment. Cloning and sequencing of bacterial 16S rRNA gene fragments further indicated a host-related effect since the two detritivores differed in terms of predominant bacterial taxa: diplopods harboured mainly representatives of the phyla Bacteroidetes and Gammaproteobacteria. In contrast, the genus Vibrio was found for the amphipod host across all feeding conditions.

Fungal propagules and DNA in feces of two detritus-feeding amphipods.

Aquatic shredders (leaf-eating invertebrates) preferentially ingest and digest leaves colonized by aquatic hyphomycetes (fungi). This activity destroys leaf-associated fungal biomass and detritial resources in streams. Fungal counter-adaptations may include the ability to survive passage through the invertebrate's digestive tract. When fecal pellets of Gammarus tigrinus and Hyalella azteca were incubated with sterile leaves, spores of nine (G. tigrinus) and seven (H. azteca) aquatic hyphomycete species were subsequently released from the leaves, indicating the presence of viable fungal structures in the feces. Extraction, amplification, and sequencing of DNA from feces revealed numerous fungal phylotypes, two of which could be assigned unequivocally to an aquatic hyphomycete. The estimated contributions of major fungal groups varied depending on whether 18S or ITS sequences were amplified and cloned. We conclude that a variable proportion of fungal DNA in the feces of detritivores may originate from aquatic hyphomycetes. Amplified DNA may be associated with metabolically active, dormant, or dead fungal cells.

Effects of repeated field applications of two formulations of Bacillus thuringiensis var. israelensis on non-target saltmarsh invertebrates in Atlantic coastal wetlands.

Bacillus thuringiensis var. israelensis (Bti) is commonly used for selective control of larval populations of mosquitoes in coastal wetlands. A two year-study was implemented to investigate whether repeated treatments with Bti applied either as a liquid (VectoBac® 12AS) or a water-dispersible granule (VectoBac® WG) formulation may affect the abundance and diversity of non-target aquatic invertebrates in saltmarsh pools. Taxonomic composition of the invertebrate communities was typical of brackishwater intermittent ecosystems, with a dominance of annelids, crustaceans and nematocerans. Conditions were contrasted between the two years of the survey, both in terms of annual cumulative rainfall and rainfall distribution throughout the year. As a consequence, the hydroperiod and some other environmental characteristics associated with pool drying played a major role in the dynamics of the invertebrate community. In summer 2006, pool drying reduced the abundance of the polychaete worm Nereis diversicolor, of the amphipod crustacean Corophium volutator and of chironomid larvae. These taxa were able to recolonize rapidly the pools after flooding in September 2006. In 2007, rainfall was more regularly distributed across the year, and the pools did not get dry. Hydrozoans, Chironomini and Orthocladiinae larvae, and oligochaetes were more abundant in treated than in control pools, especially in VectoBac® WG-treated pools. No adverse effects of the treatments were shown on the abundance of N. diversicolor, C. volutator and midge larvae, suggesting that the availability of these food sources for birds was not negatively affected by Bti applications. It is concluded that, as currently performed in Western France coastal wetlands, land-based treatments of saltmarsh pools for larval mosquito control with Bti, used either as VectoBac® 12AS or VectoBac® WG, did not adversely impact non-target aquatic invertebrate communities.

Cellular Immune Response of an Endemic Lake Baikal Amphipod to Indigenous Pseudomonas sp.

Studies of invertebrates have shown that the internal environment of crustaceans is not always sterile in normal conditions, and in many species, it can be populated by microorganisms even in the absence of any visible pathological processes in the body. This observation raises the question of whether genetically modified indigenous hemolymph microorganisms can be used for biotechnological purposes inside the crustacean either as local producers of some compounds or as sensors to physiological parameters. In this study, we tested the ability of the bacteria isolated from the hemolymph of the amphipod Eulimnogammarus verrucosus to hide from the cellular immune response of the host as the most important feature for their potential long-term application in vivo. 16S rDNA amplicon sequencing revealed five common bacterial genera in all analyzed samples of the amphipod hemolymph, among which Pseudomonas is most easily subjected to genome modification and, thus, the most prospective for biotechnological application. Cultivation of Pseudomonas gave us a number of strains undoubtedly derived from the amphipod hemolymph, and one of them (belonging to the Pseudomonas fluorescens group) was chosen for further tests. The primary culture of amphipod hemocytes was used to analyze the immunogenicity of the strain and showed a pronounced reaction of the immune cells to a high amount of the bacteria within six hours. This result indicates that modulation of cellular immune response to metabolically active bacterial cells is not mandatory for the survival and wide distribution of these microorganisms in the hemolymph of numerous amphipod individuals.