The skin microbiome of elasmobranchs follows phylosymbiosis, but in teleost fishes, the microbiomes converge.

BACKGROUND: The vertebrate clade diverged into Chondrichthyes (sharks, rays, and chimeras) and Osteichthyes fishes (bony fishes) approximately 420 mya, with each group accumulating vast anatomical and physiological differences, including skin properties. The skin of Chondrichthyes fishes is covered in dermal denticles, whereas Osteichthyes fishes are covered in scales and are mucous rich. The divergence time among these two fish groups is hypothesized to result in predictable variation among symbionts. Here, using shotgun metagenomics, we test if patterns of diversity in the skin surface microbiome across the two fish clades match predictions made by phylosymbiosis theory. We hypothesize (1) the skin microbiome will be host and clade-specific, (2) evolutionary difference in elasmobranch and teleost will correspond with a concomitant increase in host-microbiome dissimilarity, and (3) the skin structure of the two groups will affect the taxonomic and functional composition of the microbiomes. RESULTS: We show that the taxonomic and functional composition of the microbiomes is host-specific. Teleost fish had lower average microbiome within clade similarity compared to among clade comparison, but their composition is not different among clade in a null based model. Elasmobranch's average similarity within clade was not different than across clade and not different in a null based model of comparison. In the comparison of host distance with microbiome distance, we found that the taxonomic composition of the microbiome was related to host distance for the elasmobranchs, but not the teleost fishes. In comparison, the gene function composition was not related to the host-organism distance for elasmobranchs but was negatively correlated with host distance for teleost fishes. CONCLUSION: Our results show the patterns of phylosymbiosis are not consistent across both fish clades, with the elasmobranchs showing phylosymbiosis, while the teleost fish are not. The discrepancy may be linked to alternative processes underpinning microbiome assemblage, including possible historical host-microbiome evolution of the elasmobranchs and convergent evolution in the teleost which filter specific microbial groups. Our comparison of the microbiomes among fishes represents an investigation into the microbial relationships of the oldest divergence of extant vertebrate hosts and reveals that microbial relationships are not consistent across evolutionary timescales. Video abstract.

Metarhizium brunneum infection dynamics differ at the cuticle interface of susceptible and tolerant morphs of Galleria mellonella.

In order for entomopathogenic fungi to colonize an insect host, they must first attach to, and penetrate, the cuticle layers of the integument. Herein, we explored the interactions between the fungal pathogen Metarhizium brunneum ARSEF 4556 and two immunologically distinct morphs, melanic (M) and non-melanic (NM), of the greater wax moth Galleria mellonella. We first interrogated the cuticular compositions of both insect morphs to reveal substantial differences in their physiochemical properties. Enhanced melanin accumulation, fewer hydrocarbons, and higher L-dihydroxyphenylalanine (DOPA) decarboxylase activity were evident in the cuticle of the M larvae. This "hostile" terrain proved challenging for M. brunneum - reflected in poor conidial attachment and germination, and elevated expression of stress-associated genes (e.g., Hsp30, Hsp70). Lack of adherence to the cuticle impacted negatively on the speed of kill and overall host mortality; a dose of 107 conidia killed ~30% of M larvae over a 12-day period, whereas a 100-fold lower dose (105 conidia) achieved a similar result for NM larvae. Candidate gene expression patterns between the insect morphs indicated that M larvae are primed to "switch-on" immunity-associated genes (e.g., phenoloxidase) within 6-12 h of conidia exposure and can sustain a "defense" response. Critically, M. brunneum responds to the distinct physiochemical cues of both hosts and adjusts the expression of pathogenicity-related genes accordingly (e.g., Pr2, Mad1, Mad2). We reveal previously uncharacterized mechanisms of attack and defence in fungal-insect antibiosis.

Ectoparasitism and infections in the exoskeletons of large fossil cingulates.

Studies on paleopathological alterations in fossil vertebrates, including damages caused by infections and ectoparasites, are important because they are potential sources of paleoecological information. Analyzing exoskeleton material (isolated osteoderms, carapace and caudal tube fragments) from fossil cingulates of the Brazilian Quaternary Megafauna, we identified damages that were attributed to attacks by fleas and dermic infections. The former were compatible with alterations produced by one species of flea of the genus Tunga, which generates well-delimited circular perforations with a patterned distribution along the carapace; the latter were attributable to pathogenic microorganisms, likely bacteria or fungi that removed the ornamentation of osteoderms and, in certain cases, generated craters or pittings. Certain bone alterations observed in this study represent the first record of flea attack and pitting in two species of large glyptodonts (Panochthus and Glyptotherium) and in a non-glyptodontid large cingulate (Pachyarmatherium) from the Quaternary of the Brazilian Intertropical Region. These new occurrences widen the geographic distribution of those diseases during the Cenozoic and provide more evidence for the co-evolutionary interaction between cingulates and parasites registered to date only for a small number of other extinct and extant species.

Highly specific host-pathogen interactions influence Metarhizium brunneum blastospore virulence against Culex quinquefasciatus larvae.

Entomopathogenic fungi are potential biological control agents of mosquitoes. Our group observed that not all mosquitoes were equally susceptible to fungal infection and observed significant differences in virulence of different spore types. Conidiospores and blastospores were tested against Culex quinquefasciatus larvae. Blastospores are normally considered more virulent than conidia as they form germ tubes and penetrate the host integument more rapidly than conidia. However, when tested against Cx. quinquefasciatus, blastospores were less virulent than conidia. This host-fungus interaction was studied by optical, electron and atomic force microscopy (AFM). Furthermore, host immune responses and specific gene expression were investigated. Metarhizium brunneum (formerly M. anisopliae) ARSEF 4556 blastospores did not readily adhere to Culex larval integument and the main route of infection was through the gut. Adhesion forces between blastospores and Culex cuticle were significantly lower than for other insects. Larvae challenged with blastospores showed enhanced immune responses, with increased levels of phenoloxidase, glutathione-S-transferase, esterase, superoxide dismutase and lipid peroxidase activity. Interestingly, M. brunneum pathogenicity/stress-related genes were all down-regulated in blastospores exposed to Culex. Conversely, when conidia were exposed to Culex, the pathogenicity genes involved in adhesion or cuticle degradation were up-regulated. Delayed host mortality following blastospore infection of Culex was probably due to lower adhesion rates of blastospores to the cuticle and enhanced host immune responses deployed to counter infection. The results here show that subtle differences in host-pathogen interactions can be responsible for significant changes in virulence when comparing mosquito species, having important consequences for biological control strategies and the understanding of pathogenicity processes.

Prevalence of the genus Cladosporium on the integument of leaf-cutting ants characterized by 454 pyrosequencing.

The relationship of attine ants with their mutualistic fungus and other microorganisms has been studied during the last two centuries. However, previous studies about the diversity of fungi in the ants' microenvironment are based mostly on culture-dependent approaches, lacking a broad characterization of the fungal ant-associated community. Here, we analysed the fungal diversity found on the integument of Atta capiguara and Atta laevigata alate ants using 454 pyrosequencing. We obtained 35,453 ITS reads grouped into 99 molecular operational taxonomic units (MOTUs). Data analysis revealed that A. capiguara drones had the highest diversity of MOTUs. Besides the occurrence of several uncultured fungi, the mycobiota analysis revealed that the most abundant taxa were the Cladosporium-complex, Cryptococcus laurentii and Epicoccum sp. Taxa in the genus Cladosporium were predominant in all samples, comprising 67.9 % of all reads. The remarkable presence of the genus Cladosporium on the integument of leaf-cutting ants alates from distinct ant species suggests that this fungus is favored in this microenvironment.

Cloning, expression and characterization of Ostrinia furnacalis serpin1, a regulator of the prophenoloxidase activation system.

Serine protease inhibitors of the serpin superfamily are regulators of proteases involved in a variety of physiological processes including immune responses. In this study, we have isolated a full-length serpin cDNA from Ostrinia furnacalis. The 1188 bp open reading frame encodes a 395-residue protein with a theoretical molecular mass of 43.3 kDa and an isoelectric point of 4.92. Ofserpin1 contains a putative signal peptide followed by a conserved domain including a reactive center loop (RCL) with a hinge region (E(344) to S(353)) and a predicted P1-P1' cleavage site (Leu(360)-Ser(361)). Ofserpin1 mRNA and protein were detected in all the tested tissues, particularly in hemocytes and integument. The recombinant protein inhibited chymotrypsin and trypsin in a dose-dependent manner, and were significantly cleaved by the enzyme trypsin and chymotrypsin. Ofserpin1 impeded the prophenoloxidase activation cascade by 45.6% at 16.5 µg, and affected activity of prophenoloxidase activating protease. Levels of Ofserpin1 transcripts in the integument were higher than those in hemocytes, fat body and midgut. After an immune challenge with Staphylococcus aureus and Escherichia coli, the relative mRNA levels of Ofserpin1 decreased in 2-10h post-infection (hpi) in integument and hemocytes compared to the untreated control. Our results suggested that Ofserpin1 has serine protease inhibitory activity and is likely involved in the regulation of prophenoloxidase activation system in O. furnacalis.

Yeasts found on an ephemeral reproductive caste of the leaf-cutting ant Atta sexdens rubropilosa.

Winged males of leaf-cutting ants are considered an ephemeral reproductive caste only produced before the mating flight season. Although much is known about the yeast diversity found in fungus gardens of attine ants, no study has focused on the yeasts associated with males of leaf-cutting ants. Here, we surveyed the yeasts on the integuments of males of Atta sexdens rubropilosa and assessed their potential role in the attine ant-microbe symbiosis. Using culture-dependent techniques, we found yeasts to be abundant on the integuments of males (54.5 %, n = 200 alates). A total of 242 yeast strains were obtained representing six orders, ten genera and 25 species. Strains of Aureobasidium, Cryptococcus, Hannaella and Rhodotorula were prevalent on the integuments and likely originated from the fungus garden of the parental nest or from the soil. The majority of strains (87.1 %) produced at least one of the evaluated enzymes: pectinase, polygalacturonase, cellulase, xylanase, ligninases and lipase. Aureobasidium pullulans accounted for the highest number of strains that produced all enzymes. In addition, yeasts showed the ability to assimilate the resulting oligosaccharides, supporting observations of other studies that yeasts may be involved in the plant biomass metabolism in the fungus gardens. Because winged males harbor several yeasts with putative functional roles, these fungi may take part and be beneficial in the microbial consortia of the new incipient nest.

Can insects develop resistance to insect pathogenic fungi?

Microevolutionary adaptations and mechanisms of fungal pathogen resistance were explored in a melanic population of the Greater wax moth, Galleria mellonella. Under constant selective pressure from the insect pathogenic fungus Beauveria bassiana, 25(th) generation larvae exhibited significantly enhanced resistance, which was specific to this pathogen and not to another insect pathogenic fungus, Metarhizium anisopliae. Defense and stress management strategies of selected (resistant) and non-selected (susceptible) insect lines were compared to uncover mechanisms underpinning resistance, and the possible cost of those survival strategies. We hypothesize that the insects developed a transgenerationally primed resistance to the fungus B. bassiana, a costly trait that was achieved not by compromising life-history traits but rather by prioritizing and re-allocating pathogen-species-specific augmentations to integumental front-line defenses that are most likely to be encountered by invading fungi. Specifically during B. bassiana infection, systemic immune defenses are suppressed in favour of a more limited but targeted repertoire of enhanced responses in the cuticle and epidermis of the integument (e.g. expression of the fungal enzyme inhibitor IMPI, and cuticular phenoloxidase activity). A range of putative stress-management factors (e.g. antioxidants) is also activated during the specific response of selected insects to B. bassiana but not M. anisopliae. This too occurs primarily in the integument, and probably contributes to antifungal defense and/or helps ameliorate the damage inflicted by the fungus or the host's own immune responses.

Culture-independent analysis of bacterial communities in hemolymph of American lobsters with epizootic shell disease.

Epizootic shell disease (ESD) of the American lobster Homarus americanus H. Milne Edwards, 1837 is a disease of the carapace that presents grossly as large, melanized, irregularly shaped lesions, making the lobsters virtually unmarketable because of their grotesque appearance. We analyzed the bacterial communities present in the hemolymph of lobsters with and without ESD using nested-PCR of the 16S rRNA genes followed by denaturing gradient gel electrophoresis. All lobsters tested (n = 42) had bacterial communities in their hemolymph, and the community profiles were highly similar regardless of the sampling location or disease state. A number of bacteria were detected in a high proportion of samples and from numerous locations, including a Sediminibacterium sp. closely related to a symbiont of Tetraponera ants (38/42) and a Ralstonia sp. (27/42). Other bacteria commonly encountered included various Bacteroidetes, Pelomonas aquatica, and a Novosphingobium sp. One bacterium, a different Sediminibacterium sp., was detected in 20% of diseased animals (n = 29), but not in the lobsters without signs of ESD (n = 13). The bacteria in hemolymph were not the same as those known to be present in lesion communities except for the detection of a Thalassobius sp. in 1 individual. This work demonstrates that hemolymph bacteremia and the particular bacterial species present do not correlate with the incidence of ESD, providing further evidence that microbiologically, ESD is a strictly cuticular disease. Furthermore, the high incidence of the same species of bacteria in hemolymph of lobsters may indicate that they have a positive role in lobster fitness, rather than in disease, and further investigation of the role of bacteria in lobster hemolymph is required.

Differentially expressed genes in the cuticle and hemolymph of the silkworm, Bombyx mori, injected with the fungus Beauveria bassiana.

The most important pathogenic fungus of the silkworm, Bombyx mori L. (Lepidoptera: Bombycidae), is Beauveria bassiana (Balsamo-Crivelli ) Vuillemin (Hypocreales: Clavicipitaceae), which causes significant damage to sericulture production. Therefore, diagnosing fungal disease and developing new control measures are crucial to silk production. To better understand the responsive and interactive mechanisms between the host silkworm and this fungus, variations in silkworm gene expression were investigated using the suppression subtractive hybridization method following the injection of B. bassiana conidia. Two cDNA libraries were constructed, and 140 cDNA clones were isolated. Of the 50 differentially expressed genes identified, 45 (112 clones) were identified in the forward library, and 5 (28 clones) were identified in the reverse library. Expression profiling of six of these genes by quantitative polymerase chain reaction (qPCR) verified that they were induced by the fungal challenge. The present study provides insight into the interaction between lepidopteran insects and pathogenic fungi.

Lesion bacterial communities in American lobsters with diet-induced shell disease.

In southern New England, USA, shell disease affects the profitability of the American lobster Homarus americanus fishery. In laboratory trials using juvenile lobsters, exclusive feeding of herring Clupea harengus induces shell disease typified initially by small melanized spots that progress into distinct lesions. Amongst a cohabitated, but segregated, cohort of 11 juvenile lobsters fed exclusively herring, bacterial communities colonizing spots and lesions were investigated by denaturing gradient gel electrophoresis of 16S rDNA amplified using 1 group-specific and 2 universal primer sets. The Bacteroidetes and Proteobacteria predominated in both spots and lesions and included members of the orders Flavobacteriales (Bacteriodetes), Rhodobacterales, Rhodospirillales and Rhizobiales (Alphaproteobacteria), Xanthomonadales (Gammaproteobacteria) and unclassified Gammaproteobacteria. Bacterial communities in spot lesions displayed more diversity than communities with larger (older) lesions, indicating that the lesion communities stabilize over time. At least 8 bacterial types persisted as lesions developed from spots. Aquimarina 'homaria', a species commonly cultured from lesions present on wild lobsters with epizootic shell disease, was found ubiquitously in spots and lesions, as was the 'Candidatus Kopriimonas aquarianus', implicating putative roles of these species in diet-induced shell disease of captive lobsters.

First evidence of an intimate symbiotic association between fungi and larvae in basal attine ants.

The most conspicuous ants in all of tropical America are those that belong to the tribe Attini which cultivate fungus. The objective of the present study is to verify the alterations that occur in the cuticle of the worker larvae from Myrmicocrypta, Mycetarotes and Trachymyrmex with the purpose of trying to establish the degree of this association. Attine ants from the Atta genus were used as a control group. The analysis of histological sections showed results about the distribution of the cells and tissues on the epidermis of A. sexdens rubropilosa ant larvae and Mycetarotes parallelus, Trachymyrmex fuscus and Myrmicocrypta sp. A cuticle covering the ants was observed in the larvae of all the species, and this is formed by a simple cubic epithelium, whose cells possibly change its shape to prismatic, depending on their secretory activity. Just under this epithelium large adipose cells with reserve granules in their cytoplasm were found. The presence of a space filled by granulose and acellular material was also observed, indicating that the larvae were in a molt period. The presence of fungal hyphae is observed both on the external side of the basal attine larvae as well as emitting projections to the interior of the cuticle reaching the epithelium and the adipocyte cells. Data obtained in the present study demonstrated that the fungus deposited on the surface of immature ants from attine basal species maintain a close relationship with them, once the fungus hyphae have the ability to disorganize the cuticle lamellas, penetrating the interior of the insect cells through the emission of prolongations transporting the cuticle and epithelium barriers and making substances exchanges between larvae and fungus.

Commensal bacteria play a role in mating preference of Drosophila melanogaster.

Development of mating preference is considered to be an early event in speciation. In this study, mating preference was achieved by dividing a population of Drosophila melanogaster and rearing one part on a molasses medium and the other on a starch medium. When the isolated populations were mixed, "molasses flies" preferred to mate with other molasses flies and "starch flies" preferred to mate with other starch flies. The mating preference appeared after only one generation and was maintained for at least 37 generations. Antibiotic treatment abolished mating preference, suggesting that the fly microbiota was responsible for the phenomenon. This was confirmed by infection experiments with microbiota obtained from the fly media (before antibiotic treatment) as well as with a mixed culture of Lactobacillus species and a pure culture of Lactobacillus plantarum isolated from starch flies. Analytical data suggest that symbiotic bacteria can influence mating preference by changing the levels of cuticular hydrocarbon sex pheromones. The results are discussed within the framework of the hologenome theory of evolution.

Enterobacterial microbiota on Stomoxys calcitrans external surface.

The current study evaluated the prevalence of enterobacterial agents on the external surface of the stable fly, Stomoxys calcitrans. In addition, this study investigated the presence of virulence genes of enterobacteria. Twenty different species were isolated and identified, Escherichia coli was the most frequent species isolated. The genes stx1, stx2 and/or eae for production of Shiga toxin were present in 13.04% of the E. coli samples.

Effects of epizootic shell disease in American lobster Homarus americanus determined using a quantitative disease index.

The incidence of epizootic shell disease in American lobster Homarus americanus has increased in southern New England, U.S.A., in the last decade, but few longitudinal studies have followed the disease progress in individual lobsters or demonstrated direct effects on mortality or growth. Diseased lobsters were held in the laboratory for 1 yr, and the progression of disease and its effects on molting, mortality, and growth were monitored. A quantitative disease index (QDI) was developed by measuring disease lesions in digital images of the carapace and expressing the result as a proportion of shell area. Some lobsters died due to high temperatures, but at least 13 of 55 lobsters (24%) died as a direct result of disease-related problems, mostly during molting, and there was a significant relationship between mortality and high values of the QDI. Lobsters that molted successfully were free of disease lesions, but many had exoskeletal deformities. There was no relationship between pre-molt size and disease severity, but molt increment was significantly correlated with premolt carapace length (CL) and negatively correlated with QDI. However, percentage growth was negatively correlated with QDI, but not with pre-molt CL. These significant lethal and sublethal effects of epizootic shell disease should be considered in lobster management.

Disease associated with integumentary and cloacal parasites in tadpoles of northern red-legged frog Rana aurora aurora.

A total of 6830 northern red-legged frog Rana aurora aurora tadpoles were examined under a dissecting microscope for oral disc, integumentary, and cloacal abnormalities in 13 ponds in and near Redwood National Park in northern California. Of these, 163 tadpoles were collected for histopathological investigation, including 115 randomly collected individuals, 38 collected with oral disc abnormalities, and 10 collected due to severe morbidity of unknown etiology. The tadpoles were infected with 8 parasites, including Batrachochytrium dendrobatidis (the amphibian chytrid), trematodes, leeches, and protozoa. Chytridiomycosis was detected at an overall prevalence of 6.4%, but prevalence was higher in tadpoles with oral disc lesions than in those with normal oral discs (43.5% versus 6.1%). Interestingly, infection was associated with some environmental and co-infection risk factors. Individual tadpoles possessed 0 to 5 species of parasites in varying intensities. Apiosoma sp. was the most prevalent (66%) and widespread. Tadpoles infected with B. dendrobatidis had a lower diversity of oral parasites than those uninfected. During the field portion of the study, a large number (approximately 500) of moribund and dead tadpoles was seen occurring at multiple locations within and surrounding Redwood National Park. Ten animals were collected for histological examination and a diverse protozoal infection was discovered, including some known pathogens of fish. This study is the first reporting parasitism and disease in natural populations of northern red-legged frogs.

Morphological studies on the bathyal ascidian, Megalodicopia hians Oka 1918 (Octacnemidae, Phlebobranchia), with remarks on feeding and tunic morphology.

Megalodicopia hians Oka is a solitary ascidian belonging to the family Octacnemidae inhabiting the bathyal /abyssal zone as well as other octacnemid ascidians. The phylogenetic relationship of octacnemids is open to argument because of its extraordinary morphological features due to habitat adaptation, e.g., a pharynx lacking ciliated stigmata. Aggregations of M. hians were discovered by the manned submersible Shinkai 2000 in the bathyal seafloor of Toyama Bay, Japan Sea, in 2000; this was the first in situ observation of M. hians in the Japanese coastal waters. In 2001, a total of 36 M. hians specimens were collected from the bay (592 to approximately 978 m deep). In situ observation indicated that M. hians usually opens its large oral apertures to engulf the drifting food particles in the water current. Microscopical observation of the gut contents also showed that M. hians is a non selective macrophagous feeding on small crustaceans, diatoms, detritus, and so on. Along with the position of the intestinal loop and gonad, the morphological characteristics of the tunic (integument of ascidians) suggest that M. hians is closely related to Cionidae and/or Corellidae. Some symbiotic/parasitic organisms were occasionally found in the tunic, including rod-shaped bacteria, fungi-like multicellular structure, and spawns of unknown animals.

[The enzymatic activity of fungi strains isolated from the skin and skin appendages of humans returning from the tropics]. / Aktywnosc enzymatyczna szczepów grzybów wyizolowanych ze skóry i jej przydatków od osób powracajacych z tropiku.

The aim of the work was to assess the enzymatic activity of 11 fungi strains isolated from the skin of 10 Lódz residents who had visited the tropics over 1998-1999. The strains were cultured by Jeske and Lupa of The Voivodeship Outpatient Clinic of Infectious, Parasitic and Tropical Diseases and Fungal Infections in Lódz. They were as follows: Trichophyton rubrum and Acremonium kiliense from Zambia, Myriodontium keratinophilum, Beauveria bassiana, Cladosporium herbarum, Candida famata and Trichophyton yaoundei from Sudan, Trichophyton tonsurans from Ethiopia, Trichophyton phaseliforme from Egypt, Acremonium strictum from Zimbabwe and Microsporum racemosus from Tanzania. The enzymatic activity was determined with the use of API ZYM of bioMérieux enabling 19 hydrolases to be revealed. In general, all tropical strains were characterised by a weak hydrolytic activity.