Epibiotic bacteria on the carapace of hawksbill and green sea turtles.

In this study, epibiotic bacteria on the carapace of sea turtles at three different sites in the Persian Gulf were studied. Bacterial density counted using a Scanning Electron Microscope showed that the highest (9.4 × 106 ± 0.8 × 106 cm-2) and lowest (5.3 × 106 ± 0.4 × 106 cm-2) average bacterial densities were found on the green and hawksbill sea turtles, respectively. Bacterial community analysis using Illumina 16S rRNA gene sequencing showed that Gamma- and Alpha-proteobacteria were the dominant classes on all substrates. Some genera, such as Anaerolinea, were site- and substrate-specific. In general, bacterial communities on sea turtles differed from those on the non-living substrate, stones, and exhibited lower species richness and diversity compared to the latter. Despite some similarities, the majority of bacterial communities on the two sea turtles were different. This study provides baseline information about the epibiotic bacteria of sea turtles of different species.

Evolution and Potential Function in Molluscs of Neuropeptide and Receptor Homologues of the Insect Allatostatins.

The allatostatins (ASTs), AST-A, AST-B and AST-C, have mainly been investigated in insects. They are a large group of small pleotropic alloregulatory neuropeptides that are unrelated in sequence and activate receptors of the rhodopsin G-protein coupled receptor family (GPCRs). The characteristics and functions of the homologue systems in the molluscs (Buccalin, MIP and AST-C-like), the second most diverse group of protostomes after the arthropods, and of high interest for evolutionary studies due to their less rearranged genomes remains to be explored. In the present study their evolution is deciphered in molluscs and putative functions assigned in bivalves through meta-analysis of transcriptomes and experiments. Homologues of the three arthropod AST-type peptide precursors were identified in molluscs and produce a larger number of mature peptides than in insects. The number of putative receptors were also distinct across mollusc species due to lineage and species-specific duplications. Our evolutionary analysis of the receptors identified for the first time in a mollusc, the cephalopod, GALR-like genes, which challenges the accepted paradigm that AST-AR/buccalin-Rs are the orthologues of vertebrate GALRs in protostomes. Tissue transcriptomes revealed the peptides, and their putative receptors have a widespread distribution in bivalves and in the bivalve Mytilus galloprovincialis, elements of the three peptide-receptor systems are highly abundant in the mantle an innate immune barrier tissue. Exposure of M. galloprovincialis to lipopolysaccharide or a marine pathogenic bacterium, Vibrio harveyi, provoked significant modifications in the expression of genes of the peptide precursor and receptors of the AST-C-like system in the mantle suggesting involvement in the immune response. Overall, our study reveals that homologues of the arthropod AST-systems in molluscs are potentially more complex due to the greater number of putative mature peptides and receptor genes. In bivalves they have a broad and varying tissue distribution and abundance, and the elements of the AST-C-like family may have a putative function in the immune response.

Comprehensive characterization of internal and cuticle surface microbiota of laboratory-reared F1 Anopheles albimanus originating from different sites.

BACKGROUND: Research on mosquito-microbe interactions may lead to new tools for mosquito and mosquito-borne disease control. To date, such research has largely utilized laboratory-reared mosquitoes that typically lack the microbial diversity of wild populations. A logical progression in this area involves working under controlled settings using field-collected mosquitoes or, in most cases, their progeny. Thus, an understanding of how laboratory colonization affects the assemblage of mosquito microbiota would aid in advancing mosquito microbiome studies and their applications beyond laboratory settings. METHODS: Using high throughput 16S rRNA amplicon sequencing, the internal and cuticle surface microbiota of F1 progeny of wild-caught adult Anopheles albimanus from four locations in Guatemala were characterized. A total of 132 late instar larvae and 135 2-5 day-old, non-blood-fed virgin adult females that were reared under identical laboratory conditions, were pooled (3 individuals/pool) and analysed. RESULTS: Results showed location-associated heterogeneity in both F1 larval internal (p = 0.001; pseudo-F = 9.53) and cuticle surface (p = 0.001; pseudo-F = 8.51) microbiota, and only F1 adult cuticle surface (p = 0.001; pseudo-F = 4.5) microbiota, with a more homogenous adult internal microbiota (p = 0.12; pseudo-F = 1.6) across collection sites. Overall, ASVs assigned to Leucobacter, Thorsellia, Chryseobacterium and uncharacterized Enterobacteriaceae, dominated F1 larval internal microbiota, while Acidovorax, Paucibacter, and uncharacterized Comamonadaceae, dominated the larval cuticle surface. F1 adults comprised a less diverse microbiota compared to larvae, with ASVs assigned to the genus Asaia dominating both internal and cuticle surface microbiota, and constituting at least 70% of taxa in each microbial niche. CONCLUSIONS: These results suggest that location-specific heterogeneity in filed mosquito microbiota can be transferred to F1 progeny under normal laboratory conditions, but this may not last beyond the F1 larval stage without adjustments to maintain field-derived microbiota. These findings provide the first comprehensive characterization of laboratory-colonized F1 An. albimanus progeny from field-derived mothers. This provides a background for studying how parentage and environmental conditions differentially or concomitantly affect mosquito microbiome composition, and how this can be exploited in advancing mosquito microbiome studies and their applications beyond laboratory settings.

Microbiome of the Successful Freshwater Invader, the Signal Crayfish, and Its Changes along the Invasion Range.

Increasing evidence denotes the role of the microbiome in biological invasions, since it is known that microbes can affect the fitness of the host. Here, we demonstrate differences in the composition of an invader's microbiome along the invasion range, suggesting that its microbial communities may affect and be affected by range expansion. Using a 16S rRNA gene amplicon sequencing approach, we (i) analyzed the microbiomes of different tissues (exoskeleton, hemolymph, hepatopancreas, and intestine) of a successful freshwater invader, the signal crayfish, (ii) compared them to the surrounding water and sediment, and (iii) explored their changes along the invasion range. Exoskeletal, hepatopancreatic, and intestinal microbiomes varied between invasion core and invasion front populations. This indicates that they may be partly determined by population density, which was higher in the invasion core than in the invasion front. The highly diverse microbiome of exoskeletal biofilm was partly shaped by the environment (due to the similarity with the sediment microbiome) and partly by intrinsic crayfish parameters (due to the high proportion of exoskeleton-unique amplicon sequence variants [ASVs]), including the differences in invasion core and front population structure. Hemolymph had the most distinct microbiome compared to other tissues and differed between upstream (rural) and downstream (urban) river sections, indicating that its microbiome is potentially more driven by the effects of the abiotic environment. Our findings offer an insight into microbiome changes during dispersal of a successful invader and present a baseline for assessment of their contribution to an invader's overall health and its further invasion success. IMPORTANCE Invasive species are among the major drivers of biodiversity loss and impairment of ecosystem services worldwide, but our understanding of their invasion success and dynamics still has many gaps. For instance, although it is known that host-associated microbial communities may significantly affect an individual's health and fitness, the current studies on invasive species are mainly focused on pathogenic microbes, while the effects of the remaining majority of microbial communities on the invasion process are almost completely unexplored. We have analyzed the microbiome of one of the most successful crayfish invaders in Europe, the signal crayfish, and explored its changes along the signal crayfish invasion range in the Korana River, Croatia. Our study sets the perspective for future research required to assess the contribution of these changes to an individual's overall health status and resilience of dispersing populations and their impact on invasion success.

Preparation of chitosan from waste shrimp shells fermented with Paenibacillus jamilae BAT1.

In this study, chitin extraction from shrimp shell powder (SSP) using locally isolated Paenibacillus jamilae BAT1 (GenBank: MN176658), the preparation of chitosan from the extracted chitin, and the characterization and biological activity (antimicrobial and antioxidant) of the prepared chitosan (PC) were investigated. It was determined that P. jamilae BAT1 did not have chitinase activity but showed high protease activity and protein removal potential. Optimum pH, shell concentration and incubation time for deproteinization were determined as 7.0, 60 g/L and 4 days, respectively. Addition of KH2PO4 or MgSO4 did not affect chitin extraction and deproteinization yield. The maximum yields of deproteinization, demineralization and chitin extraction yields were 87.67, 41.95 and 24.5%, respectively. The viscosity-average molecular weight of PC was determined as 1.41 × 105 g/mol. The deacetylation degree of PC (86%) was found to be higher that of commercial chitosan (CC) (78%). DPPH scavenging activity of PC (IC50 0.59 mg/mL) was higher than that of CC (IC50 3.72 mg/mL). PC was found to have higher antimicrobial activity against the bacteria E. coli and S. aureus and the yeast C. albicans when compared to CC. This is the first study on the use of the bacterium P. jamilae in biological chitin extraction.

Consistent patterns in 16S and 18S microbial diversity from the shells of the common and widespread red-eared slider turtle (Trachemys scripta).

Microbial communities associated with freshwater aquatic habitats and resident species are both critical to and indicative of ecosystem status and organismal health. External surfaces of turtle shells readily accumulate microbial growth and could carry representation of habitat-wide microbial diversity, since they are in regular contact with multiple elements of freshwater environments. Yet, microbial diversity residing on freshwater turtle shells is poorly understood. We applied 16S and 18S metabarcoding to characterize microbiota associated with external shell surfaces of 20 red-eared slider (Trachemys scripta) turtles collected from varied habitats in central and western Oklahoma, and ranging to southeast Iowa. Shell-associated microbial communities were highly diverse, with samples dominated by Bacteroidia and alpha-/gamma-proteobacteria, and ciliophoran alveolates. Alpha diversity was lower on turtle shells compared to shallow-water-associated environmental samples, likely resulting from basking-drying behavior and seasonal scute shedding, while alpha diversity was higher on carapace than plastron surfaces. Beta diversity of turtle shells was similarly differentiated from environmental samples, although sampling site was consistently a significant factor. Deinococcus-Thermus bacteria and ciliophoran alveolates were recovered with significantly higher abundance on turtle shells versus environmental samples, while bacterial taxa known to include human-pathogenic species were variably more abundant between shell and environmental samples. Microbial communities from a single, shared-site collection of the ecologically similar river cooter (P. concinna) largely overlapped with those of T. scripta. These data add to a foundation for further characterization of turtle shell microbial communities across species and habitats, with implications for freshwater habitat assessment, microbial ecology and wildlife conservation efforts.

Disentangling the assembly mechanisms of ant cuticular bacterial communities of two Amazonian ant species sharing a common arboreal nest.

Bacteria living on the cuticle of ants are generally studied for their protective role against pathogens, especially in the clade of fungus-growing ants. However, little is known regarding the diversity of cuticular bacteria in other ant host species, as well as the mechanisms leading to the composition of these communities. Here, we used 16S rRNA gene amplicon sequencing to study the influence of host species, species interactions and the pool of bacteria from the environment on the assembly of cuticular bacterial communities on two phylogenetically distant Amazonian ant species that frequently nest together inside the roots system of epiphytic plants, Camponotus femoratus and Crematogaster levior. Our results show that (a) the vast majority of the bacterial community on the cuticle is shared with the nest, suggesting that most bacteria on the cuticle are acquired through environmental acquisition, (b) 5.2% and 2.0% of operational taxonomic units (OTUs) are respectively specific to Ca. femoratus and Cr. levior, probably representing their respective core cuticular bacterial community, and (c) 3.6% of OTUs are shared between the two ant species. Additionally, mass spectrometry metabolomics analysis of metabolites on the cuticle of ants, which excludes the detection of cuticular hydrocarbons produced by the host, were conducted to evaluate correlations among bacterial OTUs and m/z ion mass. Although some positive and negative correlations are found, the cuticular chemical composition was weakly species-specific, suggesting that cuticular bacterial communities are prominently environmentally acquired. Overall, our results suggest the environment is the dominant source of bacteria found on the cuticle of ants.

Microbiome diversity and composition varies across body areas in a freshwater turtle.

There is increasing recognition that microbiomes are important for host health and ecology, and understanding host microbiomes is important for planning appropriate conservation strategies. However, microbiome data are lacking for many taxa, including turtles. To further our understanding of the interactions between aquatic microbiomes and their hosts, we used next generation sequencing technology to examine the microbiomes of the Krefft's river turtle (Emydura macquarii krefftii). We examined the microbiomes of the buccal (oral) cavity, skin on the head, parts of the shell with macroalgae and parts of the shell without macroalgae. Bacteria in the phyla Proteobacteria and Bacteroidetes were the most common in most samples (particularly buccal samples), but Cyanobacteria, Deinococcus-thermus and Chloroflexi were also common (particularly in external microbiomes). We found significant differences in community composition among each body area, as well as significant differences among individuals. The buccal cavity had lower bacterial richness and evenness than any of the external microbiomes, and it had many amplicon sequence variants (ASVs) with a low relative abundance compared to other body areas. Nevertheless, the buccal cavity also had the most unique ASVs. Parts of the shell with and without algae also had different microbiomes, with particularly obvious differences in the relative abundances of the families Methylomonaceae, Saprospiraceae and Nostocaceae. This study provides novel, baseline information about the external microbiomes of turtles and is a first step in understanding their ecological roles.

Characterizations and Fibrinolytic Activity of Serine Protease from Bacillus subtilis C10.

BACKGROUND: Fibrinolytic enzymes, such as Nattokinases from Bacillus species are known to degrade the fibrin blood clots. They belong to serine protease group having commercial applications, such as therapeutic agents and functional food formulation. OBJECTIVE: The present study reports some characteristics and fibrinolytic activity of serine protease from B. subtilis C10 strain that was isolated from shrimp shell. METHODS: Extracellular enzyme from B. subtilis C10 culture was harvested and partially purified by ammonium sulphate precipitation. Fibrinolytic activity of the enzyme was determined by zymography and measured by spectrophotometry with fibrinogen and thrombin used as substrates. The optimal temperature and pH for fibrinolytic activity were studied in the range of 31-43ºC and 5-10, respectively. The thermal and pH stability of enzyme was studied by incubating enzyme for 30 min in the same range of temperature and pH as above. The effect of some metal ions and reagents on fibrinolytic activity of enzyme was evaluated by concentrations of 5 mM and 5%, respectively. RESULTS: Zymogram analysis indicated the presence of four fibrinolytic enzymes with molecular weights of approximately 69, 67, 39 and 36 kDa. The optimal temperature and pH for enzyme activity were 37°C and 9, respectively. The thermal and pH stability ranged from 35-39°C and 8-10, respectively. Fibrinolytic activity reached a maximum value of about 400 U/mg protein after 16 h of C10 strain culture. Enzyme has been drastically inhibited by PMSF and SDS, and partially inhibited by EDTA, while Triton X-100 has significantly increased enzyme activity. Effects of ions such as Mg2+, Ca2+ and Mn2+ on enzyme were negligible, except Cu2+ and Zn2+ have strongly decreased its activity. CONCLUSION: Results from the present study suggested that enzyme obtained from B. subtilis C10 could be serine protease that has a high fibrinolytic activity up to about 400 U/mg protein at the most appropriate temperature and pH of 37ºC and 9. This activity can be improved up to 142% by incubating enzyme with 5% Triton X-100 for 30 min.

Fusarium spp. in Loggerhead Sea Turtles (Caretta caretta): From Colonization to Infection.

With the aim of evaluating the presence of Fusarium spp. in sea turtles with and without lesions and assessing the risk factors favoring colonization and/or infection, 74 loggerhead sea turtles (Caretta caretta) admitted to rescue and rehabilitation clinics in Italy were analyzed. The study compared 31 individuals with no apparent macroscopic lesions and 43 individuals with macroscopic lesions. Shell and skin samples were analyzed using Calcofluor white with 10% potassium hydroxide, standard histopathological examination, and fungal cultures. Fusarium spp. were isolated more frequently from animals with superficial lesions (39%) than from those with no macroscopic lesions (16%). Isolates from animals with superficial lesions were Fusarium solani species complex (FSSC) lineages haplotypes 9, 12, and 27 (unnamed lineages), FSSC-2 (Fusarium keratoplasticum), Fusarium oxysporum (27%), and Fusarium brachygibbosum (3%). In contrast, only F. solani haplotypes 9 and 12 were isolated from animals with no macroscopic lesions. The presence of lesions was identified as a risk factor for the occurrence of Fusarium spp. Of the 74 animals, only 7 (9.5%) scored positive on microscopic examination with Calcofluor, and histological examination of those 7 animals revealed necrosis, inflammatory cells, and fungal hyphae in the carapace and skin. The results of this study suggest that fusariosis should be included in the differential diagnosis of shell and skin lesions in sea turtles. Direct examination using Calcofluor and potassium hydroxide was not useful to diagnose the infection. Histopathological examination and fungal culture should be performed to ensure correct treatment and infection control.

Amblypygid-fungal interactions: The whip spider exoskeleton as a substrate for fungal growth.

Fungi and arthropods represent some of the most diverse organisms on our planet, yet the ecological relationships between them remain largely unknown. In animals, fungal growth on body surfaces is often hazardous and is known to cause mortality. In contrast, here we report the presence of an apparently non-harmful mycobiome on the cuticle of whip spiders (Arachnida: Amblypygi). The associations are not species-specific and involve a diversity of fungal species, including cosmopolitan and local decomposers as well as entomopathogens. We discuss the ecology of the detected fungal species and hypothesize that the thick epicuticular secretion coat of whip spiders (the cerotegument) promotes fungal growth. It is possible that this relationship is beneficial towards the host if it leads to parasite control or chemical camouflage. Our findings, which are the first from this arthropod lineage, indicate that non-pathogenic interactions between arthropods and fungi may be much more widespread than predicted and call for more studies in this area.

Monomorium ant is a carrier for pathogenic and potentially pathogenic bacteria.

OBJECTIVES: Household ants are regarded as a major household pest and their close association with microorganisms and people means that they may constitute a disease risk. Our study is the first to provide information on the pathogenicity of Monomorium spp. a common insect in Kuwait by quantifying and identifying the exoskeleton bacterial burden. Samples of Monomorium were collected in June from indoor and outdoor sites of 30 houses located in two residential districts. RESULTS: The study identified a total of 16 different species of Gram-negative bacteria of which the indoor isolates were 75% greater in species count than the outdoor samples. Indoor isolates identified from both districts were more frequent than the outdoors and similar trends were obtained for a single district. Outdoor ant samples on the other hand carried a high percentage of bacteria but with less diversity in both districts. There was a significant variability in bacterial species in relation to sample sources, indoor and outdoor, and discrete geographical location. The presence of a high percentage of pathogenic and potentially pathogenic bacteria indoor poses a great threat to domestic households, which would be further exacerbated in places with poor standards of hygiene.

Emydomyces testavorans, a New Genus and Species of Onygenalean Fungus Isolated from Shell Lesions of Freshwater Aquatic Turtles.

The fungal order Onygenales includes many pathogens of humans and animals, and recent studies have shown some onygenalean fungi to be significant emerging pathogens of reptiles. Although many of these fungi have similar morphological features in histologic tissue sections, recent molecular analyses have revealed a genetically complex and diverse group of reptile pathogens comprising several genera, most notably Nannizziopsis, Ophidiomyces, and Paranannizziopsis Infections by members of these genera have been previously reported in a variety of reptile species, including crocodilians, lizards, snakes, and tuataras, with negative impacts on conservation efforts for some reptiles. Despite the well-documented pathogenicity of these fungi in all other extant reptile lineages, infection has not yet been reported in aquatic turtles. In this study, we report the isolation of an onygenalean fungus associated with shell lesions in freshwater aquatic turtles. The morphologic and genetic characteristics of multiple isolates (n = 21) are described and illustrated. Based on these features and results of a multigene phylogenetic analysis, a new genus and species, Emydomyces testavorans, are proposed for these fungi isolated from turtle shell lesions.

Shell repair and the potential microbial causal in a shell disease of the pearl oyster Pinctada fucata.

The pearl oyster Pinctada fucata is famous for producing luxurious pearls. As filter feeders, they are confronted with various infectious microorganisms. Despite a long history of aquaculture, diseases in P. fucata are not well studied, which limits the development of the pearl industry. We report here a shell disease in P. fucata and a study of the shell repair processes. Scanning electron microscopy (SEM) revealed that the nacreous layer gradually recovered from disordered CaCO3 deposition, accompanied by a polymorphic transition from a calcite-aragonite mixture to an aragonite-dominant composition, as revealed by X-ray diffraction analysis. SEM also showed that numerous microbes were embedded in the abnormal shell layers. Similar indications were induced by a high concentration of microbes injected into the extrapallial space, suggesting the potential pathogenic effect of uncontrolled microbes. Furthermore, hemocytes were found to participate in pathogens resistance and might promote shell repair. These results further our understanding of pathogen-host interactions in pearl oysters and have implications for biotic control in pearl aquaculture.

Rising Temperatures, Molting Phenology, and Epizootic Shell Disease in the American Lobster.

Phenological mismatch-maladaptive changes in phenology resulting from altered timing of environmental cues-is an increasing concern in many ecological systems, yet its effects on disease are poorly characterized. American lobster (Homarus americanus) is declining at its southern geographic limit. Rising seawater temperatures are associated with seasonal outbreaks of epizootic shell disease (ESD), which peaks in prevalence in the fall. We used a 34-year mark-recapture data set to investigate relationships between temperature, molting phenology, and ESD in Long Island Sound, where temperatures are increasing at 0.4°C per decade. Our analyses support the hypothesis that phenological mismatch is linked to the epidemiology of ESD. Warming spring temperatures are correlated with earlier spring molting. Lobsters lose diseased cuticle by molting, and early molting increases the intermolt period in the summer, when disease prevalence is increasing to a fall peak. In juvenile and adult male lobsters, September ESD prevalence was correlated with early molting, while October ESD prevalence was correlated with summer seawater temperature. This suggests that temperature-induced molting phenology affects the timing of the onset of ESD, but later in the summer this signal is swamped by the stronger signal of summer temperatures, which we hypothesize are associated with an increased rate of new infections. October ESD prevalence was ∼80% in years with hot summers and ∼30% in years with cooler summers. Yearly survival of diseased lobsters is <50% that of healthy lobsters. Thus, population impacts of ESD are expected to increase with increasing seawater temperatures.

Early skeletal colonization of the coral holobiont by the microboring Ulvophyceae Ostreobium sp.

Ostreobium sp. (Bryopsidales, Ulvophyceae) is a major microboring alga involved in tropical reef dissolution, with a proposed symbiotic lifestyle in living corals. However, its diversity and colonization dynamics in host's early life stages remained unknown. Here, we mapped microborer distribution and abundance in skeletons of the branching coral Pocillopora damicornis from the onset of calcification in primary polyps (7 days) to budding juvenile colonies (1 and 3 months) growing on carbonate and non-carbonate substrates pre-colonized by natural biofilms, and compared them to adult colonies (in aquarium settings). Primary polyps were surprisingly already colonized by microboring filaments and their level of invasion depended on the nature of settlement substrate and the extent of its pre-colonization by microborers. Growth of early coral recruits was unaffected even when microborers were in close vicinity to the polyp tissue. In addition to morphotype observations, chloroplast-encoded rbcL gene sequence analyses revealed nine new Ostreobium clades (OTU99%) in Pocillopora coral. Recruits and adults shared one dominant rbcL clade, undetected in larvae, but also present in aquarium seawater, carbonate and non-carbonate settlement substrates, and in corals from reef settings. Our results show a substratum-dependent colonization by Ostreobium clades, and indicate horizontal transmission of Ostreobium-coral associations.

Antimicrobial peptide AMPNT-6 from Bacillus subtilis inhibits biofilm formation by Shewanella putrefaciens and disrupts its preformed biofilms on both abiotic and shrimp shell surfaces.

Shewanella putrefaciens biofilm formation is of great concern for the shrimp industry because it adheres easily to food and food-contact surfaces and is a source of persistent and unseen contamination that causes shrimp spoilage and economic losses to the shrimp industry. Different concentrations of an antimicrobial lipopeptide, the fermentation product of Bacillus subtilis, AMPNT-6, were tested for the ability to reduce adhesion and disrupt S. putrefaciens preformed biofilms on two different contact surfaces (shrimp shell, stainless steel sheet). AMPNT-6 displayed a marked dose- and time-dependent anti-adhesive effect>biofilm removal. 3MIC AMPNT-6 was able both to remove biofilm and prevent bacteria from forming biofilm in a 96-well polystyrene microplate used as the model surface. 2MIC AMPNT-6 prevented bacteria from adhering to the microplate surface to form biofilm for 3h and removed already existing biofilm within 24h. Secretion of extracellular polymeric substances incubated in LB broth for 24h by S. putrefaciens was minimal at 3× MIC AMPNT-6. Scanning electron microscopy showed that damage to S. putrefaciens bacteria by AMPNT-6 possibly contributed to the non-adherence to the surfaces. Disruption of the mature biofilm structure by AMPNT-6 contributed to biofilm removal. It is concluded that AMPNT-6 can be used effectively to prevent attachment and also detach S. putrefaciens biofilms from shrimp shells, stainless steel sheets and polystyrene surfaces.

Denitrification potential of the eastern oyster microbiome using a 16S rRNA gene based metabolic inference approach.

The eastern oyster (Crassostrea virginica) is a foundation species providing significant ecosystem services. However, the roles of oyster microbiomes have not been integrated into any of the services, particularly nitrogen removal through denitrification. We investigated the composition and denitrification potential of oyster microbiomes with an approach that combined 16S rRNA gene analysis, metabolic inference, qPCR of the nitrous oxide reductase gene (nosZ), and N2 flux measurements. Microbiomes of the oyster digestive gland, the oyster shell, and sediments adjacent to the oyster reef were examined based on next generation sequencing (NGS) of 16S rRNA gene amplicons. Denitrification potentials of the microbiomes were determined by metabolic inferences using a customized denitrification gene and genome database with the paprica (PAthway PRediction by phylogenetIC plAcement) bioinformatics pipeline. Denitrification genes examined included nitrite reductase (nirS and nirK) and nitrous oxide reductase (nosZ), which was further subdivided by genotype into clade I (nosZI) or clade II (nosZII). Continuous flow through experiments measuring N2 fluxes were conducted with the oysters, shells, and sediments to compare denitrification activities. Paprica properly classified the composition of microbiomes, showing similar classification results from Silva, Greengenes and RDP databases. Microbiomes of the oyster digestive glands and shells were quite different from each other and from the sediments. The relative abundance of denitrifying bacteria inferred by paprica was higher in oysters and shells than in sediments suggesting that oysters act as hotspots for denitrification in the marine environment. Similarly, the inferred nosZI gene abundances were also higher in the oyster and shell microbiomes than in the sediment microbiome. Gene abundances for nosZI were verified with qPCR of nosZI genes, which showed a significant positive correlation (F1,7 = 14.7, p = 6.0x10-3, R2 = 0.68). N2 flux rates were significantly higher in the oyster (364.4 ± 23.5 µmol N-N2 m-2 h-1) and oyster shell (355.3 ± 6.4 µmol N-N2 m-2 h-1) compared to the sediment (270.5 ± 20.1 µmol N-N2 m-2 h-1). Thus, bacteria carrying nosZI genes were found to be an important denitrifier, facilitating nitrogen removal in oyster reefs. In addition, this is the first study to validate the use of 16S gene based metabolic inference as a method for determining microbiome function, such as denitrification, by comparing inference results with qPCR gene quantification and rate measurements.

Isolation of proteolytic bacteria from mealworm (Tenebrio molitor) exoskeletons to produce chitinous material.

The use of insects as a source of protein is becoming an important factor for feeding an increasing population. After protein extraction for food use, the insect exoskeleton may offer the possibility for the production of added value products. Here, the aim was to isolate bacteria from the surface of farmed mealworms (Tenebrio molitor Linnaeus, 1758) for the production of chitinous material from insect exoskeletons using microbial fermentation. Isolates were screened for proteases and acid production that may aid deproteination and demineralisation of insects through fermentation to produce chitin. Selected isolates were used single-step (isolated bacteria only) or two-step fermentations with Lactobacillus plantarum (DSM 20174). Two-step fermentations with isolates from mealworm exoskeletons resulted in a demineralisation of 97.9 and 98.5% from deproteinated mealworm fractions. Attenuated total reflectance-Fourier- transform infrared spectroscopy analysis showed that crude chitin was produced. However, further optimisation is needed before the process can be upscaled. This is, to our knowledge, the first report using microbial fermentation for the extraction of chitin from insects.

What determines sclerobiont colonization on marine mollusk shells?

Empty mollusk shells may act as colonization surfaces for sclerobionts depending on the physical, chemical, and biological attributes of the shells. However, the main factors that can affect the establishment of an organism on hard substrates and the colonization patterns on modern and time-averaged shells remain unclear. Using experimental and field approaches, we compared sclerobiont (i.e., bacteria and invertebrate) colonization patterns on the exposed shells (internal and external sides) of three bivalve species (Anadara brasiliana, Mactra isabelleana, and Amarilladesma mactroides) with different external shell textures. In addition, we evaluated the influence of the host characteristics (mode of life, body size, color alteration, external and internal ornamentation and mineralogy) of sclerobionts on dead mollusk shells (bivalve and gastropod) collected from the Southern Brazilian coast. Finally, we compared field observations with experiments to evaluate how the biological signs of the present-day invertebrate settlements are preserved in molluscan death assemblages (incipient fossil record) in a subtropical shallow coastal setting. The results enhance our understanding of sclerobiont colonization over modern and paleoecology perspectives. The data suggest that sclerobiont settlement is enhanced by (i) high(er) biofilm bacteria density, which is more attracted to surfaces with high ornamentation; (ii) heterogeneous internal and external shell surface; (iii) shallow infaunal or attached epifaunal life modes; (iv) colorful or post-mortem oxidized shell surfaces; (v) shell size (<50 mm2 or >1,351 mm2); and (vi) calcitic mineralogy. Although the biofilm bacteria density, shell size, and texture are considered the most important factors, the effects of other covarying attributes should also be considered. We observed a similar pattern of sclerobiont colonization frequency over modern and paleoecology perspectives, with an increase of invertebrates occurring on textured bivalve shells. This study demonstrates how bacterial biofilms may influence sclerobiont colonization on biological hosts (mollusks), and shows how ecological relationships in marine organisms may be relevant for interpreting the fossil record of sclerobionts.