The microbiome influences a variety of host-environment interactions, and there is mounting evidence of its significant role in biological invasions. During invasion, shifts in microbial diversity and function can occur due to both changing characteristics of the novel environment and physiological condition of the host. The signal crayfish (Pacifastacus leniusculus) is one of the most successful crayfish invaders in Europe. During range expansion, its populations often exhibit differences in many traits along the invasion range, including sex-composition, size-structure and aggressiveness, but to date it was not studied whether crayfish traits can also drive changes in the host microbiome. Thus, we used 16S rRNA gene amplicon sequencing to examine the effects of host-related traits, namely total length (TL), body condition index (FCF), hepatosomatic index (HSI) and sex on the microbial diversity of the signal crayfish. We examined both external (exoskeletal) and internal (intestinal, hepatopancreatic, hemolymph) microbiomes of 110 signal crayfish individuals from four sites along its invasion range in the Korana River, Croatia. While sex did not exhibit a significant effect on the microbial diversity in any of the examined tissues, exoskeletal, intestinal and hemolymph microbial diversity significantly decreased with increasing crayfish size. Additionally, significant effects of signal crayfish condition (FCF, HSI) on microbial diversity were recorded in the hepatopancreas, a main energy storage organ in crayfish that supports reproduction and growth and also regulates immune response. Our findings provide a baseline for evaluating the contribution of microbiome to an invader's overall health, fitness and subsequent invasion success.

Abstract The activity of the microbiome of fish mucosae provides functions related to immune response, digestion, or metabolism. Several biotic and abiotic factors help maintaining microbial homeostasis, with disruptions leading to dysbiosis. Diseases and antibiotic administration are known to cause dysbiosis in farmed fish. Pathogen infections greatly affect the production of gilthead seabream, and antibiotic treatment is still frequently required. Here, we employed a 16S rRNA high-throughput metataxonomics approach to characterize changes in the gut, skin, and gill microbiomes occurring due to infection with Photobacterium damselae subsp. piscicida and subsequent antibiotic treatment with oxytetracycline (OTC), as well as during recovery. Although microbiota response differed between studied tissues, overall changes in composition, diversity, structure, and predicted function were observed in all mucosae. The skin and gill microbiomes of diseased fish became largely dominated by taxa that have been frequently linked to secondary infections, whereas in the gut the genus Vibrio, known to include pathogenic bacteria, increased with OTC treatment. The study highlights the negative impacts of disease and antibiotic treatment on the microbiome of farmed fish. Our results also suggest that fish transportation operations may have profound effects on the fish microbiome, but further studies are needed to accurately evaluate their impact.

Winemaking is a well-known process comprising several steps to produce must and wine. Grape marc is a byproduct of wine production that can be vermicomposted and used as organic fertiliser. Grape marc vermicompost has a richer and more stable microbiome than grape marc alone and when added to the soil of vineyards it can improve grape production and wine quality. We compared Albariño must and wine microbiotas from grapevines treated with vermicompost derived from Albariño grape marc and controls (standard fertilisation). We hypothesised that observed microbial changes are connected to improved organoleptic properties observed in fertilised must and wine. Treated Albariño vines showed increased grape production and the final wine showed improved organoleptic properties. Metataxonomic analyses of the 16S rRNA and ITS gene regions showed that the Albariño must and wine microbiome varied in their taxonomic composition. Must bacteriotas showed no significant (p < 0.05) variation in alpha or beta-diversity, while wine bacteriotas and must and wine mycobiotas showed significant differences in richness and evenness, as well as in microbial structure (beta-diversity) between treated and control grapevines. Must and wine bacteriotas also showed significant (p < 0.05) changes in their predicted metabolic pathways. Our study suggests that changes in the abundance of specific bacterial and fungal taxa and the metabolic processes they carry out during Albariño winemaking can improve the productivity of the grapevine and the organoleptic properties of the wine.

Winemaking is a well-known process that includes several steps in the production of grape must and wine. Grape marc, or bagasse, is a byproduct of wine production that can be vermicomposted and used as organic fertilizer. Grape marc vermicompost has microbial communities that are richer and more stable than grape marc alone, and its addition to a vineyard’s terroir can improve grape yields and wine quality. Here we compare the must and wine microbiota of Mencía from grapevines treated with and without (standard fertilization) vermicompost derived from Mencía grape marc. Mencía is a high-quality red wine broadly grown in Galicia, Spain, and is appreciated for its fresh acidity and fruity flavors. When Mencía grapevines are treated with vermicompost derived from its grape marc, Mencía vines increase their grape production, and the final wine improves its organoleptic properties. Metataxonomic analyses of the bacterial 16S rRNA and fungal ITS gene regions showed that Mencía must and wine have the distinct taxonomic composition (phyla, genera and ASVs—amplicon sequence variants) of bacterial and fungal groups. Must and wine bacteriotas and mycobiotas show no significant variation in alpha-diversity, while wine bacteriotas and mycobiotas show significant differences in microbial structure (beta-diversity) between treated and control grapevines. Likewise, the functional diversity and predicted metabolic pathways (biosynthesis, degradation/utilization/assimilation, generation of precursor metabolites and energy, macromolecule modification and superpathways) of the must and wine microbiota also show significant changes. Our study proposes that changes in the abundance of microbial taxa and the metabolic processes they undergo during winemaking may improve Mencía’s organoleptic properties and productivity.

Vermicomposting is the process of organic waste degradation through interactions between earthworms and microbes. A variety of organic wastes can be vermicomposted, producing a nutrient-rich final product that can be used as a soil biofertilizer. Giving the prolific invasive nature of the Australian silver wattle Acacia dealbata Link in Europe, it is important to find alternatives for its sustainable use. However, optimization of vermicomposting needs further comprehension of the fundamental microbial processes. Here, we characterized bacterial succession during the vermicomposting of silver wattle during 56 days using the earthworm species Eisenia andrei. We observed significant differences in α- and β-diversity between fresh silver wattle (day 0) and days 14 and 28, while the bacterial community seemed more stable between days 28 and 56. Accordingly, during the first 28 days, a higher number of taxa experienced significant changes in relative abundance. A microbiome core composed of 10 amplicon sequence variants was identified during the vermicomposting of silver wattle (days 14 to 56). Finally, predicted functional profiles of genes involved in cellulose metabolism, nitrification, and salicylic acid also changed significantly during vermicomposting. This study, hence, provides detailed insights of the bacterial succession occurring during vermicomposting of the silver wattle and the characteristics of its final product as a sustainable plant biofertilizer.

The microbiota of fish skin is the primary barrier against disease; however, it is highly dynamic being modulated by several factors. In fish aquaculture, disease outbreaks occur mainly during early-life stages with high associated losses. Antibiotic treatments sometimes remain as the best option to control bacterial diseases, despite many reported negative impacts of its use on fish and associated microbiota. Notwithstanding, studies monitoring the effects of disease and antibiotic treatment on the microbiota of fingerlings are scarce. We used a 16S rRNA metataxonomic approach to assess the impact of a mixed infection with Photobacterium damselae ssp. piscicida and Vibrio harveyi and subsequent antibiotic treatment with flumequine, on the skin microbiota of farmed seabass Dicentrarchus labrax fingerlings. Both disease and antibiotic treatment led to a significant increase in bacterial diversity and core microbial communities and impacted microbial structure. Dysbiosis was confirmed by changes in the abundance of potential pathogenic and opportunistic bacterial taxa. Skin bacterial metabolic function was also significantly affected by flumequine administration, suggesting a detriment to fish skin health. Our results add to an increasing body of literature, showing how fish microbiome response to disease and antibiotics is not easily predicted.

Background: Important changes in microbial composition related to sexual maturation have been already reported in the gut of several vertebrates including mammals, amphibians and fish. Such changes in fish are linked to reproduction and growth during developmental stages, diet transitions and critical life events. We used amplicon (16S rRNA) high-throughput sequencing to characterize the skin and gill bacterial microbiota of farmed seabass and seabream belonging to three different developmental age groups: early and late juveniles and mature adults. We also assessed the impact of the surrounding estuarine water microbiota in shaping the fish skin and gill microbiota. Results: Microbial diversity, composition and predicted metabolic functions varied across fish maturity stages. Alpha-diversity in the seabass microbiota varied significantly between age groups and was higher in older fish. Conversely, in the seabream, no significant differences were found in alpha-diversity between age groups. Microbial structure varied significantly across age groups; moreover, high structural variation was also observed within groups. Different bacterial metabolic pathways were predicted to be enriched in the microbiota of both species. Finally, we found that the water microbiota was significantly distinct from the fish microbiota across all the studied age groups, although a high percentage of ASVs was shared with the skin and gill microbiotas. Conclusions: We report important microbial differences in composition and potential functionality across different ages of farmed seabass and seabream. These differences may be related to somatic growth and the onset of sexual maturation. Importantly, some of the inferred metabolic pathways could enhance the fish coping mechanisms during stressful conditions. Our results provide new evidence suggesting that growth and sexual maturation have an important role in shaping the microbiota of the fish external mucosae and highlight the importance of considering different life stages in microbiota studies.

The microbiota of fish skin, the primary barrier against disease, is highly dynamic and modulated by several factors. In fish aquaculture, disease outbreaks occur mainly during early-life stages, with associated high economic losses. Antibiotic treatments sometimes remain the best option to control bacterial diseases, despite many reported negative impacts of its use on fish and associated microbiota. Notwithstanding, studies monitoring the effects of disease and antibiotic treatment on the microbiota of fingerlings are scarce. We sequenced the bacterial 16S rRNA V4 gene region using a metabarcoding approach to assess the impact of a mixed infection with Photobacterium damselae ssp. piscicida and Vibrio harveyi and subsequent antibiotic treatment with flumequine, on the skin microbiota of farmed seabass (Dicentrarchus labrax) fingerlings. Both infection and antibiotic treatment led to a significant increase in bacterial diversity and core microbial communities and impacted microbiome structure. Dysbiosis was confirmed by changes in the abundance of potential pathogenic and opportunistic bacterial taxa. Skin bacterial metabolic function was also significantly affected by flumequine administration, suggesting a detriment to fish skin health. Our results add to an increasing body of literature, showing how fish microbiome response to infection and antibiotics cannot be easily predicted.

Fish microbiota are intrinsically linked to health and fitness, but they are highly variable and influenced by both biotic and abiotic factors. Water temperature particularly limits bacterial adhesion and growth, impacting microbial diversity and bacterial infections on the skin and gills. Aquaculture is heavily affected by infectious diseases, especially in warmer months, and industry practices often promote stress and microbial dysbiosis, leading to an increased abundance of potentially pathogenic bacteria. In this regard, fish mucosa health is extremely important because it provides a primary barrier against pathogens. We used 16 rRNA V4 metataxonomics to characterize the skin and gill microbiota of the European seabass, Dicentrarchus labrax, and the surrounding water over 12 months, assessing the impact of water temperature on microbial diversity and function. We show that the microbiota of external mucosae are highly dynamic with consistent longitudinal trends in taxon diversity. Several potentially pathogenic genera (Aliivibrio, Photobacterium, Pseudomonas, and Vibrio) were highly abundant, showing complex interactions with other bacterial genera, some of which with recognized probiotic activity, and were also significantly impacted by changes in temperature. The surrounding water temperature influenced fish microbial composition, structure and function over time (days and months). Additionally, dysbiosis was more frequent in warmer months and during transitions between cold/warm months. We also detected a strong seasonal effect in the fish microbiota, which is likely to result from the compound action of several unmeasured environmental factors (e.g., pH, nutrient availability) beyond temperature. Our results highlight the importance of performing longitudinal studies to assess the impact of environmental factors on fish microbiotas.

Damselfishes of the genus Stegastes are some of the most abundant fish inhabiting shallow reef habitats. Although Stegastes play an important role in promoting primary production in reef ecosystems, their territories are known to favor the occurrence of bacterial pathogens. However, no studies so far have examined the microbiome composition of Caribbean Stegastes species. Here, we characterized the skin bacterial microbiome of juveniles and adults of Stegastes leucostictus and Stegastes adustus collected from La Parguera, Puerto Rico, and St. Thomas, US Virgin Islands. Our results showed that bacterial communities residing on the skin of Stegastes were related to taxonomy, environment and ontogeny. These results are in line with previous knowledge on the dynamics of fish skin microbiomes.

Abstract Background Important changes in microbiome composition related to sexual maturation have been already reported in the gut of several vertebrates including mammals, amphibians and fish. Such changes in fish are linked to reproduction and growth during developmental stages, diet transitions and critical life events. We used amplicon (16S rRNA) high-throughput sequencing to characterize the skin and gill bacterial microbiomes of farmed seabass and seabream belonging to three different developmental age groups: early and late juveniles and mature adults. We also assessed the impact of the surrounding estuarine water microbiome in shaping the fish skin and gill microbiomes.Results Microbiome diversity, composition and potential metabolic functions varied across fish maturity stages. Alpha-diversity in the seabass microbiome varied significantly between age groups and was higher in older fish. Conversely, in the seabream, no significant differences were found in alpha diversity between age groups, although it was higher in the skin of juveniles. Microbiome structure varied significantly across age groups. Different bacterial metabolic pathways were predicted to be enriched in the microbiomes of both species. Finally, we found that the water microbiome is significantly distinct from all the fish microbiomes across the studied age groups, although a high percentage of ASVs is shared with the skin and gill microbiomes.Conclusions We report important microbial differences in composition and potential functionality across the different ages of farmed seabass and seabream. These differences may be related to somatic growth and the onset of sexual maturation. Importantly, some of the inferred metabolic pathways could enhance the host coping mechanisms during stressful conditions. Our results provide new evidence suggesting that growth and sexual maturation have an important role in shaping the external mucosa microbiomes of fish and highlight the importance of considering different life stages in microbiome studies.

The broadstripe cleaning goby Elacatinus prochilos has two alternative ecotypes: sponge-dwellers, which live in large groups and feed mainly upon nematode parasites; and coral-dwellers, that live in small groups or in solitude and behave as cleaners. Recent studies focusing on the skin and gut microbiomes of tropical fish showed that microbial communities are influenced mainly by diet and host species. Here, we compare the skin and gut microbiomes of the Caribbean broadstripe cleaning goby E. prochilos alternative ecotypes (cleaners and non-cleaners) from Barbados and predict that different habitat use and behavior (cleaning vs. non-cleaning) will translate in different bacterial profiles between the two ecotypes. We found significant differences in both alpha- and beta-diversity of skin and gut microbiomes belonging to different ecotypes. Importantly, the skin microbiome of obligate cleaners showed greater intra-sample diversity and harbored a significantly higher prevalence of potential fish pathogens. Likewise, potential pathogens were also more prevalent in the gut of obligate cleaners. We suggest that habitat use, diet, but also direct contact with potential diseased clientele during cleaning, could be the cause for these patterns.

The mucosal surfaces of fish harbour microbial communities that can act as the first-line of defense against pathogens. Infectious diseases are one of the main constraints to aquaculture growth leading to huge economic losses. Despite their negative impacts on microbial diversity and overall fish health, antibiotics are still the method of choice to treat many such diseases. Here, we use 16 rRNA V4 metataxonomics to study over a 6 week period the dynamics of the gill and skin microbiomes of farmed seabass before, during and after a natural disease outbreak and subsequent antibiotic treatment with oxytetracycline. Photobacterium damselae was identified as the most probable causative agent of disease. Both infection and antibiotic treatment caused significant, although asymmetrical, changes in the microbiome composition of the gills and skin. The most dramatic changes in microbial taxonomic abundance occurred between healthy and diseased fish. Disease led to a decrease in the bacterial core diversity in the skin, whereas in the gills there was both an increase and a shift in core diversity. Oxytetracycline caused a decrease in core diversity in the gill and an increase in the skin. Severe loss of core diversity in fish mucosae demonstrates the disruptive impact of disease and antibiotic treatment on the microbial communities of healthy fish.

Background: The Apicomplexa from aquatic environments are understudied relative to their terrestrial counterparts, and the seminal work assessing the phylogenetic relations of fish-infecting lineages is mostly based on freshwater hosts. The taxonomic uncertainty of some apicomplexan groups, such as the coccidia, is high and many genera were recently shown to be paraphyletic, questioning the value of strict morphological and ecological traits for parasite classification. Here, we surveyed the genetic diversity of the Apicomplexa in several commercially valuable vertebrates from the North-East Atlantic, including farmed fish. Results: Most of the sequences retrieved were closely related to common fish coccidia of Eimeria, Goussia and Calyptospora. However, some lineages from the shark Scyliorhinus canicula were placed as sister taxa to the Isospora, Caryospora and Schellakia group. Additionally, others from Pagrus caeruleostictus and Solea senegalensis belonged to an unknown apicomplexan group previously found in the Caribbean Sea, where it was sequenced from the water column, corals, and fish. Four distinct parasite lineages were found infecting farmed Dicentrarchus labrax or Sparus aurata. One of the lineages from farmed D. labrax was also found infecting wild counterparts, and another was also recovered from farmed S. aurata and farm-associated Diplodus sargus. Conclusions: Our results show that marine fish apicomplexans are diverse, and we highlight the need for a more extensive assessment of parasite diversity in this phylum. Additionally, parasites recovered from S. canicula were recovered as basal to their piscine counterparts reflecting hosts phylogeny.

This study summarizes the data of multiple surveys carried out at the Isalo Massif (southwestern Madagascar), both within and outside the National Park limits between 2004 and 2014, providing an updated list of the amphibians and non-avian reptiles present within the area. We used an integrative taxonomic approach, combining morphological and molecular data sets to identify collected samples as either described species, or as undescribed candidate species. We here report on one new amphibian candidate species (Mantidactylus sp. aff. multiplicatus Ca65 "Isalo"), and two new amphibian distribution records (Mantella sp. aff. expectata "South", Scaphiophryne sp. aff. calcarata Cao(2)) compared to the former inventories of the area. Additionally, we provide molecular reference sequences for 23 of the 24 amphibian taxa and for 40 of the 47 reptile taxa known to occur in the area, combining all available sources. With this study, we validate Isalo as an important centre of biodiversity and micro-endemism within Madagascar, with four described endemic amphibian (Gephyromantis azzurrae, Mantella expectata, Mantidactylus noralottae, and Scaphiophryne gottlebei) and one reptile species (Tmchylepis nancycoutuae). Of the taxa here listed for Isalo, seven amphibians and six reptiles are new candidate species, and among them at least one amphibian (Mantidactylus sp. aff. multiplicatus Ca65 "Isalo") and three reptiles (Lygodactylus sp. all tuberosus Caoz "Isalo", Paroedura sp. aff. bastardi Caoz "[sale' and P sp. aff. bastardi Cao(3) "Ilakaka") are currently known only from Isalo. The unique biodiversity of the Isalo Massif was likely favoured by the confluence of different biomes in the area and/or its role as a refugium during palaeoclimatic oscillations. Our study highlights the crucial importance of applying molecular taxonomic identification in updating the species lists even of previously well surveyed areas, and of maintaining and increasing museum voucher collections as a permanent and accessible testimony of the biodiversity inventory.

There is substantial evidence showing that the microbiome of teleosts plays a key role in host health and wellbeing. Aquaculture practices increase the risk of dysbiosis (i.e. microbial imbalance), which is known to facilitate pathogen infections. The skin and gills are the primary defense organs against pathogens, thus, characterizing their microbiome composition in farmed fish is pivotal for detecting potential alterations that may lead to disease susceptibility. Here, we assessed the skin and gill microbiomes of two of the most important adult fish species farmed in southern Europe, the seabass and the seabream, during winter months. We coupled next-generation sequencing (MiSeq) of the 16S rRNA V4 region with the DADA2 bioinformatic pipeline to assess microbial composition and structure. Variation in microbial alpha-diversity (intra-sample) and taxa proportions were assessed using analysis of variance. Differences in beta-diversity (between-sample) were tested using permutational multivariate analysis of variance. Microbiomes of both tissues (n = 30 per species) identified 19 bacteria phyla, dominated by the phyla Proteobacteria (44-68%) and Bacteroidetes (15-37%); the families Flavobacteriaceae (11-28%), Rhodobacteraeae (4-8%) and Vibrionaceae (2-17%); and the genera Rubritalea (4-13%), Pseudomonas (4-8%) and the NS3a marine group (4-12%). Mean relative proportion of these taxa, some alpha-diversity indices and all beta-diversity distances varied significantly between tissues within and between species. ASVs belonging to the genera Polaribacter and Vibrio, which include several species that are pathogenic, were detected in the core microbiomes of seabass or seabream.

The genus Quedenfeldtia is composed of two species, Q. moerens and Q. trachyblepharus, both endemic to the AtlasMountains region of Morocco. Previous studies recovered two main genetic lineages within each Quedenfeldtia species, although sampling did not cover a substantial portion of their known distribution. In this study we collected individuals from previously unsampled localities of Quedenfeldtia and carried out genetic analyses in order to assess the range of previously identified lineages and the occurrence of additional lineages. Phylogenetic reconstruction based on both mitochondrial (12S and ND4 + tRNA) and nuclear (MC1R) markers revealed that while the new individuals of Q. moerens belong to previously described lineages, two new lineages of Q. trachyblepharus were uncovered from the northern and southern parts of the range. Genetic divergence of these new lineages (8-9% ND4 + tRNA p-distance) was higher than values observed between other lizard sister species. In the future a thorough morphological assessment is needed to complement this study and allow a taxonomic revision of these taxa. The results of this study highlight the importance of biodiversity assessments in mountainous regions characterized by high endemicity but which are difficult to access.

Studies of biodiversity in the Maghreb have revealed high genetic diversity and divergent genetic lineages among many taxa including squamates. Geographic barriers such as the Atlas Mountains are one of the key factors promoting genetic differentiation and the high levels of endemism. The lizard-fingered gecko Saurodactylus brosseti is endemic to Morocco. Its range includes both sides of the Atlas Mountains, and although high diversity was previously identified within the species, much of the range was unsampled. To understand the evolutionary and biogeographical history of this species, we used mitochondrial and nuclear DNA sequence data from 64 populations sampled across the entire species range. We employed phylogenetic methods based on gene trees and species trees as well as a time calibrated Bayesian genealogy and coalescent species delimitation approaches. We uncovered four highly divergent and allopatric mitochondrial lineages that did not share haplotypes at variable nuclear loci, suggesting the four groups have been evolving independently since the Miocene, according to our molecular dating estimates. These results coupled with the geographic pattern of genetic diversity suggest a possible role of the Atlas Mountains for the divergence observed between the four lineages of S. brosseti , while each lineage probably later underwent several episodes of fragmentation followed by (re-) expansion during Pleistocene climatic oscillations. Bayesian species delimitation analysis indicates that the four lineages may well be distinct species but we suggest that detailed morphological analyses are needed prior to taxonomic changes. The four lineages represent ancient independent evolutionary units, and deserve conservation management as distinct entities.

Seafood mislabeling is a widespread problem even in Europe where the food market is highly regulated. In the present study, we analyzed the accuracy of seafood labeling in five Portuguese supermarket chains through DNA barcode. Our results show that approximately 19% of examined samples were mislabeled and that the consumer's trust is misplaced. As mislabeling was greater in invertebrate species, we recommend that future studies led by government agencies and scientists aiming to test labeling accuracy should expand from finfish to include invertebrates such as crustaceans and bivalves.