Oyster disease in a changing environment: Decrypting the link between pathogen, microbiome and environment

© 2018 Elsevier Ltd Shifting environmental conditions are known to be important triggers of oyster diseases. The mechanism(s) behind these synergistic effects (interplay between host, environment and pathogen/s) are often not clear, although there is evidence that shifts in environmental conditions can affect oyster immunity, and pathogen growth and virulence. However, the impact of shifting environmental parameters on the oyster microbiome and how this affects oyster health and susceptibility to infectious pathogens remains understudied. In this review, we summarise the major diseases afflicting oysters with a focus on the role of environmental factors that can catalyse or amplify disease outbreaks. We also consider the potential role of the oyster microbiome in buffering or augmenting oyster disease outbreaks and suggest that a deeper understanding of the oyster microbiome, its links to the environment and its effect on oyster health and disease susceptibility, is required to develop new frameworks for the prevention and management of oyster diseases

Simulated Marine Heat Wave Alters Abundance and Structure of Vibrio Populations Associated with the Pacific Oyster Resulting in a Mass Mortality Event

© 2018, Springer Science+Business Media, LLC, part of Springer Nature. Marine heat waves are predicted to become more frequent and intense due to anthropogenically induced climate change, which will impact global production of seafood. Links between rising seawater temperature and disease have been documented for many aquaculture species, including the Pacific oyster Crassostrea gigas. The oyster harbours a diverse microbial community that may act as a source of opportunistic pathogens during temperature stress. We rapidly raised the seawater temperature from 20 °C to 25 °C resulting in an oyster mortality rate of 77.4%. Under the same temperature conditions and with the addition of antibiotics, the mortality rate was only 4.3%, strongly indicating a role for bacteria in temperature-induced mortality. 16S rRNA amplicon sequencing revealed a change in the oyster microbiome when the temperature was increased to 25 °C, with a notable increase in the proportion of Vibrio sequences. This pattern was confirmed by qPCR, which revealed heat stress increased the abundance of Vibrio harveyi and Vibrio fortis by 324-fold and 10-fold, respectively. Our findings indicate that heat stress-induced mortality of C. gigas coincides with an increase in the abundance of putative bacterial pathogens in the oyster microbiome and highlights the negative consequences of marine heat waves on food production from aquaculture

Occurrence and dynamics of potentially pathogenic vibrios in the wet-dry tropics of northern Australia

Bacteria from the Vibrio genus are a ubiquitous component of coastal and estuarine ecosystems with several pathogenic Vibrio species displaying preferences for warm tropical waters. We studied the spatial and temporal abundance of three key human potential pathogens V. parahaemolyticus, V. cholerae and V. vulnificus in northern tropical Australia, over the wet and dry seasons, to identify environmental parameters influencing their abundance. Quantitative PCR (qPCR) analysis revealed that V. parahaemolyticus occurred more frequently and in higher abundance than V. cholerae and V. vulnificus across all locations examined. All three species were more abundant during the wet season, with V. parahaemolyticus abundance correlated to temperature and conductivity, whereas nutrient concentrations and turbidity best explained V. vulnificus abundance. In addition to these targeted qPCR analyses, we assessed the composition and dynamics of the entire Vibrio community using hsp60 amplicon sequencing. Using this approach, 42 Vibrio species were identified, including a number of other pathogenic species such as V. alginolyticus, V. mimicus and V. fluvialis. The Vibrio community was more diverse in the wet season, with temperature and dissolved oxygen as the key factors governing community composition. Seasonal differences were primarily driven by a greater abundance of V. parahaemolyticus and V. vulnificus during the wet season, while spatial differences were driven by different abundances of V. harveyi, V. campbellii, V. cholerae and V. navarrensis. When we related the abundance of Vibrio to other bacterial taxa, defined using 16S rRNA gene amplicon sequencing, V. parahaemolyticus was negatively correlated to several taxa, including members of the Rickettsiales and Saccharimonadales, while V. vulnificus was negatively correlated to Rhobacteriaceae and Cyanobiaceae. In contrast, V. alginolyticus, V. harveyi and V. mediterranei were all positively correlated to Cyanobacteria. These observations highlight the dynamic nature of Vibrio communities and expands current understanding of the processes governing the occurrence of potentially pathogenic Vibrio spp. in tropical coastal ecosystems

A New High Throughput Sequencing Assay for Characterizing the Diversity of Natural Vibrio Communities and Its Application to a Pacific Oyster Mortality Event

© Copyright © 2019 King, Siboni, Kahlke, Green, Labbate and Seymour. The Vibrio genus is notable for including several pathogens of marine animals and humans, yet characterization of Vibrio diversity using routine 16S rRNA sequencing methods is often constrained by poor resolution beyond the genus level. Here, a new high throughput sequencing approach targeting the heat shock protein (hsp60) as a phylogenetic marker was developed to more precisely discriminate members of the Vibrio genus in environmental samples. The utility of this new assay was tested using mock communities constructed from known dilutions of Vibrio isolates. Relative to standard and Vibrio-specific 16S rRNA sequencing assays, the hsp60 assay delivered high levels of fidelity with the mock community composition at the species level, including discrimination of species within the Vibrio harveyi clade. This assay was subsequently applied to characterize Vibrio community composition in seawater and delivered substantially improved taxonomic resolution of Vibrio species compared to 16S rRNA analysis. Finally, this assay was applied to examine patterns in the Vibrio community within oysters during a Pacific oyster mortality event. In these oysters, the hsp60 assay identified species-level Vibrio community shifts prior to disease onset, pinpointing V. harveyi as a putative pathogen. Given that shifts in the Vibrio community can precede, cause, and follow disease onset in numerous marine organisms, there is a need for an accurate high throughput assay for defining Vibrio community composition in natural samples. This Vibrio-centric hsp60 sequencing assay offers the potential for precise high throughput characterization of Vibrio diversity, providing an enhanced platform for dissecting Vibrio dynamics in the environment

Pearl Oyster Bacterial Community Structure Is Governed by Location and Tissue-Type, but Vibrio Species Are Shared Among Oyster Tissues.

Diseases of bivalves of aquacultural importance, including the valuable Australian silver-lipped pearl oyster (Pinctada maxima), have been increasing in frequency and severity. The bivalve microbiome is linked to health and disease dynamics, particularly in oysters, with putative pathogens within the Vibrio genus commonly implicated in oyster diseases. Previous studies have been biased toward the Pacific oyster because of its global dominance in oyster aquaculture, while much less is known about the microbiome of P. maxima. We sought to address this knowledge gap by characterizing the P. maxima bacterial community, and we hypothesized that bacterial community composition, and specifically the occurrence of Vibrio, will vary according to the sampled microenvironment. We also predicted that the inside shell swab bacterial composition could represent a source of microbial spillover biofilm into the solid pearl oyster tissues, thus providing a useful predictive sampling environment. We found that there was significant heterogeneity in bacterial composition between different pearl oyster tissues, which is consistent with patterns reported in other bivalve species and supports the hypothesis that each tissue type represents a unique microenvironment for bacterial colonization. We suggest that, based on the strong effect of tissue-type on the pearl oyster bacterial community, future studies should apply caution when attempting to compare microbial patterns from different locations, and when searching for disease agents. The lack of association with water at each farm also supported the unique nature of the microbial communities in oyster tissues. In contrast to the whole bacterial community, there was no significant difference in the Vibrio community among tissue types nor location. These results suggest that Vibrio species are shared among different pearl oyster tissues. In particular, the similarity between the haemolymph, inside shell and solid tissues, suggests that the haemolymph and inside shell environment is a source of microbial spillover into the oyster tissues, and a potentially useful tool for non-destructive routine disease testing and early warning surveillance. These data provide important foundational information for future studies identifying the factors that drive microbial assembly in a valuable aquaculture species

Parameterized Directed kk-Chinese Postman Problem and kk Arc-Disjoint Cycles Problem on Euler Digraphs

In the Directed $k$-Chinese Postman Problem ($k$-DCPP), we are given a connected weighted digraph $G$ and asked to find $k$ non-empty closed directed walks covering all arcs of $G$ such that the total weight of the walks is minimum. Gutin, Muciaccia and Yeo (Theor. Comput. Sci. 513 (2013) 124--128) asked for the parameterized complexity of $k$-DCPP when $k$ is the parameter. We prove that the $k$-DCPP is fixed-parameter tractable. We also consider a related problem of finding $k$ arc-disjoint directed cycles in an Euler digraph, parameterized by $k$. Slivkins (ESA 2003) showed that this problem is W[1]-hard for general digraphs. Generalizing another result by Slivkins, we prove that the problem is fixed-parameter tractable for Euler digraphs. The corresponding problem on vertex-disjoint cycles in Euler digraphs remains W[1]-hard even for Euler digraphs

Regional and oyster microenvironmental scale heterogeneity in the Pacific oyster bacterial community.

Different organs of a host represent distinct microenvironments resulting in the establishment of multiple discrete bacterial communities within a host. These discrete bacterial communities can also vary according to geographical location. For the Pacific oyster, Crassostrea gigas, the factors governing bacterial diversity and abundance of different oyster microenvironments are poorly understood. In this study, the factors shaping bacterial abundance, diversity and composition associated with the C. gigas mantle, gill, adductor muscle, and digestive gland were characterised using 16S (V3-V4) rRNA amplicon sequencing across six discrete estuaries. Both location and tissue-type, with tissue-type being the stronger determinant, were factors driving bacterial community composition. Bacterial communities from wave-dominated estuaries had similar compositions and higher bacterial abundance despite being geographically distant from one another, possibly indicating that functional estuarine morphology characteristics are a factor shaping the oyster bacterial community. Despite the bacterial community heterogeneity, examinations of the core bacterial community identified Spirochaetaceae bacteria as conserved across all sites and samples. Whereas members of the Vulcaniibacterium, Spirochaetaceae and Margulisbacteria, and Polynucleobacter were regionally conserved members of the digestive gland, gill, and mantle bacterial communities respectively. This indicates that baseline bacterial community profiles for specific locations are necessary when investigating bacterial communities in oyster health

Convergence and divergence in the evolution of cat skulls: temporal and spatial patterns of morphological diversity

Background: Studies of biological shape evolution are greatly enhanced when framed in a phylogenetic perspective. Inclusion of fossils amplifies the scope of macroevolutionary research, offers a deep-time perspective on tempo and mode of radiations, and elucidates life-trait changes. We explore the evolution of skull shape in felids (cats) through morphometric analyses of linear variables, phylogenetic comparative methods, and a new cladistic study of saber-toothed cats. Methodology/Principal Findings: A new phylogenetic analysis supports the monophyly of saber-toothed cats (Machairodontinae) exclusive of Felinae and some basal felids, but does not support the monophyly of various sabertoothed tribes and genera. We quantified skull shape variation in 34 extant and 18 extinct species using size-adjusted linear variables. These distinguish taxonomic group membership with high accuracy. Patterns of morphospace occupation are consistent with previous analyses, for example, in showing a size gradient along the primary axis of shape variation and a separation between large and small-medium cats. By combining the new phylogeny with a molecular tree of extant Felinae, we built a chronophylomorphospace (a phylogeny superimposed onto a two-dimensional morphospace through time). The evolutionary history of cats was characterized by two major episodes of morphological divergence, one marking the separation between saber-toothed and modern cats, the other marking the split between large and small-medium cats. Conclusions/Significance: Ancestors of large cats in the ‘Panthera’ lineage tend to occupy, at a much later stage, morphospace regions previously occupied by saber-toothed cats. The latter radiated out into new morphospace regions peripheral to those of extant large cats. The separation between large and small-medium cats was marked by considerable morphologically divergent trajectories early in feline evolution. A chronophylomorphospace has wider applications in reconstructing temporal transitions across two-dimensional trait spaces, can be used in ecophenotypical and functional diversity studies, and may reveal novel patterns of morphospace occupation