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Keywords:

  • Bathymodiolus;
  • black yeast;
  • Capronia;
  • Chaetothyriales;
  • Fiji Basin;
  • Herpotrichiellaceae;
  • marine diseases

Abstract

  1. Top of page
  2. Abstract
  3. Problem
  4. Material and Methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgements
  9. References

Mass mortalities due to disease are important determinants of population and community structure in marine ecosystems, but the speed at which an epizootic may sweep through a population, combined with rapid selection for disease-resistant stocks, can mask the ecological impact of disease in all but the most closely monitored populations. We document an emergent epizootic event in the deep sea that is occurring in mussels (Bathymodiolus brevior) at the Mussel Hill hydrothermal vent in Fiji Basin and we identify the causal agent as a black yeast (order Chaetothyriales) that elicits a pronounced host immune response and is associated with tissue deterioration. The yeast was not observed in other invertebrate taxa (the gastropods Ifremeria nautilei, Alviniconcha aff. hessleri; the limpets Lepetodrilus schrolli, Symmetromphalus aff. hageni; the polychaetes Branchipolynoe pettiboneae, Amphisamytha cf. galapagensis) associated with the mussel bed, nor in mussels (Bathymodiolus brevior) collected from adjacent Lau Basin mussel beds. Massive mussel mortality resulting from the fungal infection is anticipated at the Mussel Hill site in Fiji Basin; we expect that epizootic outbreaks in dense invertebrate communities have the potential to be major determinants of community structure in deep-sea chemosynthetic ecosystems. The possibility that submersible assets may serve as vectors for transport of the fungus warrants further attention.


Problem

  1. Top of page
  2. Abstract
  3. Problem
  4. Material and Methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgements
  9. References

Due to the generally low biomass of organisms in the deep sea, the relative inaccessibility of deep-sea environments, and the lack of economically valued species, disease agents and the ecological impact of diseases in deep-sea ecosystems are more poorly studied than they are in coastal systems. Even at deep-sea hydrothermal vents and cold seeps, where dense communities of metazoan organisms have been the focus of a large number of research expeditions in recent years, pathogens are rarely considered as causes of mortality or important factors in determining community structure, although there are recent exceptions (Powell et al. 1999; Terlizzi et al. 2004; Ward et al. 2004; Mills et al. 2005). In shallow-water ecosystems, climate variability and human activities (including transport of pathogens and habitat degradation) have been implicated in disease outbreaks (Harvell et al. 1999; Lafferty et al. 2004). There is little reason to suspect that these factors are operating in deep-sea systems at present, although inoculation of naïve populations with submersible-transmitted pathogens is not impossible. We report the discovery of a large number of diseased mussels at a deep-sea hydrothermal vent in Fiji Basin and we present the histological, ultrastructural, and molecular phylogenetic characterization of a fungus associated with tissue pathology and a strong molluscan immune response.

Material and Methods

  1. Top of page
  2. Abstract
  3. Problem
  4. Material and Methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgements
  9. References

Mussels (Bathymodiolus brevior) were collected using the Remotely Operated Vehicle Jason II during multiple lowerings at the Mussel Hill hydrothermal vent field (16°59.4′ S, 173°54.9′ E, 1990 m; Fig. 1), Fiji Basin, in May 2005. Mussels were also collected from a separate but nearby (within 70 m of Mussel Hill) mussel bed at approximately the same depth, and from mussel beds in the adjacent Lau Basin [mussels pooled from Kilo Moana (20°3.0′ S, 176°7.8′ W, 2620 m), ABE (20°45.60′ S, 176°11.40′ W, 2145 m), and Tu'i Malila (21°59.4′ S, 176°34.20 W, 1885 m) mussel beds].

image

Figure 1.  Fiji Basin and Lau Basin collection sites in the southwestern Pacific. MH (Mussel Hill), KM (Kilo Moana), ABE, and TM (Tu'i Malila) are names of active hydrothermal fields. NZ, New Zealand.

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Mussel tissues from subsamples of Fiji Basin (26 of 57 individuals from a single Mussel Hill sample, 13 individuals from White Lady) and Lau Basin (26 individuals collected as samples of opportunity and not labeled by the specific site) were processed for histological studies (fixation in 10% buffered formalin; storage in 70% ethanol); additional Fiji mussel tissue was processed for transmission electron microscopy [TEM; 2% glutaraldehyde in 0.1 m phosphate buffer (pH 7.4) with 0.25 m sucrose, followed by a post-fix in buffered 1% osmium tetroxide (OsO4) and storage in 70% ethanol], and molecular investigations (95% ethanol). To determine whether the fungus was specific to mussels, we also sectioned and examined tissues of two large symbiont mollusk species living adjacent to or within the mussel bed [the hairy gastropod Alviniconcha aff. hessleri (n = 1 individual) and the black snail, Ifremeria nautilei (n = 10 individuals)] and tissues of four macrofaunal invertebrate species (n =10 individuals per species) living in association with the mussels [polynoid scale worms that live in the mantle cavity of the mussel (Branchipolynoe pettiboneae) obtained from diseased mussels, two numerically dominant limpet species (Lepetodrilus schrolli, Symmetromphalus aff. hageni), and the numerically dominant polychaete species (Amphisamytha cf. galapagensis)].

For each individual, histological sections (4–6 μm) were cut from paraffin-embedded tissues, and slide sets (three slides, each typically with multiple tissue sections) were stained with hematoxylin and eosin (H&E; Stevens 1990) or a modified Gomori Methanamine Silver (GMS) stain (Sigma-Aldrich), or left unstained for fluorescent in situ hybridizations [FISH, for which Colorfrost®/Plus (Fisher Scientific) glass slides were used]. For large mollusk species (mussels, hairy gastropods, and black snails), selected tissues were processed for each individual [foot, gill, mantle, visceral mass (including digestive diverticula and gonad)]; smaller species (limpets and polychaetes) were sectioned whole. For TEM studies, tissues were embedded in Embed 812 epoxy resin, cut (∼90 nm), and stained with lead citrate.

Following DNA extraction using a DNeasy Tissue Kit (QIAGEN, Valencia, CA, USA), a ‘universal’ polymerase chain reaction assay (PCR) designed to amplify protistan or fungal (but also bacterial) SSU rDNA from metazoans (Carnegie et al. 2003; Bower et al. 2004) was applied to fungus-infected Bathymodiolus brevior to generate presumptive fungal SSU rDNA amplicons. Amplification products were cloned, and 16 clones were sequenced on an Applied Biosystems (Foster City, CA, USA) Genetic Analyzer 3100.

To link presumptive fungal SSU rDNA amplicons to the parasite observed histologically, we modified a FISH protocol developed for the oyster parasite Mikrocytos mackini (Carnegie et al. 2003), replacing M. mackini-specific probes with AlexaFluor 488-labeled (Invitrogen Corp., Carlsbad, CA, USA) presumptive fungal-specific probes (BATHPAR-A: 5′-TTAGGAGGATAGATCGGC-3′; BATHPAR-B: 5′-CCAGTGAAGGGCATAGGG-3′). A competitive control experiment to assess the specificity of fungal parasite-specific probe binding included four treatments: a no probe control (25 μl of hybridization buffer only), a standard experimental treatment (parasite-specific probes each at 10 ng μl−1), a competitive negative control (parasite-specific probes each at 10 ng μl−1, plus unlabeled versions of these probes at 200 ng μl−1), and a competitive positive control [parasite-specific probes each at 10 ng μl−1, plus an unlabeled, non-specific oligonucleotide (a probe for a Bonamia sp., a parasite of the oyster Ostreola equestris, chosen simply for convenience) at 800 ng μl−1]. All treatments except the no-probe control were run in duplicate.

Results

  1. Top of page
  2. Abstract
  3. Problem
  4. Material and Methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgements
  9. References

Bathymodiolus brevior mussels from Mussel Hill (Fig. 2a) presented a range of disease states, from apparently healthy individuals with normal creamy coloration, fleshy tissues, and well-developed gonads, to mussels with scattered spots of brownish discoloration of mantle tissues (Fig. 2b), to progressively more discolored bodies, and, in the most dramatic cases, to black-body stages (Fig. 2c). Black-body mussels had a distinctive odor, being neither that of hydrogen sulfide, as is typical for mussels from hydrothermal vents, nor that of putrescence.

image

Figure 2. Bathymodiolus brevior. a: Mussel beds at Mussel Hill, Fiji Basin. Note the occasional paired empty mussel shells (arrow-heads) and area of broken shells on the left hand side of the photo. Ellipse surrounds the type of mussel bed targeted for sampling, i.e., a deep stack of living mussels. Scale bar = 10 cm (foreground). b: Brown-spot stage. A few of the brown spots are highlighted with black arrowheads. c: Black-body stage. Scale bars b and c = 1 cm.

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Prevalence of black-body mussels in a quantitative sample of 57 individuals from the Mussel Hill hydrothermal-vent site was 23%; prevalence of combined brown-spot through black-body individuals was 55%. Only a single mortality (i.e., paired empty shells) was noted in this sample, but empty shells of mussels can readily be seen in video and digital images (Fig. 2a) of the mussel beds.

Histological examination of brown-spots on mantle tissues revealed the presence of dense populations of thick-walled yeast cells (Fig. 3a), often budded to form short-chained blastoconidia (budding yeast cells) up to 15 μm in length (Fig. 3b). Pseudohyphae (string of cells with complete septae resulting from budding of blastoconidia) were not observed. Brown spots were also characterized by intense hemocytic infiltration, a typical molluscan response to infection (Fig. 3a). Storage connective tissues of uninfected mantle were dominated by large cells filled with eosinophilic granules (lipids and/or glycogen; Fig. 3c); connective tissues of brown-spot mantle were depleted in granular storage materials (Fig. 3a). Connective tissues surrounding the gonad and digestive diverticula of brown-spot mussels were also infected with fungal blastoconidia and infiltrated by hemocytes.

image

Figure 3.  Pathology associated with fungal cells in Bathymodiolus brevior mussels from a Fiji Basin hydrothermal vent. a: Brown-spot mantle tissue; note lack of granular connective tissue cells and presence of numerous nuclei of mussel hemocytes [hematoxylin and eosin (H&E); scale = 20 μm]. b: Brown-spot mantle tissue; arrow points to an example of a blastoconidium (H&E; scale = 10 μm). c: Normal mantle tissue (H&E; scale = 20 μm). d: Black-body visceral mass with dense, dark-staining fungal blastoconidia (modified Gomori silver stain; scale = 20 μm). Light-brown granules in epithelial cells of the digestive diverticula are spherocrystals involved in sequestration of metals. e: Atrophied gonad of a black-body individual (H&E; scale = 20 μm). f: Well-developed gonad of an uninfected individual (H&E; scale = 20 μm). g: Gill filaments of a brown-spot individual, illustrating fungal-filled connective tissue (arrow) within water tubule (H&E; scale = 100 μm). h: Hemocyte-ringed fungal cells (arrow) in connective tissue of a superficially healthy mussel (H&E; scale = 10 μm). dd, tubule of digestive diverticula; s, developing sperm in reproductive acini.

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In black-body mussels, blastoconidia were abundant in connective tissue associated with the mantle and visceral mass (Fig. 3d). In the most severe infection examined, reproductive acini of the mussel were greatly reduced and the overall condition of the tissue in the visceral mass was extremely degraded (Fig. 3e) relative to healthy individuals (Fig. 3f). Gill filaments of black-body mussels were variable in condition within an individual, but the majority of gill filaments were blemished by macroscopically conspicuous dark bodies that corresponded microscopically to locally dense aggregations of fungal material within connective tissue in water tubules between gill filaments (Fig. 3g). In one instance, several patches of fungal cells in the visceral mass of an otherwise healthy individual were ringed by hemocytes (Fig. 3h). Bacteriocytes of some black-body gill filaments were, relative to those of asymptomatic, apparently healthy individuals, reduced in size and greatly reduced in the area of the apical portion of the cells that contain chemoautotrophic endosymbionts.

Fungal cells were not observed in the gonadal acini or epithelia of the digestive diverticula, mantle, or gills, and were relatively rare in muscle tissue (notably the foot and heart), except in the most severe infections. This distribution suggests that while fungal cells readily spread through mussel tissues, their entry into epithelial cells is blocked by an unknown mechanism.

General deterioration of mussel condition was evident in a significantly lower tissue dry weight–shell length relationship in black-body mussels compared with healthy individuals (ANCOVA, P < 0.05). Of five mussels labeled as ‘healthy’ (i.e., no patent tissue discoloration) and subsequently examined histologically, two were infected by the fungus, indicating that the total prevalence of the fungus in the population was >58%.

Blastoconidia reside intracellularly within mussel connective tissues. The simple ultrastructure of a section of a budding cell (Fig. 4) includes a thick cell wall, a nucleus, mitochondria, possible spindle polar bodies, and a large electron-dense vacuole. The cell wall is three layered, with a fibrous outer layer, an electron-opaque middle layer, and an electron-transparent inner layer, and the cell wall of the bud is continuous with that of the mother cell.

image

Figure 4.  TEM observations of interfilamental connective tissue of gills, Bathymodiolus brevior. a: Healthy tissue. b: Fungal-infected tissue (arrowheads: fungal cells). c: Budding fungal cell; a pair of spindle polar bodies may be present just to the left of the nucleus (n); mitochondria are not evident at this level of magnification. Mussel cells: Nu, nucleus. Fungal cells: n, nucleus; v, vacuole; w, cell wall. Scale bars: 5 μm.

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Molecular characterization of the fungus suggests an affinity to the ascomycete class Chaetothyriomycetes, order Chaetothyriales, members of which are commonly known as the ‘black yeasts’. The numerically dominant insert in the pool (7/16 clones) was a 579-bp amplicon (GenBank DQ314803) that is 99% similar by GenBank BLAST search to fungi in the genus Capronia (order Chaetothyriales, family Herpotrichiellaceae; Fig. 5a). The Capronia-like SSU rDNA was linked to the fungus observed histologically by in situ hybridization (Figs 5b and 6).

image

Figure 5.  a: Unrooted neighbor-joining analysis indicating the affinity of the Bathymodiolus parasite SSU rDNA sequence (bold) to the Chaetothyriales. Sequences from the GenBank database were selected and ordinal assignments (orders Chaetothyriales, Onygenales, and Eurotiales) were constructed following Sterflinger et al. (1999). Analysis of Clustal-aligned sequences was performed in PAUP v4.0. b: Fluorescent in situ hybridization links the Capronia-like SSU rDNA amplified from infected mussels to fungal blastoconidia (green fluorescence) in mussel tissues. Scale bar: 5 μm.

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image

Figure 6.  Evaluation of the specificity of the fungal-parasite-specific FISH assay using competitive controls. a: No-probe treatment. b: Experimental fungal parasite-specific probe treatment. c: Competitive negative control treatment. d: Competitive positive control treatment. Abolition of labeled, specific probe binding in the competitive negative treatment, but not in the competitive positive control treatment, indicates that probes bound to the target sites for which they were designed, rather than haphazardly within the fungal genome. Scale bars: 10 μm.

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Of 13 mussels collected from the vent field ∼70 m from Mussel Hill, one individual had a moderate fungal infection, indicating that the fungus is not restricted to the nearby Mussel Hill site. In contrast, no fungal infections were detected in mussels examined histologically (27 individuals for H&E and GMS silver stain, and 10 individuals for FISH) from the Lau Basin mussel bed. The fungus was not detected in any of the other invertebrate species examined (Alviniconcha aff. hessleri, Ifremeria nautilei, Lepetodrilus schrolli, Symmetromphalus aff. hageni, Amphisamytha galapagensis, Branchipolynoe pettiboneae) that were collected with samples of fungal-infected mussels from Fiji Basin.

Discussion

  1. Top of page
  2. Abstract
  3. Problem
  4. Material and Methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgements
  9. References

Disease has largely been ignored as an ecological forcing function in deep-sea ecosystems based on chemoautotrophic primary production, in favor of readily observed effects of physical factors [temperature and vent fluid chemistry; Luther et al. (2001)] and other biological interactions [competition, predation, recruitment, inhibition; e.g., Levesque et al. (2003), Micheli et al. (2002), Hunt et al. (2004), Mullineaux et al. (2003)]. Parasitic infections are also likely to play important roles in population dynamics of vent and seep species. For example, mussel populations at Gulf of Mexico seeps infected with bucephalid nematodes were reproductively compromised based on atrophy of the gonadal tissue (Powell et al. 1999). A viral-like inclusion in digestive diverticula of mussels from the Blake Ridge seep site is also associated with tissue pathology and may have an impact on local population structure (Ward et al. 2004).

Molluscan diseases are often not conspicuous at the macroscopic level, making them difficult to diagnose in the field. The characteristic brown-spot and black-body gross presentations of mussel tissues resulting from fungal infections make it easier to diagnose this mussel disease when it is in advanced stages. The unusual coloration and odor of black-body mussels are consistent with melanization and the production of phenolic compounds, either as an immune response on the part of the mussel (Renwrantz et al. 1996; Nappi & Ottaviani 2000) or as a virulence factor on the part of the fungus (Jacobson 2000), but the cause of the discoloration and odor remain to be determined.

Fungi are generally considered to be opportunists, infecting animals that are somehow physiologically compromised, either by other disease agents or by deteriorating environmental conditions (Guarro et al. 1999). Shimmering water and elevated temperatures (8 °C) typical of mussel beds (Bathymodiolus brevior) in the Lau and Fiji Basins were recorded among Mussel Hill mussels. There was thus no obvious evidence of waning hydrothermal activity at the time of sampling. Furthermore, tissue condition and chemoautotrophic endosymbiont population density within bacteriocytes of uninfected mussels at Mussel Hill matched those of mussels from hydrothermally robust sites elsewhere. While we cannot eliminate the possibility that fungal infections of the mussels were secondary or were facilitated by concurrent infections of other pathogens, identification of fungi in otherwise healthy individuals makes it clear that the fungus is not a strict saprophyte.

The phylogenetic affinity of the fungus and its apparent specificity to mussels in Fiji Basin is intriguing. Representatives of the family Herpotrichiellaceae to which the fungus belongs include shallow-water marine [e.g., Capronia ciliomaris (Au et al. 1999)], freshwater aquatic, and terrestrial species, and opportunistic parasitic species of plants and animals, including humans [e.g., Exophiala species Guarro et al. 1999)]. The ultrastructure of the mussel fungus is similar to that described for other black yeasts, including the pathogenic ascomycetous yeast Exophiala dermatitidis (Yamaguchi et al. 2003).

The severity of the epizootic event at Mussel Hill is underscored by the fact that fungal prevalence exceeds that of all but the worst outbreak of aspergillosis (at a prevalence of 60%) in sea fans of the Florida Keys (Kim & Harvell 2004). Epizootic ‘black-body’ events in shallow-water mussels or other bivalves are rare; the most recent reference to a black-body epizootic that we have found dates back to the early 1900s, reported in mussels from the coast of Holland (Dollfus 1923). The disease agent from the Holland outbreak was not identified during histological and microbiological examination of specimens at the time. Mycoses in general seem to be uncommon in bivalves. In a synopsis of mycotic diseases in marine invertebrates, only five fungal pathogens were listed as occurring in bivalves, none of them reported in mytilids (Noga 1990).

Although there was no direct evidence of mortality that could be unambiguously linked to the fungal infection (no moribund mussels were collected), high prevalence of the fungus within the population, progressive and pervasive connective tissue degradation, together with decreased volume of bacteriocytes and symbiotic bacteria associated with the black-body stage of the disease, suggest that massive mussel mortality is imminent within the Mussel Hill vent field. Mussels and their associated chemoautotrophic endosymbionts dominated the Mussel Hill site in 2005 and thus represent the largest standing crop of primary producers (endosymbiotic bacteria) and consumers (mussels) within the ecosystem. Populations of macro- and micro-invertebrates (i.e., gastropods, polychaetes, crustaceans, etc.) also live among the mussels. Die-off of mussels will substantially alter the trophic and taxonomic structure of the community at this site, although the precise nature of these changes is difficult to predict for this relatively remote and unstudied hydrothermal system.

The fungus was not detected in mussel tissues examined from Lau Basin, indicating that the parasite was not at epizootic proportions at the time of sampling. It may be lurking in the population at much lower prevalence. Based on our sample size of 26 individuals from an estimated host population size on the order of 10,000 to 100,000 or more individuals, the fungal prevalence is ≤10% (Anderson & Barney 1991).

The discovery of an on-going epizootic among mussels at a deep-sea hydrothermal vent likely represents a rare example of a natural disease outbreak documented in the early stages of the epizootic, prior to the onset of extensive mortality. Populations of Bathymodiolus brevior occur throughout back-arc basins of the southwest Pacific. Given that yeasts in general are resistant to desiccation and other forms of stress, and that surfaces (including sample collection boxes) and ballast waters of submersible assets are not routinely treated to prevent the spread of microorganisms from one site to another, submersible assets may serve as vectors for this mussel pathogen as vehicles are moved from infected to uninfected vent fields, unless appropriate precautions against vector transport are implemented.

Conclusions

  1. Top of page
  2. Abstract
  3. Problem
  4. Material and Methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgements
  9. References

Mussels from deep-sea hydrothermal vents in Fiji Basin are infected with a Capronia-like black yeast, a fungus that elicits an intense hemocytic immune response from the mussel. Massive mussel mortality at Mussel Hill is predicted, based on the severe tissue degradation associated with the prevalent brown-spot to black-body stages of the disease within the mussel population, but the test of this hypothesis requires a return visit to the site. The fungus so far appears to be specific to mussels and was not detected in mussels of the same species collected the same month from the adjacent Lau Basin.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Problem
  4. Material and Methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgements
  9. References

We thank Chief Scientist R. Vrijenhoek and the shipboard nautical, technical, and scientific parties of the R/V Melville/ROV JASON II cruise TUIM06 for assistance in collection of samples. C. Fisher and K. Zelnio generously provided mussel samples from Lau Basin (Lau field program supported by NSF OCE 0240985 to C. Fisher). We thank J. Shields for discussions about the disease agent. E. Blake, C. Logan, H. Heiser, and A. Barton assisted with sample processing, K. Johnson with DNA sequencing. This study was supported by an NSF grant to CLVD (OCE 0350554).

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  1. Top of page
  2. Abstract
  3. Problem
  4. Material and Methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgements
  9. References
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