Bacterial communities associated with lesions of shell disease in the American lobster, Homarus americanus Milne-Edwards.
Article Type: Report
Subject: American lobster (Diseases)
Shells (Diseases)
Anaerobic infections (Research)
Authors: Chistoserdov, Andrei Y.
Quinn, Robert A.
Gubbala, Sai Laxmi
Smolowitz, Roxanna
Pub Date: 06/01/2012
Publication: Name: Journal of Shellfish Research Publisher: National Shellfisheries Association, Inc. Audience: Academic Format: Magazine/Journal Subject: Biological sciences; Zoology and wildlife conservation Copyright: COPYRIGHT 2012 National Shellfisheries Association, Inc. ISSN: 0730-8000
Issue: Date: June, 2012 Source Volume: 31 Source Issue: 2
Topic: Event Code: 310 Science & research
Geographic: Geographic Scope: United States Geographic Code: 1USA United States
Accession Number: 302109295
Full Text: ABSTRACT Shell disease in Crustacea is a widely recognized syndrome having a polymicrobial etiology, and manifesting itself as lesions of the shell with a variable structure and shell location. We characterized major members of bacterial communities in epizootic shell disease lesions of the American lobster (Homarus americanus, Milne Edwards) and compared these communities with the ones found in study cases of impoundment and enzootic shell disease. Bacteria belonging to several Flavobacteriaceae genera (Aquimarina, Tenacibaculum, Polaribacter, Maribacter, Cellulophaga) within the phylum Bacteroidetes appear to have particular attraction to lobster lesions. The most prominent Bacteroidetes in lobster lesions were representatives of the genus Aquimarina sp., but only Aquimarina 'homaria' was detected in all analyzed lesions of epizootic, impoundment, and enzootic shell disease. It was found on 45% of surfaces unaffected by shell disease, but in smaller numbers compared with lesions. Alphaproteobacteria represent the most diverse class of proteobacteria found in both lesions and on unaffected surfaces. Three bacteria of this class appear to be ubiquitous in shell disease lesions, but only one specific alphaproteobacterium tentatively assigned to the genus Thalassobius (herein designated as 'Thalassobius' sp.) was present in all analyzed lesions of epizootic, impoundment, and enzootic shell disease. A ubiquitous gammaproteobacterium called "Candidatus Homarophilus dermatus' was also prevalent in lesions, but just as commonly it was associated with surfaces unaffected by shell disease. The bacteria A. 'homaria' and 'Thalassobius' sp. are dominant and appear obligatory in lobster shell lesions, and are only occasionally detected on unaffected surfaces, which serve as intermediate reservoirs for the two potential pathogens. Therefore, these two bacteria stand out as potential shell-disease pathogens.

KEY WORDS: epizootic shell disease, Aquimarina 'homaria, denaturing gradient gel electrophoresis, impoundment shell disease, enzootic shell disease, American lobster, Homarus americanus

INTRODUCTION

Shell diseases in crustaceans are a widely recognized syndrome manifesting as lesions of the shell (Fig. 1) with a variable structure and shell location and, in most cases, an unknown but diverse etiology. A shell disease of major concern is epizootic shell disease (ESD) of lobsters, which has been the most prevalent disease of the American lobsters (Homarus americanus, Milne Edwards) in recent years (Castro & Angell 2000). In retrospect, the first outbreak of this disease was seen as early as 1981 (Glenn & Pugh 2005). Prevalence of ESD in the years 1996 and 1997 was relatively low (0-5.6%) in southern New England, but in recent years, the percentage of diseased lobsters increased to 20-40% in the inshore populations. The disease most severely affects ovigerous female lobsters (Estrella 1991).

Although the disease is most often not fatal (Stevens 2009), commercial implications like decreased marketability resulting from nonaesthetic appearance have severe economic impacts on lobster fisheries, wholesale and retail. The economy of the lobster fisheries of New York, Connecticut, Rhode Island, and Massachusetts was particularly hard hit. Although occasionally lobsters with shell disease are collected in Maine (Chistoserdov et al. 2005a), Maine fisheries are considered to be free from ESD. The main clinical signs of this type of disease include melanized lesions that are symmetric and leave pillars of eroded chitin when the cuticular section is examined (Smolowitz et al. 2002, Smolowitz et al. 2005) (Fig. 1B). The disease is characterized by moderate to deep erosions on the carapace, which in severe stages may spread to the other parts of the lobster. Erosions, at least initially, are not associated with structures of the shell such as setal pores or tegumental glands (Smolowitz et al. 2005). Environmental factors contributing to ESD are still poorly understood, but temperature (Quinn et al. 2012a, Tlusty & Metzler 2012), alkyl phenols (Laufer et al. 2005) and other environmental contaminants (Wise 2005), and poor diet (Tlusty et al. 2008) have been examined and implicated.

The etiology of ESD is considered to be bacterial. Bacterial culture (Chistoserdov et al. 2005b) and molecular-based techniques (Chistoserdov et al. 2005a) demonstrated a moderately complex community in ESD lesions with several prominent and ubiquitous members, including a novel member of the genus Aquimarina (Aquimarina 'homaria'), a specific Pseudoalteromonas sp. (Pseudoalteromonas graeilis), and a Rhodobacteraceae alphaproteobacterium closely related to the genera Thalassobius and Jannaschia (formerly known as 'Marinosulfuromonas' sp. (Chistoserdov et al. 2009)). This bacterium is herein called 'Thalassobius' sp. ESD lesions also contain a diverse eukaryotic fauna but they are not considered as a primary agent of infection (Hsu & Smolowitz 2003, Smolowitz et al. 2005, Quinn et al. 2009).

Impoundment shell disease (ISD), the second most important form of shell disease of the American lobster, is seen primarily in lobsters held in impoundments during the winter months. Early forms of the lesions are symmetric and are centered on the setal cores of the dorsum of the animal. The important characteristics of the disease, which contrast with ESD, are that the lesions are round, melanized, appear as scooped out depressions on the eroded cuticle, and are symmetric bilaterally. In the severe form of the disease, the lesions coalesce and spread on the entire surface of the carapace (Fig. 1C). The disease is seen as melanized brown to black erosions that commence with the removal of epicuticle, extend through the exocuticle, then the calcified endocuticle, and, in extreme cases, penetrate the noncalcified endocuticle to the internal tissues (Smolowitz et al. 1992). The disease initiates as a result of several factors. Prince et al. (1995) have described the disease propagation as the result of dietary deficiencies. Degraded water quality conditions can also be a contributing factor to the spread of the disease (Smolowitz et al. 1992). Sindermann (I 991), in his studies, stated that disturbances in metabolism can be seen externally as shell disease when the animal cannot maintain normal chitin deposition. Several factors including pollution and environmental contamination (Friedman et al. 2000, Young & Pearce 1975) have been attributed to the cause and the spread of ISD.

[FIGURE 1 OMITTED]

Hess (1937) first reported that chitinolytic bacteria are involved in the shell lesions of the American lobster cultivated in the pounds. Many investigators have documented that the etiology of impoundment shell disease seen in the American lobster is bacterial, and that several kinds of bacteria are present in the shell lesions, including Vibrio, Aeromonas, Beneckea, and Pseudomonas spp. (Fischer 1977, Malloy 1978, Stewart 1980, Getchell 1989). The role of chitinolytic bacteria in the development of impoundment shell disease is supported further by pathological changes observed histologically, where their action leaves no cuticular matrix in a section of carapace. This is in a stark contrast to the chitinous pillars of eroded cuticle that remain in the erosions of lobsters with ESD (Smolowitz et al. 2005).

The third form of the disease is burn spot (BSSD) or rust disease, in which lesion initiation is attributed to several different fungi (Stewart 1980) and/or bacteria (Rosen 1970, Sindermann 1979). This type of shell disease was reported by Ziskowski et al. (1996) in the offshore, New York Bight region around the 106-mi sewage sludge disposal site. According to Ziskowski et al. (1996), this disease could be used as an indicator for degraded environmental conditions. However, both grossly and histologically, BSSD in animals collected from various locations immediately offshore of the east coast is consistent in most cases with focal trauma to the carapace and not with primary infectious causes (Smolowitz et al. 2005, Cobb & Castro 2006). Other shell disease types include diet-induced shell disease (DISD), in which lesions resemble those of BSSD superficially, but have so far been described only for juvenile lobsters kept in captivity (Tlusty et al. 2008). Enzootic or endemic shell disease (EnSD) has been identified in fished lobsters for many years, but was only recently recognized formally and named (Cobb & Castro 2006). It is characterized by light to moderate pitting of the lobster shell (Estrella 1984, Cobb & Castro 2006), and in this respect may be an analog of ISD in the wild (Fig. 1A). It is also possible that BSSD is just a form of EnSD when only one or few small round lesions are present.

The goal of this work was to ascertain the composition of the microbial communities associated with lesions of various forms of shell disease. We based our analyses on molecular techniques because it has been well established that less than 1% of the bacteria present in the environment are cultivable (Muyzer 1999). Our method of choice was denaturing gradient gel electrophoresis (DGGE) of PCR-amplified portions of 16S rDNA generated from 2 universal and one group-specific primer set. This technique offers a great potential to analyze multiple samples rapidly and simultaneously, yet data generated on microbial diversity are not confounded by the socalled "rare biosphere," which remains mostly undescribed (Muyzer 1999).

MATERIALS AND METHODS

Animals and Sampling of Lobsters

"Unaffected" lobsters refer to animals with no signs of shell disease, DNA isolated from the lesions and unaffected surfaces of lobsters from various locations and with different forms of shell disease are listed in Table 1. Not all lobsters were used for all analyses. Almost all lobsters were from approximately the same age group (one from Rhode Island was probably 2 y younger and weighed -0.5 lb), and were comparable in size (i.e., "1.5 lb" category). At the time of sampling, all animals were rinsed with sterile seawater over the entire carapace. Shell sampling for both the affected and unaffected carapace consisted of scraping with a sterile razor blade and collecting the scraped material in sterile 50-mM Tris/EDTA buffer (pH, 8.8). Egg white lysozyme (Amresco, Solon, OH) was added to the shell samples to a final concentration of 1 mg/mL, and the mixture was then incubated at 37[degrees]C for 30 min. A sodium dodecyl sulfate solution was added to a 2% final concentration, followed by the addition of proteinase K (Fisher Bioreagents, Fair Lawn, N J) at 1.25 mg/mL and incubated at 50[degrees]C for 15 rain. The samples were then subjected to 3 freeze-thaw cycles at 50[degrees]C then 80[degrees]C and bead beating in a Mini-Beadbeater-8 with 0.1 mm Zirconia/Silica Beads (BioSpec Products, Bartlesville, OK). DNA was extracted using, sequentially, phenol and then chloroform. A volume of phenol (Sigma-Aldrich Inc., St. Louis, MO) equal to that of the aqueous phase was added, the 2 were shaken vigorously, then centrifuged for 5 rain at 5,000 rpm, and the aqueous phase was removed. An equal volume of chloroform (FisherBiotech, Fair Lawn, N J) was added to the aqueous phase, and the resulting emulsion was spun at 5,000 rpm for 15 min. DNA was precipitated from this aqueous phase by adding 1/10 volume of 3-M sodium acetate and 2.2x volume of 100% cold ethanol. The sample was then frozen overnight at -80[degrees]C and spun at 5,000 rpm for 35 min in an Eppendorff microcentrifuge. The DNA pellet was then washed with 70% ethanol, dried, and resuspended in 200 [micro]L sterile dd[H.sub.2]O.

PCR Amplification

For DGGE analysis, the fragment of 16S rRNA gene containing the variable regions V3, V4, and V5 was amplified using the universal primer set 341FM-GC (5'-CCTA CGGGDGGCWGCAG-3'; Escherichia coli position, 341 bp) and 907RM (5'-CCGYCWATTCMTTTGAGTTT-3'; E. coli position, 907 bp) adjusted from Muyzer et al. (1998). The forward primer was modified by the addition of a 40-bp GC-rich sequence (Muyzer et al. 1993). This PCR contained 25 [micro]L GoTaq Green Master Mix (Promega, Madison, WI), 1.5 [micro]M of the forward and 0.5 [micro]M of the reverse primers, an additional 1.0 mM Mg[Cl.sub.2] and 1.5 [micro]L template (quantities of a template were variable, but, on average, were ~10 ng for lesions and 1 ng for the melanized spot) in a 50-[micro]L reaction volume. PCR was carried out under a touchdown protocol that consisted of 5 rain at 95[degrees]C, followed by 20 cycles of 1 rain at 95[degrees]C, 1 min at 65[degrees]C down to 55[degrees]C (touchdown, -0.5[degrees]C per cycle), and 3 min at 72[degrees]C, followed by 15 cycles of 1 min at 95[degrees]C, 1 min at 55[degrees]C, and 3 rain at 72[degrees]C, and was concluded with a final extension of 7 min at 72[degrees]C. For better resolution of the proteobacterial members of microbial communities, the primers 27F-GC (5'-AGAGTTTGATCMTGGCTCAG-3') and 355R (5'-GCTGCCTCCCGTAGGAGT-3') were used to amplify the variable regions V1 and V2 of the bacterial 16S rRNA genes of a randomly selected subset of 13 lobsters (chosen to cover all locations). The forward primer was modified by addition of a 40-bp GC-rich sequence (Muyzer et al. 1993). This PCR contained 25 [micro]L GoTaq Green Master Mix (Promega), 0.5 [micro]M of the forward and 0.5 [micro]M of the reverse primers, and 1.5 [micro]L template in a 50-[micro]L reaction volume. PCR was carried out under a thermocycling protocol that consisted of 5 min at 95[degrees]C, followed by 30 cycles of 30 sec at 95[degrees]C, 30 sec at 59[degrees]C, and 30 sec at 72[degrees]C, and was concluded with a final extension of 5 rain at 72[degrees]C. The Bacteroidetes communities were amplified with the same forward primer (341FM-GC), but with the Bacteroidetes-specific reverse primer CFB721R (5'-CTGCCTTCGCAATCGG3'), modified from Weller et al. (2000) to widen the number of Bacteroidetes sequences it could amplify. The reaction conditions for this primer set were an initial denaturation step at 95[degrees]C for 3 min followed by 30 cycles of 95[degrees]C for 30 sec, 57[degrees]C for 45 sec, and an extension at 72[degrees]C for 45 sec. The reaction was concluded with a final extension at 72[degrees]C for 3 min. For the specific PCR for A. 'homaria,' novel primers were designed on the basis of its unique 16S rDNA sequences. These primers did not match any other sequence identified in ESD lesions or any sequence deposited in the Ribosomal Database Project database or GenBank. The primers for A. 'homaria' were Ahoml90F (5'-TAGTATCMAAGACA GCMTTGTTTTATG-3') and Ahom470R (5'-CCTTATTCG TAGAGTACCGTCAGAGTAT-3'). The reaction conditions were an initial denaturation step of 95[degrees]C for 10 rain followed by 40 cycles of 95[degrees]C for 15 sec, annealing at 50[degrees]C for 30 sec, and an extension at 72[degrees]C for 30 sec. The PCR products were then run in a 1% agarose gel and visualized after staining with ethidium bromide (0.1 mg/mL) on a transilluminator.

Denaturing Gradient Gel Eleetrophoresis and Band Processing

DGGE was carried out using a CBS Scientific DGGE system (CBS Scientific Co., Del Mar, CA) in 1x Tris-acetate EDTA buffer (pH, 7.8; FisherBiotech) at 60[degrees]C. All DGGE gels were 6% polyacrylamide of dimensions 20 x 17.6 cm, 1.5 mm thick, and contained an increasing denaturant concentration (7 M urea and 40% formamide is 100% denaturant)of 20-80% for gels using the 341F-907R primers, 20-65% using the 27F-355R primers, and 30-65% for gels using the 341F-CFB721R primers. A total of 50 [micro]L of each PCR was loaded and run in DGGE gels. The electrophoresis was carried out at 80 V for 14 h. After electrophoresis, the gel was stained with ethidium bromide (0.1 mg/mL) for 20 min and visualized using a transilluminator. Bands of interest were excised from the gel using a sterile razorblade and placed into a microcentrifuge tube with 0.2 g sterile, 2-mm glass beads (Biospec Products) and 500 [micro]L dd[H.sub.2]O. The excised acrylamide/bead mixture was then bead beaten in a Mini-Beadbeater (Biospec Products, Inc.) at high speed for 3 rain. The sample was then kept at 4[degrees]C overnight to allow diffusion of DNA.

DNA Sequencing

A 1-[micro]L aliquot of the aqueous portion of the homogenized acrylamide band was reamplified with the same initial primer set from which it was derived, but using a forward primer without a GC clamp. The reactions contained 25 [micro]L GoTaq Green Master Mix, 0.5/aM of each forward and reverse primer, and 1.5[micro]L of the DNA sample. The thermocycling conditions were the same as outlined for the same DGGE amplifications. The PCR product was then purified with a Wizard SV Gel and PCR Clean Up System (Promega). The purified product was sequenced using the forward primers and a BigDye terminator cycle sequencing kit, version 3.1, on an Applied Biosystems 3130 DNA sequencer (Applied Biosystems, Foster City, CA). Sequences were searched against the GenBank database using the BLAST tool to determine the best sequence matches, and then against the Ribosome Database Project (RDP) database to determine appropriate phylogeny and identity. Nucleotide sequences were deposited in GenBank with accession nos. JF904894-JF904934. Computer analyses of DNA sequences was carried out using Lasergene 5.3 from DNA*Star (Madison, WI). Partial 16S rRNA gene sequences were compared with sequences in the GenBank database using BLASTn (Altschul et al. 1997) and in RDP (http://rdp.cme.msu.edu/) release 10 using the Seqmatch utility to determine their approximate phylogenetic affiliations. They were then aligned using the MegAlign program from the Lasergene package. The sequences were checked for possible chimeras using the CHIMERA_CHECK program (release 8.1, RDP; rdp.cme.msu.edu).

RESULTS

Bacterial Communities on Surfaces from Lobsters with No Signs of Shell Disease

Bacterial DNA yields from unaffected surfaces were variable but very low (Chistoserdov et al. 2005b) and, as a result, amplification of 16S rDNA with the 341FM-GC-907RM primer set from unaffected carapace DNA samples was poor. From all unaffected lobsters listed in the Table 1, we managed to amplify 16S rDNA in appreciable quantities only from 2 samples, either no amplification was observed or the quantities generated were too small to be loaded on a DGGE gel. However, 12 of 13 selected samples (Fig. 2A) were amplified successfully with the 27F-GC-355R primer set. Unaffected lobster surfaces shared very similar microbial communities even though the animals came from locations as far apart as Canada and eastern Long Island Sound (Fig. 2, Table 2). The few unique bands that were present were not specific to a sampling location. The communities on unaffected carapaces were dominated by members of alphaproteobacteria with at least 5 species (a Thalassobacter sp., 2 species of Loktanella, an Ahrensia sp., and 1 unclassified Rhodobacteraceae sp.), 3 of which (Thalassobacter sp., Ahrensia sp., and Loktanella species 2) were found in the majority of samples. Gammaproteobacteria were represented by 2 species, a novel bacterium detected in all lobsters, which could not be assigned to any currently existing gamma-proteobacterial order (herein called 'Candidatus Homarophilus dermatus'), and a second species belonging to genus Leucothrix, which was detected in 6 lobsters. Two unclassified Actinomycete species related distantly to the family Lamiaceae were found on 2 lobsters each. Analysis with the 27F-GC-355R primer set indicated that Bacteroidetes were not prevalent on unaffected carapaces. They were represented by 3 species, of which only one, a Pibocella sp., was present in more than half of the 13 lobsters. Because the 27F-GC-355R primer set amplified 16S rDNA poorly from the Bacteroidetes phylum, and any bands produced were blurry (Fig. 2A), a Bacteroidetes-specific primer set was also used to investigate this group more completely. Amplification with the Bacteroidetes-specific primers was successful for only 9 of 13 randomly selected samples, and of 9 positive amplification reactions, only 5 produced strong bands in DGGE gels (Fig. 2B). In addition to Bacteroidetes taxa identified with a universal primer set, 3 more Flavobacteriaceae bacteria turned out to be common on unaffected surfaces, and, surprisingly, Pibocella sp. was not among them. The bacterium A. 'homaria' was not detected on unaffected carapaces by any DGGE-based method, although other Aquimarina spp., including Aquimarina muelleri, were encountered in 3 samples. Because A. 'homaria' is suspected to be involved in ESD (Chistoserdov et al. 2009), we amplified its 16S rDNA with A. 'homaria'-specific primers, which are expected to provide a much higher sensitivity detected in the ESD lesions. All tested ISD lesions communities also contained a unique Tenacibaculum sp.

[FIGURE 2 OMITTED]

[FIGURE 3 OMITTED]

Bacterial Communities in EnSD Lobsters

Obtaining lobsters with EnSD is difficult, because the disease is very rare in the wild; therefore, only 2 samples were available for the project. Microbial communities in these lesions of both animals contained A. 'homaria,' Flavobacteriaceae species 1, and 'Candidatus Homarophilus dermatus'--all 3 were previously seen in other types of lesions (Fig. 5). Unique members of EnSD bacterial communities found in both samples included Flavobacteriaceae species 3, Maribacter species 2, Rhodobacteraceae species 4, and 'Candidatus Kopriimonas aquarianus' (Table 5).

DISCUSSION

Existing classification of shell disease forms is based on either ecosystems from which a given form of the disease was recovere--wild (ESD) versus pounds (ISD) versus aquaria (DISD)--or frequencies of the occurrence in the wild populations (epizootic versus enzootic). Grossly, ESD stands apart from other forms of shell disease in that its lesions are not associated with specific structures on the shell and they cover large carapace surfaces asymmetrically. In all other forms of shell disease, lesions appear to be round, and, at least for ISD, these initial lesion loci have been associated with setal pores and may, in time, spread to cover larger surfaces of the carapace symmetrically (Smolowitz et al. 1992). The goal of this work was to characterize major members of bacterial communities in ESD lesions and compare these communities with those found in lesions of other shell disease forms.

Three different primer sets were used for this analysis because of the discovery of primer bias with the 27F-GC-355R universal primers and low sensitivity of the 341F-GC907R. Our previous study using some of the same lobsters showed that unaffected lobster carapace has less than 106 bacterial cells/[cm.sup.2], and bacterial DNA yields from unaffected surfaces were very low (Chistoserdov et al. 2005b). Accordingly, in this study, amplification of 16S rDNA with the 341FM-GC907RM primer set from unaffected carapace DNA was only successful for 2 samples. In most cases, either no amplification was observed or the quantities generated were too small to be loaded on a DGGE gel. However, amplification with the 27F-GC-355R primer set increased our sensitivity drastically. The lower sensitivity of the 341FM-GC-907RM primer set is likely the result of the multiple redundancies designed to broaden its amplification to all known bacterial groups, and there are more redundancies in this primer set than in the 27F-GC-355R primers. Furthermore, a Bacteroidetes-specific primer set (341-GC-721R) was used because it was determined that the more sensitive universal primers (27F-GC-355R) were not detecting this phylum efficiently. It has been well established that universal primers amplify 16S rDNA selectively and differentially from any given complex microbial community, even if in silico analyses indicates amplification of 16S rDNA from nearly all community members (SunHee et al. 2009). Thus, these primer sets were used to maximize sensitivity and minimize bias toward major groups in shell-disease lesions, and future studies should follow similar protocols.

Bacteroidetes

Flavobacteriaceae genera Aquimarina, Tenacibaculum, Polaribacter, Maribacter, and Cellulophaga have particularly high prevalence in lobster lesions. The most prominent Bacteroidetes in lobster lesions were representatives of the genus Aquimarina but only A. 'homaria' was detected in all analyzed lesions of ESD, ISD, and EnSD. It was, on occasion, found (45% analyzed samples) on unaffected surfaces but in much smaller numbers. A second species of Aquimarina (closely related to A. muelleri) was detected in most but not all lesions. Other Aquimarina species (Aquimarina latercula and Aquimarina intermedia) were present in lesions only occasionally. Similar results were obtained by Meres et al. (2012), who detected the same 3 Aquimarina spp. in both lesions and unaffected surfaces.

Originally, bacteria comprising genus Aquimarina were isolated from the Sea of Japan, which Nedashkovskaya et al. (2005) described as A. muelleri. Later, the same group (Nedashkovskaya et al. 2006) renamed and amended the genus Aquimarina with 3 more species: a newly described A. intermedia, a reclassified A. latercula (formerly known as Stanierella (Cytophaga) latercula) and Aquimarina brevivitae (formerly known as Gaetbulimicrobium brevivitae). Aquimarina macrocephali, recently isolated from sediment near a sperm whale carcass (Miyazaki et al. 2010), is the closest relative of A. 'homaria'.

Apart from the isolation and presence in marine sources and their ability to degrade various polymers of marine origin, little can be said about the physiological properties of Aquimarina spp. The bacterium A. intermedia was isolated from a sea urchin, and A. brevivitae was isolated from tidal flats. Not many details are available for the precise natural habitats of A. latercula and A. muelleri. We have shown previously that both A. muelleri and A. 'homaria' actively degrade raw chitin (Chistoserdov et al. 2005a, Chistoserdov et al. 2005b). The only other work that indicates the relevance of Aquimarina spp. to the shell diseases in crustaceans was that by Shultze et al. (2006). They identified the presence of several Aquimarina strains associated with the algae and the seawater in a marine hatchery in western Canada involved in rearing of several species of marine invertebrates. According to our sequence analysis, all strains from the work of Shultze et al. (2006) belong to A. muelleri (data not shown).

Flavobacteriaceae in ESD lesions are much more diverse than in other type of lesions. In addition to 8 major bands, several minor ubiquitous bands are present in Figure 3C. They may represent minor members of the Bacteroidetes communities or may be chimeric sequences. For example, Flavobacteriaceae species 1 sequence was shown to be chimeric, because specific primers designed against this sequence fail to amplify it from lesion community DNA (Quinn et al. 2012b). Because this is the only Bacteroidetes sequence found ubiquitously on unaffected surfaces and in lesions, it must be a chimera generated from bacteria commonly associated with lobsters.

Proteobacteria

Alphaproteobacteria represent the most diverse class of proteobacteria found in both lesions and unaffected surfaces. Three bacteria of this class appear to be ubiquitous in ESD lesions: the 'Thalassobius' sp., Sulfitobacter sp., and unclassified Rhodobacteraceae species 3 of which only 'Thalassobius' sp. was also present in ISD and EnSD lesions. Meres et al. (2012) also found an alphaproteobacterium common in both lesions and unaffected carapaces of lobsters with ESD, which they assigned to Jannaschia, a sister genus related closely to 'Thalassobius'. It is interesting that DISD lesions, in addition to A. 'homaria,' also harbored 2 ubiquitous Rhodobacteraceae: a unique species different from any other found associated with lobsters and 'Candidatus Kopriimonas aquarianus', which is the same bacterium detected in EnSD lesions in this study (Quinn et al. 2012b).

[FIGURE 4 OMITTED]

Two gammaproteobacteria, P. 'gracilis' and 'Candidatus Homarophilus dermatus', were especially prevalent in shelldisease lesions. The bacterium P. 'gracilis' has been shown to be associated with ESD lesions and has been shown to be chitinivorous (Chistoserdov et al. 2005b). Although ubiquitous in Long Island Sound and Buzzards Bay lobsters, it was not found in the Kittery, ME, samples, suggesting that its association with shell disease lesions does not have an obligatory nature. Moreover, during laboratory infection experiments, P. 'gracilis', unlike the 'Thalassobius' sp. and A. 'homaria,' did not infect mechanically breached carapace (Quinn et al. 2012a). The second gammaproteobacterium, 'Candidatus Homarophilus dermatus', found on unaffected surfaces and in all lesions is a bacterium only distantly related to but clearly distinct from the genus Cardiobacterium, in fact, it likely represents a novel order. Its closest relatives (at the genus family level) are bacteria isolated from the upper respiratory tract of marine mammals submitted by Johnson and colleagues to the GenBank (Bacterial Diversity of the Bottlenose Dolphin Upper Respiratory Tract, direct GenBank submission EU491713). Most likely, it corresponds to Cardiobacterium indentified by Meres et al. (2012).

[FIGURE 5 OMITTED]

CONCLUSIONS

Three bacteria--A. 'homaria,' 'Thalassobius' sp., and 'Candidatus Homarophilus dermatus'--were found to be associated consistently with shell-disease lesions of the American lobster. Of these, only Candidatus appears to be a bacterium commonly associated with both lesions and unaffected carapace surfaces. The latter becomes more dominant in lesions, particularly those for ISD and EnSD, in comparison with unaffected surfaces. Interestingly, it was not detected in either unaffected surfaces or DISD lesions of juvenile lobsters held in aquaria (Quinn et al. 2012b), suggesting that it is a common inhabitant of the shell of adult lobsters or exclusively wild lobsters. The bacteria A. 'homaria' and 'Thalassobius' sp. are dominant and appear obligatory in lobster shell lesions, but are detected only occasionally on unaffected surfaces. Therefore, these 2 bacteria stand out as potential shell-disease pathogens. Meres et al. (2012), using a pyrosequencing approach, detected these same (or at least closely related) 3 dominant bacteria and 55 other bacteria that play a role in discriminating between the disease and no sign of disease states. Using discriminant analysis, they concluded that the presence of Aquimarina spp. does not discriminate significantly between the diseased state and the healthy state. It is important to note that Meres et al. (2012) did not use individual Aquimarina species as independent entities in their discriminant analyses (i.e., the whole Aquimarina spp. group was treated as one), and they used the 27F-355R primer set, which, as this study has demonstrated, does not amplify Bacteroidetes efficiently. Moreover, the amplification step of multiple, closely related Flavobacteriaceae sequences (Tenacibaculum, Aquimarina, Maribacter, Cellulophaga, and as-yet-unclassified Flavobacteriaceae) present in lesions and unaffected shell microbial communities prior to pyrosequencing will most likely lead to multiple chimeric sequences that further confound the discriminant analyses. Similar problems may apply to the 'Thalassobius'--Jannaschia groups because of the vast abundance of marine Rhodobacteraceae on both unaffected and diseased shells.

Bell et al. (2012) did not find significant differences in the bacterial community composition between unaffected surfaces and lesions using the T-RFLP approach. However, one of the primers used in the study by Bell et al. (2012) does not amplify 16S rDNA efficiently from Flavobacteriaceae, a family found to be one of the most diverse and abundant in lesions and unaffected shells by Meres et al. (2012) and this study.

A limited number of samples with ISD (3 samples) and EnSD (2 samples) versus ESD (39 samples) and the limited number of sources from which these samples were obtained (1 for ISD, 1 for EnSD, and 4 for ESD) allow for limited generalizations about the differences between bacterial communities in lesions of various form of shell disease. The communities in the ISD and EnSD lesions appear simpler than that found in ESD lesions, but the major microbial players were all present. The microbial community in DISD lesions (Quinn et al. 2012b) was also more complex than that in ISD and EnSD lesions, and very different from that of other forms of shell disease. The bacterium A. 'homaria' was the only one of the 3 common ESD lesion bacteria found in DISD lesions.

Although we cannot identify specific pathogens without verification of Koch's postulates, the ubiquity and abundance of the 2 specific bacteria (A. 'homaria" and 'Thalassobius' sp.) in lesions is suggestive. It has been hypothesized that some form of damage to or weakening of the outer carapace (i.e., epicuticle), such as mechanical trauma, nutritional deficiency, or chemical damage, is needed for the development of shell disease (e.g., Castro et al. 2006, Tlusty & Metzler 2012, Tlusty et al. 2008, Laufer et al. 2012, Quinn et al. 2012a). This damage would lead to the colonization by various nonspecific shell-degrading bacteria, including those that normally reside on unaffected shell surfaces. However, this hypothesis does not explain completely the ubiquity of exactly the same 2 bacteria, A. 'homaria' and 'Thalassobius' sp., in all lesions. We predict that when a compromised host is not exposed to these 2 microorganisms (and 55% of unaffected lobsters that we tested lack at least A. 'homaria'), ESD shell lesions will not develop. The unaffected shell then serves as either one of the reservoirs for the 2 potential copathogens or their presence is merely transitory. We also speculate that an initial invasion by A. 'homaria' (Quinn et al. 2012b) and, in some forms of shell disease, the 'Thalassobius' sp., facilitates increased invasions by secondary organisms, including those normally found on the unaffected shell (Tenacibaculum sp., 'Candidatus Homarophilus dermatus' and some Rhodobacteraceae) and from the water column (P. 'gracilis' and marine Rhodobacteraceae) or sediment (Flavobacteriaceae), resulting in increased degeneration of the carapace. Perhaps the type of lesion that develops is dependent on the succession of microorganisms that join A. 'homaria' in the polymicrobial community.

Further research should be conducted to characterize environmental distribution and quantitative analyses of the bacterial flora found on the carapace as well as in various types of lesions. Furthermore, the lobster "biofilm" of unaffected carapace should be studied at various molt stages to determine when certain microorganisms colonize the lobster surface to understand more completely their role in shell disease versus normal biofilm progression. Verification of Koch's postulates would lead to a much deeper understanding of the disease and its transmission.

DOI: 10.2983/035.031.0205

ACKNOWLEDGMENTS

A gift of lobsters for this research by Thomas Angell (RI Department of Environmental Management), Kathy Castro (University of Rhode Island Fisheries Center), Bruce Estrella (MA Division of Marine Fisheries), Penelope Howell (Connecticut DEP), David Kaselouskas (Maine lobster fisherman), Carl LoBue (New York DEC, presently at the Nature Conservancy), Carl Wilson (ME Department of Marine Resources), is greatly appreciated.

This work was supported by the National Marine Fisheries Service as the "New England Lobster Research Initiative: Lobster Shell Disease" under NOAA grant NA06NMF4720100 to the University of Rhode Island Fisheries Center. The views expressed herein are those of the authors and do not necessarily reflect the views of NOAA or any of its sub-agencies. The US Government is authorized to produce and distribute reprints for government purposes, notwithstanding any copyright notation that may appear hereon.

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* Corresponding author. E-mail: ayc6160@louisiana.edu

ANDREI Y. CHISTOSERDOV, (1)* ROBERT A. QUINN, (1) SAI LAXMI GUBBALAt AND ROXANNA SMOLOWITZ (2)

(1) Department of Biology, University of Louisiana at Lafayette, 400 East St. Mary Blvd., Lafayette, LA 70504; (2) Department of Biology and Marine Biology, Roger Williams University, One Old Ferry Road, Bristol, RI 02809
TABLE 1.
Summary table of lobsters sampled by locations and
health status.

                   Shell Disease   Animals   Lobster
Location           Type              (n)     Identification

Eastern Long       Epizootic         14      1-4, 6-15
  Island Sound, NY
  (ELIS)
Central Long       Epizootic          5      15, 27-30
  Island Sound, NY
  (CLIS)
Buzzards Bay,      Epizootic         10      16-25
  MA (BB)
Kittery, ME (KME)  Epizootic         10      31-40
                   No signs of dise   3      41-43
Canada (CAN)       No signs of dise   3      44-46
Maine Aquarium     Impoundment        4      48-51
  (AME)
Rhode Island       Enzootic           2      56-57
  Trawl Survey (RITNo signs of dise   3      55,58-59
Rhode Island,      No signs of dise  20      88-107
  Narragansett
  Bay (RINB)

TABLE 2.
List and inferred phylogenies of common bacteria detected on lobsters
with no signs of shell disease.

                                                 Presence in DGGE Gels
                                                 and Amplified with
                                                 Percentage of the
                                                 Following Primer Set

Band   Nearest Blast Hit       Identity with     27F-GC/
       (accession no.)        Closest Relative     355R
                              for 27F-GC/355R     n = 13
                                  Product

C2     Uncultured bacterium           99             0
       clone EPR3968-08a-
       Bc46 (EU491713)

C4     Uncultured bacterium           96             12
       clone D8A8_187,
       (FJ959833)

C6     Tenacibaculum soleae           97             0
       (T) LL04 12.1.7
       (NR042630)

H1     Uncultured alpha               97             12
       proteobacterium
       LSBS-100, (AM745987)

H2     Uncultured bacterium           94             2
       clone 8910M7G#55
       (GU197410)

H3     Uncultured bacterium           97             2
       HAT3_417 (AB4776195)

H4     Uncultured alpha               98             5
       proteobacterium clone
       u22 (GQ472782)

H5     Uncultured bacterium           96             8
       clone SAV02C09
       (EU542143)

H6     Ahrensia kielensis             97             11
       strain D4006
       (FJ161247)

H7     Uncultured bacterium           96             2
       clone V2t17
       (FM165278)

H8     Uncultured bacterium           91             2
       clone W3-F09
       (FJ930752)

H9     Uncultured bacterium           98             6
       clone M0302D3
       (GU996583)

H10    Acidimicrobidae                90             2
       bacterium YM18-244
       (AB360344)

H11    Pibocella pond                 94             8
       strain KMM 6031 (NR
       025821)

H12    Uncultured bacterium           92             5
       clone PB 89
       (AM921301)

H13    Uncultured bacterium           98             0
       clone TetIect2H3
       (EU290243)

H14    Tenacibaculum                  96             0
       lutimaris strain
       ZS23 16S (HQ538772)

H15    Cellulophaga sp. Q             95             0
       Y3 (DQ311648)

                              Presence in DGGE Gels
                              and Amplified with
                              Percentage of the
                              Following Primer Set

Band   Nearest Blast Hit      341FM-GC/
       (accession no.)         CFB721R
                               n = 13

C2     Uncultured bacterium       4
       clone EPR3968-08a-
       Bc46 (EU491713)

C4     Uncultured bacterium
       clone D8A8_187,
       (FJ959833)

C6     Tenacibaculum soleae        3
       (T) LL04 12.1.7
       (NR042630)

H1     Uncultured alpha           NA
       proteobacterium
       LSBS-100, (AM745987)

H2     Uncultured bacterium       NA
       clone 8910M7G#55
       (GU197410)

H3     Uncultured bacterium       NA
       HAT3_417 (AB4776195)

H4     Uncultured alpha           NA
       proteobacterium clone
       u22 (GQ472782)

H5     Uncultured bacterium       NA
       clone SAV02C09
       (EU542143)

H6     Ahrensia kielensis         NA
       strain D4006
       (FJ161247)

H7     Uncultured bacterium        2
       clone V2t17
       (FM165278)

H8     Uncultured bacterium       NA
       clone W3-F09
       (FJ930752)

H9     Uncultured bacterium       NA
       clone M0302D3
       (GU996583)

H10    Acidimicrobidae            NA
       bacterium YM18-244
       (AB360344)

H11    Pibocella pond              1
       strain KMM 6031 (NR
       025821)

H12    Uncultured bacterium       NA
       clone PB 89
       (AM921301)

H13    Uncultured bacterium        3
       clone TetIect2H3
       (EU290243)

H14    Tenacibaculum               3
       lutimaris strain
       ZS23 16S (HQ538772)

H15    Cellulophaga sp. Q          3
       Y3 (DQ311648)

Band   Nearest Blast Hit       RDP Phylogeny
       (accession no.)

C2     Uncultured bacterium    Bacteroidetes; Flavobacteria;
       clone EPR3968-08a-      Flavobacteriales; unclassified
       Bc46 (EU491713)         Flavobacteriaceae, species 1

C4     Uncultured bacterium    Proteobacteria;
       clone D8A8_187,         Gammaproteobacteria,
       (FJ959833)              unclassified species 1
                               ('Candidatus Homarophilus
                               dermatus')

C6     Tenacibaculum soleae    Bacteroidetes, Flavobacteria,
       (T) LL04 12.1.7         Flavobacteriales,
       (NR042630)              Flavobacteriaceae,
                               Tenacibaculum soleae

H1     Uncultured alpha        Proteobacteria,
       proteobacterium         Alphaproteobacteria,
       LSBS-100, (AM745987)    Rhodobacterales,
                               Rhodobacteraceae,
                               Thalassobacter sp.

H2     Uncultured bacterium    Bacteroidetes; Flavobacteria;
       clone 8910M7G#55        Flavobacteriales; unclassified
       (GU197410)              Cryomorphaceae, species I

H3     Uncultured bacterium    Proteobacteria, unclassified
       HAT3_417 (AB4776195)    species 2

H4     Uncultured alpha        Proteobacteria,
       proteobacterium clone   Alphaproteobacteria,
       u22 (GQ472782)          Rhodobacterales,
                               Rhodobacteraceae,
                               Loktanella species 1

H5     Uncultured bacterium    Proteobacteria,
       clone SAV02C09          Alphaproteobacteria,
       (EU542143)              Rhodobacterales,
                               Rhodobacteraceae,
                               Loktanella species 2

H6     Ahrensia kielensis      Proteobacteria,
       strain D4006            Alphaproteobacteria,
       (FJ161247)              Rhodobacterales,
                               Rhodobacteraceae,
                               Ahrensia sp.

H7     Uncultured bacterium    Bacteroidetes; Flavobacteria;
       clone V2t17             Flavobacteriales; unclassified
       (FM165278)              Flavobacteriaceae, species 2

H8     Uncultured bacterium    Actinobacteria;
       clone W3-F09            Acidimicrobiales;
       (FJ930752)              unclassified Lamiaceae,
                               species 1

H9     Uncultured bacterium    Proteobacteria,
       clone M0302D3           Gammaproteobacteria,
       (GU996583)              Thiotrichales,
                               Thiotrichaceae,
                               Leucothrix sp.

H10    Acidimicrobidae         Actinobacteria;
       bacterium YM18-244      Actinobacteria;
       (AB360344)              Acidimicrobiales;
                               unclassified Lamiaceae,
                               species 2

H11    Pibocella pond          Bacteroidetes, Flavobacteria,
       strain KMM 6031 (NR     Flavobacteriales,
       025821)                 Flavobacteriaceae,
                               Pibocella sp.

H12    Uncultured bacterium    Proteobacteria;
       clone PB 89             Alphaproteobacteria;
       (AM921301)              Rhodobacterales; unclassified
                               Rhodobacteraceae, species 1

H13    Uncultured bacterium    Bacteroidetes; Flavobacteria;
       clone TetIect2H3        Flavobacteriales; unclassified
       (EU290243)              Flavobacteriaceae,
                               species 3

H14    Tenacibaculum           Bacteroidetes, Flavobacteria,
       lutimaris strain        Flavobacteria les,
       ZS23 16S (HQ538772)     Flavobacteriaceae,
                               Tenacibaculum species 1

H15    Cellulophaga sp. Q      Bacteroidetes, Flavobacteria,
       Y3 (DQ311648)           Flavobacteriales,
                               Flavobacteriaceae,
                               Cellulophaga species I

Band   Nearest Blast Hit       Gene Bank
       (accession no.)         Accession
                               No.

C2     Uncultured bacterium    JF904898
       clone EPR3968-08a-
       Bc46 (EU491713)

C4     Uncultured bacterium    JF904902
       clone D8A8_187,
       (FJ959833)

C6     Tenacibaculum soleae    JF904904
       (T) LL04 12.1.7
       (NR042630)

H1     Uncultured alpha        JF904915
       proteobacterium
       LSBS-100, (AM745987)

H2     Uncultured bacterium    JF904916
       clone 8910M7G#55
       (GU197410)

H3     Uncultured bacterium    JF904917
       HAT3_417 (AB4776195)

H4     Uncultured alpha        JF904918
       proteobacterium clone
       u22 (GQ472782)

H5     Uncultured bacterium    JF904919
       clone SAV02C09
       (EU542143)

H6     Ahrensia kielensis      JF904920
       strain D4006
       (FJ161247)

H7     Uncultured bacterium    JF904921
       clone V2t17
       (FM165278)

H8     Uncultured bacterium    JF904922
       clone W3-F09
       (FJ930752)

H9     Uncultured bacterium    JF904923
       clone M0302D3
       (GU996583)

H10    Acidimicrobidae         JF904924
       bacterium YM18-244
       (AB360344)

H11    Pibocella pond          JF904925
       strain KMM 6031 (NR
       025821)

H12    Uncultured bacterium    JF904926
       clone PB 89
       (AM921301)

H13    Uncultured bacterium    JF904927
       clone TetIect2H3
       (EU290243)

H14    Tenacibaculum           JF904928
       lutimaris strain
       ZS23 16S (HQ538772)

H15    Cellulophaga sp. Q      JF904929
       Y3 (DQ311648)

Band designations correspond to Figures 2, 3, 4, and 5.

TABLE 3.
List and inferred phylogenies of bacteria detected in ESD lesions.

                               Presence in DGGE Gels and Amplified
                               with the Following Primer Sets

Band   Nearest                     n = 39         n = 11
       Blast Hit               341FM-GC/907RM   27F-GC/355R
       (accession no.)

C1     Aquimarina                    39              8
       macrocephali
       (AB517144)

C2     Uncultured                    11              0
       bacterium clone
       EPR3968-08a-Bc46
       (EU491713)

C3     Octadecabacter sp.            11             11
       KS9-8 (FJ889525)

C4     Uncultured bacterium          9               9
       clone D8A8_187
       (FJ959833)

C5     Aquimarina muelleri           3               0
       strain 0-005
       (DQ812535)

C6     Tenacibaculum soleae          0               0
       (T) LL04 12.1.7
       (NR042630)

C7     Flavobacteriaceae             0               0
       bacterium ACEMC
       17-9 (FM 162927)

E1     Uncultured                    9               0
       Bacteroidetes
       bacterium clone
       131,677 (AY922211)

E2     Uncultured gamma              9               0
       proteobacterium
       PICO pp37
       (AJ969456)

E3     Pseudoalteromonas             29              4
       elyakovii strain
       ZH7 (HQ538749)
       Pseudoalteromonas
       "gracilis"
       (AF038846)

E4     Uncultured bacterium          0              11
       clone B11_10.4_1
       (FJ716885)

E5     Uncultured                    0              11
       Octadecabacter sp.
       clone Y-D5 16S
       (HQ727235)

E6     Maribacter sp.                0               0
       W-15 (HM584719)

E7     Cellulophaga fucicola         0               0
       strain WH10-2-3
       (HM473184)

E8     Polaribacter sp.              0               0
       JO 18
       (FJ425212)

                               Presence in DGGE Gels and Amplified
                               with the Following Primer Sets

Band   Nearest                      n = 14
       Blast Hit               341FM-GC/CFB721R
       (accession no.)

C1     Aquimarina                     14
       macrocephali
       (AB517144)

C2     Uncultured                     11
       bacterium clone
       EPR3968-08a-Bc46
       (EU491713)

C3     Octadecabacter sp.             NA
       KS9-8 (FJ889525)

C4     Uncultured bacterium           NA
       clone D8A8_187
       (FJ959833)

C5     Aquimarina muelleri            11
       strain 0-005
       (DQ812535)

C6     Tenacibaculum soleae           9
       (T) LL04 12.1.7
       (NR042630)

C7     Flavobacteriaceae              9
       bacterium ACEMC
       17-9 (FM 162927)

E1     Uncultured                     10
       Bacteroidetes
       bacterium clone
       131,677 (AY922211)

E2     Uncultured gamma               NA
       proteobacterium
       PICO pp37
       (AJ969456)

E3     Pseudoalteromonas              NA
       elyakovii strain
       ZH7 (HQ538749)
       Pseudoalteromonas
       "gracilis"
       (AF038846)

E4     Uncultured bacterium           NA
       clone B11_10.4_1
       (FJ716885)

E5     Uncultured                     NA
       Octadecabacter sp.
       clone Y-D5 16S
       (HQ727235)

E6     Maribacter sp.                 12
       W-15 (HM584719)

E7     Cellulophaga fucicola          8
       strain WH10-2-3
       (HM473184)

E8     Polaribacter sp.               11
       JO 18
       (FJ425212)

Band   Nearest                 RDP                         GenBank
       Blast Hit               Phylogeny                   Accession
       (accession no.)                                     No.

C1     Aquimarina              Bacteroidetes,              JF904894,
       macrocephali            Flavobacteria,              JF904895,
       (AB517144)              Flavobacteriales,           JF904896
                               Flavobacteriaceae,
                               Aquimarina
                               (A. 'homaria')

C2     Uncultured              Bacteroidetes;              JF904897,
       bacterium clone         Flavobacteria;              JF904898
       EPR3968-08a-Bc46        Flavobacteriales;
       (EU491713)              unclassified
                               Flavobacteriaceae,
                               species 1

C3     Octadecabacter sp.      Proteobacteria;             JF904899,
       KS9-8 (FJ889525)        Alphaproteobacteria;        JF904900
                               Rhodobacterales;
                               unclassified
                               Rhodobacteraceae,
                               species 2
                               ('Thalassobius' sp.)

C4     Uncultured bacterium    Proteobacteria;             JF904901,
       clone D8A8_187          Gammaproteobacteria,        JF904902
       (FJ959833)              unclassified species 1
                               ('Candidatos Homarophilus
                               dermatus')

C5     Aquimarina muelleri     Bacteroidetes,              JF904903
       strain 0-005            Flavobacteria,
       (DQ812535)              Flavobacteriales,
                               Flavobacteriaceae,
                               Aquimarina sp.

C6     Tenacibaculum soleae    Bacteroidetes,              JF904904
       (T) LL04 12.1.7         Flavobacteria,
       (NR042630)              Flavobacteriales,
                               Flavobacteriaceae,
                               Tenacibaculum soleae

C7     Flavobacteriaceae       Bacteroidetes,              JF904905
       bacterium ACEMC         Flavobacteria,
       17-9 (FM 162927)        Flavobacteriales,
                               Flavobacteriaceae,

                               Tenacibaculum species 2
E1     Uncultured              Bacteroidetes,              JF904906,
       Bacteroidetes           Flavobacteria,              JF904907
       bacterium clone         Flavobacteriales,
       131,677 (AY922211)      Flavobacteriaceae,
                               Polaribacter sp.

E2     Uncultured gamma        Proteobacteria,             JF904908
       proteobacterium         Gammaproteobacteria,
       PICO pp37               Alteromonadales,
       (AJ969456)              Psychromonadaceae,
                               Psychromonas sp.

E3     Pseudoalteromonas       Proteobacteria,             JF904909
       elyakovii strain        Gammaproteobacteria,
       ZH7 (HQ538749)          Alteromonadales,
       Pseudoalteromonas       Pseudoalteromonadaceae,
       "gracilis"              Pseudoalteromonas sp.
       (AF038846)              (P. 'gracilis')

E4     Uncultured bacterium    Proteobacteria;             JF904910
       clone B11_10.4_1        Alphaproteobacteria;
       (FJ716885)              Rhodobacterales;
                               unclassified
                               Rhodobacteraceae,
                               species 3

E5     Uncultured              Proteobacteria,             JF904911
       Octadecabacter sp.      Alphaproteobacteria,
       clone Y-D5 16S          Rhodobacterales,
       (HQ727235)              Rhodobacteraceae,
                               Sulfztobacter sp.

E6     Maribacter sp.          Bacteroidetes,              JF904912
       W-15 (HM584719)         Flavobacteria,
                               Flavobacteriales,
                               Flavobacteriaceae,
                               Maribacter species 1

E7     Cellulophaga fucicola   Bacteroidetes,              JF904913
       strain WH10-2-3         Flavobacteria,
       (HM473184)              Flavobacteriales,
                               Flavobacteriaceae,
                               Cellulophaga species 2

E8     Polaribacter sp.        Bacteroidetes,              JF904914
       JO 18                   Flavobacteria,
       (FJ425212)              Flavobacteriales,
                               Flavobacteriaceae,
                               Polaribacter sp.

Band designations correspond to Figures 2, 3, 4, and 5.

TABLE 4.
List and inferred phylogenies of bacteria detected in ISD lesions.
Presence in DGGE Gels and Amplified
with the Following Primer sets

Band   Nearest Blast              n = 4           n = 3

       Hit (accession no.)    341FM-GC/907RM   27F-GC/355R

C1     Aquimarina                   4               2
       macrocephali
       (AB517144)

C2     Uncultured                   0               0
       bacterium clone
       EPR3968-08a-Bc46
       (EU491713)

C3     Octadecabacter               0               3
       sp. KS9-8
       (FJ889525)

C4     Uncultured bacterium         4               3
       clone D8A8_187
       (FJ959833)

C5     Aquimarina                   0               0
       muelleri strain
       0-005 (DQ812535)

C6     Tenacibaculum                0               0
       soleae (T)
       LL04 12.1.7

       (NR042630)

I1     Loktanella                   0               2
       koreensis strain
       GA2-M3 (DQ344498)

12     Tenacibaculum                0               0
       ovolyticum
       clone SE1 (AY771741)

Band   Nearest Blast               n = 3

       Hit (accession no.)    341FM-GC/CFB721R

C1     Aquimarina                    3
       macrocephali
       (AB517144)

C2     Uncultured                    3
       bacterium clone
       EPR3968-08a-Bc46
       (EU491713)

C3     Octadecabacter                NA
       sp. KS9-8
       (FJ889525)

C4     Uncultured bacterium          NA
       clone D8A8_187
       (FJ959833)

C5     Aquimarina                    2
       muelleri strain
       0-005 (DQ812535)

C6     Tenacibaculum                 2
       soleae (T)
       LL04 12.1.7
       (NR042630)

I1     Loktanella                    NA
       koreensis strain
       GA2-M3 (DQ344498)

12     Tenacibaculum                 3
       ovolyticum
       clone SE1 (AY771741)

Band   Nearest Blast          RDP Phylogeny

       Hit (accession no.)

C1     Aquimarina             Bacteroidetes, Flavobacteria,
       macrocephali           Flavobacteriales,
       (AB517144)             Flavobacteriaceae,
                              Aquimarina (A. 'homaria')

C2     Uncultured             Bacteroidetes; Flavobacteria;
       bacterium clone        Flavobacteriales; unclassified
       EPR3968-08a-Bc46       Flavobacteriaceae, species 1
       (EU491713)

C3     Octadecabacter         Proteobacteria;
       sp. KS9-8              Alphaproteobacteria;
       (FJ889525)             Rhodobacterales;
                              unclassified
                              Rhodobacteraceae,
                              species 2
                              ('Thalassobius' sp.)

C4     Uncultured bacterium   Proteobacteria;
       clone D8A8_187         Gammaproteobacteria,
       (FJ959833)             unclassified species 1
                              ('Candidates
                              Homarophilus dermatus')

C5     Aquimarina             Bacteroidetes, Flavobacteria,
       muelleri strain        Flavobacteriales,
       0-005 (DQ812535)       Flavobacteriaceae,
                              Aquimarina sp.

C6     Tenacibaculum          Bacteroidetes, Flavobacteria,
       soleae (T)             Flavobacteriales,
       LL04 12.1.7            Flavobacteriaceae,
       (NR042630)             Tenacibaculum soleae

I1     Loktanella             Proteobacteria,
       koreensis strain       Alphaproteobacteria,
       GA2-M3 (DQ344498)      Rhodobacterales,
                              Rhodobacteraceae,
                              Loktanella species 3
12     Tenacibaculum          Bacteroidetes, Flavobacteria,
       ovolyticum             Flavobacteriales,
       clone SE1 (AY771741)   Flavobacteriaceae,
                              Tenacibaculum species 2

Band   Nearest Blast          GenBank
                              Accession
                              No.
       Hit (accession no.)

C1     Aquimarina             JF904894,
       macrocephali           JF904895,
       (AB517144)             JF904896

C2     Uncultured             JF904897,
       bacterium clone        JF904898
       EPR3968-08a-Bc46
       (EU491713)

C3     Octadecabacter         JF904899,
       sp. KS9-8              JF904900
       (FJ889525)

C4     Uncultured bacterium   JF904901,
       clone D8A8_187         JF904902
       (FJ959833)

C5     Aquimarina             JF904903
       muelleri strain
       0-005 (DQ812535)

C6     Tenacibaculum          JF904904
       soleae (T)
       LL04 12.1.7
       (NR042630)

I1     Loktanella             JF904930
       koreensis strain
       GA2-M3 (DQ344498)

12     Tenacibaculum          JF904931
       ovolyticum
       clone SE1 (AY771741)

Band designations correspond to Figures 2, 3, 4, and 5.

TABLE 5.
List and inferred phylogenies of bacteria detected in EnSD lesions.

                                Presence in DGGE Gels and Amplified
                                with the Following Primer Sets

Band   Nearest                      n = 2           n = 2
       Blast Hit                341FM-GC/907RM   27F-GC/355R
       (accession no.)

C1     Aquimarina                     2               2
       macrocephali
       (AB517144)

C2     Uncultured bacterium           1               0
       clone EPR3968-08a-Bc46
       (EU491713)

C3     Octadecabacter sp.             0               1
       KS9-8
       (FJ889525)

C4     Uncultured bacterium           2               2
       clone D8A8-187
       (FJ959833)

C5     Aquimarina muelleri            0               0
       strain
       0-005 (DQ812535)

C7     Flavobacteriaceae              1               0
       bacterium ACEMC 17-9
       (FM 162927)

N1     Uncultured bacterium           0               2
       clone
       PropaneSIP20-6-30
       (GU584816)

N2     Koprumonas                     0               2
       byunsanensis strain
       KOPRI 13,522
       (DQ 167245)

N3     Uncultured                     0               0
       Bacteroidetes clone
       MSB-3B9 (DQ811908)

N4     Maribacter sp.                 0               0
       MOLA 57 (AM990832)

                                Presence in DGGE Gels and Amplified
                                with the Following Primer Sets

Band   Nearest                       n = 2
       Blast Hit                341FM-GC/CFB721R
       (accession no.)

C1     Aquimarina                      2
       macrocephali
       (AB517144)

C2     Uncultured bacterium            2
       clone EPR3968-08a-Bc46
       (EU491713)

C3     Octadecabacter sp.              NA
       KS9-8
       (FJ889525)

C4     Uncultured bacterium            NA
       clone D8A8-187
       (FJ959833)

C5     Aquimarina muelleri             1
       strain
       0-005 (DQ812535)

C7     Flavobacteriaceae               1
       bacterium ACEMC 17-9
       (FM 162927)

N1     Uncultured bacterium            NA
       clone
       PropaneSIP20-6-30
       (GU584816)

N2     Koprumonas                      NA
       byunsanensis strain
       KOPRI 13,522
       (DQ 167245)

N3     Uncultured                      2
       Bacteroidetes clone
       MSB-3B9 (DQ811908)

N4     Maribacter sp.                  2
       MOLA 57 (AM990832)

Band   Nearest                  RDP Phylogeny
       Blast Hit
       (accession no.)

C1     Aquimarina               Bacteroidetes, Flavobacteria,
       macrocephali             Flavobacteriales,
       (AB517144)               Flavobacteriaceae,
                                Aquimarina (A. 'homaria')

C2     Uncultured bacterium     Bacteroidetes; Flavobacteria;
       clone EPR3968-08a-Bc46   Flavobacteriales; unclassified
       (EU491713)               Flavobacteriaceae, species 1

C3     Octadecabacter sp.       Proteobacteria;
       KS9-8                    Alphaproteobacteria;
       (FJ889525)               Rhodobacterales; unclassified
                                Rhodobacteraceae, species 2
                                (Thalassobius' sp.)

C4     Uncultured bacterium     Proteobacteria;
       clone D8A8-187           Gammaproteobacteria,
       (FJ959833)               unclassified species 1
                                (Candidatos Homarophilus
                                dermatus')

C5     Aquimarina muelleri      Bacteroidetes, Flavobacteria,
       strain                   Flavobacteriales,
       0-005 (DQ812535)         Flavobacteriaceae,
                                Aquimarina sp.

C7     Flavobacteriaceae        Bacteroidetes, Flavobacteria,
       bacterium ACEMC 17-9     Flavobacteriales,
       (FM 162927)              Flavobacteriaceae,
                                Tenacibaculum species 1

N1     Uncultured bacterium     Proteobacteria;
       clone
       PropaneSIP20-6-30        Alphaproteobacteria;
       (GU584816)               Rhodobacterales;
                                unclassified
                                Rhodobacteraceae, species 4

N2     Koprumonas               Proteobacteria,
       byunsanensis strain      Alphaproteobacteria,
       KOPRI 13,522             Rhodobacterales,
       (DQ 167245)              Rhodobacteraceae,
                                Kilionella ('Candidates
                                Kopriimonas aquarianus')

N3     Uncultured               Bacteroidetes; Flavobacteria;
       Bacteroidetes clone      Flavobacteriales;
       MSB-3B9 (DQ811908)       unclassified
                                Flavobacteraceae, species 3

N4     Maribacter sp.           Bacteroidetes, Flavobacteria,
       MOLA 57 (AM990832)       Flavobacteriales,
                                Flavobacteriaceae,
                                Maribacter species 2

Band   Nearest                  GenBank
       Blast Hit                Accession
       (accession no.)          No.

C1     Aquimarina               JF904894,
       macrocephali             JF904895,
       (AB517144)               JF904896

C2     Uncultured bacterium     JF904897,
       clone EPR3968-08a-Bc46   JF904898
       (EU491713)

C3     Octadecabacter sp.       JF904899,
       KS9-8                    JF904900
       (FJ889525)

C4     Uncultured bacterium     JF904901,
       clone D8A8-187           JF904902
       (FJ959833)

C5     Aquimarina muelleri      JF904903
       strain
       0-005 (DQ812535)

C7     Flavobacteriaceae        JF904905
       bacterium ACEMC 17-9
       (FM 162927)

N1     Uncultured bacterium     N/A
       clone
       PropaneSIP20-6-30
       (GU584816)

N2     Koprumonas               JF904932
       byunsanensis strain
       KOPRI 13,522
       (DQ 167245)

N3     Uncultured               JF904933
       Bacteroidetes clone
       MSB-3B9 (DQ811908)

N4     Maribacter sp.           JF904934
       MOLA 57 (AM990832)

Band designations correspond to Figures 2, 3, 4, and 5.
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