Stephen A. Bullard

Diversity of the skin microbiota of fishes: evidence for host species specificity

Skin microbiota of Gulf of Mexico fishes were investigated by ribosomal internal spacer analysis (RISA) and 16S rRNA gene sequencing. A total of 102 fish specimens representing six species (Mugil cephalus, Lutjanus campechanus, Cynoscion nebulosus, Cynoscion arenarius, Micropogonias undulatus, and Lagodon rhomboides) were sampled at regular intervals throughout a year. The skin microbiota from each individual fish was analyzed by RISA and produced complex profiles with 23 bands on average. Similarities between RISA profiles ranged from 97.5% to 4.0%. At 70% similarity, 11 clusters were defined, each grouping individuals from the same fish species. Multidimensional scaling and analysis of similarity correlated the RISA-defined clusters with geographic locality, date, and fish species. Global R values indicated that fish species was the most indicative variable for group separation. Analysis of 16S rRNA gene sequences (from pooled samples of 10 individual fish for each fish species) showed that the Proteobacteria was the predominant phylum in skin microbiota, followed by the Firmicutes and the Actinobacteria. The distribution and abundance of bacterial sequences were different among all species analyzed. Aeribacillus was found in all fish species representing 19% of all clones sequenced, while some genera were fish species-specific (Neorickettsia in M. cephalus and Microbacterium in L. campechanus). Our data provide evidence for the existence of specific skin microbiota associated with particular fish species.

KUDOA HYPOEPICARDIALIS N. SP. (MYXOZOA: KUDOIDAE) AND ASSOCIATED LESIONS FROM THE HEART OF SEVEN PERCIFORM FISHES IN THE NORTHERN GULF OF MEXICO

Kudoa hypoepicardialis n. sp. infects the space between the epicardium and the compact myocardium and, in intense infections, the pericardial chamber of man-of-war fish (Nomeus gronovii) (Nomeidae) (the type host), blue runner (Caranx crysos) (Carangidae), Warsaw grouper (Epinephelus nigritus) (Serranidae), Atlantic tripletail (Lobotes surinamensis) (Lobotidae), northern red snapper (Lutjanus campechanus) (Lutjanidae), black drum (Pogonias cromis) (Sciaenidae), and bluefish (Pomatomus saltatrix) (Pomatomidae) in the northern Gulf of Mexico. This is the first report of a Kudoa sp. from the heart of a fish in the Gulf of Mexico, and of these hosts, only the bluefish was previously identified as a host for a species of Kudoa. Spores of the new species varied slightly in size among these hosts but were regarded as conspecific based on their nearly identical (99.9%) small-subunit (SSU) ribosomal DNA (rDNA) sequence. The new species differs both from the 4 nominal species of Kudoa reported from fishes in the Gulf of Mexico and from K. pericardialis, an allopatric species that infects the pericardial cavity, by the combination of having a large spore, a small polar capsule, and a polar filament with a single coil. The new species is morphologically and genetically most similar to K. shiomitsui, an allopatric species that infects the heart and pericardial cavity, but is distinguished from it based on a 4.2% difference in the SSU rDNA sequence. Heart lesions primarily were restricted to the vicinity of plasmodia and included a layer of fibrinous inflammation characterized by lymphocytes, macrophages, and granulomas as well as epithelioid encapsulations around plasmodia. Heavily infected hosts had melanin-like deposits and adipose cells beneath the epicardium, and the epicardium was discontinuous and apparently breached by plasmodia in some regions. Cardiac muscle, gill, liver, spleen, intestine, and kidney were normal.

Characterization of a diversity of tetraphyllidean and rhinebothriidean cestode larval types, with comments on host associations and life-cycles.

Life-cycles of marine tapeworms of the orders Tetraphyllidea and Rhinebothriidea are poorly known primarily because their larvae typically lack species level, taxonomically distinguishing adult characteristics and using morphology they can be identified to genus, family or order only. This large-scale study conducted in the northern Gulf of Mexico includes adult cestodes (25 species) collected from sharks and rays (Elasmobranchii, eight species) and larval cestodes (27 species) collected from teleosts (Neopterygii, 46 species), bivalves and gastropods (Mollusca, 24 species), and shrimps (Crustacea, five species), comprising a phylogenetically (75 species in three phyla, 14 orders and 46 families) and ecologically (e.g., benthic, epibenthic, pelagic, euryhaline, stenohaline) diverse array of hosts of larval cestode. Molecular biology and morphology informed larval identification and facilitated the circumscription of suites of morphological features representing distinct larval types (i.e., collective groups). A total of 198 specimens comprising adult and larval tetraphyllideans and rhinebothriideans assigned to 12 genera were characterized for the partial (D1-D3) lsrDNA gene and analyzed separately and in combination with data derived from species belonging to an additional 21 genera available from GenBank. Eight larval types were identified and matched to one or several genera of Tetraphyllidea or Rhinebothriidea; morphological variation within these larval types was also documented. In combination with published reports of unique larval morphologies, 15 larval types were established and a key to their larvae presented. Overall, teleosts figured prominently in the life-cycles of tetraphyllideans and rhinebothriideans. Intermediate host specificity at the level of cestode genus was euryxenous, but limited host records suggest that host specificity at the level of cestode species may be more strict. To our knowledge, this is the first published study that approaches the elucidation of marine tapeworm life-cycles by incorporating morphological, molecular biological and phylogenetic methods using specimens collected on a regional scale and from wild-caught hosts from four metazoan phyla.

Catfish hybrid Ictalurus punctatus × I. furcatus exhibits higher resistance to columnaris disease than the parental species

We present experimental data on susceptibility to columnaris disease, caused by the bacterium Flavobacterium columnare, in hybrid catfish (female channel catfish Ictalurus punctatus × male blue catfish I. furcatus) (C×B). Under our experimental conditions, C×B hybrids were significantly more resistant to columnaris disease caused by the highly virulent strain of F. columnare BGFS-27 (genomovar II) than channel catfish and blue catfish. Channel and blue catfish cumulative mortalities after immersion challenge were 74 and 87%, respectively, whereas mortality in the C×B hybrid was 31%. Susceptibility to the strain ARS-1 (genomovar I) was lower among all catfishes, although channel catfish was the least resistant species at 32% cumulative mortality. By contrast, C×B hybrid and blue catfishes were strongly resistant to the ARS-1 strain, with <10% mortality. Our data suggest enhanced disease resistance of the C×B hybrid to columnaris disease.

Adhesion dynamics of Flavobacterium columnare to channel catfish Ictalurus punctatus and zebrafish Danio rerio after immersion challenge

The adhesion dynamics of Flavobacterium columnare to fish tissues were evaluated in vivo by immersion challenge followed by bacterial plate count and confirmatory observations of gill-adhered bacterial cells using scanning electron microscopy. Adhesion of F. columnare genomovar I (ARS-1) and II (BGFS-27) strains to skin and gill of channel catfish Ictalurus punctactus and gill of zebrafish Danio rerio was compared. At 0.5 h post-challenge, both strains adhered to gill of channel catfish at comparable levels (10(6) colony forming units [CFU] g(-1)), but significant differences in adhesion were found later in the time course. Channel catfish was able to effectively reduce ARS-1 cells on gill, whereas BGFS-27 persisted in gill beyond the first 24 h post-challenge. No significant difference was found between both strains when adhered to skin, but adhered cell numbers were lower (10(3) CFU g(-1)) than those found in gill and were not detectable at 6 h post-challenge. Adhesion of BGFS-27 cells to gill of zebrafish also occurred at high numbers (> 10(6) CFU g(-1)), while only < 10(2) CFU g(-1) of ARS-1 cells were detected in this fish. The results of the present study show that particular strains of F. columnare exhibit different levels of specificity to their fish hosts and that adhesion to fish tissues is not sufficient to cause columnaris disease.

TWO NEW SPECIES OF CARDICOLA (DIGENEA: SANGUINICOLIDAE) IN DRUMS (SCIAENIDAE) FROM MISSISSIPPI AND LOUISIANA

Two new species of Cardicola (Digenea: Sanguinicolidae) are described from the heart of drums (Perciformes: Sciaenidae) in and off Mississippi and Louisiana. Cardicola palmeri n. sp. infects the black drum, Pogonias cromis, and, in mature individuals, is distinguished from its congeners by the combination of a body 1.9–2.9 times longer than wide, an esophagus 44–52% of the body length, posterior ceca 1.0–1.8 times longer than the anterior ones, a gland encircling the seminal vesicle that is 30–37% of maximum body width in diameter, and a sinistral and nearly medial male pore. Cardicola currani n. sp. infects the red drum, Sciaenops ocellatus, and is distinguished from its congeners by the combination of posterior ceca 1.8–4.2 times longer than anterior ones, a male pore located well posterior to the ootype, a female pore and transverse metraterm located just anterior to the level of the ootype, and a medial and posttesticular ovary located at the level of the terminal ends of the posterior ceca. No adult blood fluke had been described previously from the northern Gulf of Mexico west of Florida. Each of the 2 flukes infected only 1 host species despite the hosts being sympatric.

High Numbers of Vibrio vulnificus in Tar Balls Collected from Oiled Areas of the North-Central Gulf of Mexico Following the 2010 BP Deepwater Horizon Oil Spill

The Deepwater Horizon Oil Spill was the largest oil spill in USA history releasing approximately 4.9 million barrels of crude oil into the Gulf of Mexico. Soon after the spill started, tar balls and other forms of weathered oil appeared in large numbers on beaches in Mississippi and Alabama. In this study, we analyzed tar balls for total aerobic bacterial (TAB) counts and also for the presence of Vibrio vulnificus, a human pathogen known to be abundant in the Gulf Coast environment and capable of causing severe wound infections by contact with contaminated surfaces. Our results showed that TAB counts were significantly higher in tar balls than in sand and seawater collected at the same location. In addition, V. vulnificus numbers were 10× higher in tar balls than in sand and up to 100× higher than in seawater. Densities of V. vulnificus were higher than 105 colony forming units/g of tar ball in all samples analyzed. Our data suggest that tar balls can act as reservoirs for bacteria including human pathogens.

Historical account of the two family-group names in use for the single accepted family comprising the “fish blood flukes”

The family-group name for the “fish blood flukes” is unstable, with both “Aporocotylidae Odhner, 1912” and “Sanguinicolidae von Graff, 1907” in use for the single family. Although “Sanguinicolidae von Graff, 1907” (or. Graff, 1907.) has been a widely-accepted family-group name for the fish blood flukes subsequent to Yamaguti’s 1954 and 1958 synoptical publications (“Systema Helminthum”), a critical examination of the relevant literature, much of it published in German during 1900 through 1926, reveals that “Aporocotylidae Odhner, 1912” is the earliest available family-group name for these flukes. The name Aporocotylidae, moreover, was in wide usage by alpha taxonomists before 1954 and by several authors between 1954 and the present time. We speculate that the recent long-standing uncertainty about the earliest available family-group name primarily stems from the (1) logistics of Ludwig von Graff’s tome published in 1904–1908, (2) bibliographic confusion between that work and another Graff work published in 1907 (both of which treat Sanguinicola but not Aporocotyle), (3) initial ambiguity regarding the phylogenetic relatedness of the first four aporocotylid species that were named, (4) lack of consensus on the status of Aporocotylidae and Sanguinicolidae and the genera included within them, and (5) misleading application of. Graff, 1907. to Sanguinicolidae by Poche in 1926, Fuhrmann in 1928, Yamaguti in 1954 and 1958, and subsequent review articles that treated fish blood flukes. Under the International Code of Zoological Nomenclature (ICZN, Article 8.3), “Sanguinicolidae” was not made available by Graff because he disclaimed the name in the same, and only, work wherein he used the name (ICZN, Article 8.3). Sanguinicolidae was first made available in 1926 by Poche, who referenced Graff’s 1907 work. Hence, “Poche, 1926” comprises the correct authority and date for that family-group name, not “von Graff, 1907” or “Graff, 1907”. Since we presently accept only a single family for all fish blood flukes and abide by the Principal of Priority (ICZN, Article 23), we herein regard Sanguinicolidae Poche, 1926 as a junior subjective synonym of Aporocotylidae Odhner, 1912.

Cardicola forsteri (Digenea: Sanguinicolidae) from the Heart of a Northern Bluefin Tuna, Thunnus thynnus (Scombridae), in the Northwest Atlantic Ocean

Abstract We report a specimen of Cardicola forsteri Cribb, Daintith, and Munday, 2000 (Digenea: Sanguinicolidae) from the lumen of the heart of a northern bluefin tuna, Thunnus thynnus (Linnaeus, 1758) (Scombridae), that was 218 cm in total length (TL) and caught in the northwest Atlantic Ocean 12 km south of Cape Lookout, North Carolina. The hearts of 12 similarly sized northern bluefin tuna (127–262 cm TL) from Georges Bank, northwest Atlantic Ocean, were not infected. This is the first report of C. forsteri from a wild host and of a sanguinicolid from any scombrid in the northwest Atlantic Ocean. Bluefin tuna is cultured in sea cages because of its highly prized flesh, and the fact that a blood fluke infects this host is significant because some blood flukes have been identified as serious pathogens of cage-cultured fish.

Community Structure of Skin Microbiome of Gulf Killifish, Fundulus grandis, Is Driven by Seasonality and Not Exposure to Oiled Sediments in a Louisiana Salt Marsh

Mucus of fish skin harbors complex bacterial communities that likely contribute to fish homeostasis. When the equilibrium between the host and its external bacterial symbionts is disrupted, bacterial diversity decreases while opportunistic pathogen prevalence increases, making the onset of pathogenic bacterial infection more likely. Because of that relationship, documenting temporal and spatial microbial community changes may be predictive of fish health status. The 2010 Deepwater Horizon oil spill was a potential stressor to the Gulf of Mexico’s coastal ecosystem. Ribosomal intergenic spacer analysis (RISA) and pyrosequencing were used to analyze the bacterial communities (microbiome) associated with the skin and mucus of Gulf killifish (Fundulus grandis) that were collected from oiled and non-oiled salt marsh sites in Barataria Bay, LA. Water samples and fin clips were collected to examine microbiome structure. The microbiome of Gulf killifish was significantly different from that of the surrounding water, mainly attributable to shifts in abundances of Cyanobacteria and Proteobacteria. The Gulf killifish’s microbiome was dominated by Gammaproteobacteria, specifically members of Pseudomonas. No significant difference was found between microbiomes of fish collected from oiled and non-oiled sites suggesting little impact of oil contamination on fish bacterial assemblages. Conversely, seasonality significantly influenced microbiome structure. Overall, the high similarity observed between the microbiomes of individual fish observed during this study posits that skin and mucus of Gulf killifish have a resilient core microbiome.