Submitted 20 January 2017Accepted 10 January 2018Published 15 February 2018

Corresponding authorEdna J Marquezejmarqueunaleduco

Academic editorMauricio Rodriguez-Lanetty

Additional Information andDeclarations can be found onpage 12

DOI 107717peerj4307

Copyright2018 Cuartas et al

Distributed underCreative Commons CC-BY 40

OPEN ACCESS

Metagenomic analysis of orange coloredprotrusions from the muscle of QueenConch Lobatus gigas (Linnaeus 1758)Jaison H Cuartas1 Juan F Alzate2 Claudia X Moreno-Herrera1 andEdna J Marquez1

1 Facultad de Ciencias Laboratorio de Biologiacutea Molecular y Celular Universidad Nacional de ColombiaMedelliacuten Antioquia Colombia

2 Facultad de Medicina Centro Nacional de Secuenciacioacuten Genoacutemica Universidad de Antioquia MedelliacutenAntioquia Colombia

ABSTRACTThe endangeredmarine gastropod Lobatus gigas is an important fishery resource in theCaribbean regionMicrobiological and parasitological research of this species have beenpoorly addressed despite its role in ecological fitness conservation status andpreventionof potential pathogenic infections This study identified taxonomic groups associatedwith orange colored protrusions in the muscle of queen conchs using histologicalanalysis 454 pyrosequencing and a combination of PCR amplification and automatedSanger sequencing The molecular approaches indicate that the etiological agent of themuscle protrusions is a parasite belonging to the subclass Digenea Additionally thescope of the molecular technique allowed the detection of bacterial and fungi clades inthe assignment analysis This is the first evidence of a digenean infection in the muscleof this valuable Caribbean resource

Subjects Biodiversity Genomics Marine Biology ParasitologyKeywords Microbiology Parasitic infection 454 pyrosequencing Bioinformatics Trematode

INTRODUCTIONThe queen conch Lobatus gigas is an endangeredmarine gastropod of great socioeconomiccultural and ecological importance in the Caribbean region This species was included inAppendix II of the Convention on International Trade in Endangered Species ofWild Faunaand Flora (CITES) in 1992 and the Red List of the International Union for Conservation ofNature (IUCN) in 1994 Despite these regulations natural stocks of this species continue todecline (Theile 2001 Aldana 2003) likely by the loss of breeding habitats and detrimentalhuman activities such as overfishing (Glazer amp Quintero 1998 Aldana 2003)

Compared with studies in basic biology (Randall 1964) fisheries (Brownell amp Stevely1981 Theile 2001 Prada et al 2009) and genetics (Mitton Berg Jr amp Orr 1989 Tello-Cetina Rodriacuteguez-Gil amp Rodriacuteguez-Romero 2005 Zamora-Bustillos et al 2011 Maacuterquez etal 2013) parasitological and microbial studies of L gigas are less explored (Acosta et al2009 Aldana et al 2011 Rodriguez Hariharan amp Nimrod 2011 Peacuterez et al 2014) So faronly one parasitic infection with Apicomplexa coccidian protozoon has been reportedin L gigas (Baqueiro et al 2007 Aldana et al 2009 Aldana et al 2011 Gros Frenkiel amp

How to cite this article Cuartas et al (2018) Metagenomic analysis of orange colored protrusions from the muscle of Queen Conch Lo-batus gigas (Linnaeus 1758) PeerJ 6e4307 DOI 107717peerj4307

Aldana 2009 Volland et al 2010) Similarly only three published studies report theassociation between L gigas and bacteria of the family Vibrionaceae (Acosta et al 2009)the phyla Firmicutes Proteobacteria Actinobacteria (Peacuterez et al 2014) as well as potentialbacterial pathogens (Rodriguez Hariharan amp Nimrod 2011) Two recent investigationshave also studied the symbiotic association of L gigas with dinoflagellates of the genusSymbiodinium (Banaszak Ramos amp Goulet 2013 Garciacutea Ramos amp Banaszak 2014)

Moreover an unknown etiological agent sporadically produces orange coloredprotrusions in the muscle of L gigas in the Colombian San Andres archipelago However itremains to be elucidated whether such lesions are caused by different agents and posteriorlycolonized by pigment-producing microorganisms or digenean infections as found inother marine gastropods Specifically the infections of Cercaria parvicaudata and Renicolaroscovitahave been reported to produce orangelemon colored sporocysts in different tissuesof Littorina snails (Stunkard 1950 Galaktionov amp Skirnisson 2000) whereas Renicolathaidus has been found infecting Nucella lapillus (Galaktionov amp Skirnisson 2000) Thesetrematodes C parvicaudata R roscovita and R thaidus are considered synonymous basedon morphological similarities and cercariae size parameters (Werding 1969) Similarlylemon-cream to orange colored sporocysts are produced by the congeners Renicola splsquolsquopolychaetophilarsquorsquo and Renicola sp lsquolsquomartinirsquorsquo in infections of the gonad and digestiveglands in Cerithidea californica (Hechinger amp Miura 2014)

This work studied the presence of parasites bacteria and fungi in orange coloredprotrusions in the muscle of Colombian Caribbean queen conchs This was achieved byusing histological analysis and molecular approaches based on 454 FLX and capillaryautomated sequencing using an ABI PRISM 3100 Genetic Analyzer (Applied BiosystemsFoster City CA USA) This 454 FLX next-generation platform (Roche Basel Switzerland)permits high-throughput identification of hundreds of samples at reasonable cost andtime consumption (Mardis 2008) This approach allows functional analysis of sequencingdata sets for comparative analysis of microbiome diversity of orange colored protrusionsfound in the muscle of L gigas by using metagenomic taxonomical classifiers (Huson et al2007 Huson et al 2011) This information is required for queen conch conservation andmanagement strategies of potential pathogenic infections for human beings

MATERIALS AND METHODSOrange colored protrusions were taken from three pieces of frozen muscle from onespecimen of L gigas processed for food trading in the Colombian Caribbean San Andresarchipelago (between 12ndash16N and 78ndash82W) These samples were provided by theGobernacioacuten del Archipieacutelago de San Andreacutes Providencia y Santa Catalina through thescientific cooperation agreement 0832012

Since the etiological agent of these orange colored protrusions was unknown weused three approaches to elucidate the origin of these lesions (1) histological analysis(2) 454 pyrosequencing of one whole genome shotgun library and (3) automated capillarysequencing (Sanger) of PCR amplified products to confirm the results provided by themetagenomic analysis For histological analysis samples from orange colored muscle

Cuartas et al (2018) PeerJ DOI 107717peerj4307 220

were fixed in 10 neutral phosphate-buffered formalin The samples were prepared forhistological examination by paraffin wax techniques and stained with hematoxylin andeosin following standard protocols (Garciacutea del Moral 1993 Prophet et al 1995)

Due to scarcity of samples the orange protrusions were pooled and ground with liquidnitrogen to extract the genomic DNA using the commercial DNAeasy Blood amp Tissue Kit(Qiagen Hilden Germany) according tomanufacturer recommendations Sample poolingwas performed to obtain high-quantity and high-quality DNA required for the generationof the genomic library Purified DNA from the pooled sample was sequenced using the454 Whole Genome Shotgun strategy according to standard protocols recommended by454 GS FLX platform (Roche Basel Switzerland) at the Centro Nacional de SecuenciacioacutenGenoacutemica Universidad de Antioquia (Margulies et al 2005) The obtained raw reads wereend polished of low-quality regions with the toolkit PRINSEQ lite (Schmieder amp Edwards2011) and assembled using MIRA3 v34 software (Chevreux Wetter amp Suhai 1999)

Classification of assembled contigs was carried out using the BLAST algorithm againstnucleotide and protein non-redundant databases of the NCBI with further computation ofthe taxonomic position of the assembled dataset with MEGAN software v553 (Huson etal 2011) This metagenomic software uses a Lowest Common Ancestor-based algorithmthat assigns each contig to taxa such that the taxonomical level of the assigned taxon reflectsthe level of conservation of the sequence (Huson et al 2007) Then species-specific andwidely conserved sequences were assigned to particular taxa as described by Huson et al(2007) The contigs were classified using a bit-score threshold of 50 retaining only thosehits that were within 10 of the best hit for a contig Additionally the E-value confidencecriterion was set at 1Endash15 even though a threshold value of 1Endash04 is considered a goodmatch (De Wit et al 2012) Only contig alignment lengths above 100 nucleotides forBLASTN comparison or 100 amino acids for BLASTX comparisons were included in theassignment analysis These analyses comparing DNA or protein sequences were carriedout independently

Furthermore protein analysis assignments were classified to the proper taxonomic levelaccording to Monzoorul Haque et al (2009) who empirically proposes identity thresholdsfor restricting the assignments Alignments having identities in ranges of 61ndash65 56ndash6051ndash55 and 41ndash50 were conservatively restricted in the level of family order classand phylum respectively The identity threshold of 66ndash100 was used for restrictingthe assignment of contigs to either species or genus or family levels Additionally thetaxonomic level within this identity range was distinguished by the difference between thetwo alignment parameters the percentage of identities and positives

Moreover a 1000 bp fragment of the mitochondrial cytochrome c oxidase I gene wasamplified by PCR following conditions reported by Leung et al (2009) and primersdescribed by Bowles et al (1993) (JB3 5prime-TTTTTTGGGCATCCTGAGGTTTAT-3prime) andKraacutelovaacute-Hromadovaacute (2008) (tremcox1rrnl 5prime AATCATGATGCAAAAGGTA-3prime) Thissequence was used instead of ribosomal genes since the bioinformatic analysis indicateda high enrichment of molluscan and some fungi ribosomal sequences which limited theamplification of the helminth sequences of 18S and 28S ribosomal genes (data not shown)The cytochrome c oxidase I amplicon was sequenced by automated Sanger method using

Cuartas et al (2018) PeerJ DOI 107717peerj4307 320

an ABI PRISM 3100 Genetic Analyzer (Applied Biosystems Foster City CA USA) andcompared by BLASTN against the NCBI nucleotide database to look for sequence matchesof reported organisms

Finally a Bayesian tree was constructed using the sequence obtained fromorange coloredprotrusions and published sequences of Platyhelminthes Bayesian tree construction wasperformed using MrBayes (MB) V32 (Ronquist et al 2012) setting the GTR+I+G4substitution model estimated by the software IQ-TREE with 1000000 generationssampled every 1000 generations and the other analysis parameters as default values Theconvergence of the Markov Chain Monte Carlo iterations was assessed with the PotentialScale Reduction Factor (PSRF = 1 Gelman amp Rubin 1992) and the standard deviation ofsplit frequencies (0001)

RESULTSAssembly and metagenomic approachThe massive shotgun sequencing generated 515368 reads with an average length of 279 bpthat were cleaned and then assembled using MIRA software into 5180 contigs Taxonomicclassification of the contigs was carried out using the software MEGAN For this analysisall the contigs were compared with the NCBIrsquos non-redundant protein database usingthe software BLASTX With this strategy 1588 (307) contigs were assigned to taxa(Bacteria 412 Eukaryota 1157 other 19) 866 (167) were unassigned and 2726(526) presented no hits As expected the Eukaryota group was dominant due to theorigin of the sample Furthermore the group Gastropoda was frequently found in thisanalysis (186 hits) although many sequences remained unclassified due to the poorrepresentation of these organisms in the public databases Many bacterial sequences werealso identified 19 were assigned to the fungi group and 22 sequences were assigned to theTrematoda category No viral or protozoa sequences were detected

Following the MEGAN pipeline with nucleotide comparisons using BLASTN andthe ntnr database results were poorly classified One contig was assigned to the root462 (89) to particular taxa (Bacteria 267 Eukaryota 191 other 4) 32 (06) wereunassigned and 4685 (905) had no hits At the nucleotide level most of the sequenceswere left unclassified This reflects the lack of sequences in the databases of closely relatedorganisms to the ones reported in this research

Bacteria and fungi associated with orange colored protrusionsThe ranges for the confidence criterion represented by the E-value similarity and identityfor protein comparisons are shown in Table 1 Bacteria assignments included the classGammaproteobacteria and the phylum Firmicutes which includes the orders Bacillales andLactobacillales (Fig 1) Specifically the classGammaproteobacteria showed 322 assignmentsfor Psychrobacter exhibiting hits to several types of proteins with strains of Psychrobactersp (Fig 1 Table 1) The identity criterion for Psychrobacter sp ranged from 67 to 100(Table 1) Similarly nucleotide sequences showed hits for different genomic regions ofPsychrobacter sp strains and congeners displaying identities ranging from 72 to 99and alignment lengths from 104 bp to 1061 bp (Table 2)

Cuartas et al (2018) PeerJ DOI 107717peerj4307 420

Table 1 Diversity content in bacteria and fungi clades found in a pooled sample of orange colored protrusions from L gigasmuscle usingtranslated contig sequences and the taxonomic classifier MEGAN

Ranges

Contig Organism Gene E-value Positives()

Identities()

Length(aa)

Assignmenta

8 Psychrobacter sp binding protein kinase transporter adap-tor hypothetical proteins membrane pro-tein glycosylase

0000E+001325Endash30

96 100 96 100 113 259 Species

11 Psychrobacter sp dehydrogenase hypothetical proteinscatalase cytoplasmic protein propionasetransferase chaperone deaminase mem-brane protein

0000E+004451Endash73

94 100 90 99 105 306 Genus

2 Psychrobacter sp channel protein hypothetical protein 1189Endash1074390Endash77

77 83 67 73 183 242 Family

2 Carnobacteriumjeotgalil

replication initiator phosphorylase 5120Endash934347Endash87

99 100 99 100 135 138 Species

2 Carnobacteriumspl

hypothetical protein integrase 2691Endash1274938Endash58

100 100 100 100 109 183 Species

1 Carnobacteriumspl

integrase 1940Endash61 86 80 129 Genus

1 Carnobacteriumspl

hypothetical protein 2200Endash69 94 86 158 Family

2 Lactobacillusjenseniil

hypothetical protein 2022Endash782550Endash60

73 85 68 81 166 169 Genus

1 Enterococcus fae-calisl

hypothetical protein 3700Endash45 68 44 172 Phylum

7 Brochothrix ther-mosphactab

kinase transcriptional regulator trans-porter ribosomal protein reductase hy-pothetical proteins

3514Endash1629715Endash69

100 100 100 100 180 248 Species

6 Brochothrix ther-mosphactab

dehydrogenase hypothetical proteinstransposase transferase

1918Endash1481431Endash53

76 100 71 99 120 243 Genus

1 Planococcusantarcticusb

hypothetical protein 2650Endash55 90 80 106 Family

1 Bacillus cytotoxi-cusb

synthetase 7050Endash164 83 71 223 Family

1 Lactococcus lactissubsp Lactisl

replication protein 6020Endash91 81 62 164 Family

1 Staphylococcusaureusb

hypothetical protein 2050Endash32 71 64 104 Family

1 Fusarium oxyspo-rum

glutamine-rich protein 1064Endash16 56 41 243 Phylum

1 Fusarium oxyspo-rum

glutamine-rich protein 2900Endash16 85 82 243 Genus

1 Neurosporatetrasperma

hypothetical protein 1426Endash58 92 90 107 Genus

NotesaThe assignments were classified to the taxonomic level according toMonzoorul Haque et al (2009) bBacillales lLactobacillales

Cuartas et al (2018) PeerJ DOI 107717peerj4307 520

Figure 1 Phylogenetic diversity of translated contigs from orange colored protrusions of Lobatus gigas computed byMEGAN The nodes of thecladogram represent the assigned taxa and the numbers indicate the relative abundance of assigned contigs

Full-size DOI 107717peerj4307fig-1

On the other hand 18 assignments for the order Lactobacillales (Fig 1) showed hitsfor diverse proteins exhibiting similarities and identities up to 100 for Carnobacteriumjeotgali and Carnobacterium sp (Table 1) We also found hits for proteins of Lactobacillusjensenii and Enterococcus faecalis displaying identities above 68 and 44 respectively(Table 1) Similarly nucleotide analysis showed matches for genome regions and plasmidsof Carnobacterium sp displaying identities above 82 (Table 2) Additionally the singlehits for a plasmid and a genome fragment of Enterococcus casseliflavus and Enterococcusfaecalis exhibited identities of 81 and 73 respectively (Table 2)

A total of 28 contigs were assigned to different Bacillales bacteria within the phylumFirmicutes (Fig 1) specifically Brochothrix thermosphacta showed hits for several proteinsexhibiting identities up to 100 (Table 1) Planococcus antarcticus Bacillus cytotoxicusLactococcus lactis subsp Lactis and Staphylococcus aureus showed identity ranges from 62to 80 (Table 1) Furthermore the nucleotide analysis showed hits for diverse bacteriabelonging to genus Listeria Bacillus and Paenibacillus (Table 2)

Only three out of 19 assignments to fungi clades satisfied the selection parameters twohits supported the taxonomical levels of phylum and genus for Fusarium oxysporum andone hit classified to the genus taxonomical level for Neurospora tetrasperma (Table 1) Inaddition nucleotide analysis (Table 2) showed three assignments forMrakia frigida (rRNAgenes two hits) and Togninia minima (putative polyubiquitin protein mRNA one hit)

A parasite associated with orange colored protrusionsThe histological approach showed a tissue lesion characterized by the aggregation ofhemocytes (cells endowed with phagocytic and immune-related functions) inside isolatedfoci surrounded by smooth muscle fibers and a basophilic tissue contiguous to a lamellated

Cuartas et al (2018) PeerJ DOI 107717peerj4307 620

Table 2 Diversity content in bacteria and fungi clades found in a pooled sample of orange colored protrusions from L gigasmuscle using nu-cleotide contig sequences and the taxonomic classifier MEGAN

Ranges

Contig Organism Gene E-value Identities () Length (bp)

1 Psychrobacter sp pRWF101 0000E+00 98 9931 Psychrobacter sp gf 0000E+00 99 8156 Psychrobacter sp p gf 0000E+00 7247Endash59 90 96 176 6791 Psychrobacter sp gf 0000E+00 88 11627 Psychrobacter sp p gf 0000E+00 3877Endash131 80 85 520 9143 Psychrobacter sp gf 4648Endash143 2430Endash35 76 79 288 7481 Psychrobacter cryohalolentis gf 0000E+00 93 10473 Psychrobacter cryohalolentis p gf 5595Endash138 6961Endash40 92 95 112 3524 Psychrobacter cryohalolentis gf 3316Endash153 1041Endash103 83 84 419 5373 Psychrobacter cryohalolentis gf 1626Endash92 3574Endash74 75 78 524 7001 Psychrobacter arcticus gf 0000E+00 91 6461 Psychrobacter arcticus gf 5090Endash120 94 3071 Psychrobacter arcticus gf 8830Endash47 91 14811 Psychrobacter arcticus gf 0000E+00 3900Endash148 81 88 611 106111 Psychrobacter arcticus gf 0000E+00 6195Endash25 80 89 104 6037 Psychrobacter arcticus gf 1962Endash172 1181Endash24 72 79 224 9781 Carnobacterium spl gf 0000E+00 94 24221 Carnobacterium spl pWNCR9 0000E+00 92 14661 Carnobacterium spl gf 0000E+00 96 12441 Carnobacterium spl gf 0000E+00 98 10293 Carnobacterium spl p gf 0000E+00 1117Endash155 95 98 321 6246 Carnobacterium spl p gf 0000E+00 8843Endash57 82 88 264 8971 Enterococcus casseliflavusl pTnpA 4680Endash85 81 4641 Enterococcus faecalisl gf 3310Endash37 73 5371 Listeria grayib 23S rRNA 000E+00 90 12631 Listeria welshimerib 23S rRNA 1150Endash176 88 5391 Listeria monocytogenesb gf 3850Endash103 74 9021 Listeria innocuab gf 7870Endash109 76 7541 Bacillus megateriumb gf 5410Endash170 78 9081 Bacillus toyonensis b gf 1430Endash66 74 622

1 Bacillus cereusb gf 2570Endash17 78 1741 Paenibacillus larvaeb pPL374 1110Endash170 100 3351 Uncultured compost bacteriumb 16S rRNA 0000E+00 99 4361 Mrakia frigida 25S rRNA 0000E+00 100 14291 Mrakia frigida 18S rRNA 0000E+00 99 17931 Togninia minima protein mRNA 3407Endash28 90 3261

NotesbBacillales lLactobacillalesgf genome fragment p plasmid rRNA ribosomal fragment

Cuartas et al (2018) PeerJ DOI 107717peerj4307 720

Figure 2 Histological sections of orange-colored protrusions in the muscle of Lobatus gigas The le-sions showed hemocytes stained purplish-blue and smooth muscle fibers pink-red in color (A) Presenceof lamellated membrane (arrowhead) (40times) (B) Granulation process (arrowhead) (100times)

Full-size DOI 107717peerj4307fig-2

membrane (Fig 2A) Additionally some lesions exhibit interstitial immunocyte inclusionswith morphology similar to a granulation process (Fig 2B) Although the histologicalapproach did not allow for detectection of key features for identification the microscopicimages showed structures around 055 mm in diameter which are compatible withimmature developmental stages of a trematode

Furthermore the metagenomic analysis assigned 22 contigs to the trematode parasitesclade Specifically these contigs had hits to an endonuclease-reverse transcriptase ofSchistosoma mansoni (17) and Schistosoma japonicum (4) showing identities above 46and 42 respectively Similarly in the nucleotide analysis seven contigs showed identitiesabove 71 for different regions of two chromosomes of S mansoni (Table 3)

We successfully amplified and sequenced a 740 bp region that confirmed the presence oftrematode DNA in the L gigas tissue (GenBank accession number KR092371) Moreoverthis sequence clustered in a basal position to the suborder Xiphidiata (Trematoda Digenea)which encompasses Renicola and Helicometrina genera (posterior probability 098 Fig 3)Additionally the BLASTN results showed hits for some members of Xiphidiata suchas Helicometrina labrisomid (query coverage 89 identity 77) Renicola cerithidicola(query coverage 70 identity 78) Synthesium pontoporiae (query coverage 42identity 83) and Haematoloechus sp (query coverage 39 identity 77)

DISCUSSIONIn this study three approaches including histological analysis 454 pyrosequencing andautomated Sanger amplification of the cytochrome c oxidase I gene were used to explore thepotential causal agent of orange colored protrusions in the muscle of L gigas Identificationby histology was limited since no characteristic structures were detected in the sampleAlso several contigs had no hits for proteins (sim52) or nucleotide sequences (sim90)indicating a lack of information on such sequences in reference databases This explanationis plausible since the current protein sequence reference databases cover only a small

Cuartas et al (2018) PeerJ DOI 107717peerj4307 820

Table 3 Diversity content in the trematoda clade found in a pooled sample of orange colored protrusions from L gigasmuscle using contig se-quences and the taxonomic classifier MEGAN

Ranges

Contig Organism Gene E-value Positives()

Identities()

Length Assignment a

Translated contig sequences2 Schistosoma japonicum endonuclease-reverse transcriptase 3637Endash61

6062Endash4275 76 64 64 141 165 Family

2 Schistosoma japonicum endonuclease-reverse transcriptase 2102Endash1221995Endash56

61 63 42 43 262 489 Phylum

5 Schistosoma mansoni endonuclease-reverse transcriptase 3147Endash1524075Endash45

77 81 61 67 155 345 Family

5 Schistosoma mansoni endonuclease-reverse transcriptase 2497Endash1722424Endash61

71 74 56 59 204 346 Order

4 Schistosoma mansoni endonuclease-reverse transcriptase 0000E+009203Endash89

69 70 52 52 275 695 Class

3 Schistosoma mansoni endonuclease-reverse transcriptase 1343Endash661719Endash51

65 68 46 50 193 262 Phylum

Nucleotide contig sequences1 Schistosoma mansoni chromosome fragment W 1320Endash20 80 80 161 ndash5 Schistosoma mansoni chromosome fragments 1640Endash55

7006Endash2771 73 71 73 649 763 ndash

1 Schistosoma mansoni chromosome fragment 4 1990Endash19 77 77 199 ndash

NotesaThe assignments were classified to the taxonomic level according toMonzoorul Haque et al (2009)

fraction of the biodiversity believed to be present in the environment (Wu et al 2009)Despite these limitations the alignment lengths of the contigs (ge100 nucleotides or aminoacids) and the bit-scores (50) used in this research ensure a reasonable level of confidencein the taxonomic assignments (Huson et al 2007)

Bacteria and fungi associated with orange colored protrusionsThe scope of the massive sequencing approach allowed the detection of some bacteriapreviously reported as microbiota associated with L gigas (Acosta et al 2009 Peacuterez etal 2014) as well as new reports For instance Psychrobacter sp was found in the L gigasmuscle in both nucleotide and protein analyses (Tables 1 and 2) This outcome corroboratesprevious studies that found Psychrobacter sp in environmental (Acosta et al 2009 Peacuterez etal 2014) and tissue (Peacuterez et al 2014) samples from L gigas

However this study also found bacteria and fungi that have not been reported so farin L gigas Specifically homologous protein and nucleotide sequences of species (egCarnobacterium jeotgali) family and genus of Carnobacterium sp were detected in theL gigasmuscle (Tables 1 and 2) Carnobacterium strains have been reported to inhabit livefish and a variety of seafood dairy and meat (Leisner et al 2007)

In addition this research found homologous protein and nucleotide sequences of genusBacillus and Enterococcus in the affected tissue of L gigas Bacillus species have ubiquitousdistribution inhabiting different environments such as soils rocks vegetation foods and

Cuartas et al (2018) PeerJ DOI 107717peerj4307 920

Figure 3 Bayesian tree obtained from cytochrome c oxidase I gene sequences of orange-colored protrusions from the muscle of L gigas (OPML)and GenBank Platyhelminthes sequences P macrorchis (JN5920391)M sebastis (NC_0090551) E multilocularis (AB0184402) E granulosus(AF2976171) T saginata (AY1958581) T solium (AY2118801) T pisiformis (GU5690961) S mansoni (AF2166981) S japonicum (AF2158601) S chasanensis (KU7573081) P saginatum (KX0978551) F hepatica (AF2166971) F gigantica (KF5433421) H labrisomi (KJ9960091) R pin-guis (KU5637241) R sternae (KU5637231) R lari (KU5637271) R cerithidicola (KF5125731) R buchanani (KF5125721) Renicola sp lsquopoly-chaetophilarsquo (KF5125511) and Renicola sp lsquomartinirsquo (KF5125601)

Full-size DOI 107717peerj4307fig-3

waters (Nicholson 2002) Similarly the ubiquitous nature of enterococci determines theirfrequently being found in foods as contaminants although their predominant habitat ishuman and animal gastrointestinal tracts (Giraffa 2002) However they also occur in soilsurface waters vegetables and fermented foods such as sausages meat and cheese (Giraffa2002 Foulquieacute et al 2006)

Furthermore another bacteria present in the sample was Brochothrix thermosphacta asit was assigned to bacterial species or genus taxonomical levels according to MonzoorulHaque et al (2009) This bacterium closely related to Listeria is a non-proteolytic foodspoilage organism in prepacked meats and fish products (Gardner 1981 Lannelongue etal 1982 Pin Garciacutea de Fernando amp Ordoacutentildeez 2002) In addition some Listeria hits weredetected in the nucleotide analysis although the identity values did not allow speciesidentification This result is concordant with studies that have isolated Listeria membersfrom freshwater and marine environments (Colburn et al 1990 El Marrakchi Boumrsquohandiamp Hamama 2005)

Metagenomic analysis also showed some fungi assignments related to FusariumNeurospora Togninia andMrakia Both Fusarium andNeurospora exhibit wide distribution

Cuartas et al (2018) PeerJ DOI 107717peerj4307 1020

including humid tropical and subtropical marine environments (Steele 1967 TurnerPerkins amp Fairfield 2001 Babu et al 2010 Summerell et al 2010 Jebaraj et al 2012Saravanan amp Sivakumar 2013 Kumar Gousia amp Latha 2015) Specifically some Fusariumspecies are associated with infections in crustaceans and cultivated fishes (Hatai 2012)whereas other species are endosymbionts of some seaweeds (Suryanarayanan 2012) corals(Raghukumar amp Ravindran 2012) and some sea sponges (Houmlller et al 2000 Wang Li ampZhu 2008 Liu et al 2010 Paz et al 2010)

In contrast Togninia and Mrakia show more restricted distributions For instanceTogninia comprises pathogenic fungi responsible for the development of wood diseasesand some strains have been isolated from submerged wood from streams lakes pondsreservoirs and ditches (Hu Cai amp Hyde 2012) Likewise several Mrakia species have beenisolated from icy environments including meltwaters from glaciers and permafrost inAntarctica (Hua et al 2010 Pathan et al 2010 Carrasco et al 2012 Zhang et al 2012Tsuji et al 2013a Tsuji et al 2013b) Argentina (Brizzio et al 2007 De Garcia Brizzioamp Broock 2012) the Qinghai-Tibet Plateau (Su et al 2016) Italy (Turchetti et al 2008Branda et al 2010 Thomas-Hall et al 2010) and the Arctic (Pathan et al 2010)

Considering that several of the new bacteria reports are related to food microorganismswe hypothesized that they might grow under environmental or freezing conditions insteadof being native microbiota Fungi findings suggest an environmental source however sincesome species of Fusarium and Neurospora produce orange spores (Davis amp Perkins 2002Hatai 2012) the colored protrusions found in L gigas may be due to an opportunisticor primary fungal infection Thus the role of bacteria and fungi in the muscle of L gigasand their relationship with the lesion native microbiota or the environment remains to beexplored

A parasite associated with orange colored protrusionsHistology showed evidence of a membrane that is consistent with a syncytium enclosing amulticellular parasite a mollusk inflammatory response elicited by hemocytes (De Vico ampCarella 2012) Moreover such membranes are also compatible with the wall layers of thelife cycle stage of Platyhelminthes suggesting a possible infection by trematodes that infectother mollusks (Cake 1976 Sorensen amp Minchella 2001) This finding was supported bythe metagenomics analysis that showed sequences homologous to an endonuclease-reversetranscriptase of some species of trematodes like Schistosoma (Table 3) This result is expectedsince highly repetitive sequences such non-LTR retrotransposons with an estimated copynumber going up to 24000 are more likely to be detected in whole genome shotgunamplification (DeMarco et al 2005) Since databases of protein and nucleotide sequencesare currently enriched with Schistosoma but exhibit a poor representation ofmostmembersof Trematoda these assignments require cautioned interpretation Moreover the lack ofinformation in reference databases for most of the sequences from the studied sample(sim90 of nucleotide sequences andsim52 of proteins) explains the relatively low numberof hits for the parasite compared with Trematoda bacteria and fungi taxa Although theseassignments are biased by the nucleotide and protein sequences available in the NCBI

Cuartas et al (2018) PeerJ DOI 107717peerj4307 1120

databases it supports the histological finding that the protrusions may be caused by atrematode

The Bayesian tree supported the last result due to clustering of the sample in a basalposition to the suborder Xiphidiata (Trematoda Digenea) which includes Renicola speciesthat produce colored pigments (Stunkard 1950 Galaktionov amp Skirnisson 2000) Thephylogenetic relationships with Xiphidiata were consistent with the BLASTN analysisthat revealed genetic similitudes between the sequence found in this study and RenicolaHelicometrina Synthesium and Haematoloechus although its genetic distance with othermembers of these genera remains to be determined due to the lack of information forcytochrome c oxidase I and endonuclease-reverse transcriptase sequences of these taxa

The molecular findings let us hypothesize about the structures approximately 055 mmin diameter found in the snail muscle tissue although histology did not allow detection ofkey features for its identification According to the life cycle described for Xiphidiata themicroscopic life cycle stage found in the muscle of L gigas could be sporocysts which aredescribed to preferentially infect gonads and digestive glands but can also disperse to othertissues in the form of more sporocysts or rediae (Cribb et al 2003) Based on the structurersquossize other stages such as metacercaria seems unlikely at least in Renicola as they exhibit012 to 016 mm in diameter (Stunkard 1964) However the evidence presented here isnot enough to conclude which parasitic stage was observed within the colored lesions

In conclusion this study found evidence of a trematode infection as well the presence offungi and bacteria in the protrudedmuscle of L gigas which provides novel information forthe parasitology and microbiology of this species This first insight of a trematode infectionin L gigas is a baseline to expand the toolset to identify these organisms the trematode lifecycle environmental conditions that trigger its appearance and epidemiological aspectsregarding the host and possible effects on human health

ACKNOWLEDGEMENTSThe authors thank the Laboratorio de Patologiacutea Animal and Centro de SecuenciacioacutenGenoacutemica Universidad de Antioquia for assistance and services provided The authorsalso thank to the anonymous reviewers for their comments which substantially improvedthe final version of this article

ADDITIONAL INFORMATION AND DECLARATIONS

FundingThis work was funded by the Universidad Nacional de Colombia Sede Medelliacuten-theGobernacioacuten del Archipieacutelago de San Andreacutes Providencia y Santa Catalina Grant201010011420 (Scientific Cooperation Agreement 0832012) Convocatoria Nacionalpara el Programa Joacutevenes Investigadores Colciencias No525-2012-Identificacioacuten deparaacutesitos asociados al caracol pala Strombus gigas en la Reserva de Biosfera Seaflower(Jaison H Cuartas) and Programa de sostenibilidad 2016-2017 Grupo de ParasitologiacuteaVicerrectoriacutea de investigacioacuten Universidad de Antioquia The funders had no role in studydesign data collection and analysis decision to publish or preparation of the manuscript

Cuartas et al (2018) PeerJ DOI 107717peerj4307 1220

Grant DisclosuresThe following grant information was disclosed by the authorsUniversidad Nacional de ColombiaSede Medelliacuten-the Gobernacioacuten del Archipieacutelago de San AndreacutesProvidencia y Santa Catalina 201010011420Scientific Cooperation Agreement 0832012Convocatoria Nacional para el Programa Joacutevenes Investigadores Colciencias No525-2012-Identificacioacuten de paraacutesitos asociados al caracol pala Strombus gigas en la Reserva de BiosferaSeaflower (Jaison H Cuartas)Programa de sostenibilidad Vicerrectoriacutea de investigacioacuten Universidad de Antioquia

Competing InterestsThe authors declare there are no competing interests

Author Contributionsbull Jaison H Cuartas Juan F Alzate Claudia X Moreno-Herrera and Edna J Marquezconceived and designed the experiments performed the experiments analyzed the datacontributed reagentsmaterialsanalysis tools wrote the paper prepared figures andortables reviewed drafts of the paper

EthicsThe following information was supplied relating to ethical approvals (ie approving bodyand any reference numbers)

The muscle samples of Lobatus gigaswere provided by the Gobernacioacuten del Archipieacutelagode San Andreacutes Providencia y Santa Catalina through the scientific cooperation agreement 0832012

DNA DepositionThe following information was supplied regarding the deposition of DNA sequences

GenBank accession KR092371

Data AvailabilityThe following information was supplied regarding data availability

The raw data has been supplied as a Supplemental File

Supplemental InformationSupplemental information for this article can be found online at httpdxdoiorg107717peerj4307supplemental-information

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Aldana D Frenkiel L Bruleacute T Montero J Baqueiro E 2011 Occurrence of Apicomplexa-like structures in the digestive gland of Strombus gigas throughout the CaribbeanJournal of Invertebrate Pathology 106174ndash178 DOI 101016jjip201009004

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Banaszak AT Garciacutea RamosM Goulet TL 2013 The symbiosis between the gastropodStrombus gigas and the dinoflagellate Symbiodinium an ontogenic journey frommutualism to parasitism Journal of Experimental Marine Biology and Ecology449358ndash365 DOI 101016jjembe201310027

Baqueiro E Frenkiel L Zetina A Aldana D 2007 Coccidian (Apicomplexa) parasiteinfecting Strombus gigas Linneacute 1758 digestive gland Journal of Shellfish Research26319ndash321 DOI 1029830730-8000(2007)26[319CAPISG]20CO2

Bowles J HopeM TiuWU Liu X McManus DP 1993 Nuclear and mitochondrialgenetic markers highly conserved between Chinese and Philippine Schistosomajaponicum Acta Tropica 55217ndash229 DOI 1010160001-706X(93)90079-Q

Branda E Turchetti B Diolaiuti G Pecci M Smiraglia C Buzzini P 2010 Yeast andyeast-like diversity in the southernmost glacier of Europe (Calderone GlacierApennines Italy) FEMS Microbiology Ecology 72354ndash369DOI 101111j1574-6941201000864x

Brizzio S Turchetti B De Garciacutea V Libkind D Buzzini P Van BroockM 2007Extracellular enzymatic activities of basidiomycetous yeasts isolated from glacialand subglacial waters of northwest Patagonia (Argentina) Canadian Journal ofMicrobiology 53519ndash525 DOI 101139W07-010

Brownell WN Stevely JM 1981 The biology fisheries and management of the QueenConch Strombus gigasMarine Fisheries Review 43(7)1ndash12

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CarrascoM Rozas JM Barahona S Alcaiacuteno J Cifuentes V Baeza M 2012 Diversityand extracellular enzymatic activities of yeasts isolated from King George Island thesub-Antarctic region BMCMicrobiology 12251ndash260DOI 1011861471-2180-12-251

Chevreux BWetter T Suhai S 1999 Genome sequence assembly using trace signals andadditional sequence information In Computer science and biology proceedings of theGerman conference on bioinformatics (GCB) 45ndash56

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Davis RH Perkins DD 2002 Neurospora a model of model microbes Nature ReviewsGenetics 3397ndash403 DOI 101038nrg797

De Garcia V Brizzio S Van BroockMR 2012 Yeasts from glacial ice of PatagonianAndes Argentina FEMS Microbiology Ecology 82540ndash550DOI 101111j1574-6941201201470x

DeMarco R Machado AA Bisson-Filho AW Verjovski-Almeida S 2005 Identificationof 18 new transcribed retrotransposons in Schistosoma mansoni Biochemical andBiophysical Research Communications 333230ndash240 DOI 101016jbbrc200505080

De Vico G Carella F 2012Morphological features of the inflammatory response in mol-luscs Research in Veterinary Science 931109ndash1115 DOI 101016jrvsc201203014

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El Marrakchi A Boumrsquohandi N Hamama A 2005 Performance of a new chromogenicplating medium for the isolation of Listeria monocytogenes from marine environ-ments Letters in Applied Microbiology 4087ndash91DOI 101111j1472-765X200401651x

Foulquieacute MR Sarantinopoulos P Tsakalidou E De Vuyst L 2006 The role and appli-cation of enterococci in food and health International Journal of Food Microbiology1061ndash24 DOI 101016jijfoodmicro200506026

Galaktionov KV Skirnisson K 2000 Digeneans from intertidal molluscs of SW IcelandSystematic Parasitology 4787ndash101 DOI 101023A1006426117264

Garciacutea del Moral R 1993 Laboratorio de Anatomiacutea Patoloacutegica Madrid McGraw-HillInteramericana de Espantildea

Garciacutea RamosM Banaszak AT 2014 Symbiotic association between symbiodinium andthe gastropod Strombus gigas larval acquisition of symbiontsMarine Biotechnology16193ndash201 DOI 101007s10126-013-9536-x

Gardner GA 1981 Brochothrix thermosphacta (Microbacterium thermosphactum) in thespoilage of meats a review In Roberts TA Hobbs GA Christian JHB Skovgaard Neds Psychrotrophic microorganisms in spoilage and pathogenicity London AcademicPress 139ndash173

Gelman A Rubin DB 1992 Inference from iterative simulation using multiple se-quences Statistical Science 7457ndash472DOI 101214ss1177011136

Giraffa G 2002 Enterococci from foods FEMS Microbiology Reviews 26163ndash171DOI 101016S0168-6445(02)00094-3

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Gros O Frenkiel L Aldana D 2009 Structural analysis of the digestive gland of theQueen Conch Strombus gigas Linnaeus 1758 and its intracellular parasites Journalof Molluscan Studies 7559ndash68 DOI 101093molluseyn041

Hatai K 2012 Diseases of fish and shellfish caused by marine fungi In Raghukumar Ced Biology of marine fungi Berlin Springer-Verlag Berlin Heidelberg 15ndash52

Hechinger RF Miura O 2014 Two lsquolsquonewrsquorsquo renicolid trematodes (Trematoda DigeneaRenicolidae) from the California horn snail Cerithidea californica (Haldeman 1840)(Gastropoda Potamididae) Zootaxa 3784559ndash574 DOI 1011646zootaxa378455

Houmlller UWright AD Mattheacutee GF Konig GM Draeger S Aust H-J Schulz B 2000Fungi from marine sponges diversity biological activity and secondary metabolitesMycological Research 1041354ndash1365 DOI 101017S0953756200003117

HuD-M Cai L Hyde KD 2012 Three new ascomycetes from freshwater in ChinaMycologia 1041478ndash1489 DOI 10385211-430

HuaMX Chi Z Liu GL Buzdar MA Chi ZM 2010 Production of a novel and cold-active killer toxin byMrakia frigida 2E00797 isolated from sea sediment in Antarc-tica Extremophiles 14515ndash521 DOI 101007s00792-010-0331-6

Huson DH Auch AF Qi J Schuster SC 2007MEGAN analysis of metagenomic dataGenome Research 17377ndash386 DOI 101101gr5969107

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Jebaraj CS Forster D Kauff F Stoeck T 2012 Molecular diversity of fungi from marineoxygen-deficient environments (ODEs) In Raghukumar C ed Biology of marinefungi Berlin Heidelberg Springer-Verlag 1899ndash1208

Kraacutelovaacute-Hromadovaacute I SpakulovaacuteM Horaacuteckovaacute E Turcekovaacute L Novobilskyacute ABeck R Koudela B Marinculić A Rajskyacute D PybusM 2008 Sequence analysisof ribosomal and mitochondrial genes of the giant liver fluke Fascioloides magna(Trematoda Fasciolidae) intraspecific variation and differentiation from Fasciolahepatica Journal of Parasitology 9458ndash67 DOI 101645GE-13241

Kumar KA Gousia SK Latha JNL 2015 Evaluation of biological activity of secondarymetabolites of Neurospora crassa from Machilipatnam Sea Water Research Journal ofMicrobiology 10377ndash384 DOI 103923jm2015377384

LannelongueM HannaMO Finne G Nickelson IIR Vanderzant C 1982 Storagecharacteristics of finfish fillets (Archosargus probatocephalus) packaged in modifiedgas atmospheres containing carbon dioxide Journal of Food Protection 45440ndash444DOI 1043150362-028X-455440

Leisner JJ Laursen BG Preacutevost H Drider D Dalgaard P 2007 Carnobacteriumpositive and negative effects in the environment and in foods FEMS MicrobiologyReviews 31592ndash613 DOI 101111j1574-6976200700080x

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Leung TLF Donald KM Keeney DB Koehler AV Peoples RC Poulin R 2009 Trema-tode parasites of Otago Harbour (New Zealand) soft-sediment intertidal ecosystemslife cycles ecological roles and DNA barcodes New Zealand Journal of Marine andFreshwater Research 43857ndash865 DOI 10108000288330909510044

LiuWC Li CQ Zhu P Yang JL Cheng KD 2010 Phylogenetic diversity of culturablefungi associated with two marine sponges Haliclona simulans and Gelliodes carnosacollected from the Hainan Island coastal waters of the South China Sea FungalDiversity 421ndash15 DOI 101007s13225-010-0022-8

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Monzoorul HaqueM Ghosh TS Komanduri D Mande SS 2009 SOrt-ITEMSsequence orthology based approach for improved taxonomic estimation of metage-nomic sequences Bioinformatics 251722ndash1730 DOI 101093bioinformaticsbtp317

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Ronquist F TeslenkoM Vander Mark P Ayres DL Darling A Houmlhna S LargetB Liu L SuchardMA Huelsenbeck JP 2012MrBayes 32 efficient Bayesianphylogenetic inference and model choice across a large model space SystematicBiology 61539ndash542 DOI 101093sysbiosys029

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Steele CW 1967 Fungus populations in marine waters and coastal sands of the Hawai-ian Line and Phoenix Islands Pacific Science 21317ndash331

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Su Y Jiang XWuWWangM HamidMI XiangM Liu X 2016 Genomic tran-scriptomic and proteomic analysis provide insights into the cold adaptationmechanism of the obligate psychrophilic fungusMrakia psychrophila G3Genes|Genomes|Genetics 63603ndash3613 DOI 101534g3116033308

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Suryanarayanan TS 2012 Fungal endosymbionts of seaweeds In Raghukumar C edBiology of marine fungi Berlin Springer-Verlag Berlin Heidelberg 53ndash70

Tello-Cetina JA Rodriacuteguez-Gil LA Rodriacuteguez-Romero F 2005 Geneacutetica poblacionaldel caracol rosado Strombus gigas en la Peniacutensula de Yucataacuten implicaciones para sumanejo y pesqueriacutea Ciencias Marinas 31379ndash386 DOI 107773cmv31i256

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Tsuji M Singh SM Yokota Y Kudoh S Hoshino T 2013b Influence of initial pHon ethanol production by the antarctic basidiomycetous yeastMrakia blollopisBioscience Biotechnology and Biochemistry 772483ndash2485 DOI 101271bbb130497

Turchetti B Buzzini P Goretti M Branda E Diolaiuti G DrsquoAgata C Smiraglia CVaughan-Martini A 2008 Psychrophilic yeasts in glacial environments of Alpineglaciers FEMS Microbiology Ecology 6373ndash83 DOI 101111j1574-6941200700409x

Turner BC Perkins DD Fairfield A 2001 Neurospora from natural populations aglobal study Fungal Genetics and Biology 3267ndash92 DOI 101006fgbi20011247

Volland JM Gros O Frenkiel L Aldana D 2010 Apicomplexan parasite in the digestivegland of various species of the family Strombidae Strombus costatus S gigas andS pugilis Gulf and Caribbean Fisheries Institute 430ndash432

Wang G Li Q Zhu P 2008 Phylogenetic diversity of culturable fungi associated with theHawaiian Sponges Suberites zeteki and Gelliodes fibrosa Antonie Van Leeuwenhoek93163ndash174 DOI 101007s10482-007-9190-2

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