Microbiome reduction and endosymbiont gain from a switch in sea urchin life history.

Animal gastrointestinal tracts harbor a microbiome that is integral to host function, yet species from diverse phyla have evolved a reduced digestive system or lost it completely. Whether such changes are associated with alterations in the diversity and/or abundance of the microbiome remains an untested hypothesis in evolutionary symbiosis. Here, using the life history transition from planktotrophy (feeding) to lecithotrophy (nonfeeding) in the sea urchin Heliocidaris, we demonstrate that the lack of a functional gut corresponds with a reduction in microbial community diversity and abundance as well as the association with a diet-specific microbiome. We also determine that the lecithotroph vertically transmits a Rickettsiales that may complement host nutrition through amino acid biosynthesis and influence host reproduction. Our results indicate that the evolutionary loss of a functional gut correlates with a reduction in the microbiome and the association with an endosymbiont. Symbiotic transitions can therefore accompany life history transitions in the evolution of developmental strategies.

Mass mortality of collector urchins Tripneustes gratilla in Hawai`i.

As grazers, sea urchins are keystone species in tropical marine ecosystems, and their loss can have important ecological ramifications. Die-offs of urchins are frequently described, but their causes are often unclear, in part because systematic examinations of animal tissues at gross and microscopic level are not done. In some areas, urchins are being employed to control invasive marine algae. Here, we describe the pathology of a mortality event in Tripneustes gratilla in Hawai`i where urchins were translocated to control invasive algae. Although we did not determine the cause of the mortality event, our investigation indicates that animals died from inflammation of the test and epidermal ulceration, followed by inability to maintain coelomic fluid volume, colonization of coelomic fluid by opportunists (diatom, algae), and inappetence. Parasites, bacteria, fungi, and viruses were not evident as a primary cause of death. Pathology was suggestive of a toxin or other environmental cause such as lack of food, possibilities that could be pursued in future investigations. These findings highlight the need for caution and additional tools to better assess health when translocating marine invertebrates to ensure maximal biosecurity.

Febrile illness after a beach day: a case of salmonella bacteremia from oral exposure to a sand dollar.

This case report discusses a rare presentation of salmonella bacteremia after an oral exposure to a sand dollar in a pediatric patient. A 2-year-old Hispanic male presented to the emergency department with a chief complaint of diarrhea and fever for 8 days after a family trip to Destin beach, Florida, during the sea turtle nesting season. The symptoms began a day after the patient took a bite on a sand dollar found on the beach that caused a small wound inside his cheek. The laboratory testing done in the emergency department was remarkable for blood and stool culture testing positive for non-typhoid salmonella. The sand dollar is a type of a sea urchin commonly found on sandy beaches and consists of an anatomical filtration system to consume sandy water. It could be inferred that the increased presence of sea turtles during the time of the patient's visit to Destin beach could have led to higher levels of salmonella in the seawater and consequently in the sand dollar, possibly leading to the inoculation of salmonella in this patient. To the best of our knowledge, this is a first case report that links oral exposure of the sand dollar to invasive salmonellosis.

Altercrasins A⁻E, Decalin Derivatives, from a Sea-Urchin-Derived Alternaria sp.: Isolation and Structural Analysis Including Stereochemistry.

In order to find out the seeds of antitumor agents, we focused on potential bioactive materials from marine-derived microorganisms. Marine products include a number of compounds with unique structures, some of which may exhibit unusual bioactivities. As a part of this study, we studied metabolites of a strain of Alternaria sp. OUPS-117D-1 originally derived from the sea urchin Anthocidaris crassispina, and isolated five new decalin derivatives, altercrasins A-E (1-5). The absolute stereostructure of altercrasins A (1) had been decided by chemical transformation and the modified Mosher's method. In this study, four decalin derivatives, altercrasins B-E (2-5) were purified by silica gel chromatography, and reversed phase high-performance liquid chromatography (RP HPLC), and their structures were elucidated on the basis of 1D and 2D nuclear magnetic resonance (NMR) spectroscopic analyses. The absolute configuration of them were deduced by the comparison with 1 in the NMR chemical shifts, NOESY correlations, and electronic circular dichroism (ECD) spectral analyses. As a result, we found out that compound pairs of 1/2 and 4/5 were respective stereoisomers. In addition, their cytotoxic activities using murine P388 leukemia, human HL-60 leukemia, and murine L1210 leukemia cell lines showed that 4 and 5 exhibit potent cytotoxicity, in especially, the activity of 4 was equal to that of 5-fluorouracil.

A new flavonoid derivative and a new 5-hydroxyanthranilic acid derivative from the sea urchin-derived Streptomyces sp. HDa1.

One new flavonoid derivative flavoside A (1), one new 5-hydroxyanthranilic acid derivative crassilin (2), along with the known angucyclinone PD116740 (3) and oxachelin (4), was isolated from the EtOAc extract of the fermentation broth of the sea urchin (Anthocidaris crassispina)-derived actinobacterium, Streptomyces sp. HD01. The structures of these compounds were established on the basis of their HR-ESI-MS and NMR spectroscopic data. All of these compounds were assessed for their antibacterial activity.

Toxicological Investigations on the Sea Urchin Tripneustes gratilla (Toxopneustidae, Echinoid) from Anaho Bay (Nuku Hiva, French Polynesia): Evidence for the Presence of Pacific Ciguatoxins.

The sea urchin Tripneustes gratilla (Toxopneustidae, Echinoids) is a source of protein for many islanders in the Indo-West Pacific. It was previously reported to occasionally cause ciguatera-like poisoning; however, the exact nature of the causative agent was not confirmed. In April and July 2015, ciguatera poisonings were reported following the consumption of T.gratilla in Anaho Bay (Nuku Hiva Island, Marquesas archipelago, French Polynesia). Patient symptomatology was recorded and sea urchin samples were collected from Anaho Bay in July 2015 and November 2016. Toxicity analysis using the neuroblastoma cell-based assay (CBA-N2a) detected the presence of ciguatoxins (CTXs) in T.gratilla samples. Gambierdiscus species were predominant in the benthic assemblages of Anaho Bay, and G.polynesiensis was highly prevalent in in vitro cultures according to qPCR results. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses revealed that P-CTX-3B was the major ciguatoxin congener in toxic sea urchin samples, followed by 51-OH-P-CTX-3C, P-CTX-3C, P-CTX-4A, and P-CTX-4B. Between July 2015 and November 2016, the toxin content in T.gratilla decreased, but was consistently above the safety limit allowed for human consumption. This study provides evidence of CTX bioaccumulation in T.gratilla as a cause of ciguatera-like poisoning associated with a documented symptomatology.

Anthocidins A⁻D, New 5-Hydroxyanthranilic Acid Related Metabolites from the Sea Urchin-Associated Actinobacterium, Streptomyces sp. HDa1.

Four new 5-hydroxyanthranilic acid related compounds, named anthocidins A⁻D (1⁻4), two known analogues n-lauryl 5-hydroxyanthranilate (5) and isolauryl 5-hydroxyanthranilate (6), together with benzamide (7), 3-hydroxy-4-methoxycinnamamide (8), and (3S-cis)-hexahydro-3-[(3,4-dihydroxyphenyl)methyl]pyrrolo[1,2-a]pyrazine-1,4-dione (9), were isolated from the fermentation broth of the marine-derived actinomycete, Streptomyces sp. HDa1, which was isolated from the gut of a sea urchin, Anthocidaris crassispina, collected from Hainan Island, China. The structures of these secondary metabolites were elucidated on the basis of their 1D and 2D-NMR and mass spectroscopic data, and anthocidin A was confirmed by single-crystal X-ray diffraction with Cu Kα radiation. Anthocidins A⁻D (1⁻4) feature an acetyl group substitution at the amino group and varying alkyl side chains at the carboxyl group of 5-hydroxyanthranilic acid, and compound 5 was isolated as a natural product for the first time. The cytotoxic and antibacterial activity of compounds 1⁻9 were evaluated.

Establishing a Diagnostic Technique for Coelomocentesis in the Long-Spined Sea Urchin Diadema antillarum.

Coelomic fluid aspiration has been utilized in echinoderms in research and clinical settings. Detailed procedural descriptions for coelomic fluid sampling in sea urchins (class Echinoidea) are lacking, and samples are prone to contamination. The objectives of this study were to (1) standardize a technique for coelomic fluid collection in long-spined sea urchin Diadema antillarum that optimizes the diagnostic quality of the sample utilizing diagnostic imaging, (2) identify coelomic fluid bacterial isolates (using Biolog GEN III MicroLog and 16s rDNA sequencing), and (3) compare positive cultures to animal weight, holding time prior to sampling, water temperature, and gross fluid appearance. Seventy Diadema antillarum from the Florida Keys collected in two groups (March and September 2015) were utilized. Positive cultures for bacterial contamination were identified in 5% and 44%, respectively, of animals in the sampling groups. Vibrio spp. was the predominant genus identified. Positive cultures were more frequent in the group with smaller-sized animals, increased holding times, and elevated water temperatures. Deviation from clear-pink gross coelomic fluid appearance did not reliably predict bacterial contamination. A standardized technique for coelomocentesis was defined. The use of the proposed coelomocentesis methodology may facilitate improved health evaluations of sea urchins and may be applicable to research, conservation efforts, and disease investigations.

Cell-mediated immune response of postmetamorphic sea urchin juveniles against infectious stages of diatom Cylindrotheca closterium (Bacillariophyceae).

In this study, successive infectious stages by diatom Cylindrotheca closterium (Bacillariophyceae) are described for the first time during the early development of sea urchin at low temperature (8°C). Diatom cell-types enclosed or not by typical theca were capable of infection. As an immune response, red spherulocytes and amoebocytes migrated towards infested areas and restrained the infection spreading over shells in 2- and 3-month old urchins. Only amoebocyte cells appeared to be involved in the immune reaction of 1-month old specimens which turned out to be a less effective fence to stop infestation. The effectiveness of the inflammatory process caused by diatoms within sea urchin spines seemed to vary as a function of urchin age, cohort's type and individual cell pool.

Faecal contamination of echinoderms: first report of heavy Escherichia coli loading of sea urchins from a natural growing area.

UNLABELLED: Although little evidence existed to support that view, European countries and in particular France, have regarded echinoderms, including sea urchins, as low risk in terms of feacal contamination. It is hypothesized that the sea urchins mode of feeding, which is based on grazing and differs from bivalve molluscs, would prevent it from concentrating high levels of Escherichia coli. Here, we monitored E. coli levels in sea urchins (Paracentrotus lividus) and in filter-feeder mussels (Mytillus galloprovincialis), collected concurrently from the same natural area over a 1-year period to verify this assumption. Sea urchins were collected on the seafloor, whereas mussels were collected from the water column at a depth of 4 m. Our results showed heavy bacterial loading of sea urchins in a natural growing environment. Moreover, we highlighted that E. coli contamination of sea urchins could, in certain conditions, be higher than those detected in filter-feeding mussels collected at the same location. Finally, the results showed a significant correlation between rainfall and E. coli concentrations in sea urchins, suggesting that the bacterial safety of sea urchin could be linked to the quality of the surrounding water. SIGNIFICANCE AND IMPACT OF THE STUDY: The European regulation requires competent authorities to monitor the sanitary status of shellfish, including live echinoderms, through faecal indicator organisms. In the French Mediterranean, sea urchin production is significant. Until now, as no data showed significant E. coli contamination levels, no monitoring programs focused on this species. This study demonstrates that sea urchins are more vulnerable to faecal contamination than previously hypothesized, especially during heavy rainfall. In consequence, the European authority general approach to microbiological management of shellfish should be applied to sea urchins.

Chryseobacterium echinoideorum sp. nov., isolated from sea urchins (Tripneustes gratilla).

A Gram-stain-negative, aerobic, non-motile, rod-shaped, flexirubin-producing bacterium, designated strain CC-CZW010T, was isolated from the edible sea urchin Tripneustes gratilla in Penghu Island, Taiwan. The isolate grew optimally at pH 7.0 and 30 °C in the presence of 2 % (w/v) NaCl. The most closely related strains in terms of 16S rRNA gene sequence similarity were Chryseobacterium taihuense NBRC 108747T (97.6 %) and Chryseobacterium aquaticum KCTC 12483T (96.7 %). Phylogenetic analyses based on 16S rRNA gene sequences revealed a distinct taxonomic position attained by strain CC-CZW010T with respect to other species of the genus Chryseobacterium. Strain CC-CZW010T possessed iso-C15 : 0, anteiso-C15 : 0, iso-C17 :  0 3-OH, summed feature 3 (comprising C16 : 1ω7c/C16 : 1ω6c) and summed feature 9 (comprising C16 :  0 10-methyl/iso-C17 : 1ω9c) as predominant fatty acids. The major polar lipid profile consisted of phosphatidylethanolamine, two unidentified lipids and five aminolipids. The polyamine pattern contained the major compound sym-homospermidine. Menaquinone 6 (MK-6) was the predominant respiratory quinone, and the G+C content of the genomic DNA was 36.4 mol%. According to distinct phylogenetic, phenotypic and chemotaxonomic features, strain CC-CZW010T represents a novel species of the genus Chryseobacterium, for which the name Chryseobacterium echinoideorum sp. nov. is proposed. The type strain is CC-CZW010T ( = BCRC 80786T = JCM 30470T).

Vitellibacter echinoideorum sp. nov., isolated from a sea urchin (Tripneustes gratilla).

A Gram-stain-negative, aerobic, rod-shaped, yellow-pigment-producing bacterium (designated strain CC-CZW007(T)) was isolated from seafood samples (sea urchins) at Penghu Island in Taiwan. Strain CC-CZW007(T) grew optimally at pH 7.0 and 30 °C in the presence of 3% (w/v) NaCl. The novel strain shared highest 16S rRNA gene sequence similarity to Vitellibacter vladivostokensis JCM 11732(T) (96.8%), Vitellibacter soesokkakensis KCTC 32536(T) (96.4%), Vitellibacter nionensis KCTC 32420(T) (95.8%) and Vitellibacter aestuarii JCM 15496(T) (95.6%) and lower sequence similarity to members of other genera. Phylogenetic analyses based on 16S rRNA genes revealed a distinct taxonomic position attained by strain CC-CZW007(T) with respect to other species of the genus Vitellibacter. The major fatty acids were iso-C15 : 0 and iso-C17 : 0 3-OH. The polar lipid profile was composed of major amounts of phosphatidylethanolamine, unidentified lipids and aminolipids; a moderate amount of aminophospholipid was also detected. The DNA G + C content was 34.7 mol%. The predominant quinone system was menaquinone (MK-6). On the basis of polyphasic taxonomic evidence presented here, strain CC-CZW007(T) is proposed to represent a novel species within the genus Vitellibacter, for which the name Vitellibacter echinoideorum sp. nov. is proposed. The type strain is CC-CZW007(T) ( = BCRC 80886(T) = JCM 30378(T)).

Nocardioides echinoideorum sp. nov., isolated from sea urchins (Tripneustes gratilla).

A Gram-stain-positive, aerobic, non-motile, rod-shaped, yellow-pigment-producing bacterium, (designated strain CC-CZW004T), was isolated from seafood samples (sea urchins) at Penghu Island in Taiwan. Strain CC-CZW004T grew optimally at pH 7.0 and 30 °C in the presence of 3 % (w/v) NaCl. Phylogenetic analyses based on 16S rRNA genes revealed a distinct taxonomic position attained by strain CC-CZW004T with respect to other members of the genus Nocardioides. The novel strain shared highest 16S rRNA gene sequence similarities to Nocardioides daejeonensis JCM 16922T (96.4 %), Nocardioides pacificus JCM 19260T (96.3 %), and Marmoricola scoriae JCM 17444T (96.2 %). The major fatty acids of strain CC-CZW004T consisted of C17:0, C16:1ω5c, C17:1ω8c`, iso-C16:0 and C19:1ω11c/C19:1ω9c (summed feature 6). The diagnostic diamino acid in the cell wall was ll-2,6-diaminopimelic acid. The polar lipid profile was composed of major amounts of phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol and aminophospholipid. The DNA G+C content was 73.3 mol%. The predominant quinone system was menaquinone (MK-8). On the basis of polyphasic taxonomic evidences presented here, strain CC-CZW004T represents a novel species of the genus Nocardioides, for which the name Nocardioides echinoideorum sp. nov. is proposed. The type strain is CC-CZW004T ( = BCRC 16974T = JCM 30276T).

Echinimonas agarilytica gen. nov., sp. nov., a new gammaproteobacterium isolated from the sea urchin Strongylocentrotus intermedius.

A novel Gram-negative, facultatively anaerobic and motile bacterial strain, designated KMM 6351(T), was isolated from the sea urchin Strongylocentrotus intermedius and examined using a polyphasic taxonomic approach. A phylogenetic analysis based on 16S rRNA gene sequencing revealed that the strain formed a distinct phyletic line in the class Gammaproteobacteria and was most closely related to the genera Aliivibrio, Photobacterium and Vibrio. Strain KMM 6351(T) grows at 4-40 °C and with 0.5-12 % NaCl and decomposes aesculin, agar, gelatin, starch, chitin and DNA. The DNA G+C content of the strain was determined to be 46.1 mol%. The prevalent fatty acids were found to be C(16:0), C(18:1) ω7c, C(12:0) 3-OH and summed feature 3 (comprising C(16:1) ω7c and/or iso-C(15:0) 2-OH fatty acids). The major polar lipids were determined to be diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and an unidentified aminolipid. The predominant ubiquinone was found to be Q-8. The results of the phenotypic, chemotaxonomic and genotypic analyses clearly indicated that the novel strain should be assigned to a new genus and species within the class γ-Proteobacteria for which the name Echinimonas agarilytica gen. nov., sp. nov. is proposed. The type strain is KMM 6351(T) (=KCTC 22996(T) = LMG 25420(T)).

Profile and multidrug resistance determinants of Chryseobacterium indologenes from seawater and marine fauna.

The aim of this study was to investigate the prevalence and genetic basis of multidrug resistance in Chryseobacterium indologenes from seawater and marine invertebrates used for human consumption, in Kastela Bay, Adriatic Sea, Croatia. Out of 16 samples of seawater, Mediterranean mussel (Mytilus galloprovincialis Lam.), Rayed Mediterranean limpets (Patella caerulea L.) and Purple sea urchins (Paracentrotus lividus Lam.) collected, 15 were positive for C. indologenes. In total, 41 isolates were randomly selected and tested for antibiotic susceptibility by disc-diffusion and broth microdilution methods. PCR was used to detect alleles encoding extended-spectrum (ESBLs) and metallo-ß-lactamases (MBLs). The clonality of ß-lactamase-producing strains was evaluated by random amplified polymorphic DNA (RAPD) analysis. All C. indologenes isolates showed multiple resistance to at least 9 out of 16 antibiotics tested. Lowest resistance rates were found for piperacillin (9.7 %) and ciprofloxacin (24.4 %), whereas only piperacillin/tazobactam and trimethoprim/sulfamethoxazole showed 100 % activity. More than half of isolates carried bla (IND)-type gene, including 2 isolates carrying bla (IND-2) and 21 carrying bla (IND-7), that was identified as a major MBL genotype in isolates from Adriatic Sea. RAPD typing of IND-producing isolates revealed 6 major groups with no predominant clone in population. The presence of multidrug resistant and IND-producing C. indologenes in marine environment, including marine fauna, pose a risk for transmitting this opportunistic pathogen to humans through recreation or consummation of seafood. In addition, the antibiotic susceptibility test results have practical relevance for empirical treatment of C. indologenes infections.

Spiculisporic acids B­D, three new γ-butenolide derivatives from a sea urchin-derived fungus Aspergillus sp. HDf2.

Three new γ-butenolide derivatives 1–3, named spiculisporic acids B–D, were isolated from the culture of Aspergillus sp. HDf2, a marine-derived fungus that resides in the sea urchin, Anthocidaris crassispina. The structures of 1–3 were elucidated on the basis of spectroscopic methods, including MS and 2D NMR techniques. Their in vitro cytotoxic activities against two cell lines (SGC-7901, human gastric adenocarcinoma and SPC-A-1, human lung adenocarcinoma) and inhibitory activities against Staphylococcus aureus ATCC 51650 were investigated.

Bacillus hemicentroti sp. nov., a moderate halophile isolated from a sea urchin.

A novel Gram-staining-positive, moderately halophilic, facultatively alkaliphilic, non-motile, catalase-positive, oxidase-negative, endospore-forming, facultatively anaerobic rod, designated JSM 076093(T), was isolated from a sea urchin (Hemicentrotus pulcherrimus) collected from Naozhou Island in the South China Sea. Growth occurred with 0.5-25% (w/v) NaCl (optimum 5-8%) and at pH 6.0-10.5 (optimum pH 8.0) and 5-40 °C (optimum 30-35 °C). meso-Diaminopimelic acid was present in the cell-wall peptidoglycan. The predominant respiratory quinone was menaquinone 7 and the polar lipids consisted of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol and one unidentified phospholipid. The major cellular fatty acids (>10% of the total) were anteiso-C(15:0), anteiso-C(17:0), iso-C(16:0) and iso-C(14:0). The genomic DNA G+C content was 38.8 mol%. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain JSM 076093(T) belonged to the genus Bacillus and was related most closely to Bacillus hwajinpoensis SW-72(T) (99.1% 16S rRNA gene sequence similarity) and Bacillus algicola KMM 3737(T) (97.3%). The combination of results from the phylogenetic analysis, DNA-DNA hybridization and phenotypic and chemotaxonomic characterization supported the conclusion that strain JSM 076093(T) represents a novel species of the genus Bacillus, for which the name Bacillus hemicentroti sp. nov. is proposed, with JSM 076093(T) (=DSM 23007(T)=KCTC 13710(T)) as the type strain.

Comparative proteomic analysis of a sea urchin (Heliocidaris erythrogramma) antibacterial response revealed the involvement of apextrin and calreticulin.

Echinoderms evolved early in the deuterostome lineage, and as such constitute model organisms for comparative physiology and immunology. The sea urchin genome sequence (Strongylocentrotus purpuratus) revealed a complex repertoire of genes with similarities to the immune response genes of other species. To complement these genomic data, we investigated the responses of sea urchins to the injection of bacteria using a comparative proteomics approach on a closely related species. In the sea urchin, Heliocidaris erythrogramma, the relative abundance of many proteins was altered in response to the injection of both bacteria and saline, suggesting their involvement in wounding responses, while others were differentially altered in response to bacteria only. The identities of 15 proteins that differed in relative abundance were determined by mass spectrometry. These proteins revealed a significant modification in energy metabolism in coelomocytes towards the consumption of glutamate and the production of NADPH after injection, as well as an increased concentration of cell signalling molecules, such as heterotrimeric guanine nucleotide-binding protein. The injection of bacteria specifically increased the abundance of apextrin and calreticulin, suggesting that these two proteins are involved in the sequestration or inactivation of bacteria.

New diketopiperazine derivatives isolated from sea urchin-derived bacillus sp.

Two new diketopiperazine derivatives, bacillusamides A (1) and B (2), have been isolated from the EtOAc extract of the sea urchin-derived Bacillus sp. along with the known cyclo(-L-pro-L-val-) (3), cyclo(-L-pro-L-tyr-) (4), cyclo(-L-pro-L-phe-) (5). These structures were elucidated by extensive spectroscopic methods. Furthermore, the absolute configurations of the amino acid residues were determined using Marfey's method. Compound 1 displayed weak antifungal activity against Aspergillus niger.

Mannose-binding C-type lectins as defense molecules on the body surface of the sea urchin Pseudocentrotus depressus.

We found that the extract of the body wall of the sea urchin, Pseudocentrotus depressus, agglutinate Escherichia coli and is inhibited by mannose. A mannose-binding protein of 22 kDa was purified via affinity chromatography using mannose-agarose. Amino acid sequences obtained by Edman degradation and liquid chromatography quadrupole time-of-flight mass spectrometry followed by de novo sequencing suggested that the protein is a C-type lectin. Products of PCR with a degenerate primer pair and of RACE PCR for the cDNA of the 22 kDa protein were sequenced and produced two full-length cDNA sequences encoding C-type lectins. These two lectins, named P. depressus mannose-binding C-type lectin (PdMBCL) 1 and 2 are composed of 187 and 189 amino acid residues, including signal peptides, respectively, and share 86% identity in their mature form. PdMBCLs agglutinated Lactococcus garvieae, a Gram-positive fish pathogen. Reverse transcription PCR showed that both the genes for the PdMBCLs were expressed in the body wall and in other tissues. Furthermore, the lectins were detected from a rinse of the body surface. Taken together, the present study showed that PdMBCLs function as anti-microbial agents on the body surface of P. depressus.