Delroisse J et al. (2020), Epidemiology of a SKin Ulceration Disease (SKUD...

ECB-ART-49419

Sci Rep 2020 Dec 17;101:22150. doi: 10.1038/s41598-020-78876-0.

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Epidemiology of a SKin Ulceration Disease (SKUD) in the sea cucumber Holothuria scabra with a review on the SKUDs in Holothuroidea (Echinodermata).

Delroisse J , Van Wayneberghe K , Flammang P , Gillan D , Gerbaux P , Opina N , Todinanahary GGB , Eeckhaut I .

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Aquacultivated sea cucumbers often suffer from SKin Ulceration Diseases (SKUDs). SKUDs have been observed in six holothuroid species from nine countries. All SKUDs present a similar symptom-the skin ulceration-and can be induced by bacteria, viruses, or abiotic factors. We here provide an update on SKUDs in holothuroids and analyse the case of the SKUD observed in Holothuria scabra in Madagascar. Field observations revealed a seasonality of the disease (i.e. wintertime maximum peak). Morphological analyses of integument ulcers showed that sea cucumbers react by forming a collagen fibre plug. Metagenomic analyses revealed a higher proportion of Vibrionaceae (Gammaproteobacteria) in ulcers in comparison to the healthy integument of the same individuals. Experimental infection assays were performed with ulcer crude extracts and bacteria isolated from these extracts (e.g. Vibrio parahaemolyticus) but did not significantly induce skin ulceration. Our results suggest that the disease is not induced by a pathogen or, at the very least, that the pathogen is not found within the ulcers as the disease is not transmissible by contact. An initial cause of the SKUD in Madagascar might be the repeated and prolonged exposures to cold temperatures. Opportunistic bacteria could settle in the dermis of ulcerated individuals and promote the ulcer extension. We propose a general nomenclature for SKUDs based on the acronym of the disease, the affected sea cucumber species (e.g. Hs for Holothuria scabra), the concerned region using an ISO code 3166-2 (e.g. MG for Madagascar), the description date (e.g. 20 for the year 2020), and, when known, the inducing agent (first letter of the general taxon, b for bacteria, v for virus in currently known cases; a a if it is an abiotic inducing parameter; nothing if the inducing cause has not been precisely identified). The disease described in this work will be designated under the name SKUD Hs-MG-20.

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	Figure 1. Holothuriculture in Toliara. (A) Pens from the company Indian Ocean Trepang at « La Mangrove » during low tide, (B) Collectors at « La Mangrove », (C) SKUD-affected collection at « La Mangrove », (D) H. scabra buried in the sediment, (E) Freshly collected sea cucumbers, (F) SKUD-affected sea cucumbers.
	Figure 2. Phenotype characterisation of SKUD stages in H. scabra. (A) Stage I, (B) Stage II, (C) Stage III, (D) Stage IV.
	Figure 3. (A) Distribution of SKUD occurrence within IOT pens (Belaza, Toliara, Madagascar) between April 2016 and October 2017. The figure shows the monthly number of total sick Holothuria scabra individuals recorded in different IOT pens. The colour-code corresponds to the different pens that were investigated. The dotted line shows the evolution of average daytime temperatures measured in IOT pens (surface water). (B) Proportion of stages I/II and stages III/IV SKUD-affected Holothuria scabra sea cucumbers during selected months (see Fig. 2 for the description of the stages). (C) Organisation of IOT pens investigated in the present study. The map was obtained from Google map (https://www.google.be/maps, accessed in May 2020, Images 2020 Maxar Technologies, Images 2020 CNES/Airbus).
	Figure 4. Histology of healthy and SKUD-affected Holothuria scabra sea cucumber integuments. (A–C), (E–I) Masson’s Trichrome staining. (A–C) healthy integument, (D–I) SKUD-affected integument. Legend: Epi epidermis, LCT loose connective tissue, DCT dense connective tissue, Ucl ulcer.
	Figure 5. The fine external structure of healthy and SKUD-affected Holothuria scabra sea cucumber integument. (A,B) healthy integument, (A) external surface of the integument, (B) connective tissue present under the epidermis (transverse section). (C–H) SKUD-affected integument. (C–H) external surface of the ulcerated integument. (E–H) Ossicles, bacteria and diatoms visible at the ulcer surface. Legend: b bacteria, c cuticle, Cf collagen fiber, d diatom, pfe predicted flagellated eukaryotes, Os ossicle.
	Figure 6. TEM observations of the dorsal integument of healthy and SKUD-affected Holothuria scabra. (A–C) healthy integument; (D–F) SKUD-affected integument. The upper portion of the body wall showing the thin cell processes of covering cells and the loose connective tissue with the perikaria of basal cells, covering cells, type I mucocytes (A; arrows show the thin epidermal processes and asterisks underline the various collagen fibre orientations); pigment cells (B); and a detailed view of first micrometres from the surface showing the nanometric epidermal covering and the orientation of collagen fibres (C; arrows show the hemidesmosomes). SKUD-affected integument shows degrading cell clusters with vacuolated fragmented cells (D); degrading cells into the loose connective tissue (E); and invading bacteria (black asterisks) and degrading cell material with electron-dense crystalline bodies (white asterisks) (F). Legend: Bc basal cell, Cc covering cell, EpP epidermal process, LCT loose connective tissue, MuI mucocyte cell of type I, MuII mucocyte cell of type II, Np nerve process, Ol ossicle lacunae, space of dissolved ossicle, Pc pigment cell, VFC vacuolated fragmented cell.
	Figure 7. Schematic view of the fine structure of the Holothuria scabra dorsal integument. This schematic representation is based on TEM observations. Legend: Bs basal cell, Cc covering cell, Cf collagen fiber, H hemidesmosome, Mu I Type I mucocyte, Mu II Type II mucocyte, Np nerve process, Ol ossicle lacunae, Pc pigment cell.
	Figure 8. The relative proportion of Bacteria within healthy integument and ulcerated integument of two SKUD-affected Holothuria scabra sea cucumber individuals. (A) Bacteria, (B) Proteobacteria, Gammaproteobacteria, (D) Alphaproteobacteria. (MG-RAST parameter: E-value > e−10, Percentage Identity > 70%, Alignment length > 15, Minimum Abundance = 50, Representative hits, database: RefSeq). See supplementary Table S1 for statistical analyses.
	Figure 9. Attempted induction of SKUD in Holothuria scabra: qualitative pilot experiment performed to test a potential induction of the SKUD via a direct contact between healthy sea cucumbers and SKUD-affected sea cucumbers. (A) Inoculation of 10 scarified healthy individuals. (B) Inoculation of 10 non-scarified healthy individuals. Observations were performed for 10 days.
	Figure 10. Attempted induction of SKUD in Holothuria scabra via the inoculation of cultured bacteria on the dorsal integument: three cultured bacteria, isolated from SKUD ulcers, were inoculated on artificial scarifications performed on the dorsal integument of the sea cucumbers. (A) Integument scarification/ulceration coverage observed for SKUD-ulcers (purple line) and artificial scarifications (blue line) in SKUD-affected sea cucumbers (controls). (B) Integument scarification/ulceration coverage observed in scarified healthy sea cucumbers (controls). (C) Integument scarification/ulceration coverage observed after inoculation of Vibrio sp. SKUD-MD-20 C1 (culture 1). (D) Integument scarification/ulceration coverage observed after inoculation of Vibrio variabilis SKUD-MD-20 C2 (culture 2). (E) Integument scarification/ulceration coverage observed after inoculation of Vibrio parahaemolyticus SKUD-MD-20 C3 (culture 3) (F) Integument scarification/ulceration coverage observed after inoculation of filtered sea-water (negative controls).

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