Yves Samyn1,2, Didier Vanden Spiegel4, Anne Franklin1,2, Arnaud Réveillon1,2, Hendrik Segers1,3 and Jackie Van Goethem1,2
1Royal Belgian Institute of Natural Sciences, Rue Vautier 29, B-1000 Brussels, Belgium; 2Belgian Focal Point to the GTI; 3Belgian Biodiversity Platform;4 Royal Museum for Central Africa, Leuvensesteenweg 13, B-3080 Tervuren, Belgium
Alessandro Lovatelli, in his capacity as Fishery Resources Officer (Aquaculture) of the Inland Water Resources and Aquaculture Service (FIRI), FAO Fisheries Department, as well as principal editor for the present ASCAM proceedings, asked us to devote some words on how sea cucumbers should be optimally preserved for taxonomic identification. It is with pleasure that we respond to his request and take advantage of the opportunity to frame this preservation protocol in the broader context of capacity building in taxonomy and collection management. This will be done by briefly outlining the history and scope of the Global Taxonomy Initiative (GTI) as well as Belgiums contribution to it.
CBD, GTI and the Belgian contribution to it all
The United Nations Convention on Biological Diversity (CBD) was adopted at the 1992 Earth Summit in Rio de Janeiro, Brazil. Conservation of biodiversity, sustainable use of its components and fair and equitable sharing of the benefits arising from the use of genetic resources, the three goals of the CBD, became prime points on the political agenda of contracting parties (188 Parties in May 2004). However, through the Conference of the Parties (COP), it was quickly recognized that the standing capacity to identify and monitor biodiversity are, and will continue to be, the limiting factors that allow successful and effective implementation of the three CBD objectives (see also Article 7 of the Convention text).
Taxonomy and collection management were recognized to be important keys. The Global Taxonomy Initiative (GTI), one of the cross-cutting issues established by the COP, is the operational vehicle that must ensure that a satisfying amount of taxonomic capacity is installed, and this in all parts of the world, including the mega-diverse countries of the South where the paucity of taxonomic capacity is striking. As such, the GTI tends to remove or at least severely reduce the so-called taxonomic impediment - in other words, the knowledge gaps in our taxonomic system (including those associated with genetic systems), the shortage of trained taxonomists and curators, and the impact these deficiencies have on our ability to conserve, use and share the benefits of our biological diversity. More information on the GTI is available at: http://www.biodiv.org/programmes/cross-cutting/taxonomy/.
Belgium fully endorses the operational programme of work of the GTI (COP decision VI/8) and has designated the Royal Belgian Institute of Natural Sciences as National Focal Point to the GTI. This Focal Point approaches Belgiums GTI obligations by being: (i) an information centre; (ii) a facilitation centre, (iii) a partnering centre and, (iv) a tutoring centre.
Belgium has chosen to be pro-active in ensuring maximal implementation of the operational programme of work of the Global Taxonomy Initiative. The Belgian National Focal Point, in close cooperation with other Belgian Institutes such as the Royal Museum for Central Africa and the National Botanic Garden, and funded by the Belgian Development Cooperation, has designed an operative strategy that aims at constructing positive feedback-loops in capacity building for taxonomy and collection management, inter alia, for sea cucumbers.
The core of the Belgian approach is embedded in building transparent bilateral and multilateral synergies that not only promote scientific partnerships, collection valuations and optimal resources utilizations, but also envisages maximal supply of information and tuition. The modus to attain this goal is twofold:
the first approach adopts a top-down tactic: established taxonomists from the Focal Point identify important taxonomic impediments and tackle these by carrying out an in situ research project that incorporates clear-cut capacity human and/or institutional capacity building;
the second approach works bottom-up: interested parties from developing countries identify key taxonomic or collection management needs. Through an external call for proposals, the Focal Point, in close cooperation with other competent Belgian taxonomic bodies, makes available its expertise, collections and collection-based information to meet the need.
Mr Lovatellis request for an adequate preservation protocol for holothuroids complements this dichotomous approach by addressing a most crucial issue: i.e. how to expedite taxonomic research without wasting precious natural resources. Hence, we hope that the protocol below can serve as an important drive to identify and monitor this ecologically and economically important group.
Building a representative sea cucumber collection and the protocol to preserve specimens for taxonomic identification
1. Legal framework - Prior to commencing with the construction of a reference collection, make sure that you possess all the necessary legal authorisations to sample in the region. When specimens need to be transported (e.g. to specialists for identification), wrap them in alcohol impregnated cotton. Pack in sealable plastic bags and add a little more alcohol. Pack in one or two additional plastic bags so that no alcohol can leak out. Make sure that, prior to such transport, you respect all international agreements, as some airline companies will not transport samples in alcohol. If this is the case, overseas transport by ship is your only alternative.
2. Getting specimens - In the past, holothuroid specimens were gathered by handpicking in the intertidal zone or by dredging in deeper waters. With the advent of SCUBA diving, selective sampling at greater depth in shallow waters became feasible. This technological innovation greatly augments the effectiveness of sampling. Moreover, SCUBA diving allows observation of a species in situ, thus greatly improving our understanding of the ecology of the species. As such, information on life habitus, natural habitat, and even abundance can be obtained. Upon collection, individuals are best kept separate from each other, for instance by using plastic bags. Such physical separation of live specimens minimises potential antagonistic reaction between any two individuals (e.g. expulsion of Cuvierian tubules, auto-evisceration) and thus greatly augments the taxonomic value of the specimens. When and wherever possible, sampling should be done both at night as well as during the day, as it has been well documented that some species appear only during the night. Sampling in as many representative habitats as possible (for instance on the outer reef, but also in the associated coral reef lagoon as well as in the adjacent seagrass bed and mangrove forest) significantly increases the completeness of the survey. Moreover, as holothuroids often have a fugitive behaviour, turning stones and sieving substrate (especially for individuals smaller than 1 cm) frequently exposes additional specimens.
3. Number of specimens required - Although there is no rule as to the number of specimens that needs to be collected per species, it is important to realize that at least two to three specimens per species will be needed to ensure a reliable identification of the species. Identification of holothuroids depends to a large extent on the shape, the size and the distribution of the ossicles in the different body parts. As it is documented that during growth ossicle assemblage in some species can drastically change, specimens belonging to different size classes may present different ossicles. Whenever possible, it is thus strongly recommended that growth series of a single species (encompassing at least one small, one medium and one large individual) are collected. Only such growth series will enable competent taxonomists to assess intraspecific variation and as such avoid unnecessary splitting. If you have collected a large number of specimens belonging to the same species, it is ideal to deposit the voucher material in different museums. In this way, not only can your work be built upon in further studies, but you will benefit from partnerships with these museums. The same rule of thumb applies to eventual new species; distributing paratypes over different museums will not only ensure that the species is fully available for further study, it will also stimulate museums to work in partnership with you in the future.
4. Relaxation / anaesthetisation of collected specimens - Holothuroid specimens, at all times, should be adequately anaesthetized prior to preservation as otherwise the preservative will cause contraction and, at times, even evisceration of the specimen that then renders the specimen unfit for taxonomic purposes. Several methods exist to relax a holothuroid, but the most commonly used one today is anaesthetization with magnesium chloride (Epson salts as available in virtually every drug store can also be utilized). The animals are left to expand and extend their tentacles in a container of seawater, and then magnesium chloride (MgCl2) is added till the concentration is roughly 5 %. The animals should be left in the anaesthetizing solution until they cease to respond to probing, but they should not be allowed to die in the anaesthetic as tissue breakdown by autolysis can set in very quickly (for instance this is often the case with holothuroids from the family Stichopodidae; here placing the container in a fridge can help slow down autolysis). A well relaxed holothuroid, with its tentacles and, if present, tube feet fully extended, will greatly facilitate a taxonomists job. At this stage in the protocol, making pictures of the specimen will help with later description.
5. Preservation of collected specimens - When the animals are adequately anaesthetized the specimens can be place in a fixative fluid. Here again several fixatives can be used, but it should be remembered that the most important taxonomic characters of a holothuroid are its calcareous ossicles. Such ossicles can dissolve if the solution is acid (pH below 7). An acid preservative like unbuffered formalin (10 %) will quickly dissolve the ossicles in the different body parts. The preservation protocol commonly utilised is the following: (i) preserve for 1 to several days in 10 % buffered formalin solution, (ii) replace the formalin with a buffered alcohol solution of 70-80 %; (iii) after a couple of days, refresh the alcohol with a 70 % buffered alcohol solution for permanent storage. When dealing with large and/or stout specimens, it is generally useful to also inject in step (i) concentrated buffered formalin in the coelomic cavity (this can be done with a hypodermic needle; use a volume of approximately 1/10 th of the estimated volume of the coelomic cavity).
6. Sea cucumbers as micro-ecosystems - Holothuroids can be seen as micro-ecosystems as they often are host to a diverse assemblage of symbionts which range from turbellarian worms living in the coelomic cavity, scaled polychaetes foraging on the epidermis, crustaceans and molluscs between the tentacles and around the anus, and even fish living in the respiratory trees (making echinoderms the only invertebrates that host a vertebrate). Upon relaxation and preservation it is little trouble to also collect and properly label these associates.
Further information available from:
Dr Yves Samyn
CBD National Focal Point - Global Taxonomy Initiative (GTI)
Royal Belgian Institute of Natural Sciences
Rue Vautier 29
B-1000 Brussels, Belgium
Figure 1. Left: making notes on Thelenota ananas (Jaeger, 1833) as found in the Mombasa Marine National Park, Kenya. (Photo: B. Van Bogaert).
Figure 2. Relaxation and preservation of sea cucumber specimens. (a) specimens are kept separate from each other using plastic bags - pictured is a specimen of Bohadschia subrubra (Quoy & Gaimard, 1833) collected in the field (make sure the bags contain enough seawater);(b) field lab with separate containers for relaxation and preservation; (c) relaxation and anaesthetization with approximately 5% magnesium chloride (some extra magnesium chloride salt is added as the animal showed no signs of relaxation after 1 hour); (d) specimens of Neostichopus grammatus (H.L. Clark, 1923) -packed for temporarily storage in 70-80% alcohol (note that the alcohol extracts the coloration pigments) (Photo: I. Tallon).
 Formalin usually can be
purchased as a 40 % aqueous solution of the gas formaldehyde (a 10 % formalin
solution thus equals a 4 % formaldehyde solution). To make 10 % formalin
solution one adds 9 parts of seawater to 1 part commercial formalin.
Formaldehyde solutions tend to be acidic; to neutralize gently add saturated
borax solution (Na2B4O7 or
Na2B4O.4H2O) until the pH is around 8 (use
 The alcohol used for preservation is ethanol. This does not have to be pure ethanol, which can be expensive. For routine preservation (i.e. not for preserving tissue intended for molecular studies, here pure ethanol should be used) alcohol in the form of methylated spirits is commonly used. To buffer the alcohol, gently add saturated borax solution until the pH is around 8 (use indicator sticks).