Deep-Sea Coral Collection Protocols - NOAA's Coral Reef ...

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Although the focus of this chapter is reproduction inthe deepwater scleractinia, members of thegorgonacea, antipatharia and stylasterina also fallunder the definition of colonial corals, and all maycomprise significant components of deepwater coralecosystems. Apart from the stylasterina, these ordersall have the same basic bauplan, with simple polypsthat contain the gametogenic material.A study of Antipathes fiordensis from New Zealand(Parker et al 1997) showed that like many colonialscleractinians, this black coral species isgonochoristic, with an approximate 1:1 sex ratio and aseasonal reproductive cycle resulting in broadcastFig. 16c. Eggs at the base of a bamboo coral polypspawning of gametes. The gametes originate in theendoderm (gastrodermis) of the mesenteries but,unlike scleractinians, the gametes do not migrate to the mesoglea to continue development. Aninvestigation into gametogenic cycles of 6 common gorgonian species in Biscayne Bay Florida(Fitzsimmons-Sosa et al. 2004) showed that all species were gonochoristic, five showed reproductiveseasonality and sex ratios were either 1:1 or 1:2 male: female. As with the hexacorals, gametes of theseoctocorals also develop within the mesenteries of the polyps (Fig. 16c).The order Stylasterina (phylum Cnidaria, class Hydrozoa) are a group of calcareous encrusting orbranching colonial species often referred to as hydrocorals. Unlike scleractinians, there is no informationin the literature indicating that the survival and reattachment of branchfragments (fragmentation) is amechanism of new colony formation in hydrocorals. The male and female reproductive structures orgonophores develop inepidermally-lined cavities calledampullae, which are usually present as superficialhemispheres, but occasionally they are completelysubmerged (internal) in the coenosteum. They aregenerally smaller and more numerous in male colonies.Stylasterines are gonochoristic, with internal fertilizationand embryonic development. Planular larvae are releasedvia efferent ducts, which are usually seen as small irregularpores on or near the ampullae. A study of reproduction inAlaskan hydrocorals (Brooke 2004), showed a range ofdevelopmental stages in all species studied. This implieseither continuous reproduction or an extended nonsynchronousdevelopmental season. Reproductivestrategy has implications for habitat recovery potential; forexample, a species with a longer planktonic phase may bemore able to recover from large scale damage than onewith a very short larval phase, or those that can form newcolonies from fragmented branches may recover quicklyfrom small scale physical damage.15Fig. 16d. Longitudinal section of Metallogorgiamelanotrichos polyp with developing oocytes
To fully describe gametogenic cycles, samples should be collected monthly over multiple years.However, the logistics and expense of working in the deep sea often prohibit such rigorous samplingregimes. Small fragments (10-20 polyps) should be taken from several different colonies, preferably atleast 10. In a gonochoristic species with equal sex ratios, this will provide approximately 5 replicatefemales, which is usually sufficient for statistical analysis, but more samples would give better statisticalpower if logistics permit. Males can be used to describe stages of gonad development, but femaleoocyte size frequency distribution is easier to quantify and is generally used to describe the gametogeniccycle. Samples should be fixed while alive and in good condition. Stressed corals often producemucous, which can inhibit fixation so ideally, a cold room would be available to allow the corals torecover from the stress of collection and exposure to warm surface water. If not, then fix the samples assoon as they come on board. Dying or decaying tissue is not useful.There are many different fixatives that can be used; the most readily available effective fixative to use is10% formalin in seawater, which is made by diluting full-strength formaldehyde (37%) in a 1:10 ratiowith seawater. This is not recommended for fine cytological examination, but works sufficiently well todetermine gametogenic status. Tissues should be left in this fixative for at least 36 hours beforethoroughly rinsing in distilled water and transferring to70% ETOH for storage. Hellys solution (Barszczand Yevich 1975) is a better fixative for corals but is a little more complicated. A stock solution of 100gm potassium dichromate and 200 gm zinc chloride in 4 L seawater can be made prior to sampling, butthe addition of the formaldehyde (200ml) should only be made when needed since the full mixture isonly good for 24 hours. The samples should remain in the solution for 16-20 hours then thoroughlyrinsed in freshwater and transferred to 70% ETOH for storage. Another alternative is ‘Z-fix’ (AnatechLtd.), which also contains formaldehyde, and is simply mixed with seawater prior to use.<strong>Coral</strong>s need to be decalcified before further processing. A solution of 10% hydrochloric acid withEDTA buffer takes between 1 and 12 hours to decalcify, depending on the sample. An acid change maybe needed for heavily calcified skeletons. After rinsing thoroughly in running freshwater, tissues aredehydrated through a series of ethanol concentrations (70-100%) and then transferred to a clearingagent such as toluene. The tissues are then embedded in paraffin wax, cut into 8µm sections with amicrotome blade and stained using Mayer’s Haematoxylin/Eosin B before mounting on microscopeslides. This combination of stains will show lipids (vitellogenic oocytes) as bright pink and DNA(primordial germ cells as testes) as purple. Oocyte diameter can be measured using a compoundmicroscope with an ocular micrometer or images of oocytes can taken using a digital camera andsubsequently measured with image analysis software. There will be some shrinkage of the oocyte duringthe histological processing, so all measurements are “relative”. A minimum of 50 (preferably 100)oocytes should be measured per polyp; however this is not always possible. This basic process can beapplied to any of the deepwater colonial corals, but for those species with large yolky eggs, paraffinpenetration can be improved by using a series of toluene and paraffin solutions.A full treatise on histological techniques is beyond the scope of this chapter but recommended furtherreading includes Carson, 1997, Kiernan, J.A. 2003.16
Page 1 and 2: DEEP-SEA CORAL COLLECTION PROTOCOLS
Page 3 and 4: DEEP-SEA CORAL COLLECTION PROTOCOLS
Page 5 and 6: Table of ContentsForward ………
Page 8 and 9: Deep-sea coral habitat and rangeby
Page 10 and 11: Branching patterns in octocoralsby
Page 12 and 13: Fig. 9. Examples of deep-water cora
Page 14 and 15: Sclerites in octocoralsby Stephen D
Page 16 and 17: Ahermatypic scleractinians of the w
Page 18 and 19: Reproduction in deep-sea colonial c
Page 22 and 23: Genetic methods for species identif
Page 24 and 25: Handling procedures adopted by the
Page 26 and 27: Specimen deterioration. We have see
Page 28 and 29: Documenting deep-sea coral collecti
Page 30 and 31: Video protocol (cont.)Video transec
Page 32 and 33: John Reed's August 22 article on St
Page 34 and 35: CollectingThere is a broad range of
Page 36 and 37: Tissue preservationa. Do not let sp
Page 38 and 39: Some preservation techniques for (d
Page 40 and 41: LabelingThe significance of proper
Page 42 and 43: References and further readingAbbia
Page 44 and 45: Etnoyer, P., and L. Morgan. 2005. H
Page 46 and 47: McFadden, C.S., and M.B. Hutchinson
Page 48 and 49: Van Oppen, M.J.H., B.L. Willis, and
Page 50 and 51: Appendix 1. Classification of the D
Page 52 and 53: Appendix 2. Deep-sea coral Panel of
Page 54: Appendix 3. Deep-sea corals onlineN

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