Molluscan Research: Techniques for collecting, handling, preparing ...

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26 Extraction of radulae from micromolluscs The radulae of micromolluscs can be very small, often making manipulation daunting. However, with some practice and patience, radulae a fraction of a millimetre long can routinely be successfully mounted. If possible, use an adult specimen unless an ontogenetic study is specifically carried out, as the radulae of many species change morphology with age (Warén 1990). In some cases so-called generic characters are obtainable only from adult radulae. It is recommended that specimens used for radular preparation should be photographed prior to radular extraction, especially if there is any doubt as to identity. The shell may be destroyed when attempting to remove the body and, if chemical treatment is used, tissue-dissolving agents contribute to the deterioration of the shell. In some cases, species-level identification requires the observation of minute details such as protoconch microsculpture that cannot be observed with a light microscope and necessitates the use of SEM. While the radula can be dissected from larger microgastropods, there is a danger of damaging it. A safer method is to dissolve the buccal mass or even the entire animal. There are a number of methods used for dissolving the tissue surrounding the radula. The simplest and quickest methods can be used for most gastropods. Gentler, more time consuming and more complicated methods may be necessary for some of the groups with delicate radulae or radular membranes. The latter include: • Patellogastropoda. Damaged by strongly alkaline agents; mineralised cusps fall off and the remaining parts are partly dissolved. • Monoplacophora. Teeth damaged by strong alkali. GEIGER ET AL. (2007) MOLLUSCAN RESEARCH, VOL. 27 • Lepetelloidea. Teeth may get distorted and crack in strong KOH. • Vetigastropoda. o In some with thin and slender teeth (e.g., calliostomatids, trochaclids), the teeth become softer and tend to stick together. These should, after rinsing and cleaning, be soaked in 50% ethanol and mounted in at least 80% ethanol, to reduce the risk of the teeth sticking together. In ethanol the teeth become stiffer and there is less surface tension. o In some fissurellids (Cosmetalepas) the teeth fall off the radular membrane when treated with strong alkali. We advise against trying to dissolve the animal inside the shell. Strong NaOH or KOH will damage the organic matrix in the shell (Strasoldo 1991) and the remaining hydroxide will react with aerial carbon dioxide, to form a crystalline or powdery coating that cannot be removed (Fig. 10). However, with some sturdy gastropods, such as marginellids, no adverse effects have been reported (Coovert and Coovert 1987) and short periods of maceration of tissue inside the shell can be tried. Shells of more fragile gastropods, such as scissurellids, will break when sonicated after such treatment, whereas they are stable before radular extraction. Shells exposed to tissue dissolving agents also deteriorate over time and can be completely broken down in as little as 10 years, while enzymes in detergents can destroy a shell in a few hours, due to the low pH. Proteinase K, commonly used in DNA extraction from tissues, is most destructive to the shell. Whereas hydroxide treatment usually leaves a recognisable shell behind, proteinase K will fragment shells. FIGURE 10. A. Protoconch of shell (Scissurellidae) after sodium hydroxide treatment. B. Similar protoconch without hydroxide treatment. Note the tunnelling and recrystallisation in A. Scale bars = 100 µm. Images DLG. Standard method The tissue of the body can be dissolved in 5–40% NaOH, KOH, bleach, or by using proteinase K as part of DNA extraction. Sodium dodecyl sulphate (SDS = sodium lauryl sulphate) provides a further alternative. The hydroxide concentration indicated in the literature is quite variable; higher concentrations are advocated by those who like to speed up the dissolution of the tissue (e.g., Coovert and
TECHNIQUES FOR STUDYING SMALL MOLLUSCAN SPECIMENS 27 Coovert 1987), which can be additionally accelerated by heating specimens up to 100°C. Such drastic methods are usually not necessary for micromolluscs and usually lower hydroxide concentrations (5–10%) and no or low heat (30– 50°C) are suitable. Lindberg (1977) noted adverse effects of heating on patellogastropod radulae, particularly the contraction of the radular membrane and also the separation of teeth from the radular membrane. Bleach offers a low cost option but dissolving power varies from brand to brand. Test treatments should be carried out before risking rare material. Some brands of bleach dissolve soft tissue in a shorter time and cause fewer artefacts on radular teeth than KOH. Some of us consider bleach too aggressive and favour the inexpensive SDS, while others (AW, WFP) prefer KOH, as the specimen can be left in the solution for several days if necessary. With dissected patellogastropod radulae, to avoid destruction TS and AW add bleach drop by drop until maceration is visible. Never place paper identifying tags in the extraction solution, because crystal deposits will form on the radula (e.g., Geiger 1999: figs. 11, 12). It is not clear what the chemical composition of the deposit is, but it may be formed as a precipitate from the hydroxide and the filler substance used in most papers. Always label the containers on the outside. Manipulation of the radula and the body can be achieved by a variety of implements; forceps and needles, paint brushes, Pasteur and Eppendorf pipettes (see Tools above). Gentle methods. Patellogastropod radulae can be extracted by dissolving the head or body in a large quantity of very weak (ca 0.1%) KOH at room temperature. A small body requires several days, but the radula is not damaged. The body will not dissolve completely, but after a few days the buccal mass with the radula can usually be dissected out with no great risk of damage. After removal, the radula should be soaked even longer in a new bath of the same solution to remove most of the soft tissue, although usually some remains. If necessary, the remainder can be removed by quickly dipping the radula in lukewarm, diluted (1:3–1:5) commercial bleach for a few seconds and then vigorously rinsing it. Make sure that the radula can be retrieved quickly if accidentally dropped into the bleach. A strong solution of sodium lauryl sulphate is very gentle but takes a few days. It is difficult to get the radula completely clean but rinses in hot water or diluted bleach as above can be used. For larger radulae a fine paint brush can be used as a starter. Risso-Domingue (1961) discussed other amines useful for radular extraction, particularly in cases where the radular membrane is weak and hydroxide treatment results in teeth becoming isolated, rather than remaining attached to the radular membrane. Maceration For the maceration process, three different types of vessels can be used. All dishes should be made of glass; plastic charges statically and metal surfaces produce precipitates with hydroxide solutions. • Medium specimens (2–6 mm). Covered square embryo bowl is suitable, filled to about half its depth. When moving, be careful to avoid the fluid entering between the bowl and the lid. • Small (< 2 mm). Use a depression slide, 5–6 mm thick, with a depression as deep as possible and approximately 15 mm diameter. As a lid use a depression slide with a wider depression of any depth (as described above). Never use a regular, flat slide or cover slip because condensation will enter the space between the lid and bottom and the two slides will stick together and sometimes also contact the fluid in the bowl. Avoid jerky movements (and hence slop) when transporting the slide sandwich, or when removing the lid. Eppendorf tubes are not recommended, despite the advantages of a tight fitting lid and the possibility of spinning down the solids (i.e., radula and shell), because it is very difficult to remove the radula from the narrow tube. Heat incubation is best accomplished in a small incubator that allows for precise (within 2–3°C) temperature control. Cabinet incubators, slide warmer plates and dry bath incubators are suitable. The incubator should be situated close to the microscope preparation area to minimise risks associated with transport. It is important to cover the container during maceration, particularly if heating the specimen, because crystals of sodium carbonate (flocculent material of Mikkelsen 1985) may otherwise form due to absorption of carbon dioxide from the air. These do not redissolve when adding small amounts of water and make it very difficult to find a tiny radula. The formation of insoluble crystals has been observed particularly when macerating the animal within the shell, but here a calcium compound may be involved. Sometimes different water-soluble crystals are formed after heating. A number of chemicals can be used for maceration. Among the hydroxides, KOH is better than NaOH since it is less hygroscopic and reacts less quickly with aerial carbon dioxide. Always use analytic grade and preferably the kind that comes as small spheres or half-spheres because the powdered form reacts faster with aerial carbon dioxide. Do not use a stock solution but prepare it fresh in situ with distilled water, to avoid unnecessary precipitates. The KOH tablets should be semitransparent-porcellanous; they become white and dull with age because they react with aerial carbon dioxide. Thus, keep the KOH in an airtight container. Proteinase K is a safe cleaning agent of radulae from fresh, frozen, or alcohol fixed material and will not result in any damage to the radula (cf., prolonged exposure to hydroxides). Radula and operculum can be collected from the filter of spin columns (Fig. 11). Cut the spin column just above the plastic ring retaining the filter and carefully examine the filter as well as the plastic wall holding the filter under a stereomicroscope. If the pieces cannot be found,
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