TECHNIQUES

1. Introduction

Our knowledge of shallow water sponges of the British Isles is far from complete, and so users of this guide are encouraged to assist in their study. The techniques involved include observation, collection, photography, recording, preservation, slide preparation, and identification, and these are described below.

2. Observation

Marine sponges are widely distributed in the seas of the world, from intertidal to hadal depths. In this guide to sponges of Britain and Ireland, we are concerned only with those species occurring in shallow waters - i.e. down to the continental shelf (200m), but in practice a limit of 50m is imposed by the restrictions of compressed air diving. There is little doubt that this guide would not have evolved without the use of scuba-diving, which has enabled in situ observations of living sponges to be made.

Sublittorally, sponges are found mainly on hard rock substrates, but can occur on algae, or even loose on stable sediment bottoms. Massive or erect sponges are usually easy to spot, whereas encrusting sponges can be more cryptic owing to their small size and/or location. Some species tend to avoid the light and prefer to live in crevices or under overhangs. On the shore sponges occur in niches that remain wet or damp, and the undersides of large boulders are favoured habitats. On a geographical level, the greatest sponge diversity occurs on the coasts of Ireland and south-west Britain. Diversity decreases northwards on the west coast of Scotland, and eastwards along the Channel. The least diverse region is the North Sea, but some boreal species are found only there. On a local level, diversity and abundance can be variable and unpredictable. Different depth zones have different faunas, but all zones from the sublittoral fringe down to the lower circalittoral have their characteristic species, and are worth visiting. Highest diversity often occurs in areas of moderate tidal streams and some degree of shelter from wave action, below 25m depth. Caves and overhangs where natural light is reduced are particularly rich habitats.

In situ observations are extremely valuable, and an underwater recording slate or shore note book should be used to collect as much information on the following topics as is practical - form, size, colour, surface, apertures, depth, microhabitats and abundance.

3. Collection

The actual collection of specimens should not be undertaken lightly. Whilst the advances in our understanding reflected in this guide could not have been made without collecting, the interests of conservation should be kept firmly in mind. Many species are slow growing and long lived, possibly 50 years old or more, and some are locally, or even nationally, rare.

The following strategy is recommended.

3.1. Prior to collection, record as much in situ information as possible (see previous section), and take one or more photographs (see next section).

3.2. Only collect if the specimen is to be fully "processed" in the ways outlined here. Collection cannot be justified unless it results in the maximum amount of information possible being gained, and the specimen being kept for posterity.

3.3. Only collect a few specimens per dive/shore trip. To fully process more than this tends to take an unacceptable length of time and may lead to confusion.

3.4. Only collect the whole specimen if this gives essential information about its form - otherwise only collect a representative part, to include basal and surface tissue. (The part left behind will continue to grow.)

3.5. With crusts, collect the substrate if possible. Sponges often overgrow barnacles or saddle-oysters and these are more easily removed than rock surfaces. This ensures that the basal skeleton is included in the specimen.

3.6. Spicule contamination can be avoided if each specimen is placed in a separate container as collected, numbered polythene bags are ideal when diving. A series of numbered zip top bags can be prepared beforehand and stored around the periphery of an underwater flashgun, held by a rubber band. They are then available in numerical order and can be matched up with the photographs taken.

4. Photography

Good quality underwater photography can give essential information concerning shape, colour and microhabitat of the living sponge in situ. If possible, take pictures of the whole sponge, to include habitat information, and close-ups to show surface characteristics.

Additional valuable information can also be obtained by photographing collected specimens on land. This should include a 3 x 1cm tag with the accession number, and a penny piece, or ruler, for scale. If in adverse field conditions (e.g. on a hard boat), photograph the specimens out of water, against a black background. Otherwise use a black container of seawater using flash or better still 2 flashes to minimise excessive shadow. One of the flashes should be a key light and the other a fill in. The fill in should be placed at twice the distance of the key flash if both are of the same intensity.

If using film colour transparencies should always be used, and two of each shot taken to increase the likelihood of a good result and to allow one for loan if appropriate. Digital photography has now of course made it easy to check if the pictures are correctly lit and exposed. A digital SLR with macro lens is recommended but good results can also be obtained from some compact digital cameras with macro capability.

A good photograph can enormously enhance the value of a collected specimen. Photography has been one of the major "tools" in the evolution of this guide, and its value cannot be overstated. Those wishing to learn underwater photography should acquire a suitable book, such as EDGE, M. 2006. The Underwater Photographer : Digital And Traditional Techniques..

5. Recording

A significant contribution to this Guide has been the many records accumulated in the last few years. Although the process is time consuming, the completion of accurate, detailed and comprehensive records is essential for advancing our knowledge of sponges.

Records of four types are required, probably completed at different stages.

5.1. Site information. (see section 9 below on habitat recording).

5.2. Information from in situ observation - see section 2 above.

5.3. Details of the living specimen, immediately following collection. Make use of the terms in the glossary section of this guide.

5.4. Information from the specimen following preservation - see sections 7 and 8 below.

All the data that could be collected are described in detail in the Glossary and Reference Section. The terms therein should be used consistently and precisely, but one should be critical of the terminology, as this is still evolving.

6. Preservation

Sponges should be preserved as soon as possible after collection and the completion of the appropriate records. They will only keep fresh for a few hours in cool seawater - if kept overnight, aeration should be used.

Preservation is easier than for many other organisms, involving simply immersion in ethanol or isopropanol (iso-propyl alcohol), these are the preferred preservatives as they are much less damaging to DNA than the traditional industrial meths. Isopropanol (iso-propyl alcohol) is obtainable from a high street chemist, but ethanol is very restricted in its availability in most countries. Strength is not critical, as a 95% solution will become diluted with the water from the specimen. However, a large specimen may cause too much dilution, in which case the alcohol should be changed between 1 and 5 days later. Samples for DNA extraction should be cut into small pieces and the alcohol should be changed after a few hours, so as to dehydrate the specimen as completely as possible.

Before alcohol can be purchased, a Customs and Excise license is required, and this is not usually granted to private citizens. Biological institutions normally have a license, as does the Marine Conservation Society, 4 Gloucester Road, Ross-on-Wye, Herefordshire HR9 5BU.

Alternatives to the above are industrial methylated spirit (IMS - colloquially alcohol or spirit) and the ordinary methylated spirit from a hardware store. However, the former degrades DNA, and the latter is unpleasant to use, and also causes over-hardening of the specimen.

Traditionally, sponges were preserved dry. If done properly, the overall shape of the specimen will be retained, but the subsequent preparation and examination of sections for the microscope is unsatisfactory. The specimen should be dried in a gentle current of air. Seaweed biologists have been very successful extracting DNA from specimens dried and preserved in powdered silica gel and this may be a useful technique for sponges.

In the field, sponges can be preserved with alcohol in polythene bags, either the zip-top sort or sealed with a rubber band. Labels about 3 x 1cm, should be cut from a high quality writing paper, or goat-skin parchment and put into the bag immediately. Either a soft (2B) pencil, or a waterproof indian ink can be used for writing. The polythene bags can be kept in a large plastic bucket of spirit for the duration of the trip, following which the sponges can be transferred to individual jars, with fresh spirit if appropriate.

7. Slide preparation

7.1. Introduction

Microscope slide preparations are required for the examination of skeletons and spicules. By far the most useful preparation from a taxonomic point of view is a section. The arrangement of the spicules and other tissues forming the skeleton gives vital information for the identification of a sponge. Rarely will a preparation of spicules alone suffice, although it can provide useful additional information where spicules are difficult to differentiate in a section. The two types of slide preparation are described below. Note that clove oil is obtainable from high street chemist shops, and Euparol from GBI Laboratories Ltd., Shepley Industrial Estate, Audenshaw, Manchester M34 5DW.

7.2. Section preparation

a. Leave the sponge in spirit for at least 24 hours (preferably longer) to harden and dessicate.

b. Cut thin slices from the sponge; not too thin, or very few whole spicules will be included, but thin enough to allow the cover slip to sit flat on the slide. The problem with broken spicules is why hand sections are usually preferable to microtome sections. Include slices from the surface, at right angles to the surface to include surface and deeper tissue, and if the sponge has an axial structure, a longitudinal slice from the middle. Many encrusting species and some massive species (e.g. Tethyspira spinosa) have special spicule categories in the basal layer where the sponge adjoins the substratum, so ensure that this region is included. Most species have special layers at the surface so always include this in the section.

c. To cut sections, use a sharp scalpel, a cut throat razor, a safety razor blade, or an ordinary razor blade. For firm sponges a sawing action is best and for soft ones a single, decisive stroke. Sharpen the cut throat razor on a smooth oil stone frequently. Spicules are harder than steel! Small thin specimens may be best sectioned using a scalpel under a bimocular microscope.

d. Immerse the sections in absolute alcohol in a small watch-glass until they are free of water. (15 mins). If the specimens are recently collected two changes of absolute alcohol may be necessary to remove all water from the tissue.

e. Transfer the sections to clove oil in another watch-glass and leave until the sections look translucent. A milky look indicates that water remains in the sections - go back to alcohol again. Clove oil goes milky if the mounting is performed in a damp atmosphere, so don’t breathe on it! (An alternative clearing agent is xylene (xylol), which does not inhibit the setting of Canada balsam so much. Xylene has Health and Safety implications and should only be used in a fume hood. However, clove oil is more easily obtainable, and has some desiccating properties of its own).

f. Transfer the sections to a slide, and mount in a drop of Canada balsam or Euparol (which has slight desiccating properties). Carefully lower a cover slip onto the section, round ones are less prone to trap bubbles. Gently squash the cover slip down with the blunt end of a pencil. Label the slide immediately with the code number of the specimen to avoid possible confusion. Ensure the slide label identifies the orientation of the sections if you are unfamiliar with sponge sections.

g. The slide may be examined immediately under objectives up to 40X magnification but beware of disturbing the cover slip. Leave to dry for several months; the presence of clove oil increases the drying time of Canada balsam dramatically. A slightly raised temperature, such as in an airing cupboard, accelerates the process.

7.3. Spicule preparation

7.3.1. For sponges with siliceous spicules (i.e. Demospongiae).

WARNING. This method involves the use of highly dangerous concentrated nitric acid, and should only be undertaken in laboratory conditions conforming to the appropriate Health & Safety standards.

a. Select areas of sponge which are likely to have variation of spicules: i.e. base, skin, walls of internal cavity, oscular region etc.

b. Remove a small piece (0.25 cm3) from a selected area and place it in a boiling tube.

c. Gently add enough concentrated. nitric acid to cover the fragment and then if necessary, either warm over a spirit burner or Bunsen or heat in a water bath until all the tissue has dissolved.

d. When no tissue remains carefully add distilled water and agitate. Allow the tube to stand for up to 2 hours to ensure that light-weight spicules have settled to the bottom before pipetting or draining off the surplus liquid. Repeat the washing process 3 or 4 times until all the nitric acid has been removed, then wash with absolute alcohol.

f. Finally pipette off as much alcohol as possible without losing the spicules and add 1-2cc of absolute alcohol.

g. After shaking the tube spread the suspension of spicules in alcohol on to slides by means of a pipette and speed evaporation of the alcohol by passing the slides over a flame. (Careful, the alcohol will usually burn off!)

h. Mount in Araldite, Canada balsam or Euparol. On a well prepared slide the spicules should be clean and numerous but well separated.

i. Potassium hydroxide (caustic potash) or sodium hydroxide (caustic soda) is an alternative to nitric acid. N.B. These are also dangerous substances.

7.3.2. For sponges with calcareous spicules (i.e. Calcarea).

These sponges have calcareous spicules (they dissolve in acid). Caveat: adding acid to the whole sponge and observing fizzing is not conclusive evidence for the presence of calcareous spicules, because non calcareous sponges often incorporate pieces of shell in their bodies.

The method is basically the same as that for siliceous spicules but bleach or caustic potash must be used to dissolve the tissue. The spicules are very soluble in acids, even acid in the preservative (beware formalin) may have dissolved the spicules! Dissolution of the tissue is not so rapid using this method.

7.3.3 Rapid spicule preparation method for silica spicules. As in 7.3.1 this must be done in a fume cupboard and wearing eye protection.

a. Remove a very small piece of tissue, 1-2mm in size and place on a slide, hold the slide with tongs or long-nosed pliers.

b. Add a drop of concentrated nitric acid with a pasteur pipette and heat gently over a spirit burner or bunsen.

c. As soon as the acid begins to react remove from heat and add another drop or two of acid.

d. Continue to heat gently, adding a little more acid if necessary, until the sponge is dissolved completely.

e. Evaporate the acid gently to dryness using minimum heat.

f. Allow to cool for 30 seconds, then wash with a few drops of 1% hydrochloric acid.

g. Wash with water, drain, then rinse with 95% alcohol (IMS) using wash bottles.

h. Burn off the excess alcohol and heat the slide for 2 seconds.

i. Mount with a drop of Canada Balsam and coverslip.

8. Identification

8.1. Introduction

Like any learning process, naming sponges is likely to involve some mistakes on the part of those who are developing an interest in the group. Sponge identification is difficult; the highly variable nature of the growth patterns and the subtle distinctions between certain species dictate that utmost care must be exercised when arriving at the name of a sponge. This is especially so if the name is to be used in a survey report or a published work. Before embarking on an identification, the following inputs are required - the specimen, slide preparation(s), photograph(s), recorded information, a microscope, and literature.

8.2 Microscopy

For the examination of the slide preparation, a compound microscope is required. The more modern (and expensive!) models found in institutions tend to be relatively easy to use, having an integral light source, and binocular eyepieces. A range of magnifications from 40x to 400x is ideal. Those with access to the older style traditional compound microscopes could consult White, 1978 for guidance on use. This is available in booklet form, as a reprint, from The Quekett Microscopical Club, c/o British Museum (Natural History), Cromwell Road, London SW7 5BD. Also, there are many general books on microscopy (e.g. Hartley, 1979) that provide useful introductions to the subject.

8.3. Literature

Unfortunately, the literature of potential relevance to the identification of British sponges is voluminous, widespread, and often difficult to obtain. Two starting points are the bibliographies of Vosmaer (1928) and the Porifera and Archaeocyatha section of The Zoological Record. The latter is now published by BIOSIS, 21OO Arch Street, Philadelphia, Pennsylvania 191O3, USA. Systema Porifera (Hooper, J.N.A. & Van Soest, R.W.M. (eds) 2002. Systema Porifera. A guide to the classification of sponges. I-xix, 1-1708, i-xlviii. Kluwer Academic Publishers, New York.) is an invaluable guide to sponge classification to genus level. Another useful reference is Sponges of the North East Atlantic (World Biodiversity Database CD-ROM Series) Authors: R.W.M. van Soest, B. Picton & C. Morrow. Publisher ETI. This taxonomic reference work was made by Rob van Soest, Zoological Museum, University of Amsterdam and Bernard Picton and Christine Morrow, Ulster Museum, Belfast. It is a unique, comprehensive guide to shallow-water sponges occurring in the coastal waters of Western Europe between the Arctic and southern Portugal. The CD-ROM contains up-to-date information on 337 shallow-water sponges, including a fully illustrated multiple-entry key, text key and interactive distribution maps. All text is hyperlinked.

The principal authors producing work relevant to British sponges are (or were!): W. Arndt, J.S. Bowerbank, M. Burton, L. Cabioch, C. Lévi, E.O. Schmidt, J. Stephens and E. Topsent. The two main monographic works are Arndt (1935) and Bowerbank (1864-1882). However, neither are particularly easy to use, and both are taxonomically out of date, and misleading in places. Arndt is written in German, concentrating on spicule characters, and having no illustrations of skeletons or specimens. Bowerbank uses an obsolete form of language for spicules, and of necessity concentrates his description on preserved rather than living material. Those works that concentrate on specific taxonomic groups (e.g. Lévi, 1960; Topsent, 1900 and 1924), or sponges from a particular area (e.g. Burton, 1930; Cabioch, 1968) tend to be more helpful. Some references to these, as well as to papers dealing with individual genera or species, will be found on the species sheets. There is a bibliography of useful references at the end of this publication.

Marine faunas can provide good starting points, acting as check lists for sponges present in an area, as well as providing literature references. Three such are the Plymouth Marine Fauna (1957), the Roscoff Marine Fauna (Borojevic, Cabioch and Lévi, 1968), and the Lundy Marine Fauna (Hiscock, Stone & George, 1983[1984]). The latter includes non-technical descriptions of external form.

Literature can of course be consulted in the major reference libraries including the British Museum (Natural History), which runs a photocopying service (at a price!) Although it is not always apparent, some works are still in print. These, and second hand out of print works, can sometimes be obtained from specialist natural history booksellers such as Wheldon & Wesley Ltd., Lytton Lodge, Codicote, Hitchin, Herts, SG4 8TE.

The internet is becoming an invaluable source of new information on sponges. This guide is hosted at http://www.habitas.org.uk/marinelife/sponge_guide/ (with additional species accounts) and there is a world list of extant sponges at http://www.marinespecies.org/porifera/. The latest developments in DNA barcoding of sponges can be found at http://www.spongebarcoding.org/.

8.4. Methods

Traditional methods of identification have centred on the skeleton and spicules, and descriptions of these are included in this Guide. The types, sizes, abundance, organisation and orientation of spicules on the surface, in the body, and at the base of the sponge are important. However, it is also important to use the living features and photographs, and to test their value when making an identification.

Previous versions of this Guide have stated quite clearly that no reliance should be placed on any identification without microscopic examination. Whilst this still holds true, certain sponges can often be identified provisionally by sight alone, and these are indicated in the text. Several other species can be recognised as being one of a pair. This phenomenon is rather curious, because the two species are not necessarily as close to each other taxonomically as they are to different species. The pairs to watch out for are as follows:
Leucosolenia complicata - L. variabilis; Leuconia gossei - L. nivea; Suberites carnosus - S. domuncula; Polymastia boletiformis - P. mamillaris; Axinella damicornis - Stelligera rigida; Axinella infundibuliformis - Phakellia ventilabrum; Stelligera stuposa - Raspailia hispida; Halichondria bowerbanki - H. panicea (sublittoral form); Hemimycale columella - Phorbas fictitius.

It must be remembered that whilst all the commoner shallow water sponges are included in this Guide, it is by no means comprehensive. In many cases, particularly encrusting species, further literature will need to be consulted for an identification and even in the NE Atlantic there are still many undescribed species. If all else fails, the authors may be prepared to attempt identifications of limited amounts of material collected and curated to the standards outlined above.


 Picton, B.E., Morrow, C.C. & van Soest, R.W.B., 2007. [In] Sponges of Britain and Ireland
http://www.habitas.org.uk/marinelife/sponge_guide/introduction.html

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