Examination of specimens

• Observe the entire individual or colony, noting its size, shape, colour, position, orientation and form of the openings, nature of the test and its inclusions and the arrangement of colonial zooids.

• If the test is transparent it may be possible to observe the body through it. However, usually it is necessary to remove the individual or zooid from the test. Solitary specimens should be opened around the ventral midline (along the endostyle), either by opening the whole body including the test, or by removing the body from the test before making the incision. The cut is made (with sharp scissors, one blade inside the branchial sac) from siphon to siphon (the long way around). Specimens can be pinned out, and the branchial sac examined before gently removing it from the body wall, by severing the connectives, so that gut and gonads and other structures embedded in or on the pallial body wall can be seen (see Fig. 27.7).

• Colonial zooids can be examined in water or in a drop of glycerol on a slide after their removal from the test. The pharynx can often be opened along the endostyle by inserting the very sharp tip of fine scissors or watchmaker's forceps into the branchial sac and cutting it open against a hard surface. A drop of stain in the water or glycerol often makes structures less obscure. Smaller aplousobranch zooids may

han c

Figure 27.7 Morphology of a phlebobranch and stolidobranch ascidian, A-B, respectively. Each opened along the mid ventral line and with the branchial sac largely removed to show structures embedded in the pallial body wall (semidia-grammatic): an, anus; ap, atrial aperture; b1-2, branchial tentacles; bap, branchial aperture; bf, branchial fold; bs, branchial sac; bw, body wall; dt, dorsal tubercle with opening of neural gland; dl, dorsal lamina; e1-2, endocarp; end, endostyle; h, heart; ilv, internal longitudinal vessel; int, intestine; k, kidney; l, liver lobules; oes, oesophagus; pb, pero-branchial cavity; r, rectum; st, stomach; t, test; tv, test vessel. (Figure: Kott 1985.)

Figure 27.7 Morphology of a phlebobranch and stolidobranch ascidian, A-B, respectively. Each opened along the mid ventral line and with the branchial sac largely removed to show structures embedded in the pallial body wall (semidia-grammatic): an, anus; ap, atrial aperture; b1-2, branchial tentacles; bap, branchial aperture; bf, branchial fold; bs, branchial sac; bw, body wall; dt, dorsal tubercle with opening of neural gland; dl, dorsal lamina; e1-2, endocarp; end, endostyle; h, heart; ilv, internal longitudinal vessel; int, intestine; k, kidney; l, liver lobules; oes, oesophagus; pb, pero-branchial cavity; r, rectum; st, stomach; t, test; tv, test vessel. (Figure: Kott 1985.)

need to be stained, cleared and mounted for light microscopy.

• Often it is easier to remove zooids from a section cut vertically from the colony and laid on its side so that the arrangement of the zooids and the common cloacal cavities can be observed. Vertical handcut sections of didemnid species will need to be decalcified (in 3% HCl). When all the spicules are dissolved, the whole section, with the zooids in place, can be washed (to neutralise it), stained, cleared and mounted in the usual way.

• Colonial species of all suborders are viviparous and the larvae being brooded in the colonies often are found in the atrial cavities of the zooids, or in brood pouches attached to the thorax or abdomen of zooids or lying free in the test. Very few solitary (phlebobranch or stolidobranch) species are viviparous. The exceptions are some species of Agnezia, Molgula and Polycarpa. Larvae need to be stained, cleared and mounted for their structures to be seen.

Calcareous spicules of Didemnidae and some Poly-citoridae are best examined by scanning electron microscopy. Prepare the stub by wiping a thin smear of glue over it with a finger tip and letting it dry thoroughly. Then dehydrate a small strip of test containing spicules (remove any zooids or other foreign matter) by holding it in the tip of stainless steel forceps and incinerating it in a hot flame (e.g. over a bunsen burner). When it is completely white, dip the completely dehydrated strip into a drop of absolute alcohol. The spi-cules will be spread by surface tension in the drop of alcohol, which evaporates immediately leaving the spi-cules spread over the surface of the stub. This stage is very rapid, because the alcohol must not dissolve the glue before it evaporates.

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