The shell of the ostracod Halocypris inflata (Dana, 1849) examined by the ion beam etch technique

Bombardment of the shell of the myodocopid ostracod Halocypris inflata (Dana, 1849) with charged ions (ion beam etch technique) has resulted in the elucidation of the chitin structure of the cuticle. We show that this technique has produced results that support earlier transmission electron microscopical studies and we demonstrate the importance of the ion beam etch technique not only to ostracod research but to the study of organic material in general.


INTRODUCTION
Th:e study of the organic framework of the ostracod shell was initiated by Bate & East (1 972 & 1975) when they studied Recent freshwater podocopids and marine myotlocopids. The selection of these two groups of ostracod was influenced solely by their low incidence of calcil'ication. They were thus not only easier to section but revealed their chitin structure more readily.
Throughout the entire arthropod group there can be recognised a consistency of structure that in transmission electronmicrographs (see Bate & East, 1975, pl. 4) appears as a 'feathered' arrangement off fibres that form parallel rows of horizontal fibres alternating between broader rows where the fibres are curved (PI. I , fig. 3 ) . This structure has often been considered to be an artefact produced b y obliquely sectioning a spiral arrangement of chitin sheets (Neville, 1070). Subsequently, the beautifully illustrated work of Mutvei (1974) anld of Dalingwater (1975a, b) has shown, for crust,acean cuticle, that the fibres or sheet:; of fibres, bend round between more horizontal layers.
As far a s the structure of the ostracod shell is concerned the calcified discs seen in several of our illustrations are purely incidental to our study. A brief discussion of this mode of calcification will. be made, however, in the context of Sohn & Kornicker's work (1 969) but the essential details of this unusual calcification may be read in Bate & Sheppard (1982).
The aims of the present paper are twofold: firstly, we wish to deinonstrate that the 'feathered' mici~ostructure of the ostracod shell illustrated in earlier papers by means of transmission electron micrographs is supported by the macrostructure obtained by ion beam etching of the specimen and subsequent examination using the scanning electron microscope; secondly, we wish to draw attention to the use of ion beam etching as a tool in the si.udy of organic material.

M ETH 0 DS
Ion beam etching has long been used by material scientists for thinning sections of metals and nonconductive materials prior to examination in the transmission electron microscope. Stewart ( 1962) described a method of etching material using ion beams inside the scanning electron microscope. Later, Boyde &L Stewart (1 962) used this technique to examine the structure of teeth, Echlin (1971) examined pollen by the same method and more recently Blackmore & Claugher (1983) have used a similar method but external to the microscope, in their study of pollen.
In the E.M. laboratory of the British Museum (Natural History), ion beam etching is carried out using a purpose built instrument independent of the microscope. Specimens are bombarded with a beam of ions to remove successive layers of tissue or material thus revealing the underlying structures. These are photographed under the S.E.M. and provide a comparison with the results obtained using transmission electron microscopy.
Ion beam etching is carried out in a vacuum of 10 3torr; when a DC voltage is applied to the ion gun, electrons oscillate in the electrostatic saddle-field region of the central anode, and describe long paths increasing the probability of ionization of the gas used. Ionization occurs between 3kV and lOkV, but requires greater gas pressure below 3kV. Argon is bled into the system through a needle valve and directed through the gun, the gas is ionized by the excited electrons at Ar", excited neutrals are also produced, and this constitutes the beam. The output, or ion current, is varied by adjusting the flow of argon into the gun which affects the relationship between the ion beam and the accelerating voltage. The beam is projected through a slit lOmm long by 1.5mm wide and diverges to produce a wedge-shaped configuration. Uneven etching will occur on irregular or unpolished specimens due t o shadowing but this is minimised by rotating the specimen in the beam.
In this study, ion beam etching was carried out using a Super Microlap MK2, model B306 with a B216 lOmm saddle-field ion source manufactured by Ion Tech Ltd. of Teddington. Alcohol-fixed specimens were dehydrated through graded alcohols and critical point dried before mounting on aluminium stubs with araldite. Specimens were initially etched for 15 minutes at a gun angle of 30". a voltage of SkV and a current of 3mA giving an ion beam current of 0.6uA. After etching the specimens were splutter coated with gold palladium and examined using a Cambridge S180 S.E.M. This procedurc was repeated with the same specimen for a further period o f 1.5 minutes, and three periods of 30 in i n u t es .

DISCUSSION
Chitin structure. The carapace of Halocypris injlulu (Dana, 1849) seen in TS (PI. 1, fig. 3) has a three layered 5tructure: an outer thin epicuticle covers the entire outer shell surface and forms the upraised ornamental ridges (PI. 2, fig. 1) that produce a finger-print pattern over the surface (PI. 1, fig. 1). These epicuticular ridges appear to be calcified (see Ceoscan elemental electron micrograph for Ca in Bate & Sheppard, 1982, pl. 3, fig. 1) and are resistant t o ion beam etching as are the calcite discs (PI. I , fig. 2). As shown in Bate & Sheppard ( 1 982, pl. 10, fig. 3 ) the epicuticular ridges are hollow and it is more probable that the calcification of these ridges is con tained within this space.
As can be observed from the transmission electronmicrograph, the epicuticlc and the ridges produced d o not extend down into the shell matrix. This is an important point to make otherwise the parallel lines seen at depth could be misinterpreted; in fact they are produced by the chitin sheets coming together. The epicuticular ridges, although not homologous with the ornamental ridges described by Okada (1 982), could reflect the subsurface outline of the epidermal cells but this is conjectural at this stage. Exocuticle. This is a layer of unstructured (as presently considered) chitin fibres lying just beneath the epicuticle (PI. 1, fig. 3), it appears as a rather dense layer when revealed by ion etching (PI. 1, fig. 4; PI. 2, fig. 1). In transmission e!ectronmicrographs the exocuticle has a more open structure than the 'feathered' endocuticle. In fact in some species of Myodocopida calcification of the carapace appears to be largely limited to the exocuticle (see Bate & Sheppard, 1982, pl. 3, fig. 5; pl. 7, figs. I , 2). In Hulocypris inflatu, however, this is not so, the calcite discs cutting down through the exo-and endocuticles (Bate & Sheppard, 1982, pl. 4, fig. 4; pl. 5, fig. 1). Endocuticle. In transmission electronmicrographs the endocuticle appears as a 'feathered' arrangment of fibres that curve round (dark layers) between rows o f more compacted horizontal layers (light layers). O u r illustration of this structure does not extend through the complete thickness of the endocuticle and the reader is referred to Bate & Sheppard ( 1 982, pl. 6, fig. 1 ) where it may be seen that the alternating light and dark layers come closer together and are tightly compacted immediately overlying the epidermal cells. Although our illustration of this layer (PI. 2, fig. 6 ) is photographed 4 +:iquely and the material has suffered some collapse due t o ion beam etch thinning of the tissue, we nevertheless consider that the structure illustrated gives some idea as to the compaction o f the tissue at the inner surface (base) of the shell. Part of this inner layer of the endocuticle has been considered (Bate & Sheppard. 1982, p. 29) to represent new shell tissue produced by the epidermal cells prior to the moulting of the 'old' shell.
The main thickness o f the endocuticle is taken up by Explanation of Plate 1     widely spaced layers that in transverse section appear as fibres of chitin. Our scanning electron micrographs of ion beam etched tissue reveal only the chitin macrostructure of sheets of chitin curving between rows of horizontal layers (PI. 1, fig. 5). The fibres shown in P1. 1, fig. 3 indicate the microstructure of these chitin sheets (Fig. 1). These sheets of chitin (themselves probably formed of fibres as seen in Mutvei (1 974)        Calcification. The calcification observed in Halocypris inflata (and subsequently seen in some myodocopids collected in the Arabian Gulf) contrasts with the uniform layer calcification seen in the myodocopids Conchoecia valdiviae and Codonocera polygonia (see Bate & Sheppard, 1982) in that it takes the form of discs of calcium carbonate that sometimes coalesce to produce larger patches of calcification (Pl. 1, fig. 1). Sohn & Kornicker (1 969) demonstrated that by taking non-calcified myodocopids (preserved in alcohol) and immersing them in either ordinary tap water, distilled water, sea water or artificial sea water, sphaerulites of calcium carbonate would develop within the shell. The sphaerulites so formed, however, are not comparable with the substantial calcite discs present here in Halocypris inflata. Indeed, the calcification present in this species did not develop in the laboratory as the ostracods have been stored in alcohol since they were trawled from the Atlantic during one of the scientific cruises of HMS Discovery. More rarely, some individuals are completely uncalicified and we have considered that this could be due to the ostracod having been caught and killed just after moulting and before it had time to calcify its new shell. As with all E.M. techniques, ion beam etching is not without its problems. Results achieved are subject to correct interpretation for the fullest contribution to be made from them. One of the problems involved the evenness with which the specimens were etched. The curvature of the ostracod, its eccentric position on the stub and the non-uniform intensity of the beam produced varying depths within the shell being reached during the same etching period. Some areas were almost entirely etched away (Pl. 2, fig. 6) with collapse of the surface and compression of the structural elements occuring. Some heat is also generated from the etching process and this is dissipated through the specimen. Characteristic distortion of structure is observed after prolonged etching ( 2 hours or more) of various different specimens. It is not known if local heating causes these effects but this could be a contributary factor.

CONCLUSIONS
The chitin matrix of the shell in Halocypris inflata is demonstrated to consist of an outer exocuticle composed of apparently non-aligned fibres and an inner endocuticle composed of parallel rows of curved sheets of chitin. The more compact layers of the endocuticle are formed by the fusing of these sheets of chitin. The 'feathered' microstructure previously demonstrated only by T.E.M. is thus mirrored by the macrostructure seen in the 'solid' using the ion beam etch technique.
An outer organic layer, the epicuticle, covers the entire outer lamella of the carapace. This epicuticle is upraised to produce a series of parallel ridges that, in transverse section, are hollow. As the epicuticular ridges are calcified, in those specimens examined, it is considered that the calcification is restricted to the space within the ridge. These ridges are not continued at depth within the shell (Bate & Sheppard, 1982, pl. 10, fig. 3).
The ion beam etch technique is shown to be capable of supporting evidence for structures previously observed in transverse section and to have considerable potential in elucidating the sub-surface structures of organic material to varying depths and in a controllable manner. The technique has proven its potential for ostracod research and its use in other areas of biology is currently being explored. Given further testing and experience as to duration of etching, this technique could prove of use to a wide variety of disciplines.