The ultrastructure of the completed cuticle is given in the accompanying paper (Bron, Shinn & Sommerville, this volume). The size of the chalimus II prevents the use of cuticle rigidity / appearance under light microscopy as a criterion for assessing the classical moult stages of Drach Tchernigovtzeff (1967). The aim of this work has therefore been to try to associate electron microscope observations made here with those of other authors working on species where such ultrastructural changes may be more formally linked to visual observations. Because of the short duration of the moult cycle and the variability of individual developmental progress seen, no attempt could be made to apply a time course to the morphological events described.
The moult cycle involves the entire cuticle inclusive of the buccal, foregut and hindgut cuticles. The newly moulted individual is recognisably paler than older animals, which is suggestive of subsequent cuticular tanning. At moult stage A/B (“metecdysis”), following the moult from the chalimus I stage (moult stage E), the procuticle in most areas comprises a reduced number of laminae although the epicuticle is complete. The apical membrane of the epidermis is not pronounced and can not easily be distinguished as a distinct entity due to a wide electron-dense band at the interface between the epidermis and the cuticle (Fig. 1a). This dense band is likely to be associated with deposition of further procuticle following the moult and represents a zone of disorganised cuticular components.
From this point onwards, i.e. during the further metecdysial stages B-C, the procuticle is elaborated upon in terms of the addition of further laminae although these are generally not morphologically distinguishable into p1 and p2. In most areas the new laminae, whilst usually barely or not at all distinguishable from those laid down before the preceding moult, may sometimes be closer together and less diffuse in their structure. The epidermis retains the appearance of high activity at this time with the presence of abundant organelles. Stages B-C are not morphologically recognisable save for the increase in numbers of laminae displayed.
There has been no indication within the many individuals examined of a true stage C4 (“anecdysis”) in that there is no appearance of reduced activity within the epidermis that might signal cessation of cuticulogenesis. This suggests either that within the chalimus II larva, preparation for the next moult proceeds immediately following completion of the cuticle, or that this stage has a greatly curtailed duration. The first indication of entry into “proecdysis” (putative stage D0-D1) is the production by the epidermis of a layer having the appearance of a second apical (ecdysial) membrane (Fig. 1b) although this structure may represent the precursor of the new epicuticle. The formation of this layer is accompanied by the first signs of apolysis, with the old procuticle beginning to come away from the apical epidermal surface. The gap or exuvial cleft formed between the epithelium and the old procuticle is filled with diffuse material having the appearance of a fibrillar substance (Fig. 1c). The material filling the exuvial cleft is likely to represent a mixture of products including not only enzymes secreted into the cleft from the epidermis but also material resulting from the dissolution of the old cuticle. In some sections, vesicles can be seen fusing to the apical membrane which probably represent secretion of material into the cleft (Fig. 1c). Tonofibrils may be clearly seen crossing the exuvial cleft to their points of insertion in the old cuticle, although there is no indication that tonofibrils continue to insert in the old procuticle once the new procuticle has been laid down (Fig. 1d).
As the exuvial cleft widens, the apical membrane of the epidermis becomes more rugose. This change is particularly apparent in oblique and planar sections of the integument where microvillous protrusions of the apical membrane may be observed either as a tangentially cut line of more electron dense spots ranged along the membrane or as groups of microvilli cut transversely (Fig. 1e). During stage D2, following the completion of the new epicuticle in early D2, the first laminae of new procuticle are also laid down (late D2). Both the new epicuticle and the new procuticle are initially very similar in appearance to those of the old cuticle (Fig. 1f).
Whilst the new epicuticle is initially similar to its older counterpart, this does not remain the case. The new epicuticle, and in particular the e4 sublayer (see Bron, Shinn & Sommerville, this volume), often becomes much more highly electron dense (Fig. 2a). At the same time, the underlying transitional layer tp1 expands in volume considerably and becomes more electron dense and granular in its appearance – distinguishing it from the underlying laminae of the developing procuticle (Figs. 2b, 2c). The expansion of the tp1 layer is probably the result of material passing through from the epidermis and, more importantly, absorption of material resulting from the dismantling of the old cuticle. The expansion in volume of the tp1 layer leads to the development of a highly rugose surface for both the new epicuticle and the tp1 layer itself. The subjacent procuticle does not, however, appear to be affected although it may contain electron-lucent lacunae not present in the old procuticle. At this stage, extensive folding of the epidermal apical membrane is frequently apparent.
During the laying down of the new cuticle, large folds (macro-folds) of new cuticle secreted by epidermal invaginations also become apparent beneath the old cuticle (Figs. 2d, 2e). The scale of these folds is greater than the depth of the old cuticle and their appearance is presumed to allow for expansion of the larval volume following ecdysis.
In the latter stages of proecdysis (D4) a comparison between the number of laminae in the old and new procuticle indicates that the new procuticle usually comprises fewer laminae than the old. Similarly, the laminae that have been laid down are frequently deeper and more diffuse than those of the preceding cuticle. Approaching the point of ecdysis (Stage E), the old cuticle has lost much of its structure, with only the epicuticle appearing intact (Fig. 2f). Within the old procuticle the fibres of the laminae appear very disorganised and are no longer bound together in coherent lamellae. The exuvial cleft has widened substantially as the old cuticle comes away and is filled with material having a fibrillar appearance admixed with cuticular debris. The epidermis is still highly active at this point and still displays a highly rugose apical membrane. The new tp1 layer, whilst still expanded, is less heterogeneous in its appearance and also less electron dense, having a finely granular appearance (Fig. 2f).
Under the dissecting microscope individuals undergoing ecdysis often appear misshapen with respect to non-moulting individuals and are noticeably less mobile. The initial rupture of the cuticle occurs in the chalimus cephalothorax and the animal subsequently squeezes out through the aperture produced in this region by repeated whole-body contractions. The time taken to complete the moult is highly variable with some individuals requiring hours to fully divest themselves of the old cuticle and others completing the exercise within 10 minutes.