FURTHER STUDY RE-EXAMINING WHETHER LARVAL EPIDERMIS CAN OMIT THE SECRETION OF A PUPAL CUTICLE, USING THE SILKWORM, BOMBYX MORI

When larval tissue is exposed to a hormonal milieu lacking juvenile hormone, adult characters appear directly, omitting the pupal stage, in some insects but not in others, including Bombyx mori. An attempt was made to induce omission of pupal characters in this species by varying the stage of the larval epidermis to be tested. Pieces of larval integument taken from fourth- and fifth-instar larvae of various stages were transplanted to developing adults. Although the number of cuticle layers and the types of cuticle produced differed depending on the age of the donors, none of the pieces omitted secreting the pupal cuticle. It is concluded that the larval epidermis cannot omit secreting pupal cuticle, and that a transition of tissue competence may play an important part in the sequential appearance of larval, pupal, and adult characters.     

Cuticle secretion during larval growth in Drosophila melanogaster

The moulting cycle and growth of the larval integument of Drosophila melanogaster has been studied by light and electron microscopy. Growth during the first, second and third larval instars is accompanied by 3.0-, 3.4- and 3.7-fold increases in surface area, respectively. Growth in surface area occurs continuously during the larval stages, with no detectable relationship to the moulting cycle. Measurements of the thickness of the cuticular layers show that the endocuticle grows in thickness by apposition and in surface area by stretching. The pre-apolytic epicuticle remains at fairly constant thickness during the increase in surface area, indicating that it grows by intussusception of new components. Post-apolytic epicuticle becomes thinner and increases in surface area by stretching. The epicuticle and pre-ecdysial endocuticle are traversed by filaments, but these do not penetrate the endocuticle secreted after ecdysis. We suggest that the filaments transport breakdown products from the old cuticle inward to the epidermis for reutilization. The growth and deposition of cuticle in two larval growth mutants, lethal (2) giant larvae and Chubby Tubby, involves mechanisms similar to those found in wild-type larvae, but in Chubby Tubby the endocuticle contains inclusions which are ultrastructurally similar to dense epicuticle.     

Angiostrongylus cantonensis: Scanning Electron Microscopic Observations on the Cuticle of Moulting Larvae

Angiostrongylus cantonensis is a parasitic nematode that needs to develop in different hosts in different larval stages. Freshwater snails, such as Pomacea canaliculata, are the intermediate host, and rats are the definitive host. Periodic shedding of the cuticle (moulting) is an important biological process for the survival and development of the parasite in the intermediate and definitive hosts. However, there are few studies on the cuticle alterations between different stages of this parasite. In this study, we observed the ultrastructural appearance and changes of the cuticle of the 2nd/3rd stage larvae (L2/L3) and the 3rd/4th stage larvae (L3/L4) using a scanning electron microscope. We also first divided L2/L3 into late L2 and early L3. The late L2 lacked alae, but possessed a pull-chain-like fissure. Irregular alignment of spherical particles on the cuticle were noted compared to the L3. Alae appeared in the early L3. The old cuticle turned into a thin film-like structure which adhered to the new cuticle, and spherical particles were seen regularly arranged on the surface of this structure. Regular rectangular cavities were found on the surface of L3/L4. The caudal structure of L3/L4 was much larger than that of L3, but caudal inflation, such as seen in L4, was not observed. These results are the first to reveal the ultrastructural changes of the cuticle of A. cantonensis before and after moulting of L2/L3 and L3/L4.     

Temporal reiteration of a precise gene expression pattern during nematode development.

The nematode Caenorhabditis elegans is contained within a multifunctional exoskeleton, the cuticle, that contains a large number of distinct collagens. As the nematode proceeds from the egg through four larval stages to the adult, transition between larval stages is marked by synthesis of a new cuticle and subsequent moulting of the old one. This is a cyclically repeated developmental event, frequently described as the moulting cycle. We have examined the temporal expression of a group of six genes encoding distinct cuticular collagens. As expected, mRNA abundance for each of the six genes tested is found to oscillate, peaking once during each larval stage. Unexpectedly, the periods of abundance for each gene do not coincide, different genes being expressed at different times relative to one another within the moulting cycle. We detect a programme of temporally distinct waves of collagen gene expression, the precise pattern of which is repeated during each of the four larval stages. This multiphasic pattern of oscillating cuticular collagen gene expression indicates an unexpected complexity of temporal control during the nematode moulting cycle and has implications for collagen trimerization and cuticle synthesis.     

Pupal cuticle formation by Manduca sexta epidermis in vitro: Patterns of ecdysone sensitivity

During the larval-pupal transformation, various regions of the epidermis of Manduca sexta larvae have previously been found to require different lengths of exposure to the prothoracic glands in order to form pupal cuticle. To distinguish between requirements for differing threshold concentrations of ecdysone and those for differing durations of exposure to ecdysone, wandering stage larval epidermis was cultured in Grace's medium. When most of the thick larval cuticle was removed, the epidermis responded to concentrations of β-ecdysone of 1.0 μ/ml or greater for 4 days by forming cysts which later formed tanned pupal cuticle. No fat body or protein supplement was required. When the larval integument was explanted intact, similar requirements for cuticle formation and for tanning were found. All regions of the fifth abdominal segment required similar concentrations of β-ecdysone (0.4–0.6 μg/ml) for 4 days for 50% to form pupal cuticle, but gin trap epidermis required the least exposure to a threshold concentration of ecdysone (1.5 days in 0.9 μg/ml). The anterior dorsal intersegmental region required about 0.5 day longer, followed by the posterior intersegmental and the dorsal intrasegmental regions. Thus, the duration of exposure seemed more important. About 1 day longer of exposure to ecdysone was required for subsequent tanning of the new cuticle than for cuticle formation, yet tanning of the cuticle did not occur with prolonged exposure to ecdysone.     

Entrainment of the circadian rhythm in larval release of the crab Dyspanopeus sayi by temperature cycles

Marine and estuarine crabs brood attached eggs, which hatch synchronously releasing larvae at precise times relative to environmental cycles. The subtidal crab Dyspanopeus sayi has a circadian rhythm, in which larvae are released within the 4-h interval after the time of ambient sunset. Previous studies demonstrated that the rhythm can be entrained by the light:dark cycle. Since subtidal crabs are also exposed to temperature fluctuations, an unstudied question was whether the circadian rhythm could be entrained by the diel temperature cycle. To answer this question, ovigerous D. sayi were entrained in darkness to 2.5, 5, and 10 °C temperature cycles that were reverse in phase from the ambient temperature cycle. After entrainment, larval release times were monitored in constant conditions of temperature and darkness with a time-lapse video system. The effectiveness of a temperature cycle to shift the timing of larval release increased as the magnitude of the temperature cycle increased and as crabs were exposed to increasing numbers of entrainment cycles. However, entrainment to a 10 °C cycle only lasted 2 days in constant conditions. When crabs were entrained to a light:dark vs. a 10 °C temperature cycle, the light:dark cycle was dominant for entrainment. Nevertheless, ovigerous crabs do sense temperature cycles and in areas where daylight is too low for entrainment, temperature cycles can be used to regulate the time of larval release.     

Can larval epidermis omit the secretion of a pupal cuticle in a saturniid moth, Samia cynthia ricini?

When the larval tissue is exposed to the hormonal milieu lacking juvenile hormone, adult characters appear directly omitting the pupal stage in some insects but not in others. In Samia cynthia ricini, a species belonging to the latter group, a possible omission of pupal characters was tested by previously untried experiments. Firstly, the possibility that the larval epidermis of only some stages is capable of responding so as to omit to secrete the pupal cuticle was tested. Pieces of larval integument taken from various developmental stages were implanted into developing (pharate) adults. None of these failed to secrete the pupal cuticle. Secondly, pieces of larval integument were first implanted into brainless pupae and left there for a month to eliminate the effect of a trace of juvenile hormone which might have been carried over by the implants. They were then caused to develop, and they again secreted pupal cuticle. It is concluded that the larval epidermis cannot omit secreting pupal cuticle in this species.     

The life-cycle of the flatfish nematode Cucullanus heterochrous

Mature specimens of Cucullanus heterochrous Rudolphi, 1802 (Nematoda: Cucullanidae) were obtained from the intestine of the flounder, Platichthys flesus, from Danish waters. Eggs embryonate in seawater but do not hatch. Fully developed larvae pressed out of eggs are 430 microm long with amphids and dereids and enclosed within the cuticle of a previous larval stage. Infective larvae are believed to be in their third stage. Experimental studies showed that the polychaetes, Nereis spp., Scoloplos armiger, Brada villosa and Capitella sp., may act as intermediate hosts. In N. diversicolor the larvae increase their length to 1 mm within four weeks (15 degrees C) without moulting. Experimental infections showed that larvated eggs are not infective to fish, whereas >550 microm long larvae from polychaetes survived in 4-24 cm long flounders and plaice, Pleuronectes platessa. Third-stage larvae 550 microm to 1.1 mm long were found in the submucosa of the intestine one week post infection. At a length of about 800 microm to 1.4 mm they moult to fourth-stage larvae. Fourth-stage larvae, immature and mature worms occur in the intestine and rectum. Fourth-stage larvae and adults survived experimental transfer from one flounder to another. Similar developmental stages survived for two weeks in the intestine of experimentally infected cod, Gadus morhua.     

The period of occurrence,density, and distribution of larvae of three commercial crab species in Peter the Great Bay,Sea of Japan

Based on materials from plankton surveys carried out in 2004–2009, the period of occurrence, density, and distribution of larvae of three commercial species of crabs in the Peter the Great bay and adjacent areas of Sea of Japan were studied. The larvae of the horsehair crab Erimacrus isenbeckii (Brandt, 1848) occurred in the plankton from mid-March to early June, within the range of water temperature from −1 to 10.8°C. The larvae of the helmet crab Telmessus cheiragonus (Tilesius, 1812) appeared in the plankton in mid-April and occurred to the end of June within the temperature range from 2.8 to 13.0°C. The larvae of the snow crab Chionoecetes opilio (O. Fabricius, 1788) appeared in the plankton in mid-April as well, but some individuals sporadically occurred until early August. All the species of crabs produced one generation of larvae for their reproduction season. The terms of larval stay in plankton depended on water temperature and the duration of the pelagic period increased in colder years. In that area, the larvae of C. opilio were the most abundant (up to 41 ind./m³) and the zoea density of horsehair and helmet crabs was significantly lower (no more than 2 ind./m³). The larvae of C. opilio occurred over the entire area of the Peter the Great bay; the greatest aggregations of their early stages were observed in its southwestern open part. The maximum density of E. isenbeckii zoea was recorded in the south of the Amursky bay and in the Posyet bay. Individual larvae of T. cheiragonus occurred in the Posyet bay and in the southern part of the Amursky and Ussuriisky bays. The late-stage larvae of all crab species were concentrated in areas of the coastal circulation.     

Larval salinity tolerance of the South American salt-marsh crab, Neohelice (Chasmagnathus) granulata: physiological constraints to estuarine retention, export and reimmigration

The semiterrestrial crab Neohelice (=Chasmagnathus) granulata (Dana 1851) is a predominant species in brackish salt marshes, mangroves and estuaries. Its larvae are exported towards coastal marine waters. In order to estimate the limits of salinity tolerance constraining larval retention in estuarine habitats, we exposed in laboratory experiments freshly hatched zoeae to six different salinities (5–32‰). At 5‰, the larvae survived for a maximum of 2 weeks, reaching only exceptionally the second zoeal stage, while 38% survived to the megalopa stage at 10‰. Shortest development and negligible mortality occurred at all higher salt concentrations. These observations show that the larvae of N. granulata can tolerate a retention in the mesohaline reaches of estuaries, with a lower limit of ca. 10–15‰. Maximum survival at 25‰ suggests that polyhaline conditions rather than an export to oceanic waters are optimal for successful larval development of this species. In another experiment, we tested the capability of the last zoeal stage (IV) for reimmigration from coastal marine into brackish waters. Stepwise reductions of salinity during this stage allowed for moulting to the megalopa at 4–10‰. Although survival was at these conditions reduced and development delayed, these results suggest that already the zoea-IV stage is able to initiate the reimmigration into estuaries. After further salinity reduction, megalopae survived in this experiment for up to >3 weeks in freshwater, without moulting to juvenile crabs. In a similar experiment starting from the megalopa stage, successful metamorphosis occurred at 4–10‰, and juvenile growth continued in freshwater. Although these juvenile crabs showed significantly enhanced mortality and smaller carapace width compared to a seawater control, our results show that the late larval and early juvenile stages of N. granulata are well adapted for successful recruitment in brackish and even limnetic habitats.     

Dirofilaria immitis: the moulting of the infective larva in vitro

A number of in vitro culture systems were tested for their ability to support the development of Dirofilaria immitis infective larvae to the fourth larval stage. In cultures of medium ML-15 containing a feeder layer of Dog Sarcoma (DS) cells larvae successfully moulted and showed a small but significant increase in length. Ultrastructural observations demonstrated that the fourth-stage cuticle was synthesized in vitro and in some larvae was fully formed by 60 hours of culture. The hypodermis of moulting larvae contained numerous multi-vesicular bodies. It is concluded that the moult in vitro is a true moult and not an atypical response of the larvae to the conditions of culture.     

The thermal tolerance of crown-of-thorns (Acanthaster planci) embryos and bipinnaria larvae: implications for spatial and temporal variation in adult populations

To understand the role of sea temperature on the population biology of the crown-of-thorns sea star Acanthaster planci, the thermal window for embryonic and larval development was investigated. In two experiments, the response of embryos and larvae across 12 temperatures from 19.4 to 36.5 °C was quantified as the percentage of individuals reaching cleavage stage embryos, blastula, gastrula, early-bipinnaria, late-bipinnaria larvae or abnormal. Measurements were made at 7 times up to 72 h post-fertilisation, with the morphometrics of larvae measured in the 72-h sample. Acanthaster planci developed at temperatures between 19.4 and 33.2 °C, with a thermal window for development to the late-bipinnaria stage between 25.6 and 31.6 °C. Development rate, normal development and larval size were optimal at 28.7 °C, with development rates remaining relatively constant up to 31.6 °C. Rates of abnormality increased steadily (early embryonic stages) above 28.7 °C and was 100 % at temperatures approaching 33 °C. These experiments provide a more detailed insight into the response of A. planci developmental stages to temperature. The present day distribution of the species in eastern Australia overlap with the optimal thermal window for development to the late-bipinnaria stage (≈25–32 °C), implying a role of temperature in controlling population distributions and abundances. Despite this, short- or long-term temperature increases may not be a major modulator of the crown-of-thorns recruitment success, population dynamics and distribution in the future as no significant change in development rates, larval survival and growth occurred within this thermal window. Therefore, moderate (1–2 °C) increases in sea temperatures caused by El Niño or near-future ocean warming may not drive an increase in developmental and settlement success. Indeed, without any acclimation to warmer temperatures expected under near-future warming (+2 to 4 °C), climate change could ultimately reduce larval survival due to elevated mortality above the optimal development temperature.     

Moulting and growth in earlier and later moulters of adolescent male snow crabs (Chionoecetes opilio) (Brachyura: Majoidea) under laboratory conditions

Growth and moulting of male adolescent snow crabs were investigated in a laboratory culture experiment. The moulting season of adolescents was separated into two groups of earlier and later, and they were regarded as normal and skip moulters, respectively. The existence of male snow crab skip moulters was first demonstrated in the Sea of Japan. Although the precise moulting season of the earlier moulter could not be revealed owing to the captive method, the later moulter is suspected to moult approximately 1 year after the earlier moulter. Earlier and later moulters were not different in growth of carapace width or chela height during the pubertal and terminal moult. Our moult and growth results of earlier and later moulters will be useful for predicting recruitment to legally fishable (>90 mm carapace width) or valuable (hard-shelled adult) populations of male snow crab in the Sea of Japan.     

Induction of supernumerary larval moulting in the tobacco hornworm Manduca sexta: interaction of bisacylhydrazine ecdysteroid agonists with endogenous Juvenile Hormone

Abstract When given in a critical dietary dose range, the insecticidal bisacylhydrazine ecdysteroid agonists RH‐5849 or tebufenozide (RH‐5992) cause fifth stage Manduca sexta (L.) larvae to moult to a supernumerary sixth‐stage giant larva. The effect is dependent on exposure to the chemicals immediately after the previous ecdysis. Previous removal of the corpora allata does not interfere with the induction of premature moulting by RH‐5849 but completely prevents the formation of supernumerary larvae. The juvenilizing effect is therefore due to the interaction of the moult‐promoting effect of the ecdysteroid agonists with the high titre of endogenous Juvenile Hormone that is present just after ecdysis to the fifth stage in this insect. The ecdysteroid agonists themselves appear to have no intrinsic Juvenile Hormone‐agonist properties. Sixth‐stage larvae resulting from exposure to critical dietary concentrations of RH‐5849 are morphologically completely larval in character. When transferred to diet without the ecdysteroid agonist, they feed normally and gain weight, growing much larger than control fifth stage insects. At the end of the supernumerary stage, they cease to feed, wander in the usual way, and form a normal pupal cuticle but then die as pharate pupae without shedding the sixth‐stage larval cuticle.     

Larval Development and Metamorphosis in Sipuncula

In a brief review of development of the phylum Sipuncula, fourpatterns of development are recognized: (1) direct with no pelagicstage; (2) one larval stage, a lecithotrophic trochophore; (3)two larval stages, a lecithotrophic trochophore and a lecithotrophicpelagosphera; (4) two larval stages, a lecithotrophic trochophoreand a planktotrophic pelagosphera. Larval types and their metamorphosesare described, with special attention to the development andmorphology of the larval cuticle. In the majority of speciesstudied, the egg envelope is transformed into the larval cuticleat metamorphosis of the trochophore. The cuticle of many planktotrophicpelagosphera larvae is characterized by surface papillae ofdiverse form and pattern. The underlying cuticle in some speciesis composed of layers of fibers at right angles to one another.     

Temperature and growth in Carcinus maenas L. (Decapoda: Portunidae) larvae reared in the laboratory from hatching through metamorphosis

Larvae of Carcinus maenas L. were reared in the laboratory from hatching through metamorphosis at 9, 12, and 18°C. Dry weight (DW) and elemental contents of carbon (C), nitrogen (N), and hydrogen (H) were analysed at short intervals through successive larval moulting cycles (four zoea-stages, megalopa), and newly metamorphosed crabs. C. maenas larvae grew significantly during all instars, at all temperatures tested. Biomass (DW, C, N, H) and energy (Joule) slightly declined shortly before ecdysis in zoea stages. This terminal decrease was more distinct in the megalopa stage, where ≈39 and 83% of the maximum energy attained, was lost at 12 and 18°C, respectively. Changes of biomass and energy in successive moult cycles showed best fits to quadratic equations, whereas their maximum in successive larval instars formed exponential sequences with time. Due to parabolic growth curves, biomass and energy accumulation within single instars were discussed as maximum (MG) and effective growth (EG), considering gain both at times of maximum biomass, and shortly before ecdysis. Metamorphosing larvae achieved EG with 1137% (DW), 1195% (C), 1108% (N), 1395% (H), 1339% (Joule) at 12°C, and 1140% (DW), 1099% (C), 1133% (N), 1225% (H), 1107% (Joule) at 18°C, relative to newly hatched zoea-1. Ash content and inorganic C in newly hatched zoea-1, were 29.4% and 5.5% ash, respectively. The stoichiometric C H N method of Gnaiger & Bitterlich was used to assess protein, lipid, and carbohydrate compositions. Obviously proteins formed the major part of larval biomass (>50% DW). C: N ratios indicate that more lipid than protein was built up shortly after moulting, but relatively more protein was subsequently accumulated. Temperature effects on larval growth (MG, EG), growth rates (GR), and gross growth efficiencies (K₁) were discussed. C. maenas zoea stages accumulated energy and biomass with higher efficiencies at 18 than at 12°C. Megalopa growth seemed to be limited at 18°C, showing lower K₁ values than at 12°C. N was accumulated with higher efficiencies than C in all larval stages. Characteristic variations in larval K₁ values between premoult and ecdysis were discussed. Cumulative gross growth efficiencies (MG-related) were calculated as ≈11 and 10%, at 12 and 18°C, respectively.     

Physiological and morphological aspects of Aedes aegypti developing larvae: effects of the chitin synthesis inhibitor novaluron

Population control of the dengue vector mosquito, Aedes aegypti, is difficult due to many reasons, one being the development of resistance to neurotoxic insecticides employed. The biosynthesis of chitin, a major constituent of insect cuticle, is a novel target for population control. Novaluron is a benzoylphenylurea (BPU) that acts as a chitin synthesis inhibitor, already used against mosquitoes. However, information regarding BPU effects on immature mosquito stages and physiological parameters related with mosquito larval development are scarce. A set of physiological parameters were recorded in control developing larvae and novaluron was administered continuously to Ae. aegypti larvae, since early third instar. Larval instar period duration was recorded from third instar until pupation. Chitin content was measured during third and fourth instars. Fourth instars were processed histochemically at the mesothorax region, stained with hematoxylin and eosin (HE) for assessment of internal tissues, and labeled with WGA-FITC to reveal chitinized structures. In control larvae: i) there is a chitin content increase during both third and fourth instars where late third instars contain more chitin than early fourth instars; ii) thoracic organs and a continuous cuticle, closely associated with the underlying epidermis were observed; iii) chitin was continuously present throughout integument cuticle. Novaluron treatment inhibited adult emergence, induced immature mortality, altered adult sex ratio and caused delay in larval development. Moreover, novaluron: i) significantly affected chitin content during larval development; ii) induced a discontinuous and altered cuticle in some regions while epidermis was often thinner or missing; iii) rendered chitin cuticle presence discontinuous and less evident. In both control and novaluron larvae, chitin was present in the peritrophic matrix. This study showed quantitatively and qualitatively evidences of novaluron effects on Ae. aegypti larval development. To our knowledge, this is the first report describing histological alterations produced by a BPU in immature vector mosquitoes.     

Development of Sebastes taczanowskii (Scorpaenidae) in the Sea of Japan off Hokkaido with a key to species of larvae

The larval and juvenile stages of Sebastes taczanowskii (Japanese name: Ezo-mebaru) are described and illustrated based on 33 wild specimens [7.1–26.9 mm in body length (BL)] collected in the Sea of Japan, and eight specimens of reared larvae extruded from the one specimen of a captive pregnant female. Larvae were extruded between 4.3–5.0 mm BL and notochord flexion occurred 5.7–9.0 mm BL. Transformation from postflexion larvae to pelagic juveniles occurred between 13 and 17 mm BL. Preflexion and flexion larvae have a single melanophore row on the dorsal surface on the tail, and an internal line of melanistic dashes on the ventral side of the tail. Lateral pigmentation of postflexion and transforming larval body surfaces are light. Compared with other Japanese rockfish species, S. taczanowskii is shallow-bodied throughout both larval and juvenile stages. We provide an identification key to preflexion and flexion stage rockfish larvae found around the Japanese archipelago, and comparisons with other species. Larval and juvenile S. taczanowskii occurred in both near-shore and relatively offshore water around Shakotan Peninsula-Ishikari Bay, Hokkaido in June and July.     

Untersuchungen zur Häutungsphysiologie der dekapoden Krebse am Beispiel der StrandkrabbeCarcinus maenas

Under constant laboratory conditions, juvenile shore crabs moult at fixed intervals which depend upon their body size. During one moult every crab exhibits increases of the same relative amounts, independent of its absolute size. Basing on the predictable duration of the intermoult period, the morphological changes in the structure of the cuticle and the development of limb-buds, the intermoult period could be divided into 21 different stages. After studying the moulting rhythm in constant milieu, the influence of the following exogenous and endogenous factors upon the moulting rhythm and growth of normal and of eye-stalkless individuals was investigated: temperature, photoperiod, loss of pereiopods, feeding, and presence of larger specimens. From these investigations it became evident that the moulting rhythm is regulated by growth. The crabs are able to moult only after achieving a minimum of tissue growth. So long as this minimum growth is not achieved, a moult-inhibiting hormone is secreted and moulting is prevented. If the moult-inhibiting hormone is absent, moulting hormone is secreted and initiates a moult. Under dangerous conditions, the crabs are able to delay the next moult. Under unfavourable conditions they consume less food than normal. Therefore, the amount of tissue growth which is the necessary prerequisite for moulting is delayed, and continued release of moult-inhibiting hormone prevents the moult. Under conditions favourable for moulting, or demanding moult (e. g. after loss of many pereiopods) the crabs accelerate the moult. Temperature influences the moulting rhythm by indirect effects on the metabolic rate. During further investigations, the variation of the following parameters were determined quantitatively: content of moulting hormone in whole crabs; content of aminoacids, protein, glucose, Na⁺, K⁺, Mg⁺⁺ and Ca⁺⁺ in the hemolymph; pH and osmotic pressure in the hemolymph; and Ca⁺⁺ content in skeleton and whole crabs. All parameters mentioned — excepting pH and K⁺ content of the hemolymph — vary characteristically during the intermoult period. The titre of moulting hormone has 4 different maxima. Of all parameters, only the content of animoacids and protein in the hemolymph vary in the same way as the titre of the hormone. From these results the following conclusions are drawn: The moulting hormone not only initiates the moulting process, but controls it at several stages. Only protein metabolism seems to be under direct control of the moulting hormone which stimulates protein-synthesis. Chitin formation, regeneration, apolysis and ecdysis are indirectly controlled by the moulting hormone through protein metabolism. As in most of the other processes mentioned, the calcification of the new cuticle is not under the direct influence of the moulting hormone. The conclusion ofDigby (1966) that calcification in crabs is an electrochemical process, is confirmed.