Ontogeny of Cheliped Laterality and Mechanisms of Reversal of Handedness in the Durophagous Gazami Crab, Portunus trituberculatus.

The paired claws in Gazami crabs, Portunus trituberculatus, are bilaterally asymmetrical, and asymmetry is remarkable on the distal two segments of the first pereiopod, that is, the dactylus and propodus. Shells are exclusively cracked by use of the right chela, representing handedness. In Gazami crabs, handedness is reversed after autotomy of the right chela. Our study focused on the ontogeny of handedness and the mechanism of handedness reversal. Morphologically, asymmetry was first detected in megalopa larvae where the right propodus was significantly larger than the left, as was the canine at the base of the right dactylus. Presumably, the rate of chelagenesis differed between the left and right chelae. With these morphological features, the right chela functioned as a crusher. The crusher exerted a closing force two to three times that of the cutter. With loss of the right crusher, the left chela was bigger than the regenerated right chela and was converted to the crusher. In contrast, the performance of the regenerated right chela deteriorated compared to that of the original right crusher, and exertion of full closing force was inhibited by the more active left chela. Furthermore, crabs with two crusher chelae did not clearly show handedness. A decrease in size and performance of the regenerated right chela can be explained by a default program hypothesis. In conclusion, a difference in the chelagenesis rate results in bilateral asymmetry of the two chelipeds, and then handedness is generated by neural regulation in the thoracic ganglion innervating these claws. Since handedness is reversed after autotomy, the thoracic ganglion would not be lateralized in Gazami crabs. A default program hypothesis is proposed to explain the ontogeny of bilateral chela asymmetry and handedness reversal.

Anhydrobiosis Affects Thermal Habituation in the Bdelloid Rotifer, Adineta sp.

The bdelloid rotifer of the genus Adineta is a freshwater metazoan characterized by anhydrobiosis, a highly stable state of suspended animation induced by desiccation. This study investigated the influence of anhydrobiosis on the thermal habituation by use of an index, Activity Ratio (AR = the number of active rotifers at each experimental temperature/ number of active rotifers at the 25°C stage). In the first experiment, rotifers were divided into two groups: one group was cultivated at 25°C throughout experiment, and another group was transferred to 15°C for two days. AR was estimated during heating up to 40°C, or during cooling down to 5°C in each group. The largest difference in AR occurred at 35°C and 10°C, indicating that AR was changed depending on the pretreated medium temperature. In the next experiment, rotifers were maintained at 15°C, and were desiccated (anhydrobiosis). AR was estimated in the high temperature range (25°C to 40°C), using rotifers that had recovered from anhydrobiosis. AR was significantly different between the groups with and without desiccation, suggesting that thermal habituation at 15°C was completely cancelled by anhydrobiosis. Possible mechanisms on the influence of anhydrobiosis on the thermal habituation have been discussed in terms of neural changes and proteins.

Chela asymmetry in a durophagous crab: predominance of right-handedness and handedness reversal is linked to chela size and closing force.

The swimming crab Portunus trituberculatus is a durophagous brachyuran. Right-handed crabs are predominant, but left-handed crabs are also found in nature. Left-handedness may arise from loss of the right crusher. We examined whether heterochely (morphology) was correlated with differences in closing force (physical property) and handedness (behaviour). The closing force was stronger in larger chela with greater apodeme height and handedness resided in the chela with stronger closing force. With loss of the right chela (autotomy), handedness transitioned from the right to left chela, and all crabs were left-handed thereafter. Reversed handedness was accompanied with a reduction of size and closing force in the regenerated right chela, and growth of the original left chela. After handedness reversal, dentition on the left dactylus of the newly-converted crusher was close to that of the original right crusher, but did not attain the same shape, even after 10 moults. Left-handed crabs were significantly worse than right-handed crabs at crushing hard-shelled prey. Chela formation was symmetrical in the zoea, and heterochely and right-handedness started in the megalopa, regardless of maternal handedness. Since the left chela is capable of being the crusher, heterochely may be caused by differences in morphogenetic velocity between the right and left chelae, under a signal discriminating right from left. Right-handedness is an attribute of P. trituberculatus, that would be inheritable across generations. It is probable that right-handedness was used in the earliest durophagous crabs, and this trend has been succeeded to extant species.

Induction of hatching by chemical signals secreted by the ovigerous female of an estuarine crab Sesarma haematocheir.

Hatching of embryos in the estuarine crab Sesarma haematocheir is highly synchronized with nocturnal high tide and completes within 1 hr among all embryos incubated by the female. This highly synchronized hatching is induced by a "Hatching-Program Inducing Factor (HPIF)" released from the female. To further define the cues involved in synchronized hatching, experiments were designed to characterize this factor and to determine possible sites of release and temporal release patterns using strategies involving isolation of egg masses, perfusion, and ablation experiments on fully developed embryos that had not yet entered the hatching program. Embryo transplantations indicate that not only HPIF may be released from the branchial chamber, but that it is extraordinarily unstable, and loses activity within 15 min, which frustrates further attempts at characterization. Nevertheless, with regard to temporal release patterns, it was established that HPIF activity was detected during short periods over three consecutive nights prior to release of larvae. This activity did not explain the gated response of embryo release in this crab, which might correspond with circatidal larval release events in the field.

Purification and cDNA cloning of the ovigerous-hair stripping substance (OHSS) contained in the hatch water of an estuarine crab Sesarma haematocheir.

The egg attachment system of an estuarine crab Sesarma haematocheir is formed on the maternal ovigerous hairs just after egg laying, and slips off these hairs just after hatching. The stripping is caused by an active factor that we call OHSS (ovigerous-hair stripping substance), which is released by the embryo upon hatching. OHSS was purified, and its active form had a molecular mass of 25 kDa. The cDNA of OHSS cloned from an embryonic cDNA library was 1759 bp long, encoding 492 amino acids in a single open reading frame (ORF). The C-terminal part of the predicted protein was composed of a trypsin-like serine protease domain, with homology to counterparts in other animals of 33-38%. The predicted protein (54.7 kDa) secreted as a zymogen may be cleaved post-translationally, separating the C-terminal from the N-terminal region. The OHSS gene was expressed in the embryo at least 2 weeks before hatching. Expression was also detected in the zoea larva 1 day after hatching and in the brain of the female. However, it was not detected in the muscle, hepatopancreas or ovigerous seta of the female. Ultrastructural analysis indicated that the material investing maternal ovigerous hair, i.e. the outermost layer (E1) of the egg case, is attached at the special sites (attachment sites) arranged at intervals of 130-160 nm on the hair. It is suggested that OHSS acts specifically at these sites, lysing the bond with the coat, thus disposing of the embryo attachment system. This enables the female to prepare the next clutch of embryos without ecdysis.

A pair of rosette glands in the embryo and zoeal larva of an estuarine crab Sesarma haematocheir, and classification of the tegumental glands in the embryos of other crabs.

A pair of rosette glands (one of the tegumental glands in crustaceans) is present at the root of the dorsal spine of the thorax in mature embryos of the estuarine crab Sesarma haematocheir. Each rosette gland is spherical, 45-50 microm in diameter. This gland consists of three types of cells: 18-20 secretory cells, one central cell, and one canal cell. The secretory cells are further classified into two types on the basis of the morphology of secretory granules. There are 17-19 a cells, and only one b cell per rosette gland. An a cell contains spherical secretory granules of 2-3 microm in diameter. The granules are filled with highly electron-dense materials near the nucleus but have lower electron-density near the central cell. The secretory granules contained in the b cell have an irregular shape and are 1-1.5 microm in diameter. The density of the materials in the granules is uniform throughout the cytoplasm. The secretory granules contained in both the a and b cells are produced by the rough endoplasmic reticulum. Materials in the granules are exocytotically discharged into the secretory apparatus inside the secretory cell, sent to the extracellular channels in the central cell, and secreted through the canal cell. The rosette gland can be distinguished from the epidermal cells 2 weeks after egg-laying and the gland matures just before hatching. Materials produced by this gland are secreted after hatching and secretion continues through five stages of zoeal larvae. These rosette glands were never found in the megalopal larva. Rosette glands are found in the embryos of Sesarma spp. and Uca spp. In other crabs, tegumental glands are also found at the same position as in the embryo of S. haematocheir, but the fine structure of their glands is largely different from that of the rosette gland. On the basis of the morphology of secretory cells (a-g cell types), the tegumental glands of a variety of crab embryos can be classified into four types, including rosette glands (type I-IV). The function of these tegumental glands is not yet known, but different types of the gland seem to reflect the phylogeny of the crabs rather than differences of habitat.

Hatching controlled by the circatidal clock, and the role of the medulla terminalis in the optic peduncle of the eyestalk, in an estuarine crab Sesarma haematocheir.

Embryos attached to the female crab Sesarma haematocheir hatch synchronously within 1 h. Hatching is also synchronized near the time of the expected nocturnal high tide. These events are governed by a single circatidal clock (or pacemaker) in the female crab. The present study examined the role of the optic peduncle of the eyestalk on hatching and hatching synchrony. Surgery was performed either from the tip of the eyestalk [to remove the region of the optic peduncle from the compound eye-retina complex to the medulla interna (MI)] or from a small triangle 'window' opened on the eyestalk exoskeleton [to create lesions on the medulla terminalis (MT) of the optic peduncle]. Neither hatching nor hatching synchrony was affected by removal of the region of the optic peduncle from the compound eye-retina complex to the MI: the circatidal rhythm also remained. Removal of the MI probably caused damage to the sinus gland and the bundle of axons running from the sinus gland to the X organ. Nevertheless, maintenance of highly synchronized hatching indicates that the X organ-sinus gland system is not related to hatching. Hatching and hatching synchrony were not affected by dorsal-half cuts of the MT: the timing of hatching was not affected either. By contrast, transverse and ventral-half cuts of the MT caused severe damage to most females; hatching of many females was suppressed, while hatching of some females was either periodic, at intervals of approximately 24 h, or arrhythmic for a few days. The bundle of neuronal axons is tangled in the MT, and the axons inducing hatching pass through the ventral half of the MT. Complete incision of these axon bundles may have suppressed hatching. Incomplete incision of the axon bundle or partial damage to the neurons may have caused periodic or arrhythmic patterns of hatching. There are two possible roles for MT in hatching. One possibility is that neurons in the MT only induce hatching under the control of the circatidal pacemaker located in a site somewhere other than the optic peduncle. Another possibility is that the circatidal pacemaker is actually present in the MT. The second possibility seems more plausible. Each embryo has a special 48-49.5 h developmental program for hatching. This program could be initiated by the circatidal pacemaker in the female, and hatching synchrony may also be enhanced by the same pacemaker.

Entrainment of a semilunar rhythm by a simulated moonlight cycle in the terrestrial crab, Sesarma haematocheir.

Sesarma haematocheir is a species of terrestrial crabs inhabiting hillsides and paddy fields near the sea. Females show a semilunar rhythm of zoea-release coinciding with days of spring tides and in addition with the time of high water occurring at the nearby seacoast about dusk. Nature of environmental stimuli or zeitgebers that induce the semilunar rhythm of zoea-release was examined experimentally. In case that a population of males and females was kept under the condition of a 24-h light-dark cycle (LD 14:10) only, females showed a free-running semilunar rhythm of egg-laying and zoea-release synchronized from field conditions. On the other hand, a semilunar rhythm of egg-laying and zoea-release was entrained by the combination of the 24-h LD and a simulated 24.8-hr moonlight cycle of which the phase was shifted in relation to the natural lunar cycle. This result suggests that the 24.8-h moonlight cycle acts as a zeitgeber of a semilunar rhythm. The 12.4-h tidal cycle parallels with the 24.8-h moonlight cycle in the field. On the basis of the perception of a distinct phase relationship between the 24-h LD and the 24.8-h moonlight cycle, it is considered that crabs substitute the 24.8-h moonlight cycle for the 12.4-h cycle of tides as a zeitgeber to synchronize the phase of the semilunar rhythm with a tidal situation.

Semilunar rhythm in the zoea-release activity of the land crabs Sesarma.

The number of the land crabs Sesarma which released zoeae in a river was counted and recorded for 2 years. The number of crabs releasing zoeae reached a maximum at around every syzygy and decreased to a minimum at around every half moon, showing a semilunar rhythm (Fig. 2). This semilunar rhythm, when examined temporally, showed a peculiar pattern accurately synchronized with the lunar cycle. On the day of and during 4-5 days prior to the syzygy, the peak of zoea-release activity came just after sunset. A few days later, the peak gradually shifted to later in the evening and became flattened until temporal concentration was no longer observed. About 2 days after the half moon, the peak appeared again just after sunset (Figs. 3, 4 and 5). It is not likely that the tide itself affects the semilunar rhythm of Sesarma, but it is supposed that the lunadian factor is involved in it. The adaptive significance of this semilunar rhythm may be interpreted to mean that zoeae released in the river just after the time of spring high tides will successfully arrive at the sea and that the lunadian modification of the peak in the temporal structure will also ensure that the zoeae will be released at the time of high tides and will have a better chance of arriving at the sea than they would otherwise do.