Effects of substratum and conspecific adults on the metamorphosis of Chasmagnathus granulata (Dana) (Decapoda: Grapsidae) megalopae

The ability of marine invertebrate larvae to delay their metamorphosis in the absence of adequate environmental cues has been reported for numerous sedentary and sessile species. In the present study, the effect of various substrata and the presence of conspecific adults on the metamorphosis of a mobile species, the crab Chasmagnathus granulata, was evaluated. The duration of the megalopa stage in experiments with six different substrata and in the presence or absence of conspecific adults was compared in a laboratory study. In addition, the influence of natural substrata was compared with that of artificial substrata of similar grain size or texture. In a further experiment, the two most effective cues (natural mud and conspecific adults) were tested as single vs. combined factors. Natural mud and unidentified chemical cues from conspecific adults had the strongest accelerating effects on development duration to metamorphosis. With the exception of nylon threads (artificial filamentous substratum), none of the artificial substrata had a significant effect on the duration of the megalopa stage. Simultaneous exposure to natural mud and water containing chemical cues from conspecific adults accelerated metamorphosis more than each of these factors separately. Megalopae that were reared without a substratum (control) delayed their metamorphosis by 29% (about 3 days) compared with those in simultaneous contact with natural mud and rearing water of adult conspecifics. The results indicate that the metamorphosis of the megalopa of C. granulata is influenced by the presence or absence of environmental stimuli that are associated with the preferred adult habitat.     

Stimulation of metamorphosis in an estuarine crab, Chasmagnathus granulata (Dana, 1851): temporal window of cue receptivity

The larvae of most benthic marine invertebrate species must develop for a minimum of time in the plankton before they become competent for settlement and metamorphosis in response to stimulating external cues. In an experimental laboratory study, we identified the temporal window of cue receptivity within the moulting cycle of the megalopa stage of an estuarine crab, Chasmagnathus granulata Dana. This species shows an export strategy including an early larval transport to coastal marine waters where zoeal development takes place, followed by the return of the megalopa stage to brackish habitats where the adults live. In two series of experiments (A, B), megalopae were exposed for differential periods to a combination of metamorphosis-stimulating cues which had previously been found effective (seawater conditioned with adult odor and presence of mud). In experimental series A, these cues were added on successively later days of the moulting cycle, while series B comprised treatments in which the cues were provided from the first day (postmoult) and removed on successively later days of the moulting cycle. Each series of experiments was repeated with larvae originating from two different females (F1, F2). The average development time of megalopae kept continuously in the presence of cues (control experiments, C1) ranged in the two hatches from 9.3 to 9.6 days. In the inverse controls where no cue was added at any time (C2), megalopal development to metamorphosis took on average 11.2-12.0 days. In series A, development duration in treatments with exposure to the cues commencing within 3-4 days after moulting was not significantly different from that in the permanently exposed C1 controls. Later beginning of the exposure, by contrast, had no stimulating effect (significant delay compared to C1, no significant difference from unexposed control, C2). In series B, no significant differences in development time were observed between the C1 controls and treatments with an initial exposure for a minimum of 4 or 6 days of the moulting cycle (F1, F2, respectively). Shorter initial periods of exposure had no metamorphosis-stimulating effects (no significant difference from C2). In conclusion, our results from both experiments suggest that the megalopa stage of C. granulata is most receptive of stimulating cues during a period lasting from ca. one third to one half of the moulting cycle, which coincides with the transition between stages C (intermoult) and D₀ (early premoult) of Drach's classification system. This suggests an interaction of extrinsic stimulating cues with intrinsic (hormonal) factors involved in the control of the moulting cycle.     

Behavioural response thresholds in New Zealand crab megalopae to ambient underwater sound

A small number of studies have demonstrated that settlement stage decapod crustaceans are able to detect and exhibit swimming, settlement and metamorphosis responses to ambient underwater sound emanating from coastal reefs. However, the intensity of the acoustic cue required to initiate the settlement and metamorphosis response, and therefore the potential range over which this acoustic cue may operate, is not known. The current study determined the behavioural response thresholds of four species of New Zealand brachyuran crab megalopae by exposing them to different intensity levels of broadcast reef sound recorded from their preferred settlement habitat and from an unfavourable settlement habitat. Megalopae of the rocky-reef crab, Leptograpsus variegatus, exhibited the lowest behavioural response threshold (highest sensitivity), with a significant reduction in time to metamorphosis (TTM) when exposed to underwater reef sound with an intensity of 90 dB re 1 μPa and greater (100, 126 and 135 dB re 1 μPa). Megalopae of the mud crab, Austrohelice crassa, which settle in soft sediment habitats, exhibited no response to any of the underwater reef sound levels. All reef associated species exposed to sound levels from an unfavourable settlement habitat showed no significant change in TTM, even at intensities that were similar to their preferred reef sound for which reductions in TTM were observed. These results indicated that megalopae were able to discern and respond selectively to habitat-specific acoustic cues. The settlement and metamorphosis behavioural response thresholds to levels of underwater reef sound determined in the current study of four species of crabs, enables preliminary estimation of the spatial range at which an acoustic settlement cue may be operating, from 5 m to 40 km depending on the species. Overall, these results indicate that underwater sound is likely to play a major role in influencing the spatial patterns of settlement of coastal crab species.     

Metamorphosis in a semiterrestrial crab, Sesarma curacaoense: intra- and interspecific settlement cues from adult odors

The larvae of many marine invertebrate species are able to delay their settlement and metamorphosis in the absence of characteristic cues from the adult habitat. This phenomenon was experimentally studied in the megalopa stage of Sesarma curacaoense de Man, 1892, a semiterrestrial grapsid crab that lives in the shallow coastal mangrove habitats in the Caribbean region. Duration of the development and survival to metamorphosis to the first juvenile crab stage were compared between experimental treatments, where the water was conditioned with adult crabs (“adult-conditioned water,” ACW) and control groups reared in filtered seawater. In the experiments with larvae from two different females, development duration was significantly shorter and mortality lower in water conditioned with conspecific adults. In the two control groups, the effects of supply with an artificial substrate (nylon gauze) were tested. This comparison showed that the presence of substrate did not significantly influence the time to metamorphosis, but did reduce the mortality rate. In all later experiments, the megalopae were thus routinely provided with nylon gauze as a substrate. In each of the three subsequent replicate experiments conducted with larvae from different females, survival rate and development time to metamorphosis were compared between one control group and four treatments with ACW. The effectiveness of conspecific (S. curacaoense) adult odors as metamorphosis-stimulating cue was, in these experiments, compared with that of ACW from one congener (S. rectum) and two species belonging to different genera within the Grapsidae (Armases miersii, Chasmagnathus granulata). While the rate of survival showed inconsistent patterns among repeated experiments, the development was consistently fastest with conspecific ACW, followed by ACW from S. rectum, A. miersii and C. granulata. Only the conspecific and congeneric cues had statistically significant effects (i.e. shorter development than in the controls). These response patterns suggest that chemically similar factors (presumably pheromones) are produced by closely related species and, thus, their chemical structure may reflect phylogenetical relationships within a clade.     

Desiccation resistance in megalopae of the terrestrial hermit crab Coenobita compressus: water loss and the role of the shell

Abstract. The terrestrial hermit crab Coenobita compressus H. M ilne E dwards undergoes larval development in the sea and then moves to land as a megalopa, where it metamorphoses and remains for the rest of its life. As a small organism (generally <3 mg), in a body adapted for pelagic life, the megalopa must avoid desiccating in air to make a successful sea-to-land transition. In this study, I measured rates of water loss in 1 to 26-day-old megalopae without mollusk shells to determine if there is an improvement in desiccation resistance with age. I also exposed 26-day-old megalopae with or without shells to different relative-humidity conditions for 1 h to determine if shells worn by megalopae allow them to function in air that is not fully saturated. Megalopae without shells did not survive exposure to a relative humidity (RH) of less than 99%, while those with shells survived 52% RH. Older megalopae lost water more slowly than younger ones. However, the amount of body water explained more of the variation in water-loss rate than age; individuals with smaller body-water masses showed lower rates of absolute water loss. Though megalopae of C. compressus become less water permeable as they approach metamorphosis on land, most of their ability to avoid desiccation comes from the shell. Shell-wearing can be considered a pre-adaptation to a terrestrial life-style because shell-wearing behavior predates land invasion in hermit crabs, and desiccation-proofing in air is a novel function for shells.     

Neurogenesis in larval stages of the spider crab Hyas araneus (Decapoda,Brachyura): proliferation of neuroblasts in the ventral nerve cord

Larvae of the spider crab Hyas araneus were reared in the laboratory from hatching through to metamorphosis. Neurogenesis was recorded in the ventral nerve cord during development of successive larval stages, zoea 1, zoea 2, megalopa and crab 1. Proliferating cells were detected immunocytochemically after in vivo labelling with 5-bromo-2-deoxyuridine (BrdU) which, as a thymidine analogue, is incorporated into the DNA of dividing cells. Segmental sets of mitotically highly active neuroblasts were found in newly hatched larvae. A dorsal neuroblast, a ventral-median neuroblast, 3–6 anterior-ventral neuroblasts and 1–3 lateral neuroblasts could be distinguished in each thoracic ganglion. Significantly fewer neuroblasts were labelled in the suboesophageal ganglion as compared to the thoracic ganglia. The number of active labelled neuroblasts was high throughout zoea 1 and about 30% of zoea 2 development and then dramatically decreased towards premetamorphosis. In the newly moulted megalopa, only a reduced set of neuroblasts was labelled which ceased dividing within the first few days of megalopa development. There is an indication that, although most ganglion mother cells born by unequal division of neuroblasts may go through their final division at an early stage, certain clusters of ganglion mother cells obviously delay their final mitosis. These results are discussed with regard to neuronal integration which necessarily changes during the course of metamorphosis in relation to the altered behavioural repertoire of the larvae.     

Studies on the larval development of northeastern Atlantic and Mediterranean Procellanidae (Decapoda,Anomura). I — Redescription of the larval stages ofPorcellana platycheles (Pennant, 1777) reared under laboratory conditions

The complete larval development is described forPorcellana platycheles (Pennant) reared under laboratory conditions. The development consists of two zoeal stages and one megalopa. At 20°C and 35‰ salinity, the megalopa appeared 17–18 days after hatching. Survival was 56% from hatching to the megalopa stage. The morphological features of the zoeal and megalopa stages ofP. platycheles are compared with those of other species ofPorcellana, and a key of the known zoeal stages of the genus is given.     

Novel triphosphate phosphohydrolase activity of Clostridium thermocellum TTM, a member of the triphosphate tunnel metalloenzyme superfamily

Triphosphate tunnel metalloenzymes (TTMs) are a newly recognized superfamily of phosphotransferases defined by a unique active site residing within an eight-stranded beta barrel. The prototypical members are the eukaryal metal-dependent RNA triphosphatases, which catalyze the initial step in mRNA capping. Little is known about the activities and substrate specificities of the scores of TTM homologs present in bacterial and archaeal proteomes, nearly all of which are annotated as adenylate cyclases. Here we have conducted a biochemical and structure-function analysis of a TTM protein (CthTTM) from the bacterium Clostridium thermocellum. CthTTM is a metal-dependent tripolyphosphatase and nucleoside triphosphatase; it is not an adenylate cyclase. We have identified 11 conserved amino acids in the tunnel that are critical for tripolyphosphatase and ATPase activity. The most salient findings are that (i) CthTTM is 150-fold more active in cleaving tripolyphosphate than ATP and (ii) the substrate specificity of CthTTM can be transformed by a single mutation (K8A) that abolishes tripolyphosphatase activity while strongly stimulating ATP hydrolysis. Our results underscore the plasticity of CthTTM substrate choice and suggest how novel specificities within the TTM superfamily might evolve through changes in the residues that line the tunnel walls.     

Tetrathiomolybdate inhibition of the Enterococcus hirae CopB copper ATPase.

Tetrathiomolybdate (TTM) avidly interacts with copper and has recently been employed to reduce excess copper in patients with Wilson disease. We found that TTM inhibits the purified Enterococcus hirae CopB copper ATPase with an IC(50) of 34 nM. Dithiomolybdate and trithiomolybdate, which commonly contaminate TTM, inhibited the copper ATPases with similar potency. Inhibition could be reversed by copper or silver, suggesting inhibition by substrate binding. These findings for the first time allowed an estimate of the high affinity of CopB for copper and silver. TTM is a new tool for the study of copper ATPases.     

Multiple cues from multiple habitats: Effect on metamorphosis of the Florida stone crab, Menippe mercenaria

The Florida stone crab, Menippe mercenaria, is an economically and ecologically important species that ranges from North Carolina throughout the Caribbean and the southeastern Gulf of Mexico. However, there is little known about its early life history stages as compared to other commercially important species in the region. The goal of this research was to examine effects of putative cues on metamorphosis from the megalopa stage to the first juvenile stage. Our study investigated the effect of water-soluble exudates from four substrata, as well as natural biofilms, and exudates from adult stone crabs. In addition, the influence of natural substrata was compared to that of artificial substrata. Adult exudate had no significant effect on metamorphosis, despite a wide range of tested concentrations. In contrast, there was a significant effect on mean time to metamorphosis in experimental groups exposed to multiple cues associated with the brown alga Sargassum fluitans, rubble from stone crab habitat, the eastern oyster Crassostrea virginica, and biofilms associated with the oyster. Furthermore, we provide evidence for metamorphic responses to water-soluble chemical cues, as well as biochemical and physical cues associated with different substrata. Overall results were coherent with the relevant body of previous work on metamorphosis of brachyuran crab larvae and indicate that both physical and chemical cues are important factors in facilitating the settlement and metamorphosis of M. mercenaria larvae in juvenile nursery habitat.     

Depth regulation of crab larvae in the absence of light

Behavioral responses to gravity and hydrostatic pressure have been investigated in two species of xanthid crabs Leptodius floridanus (Gibbes) and Panopeus herbstii Milne-Edwards to determine whether such responses provide a mechanism for depth regulation in the absence of light.In laboratory experiments, the four zoea stages and one megalopa stage of each species assume a differential vertical distribution in darkness, with succeeding stages showing a deeper overall distribution. Passive sinking rates increase in succeeding zoea stages and drop to an intermediate level after the molt to the megalopa stage. All zoea stages exhibit a negative geotaxis in the absence of light; the megalopa shows a positive geotaxis. The first zoea stage of Leptodius floridanus responds to an increase in hydrostatic pressure (up to 1 atmos above ambient) with an increase in swimming rate. This pressure response is shown to be reversible and not subject to short-term acclimation. The swimming rate of the last zoea stage does not increase in response to an increase in pressure.It is concluded that the responses of these larvae to gravity and hydrostatic pressure together with their characteristic passive sinking rates provide a mechanism for depth regulation in the absence of light that varies during ontogeny.     

The capacity of crab megalopae to autotomize body appendages and the consequences upon their feeding ability–the price to pay to live another day

During the mass settlement events of brachyuran crabs, there is a significant chance of density-dependent injury in the megalopae (last larval stage) because cannibalism can occur by larger conspecifics. Laboratory observations revealed that the appendages that are more prone to injury are eyestalks, as well as first (P1) and fifth (P5) pereiopods. The ability of Carcinus maenas megalopae to autotomize these structures and the effect of such injuries in their feeding ability and metamorphosis were investigated. All tested specimens were able to autotomize one or both of their P1 and P5, but not their eyestalks. Megalopae missing a single P1, as well as one or both P5, were able to capture and ingest prey, as well as intact specimens. Megalopae with either P1 and P5 appendages or at least one damaged eyestalk failed to ingest sufficient food to reach the nutritional threshold required to successfully metamorphose.     

Comparison of low salinity tolerance in Callinectes sapidus Rathbun and Callinectes similis Williams postlarvae upon entry into an estuary

Planktonic larvae of estuarine crabs are commonly exported to the continental shelf for development and then return to coastal and estuarine areas as postlarvae (megalopae). Megalopae returning to estuaries must be adapted to survive in brackish water whereas those of coastally distributed species should not need such adaptations. We investigated 1) whether megalopae of the estuarine crab Callinectes sapidus and the coastal crab Callinectes similis undergo changes in salinity tolerance upon entry into an estuary and 2) what factors induce those changes. Megalopae were collected at a coastal site and a nearby estuarine site and exposed to a range of salinities (5, 10, 15, 20 and 30) for 6 h. Percent survival was determined after 24 h reintroduction to the collection site water. We also investigated 1) whether increased salinity tolerance was induced by reduced salinity or estuarine chemical cues, 2) the time to acclimation and 3) the salinity necessary for acclimation. C. sapidus megalopae from the estuarine site were more likely to survive exposure to low salinities than those from the coastal site. C. sapidus megalopae from the coastal site exhibited increased survival after acclimation to salinities of 27 and 23 for 12 h. Estuarine chemical cues had no effect on salinity tolerance. C. similis megalopae were less likely to survive at low salinities and did not exhibit an acclimation response upon exposure to reduced salinities. These results suggest that megalopae of C. sapidus are physiologically adapted to recruit to estuaries whereas megalopae of C. similis are unable to acclimate to low salinity conditions.     

Metamorphosis of a sesarmid river crab, Armases roberti: stimulation by adult odours versus inhibition by salinity stress

In an experimental laboratory study with the megalopa stage of Armases roberti, a freshwater-inhabiting species of crab from the Caribbean region, we evaluated the combined, potentially antagonistic effects of odours from conspecific adults and of stepwise salinity reductions (simulating upstream migration, reaching within 1 week conditions of 2‰ or freshwater). Neither of these treatments affected the rate of survival, but the duration of development to metamorphosis was significantly (by about 25%) shortened, when odours from conspecific adult crabs were present, regardless of the salinity conditions. Our results indicate that the metamorphosis-stimulating effect of chemical cues from an adult population of A. roberti is far stronger than the potentially retarding effect of increasing hypoosmotic stress. This suggests that the final phase of larval development, including the processes of settlement and metamorphosis, occurs in this species in freshwater habitats, where conspecific populations live.     

Partitioning and utilization of energy during the larval development of the xanthid crab, Rhithropanopeus harrisii (Gould)

An energy budget is constructed for the larval development of the crab Rhithropanopeus harrisii (Gould) fed nauplii of the brine shrimp Artemia salina (L.). Between the first zoeal instar and the megalopa, there is a 5.4-fold increase in caloric consumption and a 13.2-fold increase in dry weight. Weight specific energy content increases through the zoeal stages and drops at the megalopa. Rate of oxygen consumption increases steadily throughout development. Assimilation, gross growth, and net growth efficiencies increase steadily through zoeal development and drop at the megalopa. Calories put into tissue production exceed those expended via respiration in zoeal stages II–IV; the reverse is true in zoeal stage I and the megalopa.

A total energy budget has been calculated and the partitioning of energy is discussed in relation to other physiological studies on this species.     

Chronic toxicity bioassay and effect of endosulfan,HCH, copper and zinc on the survival and intermoult duration of larvae of mangrove crab Macrophthalmus erato De Man

The effects of insecticides (endosulfan, HCH) and heavy metals (copper and zinc) were studied on survival and duration of the moult cycle of larvae and megalopa of a common mangrove crab, Macrophthalmus erato, that inhabits Pitchavaram mangroves, southeast coast of India. In chronic toxicity test, percentage of survival from hatchling to megalopa stage at different concentrations of endosulfan (0.048 to 0.36 microg/L), HCH (0.32 to 2.4 microg/L), copper (9.0 to 67.5 microg/L) and zinc (15.2 to 139.0 microg/L) were 6, 12, 8 and 12 respectively. Endosulfan, HCH, copper and zinc considerably increased the total period in development from first zoea to megalopa stage from 18 days in the control to 23, 21, 20 and 22 days in the treated specimens. It is concluded that concentrations well below the observed chronic concentrations, may prove to be more deleterious to larva of this species in natural environment leading to reduction in the success of recruitment to the adult population.     

Occurrence of an alternative pathway in the larval development of the crab Chasmagnathus granulata Dana, 1851 under laboratory conditions

Larvae of the crab Chasmagnathus granulata were collected in a salt marsh located in the Lagoa dos Patos, Brazil and reared from eclosion to metamorphosis under different dietary regimes. Larvae reared individually in beakers of 40 ml and fed Tetraselmis chuii (zoea III and zoea IV), showed a supplementary stage, here designated as zoea V, with morphological characteristics intermediary between zoea IV and megalopa. No zoeae V molted to megalopa stage. To confirm the occurrence of the supplementary stage, mass cultures of larvae of C. granulata were fed Artemia sp. at high densities, we again detected the fifth zoeal instar. However, when zoeae V were individually placed in beakers and fed Artemia nauplii, they succeeded in molting into megalopae. We observed the occurrence of two types of zoeae IV — a smaller type (from which originated the zoeae V) and a larger type (which directly developed into megalopae). We conclude that stressful nutritional/environmental conditions were responsible for the occurrence of this alternative path of development.     

延龄草对LPS诱导大鼠肝损伤的保护作用及机制

目的：研究延龄草（TTM）对脂多糖（LPS）诱导大鼠氧化应激与肝损伤的保护作用。方法：SD大鼠60只,按体重随机分成TTM高、中、低剂量组、模型组、地塞米松磷酸钠（DEX）对照组及空白对照组（n=10）。TTM高、中及低剂量组按（8、4、2） g/（kg·d） TTM灌胃,模型组、DEX对照组及空白对照组灌胃等量蒸馏水,每隔5 d,TTM高、中、低剂量组、模型组、DEX对照组按1 mg/kg腹腔注射LPS,DEX对照组同时腹腔注射DEX（5 mg/kg）,空白对照组注射等量生理盐水。30 d后,测定大鼠胸腺指数、脾脏指数,对血清一氧化氮合酶（NOS）、超氧化物歧化酶（SOD）活性与一氧化氮（NO）、谷胱甘肽（GSH）、硫代巴比妥酸反应产物（TBARS）、白细胞介素6（IL-6）、IL-10及肿瘤坏死因子α（TNF-α）含量,肝组织SOD、谷胱甘肽过氧化氢酶（GSH-Px）活性与GSH、TBARS含量进行检测。结果：与模型组相比,TTM高剂量组在（19~30） d体重显著降低（P<0.05）,TTM高、中、低剂量组胸腺指数,TTM高剂量组脾脏指数显著降低（P<0.05）,TTM高、中、低剂量组血清NOS活性与TBARS、NO含量显著降低（P<0.05）,TTM高剂量组血清SOD活性及中、高剂量组GSH含量显著上升（P<0.05）,TTM高、中剂量组血清IL-6、TNF-α含量显著降低,IL-10含量显著升高（P<0.05）,TTM中、高剂量组肝脏TBARS含量显著降低,TTM各剂量组肝脏SOD活性与中、高剂量组GSH-Px活性,高剂量组GSH含量显著升高（P<0.05）。结论：TTM对LPS所致大鼠的胸腺、脾脏萎缩有一定的延缓作用,能有效降低血清中NOS活性,减少NO生成,提升SOD、GSH-Px活性与GSH含量,减轻脂质过氧化,降低IL-6、TNF-α过量分泌、提升IL-10含量,有抗炎护肝的功能。