Seawater carbonate chemistry and biological processes during experiments with spider crab Hyas araneus, 2009

Future scenarios for the oceans project combined developments of CO2 accumulation and global warming and their impact on marine ecosystems. The synergistic impact of both factors was addressed by studying the effect of elevated CO2 concentrations on thermal tolerance of the cold-eurythermal spider crab Hyas araneus from the population around Helgoland. Here ambient temperatures characterize the southernmost distribution limit of this species. Animals were exposed to present day normocapnia (380 ppm CO2), CO2 levels expected towards 2100 (710 ppm) and beyond (3000 ppm). Heart rate and haemolymph PO2 (PeO2) were measured during progressive short term cooling from 10 to 0°C and during warming from 10 to 25°C. An increase of PeO2 occurred during cooling, the highest values being reached at 0°C under all three CO2 levels. Heart rate increased during warming until a critical temperature (Tc) was reached. The putative Tc under normocapnia was presumably >25°C, from where it fell to 23.5°C under 710 ppm and then 21.1°C under 3000 ppm. At the same time, thermal sensitivity, as seen in the Q10 values of heart rate, rose with increasing CO2concentration in the warmth. Our results suggest a narrowing of the thermal window of Hyas araneus under moderate increases in CO2 levels by exacerbation of the heat or cold induced oxygen and capacity limitation of thermal tolerance.

Electric shock causes physiological stress responses in shore crabs, consistent with prediction of pain

Animal pain is defined by a series of expectations or criteria, one of which is that there should be a physiological stress response associated with noxious stimuli. While crustacean stress responses have been demonstrated they are typically preceded by escape behaviour and thus the physiological change might be attributed to the behaviour rather than a pain experience. We found higher levels of stress as measured by lactate in shore crabs exposed to brief electric shock than non-shocked controls. However, shocked crabs showed more vigorous behaviour than controls. We then matched crabs with the same level of behaviour and still found that shocked crabs had stronger stress response compared with controls. The finding of the stress response, coupled with previous findings of long-Term motivational change and avoidance learning, fulfils the criteria expected of a pain experience.

No discrimination shock avoidance with sequential presentation of stimuli but shore crabs still reduce shock exposure

Insights into the potential for pain may be obtained from examination of behavioural responses to noxious stimuli. In particular, prolonged responses coupled with long-term motivational change and avoidance learning cannot be explained by nociceptive reflex but are consistent with the idea of pain. Here, we placed shore crabs alternately in two halves of a test area divided by an opaque partition. Each area had a dark shelter and in one repeated small electric shocks were delivered in an experimental but not in a control group. Crabs showed no specific avoidance of the shock shelter either during these trials or in a subsequent test in which both were offered simultaneously; however they often emerged from the shock shelter during a trial and thus avoided further shock. More crabs emerged in later trials and took less time to emerge than in early trials. Thus, despite the lack of discrimination learning between the two shelters they used other tactics to markedly reduce the amount of shock received. We note that a previous experiment using simultaneous presentation of two shelters demonstrated rapid discrimination and avoidance learning but the paradigm of sequential presentation appears to prevent this. Nevertheless, the data show clearly that the shock is aversive and tactics, other than discrimination learning, are used to avoid it. Thus, the behaviour is only partially consistent with the idea of pain.

Erection induced by Tx2-6 toxin of Phoneutria nigriventer spider: expression profile of genes in the nitric oxide pathway of penile tissue of mice

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Massive loggerhead nest predation by ghost crabs in Boavista Island (Cape Verde): implications of the absence of large predators.

[EN] The impact of nest predators on sea turtle hatching success is highly variable depending on predator abundance and also on interactions among different predators. Food web connectivity usually makes it difficult to understand predator-prey interactions and develop efficient conservation strategies. In the Cape Verde archipelago there is an important nesting area for loggerheads where ghost crabs are the only described nest predator. We have studied the impact of ghost crabs on loggerhead nests on this threatened population as well as the efficiency of several management practices to reduce this impact.

Artificial Skin - Culturing of Different Skin Cell Lines for Generating an Artificial Skin Substitute on Cross-Weaved Spider Silk Fibres

Background: In the field of Plastic Reconstructive Surgery the development of new innovative matrices for skin repair is in urgent need. The ideal biomaterial should promote attachment, proliferation and growth of cells. Additionally, it should degrade in an appropriate time period without releasing harmful substances, but not exert a pathological immune response. Spider dragline silk from Nephila spp meets these demands to a large extent. Methodology/Principal Findings: Native spider dragline silk, harvested directly out of Nephila spp spiders, was woven on steel frames. Constructs were sterilized and seeded with fibroblasts. After two weeks of cultivating single fibroblasts, keratinocytes were added to generate a bilayered skin model, consisting of dermis and epidermis equivalents. For the next three weeks, constructs in co-culture were lifted on an originally designed setup for air/liquid interface cultivation. After the culturing period, constructs were embedded in paraffin with an especially developed program for spidersilk to avoid supercontraction. Paraffin cross-sections were stained in Haematoxylin & Eosin (H&E) for microscopic analyses. Conclusion/Significance: Native spider dragline silk woven on steel frames provides a suitable matrix for 3 dimensional skin cell culturing. Both fibroblasts and keratinocytes cell lines adhere to the spider silk fibres and proliferate. Guided by the spider silk fibres, they sprout into the meshes and reach confluence in at most one week. A well-balanced, bilayered cocultivation in two continuously separated strata can be achieved by serum reduction, changing the medium conditions and the cultivation period at the air/liquid interphase. Therefore spider silk appears to be a promising biomaterial for the enhancement of skin regeneration.

Interactions between Spider Silk and Cells - NIH/3T3 Fibroblasts Seeded on Miniature Weaving Frames

Background: Several materials have been used for tissue engineering purposes, since the ideal matrix depends on the desired tissue. Silk biomaterials have come to focus due to their great mechanical properties. As untreated silkworm silk has been found to be quite immunogenic, an alternative could be spider silk. Not only does it own unique mechanical properties, its biocompatibility has been shown already in vivo. In our study, we used native spider dragline silk which is known as the strongest fibre in nature. Methodology/Principal Findings: Steel frames were originally designed and manufactured and woven with spider silk, harvesting dragline silk directly out of the animal. After sterilization, scaffolds were seeded with fibroblasts to analyse cell proliferation and adhesion. Analysis of cell morphology and actin filament alignment clearly revealed adherence. Proliferation was measured by cell count as well as determination of relative fluorescence each after 1, 2, 3, and 5 days. Cell counts for native spider silk were also compared with those for trypsin-digested spider silk. Spider silk specimens displayed less proliferation than collagen-and fibronectin-coated cover slips, enzymatic treatment reduced adhesion and proliferation rates tendentially though not significantly. Nevertheless, proliferation could be proven with high significance (p<0.01). Conclusion/Significance: Native spider silk does not require any modification to its application as a biomaterial that can rival any artificial material in terms of cell growth promoting properties. We could show adhesion mechanics on intracellular level. Additionally, proliferation kinetics were higher than in enzymatically digested controls, indicating that spider silk does not require modification. Recent findings concerning reduction of cell proliferation after exposure could not be met. As biotechnological production of the hierarchical composition of native spider silk fibres is still a challenge, our study has a pioneer role in researching cellular mechanics on native spider silk fibres.

An examination of the techniques used to capture mangrove crabs, Ucides cordatus, in the Mamanguape River estuary, Northeastern Brazil, with implications for management.

The present research, undertaken in a mangrove swamp in northeastern Brazil (Mamanguape River Estuary), examined the factors that led to the overwhelming acceptance of the tangle-netting technique by crab harvesters in detriment to the now illegal tamping technique. Both techniques are the only ones currently used at our study site and in many other areas in Brazil, despite being prohibited by law. Data were collected through direct observations to determine capture efficiency, productivity, daily production, selectivity, and harvesting effort, and through interviews with crab harvesters, focusing on their perceptions of the capture techniques, the conditions of crab stocks and the sales price of a dozen crabs. Our results indicated that the two capture techniques did not significantly differ in terms of their efficiency or productivity, but daily production rates differed significantly, being greater using tangle-netting. The tangle-netting permits a greater harvesting effort (6 hours and 34 min) compared to tamping (4 hours and 19 min). Tangle-netting is also less selective than tamping indicated by the larger number of captured smaller specimens, including females. This results in a lower average sales price for a dozen crabs caught by tangle-netting (US$ 0.95) compared to tamping (US$ 1.02). The greater daily production of crab harvesters using the tangle-netting technique nevertheless increased their net gain, explaining their preference for this method, Given that tangle-netting results in greater harvesting pressure but lower selectivity compared to tamping, it may potentially be less sustainable. All of the crab harvesters interviewed having more than 20 years of experience (n = 34) stated they perceived that stocks of U. cordatus had become reduced over the last 20 years, together with average crab sizes. It is now important to examine the structure of the local U. cordatus population and to assess its fishery to allow evaluating whether the illegal, but prominent tangle-netting and tamping mangrove crab capture techniques are sustainable or not. We further suggest improving the dialogue between decision makers and fishermen, which barely exists to date, to initiate a discussion about possible ways of resolving the current situation of illegality of the fishermen. This will be key to achieving effective sustainable co-management of this important natural mangrove forest resource.

A survey on diversity and distribution of crabs in inter tidal and sub tidal shore of the Sistan and Baloochestan Province (Oman sea)

This project was done for identifying and survey on distribution and diversity of true crabs in inter tidal and sub tidal zones of the Gulf of Oman (Sistan and Baluchistan province) during two year from 2009 to 2010. Specimens of inter tidal zones were carried out at 10 stations and 8 stations from sub tidal. The specimens were collected by hand and dip-net from inter tidal and by trawl net from sub tidal regions, preserved in 70% alcohol and carried to the laboratory. A total of 37 species belonged to 17 families from inter tidal and 23 species belonged to 9 families from sub tidal were identified. Of which 54 species were identified up to species level. 2 species from Matutidae, 1 species from Eriphiidae, Menippidae, Pseudoziidae, Plagusidae, Varunidae, Camptandriidae, Dromiidae and Dorippidae, 2 species from Oziidae, 3 species from Epialtidae, 2 species from Majidae, 4 species from Pilumnidae, 12 species from Portunidae, 6 species from Xanthidae, 2 species from Grapsidae, 3 species from Dotillidae, 3 species from Macrophthalmidae, 3 species from Ocypodidae, 3 species from Calappidae, 2 species from Parthenopidae and 1 species from Galenidae were identified. All specimens are deposited in the Zoological Museum, University of Tehran (ZUTC). The results of the present study revealed that family Portunidae with 6 species from inter tidal and 9 species from sub tidal regions have the highest species richness among the 22 families. Maximum similarity (Sorenson's Index) was obtained among the stations Breis, Lipar, Pozm and Gordim, and minimum was obtained among the stations Chazire-Kharchang with Pasabandar, Beris, Lipar, Daria-Bozorg, Pozm and Gordim in intertidal regions. In sub tidal regions maximum similarity (Sorenson's Index) was obtained among the stations Pasa bandar with Berisand minimum was obtained among the stations Govatr with Ramin and Gordim, Ramin with Pozm. Also maximum species richness was observed at Tiss in inter tidal and Chabahar in sub tidal stations, whereas minimum was obtained at Beris, Pozm, Gordim and Lipar in inter tidal and Govatr and Pozm in sub tidal stations. Family Ocypodidae in inter tidal and Portunidae in sub tidal regions have the highest distribution. In all of the species length and Breadth of carapace showed significant relation.

Are the preference and selection patterns of hermit crabs for gastropod shells species- or site-specific?

Experimental analyses of hermit crabs and their preferences for shells are essential to understand the intrinsic relationship of the crabs' dependence on shells, and may be useful to explain their shell use pattern in nature. The aim of this study was to evaluate the effect of crab species and site on the pattern of shell use, selection, and preference in the south-western Atlantic hermit crabs Pagurus brevidactylus and Pagurus criniticornis, comparing sympatric and allopatric populations. Differently from the traditional approach to evaluate shell preference by simply determining the shell selection pattern (i.e., the number of shells of each type selected), preference was defined (according to [Liszka, D., Underwood, AJ., 1990. An experimental design to determine preferences for gastropod shells by a hermit-crab. J. Exp. Mar. Biol. Ecol., 137(1), 47-62]) by the comparison of the number of crabs changing for a particular shell type when three options were given (Cerithium atratum, Morula nodulosa, and Tegula viridula) with the number of crabs changing for this same type when only this type was offered. The effect of crab species was tested at Cabelo Gordo Beach, where P. brevidacrylus was found occupying shells of C. atratum, M. nodulosa, and T viridula in similar frequencies, whereas P. criniticornis occupied predominantly shells of C atratum. In laboratory experiments the selection patterns of the two hermit-crab species for these three gastropods were different, with P criniticornis selecting mainly shells of C atratum, and R brevidactylus selecting more shells of M. nodulosa. The shell preference was also dependent on crab species, with P. criniticornis showing a clear preference for shells of C atratum, whereas P. brevidactylus did not show a preference for any of the tested shells. The effect of site was tested for the two species comparing data from Cabelo Gordo to Preta (P brevidactylus) and Araca beaches (P. criniticornis). The pattern of shell use, selection, and preference was demonstrated to be dependent on site only for P. brevidactylus. The results also showed that the shell use pattern of P criniticornis can be explained by its preference at both sites, whereas for P. brevidactylus it occurred only at Cabelo Gordo, where the absence of preference was correlated with the similar use of the three gastropod species studied. Finally, the results showed that the shell selection pattern cannot be considered as a measure of shell preference, since it overestimates crab selectivity. (C) 2009 Elsevier B.V. All rights reserved.

Do the crabs Goniopsis cruentata and Ucides cordatus compete for mangrove propagules? A field-based experimental approach

Mangroves are under threat worldwide by deforestation, overexploitation and climate change. The availability and consumption rates of propagules influence mangrove recruitment and can play a major role in their viability and restoration potential. We assess the potential trophic competition between Goniopsis cruentata and Ucides cordatus, two dominant crab species in the New World, by experimentally comparing herbivory levels between forest stands with varying crab abundance. We hypothesize that herbivory rates (HR) of G. cruentata will be lower in mangroves where it coexists with U. cordatus than in mangroves where U. cordatus is absent. The removal of Rhizophora mangle propagules was very rapid, and HR were overall high and increased through time. However, HR did not differ significantly between mangroves with and without the potential trophic competitor U. cordatus. Our study did not support previous literature indications of food competition between these two crab species, which seem to have developed strategies for competition avoidance.

Genetic resources in wild and cultured stocks of the Asian mud crab, Scylla paramamosain

The mud crab (Scylla spp.) aquaculture industry has expanded rapidly in recent years in many countries in the Indo - West Pacific (IWP) region as an alternative to marine shrimp culture because of significant disease outbreaks and associated failures of many shrimp culture industries in the region. Currently, practices used to produce and manage breeding crabs in hatcheries may compromise levels of genetic diversity, ultimately compromising growth rates, disease resistance and stock productivity. Therefore, to avoid “genetic pollution” and its harmful effects and to promote further development of mud crab aquaculture and fisheries in a sustainable way, a greater understanding of the genetic attributes of wild and cultured mud crab stocks is required. Application of these results can provide benefits for managing wild and cultured Asian mud crab populations for multiple purposes including for commercial production, recreation and conservation and to increase profitability and sustainability of newly emerging crab culture industries. Phylogeographic patterns and the genetic structure of Asian mud crab populations across the IWP were assessed to determine if they were concordant with those of other widespread taxa possessing pelagic larvae of relatively long duration. A 597 bp fragment of the mitochondrial DNA COI gene was amplified and screened for variation in a total of 297 individuals of S. paramamosain from six sampling sites across the species’ natural geographical distribution in the IWP and 36 unique haplotypes were identified. Haplotype diversities per site ranged from 0.516 to 0.879. Nucleotide diversity estimates among haplotypes were 0.11% – 0.48%. Maximum divergence observed among S. paramamosain samples was 1.533% and samples formed essentially a single monophyletic group as no obvious clades were related to geographical location of sites. A weak positive relationship was observed however, between genetic distance and geographical distance among sites. Microsatellite markers were then used to assess contemporary gene flow and population structure in Asian mud crab populations sampled across their natural distribution in the IWP. Eight microsatellite loci were screened in sampled S. paramamosain populations and all showed high allelic diversity at all loci in sampled populations. In total, 344 individuals were analysed, and 304 microsatellite alleles were found across the 8 loci. The mean number of alleles per locus at each site ranged from 20.75 to 28.25. Mean allelic richness per site varied from 17.2 to 18.9. All sites showed high levels of heterozygosity as average expected heterozygosities for all loci ranged from 0.917 – 0.953 while mean observed heterozygosity ranged from 0.916 – 0.959. Allele diversities were similar at all sites and across all loci. The results did not show any evidence for major differences in allele frequencies among sites and patterns of allele frequencies were very similar in all populations across all loci. Estimates of population differentiation (FST) were relatively low and most probably largely reflect intra – individual variation for very highly variable loci. Results from nDNA analysis showed evidence for only very limited population genetic structure among sampled S. paramamosain, and a positive and significant association for genetic and geographical distance among sample sites. Microsatellite markers were then employed to determine if adequate levels of genetic diversity has been captured in crab hatcheries for the breeding cycle. The results showed that all microsatellite loci were polymorphic in hatchery samples. Culture populations were in general, highly genetically depauperate, compared with comparable wild populations, with only 3 to 8 alleles recorded for the same loci set per population. In contrast, very high numbers of alleles per locus were found in reference wild S. paramamosain populations, which ranged from 18 to 46 alleles per locus per population. In general, this translates into a 3 to 10 fold decline in mean allelic richness per locus in all culture stocks compared with wild reference counterparts. Furthermore, most loci in all cultured S. paramamosain samples showed departures from HWE equilibrium. Allele frequencies were very different in culture samples from that present in comparable wild reference samples and this in particular, was reflected in a large decline in allele diversity per locus. The pattern observed was best explained by significant impacts of breeding practices employed in hatcheries rather than natural differentiation among wild populations used as the source of brood stock. Recognition of current problems and management strategies for the species both for the medium and long-term development of the new culture industry are discussed. The priority research to be undertaken over the medium term for S. paramamosain should be to close the life cycle fully to allow individuals to be bred on demand and their offspring equalised to control broodstock reproductive contributions. Establishing a broodstock register and pedigree mating system will be required before any selection program is implemented. This will ensure that sufficient genetic variation will be available to allow genetic gains to be sustainably achieved in a future stock improvement program. A fundamental starting point to improve hatchery practices will be to encourage farmers and hatchery managers to spawn more females in their hatcheries as it will increase background genetic diversity in culture stocks. Combining crablet cohorts from multiple hatcheries into a single cohort for supply to farmers or rotation of breeding females regularly in hatcheries will help to address immediate genetic diversity problems in culture stocks. Application of these results can provide benefits for managing wild and cultured Asian mud crab populations more efficiently. Over the long-term, application of data on genetic diversity in wild and cultured stocks of Asian mud crab will contribute to development of sustainable and productive culture industries in Vietnam and other countries in the IWP and can contribute towards conservation of wild genetic resources.