Sublethal effects of copper on the freshwater crab Potamonautes warreni

The mass specific rates of oxygen consumption (M (O(2)) M(b)(-1)), ammonia excretion (M (NH(4)-N) M(b)(-1)) and carbon dioxide production (M (CO(2)) M(b)(-1)) were measured after 7, 14 and 21 days exposure of adult Potamonautes warreni to a sublethal concentration of 1.0 mg Cu l(-1) (15.75 micromol l(-1)). Under control (non-copper-exposed) conditions M (O(2)) M(b)(-1) was 35.7+/-8.5 micromol kg(-1)min(-1) (mean+/-S.D.), M (NH(4)-N) M(b)(-1) 2.92+/-0.26 micromol kg(-1)min(-1) and M (CO(2)) M(b)(-1) 25.6+/-9.0 micromol kg(-1)min(-1). The oxygen:nitrogen (O:N) ratio and respiratory quotient (RQ) were 24.5+/-3.0 and 0.80+/-0.06, respectively. M (O(2)) M(b)(-1) of copper-exposed crabs showed a significant increase after 7 and 14 days, but decreased significantly by 40% after 21 days. From the increased O:N ratio and RQ below 0.7, it is clear that crabs exposed to 1 mg Cul(-1) metabolize lipids during the entire 21-day exposure period. Free fatty acids in the midgut gland were determined by GC-MS, and showed increases of up to 600% in some C14 to C18 fatty acids. It is proposed that the excess lipids inhibit the pyruvate dehydrogenase complex, leading to the acceleration of the gluco- and glyco-neogenic pathways. Increased glyconeogenesis results in elevated glycogen concentrations in all tissues after 21 days. Experiments on acutely exposed P. warreni show increased incorporation of 14C-labelled lactate into glycogen.     

Pathological and physiological changes in the South African freshwater crab Potamonautes warreni calman induced by microbial gill infestations.

The impact of microbial gill infestations on the pathology and physiology of the freshwater crab Potamonautes warreni was investigated by comparison of infested and uninfested crab populations from, respectively, a polluted and an unpolluted site along the Mooi River, North West Province, South Africa. Heavy gill infestations by bacteria (70%), peritrichous ciliates such as Lagenophrys sp. (15%), Zoothamnium sp. (10%), and Epistylis sp. (5%), and motile protozoans resulted in species-specific lesions in the gill epithelia of P. warreni and physiological changes in crabs from the polluted site. Bacterial colonies enmeshed in polysaccharide-like films produced indentations of the gill cuticular surfaces and dissociation of microvillous membranes at the basal zone of epithelial cells of gill lamellae of P. warreni. Lagenophrys sp. induced large subcuticular spaces with an unfolding or resorption of the plasma membrane in the gill epithelia. The attachment of stalks of Zoothamnium and Epistylis resulted in dilation of lamellar tissues, the formation of vacuoles, and an increase in subcuticular spaces in the epithelia. Physiological changes in infested crabs included significant differences (P = 0.001) in increments of wet body mass and a reduced growth rate over time compared with uninfested crabs. The specific oxygen consumption (M(O2)) in rested infested crabs significantly increased (31.29 +/- 5.8 micromol O2/kg/min) compared with the M(O2) in uninfested crabs (27.92 +/- 5.6 micromol O2/kg/min; P = 0.009). The heart rate of infested rested P. warreni was significantly lower (40.77 +/- 13.79 beats/min; P < 0.02) than that in uninfested crabs (61.09 +/- 29.02 beats/min) but the heart rate of infested crabs increased significantly with body mass (r = 0.53, P = 0.02). These findings suggest an interrelationship among organic pollution, microbial gill infestations, and specific pathological and physiological responses in the crab host. The role of P. warreni and its microbial gill communities as bioindicators of pollution are discussed.     

Effects of anoxia on the haemolymph physiology and lactate concentrations in the freshwater crab Potamon warreni calman

• 1.1. Haemolymph lactate levels rose rapidly from 0.54 ( ± 0.39) mmol to 34.78 ( ± 4.9) mmol during 6 hr of anoxia in a N₂ atmosphere.
• 2.2. A sharp decrease in the pH from 7.478 (± 0.04) to 7.197 ( ± 0.04) and the total carbon dioxide content of the haemolymph from 13.97 (± 2.0) mmol to 6.25 (± 1.2) mmol during anoxia indicates a gross disturbance in the acid-base balance in Potamon warreni.
• 3.3. The low concentrations of succinate (98 ± 30 μmol) and alanine (5.8 ± 1.0 mmol) in the haemolymph suggest that they do not play a role as an energy source during anoxia.
• 4.4. Probably only l-( + )-lactate is produced during lactate production when P. warreni is exposed to anoxic conditions.

     

Bioaccumulation of manganese in selected tissues of the freshwater crab,Potamonautes warreni (Calman), from industrial and mine-polluted freshwater ecosystems

Manganese concentrations in water and sediments of the Bronkhorstspruit River, Nooitgedacht Dam and especially in the Natalspruit River, did not fall within stated limits for the protection of aquatic life. Tissue manganese concentrations in Potamonautes warreni from the Natalspruit River were generally higher than those in the tissues of crabs from the other two aquatic ecosystems. The highest mean manganese tissue concentration in crabs from the three systems was detected in the carapace (587± 445 µg g^-1 wet weight). It appears that the carapace in these animals acts as a sink in which manganese can be deposited, thus also playing an important role in the detoxification of manganese in these crustaceans. No seasonal or sex-related variation was detected. Body size of the crabs, however, seems to be an important influencing factor in manganese bioaccumulation. A significant increase in carapace manganese concentrations was detected with an increase in body size. However, muscle manganese concentrations were higher in the smaller groups.The concentration factors (BF) calculated for the different tissues with respect to the water were highest in the carapace and ranged from 280.9 to 742.8. The BF with respect to the sediment was comparatively low for all the tissues (0.1 to 0.7). As the manganese concentration in the tissues reflects to some extent the degree of manganese contamination of the surrounding aquatic environment, it appears that P. warreni may be useful as a potential biomonitor of manganese pollution.     

Prey preferences in captivity of the freshwater crab Potamonautes lirrangensis from Lake Malawi with special emphasis on molluscivory

Freshwater crabs play an important role for the diversification of shell morphologies in freshwater gastropods. For example, the radiation of the freshwater crab genus Platythelphusa in Lake Tanganyika is thought to have driven shell diversification of the lake’s snail fauna, promoting the evolution of thalassoid shells. No comparable thalassoid snails are known from Lake Malawi. Accordingly, it was hypothesized that the lake’s only freshwater crab, Potamonautes lirrangensis, is not a snail predator. We tested this hypothesis using feeding experiments with specimens caught in the southern part of Lake Malawi. Individual crabs were held in experimental containers offshore and were presented with various food items overnight, after which ingestion frequency was recorded. Potamonautes lirrangensis can be characterized as a scavenger that is opportunistically carnivorous. A preference for fish and snail flesh could be observed, indicating a bias toward carnivory. We observed occasional cracking of the shell in different snail species, with frequent ingestion of artificially crushed specimens, suggesting that crabs do attempt to feed on snails. However, the investigated Lake Malawi gastropods appear to be partly protected against crab predation through thick-walled and low-spired shells (especially Lanistes and Bellamya), obviating the evolution of thalassoid shells carrying rims, ridges, or spines.     

Oxygen consumption and haemocyanin function in the freshwater snail Marisa cornuarietis (L.)

• 1.1. The MO₂ for branchial respiration in adult snails increased from 0.24 mmol/l/O₂ kg/hr at 18°C to 0.83 mmol/l/O₂ kg/hr at 40°C. Q₁₀ values were 2.75 between 35 and 40°C and 1.8 between 18 and 30°C.
• 2.2. The haemocyanin (31.9 ± 5.8 mg/ml) has a high oxygen affinity (6.28 ± 0.8 at 25°C) with a reversed Bohr effect measured between a pH of 6.80 and 7.95 with gelchromatographed haemolymph, and measured between a pH of 7.34 and 8.10 for native haemolymph.
• 3.3. Growth rate is optimal between 27 and 30°C whilst at 24°C stunted growth was found.
• 4.4. At 25°C the same MO₂ values were found for aerial and aquatic respiration.

     

Equilibrium and kinetic studies of oxygen binding to the haemocyanin from the freshwater snail Lymnaea stagnalis.

The binding of oxygen by the haemocyanin of the gastropod Lymnaea stagnalis was studied by equilibrium and kinetic methods. The studies were performed under conditions in which the haemocyanin molecule was in the native state. Over the pH range 6.8-7.6, in the presence of 10mM-CaCl2 the haemocyanin bound O2 cooperatively. Over this pH range the haemocyanin molecule displayed a normal Bohr effect whereby the O2 affinity of the molecule decreased with a fall in the pH of the solution. The maximum slope of the Hill plot (hmax.) was 3.5, obtained at pH 7.5. An increase in the CaCl2 concentration from 5 to 20 mM at pH 6.8 resulted in a slight increase in the oxygen affinity, with hmax. remaining virtually unchanged. At constant pH and CaCl2 concentration, an increase in NaCl concentration from 0 to 50 mM resulted in a small decrease in O2 affinity, but a significant increase in the value of hmax. from 3.5 to 8.6. Temperature-jump relaxation experiments over a range of O2 concentrations produced single relaxation times. The dependence of the relaxation time on the reactant concentrations indicated a simple bimolecular binding process. The calculated association and dissociation rate constants for this process at pH 7.5 are 29.5 X 10(6) M-1 X S-1 and 49 S-1 respectively. The association rate constant kon was found to be essentially independent of pH and CaCl2 concentration. The dissociation rate constant, koff, however, increased with a decrease in the pH, but was also independent of CaCl2 concentration. These results indicate that the stability of the haemocyanin-O2 complex is determined by the dissociation rate constant.     

Phylogeographic patterning in a freshwater crab species (Decapoda: Potamonautidae: Potamonautes) reveals the signature of historical climatic oscillations

Aim The phylogeographic relationships among populations of the common Cape River crab, Potamonautes perlatus, are examined to investigate whether the contemporary population genetic structure is congruent with the hypothesized hydrographic evolution of drainage systems established during the Pliocene, or whether it reflects an older Miocene climatic amelioration. Location 139 samples of P. perlatus were collected from 31 populations distributed among the five major perennial drainage systems and a number of smaller catchments in the Western and Eastern Cape, South Africa. Methods Phylogeographic analysis using parsimony, maximum likelihood, minimum evolution and Bayesian inferences was employed for the 16S rRNA mtDNA gene region, while bootstrapping and posterior probabilities were used to assess the robustness of clades. In addition, nested clade analysis was performed in an attempt to disentangle the contemporary and historical factors that have sculpted genealogical relationships among conspecific populations of P. perlatus. Results Phylogenetic topologies were congruent irrespective of the evolutionary method employed. Two highly distinct reciprocally monophyletic clades characterized by marked levels of corrected sequence divergence were present, with no shared haplotypes between the two major phylogroups. Phylogroup one comprises the populations of the westward‐flowing drainages (mainly the Berg and Olifants drainages), and phylogroup two comprises all of the southward‐flowing drainages and can further be divided into two subclades – one containing the Breede River populations, and the other containing the Gamtoos and Gourits drainage systems. The nested clade analysis demonstrated restricted gene flow and long‐distance dispersal for a number of higher clade levels. The higher‐level groups and results for the total cladogram suggest either fragmentation or isolation by distance. Main conclusions Freshwater crabs are generally highly philopatric, and dispersal, although not common, has occurred historically. The westward‐flowing drainages (Berg, Olifants, Eerste, Liesbeeck and Tokai) are isolated from the southward‐flowing drainages by the Cape Fold Mountains, while the southward‐flowing drainages have a number of tributaries that extend into the low‐lying regions, allowing for gene flow between these three major drainages systems (Breede, Gamtoos and Gourits). Among the westward‐flowing drainages, a more intensive sampling regime is required to understand evolutionary relationships. Our molecular results suggest that the observed patterns pre‐date the formation of contemporary hydrographic patterns in the Cape. This suggests that an older Late Miocene event has severely impacted the contemporary population structure in this species, as recent Pliocene hydrographic boundaries do not correspond to the phylogeographic pattern observed. Conservation efforts for aquatic taxa should clearly be directed at the catchments, in an attempt to conserve biological diversity.     

Some changes in biochemical composition with sex and size of the crab,Sesarma boulengeri calman

Variations in the biochemical composition (protein and cholesterol) have been studied in the hepatopancreas and thoracic muscle of the crab, Sesarma boulengeri as a function of sex and size. Second degree polynomial regression equations are used in predictive fashion to quantify the biochemical constituents in relation to sex and size. The effect of size is expressed as a power function of body weight. A good agreement is found between the experimental data and the theoretical values based on the polynomial.There is a negative correlation between the mass of crab and the reduced mass of cholesterol and protein of the tissues in both sexes, signifying higher metabolic and growth rates in younger crabs. The cholesterol and protein contents are higher in the male indicating more growth than in the female. However, there is tendency of the muscle protein to increase in large sized female crabs suggesting that bigger sized female are structurally better suited than males.     

Neglected refugia of biodiversity: mountainous regions in Mozambique and Malawi yield two novel freshwater crab species (Potamonautidae: Potamonautes)

Phylogenetic relationships amongst the southern African freshwater crab fauna are reinvestigated following the recent collection of morphologically distinct Potamonautes specimens from remote mountainous regions in Malawi and Mozambique. Specimens were subjected to DNA sequencing of three mtDNA loci, cytochrome c oxidase subunit I (COI), 12S rRNA, and 16S rRNA and compared to the 14 described species from the region. Phylogenetic analysis using maximum parsimony and Bayesian inference revealed the presence of two novel evolutionary lineages. The phylogeny demonstrates that Potamonautes obesus (A. Milne‐Edwards, 1868) is sister to a morphologically distinct novel species from Mount Namuli in Mozambique. Two sympatric and genetically distinct species from Mount Mulanje, in Malawi (forms A and B) were recognized. Form B is sister to the large‐bodied South African riverine freshwater crabs and represents a novel lineage whereas the remaining species (form A) from Mulanje, in Malawi was sister to samples from Mounts Inago and Mabu, and in Mozambique was identified as Potamonautes choloensis (Chace, 1953). The two novel evolutionary lineages were genetically distinct and morphologically different from the described species in each of the respective regions. Two new freshwater crab species Potamonautes namuliensis sp. nov. and Potamonautes mulanjeensis sp. nov. , are described in the present study. The samples from Mount Mulanje in Malawi, and Mounts Mabu and Inago in Mozambique represent new distribution records for Potamonautes choloensis. © 2012 The Linnean Society of London, Zoological Journal of the Linnean Society, 2012, 164 , 498–509.     

Structural and functional characterization of haemocyanin from the anemone hermit crab Dardanus calidus

Oxygen-binding to haemocyanin (Hc) is generally an exothermic process, with overall enthalphy of oxygenation varying from species to species. A number of crustacean Hcs showed a null or reduced enthalphy of oxygenation, among others, the anomuran Pagurus bernhardus and Paralithodes camtscaticae possess a completely temperature-independent oxygen-binding in a wide range of temperature and pH. Functional analysis performed on purified native, hexameric and dodecameric Hc forms of the anemone hermit crab Dardanus calidus allowed to calculate the enthalphy of oxygenation values that resulted equal to -36.2, -33.8 and -26.8 kJ/mol, respectively. Thus, the temperature sensitivity of oxygen binding of D. calidus Hc is in contrast with the temperature independence reported for P. bernhardus and P. camtscaticae, suggesting a high Hc functional heterogeneity within Anomura. Functional characterization also evidenced a strong oxygen affinity modulation by protons (DeltalogP(50)/DeltapH = -0.97) and lactate [DeltalogP(50)/Deltalog(lactate) = -0.38], and a significant decrease in cooperativity by physiological concentration of lactate (n(50) from 2.8 to 1.7 at pH 7.5).     

Renewed sampling of inland aquatic habitats in southern Africa yields two novel freshwater crab species (Decapoda: Potamonautidae: Potamonautes)

A recent survey of the freshwater streams of the Mecula and Yao mountains in the Niassa province of Mozambique resulted in the discovery of a new freshwater crab species. This species is genetically and morphologically distinct from described species from Mozambique or its neighbouring countries, and is described as P otamonautes bellarussus sp. nov. In addition, a new semi‐terrestrial burrowing freshwater crab P otamonautes flavusjo sp. nov. from the Highveld of the Mpumalanga province in South Africa is described based on unique genetic and morphological characters. The phylogenetic affinities of the two new species in relation to the described eastern and southern African Potamonautes species is determined and the biogeographic implications are discussed. © 2014 The Linnean Society of London     

Phylogenetic relationships of the southern African freshwater crab fauna (Decapoda: Potamonautidae: Potamonautes) derived from multiple data sets reveal biogeographic patterning

The phylogenetic relationships among the southern African freshwater crab species were examined using partial sequence data from three mitochondrial genes (12S rRNA, 16S rRNA, and mtDNA COI) 26 morphological characters and 14 allozyme loci. The aims of the present study were firstly to determine whether freshwater crab species that live in the same geographic region share a close phylogenetic relationship. Secondly, to investigate whether hybridizing species are genetically closely related and thirdly, to test for the validity of subgenera based on the genetic data sets. Phylogenetic analysis based on sequence data revealed largely congruent tree topologies and some associations had consistently high bootstrap support, and these data did not support Bott's subgeneric divisions. The morphological data were less informative for phylogenetic reconstruction while the allozyme data generally supported patterns recovered by the sequence data. A combined analysis of all the data recovered two monophyletic clades, one comprised of small-bodied mountain stream species and the other clade consisting of large-bodied riverine species. The combined analyses reflected clear biogeographic patterning for these river crabs. In addition, there was a clear correlation between genetic distance values and the ability of sympatric species to hybridize.