The influence of temperature on the haemocyanin function and oxygen uptake of the freshwater crab Potamonautes warreni calman

• 1.1. At 35°C a maximal VO₂ value of 110 ml O₂/kg/hr was obtained with a significant decrease in the value at 40°C.
• 2.2. The Bohr-effect for P. warreni is — 0.28 and does not change significantly at 15, 25 and 35°C.
• 3.3. The ability of the crab to extract oxygen from the water medium during a single exhalation is on average 41.2% whilst the limitation diffusion (L. diff, Piiper, [1982], A Companion to Animal Physiology, pp. 49–64. Cambridge University Press.) is 0.84.
• 4.4. Compared to land and marine crabs, in P. warreni, the P_aO₂ (29.5 mm Hg) and the P_vO₂ (15.3 mm Hg) is low.

     

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.     

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.     

Cellular responses to increasing Cd concentrations in the freshwater crab,<Emphasis Type="Italic"> Potamonautes warreni</Emphasis>, harbouring microbial gill infestations

We investigated the uptake, transport, storage and defence mechanisms in the freshwater crab, Potamonautes warreni, harbouring microbial gill infestations and exposed to increasing chronic (0.2, 0.5, 1.0 mg l^–1) and acute (2.0 mg l^–1) cadmium (Cd) concentrations under controlled laboratory conditions over a period of 21 days. Transmission electron microscopy and X-ray microanalysis revealed that the microbial gill fauna was eliminated on exposure to 0.2 mg Cd²⁺ l^–1 and that Cd became increasingly adsorbed and incorporated into lamellar crystal deposits and permeated the cuticle of the gills of P. warreni. Degeneration of the apical membrane infoldings and vacuolation of epithelial cells occurred concurrently with pinocytosis, endocytosis and pronounced phagocytotic activity in the epithelia and haemal canal of the gills. Elevated Cd exposures (0.5 or 1.0 mg l^–1) resulted in the swelling and dissociation of mitochondrial outer membranes together with an increase in transport of Cu, Cl and S by haemocytes in the haemal canal to epithelial tissues depleted in these elements. Cd also accumulated in tightly coiled concentric membrane whorls in the haemal canal, whereas the highest concentrations of Cd were found within aggregates of lysosome-like bodies in cuticulin-secreting cells of the gill stem. Chronic exposure to Cd induced increased fatigue and mild uncoordinated motor activity. In contrast, at an acute exposure of 2.0 mg l^–1 over 48 h, P. warreni showed a time-specific rapid loss of motor function, although only mild cellular lesions occurred in the gill tissues. The significance of cellular changes in the gill epithelia and altered motor activity of P. warreni with increased waterborne Cd are discussed as potential biomarker responses in monitoring aquatic pollution.     

Morphological changes induced by acclimation to high pressure in the gill epithelium of the freshwater yellow eel

After acclimation either to high pressure (101 ATA) or to low temperature (9°C), the number of mucous cells within gill epithelium of freshwater eel Anguilla anguilla was significantly decreased and the density of chloride cells was significantly increased when compared to control fish (1 ATA, 19°C).     

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.     

Calcium transport and homeostasis in gill cells of a freshwater crab Dilocarcinus pagei

Crustaceans present a very interesting model system to study the process of calcification and calcium (Ca²⁺) transport because of molting-related events and the deposition of CaCO₃ in the new exoskeleton. Dilocarcinus pagei, a freshwater crab endemic to Brazil, was studied to understand Ca²⁺ transport in whole gill cells using a fluorescent probe. Cells were dissociated, all of the gill cell types were loaded with fluo-3 and intracellular Ca²⁺ change was monitored by adding Ca as CaCl₂ (0, 0.1, 0.25, 0.50, 1.0 and 5 mM), with a series of different inhibitors. For control gill cells, Ca²⁺ transport followed Michaelis–Menten kinetics with K _m = 0.42 ± 0.04 mM and V _max = 0.50 ± 0.02 μM (Ca²⁺ change × initial intracellular Ca⁻¹ × 180 s⁻¹; N = 14, r ² = 0.99). Verapamil (a Ca²⁺ channel inhibitor) and amiloride (a Na⁺/Ca²⁺ exchanger [NCX] inhibitor) completely reduced intracellular Ca²⁺ transport, while nifedipine, another Ca²⁺ channel inhibitor, did not. Vanadate, a plasma membrane Ca²⁺-ATPase inhibitor (PMCA), increased intracellular Ca²⁺ in gill cells through a decrease in the efflux of Ca²⁺. Ouabain increased intracellular Ca²⁺, similar to the effect of KB-R, a specific NCX inhibitor for Ca²⁺ in the influx mode. Alterations in extracellular [Na] in the saline did not affect intracellular Ca²⁺ transport. Caffeine, responsible for inducing Ca release from sarcoplasmic reticulum in vertebrate muscle, increased intracellular Ca²⁺ compared to control, suggesting an effect of this inhibitor in gill epithelial cells of Dilocarcinus pagei, probably through release of intracellular stores. We also demonstrate here that intracellular Ca²⁺ in gill cells of Dilocarcinus pagei was kept relatively constant in face of an extracellular Ca concentration of 50-fold, suggesting that crustaceans are able to display Ca²⁺ homeostasis through various Ca²⁺ intracellular sequestration mechanisms and/or plasma membrane Ca²⁺ influx and outflux that are highly regulatory. In summary, studies using whole gill cells are an interesting approach for working with real regulatory Ca²⁺ mechanisms in intact cells under physiological Ca levels (mM range), compared to earlier work using isolated vesicles of various epithelial cells.     

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.     

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.     

Pathological changes in the liver tissue of different aged mice with induced and physiological fibrosis

This paper presents the results of a study of developmental dynamics of pathological changes in the liver tissue of different aged mice with induced fibrosis, and also shows the role of melatonin as hepatoprotector. It was demonstrated that the toxic damage causes more severe pathological changes in the structure of liver tissue of old animals than of younger ones. The use of melatonin demonstrates a tendency to reduce the degree of liver fibrotization in young animals with induced fibrosis.     

Ultrastructural and physiological changes induced by atractylate in the canine myocardium.

The myocardial cellular response to the cardiac glycoside atractylate, a known inhibitor of mitochondrial adenine nucleotide translocation, was examined physiologically, morphologically, and morphometrically in eight dogs. An isolated in situ segment of left ventricular tissue was employed in these experiments to eliminate collateral flow and the potential for large sampling error. It was found that atractylate infusion rapidly induced physiological and morphological changes suggestive of a loss of membrane regulation of ion flux in the arteriole, capillary endothelium, and myocardial cell. Also mitochondrial changes suggestive of adenine nucleotide translocase inhibition were observed. As these responses are similar to those induced by acute myocardial ischaemia, it is suggested that ischaemia and atractylate induce cell injury by similar mechanisms.     

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.     

Cadmium induces hydrogen peroxide production and initiates hydrogen peroxide-dependent apoptosis in the gill of freshwater crab, Sinopotamon henanense

Cadmium (Cd) is a well-known toxic heavy metal that accumulates in the aquatic environment. Cd has been reported to induce oxidative damage and apoptosis. We investigated the regulation mechanism of hydrogen peroxide (H(2)O(2)) on Cd-induced apoptosis. We show that in the gills of the freshwater crab Sinopotamon henanense Cd induced apoptosis, in a time- and concentration-dependent manner, as confirmed by DNA fragmentation analysis and transmission electron microscopy. Additionally, Cd caused production of H(2)O(2) after 2h of treatment at 58mg L(-1) Cd, and significantly increased the caspase-3/8/9 activity in crabs relative to the control group. Pre-treatment with the scavenger for H(2)O(2), dimethylthiourea (DMTU) and antioxidant, N-acetyl cysteine (NAC), effectively inhibited the activities of caspase-3 and caspase-9, eventually blocked Cd-induced DNA fragmentation and the appearance of markers for apoptotic cell death. These results suggest that Cd might induce intracellular H(2)O(2) generation to trigger the crab apoptotic processes by regulating the activities of caspase enzymes.     

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.     

Disentangling the divergence and cladogenesis in the freshwater crab species (Potamonautidae: Potamonautes perlatus sensu lato) in the Cape Fold Mountains,South Africa,with the description of two novel cryptic lineages

River networks of major drainages can form barriers that shape the phylogeography of freshwater organisms, particularly those with low dispersal capabilities. Freshwater crab species' distributions can be used to examine hydrological patterns to expose historical drainage interconnectivity. We used molecular sequence data (mitochondrial and nuclear DNA) and divergence time estimations to determine the phylogeography of the freshwater crab, Potamonautes perlatus sensu lato, from six drainage systems along the Cape Fold Mountains, South Africa. Two major clades were detected: clade 1 comprised two geographically discrete haploclades occurring in southern flowing drainages, whereas clade 2 included specimens from western flowing drainages. Divergence time estimations suggested a Pleistocene (c. 2.61 Mya) divergence of P. perlatus s.l. The Pleistocene was associated with arid conditions and drainage contractions. However, it is likely that during the mesic conditions of the Pleistocene, P. perlatus s.l. migrated and diverged into contemporary patterns. We conclude that three lineages are nested within P. perlatus s.l., two representing novel species. Potamonautes perlatus sensu stricto is confined to western flowing drainages. The two novel species both occurring in southern flowing drainages are described here: P otamonautes barbarai sp. nov. occurs in the Gamtoos and Gourits Rivers and P otamonautes barnardi sp. nov. in the Breede River. © 2014 The Linnean Society of London