An analysis of fertilization in Bulinus cernicus (Gastropoda: Planorbidae)

Experiments have been carried out to determine whether cross–fertilization, multiple insemination and sperm sharing occur in Bulinus cernicus. Snails collected from Canal La Paix, Mauritius, were found to be polymorphic for glucose phosphate isomerase, and from this stock three snail colonies homozygous for GPI 2/2, GPI4/4 and GPI6/6 were selected. Thus, three markers were available to monitor cross–fertilization. Isolated snails produced egg–masses by self–fertilization 40 days post–hatching. Egg–masses produced by self–fertilizing individuals tended to be smaller than those from cross–fertilizing snails, 367 eggs per egg–mass as opposed to 5–34. The majority of snails (70%) switched rapidly to cross–fertilization after pairing. Multiple insemination by two partners was common, heterozygous progeny were produced in a random manner and did not reflect the sequence of mating. No evidence of sperm sharing was found, snails were found to be capable of passing on their own sperm while still producing eggs fertilized by sperm received from an earlier mating. Differences in mating behaviour were noted between snails representing the homozygous GPI colonies, with snails from one colony adopting the female role more readily than those from the other two, which predominantly mated as males.     

Two glutathione S-transferase isoenzymes purified from Bulinus truncatus (Gastropoda: Planorbidae)

We purified and characterized two major glutathione S-transferase isoenzymes (GST2 and GST3) from snail Bulinus truncatus (Mollusca, Gastropoda, Planorbidae) tissue. The Km with respect to 1-chloro-2, 4-dinitrobenzene (CDNB) for both isoenzymes was increased as the pH decreased. Km of both isoenzymes with respect to glutathione (GSH) doubled when the pH was increased from 6.0 to 6.5. Acid inactivated GST2 and GST3 and the two enzymes were almost inactive at pH 3.5. However, they retain the full activity for at least 20 h when incubated at pH between 6.0 and 9.0. The optimum temperature was 45 degrees C for GST2 and 50 degrees C for GST3. The half lifetime at 50 degrees C was 70 min and 45 min for GST2 and GST3 isoenzymes, respectively. Addition of 5 mM GSH to the incubation buffer increased the half life of both isoenzymes more than fourfold. The activation energy for catalyzing the conjugation of CDNB was 1.826 and 3.435 kcal/mol for GST2 and GST3, respectively. I50 values for Cibacron blue, bromosulphophthalein, indocyanine green, hematin and ethacrynic acid were 0.76 microM, 47.9 microM, 7.59 microM, 0.03 microM and 0.79 microM for GST2, and 0.479 microM, 79.4 microM, 89.1 microM, 32.4 microM and 1.15 microM for GST3, respectively. Cibacron blue and indocyanine green were non-competitive inhibitors, while hematin was a mixed inhibitor. Bromosulphophthalein was found to be a competitive inhibitor for GST2 and a mixed inhibitor for GST3.     

Observations on genetic diversity of Bulinus cernicus (Gastropoda: Planorbidae) from Mauritius

Enzyme variation has been investigated in eight populations of Bulinus cernicus from Mauritius using isoelectric focusing and starch gel electrophoresis. Of 11 loci examined for 10 enzyme systems, seven were found to be polymorphic. These were malate dehydrogenase, glucose phosphate isomerase, acid phosphatase (A and B), 3-hydroxybutyrate dehydrogenase, 6-phosphogluconate dehydrogenase and NADH-dependent diaphorase. Invariant loci were phosphoglucomutase, xanthine oxidase, fructokinase and αaL-glycerophosphate dehydrogenase. The percentage of polymorphic loci within the populations ranged from 9.1% to 54.5%. The average heterozygosity per population for all loci ranged from 0,009 to 0.203. Genetic distance values ranged from 0.01 to 0.3 with the higher values associated with comparisons of populations from the north-west and south-east of the island. A comparison of data sets for 1980 and 1986 revealed the allelic frequencies at five enzyme loci to be remarkably consistent.     

Microsatellites in the freshwater snail Bulinus globosus (Gastropoda: Planorbidae) from Zanzibar

Six microsatellite loci were isolated and characterized in Bulinus globosus, a freshwater snail with a wide distribution throughout sub‐Saharan Africa. Bulinus globosus is an intermediate host of Schistosoma haematobium, the causative agent of human urinary schistosomiasis. Microsatellites were tested using 32 snails from four populations collected from Pemba and Unguja islands of Zanzibar. The microsatellite loci displayed relatively low levels of variation, with between two and five alleles per locus. F_ST estimates indicate that gene flow is low, as has previously been suggested for other species of Bulinus.     

Heritability of male outcrossing ability in the simultaneous hermaphrodite, Bulinus truncatus (Gastropoda: Planorbidae)

The heritability of phally, a dichotomous trait defined by the functional state of the male reproductive tract, was measured in a laboratory population of the simultaneous hermaphrodite snail Bulinus truncatus by means of a breeding experiment and a selection experiment. Euphallic individuals develop a fully functional male and female tract and are capable of receiving and donating sperm. In aphallic individuals the male tract does not develop fully, preventing sperm donation. There was no evidence of a heritable component to phally in the breeding experiment, but the selection experiment demonstrated a slight heritable effect. In both experiments there was more variation in the observed proportion of euphallics than expected by chance alone and no evidence of line or family effects, implicating environmental determination of male outcrossing ability even under controlled laboratory conditions. Previous studies of populations of B. truncatus reported that the proportion of euphallics was under strong genetic control. We suggest that there may be population differences in the extent of environmental control over phally, analogous to that reported for sex determination.     

A molecular phylogenetic analysis of Bulinus (Gastropoda: Planorbidae) with conserved nuclear genes

Jørgensen, A., Madsen, H., Nalugwa, A., Nyakaana, S., Rollinson, D., Stothard, J. R. & Kristensen, T. K. A molecular phylogenetic analysis of Bulinus (Gastropoda: Planorbidae) with conserved nuclear genes. —Zoologica Scripta, 40, 126–136. Mutational saturation of inspected DNA loci and topological incongruence in the phylogenetic inferences have previously confounded attempts to resolve the evolutionary relationships within the freshwater snail genus Bulinus. Traditionally, the 37 species of Bulinus are placed within the four species groups and the evolutionary divergence between groups is substantial. With an intention to shed new light on species group relationships, the present study was designed to investigate the basal divergences in the phylogeny of Bulinus using highly conserved nuclear genes. The resolved phylogeny inferred that the four species groups of Bulinus were monophyletic and Shimodaira‐Hasegawa topology tests found them to be significantly supported. The Bulinus truncatus/tropicus species complex and Bulinus wrighti (Bulinus reticulatus group) formed a well‐supported sister‐group relationship. The Bulinus africanus species group was the sister‐group to the clade (Bulinus truncatus/tropicus + B. wrighti) with the Bulinus forskalii species group as the sister‐group to these taxa. The sister‐group relationship between Indoplanorbis and Bulinus was non‐significant and the basal clade support of Bulinus improved upon exclusion of Indoplanorbis. The finding of basal long branches of Bulinus species originating from Madagascar strongly suggests the presence of additional cryptic species and an evolutionary scenario influenced by this island’s geological vicariance from the African mainland. Speciation by polyploidy was inferred to have evolved within a clade in the Bulinus truncatus/tropicus species complex. Although the monophyletic status of each species group was firmly supported, it was difficult to establish species group concepts equally across the variations and place this precisely in a specific temporal framework.     

Experimental taxonomy of Bulinus (Gastropoda: Planorbidae): the West and North African species reconsidered, based upon an electrophoretic study of several enzymes per individual

Horizontal starch gel electrophoresis was used to score mobilities of seven different enzymes in species of Bulinus from North and West Africa. An account of the intra- and inter-population variation observed was given. Based on the data obtained it was suggested that enzymic data should be used as taxonomic characters in parallel with morphological and anatomical characters. It is suggested that enzymic characters could be used to place taxa into synonymy if the data are collected in such a way that one can write up an enzyme profile for the single individual. The nine taxa of Bulinus known to occur in North and West Africa were revised, the taxon B. jousseaumei was synonymized with B. globosus , and the two taxa B. guernei and B. rohlfsi were synonymized with B. truncatus. As a result of the revision only six species could be recognized as valid from the area, these being B. forskalii, B. globosus, B. senegalensis, B. truncatus, B. ugandae and B. umbilicatus. Finally, some enzymic characters are suggested that may be used for a reliable identification of these morphologically very difficult taxa.     

Molecular identification of schistosome intermediate hosts: case studies of Bulinus forskalii group species (Gastropoda: Planorbidae) from Central and East Africa

African freshwater snails of the genus Bulinus act as intermediate hosts for schistosomes, trematode parasites responsible for medical and veterinary forms of schistosomiasis. The relationship between these snails and their parasites is an intricate one, with particular species of snail susceptible to infection only by certain species of schistosome. In common with other self-fertile hermaphrodite gastropods, Bulinus consists of a number of closely related species complexes with restricted gene flow between populations of each taxon. Consequently, despite their medical and veterinary importance as intermediate hosts, unambiguous identification and differentiation of planorbid snails such as these remains problematic, often confounding attempts to define the distribution and evolutionary relationships of conchologically similar taxa. Here we consider how morphological methods of discrimination can be used in conjunction with molecular based approaches to improve snail identification, thereby achieving a better understanding of the epidemiology of schistosomiasis. Data are presented from Central and East African taxa which illustrate how PCR-based methods have begun to be used in combination with traditional analyses in an integrated approach to characterize the genus Bulinus , specifically the B. forskalii species group. Particular emphasis is given to the analyses of Random Amplified Polymorphic DNA (RAPDs) and the mitochondrial gene cytochrome oxidase I (COI).     

A new species of Gyraulus (Gastropoda: Planorbidae) from Ancient Lake Lugu,Yunnan-Guizhou Plateau,Southwest China

A new aberrant species of the planorbid genus Gyraulus, Gyraulus luguhuensis n. sp., is described from Lake Lugu (Lugu-hu, in Chinese), Southwest China. The generic assignment with Gyraulus is based on features of the genital anatomy that are characteristic for members of that genus, in particular the presence of a chitinized penial stylet. Gyraulus luguhuensis n. sp. differs from most other congeners by its large, thick shell with an elevated spire. Similarly, aberrant shells are known from congeners in other Ancient Lakes worldwide indicating a potentially convergent evolution of shell characteristics in exclusively lacustrine species. Gyraulus luguhuensis differs from other lacustrine Gyraulus species with similarly large shells in having a sub-terminal penis pore and an unkeeled shell.     

Species-specific primers discriminate schistosome intermediate hosts: unambiguous PCR diagnosis of Bulinus forskalii group taxa (Gastropoda: Planorbidae)

The morphological definition of taxa has proved difficult within the Bulinus forskalii group, which includes intermediate hosts of medically important Schistosoma species in West Africa. Although B. forskalii and B. senegalensis transmit different schistosome species they are conchologically similar and their distributions overlap. Randomly amplified polymorphic DNA (RAPD) allows differentiation of sibling species in the genus Bulinus, but RAPDs are difficult to standardize, impairing their value as a taxonomic tool. Hence, RAPD products diagnostic for either B. senegalensis or B. forskalii from West Africa were cloned, sequenced and a panel of species-specific primers designed. Sequencing of RAPD products identified a homology in two apparently independent RAPD loci, a problem where RAPDs are indiscriminately scored for phylogenetic analyses. Specificity of primers was confirmed by widespread sampling throughout each species' range. This approach produced a simple, robust, unambiguous PCR-based species identification strategy for this difficult group.     

Endemic species of Bulinus (Mollusca: Planorbidae) in Lake Malawi (= Lake Nyasa)

Bulinus nyassanus and B. succinoides are both endemic to Lake Malawi and for many years were known only from their shells. Recent collections of living material have made possible a study of the anatomy of both species and the application of biochemical and immunological techniques have indicated their affinities within Bulinus . Some interesting aspects of their ecology have also been revealed.     

Endemic species of Bulinus (Mollusca: Planorbidae) in Lake Malawi (= Lake Nyasa)

Bulinus nyassanus and B. succinoides are both endemic to Lake Malawi and for many years were known only from their shells. Recent collections of living material have made possible a study of the anatomy of both species and the application of biochemical and immunological techniques have indicated their affinities within Bulinus . Some interesting aspects of their ecology have also been revealed.     

On a polyploid complex of freshwater snails (Planorbidae: Bulinus) in Ethiopia

Allometric growth of the postembryonic stages of Ectobius lapponicus (Linn.) and E. panzeri Stephens is described and analysed. Alternative methods of computing the allometric growth equation are discussed and applied, with the necessary significance tests, to a large number of skeletal structures in all developmental stages of each sex. The validity of Dyar's Law and its modifications are assessed quantitatively.  

Summary:

The growth in linear dimensions of 74 exosketetal structures has been studied quantitatively in all instars of both sexes of Ectobius lapponicus and E. panzeri.
Almost all the investigated structures show some statistically significant degree of allometric growth, but relatively few of them grow by simple allometry. Some possible reasons for this are indicated.
Well-defined growth gradients exist in the appendages and along the main axis of the body. Allometric growth contours, showing sexual and specific differences, express the temporal and spatial variations in the allometric growth ratio.
The limitations of Dyar's Law and Przibram's Rule are discussed and it is shown that conformity with Dyar's Law is not improved by allowing for differences in the duration of the instars.
It is emphasized that allometric growth studies should involve the choice of a suitable reference dimension and the selection of an appropriate statistical model to which the growth data are fitted. The estimation of the allometry parameters a and b , as well as the appropriate significance tests, depend on the model chosen, though the two models compared in this study do not yield appreciably different estimates.     

Molecular phylogenetic investigations of Bulinus (Gastropoda: Planorbidae) in Lake Malawi with comments on the topological incongruence between DNA loci

Freshwater snails of the genus Bulinus, subfamily Bulininae, are widespread in Africa and the Middle East. Several species are intermediate hosts for schistosomes, mammalian blood flukes that cause schistosomiasis. The genus is split into four species groups and without recourse to further molecular DNA studies, phylogenetic relationships within and between species‐groups remain obscure. For example, the Bulinus truncatus/tropicus species complex, which cannot be adequately defined with morphological characters, appears to be monophyletic but increased taxon sampling is warranted for confirmation when this group appears to contain species with limited sequence divergence in either mitochondrial 16S rRNA or Cytochome Oxidase subunit I (COI) loci. In the present study, taxonomic sampling is increased through addition of several taxa, including B. nyassanus and B. succinoides both endemic to Lake Malawi. From DNA analyses, these two species appear basal within the B. truncatus/tropicus species complex which provides an interesting evolutionary insight into its origins. The relationship of the B. truncatus/tropicus species complex with the three other groups, however, is more problematic as incongruence between loci and computational methodologies exist. This phenomenon is likely due to mutational saturation of the COI as evidenced by the transition : transversion ratio which also results in placement of the discoidal Indoplanorbis exustus, a member of the Bulininae, within the ingroup. To resolve these more basal incongruencies between species groups inspection of more evolutionary conserved molecular loci is needed, for example, the nuclear histone H3 gene, and 18S and 28S rDNA. Louise V. Gersdorff Jørgensen, Thomas K. Kristensen and Henry Madsen, The Mandahl‐Barth Research Centre for Biodiversity and Health, DBL — Centre for Health Research and Development, Department of Veterinary Pathobiology, Faculty of Life Sciences, University of Copenhagen, Jægersborg Allé 1D, DK‐2920 Charlottenlund, Denmark. E‐mails: lgj@life.ku.dk , tkk@life.ku.dk , hmad@life.ku.dk J. Russell Stothard, Biomedical Parasitology Division, Department of Zoology, Natural History Museum, Cromwell Road, London SW7 5BD, UK. E‐mail: R.Stothard@nhm.ac.uk     

The evolution of phally polymorphism in Bulinus truncatus (Gastropoda,Planorbidae): the cost of male function analysed through life-history traits and sex allocation

In the hermaphrodite freshwater snail Bulinus truncatus, two sexual morphs, euphallic (regular hermaphrodites) and aphallic individuals without a male copulatory organ, co-occur at various ratios in natural populations. Both aphallic and euphallic individuals can reproduce by selfing, but when outcrossing aphallic individuals can only play the female role. A comparison of life-history traits and sex allocation in these two forms provides the opportunity to investigate the evolution and maintenance of sexual polymorphisms. This study was performed to test whether a reallocation of resources from the lost male function to the female function occurs in aphallic snails at the level of both sex organs (sex allocation) and life-history traits. In a first experiment we compared life-history traits over a whole life-cycle under selfing between the two sexual morphs. In a second experiment, the sex organs were weighed to test for a difference in sex allocation between the two morphs. No difference in resource allocation to female function between the two morphs was observed in either experiment. This is in contrast to patterns frequently observed in sexually polymorphic plants, and in a previous study performed on aphally in the same snail species. We discuss the genetic and physiological hypotheses that could explain these results, and their consequences for the evolution and maintenance of phally polymorphism in B. truncatus.     

Hemocytes of Bulinus truncatus rohlfsi (Mollusca: Gastropoda)

Two basic cell types occur in the hemolymph of Bulinus truncatus rohlfsi: granulocytes and hyalinocytes. Granulocytes are divided into three subtypes: (1) Granulocytes I, which account for 19% of the hemocytes, are small, young amoebocytes with 1–20 filopodia and small numbers of cytoplasmic granules, including some lysosomes; (2) granulocytes II, which account for 78% of the cells, are large, fully developed amoebocytes that possess 1–20 filopodia and many granules, both acidophilic and basophilic, including numerous lysosomes, phagosomes, and mitochondria; and (3) spent granulocytes, which are rare, have few filopodia, large accumulations of glycogen granules and prominent vacuoles in addition to lysosomes in the cytoplasm. These three subtypes of granulocytes probably represent ontogenetic stages within a single cell line. In addition, granulocytes with 40 or more filopodia and little ectoplasm, found in only 1 of 45 snails examined, probably reflect a pathologic condition. Hyalinocytes, which account for 3% of all hemocytes, are similar in size to mature granulocytes, but have few or no cytoplasmic granules and lack filopodia and glycogen granules. Total hemocyte concentration in hemolymph is 328,000 ± 188,000 cells/ml.     

Karyotype delineation in Isidora truncata (Gastropoda: Planorbidae)

Metaphase chromosomes of a single Sardinian population of Isidora truncata were examined in an attempt to provide a picture of a standard karyotype to which Sardinian populations could be referred. Several new foci were identified and mapped.