Teofânia H.D.A. Vidigal

PCR amplification of the mitochondrial DNA minisatellite region to detect Schistosoma mansoni infection in Biomphalaria glabrata snails

Schistosoma infection in Biomphalaria glabrata can be detected by either exposing snails to light to induce cercarial shedding or by squeezing them between glass slides to detect parasites in the digestive gland and other regions. The methods available are inefficient for identification of prepatent infections and do not allow the diagnosis of infection in snails that die before arriving in the laboratory. Furthermore, infection is undetectable after migration of sporocysts from the head-foot region of the snail. We examined the use of polymerase chain reaction (PCR) amplification of minisatellite repeats from Schistosoma mansoni mitochondrial DNA (mtDNA) to identify snail infection. We found that amplification of mtDNA under low stringency conditions (LS-PCR) allowed for the identification of specific S. mansoni infection in Biomphalaria snails. To confirm these results, specific amplification reactions were performed using 2 sets of primers that allowed for the diagnosis of infection and an internal control of the reaction (multiplex PCR). Results obtained using multiplex PCR demonstrated the ability of the assay to detect S. mansoni-specific infection. Thus, LS-PCR as well as specific multiplex PCR allow for the detection of prepatent infections and show high specificity for S. mansoni in comparison with other trematode infections in either living or dead snails.

Resistance to Schistosoma mansoni by transplantation of APO Biomphalaria tenagophila

Transplantation of the haematopoietic organ from Biomphalaria tenagophila (Taim strain, RS, Brazil), resistant to Schistosoma mansoni, to a highly susceptible strain (Cabo Frio, RJ, Brazil) of the same species, showed in the recipient snails resistance against the trematode, when a successful transplant occurred. The success of transplantation could be confirmed by a typical molecular marker of the Taim strain in haemocytes of the recipients (350 bp detected by PCR‐RFLP). The recipient snails which did not present the donor marker in haemocytes (unsuccessful transplantation) were infected with the parasite. The use of an atoxic modelling clay for closing the hole in the transplantation site reduced significantly the mortality caused by bleeding after transplantation procedures.

Analysis of the first and second internal transcribed spacer sequences of the ribosomal DNA in Biomphalaria tenagophila complex (Mollusca: Planorbidae)

The first and second internal transcribed spacer regions (ITS1 and ITS2) of the ribosomal DNA of Biomphalaria tenagophila complex (B. tenagophila, B. occidentalis, and B. t. guaibensis) were sequenced and compared. The alignment lengths of these regions were about 655 bp and 481 bp, respectively. Phylogenetic relationships among the Biomphalaria species were inferred by Maximum Parsimony and Neighbor-joining methods. The phylogenetic trees produced, in most of the cases, were in accordance with morphological systematics and other molecular data previously obtained by polymerase chain reaction and restriction fragment length polymorphism analysis. The present results provide support for the proposal that B. tenagophila represents a complex comprising B. tenagophila, B. occidentalis and B. t. guaibensis.

Genetic variability in Brazilian populations of Biomphalaria straminea complex detected by simple sequence repeat anchored polymerase chain reaction amplification.

Biomphalaria glabrata, B. tenagophila and B. straminea are intermediate hosts of Schistosoma mansoni, in Brazil. The latter is of epidemiological importance in the northwest of Brazil and, due to morphological similarities, has been grouped with B. intermedia and B. kuhniana in a complex named B. straminea. In the current work, we have standardized the simple sequence repeat anchored polymerase chain reaction (SSR-PCR) technique, using the primers (CA)8RY and K7, to study the genetic variability of these species. The similarity level was calculated using the Dice coefficient and genetic distance using the Nei and Li coefficient. The trees were obtained by the UPGMA and neighbor-joining methods. We have observed that the most related individuals belong to the same species and locality and that individuals from different localities, but of the same species, present clear heterogeneity. The trees generated using both methods showed similar topologies. The SSR-PCR technique was shown to be very efficient in intrapopulational and intraspecific studies of the B. straminea complex snails.

Strong spatial structure, Pliocene diversification and cryptic diversity in the Neotropical dry forest spider Sicarius cariri

The Brazilian Caatinga is part of the seasonally dry tropical forests, a vegetation type disjunctly distributed throughout the Neotropics. It has been suggested that during Pleistocene glacial periods, these dry forests had a continuous distribution, so that these climatic shifts may have acted as important driving forces of the Caatinga biota diversification. To address how these events affected the distribution of a dry forest species, we chose Sicarius cariri, a spider endemic to the Caatinga, as a model. We studied the phylogeography of one mitochondrial and one nuclear gene and reconstructed the paleodistribution of the species using modelling algorithms. We found two allopatric and deeply divergent clades within S. cariri, suggesting that this species as currently recognized might consist of more than one independently evolving lineage. Sicarius cariri populations are highly structured, with low haplotype sharing among localities, high fixation index and isolation by distance. Models of paleodistribution, Bayesian reconstructions and coalescent simulations suggest that this species experienced a reduction in its population size during glacial periods, rather than the expansion expected by previous hypotheses on the paleodistribution of dry forest taxa. In addition to that, major splits of intraspecific lineages of S. cariri took place in the Pliocene. Taken together, these results indicate S. cariri has a complex diversification history dating back to the Tertiary, suggesting the history of dry forest taxa may be significantly older than previously thought.

Characterization of Biomphalaria orbignyi, Biomphalaria peregrina and Biomphalaria oligoza by polymerase chain reaction and restriction enzyme digestion of the internal transcribed spacer region of the RNA ribosomal gene

The correct identification of Biomphalaria oligoza, B. orbignyi and B. peregrina species is difficult due to the morphological similarities among them. B. peregrina is widely distributed in South America and is considered a potential intermediate host of Schistosoma mansoni. We have reported the use of the polymerase chain reaction and restriction fragment length polymorphism analysis of the internal transcribed spacer region of the ribosomal DNA for the molecular identification of these snails. The snails were obtained from different localities of Argentina, Brazil and Uruguay. The restriction patterns obtained with MvaI enzyme presented the best profile to identify the three species. The profiles obtained with all enzymes were used to estimate genetic similarities among B. oligoza, B. peregrina and B. orbignyi. This is also the first report of B. orbignyi in Uruguay.

GENETIC VARIABILITY AND IDENTIFICATION OF THE INTERMEDIATE SNAIL HOSTS OF SCHISTOSOMA MANSONI

Studies based on shell or reproductive organ morphology and genetic considerations suggest extensive intraspecific variation in Biomphalaria snails. The high variability at the morphological and genetic levels, as well as the small size of some specimens and similarities between species complicate the correct identification of these snails. Here we review our work using methods based on polymerase chain reaction (PCR) amplification for analysis of genetic variation and identification of Biomphalaria snails from Brazil, Argentina, Uruguay and Paraguay. Arbitrarily primed-PCR revealed that the genome of B. glabrata exhibits a remarkable degree of intraspecific polymorphism. Low stringency-PCR using primers for 18S rRNA permitted the identification of B. glabrata, B. tenagophila and B. occidentalis. The study of individuals obtained from geographically distinct populations exhibits significant intraspecific DNA polymorphism, however, specimens from the same species, exhibit some species specific LSPs. We also showed that PCR-restriction fragment of length polymorphism of the internal transcribed spacer region of Biomphalaria rDNA, using Ddel permits the differentiation of the three intermediate hosts of Schistosoma mansoni. the molecular biological techniques used in our studies are very useful for the generation of new knowledge concerning the systematics and population genetics of Biomphalaria snails.

Short-term toxicity of ammonia, sodium Hydroxide and a commercial biocide to golden mussel Limnoperna fortunei (Dunker, 1857)

Macrofouling bivalves are considered an ecological and technological problem worldwide. Control measures have been researched with Limnoperna fortunei, but without success. The aim of the manuscript is to test some alternatives to regulate this harmful invasive mollusk. Mortality and behavioral response (shell gaping) of Limnoperna fortunei exposed to three chemical compounds were evaluated. Values for LC50 96h were: 0.25 (0.24-0.27)mg/L NH3-N, 11.10 (7.45-16.55) mg/L MXD-100 and 88.51 (74.61-105.01)mg/L NaOH. Reduced gaping was observed beginning at concentrations of 0.31mg/L (NH3-N), 100mg/L (MXD-100) and 160mg/L (NaOH) and increased above these values. The percentage of individuals gaping after two hours at LC50 96h differed significantly (χ(2)=79.9; DF=3; p<0.001) in MXD-100 (50%), NaOH (0%), NH3-N (96.7%) and the controls (93.3%). This study contributes to the understanding of the relationship between toxicity and behavioral effects of some toxicants in L. fortunei.

Size polymorphism in alleles of the myoglobin gene from biomphalaria mollusks.

Introns are common among all eukaryotes, while only a limited number of introns are found in prokaryotes. Globin, globin-like proteins are widely distributed in nature, being found even in prokaryotes, a wide range of patterns of intron-exon have been reported in several eukaryotic globin genes. Globin genes in invertebrates show considerable variation in the positions of introns; globins can be found without introns, with only one intron or with three introns in different positions. In this work we analyzed the introns in the myoglobin gene from Biomphalaria glabrata, B. straminea, B. tenagophila. In the Biomphalaria genus, the myoglobin gene has three introns; these were amplified by PCR, analyzed by PCR-RFLP. Results showed that the size (number or nucleotides), the nucleotide sequence of the coding gene of the myoglobin are variable in the three species. We observed the presence of size polymorphisms in intron 2, 3; this characterizes a homozygous/heterozygous profile, it indicates the existence of two alleles which are different in size in each species of Biomphalaria. This polymorphism could be explored for specific identification of Biomphalaria individuals.

First record of Corbicula largillierti (Philippi 1844) in the Paraíba River Basin and potential implications from water diversion of the São Francisco River

Corbicula largillierti is a native mollusk from China. In Brazil, this species was first recorded in the Pantanal of Mato Grosso. This short communication reports the occurrence of C. largillierti for the first time in the Paraiba river basin (Brazilian semi-arid), and also considers the risk of introduction of other molluscs invaders in this basin due to the diversion of water from the Sao Francisco River. Densities of individuals ranged from 33 to 65 ind.m-2 (maximum values of 484 ind.m-2) in coarse sediment (gravel, 2-4 mm). The diversion of waters from the Sao Francisco river can lead to the introduction of new species, enhancing ecological problems in the Paraiba river basin.