A new species of Urocleidoides Mizelle & Price 1964 (Monogenea: Dactylogyridae) parasite of Hemiodus orthonops Eigenmann & Kennedy, 1903 (Hemiodontidae) from the upper Paraná River floodplain.

During the study of ectoparasites (Platyhelminthes) of fish in the floodplain of the Upper Paraná River - Brazil, a new species of Urocleidoides, present in the gill filaments of Hemiodus orthonops (Hemiodontidae), is described using morphological description and molecular data from the mitochondrial region of cytochrome c oxidase, subunit 1 (COI) and the partial region of the 28S rDNA gene. Urocleidoides luquei n. sp. differs from all its congeners by the presence of a filament that joins the accessory piece to the base of the male copulatory organ and resembles U. paradoxus and U. surianoae. Phylogenetic analyses using molecular data revealed that U. luquei n. sp. forms a paraphyletic group concerning the other Urocleidoides species. In this way, as well as contributing to the description of a new species, we seek to encourage and contribute to the increase in research using integrative taxonomy, thus making it possible to elucidate some unresolved questions about the genus Urocleidoides.

Morphological description and molecular characterization of Ancyracanthus electrophori n. sp. (Gnathostomatoidea: Gnathostomatidae): a new nematode parasitic in the electric eel Electrophorus varii (Gymnotiformes: Gymnotidae), from the Brazilian Amazon.

A new species of Ancyracanthus, parasite of the electric eel Electrophorus varii, in the Brazilian Amazon, is described based on morphological and molecular characterization. Ancyracanthus electrophori n. sp. differs from the two congeners namely, Ancyracanthus pinnatifidus and Ancyracanthus schubarti, based on the structure of cephalic appendages, number and arrangement of caudal papillae in males, vulva very close to anus in females, eggs with smoothly mamillated shell, host taxon and geographical origin. Moreover, the new species is the first in the genus to be described with thorny cuticular rings and to be observed with the use of scanning electron microscopy (SEM). The morphology of A. pinnatifidus and A. schubarti is still poorly-known and should be revised in details; however, the separation between them and the new species was clear. Genetic characterization based on 28S rDNA and cytochrome c oxidase subunit I (cox1) mtDNA partial sequences, performed for the first time in Acyracanthus, along with phylogenetic reconstructions using both genetic markers, placed Ancyracanthus electrophori n. sp. in a suggestive basal position within Gnathostomatidae. Phylogenetic reconstructions using cox1 sequences also suggested lack of monophyly in the genera Gnathostoma and Spiroxys and, consequently, in the subfamilies Gnathostominae and Spiroxyinae. However, such results are preliminary. With the first genetic characterization and observations using SEM in Ancyracanthus, resulting in the discovery of a new species and in the expansion of the geographical occurrence of the genus to Amazonian fish, an important step towards a better understanding of these nematodes has been taken.

Extenders and cryosolutions for Grumata (Prochilodus vimboides) sperm preservation.

BACKGROUND: Prochilodus vimboides populations are being reduced in rivers due to changes in their habitat, overfishing, urbanization, and pollution. OBJECTIVE: To evaluate the effect of sperm extender solutions for short-term storage and cryosolutions for freezing sperm of Prochilodus vimboides. MATERIALS AND METHODS: For short-term storage, the sperm was diluted in 0.9% NaCl, 1.2% NaCl, 5% glucose, 5% BTS, or 6% MIII. Sperm motility was evaluated after 0, 24, 48, and 72 h of short-term storage at 4-6 degree C. For cryopreservation, sperm samples were diluted in the same extenders and factorially combined with three cryoprotectants (dimethylsulfoxide, methyl glycol, and ethylene glycol). After thawing, sperm motility and oxidative stress parameters were evaluated. RESULTS: Dilution of samples in BTS preserved sperm motility >40% for up to 48 h. Samples cryopreserved in 5% glucose and methylglycol presented higher sperm motility, lower catalase, and lipid peroxidation activities. CONCLUSION: Prochilodus vimboides sperm can be cooled for up to 48 h in an extender solution of 5% BTS and cryopreserved in 5% glucose and methyl glycol. doi.org/10.54680/fr22410110612.

Molecular characterization and identification of digenean larval stages in Aylacostoma chloroticum (Prosobranchia: Thiaridae) from a neotropical floodplain.

Digeneans (Trematoda: Digenea) are endoparasites that present a complex life cycle, generally involving an intermediate invertebrate host and a vertebrate host. There is limited information about which species of molluscs may act as intermediate hosts in the upper Paraná River floodplain (UPRF), where Aylacostoma chloroticum can be considered a potential candidate. The study of digeneans in this region is important because some of these parasites are potentially zoonotic, and, therefore, are relevant to public health. However, the correct identification of these organisms during the larval stages is difficult because of the lack of morphologically diagnostic characteristics. The objective of this study was to identify and molecularly characterize the larval stages of digeneans found in A. chloroticum in the UPRF, using the mitochondrial marker of subunit I of cytochrome c oxidase and the 28S nuclear marker. The molluscs were examined in the laboratory and three morphotypes of cercariae were found. DNA was extracted from the specimens obtained and was then amplified and sequenced. The morphotypes exhibited high genetic similarities with Pseudosellacotyla, Paralecithodendrium and Philophthalmus, indicating that these organisms belong to these genera. This is the first record of larval stages of these genera in molluscs collected in the UPRF.

Genetic relationships of Plagioscion squamosissimus (Perciformes, Sciaenidae) from five Neotropical river basins evaluated using mitochondrial atpase6/8 gene sequences.

The genetic relationships of native or introduced Plagioscion squamosissimus in five Brazilian Neotropical basins were evaluated using the mitochondrial atpase6/8 genes. Results revealed that the population of the Tocantins River basin is more basal than the native populations of the Amazon and Parnaíba River basins. Moreover, the populations of P. squamosissimus that were introduced in the São Francisco and upper Paraná River basins originated from the population of the Parnaíba River.

Molecular characterization of the species Salvinia (Salviniaceae) from the upper Paraná River floodplain.

The pteridophytes Salvinia minima, S. herzogii, and S. auriculata are among the most abundant aquatic macrophytes in the upper Paraná River floodplain. Since some species have highly similar morphological features, it is very difficult to identify members of this genus to the species level. An indication of this difficulty is a set of poorly differentiated taxa comprising S. auriculata and S. herzogii known as the 'S. auriculata complex', which is found in the Paraná River together with other Salvinia species such as S. biloba and S. molesta. Some authors have reported the existence of inter-species hybrids. Despite the complex Salvinia taxonomy, few genetic studies have been performed on purported species within the genus to resolve this complexity. The present study was conducted to determine useful molecular sequences for the discrimination of Salvinia species of the upper Paraná River floodplain. Molecular data were compared with data of other species of the genus to clarify phylogenetic relationships, employing the nucleotide sequence trnL-trnF from the chloroplast DNA. The results revealed that Salvinia populations in the upper Paraná River floodplain belong to different species and indicated that species of the S. auriculata complex may be distinguished from one another after the division of the S. minima group, corroborating results by other researchers. Although the taxonomic position of S. oblongifolia was clarified, as high closeness between S. oblongifolia and the S. auriculata complex was reported, Salvinia kinship is still not thoroughly established and further investigations in morphology and molecular diversity are required.

Genetic characterization of native and invasive Plagioscion squamosissimus (Perciformes, Sciaenidae) populations in Brazilian hydrographic basins.

The genetic diversity of invasive and native populations of Plagioscion squamosissimus (Heckel, 1840) from the Paraná, Parnaiba and Araguaia-Tocantins river basins was assessed by using Random Amplified Polymorphic DNA (RAPD) markers. Genetic data confirmed the hypothesis of low genetic variability within and among P. squamosissimus populations introduced in the upper Paraná River basin, and indicated that they likely originated from a common ancestor. Moreover, the data demonstrated that, in agreement with available historical records, the P. squamosissimus populations established in the Paraná River basin were derived from a population native to the Parnaíba River basin. The genetic data presented here are of potential future application for the management of the invasive P. squamosissimus populations and for the preservation of the genetic legacy of native fish.

Thermal conditions in freezing chambers and prediction of the thermophysiological responses of workers.

The present work is dedicated to the assessment of the cold thermal strain of human beings working within freezing chambers. To obtain the present results, both field measurements and a numerical procedure based on a modified version of the Stolwijk thermoregulation model were used. Eighteen freezing chambers were considered. A wide range of physical parameters of the cold stores, the workers clothing insulation, and the working and recovering periods were observed. The combination of these environmental and individual parameters lead to different levels of thermal stress, which were grouped under three categories. Some good practices were observed in the field evaluations, namely situations with appropriate level of clothing protection and limited duration of exposure to cold avoiding unacceptable level of hypothermia. However, the clothing ensembles normally used by the workers do not provide the minimum required insulation, which suggests the possibility of the whole body cooling for levels higher than admissible. The numerical predictions corroborate the main conclusions of the field survey. The results obtained with both methodologies clearly show that, for the low temperature of the freezing chambers, the clothing insulation is insufficient, the exposure periods are too long, and the recovering periods are inadequate. Thus, high levels of physiological strain can indeed be reached by human beings under such working environments.

Conjunctival flora, Schirmer's tear test, intraocular pressure, and conjunctival cytology in neotropical primates from Salvador, Brazil.

BACKGROUND: This study aimed to establish reference values for selected ophthalmic diagnostic tests in healthy neotropical primates from Salvador, Brazil. METHODS: A total of 73 intact adults, including Callithrix jacchus (n = 31), Callithrix penicillata (n = 8), Cebus sp. (n = 22), and Cebus xanthosternos (n = 9) were used to evaluate the normal conjunctival bacterial flora. Cebus xanthosternos (n = 12) were used to evaluate tear production with Schirmer's tear test (STT), intraocular pressure (IOP), and conjunctival cytology. RESULTS: For all animals evaluated, Gram-positive bacteria were predominant. Results of the diagnostic tests in Cebus xanthosternos were as follows: STT: 14.92 ± 5.46 mm/minutes, IOP: 19.62 ± 4.57 mmHg, and conjunctival cytology revealed intermediate squamous epithelial cells in great quantities. CONCLUSIONS: These ophthalmic reference values will be particularly useful to diagnose discrete or unusual pathological changes in the neotropical primates eye.

Effects of high-fat chow on heart tissue in acute and chronic experimental murine schistosomiasis mansoni.

This study aimed to investigate myocardial injuries in mice infected with Schistosoma mansoni and fed a high-fat chow. Sections of myocardial tissue from S. mansoni-infected mice, and controls that had been killed 9 and 17 weeks post-infection, were stained with H&E and Picrosirius red. Histopathological examination, stereological design-based method (optical disector) and morphometry (vessels, cardiomyocytes and an amount of collagen) were used. Data were analysed using two-way ANOVA. Regardless of time of infection, myocardial tissue from the infected mice fed high-fat chow showed myocarditis characterized by a higher number of inflammatory foci, several areas displaying coagulation of cardiac fibres, a greater loss of cardiomyocytes and fibroblast proliferation than in the standard chow control. Comparing infected mice from acute and chronic infections, a higher cardiomyocyte hyperplasia (P < 0·0001) and higher amounts of collagen (P < 0·05) were observed than in standard chow control. In addition, all animals fed high-fat chow showed lower numerical density and total number of cardiomyocytes (P < 0·05), thicker vessel walls and narrowed luminal intramyocardial vessels (P > 0·05) than in the standard chow control. Altogether the data supported the view that a double burden has a synergistic deleterious effect on the myocardial tissue.

Critical mass of splenic autotransplant needed for the development of phagocytic activity in rats.

When total splenectomy is inevitable, heterotopic splenic autotransplantation seems to be the only alternative to maintain the functions of the spleen. The present study was carried out to analyse the critical mass of splenic autotransplant (SAT) for the development of phagocytic activity in rats. Wistar rats were submitted to total splenectomy (TS) alone or in combination with slices of SAT ranging from an average rate of 21·9% (one slice) to 100% (five slices) of the total splenic mass implanted into the greater omentum. Sixteen weeks after the beginning of the experiment, the animals were inoculated intravenously with a suspension of Escherichia coli labelled with Tc-99m. After 20 min, the rats were killed and the liver, lung and spleen or SAT, as well as blood samples were removed to determine the percentage of labelled bacteria uptake in these tissues. As the percentage of the total splenic mass contained in the SAT increased, the bacteria remaining in the blood decreased. From the implant of 26% up to the implant of the total splenic mass (100%) there was no difference in the bacteria remaining in the blood between the healthy animals of the control group and those submitted to TS combined with SAT. This finding shows that the critical mass needed for the development of phagocytic activity of macrophages in splenic autotransplants in adult rats is 26% of the total splenic mass.

Genetic divergence between Pseudoplatystoma corruscans and Pseudoplatystoma reticulatum (Siluriformes: Pimelodidae) in the Paraná River Basin.

Pseudoplatystoma corruscans (Spix and Agassiz, 1829) and Pseudoplatystoma reticulatum (Eingenmann and Eigenmann, 1889) are large migratory catfishes of high biological importance and great commercial value in South America. Because fertile crossbreeds can be artificially produced in hatcheries, a high genetic proximity between these two Pimelodidae species is conceivable. Possible escape of crossbred specimens from pisciculture stations is a serious environmental concern. Despite their importance, knowledge of P. corruscans and P. reticulatum biology, ecology, population diversity and genetics is limited. In the present work, the genetic divergence between P. corruscans and P. reticulatum populations from the Paraná River Basin was analyzed on the basis of polymorphisms in ISSR fragments and in the hypervariable sequence of the mitochondrial DNA (mtDNA) control region. Estimates of intraspecific haplotype (h > 0.5) and nucleotide diversities (pi < 0.01) indicate that P. corruscans and P. reticulatum have survived a historical population decline, followed by a demographic expansion. The interspecific polymorphisms within the mtDNA control region and ISSR fragments were suitable as diagnostic molecular markers and could be used to discriminate the two species. A unique Pseudoplatystoma specimen, captured in the Upper Paraná River Floodplain, was identified by these DNA diagnostic markers as a hybrid P. reticulatum x P. corruscans, which possibly escaped from pisciculture. The integrity of the natural population of P. corruscans in the Upper Paraná River is at risk of genetic introgression or homogenization due to the presence of hybrids and the transposition of P. reticulatum upstream through the Canal da Piracema at Itaipu Dam. Data presented herein improve the understanding of the genetic relatedness between P. corruscans and P. reticulatum and represent potential tools for future programs of conservation and surveillance of genetic introgression events and the genetic integrity of these populations.

Sensibilidade dos espermatozoides de dourado (Salminus brasiliensis) a diferentes soluções crioprotetoras / Sensitivity of dourado (Salminus brasiliensis) spermatozoa to different cryoprotectant solutions

Em três experimentos, avaliou-se a sensibilidade dos espermatozoides de dourado (Salminus brasiliensis) a diferentes soluções crioprotetoras. No experimento 1, o sêmen foi diluído, 1:10, em 12 soluções (quatro diluidores x três crioprotetores - dimetilsulfóxido (DMSO), metilglicol ou glicerol). Metade de cada amostra foi resfriada por uma hora e a outra, criopreservada. A motilidade espermática foi avaliada imediatamente após a diluição e após o resfriamento em todas as amostras e, após o descongelamento, apenas nas amostras criopreservadas em DMSO. No experimento 2, o sêmen foi diluído, 1:5, em cinco soluções contendo DMSO e resfriado, criopreservado e avaliado como no experimento 1. No experimento 3, o sêmen foi diluído, 1:5, em quatro soluções contendo DMSO e criopreservado e avaliado quanto à motilidade e à fertilidade. Quando o sêmen foi diluído 1:10, observou-se motilidade acima de 58 por cento em todas as amostras resfriadas em DMSO e em NaCl-tris-metilglicol. Baixa motilidade foi observada nas amostras resfriadas nas outras combinações com metilglicol (5-32 por cento) ou glicerol (0-8 por cento) e naquelas criopreservadas (16-20 por cento). Todas as amostras diluídas 1:5 apresentaram motilidade de 65-72 por cento após o resfriamento e de 45-66 por cento após o descongelamento (experimentos 2 e 3). As taxas de eclosão produzidas com sêmen criopreservado, entretanto, foram baixas (17-23 por cento) em relação ao sêmen fresco (60 por cento).

The sensitivity of dourado (Salminus brasiliensis) spermatozoa to different cryoprotectant solutions was evaluated in three experiments. In experiment 1, semen was diluted, 1:10, in 12 solutions (four extenders x three cryoprotectants - dimethylsulphoxide (DMSO), methyglycol, or glycerol). Half of each sample was refrigerated for one hour while the other half was cryopreserved. Sperm motility was immediately assessed after dilution and after refrigeration in all samples, and after thawing in those cryopreserved in DMSO. In experiment 2, semen was diluted, 1:5, in five solutions containing DMSO, refrigerated, cryopreserved, and analyzed as in experiment 1. In experiment 3, semen was diluted, 1:5, in five solutions containing DMSO, cryopreserved and evaluated for motility and fertility. When semen was diluted 1:10, motility higher than 58 percent was observed in all samples refrigerated in DMSO and in NaCl-tris-methylglycol. Low motility was observed in samples refrigerated in the other combinations of methylglycol (5-32 percent) or glycerol (0-8 percent) and in those cryopreserved (16-20 percent). All samples diluted 1:5 yielded motility of 65-72 percent after refrigeration, and 45-66 percent after thawing (experiments 2 and 3). The hatching rates produced with cryopreserved semen, however, were lower (17-23 percent) compared to fresh semen (60 percent).

Genetic divergence between Pseudoplatystoma corruscans and Pseudoplatystoma reticulatum (Siluriformes: Pimelodidae) in the Paraná River Basin / Divergência genética entre Pseudoplatystoma corruscans e Pseudoplatystoma reticulatum (Pisces; Siluriformes) da Bacia do Rio Paraná

Pseudoplatystoma corruscans (Spix and Agassiz, 1829) and Pseudoplatystoma reticulatum (Eingenmann and Eigenmann, 1889) are large migratory catfishes of high biological importance and great commercial value in South America. Because fertile crossbreeds can be artificially produced in hatcheries, a high genetic proximity between these two Pimelodidae species is conceivable. Possible escape of crossbred specimens from pisciculture stations is a serious environmental concern. Despite their importance, knowledge of P. corruscans and P. reticulatum biology, ecology, population diversity and genetics is limited. In the present work, the genetic divergence between P. corruscans and P. reticulatum populations from the Paraná River Basin was analyzed on the basis of polymorphisms in ISSR fragments and in the hypervariable sequence of the mitochondrial DNA (mtDNA) control region. Estimates of intraspecific haplotype (h > 0.5) and nucleotide diversities (π < 0.01) indicate that P. corruscans and P. reticulatum have survived a historical population decline, followed by a demographic expansion. The interspecific polymorphisms within the mtDNA control region and ISSR fragments were suitable as diagnostic molecular markers and could be used to discriminate the two species. A unique Pseudoplatystoma specimen, captured in the Upper Paraná River Floodplain, was identified by these DNA diagnostic markers as a hybrid P. reticulatum x P. corruscans, which possibly escaped from pisciculture. The integrity of the natural population of P. corruscans in the Upper Paraná River is at risk of genetic introgression or homogenization due to the presence of hybrids and the transposition of P. reticulatum upstream through the Canal da Piracema at Itaipu Dam. Data presented herein improve the understanding of the genetic relatedness between P. corruscans and P. reticulatum and represent potential tools for future programs of conservation and surveillance of genetic ...

Pseudoplatystoma corruscans Spix e Agassiz, 1829 e Pseudoplatystoma reticulatum Eigenmann e Eigenmann, 1889 são peixes migratórios de grande porte, com alta importância biológica e elevado valor comercial na América do Sul. Híbridos férteis são obtidos em cativeiro e, portanto, é esperada alta proximidade genética entre essas duas espécies de Pimelodidae. Escapes de espécimes híbridos a partir de estações de piscicultura representam um sério problema ambiental. Apesar da sua importância, conhecimentos sobre a biologia, ecologia, diversidade de populações e genética de P. corruscans e P. reticulatum são escassos. No presente trabalho, foi avaliada a divergência genética entre P. corruscans e P. reticulatum da Bacia do Rio Paraná, com base em fragmentos ISSR e na seqüência D-loop do DNA mitocondrial (mtDNA). As estimativas das diversidades intra-específicas haplotípica (h > 0,5) e nucleotídica (π < 0,01) evidenciaram que P. corruscans e P. reticulatum sobreviveram a um declínio populacional histórico, seguido de expansão demográfica. Os polimorfismos interespecíficos no mtDNA e nos fragmentos ISSR foram eficientes para diagnósticos e discriminaram as duas espécies. Um espécime de Pseudoplatystoma capturado na planície de inundação do Alto Rio Paraná foi identificado com esses marcadores moleculares como híbrido P. reticulatum x P. corruscans, que possivelmente escapou de psicicultura. A integridade da população de P. corruscans no Alto Rio Paraná está ameaçada, por introgressão ou homogeneização genética, pela presença de híbridos e pela transposição para montante de P. reticulatum através do Canal da Piracema em Itaipu. Os dados apresentados constituem um avanço na compreensão do parentesco entre P. corruscans e P. reticulatum e representam ferramentas em potencial para programas de conservação biológica, incluindo o monitoramento de introgressão e de integridade genética das populações.

Sucesso do resfriamento e congelamento de sêmen de pirapitinga Brycon nattereri / Sucess of cooling and freezing of pirapitinga (Brycon nattereri) semen

Avaliaram-se protocolos de resfriamento e de criopreservação do sêmen de pirapitinga (Brycon nattereri) utilizando-se sêmen diluído em NaCl 154mM, NaCl 200mM, Saad e BTS® e resfriado por sete dias. Cinco diluidores (glicose 277mM, NaCl 154mM, NaCl 200mM, Saad e BTS®) foram combinados com dois crioprotetores (DMSO - dimetilsulfóxido e metilglicol) e usados como meio de congelamento. O sêmen diluído em cada meio foi envasado (palhetas de 0,5ml) e congelado, e a motilidade espermática avaliada após o descongelamento (60ºC, 8seg). O sêmen foi novamente congelado em palhetas com diferentes volumes (0,25 e 0,5ml) e descongelados em banho-maria em duas temperaturas (50º e 60ºC). As maiores motilidades (48 por cento) foram observadas no sêmen diluído em BTS® e resfriado por sete dias. Motilidade espermática acima de 68 por cento foram observadas no sêmen congelado em NaCl 154mM-metilglicol, BTS®-metilglicol, NaCl 200mM-DMSO e Saad-DMSO. Não houve diferença entre os volumes de palheta nem entre as temperaturas de descongelamento quanto a motilidade espermática. Assim, o sêmen de pirapitinga mantém altas taxas de motilidade quando resfriado em BTS® por até sete dias ou congelado em NaCl 154mM-metilglicol, BTS®-metilglicol, NaCl 200mM-DMSO e Saad-DMSO

Cooling and freezing protocols of pirapitinga (Brycon nattereri) semen were evaluated using semen diluted in 154mM NaCl, 200mM NaCl, Saad or BTSÕ, and cooled for seven days. Sperm motility was daily evaluated. Five extenders (277mM glucose, 154mM NaCl, 200mM NaCl, Saad and BTSÕ) were combined with two cryoprotectants (DMSO - dimethyl sulphoxide and methylglycol) to produce 10 cryosolutions. Semen was diluted in each cryosolutions, aspirated into 0.5ml straws and frozen. Sperm motility was evaluated after thawing (60ºC, 8 sec). Then, semen was frozen in straws with different volumes (0.25 and 0.5ml), and thawed under different water-bath temperatures (50º and 60ºC). Higher sperm motility (48 percent) was observed when semen was cooled in BTSÕ for seven days. Post-thawing sperm motility above 68 percent was observed when semen was frozen in 154mM NaCl-methylglycol, BTSÕ-methylglycol, 200mM NaCl-DMSO or Saad-DMSO. There was no difference on sperm motility when semen was frozen in 0.25 or 0.5ml straws and thawed in 50º or 60ºC water-bath. Thus, pirapitinga semen can be successfully cooled in BTSÕ for seven days or frozen in 154 mM NaCl-methylglycol, BTSÕ- methylglycol, 200mM NaCl-DMSO and Saad-DMSO

Diversity and genetic distance in populations of Steindachnerina in the upper Paraná river floodplain of Brazil.

Whereas four species of the genus Steindachnerina occur in the Paraná river basin, S. insculpta was the only endemic species of the region under analysis, which is the third lower section of the upper Paraná river. Among other factors, this species has been characterised by the absence of spots in the basal region of the dorsal fin. However, various specimens with this characteristic appeared in the region after the construction of the Itaipu Hydroelectric Plant in 1982. An analysis of the genetic variability of Steindachnerina populations with or without spots is provided. Specimens were collected in different sites of the floodplain of the upper Paraná river and samples were compared by random amplified polymorphic DNA (RAPD) technique and morphological analyses. Ninety-eight amplified loci with nine random primers were analysed in 19 specimens of each phenotype. Data for genetic distance showed great divergences between the two phenotypes and indicate two different species. Spotted specimens may be identified as S. brevipinna, found in the region downstream Sete Quedas Falls. The species must have overcome the geographical barrier during the building of the Itaipu hydroelectric dam that submerged the waterfalls and which became an obstacle between the upper and middle Paraná river some 150 km downstream. Since phenotypes do not share dominant alleles, absence of gene flow has been suggested.

Organic carbonate from Calotropis procera leaves.

The leaves of Calotropis procera yielded an organic carbonate (1), along with stigmasterol and beta-sitosterol, identified by spectroscopic methods.

Leishmanial antigens in the diagnosis of active lesions and ancient scars of American tegumentary leishmaniasis patients.

Cutaneous biopsies (n = 94) obtained from 88 patients with American tegumentary leishmaniasis were studied by conventional and immunohistochemical techniques. Specimens were distributed as active lesions of cutaneous leishmaniasis (n = 53) (Group I), cicatricial lesions of cutaneous leishmaniasis (n = 35) (Group II) and suggestive scars of healed mucosal leishmaniasis patients (n = 6) (Group III). In addition, active cutaneous lesions of other etiology (n = 24) (Group C1) and cutaneous scars not related to leishmaniasis (n = 10) (Group C2) were also included in the protocol. Amastigotes in Group I biopsies were detected by routine histopathological exam (30.2%), imprint (28.2%), culture (43.4%), immunofluorescence (41.4%) and immunoperoxidase (58.5%) techniques; and by the five methods together (79.3%). In Group II, 5.7% of cultures were positive. Leishmanial antigen was also seen in the cytoplasm of macrophages and giant cells (cellular pattern), vessel walls (vascular pattern) and dermal nerves (neural pattern). Positive reaction was detected in 49 (92.5%), 20 (57%) and 4 (67%) biopsies of Groups I, II and III, respectively. Antigen persistency in cicatricial tissue may be related to immunoprotection or, on the contrary, to the development of late lesions. We suggest that the cellular, vascular and neural patterns could be applied in the immunodiagnosis of active and cicatricial lesions in which leishmaniasis is suspected.

Cutaneous scars in American tegumentary leishmaniasis patients: a site of Leishmania (Viannia) braziliensis persistence and viability eleven years after antimonial therapy and clinical cure.

Two former patients treated for the cutaneous form of American tegumentary leishmaniasis were reviewed eight and 11 years, respectively, following clinical cure. We were able to isolate Leishmania parasites in a culture of material from the two scar biopsies, and in one of them the parasite was characterized as Leishmania (Viannia) braziliensis. In both cases, the histopathology revealed discreet hyperceratosis and a slight infiltrate of mononuclear cells surrounding and on the walls of the surface and deep dermal vessels. No amastigotes were seen on immunohistochemical or histopathologic examination. The Montenegro skin test result and the in vitro lymphoproliferative response to Leishmania antigen were positive, but no specific IgG and IgM antibodies were detected. Otorhinolaryngologic examination showed no macroscopic alteration in the mucosae. These findings are important for the evaluation and criteria of post-treatment cure.

Retrospective study on dengue fatal cases.

Immunohistochemical procedure (avidin biotin peroxidase complex) was applied in formalin-fixed and paraffin-embedded tissues obtained from 5 fatal cases of dengue infection associated with encephalopathy. Dengue virus antigen was demonstrated in the cytoplasm of phagocytic mononuclear cells from liver, spleen, and lung. Moreover, dengue viral antigens were here, to our knowledge, first demonstrated in the central nervous system (CNS) and numerous immunolabelled cells were found in brain sections from 3 cases. Extended immunohistochemical studies carried out in 1 case showed virus-positive cells mostly located within Virchow Robin space of medium size and small veins, infiltrating the white and grey matter, and often situated close to neurons displaying apparent cytopathic features. Furthermore, immunostaining for CD68 antigens demonstrated that most CD68+ macrophages and dengue antigen-positive cells share similar morphology and localization, suggesting a unique identity for at least part of these cells. Since in dengue fever, virus replicates mostly in cells of macrophage lineage, our results seem to indicate that infiltration of virus-infected macrophages could be one of the pathways by which viruses enter the brain in dengue encephalitis. Whether bone marrow-derived infected macrophages and viral-free particles induce CSN lesions through immune, metabolic, and/or direct viral-induced mechanisms will be essential to better understand the pathogenesis and provide new therapeutic strategies for dengue-associated encephalitis. As the evidence of tissue damage was nonspecific, the detection of virus antigen by immunoperoxidase technique appeared to be highly reliable for dengue diagnosis.