Occurrence of Cryptosporidium (Apicomplexa,Cryptosporidiidae) in Crotalus durissus terrificus (Serpentes,Viperidae) in Brazil

The objective of the present study was to investigate the prevalence of Cryptosporidium (Apicomplexa, Cryptosporidiidae) in the snake Crotalus durissus terrificus (Serpentes, Viperidae). Fifty animals were evaluated for the presence of oocysts of Cryptosporidium sp. at the time of arrival and 30 and 60 days later. Intestinal washings with saline solution (1% body weight), fecal samples, and organ scrapings were collected during the study. Oocysts were concentrated by an ether-phosphate-buffered saline sedimentation technique and then separated by a density gradient centrifugation technique. Smears were made with the sediment and submitted to modified acid-fast and auramine-rhodamine staining. Cryptosporidium-positive smears were used as controls for the experimental findings. The overall prevalence of Cryptosporidium sp. oocysts was 14%. Among the positive snakes, oocysts were detected only in the intestinal washing in two specimens, only in the feces in four specimens, and in both materials at least once in one specimen. The positive snakes were predominantly from Santa Maria da Serra city State of S?o Paulo (57.1%). We also observed that all of the examinations that presented positive results were obtained at least 27 days after the capture of the animals.     

Sperm storage in males of the snake Crotalus durissus terrificus (Crotalinae: Viperidae) in southeastern Brazil

Seasonal variations in spermatozoa numbers and in sperm motility along the vas deferens in Crotalus durissus terrificus from southeastern Brazil were analyzed. Our data demonstrate storage and motility of the spermatozoa along the vas deferens throughout the year. This is characteristic of a postnuptial reproductive cycle, usually found in snakes living in temperate climates. We describe similarities in reproductive cycle patterns found in the tropical nonhibernator C. durissus terrificus and in hibernator snakes from temperate zones. Our results show that in C. durissus terrificus, a significant difference in spermatozoa counts occurs between winter and summer. Higher numbers of spermatozoa in summer and autumn, due to intense spermiogenesis, coincides with the mating season in autumn. These data indicate that after spermiogenesis in summer, the males combine the peak of sperm storage to the period females are attractive. Mating, however, is not linked to ovulation, and the sperm is stored in the females during winter until fertilization occurs in spring. In the males, after mating, spermatozoon counts low. In spring, they gradually increase, turning again the highest in summer and autumn. During spermiogenesis in the convoluted vas deferens, spermatozoa gain motility, enhancing their performance along their way towards the distal portion.     

The Evolutionary Implications of Hemipenial Morphology of Rattlesnake Crotalus durissus terrificus (Laurent, 1768) (Serpentes: Viperidae: Crotalinae)

Most amniotes vertebrates have an intromittent organ to deliver semen. The reptile Sphenodon and most birds lost the ancestral penis and developed a cloaca-cloaca mating. Known as hemipenises, the copulatory organ of Squamata shows unique features between the amniotes intromittent organ. They are the only paired intromittent organs across amniotes and are fully inverted and encapsulated in the tail when not in use. The histology and ultrastructure of the hemipenes of Crotalus durissus rattlesnake is described as the evolutionary implications of the main features discussed. The organization of hemipenis of Crotalus durissus terrificus in two concentric corpora cavernosa is similar to other Squamata but differ markedly from the organization of the penis found in crocodilians, testudinata, birds and mammals. Based on the available data, the penis of the ancestral amniotes was made of connective tissue and the incorporation of smooth muscle in the framework of the sinusoids occurred independently in mammals and Crotalus durissus. The propulsor action of the muscle retractor penis basalis was confirmed and therefore the named should be changed to musculus hemipenis propulsor.The retractor penis magnus found in Squamata has no homology to the retractor penis of mammals, although both are responsible for the retraction of the copulatory organ.     

The development of the pit organ of Bothrops jararaca and Crotalus durissus terrificus (Serpentes, Viperidae): support for the monophyly of the subfamily Crotalinae

The development of the pit organ and the course of the lateral branch of the ethmoidal nerve of the two South American crotalines Bothrops jararaca and Crotalus durissus terrificus are described in detail using serially sectioned material from fetal stages. Serial sections of the Asian pit vipers Agkistrodon halys halys and Trimeresurus albolabris were included in this work. The viperines, as the sister‐group of the Crotalinae, and the Colubridae, Elapidae and Boidae as their outgroups were used for comparisons. New synapomorphies of the advanced subfamily Crotalinae were found: namely, absence of the foramen epiphaniale and an extracapsular course of the lateral branch of ethmoidal nerve. These results support the monophyly of the crotalines.     

PCR primers for microsatellite loci in the tiger rattlesnake (Crotalus tigris,Viperidae)

The tiger rattlesnake (Crotalus tigris) is found only in the Sonoran Desert of North America, where it inhabits rocky foothills that are increasingly being affected by urban encroachment. In order to continue a long‐term study of the demography and movements of tiger rattlesnakes around Tucson, Arizona, we identified six novel polymorphic microsatellite loci for this species. All loci exhibited high variability (five to 41 alleles). These markers are useful for studies of paternity, population structure, and assessing appropriate destinations for translocated animals.     

Synthesis of ribonucleic acid in the venom gland of Crotalus durissus terrificus (Ophidia, Reptilia) after manual extraction of the venom

1. The incorporation of [5-(3)H]uridine into RNA of the venom gland of Crotalus durissus terrificus was studied after manual extraction (;milking') of the venom. The labelled precursor was injected immediately after milking. 2. The RNA was extracted 1, 2, 4, 6 and 8h after injection of the label and analysed by sucrose-density-gradient centrifugation. 3. The sedimentation analysis showed that 18S rRNA synthesis is higher than 28S rRNA at all time-intervals. The specific radioactivities of both ribosomal components did not reach a plateau even at 8h after injection. 4. An RNA fraction was detected sedimenting between 18S rRNA and 4S tRNA and was called the 10-14S fraction. The specific radioactivity was always higher than that of both classes of rRNA and reached the maximum value at 6h of labelling. 5. The incorporation of the precursor was also studied by radioautography, which helped to elucidate the intracellular origin of the RNA analysed by sucrose-density-gradient centrifugation.     

Intraspecific variation in the venoms of the South American rattlesnake (Crotalus durissus terrificus)

The venom of eight individual Crotalus durissus terrificus snakes from the State of Minas Gerais, Brazil, in addition to pooled venom from Butantan Institute, were compared. Snakes were captured in distinct locations, some of them 600 km apart: Conselheiro Lafaiete, Entre Rios de Minas, Itauna, Itapecerica, Lavras, Patos de Minas, Paracatu, and Santo Antonio do Amparo. The crude venoms were tested for proteolytic, phospholipase A2, platelet aggregating, and hemagglutinating activities. The venoms were also analyzed by polyacrylamide gel electrophoresis (PAGE) and isoelectric focusing (IEF). Chromatographic patterns of venom proteins on both gel-filtration and anion-exchange chromatographies were also performed. All venoms presented high phospholipase A2 and platelet-aggregating activities, but only minimal hemagglutinating or proteolytic activities were found. Gel-filtration chromatography showed a characteristic profile for most venoms where four main peaks were separated, including the typical ones where convulxin and crotoxin were identified; however, peaks with high amounts of lower molecular weight proteins were found in the venoms from the Santo Antonio do Amparo location and Butantan Institute, characterizing these venoms as crotamine positive. Anion-exchange chromatographies presented a similar protein distribution pattern, although the number of peaks (up to ten) distinguished some venom samples. Consistent with these results, polyacrylamide gels that were silver stained after venom separation by PAGE or IEF presented a similar qualitative band distribution, although a quantitative heterogeneity was detected among venoms. Our results suggest that the variability found in venom components of C. d. terrificus venoms captured in Minas Gerais State may be genetically inherited and/or environmentally induced.     

Chromatin supraorganization, DNA fragmentation, and cell death in erythrocytes of the rattlesnake, Crotalus durissus terrificus (Serpentes, Viperidae), infected with the protozoan, Hepatozoon spp. (Apicomplexa, Hepatozoidae)

Forms of the protozoan of the Hepatozoon genus are detected free in the circulation and also within some of the erythrocytes of infected snakes. In healthy snakes, DNA fragmentation and cell death usually affect a few circulating erythrocytes in agreement with the long life span expected for these cells. In the present study we investigated whether infection by Hepatozoon spp. affected the incidence of DNA fragmentation and cell death in erythrocytes from the rattlesnake, Crotalus durissus terrificus. Methods such as the kinetics of Feulgen-DNA hydrolysis, and the TUNEL and comet assays, previously used for the study of chromatin organization and DNA fragmentation in erythrocytes of healthy snakes, were used. The results indicated that Hepatozoon spp. increased the DNA fragmentation and chromatin condensation typical of cell death in circulating erythrocytes of C. d. terrificus, including cells that do not harbour the parasite. The Hepatozoon infection is thus suggested to accelerate destruction of erythrocytes in the rattlesnake, not only affecting cells harbouring the parasite, but also in those without it.     

Body Size Evolution in Insular Speckled Rattlesnakes (Viperidae: Crotalus mitchellii)

Background

Speckled rattlesnakes (Crotalus mitchellii) inhabit multiple islands off the coast of Baja California, Mexico. Two of the 14 known insular populations have been recognized as subspecies based primarily on body size divergence from putative mainland ancestral populations; however, a survey of body size variation from other islands occupied by these snakes has not been previously reported. We examined body size variation between island and mainland speckled rattlesnakes, and the relationship between body size and various island physical variables among 12 island populations. We also examined relative head size among giant, dwarfed, and mainland speckled rattlesnakes to determine whether allometric differences conformed to predictions of gape size (and indirectly body size) evolving in response to shifts in prey size.

Methodology/Principal Findings

Insular speckled rattlesnakes show considerable variation in body size when compared to mainland source subspecies. In addition to previously known instances of gigantism on Ángel de la Guarda and dwarfism on El Muerto, various degrees of body size decrease have occurred frequently in this taxon, with dwarfed rattlesnakes occurring mostly on small, recently isolated, land-bridge islands. Regression models using the Akaike information criterion (AIC) showed that mean SVL of insular populations was most strongly correlated with island area, suggesting the influence of selection for different body size optima for islands of different size. Allometric differences in head size of giant and dwarf rattlesnakes revealed patterns consistent with shifts to larger and smaller prey, respectively.

Conclusions/Significance

Our data provide the first example of a clear relationship between body size and island area in a squamate reptile species; among vertebrates this pattern has been previously documented in few insular mammals. This finding suggests that selection for body size is influenced by changes in community dynamics that are related to graded differences in area over what are otherwise similar bioclimatic conditions. We hypothesize that in this system shifts to larger prey, episodic saturation and depression of primary prey density, and predator release may have led to insular gigantism, and that shifts to smaller prey and increased reproductive efficiency in the presence of intense intraspecific competition may have led to insular dwarfism.     

Thermolability of 28S ribosomal ribonucleic acid from the liver of Crotalus durissus terrificus (Ophidia, Reptilia)

Instability of 28S rRNA of Crotalus durissus terrificus liver was observed during hotphenol extraction: purified 28S rRNA is converted into an 18S RNA component by heat treatment. It was also found that ;6S' and ;8S' low-molecular-weight RNA species were released during the thermal conversion. This conversion and the release of the low-molecular-weight species were also induced by 8m-urea and 80% (v/v) dimethyl sulphoxide at 0 degrees C. Evidence is presented that this phenomenon is an irreversible process and results from the rupture of hydrogen bonds. The 18S RNA product was shown to be homogeneous by polyacrylamide-gel electrophoresis and by sucrose-density-gradient centrifugation. The base composition of the 18S RNA products obtained by heat, urea or dimethyl sulphoxide treatments was similar. The C+G content of the 18S RNA product was different from that of the native 18S rRNA, but similar to that of 28S rRNA.     

Morphological and Histochemical Study of Cephalic Glands of Bothrops jararaca (Ophidia,Viperidae)

Morphological and histochemical studies of the cell types in the cephalic glands of Bothrops jararaca have been performed. It is concluded: 1) mucous cells are found in the salivary labial, accessory glands; mucous-serous cells are found in the salivary labial, accessory and Harderian glands; serous-mucous cells are found only in the venom gland; 2) neutral mucosubstances and protein were found in the salivary labial, venom, accessory and Harderian glands; 3) hyaluronic acid was detected in the Harderian gland; 4) of the to sulfated acid mucosubstances, only chondroitin sulfate B was detected in the salivary labial and accessory glands; 5) sialic acid was detected in the salivary labial, accessory and Harderian glands.     

Semen collection and evaluation in free‐ranging Brazilian rattlesnakes (Crotalus durissus terrificus)

Two hundred‐ninety species of reptiles are estimated to need urgent action for conservation, with at least 113 threatened species worldwide. The International Union for Conservation of Nature and Natural Resources (IUCN) Red List of Threatened Species includes 80 species of snakes, with six native Brazilian species, a number likely to be an underestimation. Some authors believe that assisted reproduction would be an important tool to improve reproduction in captivity of some reptiles. An efficient technique for semen collection and evaluation is an important step in development of protocols for cryopreservation of semen or artificial insemination in snakes, contributing to the conservation of endangered species. Although these techniques are important, some basic semen parameters are described for four of the ～2,900 snake species in the world. The Brazilian rattlesnake (Crotalus durissus terrificus) was chosen as a model for semen collection in snakes because it is found quite often in Sao Paulo State. Semen was collected once from each animal by the same investigator during the mating season of this species in Brazil. After antiseptic cleansing of the skin around the cloaca, the snakes were injected subcutaneously with a dose of 15 mg/kg of 1% solution of lidocaine around the cloaca. Semen was collected with ventral massages after cloacal relaxation and directly from genital papilla inside the cloaca. A total of 28 ejaculates from 39 animals were obtained, representing collection efficiency of 71.80%. Semen volume and concentration in Brazilian rattlesnakes ranged from 3–70 µl and from 0.94–2.23 × 10⁹ spermatozoa/ml, respectively. Zoo Biol 0:1–6, 2007. © 2007 Wiley‐Liss, Inc.     

Comparison of the biodistribution of free or liposome-entrapped Crotalus durissus terrificus (South American rattlesnake) venom in mice

The local absorption rate, clearance and tissue distribution of Crotalus durissus terrificus venom, (Cdt) were examined using a two-antibody sandwich ELISA assay. We compared the biodistribution of both free or encapsulated Cdt in mice. Following subcutaneous injection of 10 microg/mouse of free Cdt (0.8 LD50), venom was detected in serum after 15 min, showed its highest level at 30 min (45+/-5 ng/ml) and was cleared from the circulation after 6 h. After 2 h of inoculation, venom was detected in the kidney (57+/-9 ng/g of tissue), spleen (18+/-4 ng/g of tissue) and brain (14+/-6 ng/g of tissue). For both subcutaneous or intravenous injection of free Cdt, venom was firstly detected in the kidney. No Cdt appeared either in the kidney, spleen, brain, or other tissues after subcutaneous inoculation of encapsulated venom even though a higher dose was used, 25 microg/mouse (2 LD50). Venom remained at the site of injection for a period of 1 week. Following intravenous injection of encapsulated venom (5 microg/mouse, 2 LD50), venom was detected in liver and spleen tissues. The biodistribution of encapsulated venom is discussed in relation to the effects of reduction of toxicity and increase of adjuvanticity.     

Tracing an invasion: landbridges, refugia, and the phylogeography of the Neotropical rattlesnake (Serpentes: Viperidae: Crotalus durissus)

Abstract Pleistocene fragmentation of the Amazonian rainforest has been hypothesized to be a major cause of Neotropical speciation and diversity. However, the role and even the reality of Pleistocene forest refugia have attracted much scepticism. In Amazonia, previous phylogeographical studies have focused mostly on organisms found in the forests themselves, and generally found speciation events to have predated the Pleistocene. However, molecular studies of open-formation taxa found both north and south of the Amazonian forests, probably because of vicariance resulting from expansion of the rainforests, may provide novel insights into the age of continuous forest cover across the Amazon basin. Here, we analyse three mitochondrial genes to infer the phylogeography of one such trans-Amazonian vicariant, the Neotropical rattlesnake (Crotalus durissus), which occupies primarily seasonal formations from Mexico to Argentina, but avoids the rainforests of Central and tropical South America. The phylogeographical pattern is consistent with gradual dispersal along the Central American Isthmus, followed by more rapid dispersal into and across South America after the uplift of the Isthmus of Panama. Low sequence divergence between populations from north and south of the Amazon rainforest is consistent with mid-Pleistocene divergence, approximately 1.1 million years ago (Ma). This suggests that the Amazonian rainforests must have become fragmented or at least shrunk considerably during that period, lending support to the Pleistocene refugia theory as an important cause of distribution patterns, if not necessarily speciation, in Amazonian forest organisms. These results highlight the potential of nonforest species to contribute to an understanding of the history of the Amazonian rainforests themselves.     

Crotalus durissus terrificus venom interferes with morphological, functional, and biochemical changes in murine macrophage

Crotalus durissus terrificus venom (Cdt) is toxic for a variety of eukaryotic cells, especially at high concentrations. However its effects on host immune cells are not well known. The purpose of this study was to determine the effect of Cdt on functional status and the mediators production in peritoneal macrophages. The effects of Cdt were analyzed in vitro and were detected using functional status of macrophages as determined by the H(2)O(2) release, spreading percentage, phagocytic index, vacuole formation, and mediators production. Several functional bioassays were employed: cytotoxicity was determined by taking the lyses percentage and the presence of hydrogen peroxide (H(2)O(2)) in macrophages, using the horseradish peroxidase-dependent oxidation of phenol red and nitric oxide (NO) in the supernatants of macrophages by the Griess reaction. The tumor necrosis factor (TNF) activity was detected by measuring its cytotoxic activity on L929 cells, and the production the level of other cytokines was assayed using enzyme-linked immunosorbent assay. In vitro studies revealed that Cdt produced (a) a discrete increase in the release of H(2)O(2) and vacuole formation; (b) a decrease in spreading percentage and in the phagocytic index; and (c) an increment in the mediators production. More pronounced increments of IL-6 and TNF were observed after 24 and 48 hours, respectively. Maximum levels of IFN-gamma and NO were observed after 96 hours. Interestingly, levels of all mediators presented a discreet decrease, as the amount of Cdt was increased. In contrast, the IL-10 levels observed for all doses studied here did not alter. The IL-6/IL-10 ratio may possibly reflect the balance of pro- and anti-inflammatory cytokines in macrophages, which may be manifested in the inflammatory status during the envenoming processes. Taken together, these data indicate that Cdt have a differential effect on macrophage activation and that this venom is a potent inhibitor of anti-inflammatory response.