Forecasting seasonal peaks in roadkill patterns for improving road management.

For several species, roadkill is not spatially aggregated on hotspots, having instead a more diffuse pattern along the roads. For such species, management measures such as road passages may be insufficient for effective mitigation, since a large part of the road crossings is likely to occur outside the influence of those structures. One complementary approach could be to implement temporary mitigation actions, such as traffic calming. This requires understanding when roadkill peaks may occur. We tested the feasibility of predicting seasonal peaks of roadkill using data from a 3-year systematic monitoring (78 surveys over ca. 960 km of roads) from eight non-flying vertebrate species from Mato Grosso do Sul, Brazil, with different body size and life history traits (ca. 6400 records from focal species). We modelled the time-series of the roadkill of these species at large scale (state level) using generalized additive mixed models (GAMMs). We used the data of the first 2 years as training datasets, and the information from the third year of surveys as testing datasets to evaluate the prediction performance of models. Overall, the models of species feed with a higher number of records were able to follow reasonably well the variations of roadkill over time, although they were not able to correctly predict the number of collisions. For species with fewer observations, the models presented a poorer goodness-of-fit and prediction ability. Our results suggest that, at least for those species with higher roadkill rates, it can be possible to forecast periods of higher probability of occurring hot-moments of mortality. Such models can provide valuable information to implement seasonal management actions.

Neutralising antibodies for Mayaro virus in Pantanal, Brazil.

The Pantanal hosts diverse wildlife species and therefore is a hotspot for arbovirus studies in South America. A serosurvey for Mayaro virus (MAYV), eastern (EEEV), western (WEEV) and Venezuelan (VEEV) equine encephalitis viruses was conducted with 237 sheep, 87 free-ranging caimans and 748 equids, including 37 collected from a ranch where a neurologic disorder outbreak had been recently reported. Sera were tested for specific viral antibodies using plaque-reduction neutralisation test. From a total of 748 equids, of which 264 were immunised with vaccine composed of EEEV and WEEV and 484 had no history of immunisation, 10 (1.3%) were seropositive for MAYV and two (0.3%) for VEEV using criteria of a ≥ 4-fold antibody titre difference. Among the 484 equids without history of immunisation, 48 (9.9%) were seropositive for EEEV and four (0.8%) for WEEV using the same criteria. Among the sheep, five were sero- positive for equine encephalitis alphaviruses, with one (0.4%) for EEEV, one (0.4%) for WEEV and three (1.3%) for VEEV. Regarding free-ranging caimans, one (1.1%) and three (3.4%), respectively, had low titres for neutralising antibodies to VEEV and undetermined alphaviruses. The neurological disorder outbreak could not be linked to the alphaviruses tested. Our findings represent strong evidence that MAYV and all equine encephalitis alphaviruses circulated in the Pantanal.

Thermal relations of dwarf caiman, Paleosuchus palpebrosus, in a hillside stream: evidence for an unusual thermal niche among crocodilians.

Body temperatures of 13 Paleosuchus palpebrosus, 7 males and 6 females, were monitored by radio-telemetry during cold periods (dry season) and warm periods (wet and dry seasons) in a stream draining into the Brazilian Pantanal. The mass of the caimans varied from 2.5 to 20.0kg, and snout-vent length from 47.5 to 95.0cm. Mean monthly body temperature was 21.6°C, and varied from 20.1 to 25.6°C throughout the year. Body temperature was correlated with air and water temperature but did not differ between males and females. Unlike all other crocodilians investigated in detail to date, the caimans did not show evidence of attempts to obtain higher body temperatures when ambient temperatures were low, and had low and generally constant temperatures in relation to the surrounding air and water throughout the year. The caimans remained in burrows during cold periods in the dry season, which may explain why they did not seek higher temperatures. Tolerance of relatively low and constant body temperatures may be a key adaptation of species of Paleosuchus, allowing them to occupy environments inhospitable to other crocodilians.

Spatial release from masking in crocodilians.

Ambient noise is a major constraint on acoustic communication in both animals and humans. One mechanism to overcome this problem is Spatial Release from Masking (SRM), the ability to distinguish a target sound signal from masking noise when both sources are spatially separated. SRM is well described in humans but has been poorly explored in animals. Although laboratory tests with trained individuals have suggested that SRM may be a widespread ability in vertebrates, it may play a limited role in natural environments. Here we combine field experiments with investigations in captivity to test whether crocodilians experience SRM. We show that 2 species of crocodilians are able to use SRM in their natural habitat and that it quickly becomes effective for small angles between the target signal source and the noise source, becoming maximal when the angle exceeds 15∘. Crocodiles can therefore take advantage of SRM to improve sound scene analysis and the detection of biologically relevant signals.

Neutralising antibodies for West Nile virus in horses from Brazilian Pantanal.

Despite evidence of West Nile virus (WNV) activity in Colombia, Venezuela and Argentina, this virus has not been reported in most South American countries. In February 2009, we commenced an investigation for WNV in mosquitoes, horses and caimans from the Pantanal, Central-West Brazil. The sera of 168 horses and 30 caimans were initially tested using a flaviviruses-specific epitope-blocking enzyme-linked immunosorbent assay (blocking ELISA) for the detection of flavivirus-reactive antibodies. The seropositive samples were further tested using a plaque-reduction neutralisation test (PRNT90) for WNV and its most closely-related flaviviruses that circulate in Brazil to confirm the detection of specific virus-neutralising antibodies. Of the 93 (55.4%) blocking ELISA-seropositive horse serum samples, five (3%) were seropositive for WNV, nine (5.4%) were seropositive for St. Louis encephalitis virus, 18 (10.7%) were seropositive for Ilheus virus, three (1.8%) were seropositive for Cacipacore virus and none were seropositive for Rocio virus using PRNT90, with a criteria of ≥ four-fold antibody titre difference. All caimans were negative for flaviviruses-specific antibodies using the blocking ELISA. No virus genome was detected from caiman blood or mosquito samples. The present study is the first report of confirmed serological evidence of WNV activity in Brazil.

Evidence of cryptic lineages within a small South American crocodilian: the Schneider's dwarf caiman Paleosuchus trigonatus (Alligatoridae: Caimaninae).

Schneider's dwarf caiman Paleosuchus trigonatus is one of the smallest living crocodilians. Due to its broad distribution, cryptic behavior, and small home range, the species is well suited for the study of phylogeographic patterns on a continental scale. Additionally, this species is under threat due to habitat loss, trade and harvest, but is considered at low conservation risk by the IUCN. In the present study we test the hypothesis that P. trigonatus is comprised of geographically structured lineages. Phylogenetic reconstructions of the mitochondrial cytochrome b gene and single locus species discovery methods revealed the existence of two well-supported lineages within P. trigonatus-an Amazonian and Guianan lineage. Fossil calibrated divergence of these lineages was estimated to have occurred in the Late Miocene (7.5 Ma). The hypothesis that the Atlantic coast drainages might have been colonized from the southeast or central Amazon is supported by demographic metrics and relatively low genetic diversity of the Coastal and upper Branco populations when compared to the Amazon basin populations. The Amazon basin lineage is structured along an east-west gradient, with a sharp transition in haplotype frequencies to the east and west of the Negro and Madeira rivers. These lineages are already under anthropogenic threat and, therefore, are conservation dependent. Recognition of these lineages will foster discussion of conservation future of P. trigonatus and these lineages.

The effect of dam construction on the movement of dwarf caimans, Paleosuchus trigonatus and Paleosuchus palpebrosus, in Brazilian Amazonia.

Run-of-the-river hydroelectric dams cause changes in seasonal inundation of the floodplains, and this may cause displacement of semi-aquatic vertebrates present before dam construction. This study evaluated the movement of crocodilians before and after the filling of the Santo Antônio hydroelectric reservoir on the Madeira River in the Brazilian Amazon, which occurred in November 2011. We radio-tracked four adult male Paleosuchus palpebrosus and four adult male Paleosuchus trigonatus before and after the formation of the reservoir between 2011 and 2013. The home ranges of the P. palpebrosus varied from < 1 km2 to 91 km2 and the home ranges of the P. trigonatus varied from < 1km2 to 5 km2. The species responded differently to time since filling and water level in weekly movement and home range. However, overall the dam appears to have had little effect on the use of space by the individuals that were present before dam construction.

Neutralizing antibodies for orthobunyaviruses in Pantanal, Brazil.

The Pantanal is a hotspot for arbovirus studies in South America. Various medically important flaviviruses and alphaviruses have been reported in domestic and wild animals in the region. To expand the knowledge of local arbovirus circulation, a serosurvey for 14 Brazilian orthobunyaviruses was conducted with equines, sheep and free-ranging caimans. Sera were tested for specific viral antibodies using plaque-reduction neutralization test (PRNT). Monotypic reactions were detected for Maguari, Xingu, Apeu, Guaroa, Murutucu, Oriboca, Oropouche and Nepuyo viruses. Despite the low titers for most of the orthobunyaviruses tested, the detection of monotypic reactions for eight orthobunyaviruses suggests the Pantanal as a region of great orthobunyavirus diversity. The present data, in conjunction with previous studies that detected a high diversity of other arboviruses, ratify the Pantanal as an important natural reservoir for sylvatic and medically important arboviruses in Brazil.

Density and Biomass Estimates by Removal for an Amazonian Crocodilian, Paleosuchus palpebrosus.

Direct counts of crocodilians are rarely feasible and it is difficult to meet the assumptions of mark-recapture methods for most species in most habitats. Catch-out experiments are also usually not logistically or morally justifiable because it would be necessary to destroy the habitat in order to be confident that most individuals had been captured. We took advantage of the draining and filling of a large area of flooded forest during the building of the Santo Antônio dam on the Madeira River to obtain accurate estimates of the density and biomass of Paleosuchus palpebrosus. The density, 28.4 non-hatchling individuals per km2, is one of the highest reported for any crocodilian, except for species that are temporarily concentrated in small areas during dry-season drought. The biomass estimate of 63.15 kg*km-2 is higher than that for most or even all mammalian carnivores in tropical forest. P. palpebrosus may be one of the World´s most abundant crocodilians.

Rickettsial infection in ticks (Acari: Ixodidae) of wild animals in midwestern Brazil.

Ticks collected in the last two decades from free-living and captive wild animals from 28 municipalities of the Brazilian state of Mato Grosso were identified and tested using molecular methods for the presence of rickettsial agents. A total of 4467 ticks (229 larvae, 1676 nymphs, 1565 males, 997 females) representing 27 ixodid species were collected from 235 species of amphibians, reptiles, birds, and mammals from three different ecoregions (Pantanal, Cerrado, and Amazonia). The species Amblyomma parkeri, Amblyomma romitii, Amblyomma varium and Ixodes luciae are reported for the first time in the state of Mato Grosso. Amongst 538 ticks tested by molecular methods for rickettsial infection, we detected 'Candidatus Rickettsia amblyommii' infecting Amblyomma cajennense sensu stricto and Amblyomma coelebs, Rickettsia sp. strain Atlantic rainforest infecting Amblyomma ovale, Rickettsia sp. strain NOD infecting Amblyomma nodosum, and 'Candidatus Rickettsia andeanae' infecting Amblyomma sculptum. Our results represent an impressive expansion of knowledge on tick fauna and rickettsiae and are essential for understanding the ecology of ticks and tick-borne diseases in the Neotropical region, particularly in midwestern Brazil.

Spatial and Temporal Variation in Reproduction of a Generalist Crocodilian, Caiman crocodilus yacare, in a Seasonally Flooded Wetland.

We monitored the number of caiman (Caiman crocodilus yacare) nests in two ranches in the Brazilian Pantanal that cover an area of about 50.000 ha for 28 years (1987-2014). The number of nests was related to combinations of rainfall, water level, and number of days with temperature below 20°C, depending on the area. Most of the variation in number of nests could not be predicted by the environmental variables, but could be represented mathematically by a sine wave. We were not able to identify any external driver and suspect that the regular fluctuations may have resulted from an intrinsic population process. Presently, ranches are used as management units under the legislation for ranching Pantanal caimans. However, although some breeding females were recaptured in the area after periods of up to 21 years, most were not recaptured near nests or in general surveys of the area, suggesting that females are not strongly philopatric and that ranches do not represent isolated demographic units.

Growth of Caiman crocodilus yacare in the Brazilian Pantanal.

We studied growth of the caiman, Caiman crocodilus yacare, in the Brazilian Pantanal for 27 years between 1987 and 2013.We recaptured 647 of 7769 C. c. yacare initially marked in an area of 50 km(2), in two ranches. We were able to determine size at age accurately for 24 male and17 female caimans that had been marked at hatching or less than 1 year old, and recaptured over periods of 5 to 24 years. The other 606 caimans were used to evaluate short-term growth rates. Age-size relationships were estimated using growth models from the Richards family of curves (full model, von Bertalanffy and monomolecular). The form of the relationships differed between analyses based on caimans of known age and analyses based on integration of growth rate on size relationships for caimans whose ages were not known. Individuals showed large variation in short-term growth rates, but data on known-age animals indicated little between-individual variability in long-term growth rates. There was evidence of a small effect of rainfall, but not temperature, on short-term growth of small caimans, but most variation in growth rates was unexplained by variables other than age and sex. Data on known-age individuals indicated that female C. c. yacare generally reach sexual maturity between 10 and 15 years of age. Because of the asymptotic relationship between age and size, deviations of observations from the model for age are larger than for size, and estimates of age at a given size have greater errors than estimates of size at a given age. Integration of growth rate on size relationships may be adequate for estimating size from age in many cases, but accurate estimates of age from size require data on known-age individuals over the size range of the species.

Serological evidence of widespread circulation of West Nile virus and other flaviviruses in equines of the Pantanal, Brazil.

A recent study reported neutralizing antibodies to West Nile virus (WNV) in horses from four ranches of southern Pantanal. To extend that study, a serosurvey for WNV and 11 Brazilian flaviviruses was conducted with 760 equines, 238 sheep and 61 caimans from 17 local cattle ranches. Among the tested equines, 32 were collected from a ranch where a neurologic disorder outbreak had been recently reported. The sera were initially screened by using a blocking ELISA and then titrated by 90% plaque-reduction neutralization test (PRNT90) for 12 flaviviruses. Employing the criterion of 4-fold greater titer, 78 (10.3%) equines were seropositive for Ilheus virus, 59 (7.8%) for Saint Louis encephalitis virus, 24 (3.2%) for WNV, two (0.3%) for Cacipacore virus and one (0.1%) for Rocio virus. No serological evidence was found linking the neurological disease that affected local equines to WNV. All caimans and sheep were negative by blocking ELISA for flaviviruses. There were no seropositive equines for Bussuquara, Iguape, Yellow fever and all four Dengue virus serotypes. The detection of WNV-seropositive equines in ten ranches and ILHV and SLEV-seropositive equines in fourteen ranches of two different sub-regions of Pantanal is strong evidence of widespread circulation of these flaviviruses in the region.

Ilheus virus isolation in the Pantanal, west-central Brazil.

The wetlands of the Brazilian Pantanal host large concentrations of diverse wildlife species and hematophagous arthropods, conditions that favor the circulation of zoonotic arboviruses. A recent study from the Nhecolândia sub-region of Pantanal reported serological evidence of various flaviviruses, including West Nile virus and Ilheus virus (ILHV). According to the age of seropositive horses, at least three flaviviruses, including ILHV, circulated in the Brazilian Pantanal between 2005 and 2009. To extend this study, we collected 3,234 adult mosquitoes of 16 species during 2009 and 2010 in the same sub-region. Mosquito pool homogenates were assayed for infectious virus on C6/36 and Vero cell monolayers and also tested for flaviviral RNA by a group-specific real-time RT-PCR. One pool containing 50 non-engorged female specimens of Aedes scapularis tested positive for ILHV by culture and for ILHV RNA by real-time RT-PCR, indicating a minimum infection rate of 2.5 per 1000. Full-length genomic sequence exhibited 95% identity to the only full genome sequence available for ILHV. The present data confirm the circulation of ILHV in the Brazilian Pantanal.

Neutralising antibodies for Mayaro virus in Pantanal, Brazil

The Pantanal hosts diverse wildlife species and therefore is a hotspot for arbovirus studies in South America. A serosurvey for Mayaro virus (MAYV), eastern (EEEV), western (WEEV) and Venezuelan (VEEV) equine encephalitis viruses was conducted with 237 sheep, 87 free-ranging caimans and 748 equids, including 37 collected from a ranch where a neurologic disorder outbreak had been recently reported. Sera were tested for specific viral antibodies using plaque-reduction neutralisation test. From a total of 748 equids, of which 264 were immunised with vaccine composed of EEEV and WEEV and 484 had no history of immunisation, 10 (1.3%) were seropositive for MAYV and two (0.3%) for VEEV using criteria of a ≥ 4-fold antibody titre difference. Among the 484 equids without history of immunisation, 48 (9.9%) were seropositive for EEEV and four (0.8%) for WEEV using the same criteria. Among the sheep, five were sero- positive for equine encephalitis alphaviruses, with one (0.4%) for EEEV, one (0.4%) for WEEV and three (1.3%) for VEEV. Regarding free-ranging caimans, one (1.1%) and three (3.4%), respectively, had low titres for neutralising antibodies to VEEV and undetermined alphaviruses. The neurological disorder outbreak could not be linked to the alphaviruses tested. Our findings represent strong evidence that MAYV and all equine encephalitis alphaviruses circulated in the Pantanal.

Neutralising antibodies for West Nile virus in horses from Brazilian Pantanal

Despite evidence of West Nile virus (WNV) activity in Colombia, Venezuela and Argentina, this virus has not been reported in most South American countries. In February 2009, we commenced an investigation for WNV in mosquitoes, horses and caimans from the Pantanal, Central-West Brazil. The sera of 168 horses and 30 caimans were initially tested using a flaviviruses-specific epitope-blocking enzyme-linked immunosorbent assay (blocking ELISA) for the detection of flavivirus-reactive antibodies. The seropositive samples were further tested using a plaque-reduction neutralisation test (PRNT90) for WNV and its most closely-related flaviviruses that circulate in Brazil to confirm the detection of specific virus-neutralising antibodies. Of the 93 (55.4 percent) blocking ELISA-seropositive horse serum samples, five (3 percent) were seropositive for WNV, nine (5.4 percent) were seropositive for St. Louis encephalitis virus, 18 (10.7 percent) were seropositive for Ilheus virus, three (1.8 percent) were seropositive for Cacipacore virus and none were seropositive for Rocio virus using PRNT90, with a criteria of > four-fold antibody titre difference. All caimans were negative for flaviviruses-specific antibodies using the blocking ELISA. No virus genome was detected from caiman blood or mosquito samples. The present study is the first report of confirmed serological evidence of WNV activity in Brazil.