Rescuing Perishable Neuroanatomical Information from a Threatened Biodiversity Hotspot: Remote Field Methods for Brain Tissue Preservation Validated by Cytoarchitectonic Analysis, Immunohistochemistry, and X-Ray Microcomputed Tomography.

Biodiversity hotspots, which harbor more endemic species than elsewhere on Earth, are increasingly threatened. There is a need to accelerate collection efforts in these regions before threatened or endangered species become extinct. The diverse geographical, ecological, genetic, morphological, and behavioral data generated from the on-site collection of an individual specimen are useful for many scientific purposes. However, traditional methods for specimen preparation in the field do not permit researchers to retrieve neuroanatomical data, disregarding potentially useful data for increasing our understanding of brain diversity. These data have helped clarify brain evolution, deciphered relationships between structure and function, and revealed constraints and selective pressures that provide context about the evolution of complex behavior. Here, we report our field-testing of two commonly used laboratory-based techniques for brain preservation while on a collecting expedition in the Congo Basin and Albertine Rift, two poorly known regions associated with the Eastern Afromontane biodiversity hotspot. First, we found that transcardial perfusion fixation and long-term brain storage, conducted in remote field conditions with no access to cold storage laboratory equipment, had no observable impact on cytoarchitectural features of lizard brain tissue when compared to lizard brain tissue processed under laboratory conditions. Second, field-perfused brain tissue subjected to prolonged post-fixation remained readily compatible with subsequent immunohistochemical detection of neural antigens, with immunostaining that was comparable to that of laboratory-perfused brain tissue. Third, immersion-fixation of lizard brains, prepared under identical environmental conditions, was readily compatible with subsequent iodine-enhanced X-ray microcomputed tomography, which facilitated the non-destructive imaging of the intact brain within its skull. In summary, we have validated multiple approaches to preserving intact lizard brains in remote field conditions with limited access to supplies and a high degree of environmental exposure. This protocol should serve as a malleable framework for researchers attempting to rescue perishable and irreplaceable morphological and molecular data from regions of disappearing biodiversity. Our approach can be harnessed to extend the numbers of species being actively studied by the neuroscience community, by reducing some of the difficulty associated with acquiring brains of animal species that are not readily available in captivity.

Genetic Basis for Variation of Metalloproteinase-Associated Biochemical Activity in Venom of the Mojave Rattlesnake (Crotalus scutulatus scutulatus).

The metalloproteinase composition and biochemical profiles of rattlesnake venom can be highly variable among rattlesnakes of the same species. We have previously shown that the neurotoxic properties of the Mojave rattlesnake (Crotalus scutulatus scutulatus) are associated with the presence of the Mojave toxin A subunit suggesting the existence of a genetic basis for rattlesnake venom composition. In this report, we hypothesized the existence of a genetic basis for intraspecies variation in metalloproteinase-associated biochemical properties of rattlesnake venom of the Mojave rattlesnake. To address this question, we PCR-amplified and compared the genomic DNA nucleotide sequences that code for the mature metalloproteinase domain of fourteen Mojave rattlesnakes captured from different geographical locations across the southwest region of the United States. In addition, the venoms from the same rattlesnakes were tested for their ability to hydrolyze fibrinogen, fibrin, casein, and hide powder azure and for induction of hemorrhage in mice. Overall, based on genomic sequencing and biochemical data, we classified Mojave rattlesnake venom into four distinct groups of metalloproteinases. These findings indicate that differences in nucleotide sequences encoding the mature proteinase domain and noncoding regions contribute to differences in venom metalloproteinase activities among rattlesnakes of the same species.

Synonyms for some species of Mexican anoles (Squamata: Dactyloidae).

We studied type material and freshly collected topotypical specimens to assess the taxonomic status of five names associated with species of Mexican Anolis. We find A. schmidti to be a junior synonym of A. nebulosus, A. breedlovei to be a junior synonym of A. cuprinus, A. polyrhachis to be a junior synonym of A. rubiginosus, A. simmonsi to be a junior synonym of A. nebuloides, and A. adleri to be a junior synonym of A. liogaster.

Complement inactivating proteins and intraspecies venom variation in Crotalus oreganus helleri.

Complement inactivating properties were detected in venom from the southern California distribution of Crotalus oreganus helleri (Southern Pacific Rattlesnake). This activity showed strong geographic bias to the San Bernardino Mountain range, and venom from this area reacted strongly with Fraction 5 antiserum (AF5). However, venoms from the San Jacinto Mountain range, which have been previously shown to contain Mojave toxin, did not inhibit complement and did not react with AF5. AF5 has been previously shown to recognize a protease in C. scutulatus venom that inactivates complement, but the identity of this protein has remained unknown. Using a functional venomic approach, utilizing two-dimensional gel electrophoresis coupled with liquid chromatography and tandem mass spectrometry (LC/MS/MS), we have identified catrocollastatin and hemorrhagic toxin II (HT-2) as the primary proteins recognized by AF5. The information we present within this manuscript further illustrates the now well-known reality of intraspecies venom variation and the challenges faced in providing comprehensive polyvalent antivenoms.