External nasal gland morphology of Eurycea bislineata (Amphibia, Urodela, Plethodontidae).

Plethodontid salamanders possess numerous courtship glands. Previous studies have shown that the glands are more prominent in male individuals than females, and often experience periods of atrophy and hypertrophy throughout the year that correlate to the nonmating and mating seasons, respectively. We sampled male and female Eurycea bislineata throughout the year to test the hypothesis that external nasal glands are courtship glands. External nasal glands are paired, branched tubular glands that extend from excretory ducts dorsal to the nares to terminal secretory units posterior to the eyes. We found that the glands hypertrophy and stain/react more intensely with histochemical procedures during the mating season. Hypertrophy of the glands is more pronounced in males, and seasonal variation in epithelial height of external nasal glands of males is significantly correlated to that of seasonal variation in mental gland epithelial height, a known courtship gland found in males, when compared throughout the year. This correlation was not as strong in females, confirming sexual dimorphism of external nasal glands in terms of seasonal variation. We found no ultrastructural differences between male and female external nasal glands. In all specimens, the glandular tubules were lined by a simple, columnar epithelium that was packed with secretory granules that often obscured other cytoplasmic contents.

Predicting antibacterial activity from snake venom proteomes.

The continued evolution of antibiotic resistance has increased the urgency for new antibiotic development, leading to exploration of non-traditional sources. In particular, snake venom has garnered attention for its potent antibacterial properties. Numerous studies describing snake venom proteomic composition as well as antibiotic efficacy have created an opportunity to synthesize relationships between venom proteomes and their antibacterial properties. Using literature reported values from peer-reviewed studies, our study generated models to predict efficacy given venom protein family composition, snake taxonomic family, bacterial Gram stain, bacterial morphology, and bacterial respiration strategy. We then applied our predictive models to untested snake species with known venom proteomic compositions. Overall, our results provide potential protein families that serve as accurate predictors of efficacy as well as promising organisms in terms of antibacterial properties of venom. The results from this study suggest potential future research trajectories for antibacterial properties in snake venom by offering hypotheses for a variety of taxa.

Analysis of snake venom composition and antimicrobial activity.

With the threat of a post-antibiotic era looming, the search for new and effective antibiotics from novel sources is imperative. Not only has crude snake venom been shown to be effective, but specific components within the venoms, such as Phospholipase A2s and l-amino acid oxidases have been isolated and demonstrated to be effective as well. Despite numerous studies being completed on snake venoms, there is a heavy bias towards utilizing the venoms from the highly toxic Elapidae and Viperidae species. Very few studies have been conducted on the less toxic, but taxonomically more diverse, Colubridae. Furthermore, an extensive review of the literature examining the efficacy and potential specificity of these venoms has not been completed. Therefore, the aims of this study were to elucidate any similarities in snake venoms as well as investigate the efficacy of snake venom antimicrobial properties towards morphologically and metabolically diverse microbial classes and the prevalence of snake species with antimicrobial properties within each snake family. The results indicate that snake venoms and their isolated components are powerful antimicrobial agents but vary in efficacy towards different microbial classes. Furthermore, due to similarities in venom composition, and limited preliminary studies, the less toxic Colubridae family may be a fruitful area of research to find novel antimicrobial agents that are less harmful to humans.

Histology of the Urogenital System in the American Bullfrog (Rana catesbeiana), with Emphasis on Male Reproductive Morphology.

Previous studies have revealed variations in the urogenital system morphology of amphibians. Recently, the urogenital system of salamanders was reviewed and terminology was synonymized across taxa. Discrepancies exist in the terminology describing the urogenital system of anurans, which prompted our group to develop a complete, detailed description of the urogenital system in an anuran species and provide nomenclature that is synonymous with those of other amphibian taxa. In Rana catesbeiana, sperm mature within spermatocysts of the seminiferous tubule epithelia and are transported to a series of intratesticular ducts that exit the testes and merge to form vasa efferentia. Vasa efferentia converge into single longitudinal ducts (Bidder's ducts) on the lateral aspects of the kidneys. Branches from the longitudinal ducts merge with genital kidney renal tubules through renal corpuscles. The nephrons travel caudally and empty into the Wöffian ducts. Similar to salamanders, the caudal portion of the kidneys (termed the pelvic kidneys in salamanders) only possesses nephrons involved in urine formation, not sperm transport. Data from the present study provide a detailed description and synonymous nomenclature that can be used to make future comparative analyses between taxa more efficient.

Methyltestosterone alters sex determination in the American alligator (Alligator mississippiensis).

Effects of xenobiotics can be organizational, permanently affecting anatomy during embryonic development, and/or activational, influencing transitory actions during adulthood. The organizational influence of endocrine-disrupting contaminants (EDC's) produces a wide variety of reproductive abnormalities among vertebrates that exhibit temperature-dependent sex determination (TSD). Typically, such influences result in subsequent activational malfunction, some of which are beneficial in aquaculture. For example, 17-αmethyltestosterone (MT), a synthetic androgen, is utilized in tilapia farming to bias sex ratio towards males because they are more profitable. A heavily male-biased hatchling sex ratio is reported from a crocodile population near one such tilapia operation in Guanacaste, Costa Rica. In this study we test the effects of MT on sexual differentiation in American alligators, which we used as a surrogate for all crocodilians. Experimentally, alligators were exposed to MT in ovo at standard ecotoxicological concentrations. Sexual differentiation was determined by examination of primary and secondary sex organs post hatching. We find that MT is capable of producing male embryos at temperatures known to produce females and demonstrate a dose-dependent gradient of masculinization. Embryonic exposure to MT results in hermaphroditic primary sex organs, delayed renal development and masculinization of the clitero-penis (CTP).

Ultrastructural analysis of spermiogenesis in the Eastern Fence Lizard, Sceloporus undulatus (Squamata: Phrynosomatidae).

Studies on reptilian sperm morphology have shown that variation exists at various taxonomic levels but studies on the ontogeny of variation are rare. Sperm development follows a generalized bauplan that includes acrosome development, nuclear condensation and elongation, and flagellar development. However, minute differences can be observed such as the presence/absence of manchette microtubules, structural organization during nuclear condensation, and presence/absence of a nuclear lacuna. The purpose of this investigation was to examine sperm development within the Sceloporus genus. The process begins with the development of an acrosomal complex from Golgi vesicles followed by nuclear condensation and elongation, which results in the presence of a nuclear lacuna. As the acrosomal complex differentiates, flagellar development commences with elongation of the distal centriole. Spermatid development culminates in a mature spermatid with a highly differentiated acrosomal complex, a condensed nucleus with a nuclear lacuna, and a differentiated flagellum. Although the overall developmental pattern is consistent with other squamate species, minute differences are observed, even within the same genus. For example there is variation in the presence/absence of an endoplasmic reticulum complex during acrosome development, presence/absence of a nuclear lacuna, and presence/absence of manchette microtubules within the three species of Sceloporus studied to date. Future studies concerning sperm morphology in closely related species will aid in our understanding of variation in sperm development and may prove to be useful in testing phylogenetic and evolutionary hypotheses.

Agkistrodon piscivorus spermatogenesis addendum: The effect of Hurricane Katrina on spermatogenesis of the western cottonmouth snake.

Recent studies detailed the spermatogenic cycle of the Western Cottonmouth Snake, Agkistrodon piscivorus and noted that spermatogenesis is bimodal, with active periods during March-June and August-October in southeastern Louisiana. However, only spermatogonia were present in September in the only specimen that was captured and the authors state that the individual "should have a high testis volume and also show spermiogenic activity." The specimen in their study was caught immediately following Hurricane Katrina outside of its normal habitat. Therefore, in order to verify their assumption, individuals were captured during September of 2008 and the testes were spermatogenically active with spermatogonia, spermatocytes, and mature spermatozoa being present in the seminiferous epithelium of the testes. These data indicate that Hurricane Katrina could have had an impact on the spermatogenic cycle in Cottonmouths, resulting in stress-induced testicular regression.

Seasonal spermatogenesis in the Mexican endemic oviparous lizard, Sceloporus aeneus (Squamata: Phrynosomatidae).

Oviparous species of Sceloporus exhibit either seasonal or continuous spermatogenesis and populations from high-elevation show a seasonal pattern known as spring reproductive activity. We studied the spermatogenic cycle of a high-elevation (2700 m) population of endemic oviparous lizard, Sceloporus aeneus, that resided south of México, D.F. Histological analyses were performed on the testes and reproductive ducts from individual lizards collected monthly. This population of S. aeneus showed a seasonal pattern of spermatogenesis, with 4 successive phases common in other lizards. These include: 1) Quiescence in August, which contained solely spermatogonia and Sertoli cells; 2) Testicular recrudescence (September-January) when testes became active with mitotic spermatogonia, spermatocytes beginning meiosis, and the early stages of spermiogenesis with spermatids; 3) Maximum testicular activity occurred from March to May and is when the largest spermiation events ensued within the germinal epithelia, which were also dominated by spermatids and spermiogenic cells; 4) Testicular regression in June was marked with the number of all germs cells decreasing rapidly and spermatogonia dominated the seminiferous epithelium. February was a transitional month between recrudescence and maximum activity. The highest sperm abundance in the lumina of epididymides was during maximum testicular activity (March-May). Thus, before and after these months fewer spermatozoa were detected within the excurrent ducts as the testis transitions from recrudescence to maximum activity in February and from maximum activity to quiescence in June. Maximum spermatogenic activity corresponds with warmest temperatures at this study site. This pattern known as spring reproductive activity with a fall recrudescence was similar to other oviparous species of genus Sceloporus.

Spermiogenesis in the imbricate alligator lizard, Barisia imbricata (Reptilia, Squamata, Anguidae).

Although the events of spermiogenesis are commonly studied in amniotes, the amount of research available for Squamata is lacking. Many studies have described the morphological characteristics of mature spermatozoa in squamates, but few detail the ultrastructural changes that occur during spermiogenesis. This study's purpose is to gain a better understanding of the subcellular events of spermatid development within the Imbricate Alligator Lizard, Barisia imbricata. The morphological data presented here represent the first complete ultrastructural study of spermiogenesis within the family Anguidae. Samples of testes from four specimens collected on the northwest side of the Nevado de Toluca, México, were prepared using standard techniques for transmission electron microscopy. Many of the ultrastructural changes occurring during spermiogenesis within B. imbricata are similar to that of other squamates (i.e., early acrosome formation, chromatin condensation, flagella formation, annulus present, and a prominent manchette). However, there are a few unique characteristics within B. imbricata spermatids that to date have not been described during spermiogenesis in other squamates. For example, penetration of the acrosomal granule into the subacrosomal space to form the basal plate of the perforatorium during round spermatid development, the clover-shaped morphology of the developing nuclear fossa of the flagellum, and the bulbous shape to the perforatorium are all unique to the Imbricate Alligator Lizard. These anatomical character differences may be valuable nontraditional data that along with more traditional matrices (such as DNA sequences and gross morphological data) may help elucidate phylogenetic relationships, which are historically considered controversial within Squamata.

The testicular sperm ducts and genital kidney of male Ambystoma maculatum (Amphibia, Urodela, Ambystomatidae).

The ducts associated with sperm transport from the testicular lobules to the Wolffian ducts in Ambystoma maculatum were examined with transmission electron microscopy. Based on the ultrastructure and historical precedence, new terminology for this network of ducts is proposed that better represents primary hypotheses of homology. Furthermore, the terminology proposed better characterizes the distinct regions of the sperm transport ducts in salamanders based on anatomy and should, therefore, lead to more accurate comparisons in the future. While developing the above ontology, we also tested the hypothesis that nephrons from the genital kidney are modified from those of the pelvic kidney due to the fact that the former nephrons function in sperm transport. Our ultrastructural analysis of the genital kidney supports this hypothesis, as the basal plasma membrane of distinct functional regions of the nephron (proximal convoluted tubule, distal convoluted tubule, and collecting tubule) appear less folded (indicating decreased surface area and reduced reabsorption efficiency) and the proximal convoluted tubule possesses ciliated epithelial cells along its entire length. Furthermore, visible luminal filtrate is absent from the nephrons of the genital kidney throughout their entire length. Thus, it appears that the nephrons of the genital kidney have reduced reabsorptive capacity and ciliated cells of the proximal convoluted tubule may increase the movement of immature sperm through the sperm transport ducts or aid in the mixing of seminal fluids within the ducts.

Observations on variation in the ultrastructure of the proximal testicular ducts of the Ground Skink, Scincella lateralis (Reptilia: Squamata).

The North American Ground Skink, Scincella lateralis, is a member of the most speciose family of lizards, the Scincidae. The only descriptions of the testicular ducts of skinks concern the light microscopy of 13 species in eight other genera. We combine histological observations with results from transmission electron microscopy on a sample of skinks collected throughout the active season. The single rete testis has squamous epithelium with a large, indented nucleus and no junctional complexes between cells or conspicuous organelles. Nuclei of sperm in the rete testis area are associated with cytoplasmic bodies that are lost in the ductuli efferentes. The ductuli efferentes have both ciliated and nonciliated cells and show little seasonal variation except for the narrowing of intercellular canaliculi when sperm are absent. When the ductus epididymis contains sperm, the anterior one-third lacks copious secretory material around luminal sperm, whereas in the posterior two-thirds sperm are embedded in a dense matrix of secretory material. Light and dark principal cells exist and both contain saccular, often distended rough endoplasmic reticula, and widened intercellular canaliculi that bridge intracellular spaces. Junctional complexes are lacking between principal cells except for apical tight junctions. Electron-dense secretory granules coalesce at the luminal border for apocrine release. The cranial end of the ductus deferens is similar in cytology to the posterior ductus epididymis. Each of the nine squamates in which the proximal testicular ducts have been studied with electron microscopy has some unique characters, but no synapomorphies for squamates as a group are recognized.

Observations on the sexual segment of the kidney of snakes with emphasis on ultrastructure in the yellow-bellied sea snake, Pelamis platurus.

The sexual segment of the kidney (SSK) is an accessory sex structure in male lizards and snakes (Squamata). We describe histology of the SSK in 12 species of snakes, including one from the basal Scolecophidia, Leptotyphlops dulcis, and from the more advanced Alethinophidia, species from the Acrochordidae (Acrochordus granulatus), Homalopsidae (Cerberus rynchops), Uropeltidae (Teretrurus sanguineus), and eight species from the Elapidae, including six species of sea snakes. We also describe the ultrastructure of the SSK of the sea snake, Pelamis platurus. The SSK of L. dulcis does not include the ureter but does include distal convoluted tubules (DCTs) and collecting ducts. In all other snakes examined, the SSK is limited to the DCTs and does not differ in histology by any consistent character. We found apparently mature individuals of several species with inactive SSKs. Hypertrophied SSKs give positive reactions for protein secretions but variable reactions for carbohydrates. Ultrastructure of the SSK of P. platurus reveals nuclei situated medially in the epithelium and mature electron dense secretory vacuoles in other areas of the cytoplasm. Product release is apocrine. Junctional complexes only occur at the luminal border, and intercellular canaliculi become widened and are open basally. No cytologically unique characters occur in the SSK of P. platurus. The ancestral condition of the SSK in squamates is the presence of simple columnar epithelium specialized for secretion of a protein + carbohydrate product that matures and is released seasonally.

Ultrastructural description of spermiogenesis within the Mediterranean Gecko, Hemidactylus turcicus (Squamata: Gekkonidae).

We studied spermiogenesis in the Mediterranean Gecko, Hemidactylus turcicus, at the electron microscope level and compared to what is known within other Lepidosaurs. In H. turcicus germ cells are connected via cytoplasmic bridges where organelle and cytoplasm sharing is observed. The acrosome develops from merging transport vesicles that arise from the Golgi and subsequently partition into an acrosomal cap containing an acrosomal cortex, acrosomal medulla, perforatorium, and subacrosomal cone. Condensation of DNA occurs in a spiral fashion and elongation is aided by microtubules of the manchette. A nuclear rostrum extends into the subacrosomal cone and is capped by an epinuclear lucent zone. Mitochondria and rough endoplasmic reticulum migrate to the posterior portion of the developing germ cell during the cytoplasmic shift and the flagellum elongates. Mitochondria surround the midpiece as the anlage of the annulus forms. The fibrous sheath begins at mitochondrial tier 3 and continues into the principal piece. Peripheral fibers associated with microtubule doublets 3 and 8 are grossly enlarged. During the final stages of germ cell development spermatids are wrapped with a series of Sertoli cell processes, which exhibit ectoplasmic specializations and differing cytoplasmic consistencies. The results observed here corroborate previous studies, which show the conservative nature of sperm morphology. However, ultrastructural character combinations specific to sperm and spermiogenesis seem to differ among taxa. Further studies into sperm morphology are needed in order to judge the relevance of the ontogenic changes recorded here and to determine their role in future studies on amniote evolution.

The sexual segment of Hemidactylus turcicus and the evolution of sexual segment location in squamata.

The kidneys of the Mediterranean Gecko, Hemidactylus turcicus (Gekkonidae), were investigated using light and electron microscopy with the primary focus placed on morphology of the sexual segment of the kidney. The nephrons of male H. turcicus are composed of five distinct regions: 1) a renal corpuscle and glomerulus, 2) a proximal convoluted tubule, 3) an intermediate segment, 4) a distal convoluted tubule, and 5) the sexual segment of the kidney/collecting duct. Female H. turcicus is similar but lack a sexual segment of the kidney. The sexual segment of the kidney is hypertrophied during the months of March through August, which corroborates previous reports of reproductive activity. During inactive months, the sexual segment of the kidney is nondiscernable from the collecting ducts. The sexual segment consists of tall columnar epithelial cells with basally positioned nuclei. Perinuclear Golgi complexes and rough endoplasmic reticulum are present. Secretory granules, which fill the apices of the epithelial cells, are electron dense and released into the lumen by a merocrine secretory process. Narrow intercellular canaliculi separate each epithelial cell and are sealed by tight junctions at the luminal aspect. Basally, leukoctyes are observed within the intercellular canaliculi and outside the basal lamina. Mast cells can be found just outside the basal lamina in close association with renal capillaries. The sexual segment of the kidney of H. turcicus is similar to that of three unrelated lizards for which ultrastructure was investigated with secretion mode being the major difference Also, H. turcicus is similar to most other lizards in that complete regression occurs during reproductive inactivity, but differs in this trait from the skink, Scincella lateralis, and most snakes which display a hypertrophied sexual segment of the kidney throughout the entire year. Although some unique similarities appear during the optimization, no direct patterns or directions are observed, and only the molecular based phylogeny resolves the ancestral condition of the Squamata as the sexual segment of the kidney being observed in the distal convoluted tubule, collecting duct, and ureter.

Proximal testicular ducts of the Mediterranean gecko (Hemidactylus turcicus).

The efferent ducts of the Mediterranean Gecko, Hemidactylus turcicus (Gekkonidae) were investigated using light and electron microscopy. The seminiferous tubules unite into a single rete testis tubule. The rete testis divides into 3-4 ductuli efferentes which all drain into the cranial portion of the ductus epididymis. All efferent ducts are most active during the months of December to August. The rete testis is composed of a simple squamous epithelium with bifurcated nuclei and a labyrinthine network of intercellular canaliculi. Ciliated and nonciliated cells are present, and more than one cilium extends from the scattered ciliated cells. The presence of small clear vesicles and widened intercellular canaliculi suggest that cells of the rete testis are responsible for intake of luminal fluids. The ductuli efferentes are composed of a simple cuboidal epithelium consisting of ciliated and nonciliated cells, and ciliated cells are the dominant cell type. During the inactive season the number of lysosomes increases and the cells become spermiophagic. The ductus epididymis is composed of a tall pseudostratified columnar epithelium with relatively scarce basal cells. No evidence for regionalization was observed. The ductus epididymis is highly secretory during the active season with numerous electron-dense secretory granules, whose glycoprotein products are released by merocrine secretion. Basally, the active epididymis has swollen intercellular canaliculi and enlarged cisternae of rough endoplasmic reticulum. During the inactive season the secretory activity decreases and membranous structures and fibrous material are observed within widened intercellular canaliculi apical to the basal cells.

Ultrastructure of spermiogenesis in the American alligator, Alligator mississippiensis (Reptilia, Crocodylia, Alligatoridae).

Testicular samples were collected to describe the ultrastructure of spermiogenisis in Alligator mississipiensis (American Alligator). Spermiogenesis commences with an acrosome vesicle forming from Golgi transport vesicles. An acrosome granule forms during vesicle contact with the nucleus, and remains posterior until mid to late elongation when it diffuses uniformly throughout the acrosomal lumen. The nucleus has uniform diffuse chromatin with small indices of heterochromatin, and the condensation of DNA is granular. The subacrosome space develops early, enlarges during elongation, and accumulates a thick layer of dark staining granules. Once the acrosome has completed its development, the nucleus of the early elongating spermatid becomes associated with the cell membrane flattening the acrosome vesicle on the apical surface of the nucleus, which aids in the migration of the acrosomal shoulders laterally. One endonuclear canal is present where the perforatorium resides. A prominent longitudinal manchette is associated with the nuclei of late elongating spermatids, and less numerous circular microtubules are observed close to the acrosome complex. The microtubule doublets of the midpiece axoneme are surrounded by a layer of dense staining granular material. The mitochondria of the midpiece abut the proximal centriole resulting in a very short neck region, and possess tubular cristae internally and concentric layers of cristae superficially. A fibrous sheath surrounds only the axoneme of the principal piece. Characters not previously described during spermiogenesis in any other amniote are observed and include (1) an endoplasmic reticulum cap during early acrosome development, (2) a concentric ring of endoplasmic reticulum around the nucleus of early to middle elongating spermatids, (3) a band of endoplasmic reticulum around the acrosome complex of late developing elongate spermatids, and (4) midpiece mitochondria that have both tubular and concentric layers of cristae.

Ultrastructure of spermiogenesis in the Cottonmouth, Agkistrodon piscivorus (Squamata: Viperidae: Crotalinae).

To date multiple studies exist that examine the morphology of spermatozoa. However, there are limited numbers of data detailing the ontogenic characters of spermiogenesis within squamates. Testicular tissues were collected from Cottonmouths (Agkistrodon piscivorus) and tissues from spermiogenically active months were analyzed ultrastructurally to detail the cellular changes that occur during spermiogenesis. The major events of spermiogenesis (acrosome formation, nuclear elongation/DNA condensation, and flagellar development) resemble that of other squamates; however, specific ultrastructural differences can be observed between Cottonmouths and other squamates studied to date. During acrosome formation vesicles from the Golgi apparatus fuse at the apical surface of the nuclear membrane prior to making nuclear contact. At this stage, the acrosome granule can be observed in a centralized location within the vesicle. As elongation commences the acrosome complex becomes highly compartmentalized and migrates laterally along the nucleus. Parallel and circum-cylindrical microtubules (components of the manchette) are observed with parallel microtubules outnumbering the circum-cylindrical microtubules. Flagella, displaying the conserved 9 + 2 microtubule arrangement, sit in nuclear fossae that have electron lucent shoulders juxtaposed on either side of the spermatids basal plates. This study aims to provide developmental characters for squamates in the subfamily Crotalinae, family Viperidae, which may be useful for histopathological studies on spermatogenesis in semi-aquatic species exposed to pesticides. Furthermore, these data in the near future may provide morphological characters for spermiogenesis that can be added to morphological data matrices that may be used in phylogenetic analyses.

Histological analysis of spermatogenesis and the germ cell development strategy within the testis of the male Western Cottonmouth Snake, Agkistrodon piscivorus leucostoma.

Cottonmouth (Agkistrodon piscivorus leucostoma) testes were examined histologically to determine the germ cell development strategy employed during spermatogenesis. Testicular tissues from Cottonmouths were collected monthly from swamps around Hammond, Louisiana. Pieces of testis were fixed in Trump's fixative, dehydrated in ethanol, embedded in Spurr's plastic, sectioned with an ultramicrotome, and stained with toluidine blue and basic fuchsin. Spermatogenesis within Cottonmouths occurs in two independent events within a single calendar year. The testes are active during the months of March-June and August-October with spermiation most heavily observed during April-May and October. To our knowledge, this is the first study that describes bimodal spermatogenesis occurring in the same year within the subfamily Crotalinae. During spermatogenesis, no consistent spatial relationships are observed between germ cell generations. Typically, either certain cell types were missing (spermatocytes) or the layering of 3-5 spermatids and/or spermatocytes within the same cross-section of seminiferous tubule prevented consistent spatial stages from occurring. This temporal pattern of sperm development is different from the spatial development found within birds and mammals, being more reminiscent of that seen in amphibians, and has now been documented within every major clade of reptile (Chelonia, Serpentes, Sauria, Crocodylia). This primitive-like sperm development, within a testis structurally similar to mammals and birds, may represent an intermediate testicular model within the basally positioned (phylogenetically) reptiles that may be evolutionarily significant.