Male Tuatara Research Articles

Initial collection, characterization, and storage of tuatara (Sphenodon punctatus) sperm offers insight into their unique reproductive system

Successful reproduction is critical to the persistence of at-risk species; however, reproductive characteristics are understudied in many wild species. New Zealand’s endemic tuatara (Sphenodon punctatus), the sole surviving member of the reptile order Rhynchocephalia, is restricted to 10% of its historic range. To complement ongoing conservation efforts, we collected and characterized mature sperm from male tuatara for the first time. Semen collected both during mating and from urine after courting contained motile sperm and had the potential for a very high percentage of viable sperm cells (98%). Scanning electron microscopy revealed a filiform sperm cell with distinct divisions: head, midpiece, tail, and reduced end piece. Finally, our initial curvilinear velocity estimates for tuatara sperm are 2–4 times faster than any previously studied reptile. Further work is needed to examine these trends at a larger scale; however, this research provides valuable information regarding reproduction in this basal reptile.

Detection of Paranannizziopsis australasiensis in tuatara (Sphenodon punctatus) using fungal culture and a generic fungal PCR

AIMS: To describe the methods used at the Animal Health Laboratory (AHL, Ministry for Primary Industries) to identify Paranannizziopsis australasiensis.METHODS: Skin biopsy samples from two adult male tuatara were submitted to the AHL in March 2014. Approximately half of each sample was processed for fungal culture and incubated on mycobiotic agar containing cycloheximide at 30°C. Following morphological examination of the culture products, DNA was extracted from suspect colonies. PCR was used to amplify the internal transcribed spacer (ITS) region of fungal rRNA using primers ITS1 and ITS4. Positive amplicons were subjected to DNA sequencing and the results were compared to published sequences. In addition, DNA was extracted from the remaining skin samples and the same PCR was carried out to compare the results.RESULTS: After 7 days of incubation, colonies morphologically resembling P. australasiensis were observed. DNA extracted from these isolates tested positive for P. australasiensis by PCR and DNA sequencing. Samples of DNA extracted directly from the infected skin samples tested negative for P. australasiensis using the generic fungal PCR.CONCLUSIONS AND CLINICAL RELEVANCE: Isolation and identification of P. australasiensis was carried out using a combination of fungal culture and molecular testing available at AHL. Results were available in significantly less time than in the past, when isolates had to be sent overseas. PCR and sequencing of fungal isolates is a valuable tool for identification of species that have few, if any, unique macroscopic or microscopic features to aid identification. Further sampling from captive and wild New Zealand reptiles will provide important information on the epidemiology of P. australasiensis, and the conservation and management implications for tuatara and other native reptile species.

Use of Ultrasonography and Computed Tomography in the Diagnosis of Panophthalmitis in a Tuatara (Sphenodon punctatus)

Abstract A 605 g, mature, male tuatara (Sphenodon punctatus) was presented with periorbital swelling, exophthalmos, and hyphema of the right eye. Ultrasound examination showed distortion of the globe, an altered lens contour, heterogeneous vitreous content, and a loss of ocular and retrobulbar tissue planes. Computed tomography demonstrated displacement of the scleral ossicles but an absence of orbital bone lesions. A decision was made to enucleate the right eye. The tuatara was premedicated with medetomidine (0.06 mg/kg) and morphine (1 mg/kg). Intravenous alfaxalone (5 mg/kg) was administered to facilitate intubation. Maintenance anesthesia was achieved using 1–2% sevoflurane. Perioperative support included intraosseous 0.45% saline with 2% dextrose (combined 50% each) at 10 ml/kg/h and thermal support via a forced-air blanket. Transpalpebral enucleation of the right eye was performed and the excised tissue submitted for histopathologic evaluation. Postoperative analgesia was provided using morphine (1 ...

Tuatara (Sphenodon) Genomics: BAC Library Construction, Sequence Survey, and Application to the DMRT Gene Family

The tuatara (Sphenodon punctatus) is of "extraordinary biological interest" as the most distinctive surviving reptilian lineage (Rhyncocephalia) in the world. To provide a genomic resource for an understanding of genome evolution in reptiles, and as part of a larger project to produce genomic resources for various reptiles (evogen.jgi.doe.gov/second_levels/BACs/our_libraries.html), a large-insert bacterial artificial chromosome (BAC) library from a male tuatara was constructed. The library consists of 215 424 individual clones whose average insert size was empirically determined to be 145 kb, yielding a genomic coverage of approximately 6.3x. A BAC-end sequencing analysis of 121 420 bp of sequence revealed a genomic GC content of 46.8%, among the highest observed thus far for vertebrates, and identified several short interspersed repetitive elements (mammalian interspersed repeat-type repeats) and long interspersed repetitive elements, including chicken repeat 1 element. Finally, as a quality control measure the arrayed library was screened with probes corresponding to 2 conserved noncoding regions of the candidate sex-determining gene DMRT1 and the DM domain of the related DMRT2 gene. A deep coverage contig spanning nearly 300 kb was generated, supporting the deep coverage and utility of the library for exploring tuatara genomics.

Squamous cell carcinoma in a tuatara (Sphenodon punctatus)

CASE HISTORY: A mature male tuatara was presented with a history of recurrent cloacal prolapse. CLINICAL AND PATHOLOGICAL FINDINGS: The prolapsed tissue included a 12 mm diameter mass, which histologically showed cords and nodules of neoplastic epithelial cells. DIAGNOSIS: The mass was diagnosed as a squamous cell carcinoma with ulceration of the overlying stratified squamous epithelium and diffuse inflammation of the surrounding dermis. CLINICAL RELEVANCE: This case presented a rare opportunity to investigate squamous cell carcinoma in a long-lived lower vertebrate species. Future recurrence or metastasis of the mass may add useful information to the current base of knowledge of the behaviour of malignant neoplasms in reptiles.

Stable carbon isotope ratios as indicators of marine versus terrestrial inputs to the diets of wild and captive tuatara (Sphenodon punctatus)

Stable carbon isotope analysis was used to examine feeding relationships of wild tuatara on Stephens Island and captive tuatara in New Zealand institutions. We first measured δ13C in three food items of wild tuatara. Pectoral muscle of fairy prions (a seabird eaten seasonally by tuatara) was significantly enriched in 13C compared with whole bodies of wild insects (darkling beetles and tree weta). Values for 813C in blood cells varied significantly among wild tuatara of different life‐history stages. Male tuatara were more enriched in 13C than were females or juveniles, suggesting that males prey more heavily on seabirds. Insect foods of captive tuatara varied dramatically in δ13C; this is attributed to differential consumption of plant material derived from the C3 and C4 photosynthetic pathways. Blood cells from four different groups of captive tuatara differed significantly in δ13C. This was perhaps related to assimilation of insects with different δ13C values, and cannot be attributed to differences in seabird predation as captive tuatara do not have access to seabirds. For wild tuatara on Stephens Island, stable carbon isotope analysis provides support for the dietary information available from behavioural observations, gut analyses and measurements of plasma composition.

Reproductive cycles of male and female tuatara (Sphenodon punctatus) on Stephens Island, New Zealand

Cycles in gonadal activity and plasma sex steroid concentrations were investigated in wild female and male tuatara on Stephens Island, New Zealand. Females nest once every four years on average. Vitellogenesis is spread over the first three years, and mating, ovulation and nesting occur in the fourth. Oviducal eggs are carried for6–8 months before nesting. Although the length of this ovarian cycle is unparalleled among oviparous reptiles, the associated cycles in plasma concentrations of sex steroids are similar to those in other reptiles. Mean plasma concentrations of oestradiol increase during vitellogenesis, peak at mating, fall rapidly before or around ovulation, are low during most of gravidity, and rise slightly during late gravidity‐nesting. Plasma concentrations of testosterone show a similarcycle. Mean plasma concentrations of progesterone are low during vitellogenesis and peak around ovulation. This periovulatory surge falls within1–2 months, and mean concentrations are low during the final 5–6 months of gravidity. Male tuatara show an annual, pre‐nuptial reproductive cycle. Mean plasma concentrations of testosterone are low during winter, rise during spring, peak during midsummer‐early autumn, when pre‐nuptial displays and mating occur, and fall in mid autumn. Limited histological data indicate that spermiogenesis occurs during midsummer‐early autumn, and also support a previous study suggesting that there may be no period of complete testicular and epididymal regression. In comparison with the pre‐nuptial cycles of other temperate‐zone reptiles, the cycle in male tuatara shows a more prolonged duration of testicular activity and of rising or elevated plasma testosterone concentrations. The prolonged reproductive cycles of male and female tuatara may be adaptations to a temperate environment with cool summers and cool, but not freezing, winters.

Effects of capture and temperature stresses on plasma steroid concentrations in male tuatara (Sphenodon punctatus)

AbstractThe effects of capture and temperature stresses on plasma concentrations of testosterone (T) and progesterone (P) were examined in wild male tuatara (Sphenodon punctatus), the only living sphenodontid reptile. Free‐roaming male tuatara on Stephens Island, New Zealand, had low levels (0.3–0.5 ng/ml) of plasma T in July (winter), and high levels (15–17 ng/ml) in January (summer). Levels in November appeared intermediate (5 ng/ml) but were measured with reference to standards prepared in human rather than tuatara plasma. These seasonal variations are consistent with available information on the annual cycle of spermatogenesis and reproductive behavior in male tuatara. In November, plasma T was unchanged in males bled at 0, 3, and 12 h after capture but had fallen significantly to 57% of the initial value by 24 h after capture (experiment 1). In January, males held in collecting bags for 3 h prior to blood sampling had levels of plasma T similar to those of free‐roaming males sampled at the same time (experiment 2). In July, an acute heat stress (12.8°C elevation in body temperature over 30 min) had no effect on plasma T (experiment 3). Significant changes in plasma P were seen prior to changes in plasma T in experiment 1: plasma P was elevated at 3, 12, and 24 h after capture. No difference in plasma P was observed between free‐roaming and captive males in experiment 2. In experiment 3, no change in plasma P occurred in response to heat stress. Free‐roaming males showed no evidence of a diurnal cycle in plasma T or plasma P in November. These data indicate that plasma T in male tuatara is unaffected by short‐term (3 h) capture; however, longer‐term (24 h) capture combined with repeated blood sampling causes a significant reduction in plasma T, as previously reported for other male reptiles. Changes in plasma P suggest that activity of the adrenal gland may be sensitive to captivity stress in this species. These data provide the first values for circulating steroid levels in free‐roaming wild male tuatara and help define conditions of captivity that influence plasma steroid concentrations, a prerequisite for further studies of steroid cycles in this species.

Seasonal changes in plasma sex steroid levels in the male tuatara, Sphenodon punctatus, from Stephens Island, New Zealand

A unique opportunity, occasioned by the sacrifice of five male tuataras ( Sphenodon punctatus), was taken to measure the circulating levels of a number of sex steroids, and other parameters, at different seasons throughout the year on Stephens Island, New Zealand. Male tuataras exhibit continuous spermatocytogenesis throughout the year (H. Saint Girons and D. G. Newman, 1987, N.Z. J. Zool. 14, 231–237) but levels of sex steroids showed significant seasonal variation nonetheless, with testosterone concentrations being positively correlated with the height of the epithelium lining the epididymal ducts. Plasma testosterone and 5α-dihydrotestosterone levels were highest in February, coincident with the mating season when spermiogenesis is maximal and epididymal ducts are hypertrophied, but DHT concentrations were generally lower and more constant throughout the year. Circulating levels of progesterone, 17β-oestradiol, and corticosterone showed no significant changes over the period of sampling which extended from February until August. The tuatara is the only surviving member of the ancient Sub-Order Rhynchocephalia, and it seems likely that testosterone functions as a male sex hormone in this species, as it does in other members of the Class Reptilia.

The reproductive cycle of the male tuatara, Sphenodon punctatus, on Stephens Island, New Zealand

Abstract The reproductive cycle of male Sphenodon punctatus is characterised by continuous sexual activity. Spermatocytogenesis is uninterrupted, though abnormal in some cells, and probably very slow in winter. Spermiogenesis is normal from mid spring to mid summer, then becomes abortive and finally ceases during the 5 or 6 coolest months. The epididymal duct, which is hypertrophied during the mating period in summer, remains partly developed during the rest of the year. The kidney has no sexual segment. Many spermatozoa are present throughout the year in the deferent ducts of males, but their number gradually decreases in winter and at the beginning of spring. The reproductive cycle of the male tuatara is similar, but not identical, to that of the few New Zealand lizards that have been studied, but differs markedly from all types of cycles, known in reptiles, from other parts of the world.