Tasmanian skinks

​Application for Scientific Permit – Available for Public Comment

Public comment on the following application for a Scientific Research (Fauna) Permit is open until 1 February 2021.

Applicant: Biological Sciences, University of Tasmania

Species/Taxon:
Carinascincus ocellatus, spotted snow skink
Carinascincus metallicus, metallic skink
Carinascincus pretiosus, Tasmanian tree skink
Carinascincus greeni, northern snow skink​
Carinascincus orocryptus, mountain skink
Carinascincus microlepidotus, southern snow skink
Pseudomoia entrecasteauxii, southern grass skink
Liopholis whitii, White's skink

Location: (where activities are to take place, including land tenure)

Carinascincus ocellatus, Miena, Orford (Private Freehold)
Carinascincus metallicus, Miena, Orford (Private Freehold)
Carinascincus pretiosus, Orford (Private Freehold), Clarence Lagoon (Central Plateau Conservation Area)
Carinascincus greeni, Pine Lake (Central Plateau Conservation Area)
Carinascincus orocryptus, Hartz Mountain National Park
Carinascincus microlepidotus, Mt Wellington Park
Pseudomoia entrecasteauxii, Orford (Private Freehold)
Liopholis whitii, Orford (Private Freehold)

Title of research: Transitions between modes of sex-determination in a changing world

Aim of project: Sex in vertebrates can be determined genetically, environmentally or by a combination of genes and environment. Genetic sex determination (GSD) is controlled by sex chromosomes and occurs at fertilisation. Individuals may contain two copies of the same sex chromosome (homogamety), or single copies of two different sex chromsomes (heterogamety). Sex chromosomes systems are either male heterogametic (known as XY heterogamety) or female heterogametic (known as ZW heterogamety).

Sex can also be determined by the environment experienced during embryonic development. An example of this is temperature dependent sex determination (TSD), which operates to determine sex after fertilisation during a temperature sensitive period of development (e.g., turtles, crocodiles and many geckos).
Recent work on other lizards reveals that sex can also be determined by a combination of genes and temperature.

Sex determination has transitioned between GSD and TSD in reptiles many times over their evolutionary history. Understanding how selection favours these transitions and the mechanisms that allow them to occur are central questions in evolutionary biology.

A long-term dataset has been used to demonstrate that the spotted snow skink (Carinascincus ocellatus) exhibits distinct differences in sex determining systems between populations located at the extreme climatic limits of its range. Whilst both populations possess sex-linked genes that suggest XY heterogamety, in a coastal population this genetic determinant can be overridden by temperature.

Over recent years snow skinks have been well-studied by researchers at the University of Tasmania and have become excellent models for studies
of sex determining systems.

The aims of our research are to:
1) elucidate the DNA changes responsible for the population differences in sex determination (GSD and TSD) in Carinascincus ocellatus.
2) quantify differences at a chromosomal level among populations that differ in sex determination (GSD and TSD)
3) perform comparisons relative to other skink species (including other Carinascincus) to infer the direction of change (e.g. GSD -> TSD).

Justification:
In addition to addressing fundamental knowledge gaps, the proposed research has strong urgency and translational relevance for conservation of biodiversity. Climate change is identified as a significant extinction risk for TSD species via the production of highly biased sex ratios. For example, male:female in the important fishery species Odontesthes bonariensis would shift to ~1:3 given a ~1.5°C increase in temperature, greatly reducing recruitment. Climate change can also override apparently stable GSD systems in central bearded dragon into TSD, with a <1°C increase in incubation temperature resulting in all-female offspring. Therefore, there is urgency to understand the molecular mechanism that enables GSD-TSD transitions, and hence exposes species to risk of sex ratio shift with climate.

Maximum likely numbers of individuals involved:

Carinascincus ocellatus, spotted snow skink, 10
Carinascincus metallicus, metallic skink, 25
Carinascincus pretiosus, Tasmanian tree skink, 25
Carinascincus greeni, northern snow skink, 25
Carinascincus orocryptus, mountain skink, 25
Carinascincus microlepidotus, southern snow skink, 25
Pseudomoia entrecasteauxii, southern grass skink, 25
Liopholis whitii, White's skink, 10

Activities undertaken and methods:

FIELD-CAPTURE
Animals are captured by either of two techniques: “fishing” or “noosing”. The fishing technique involves baiting the end of a fishing rod with a mealworm (tied on with cotton, no hooks are used). This is presented to lizards, which bite the mealworm and the lizard is raised into a bucket. With the noosing technique, a loop of fishing-line is placed around a lizard’s neck and pulled taut, and lizards are lifted into a bucket (this procedure is a common method for capturing reptiles).

Animals will be sexed in the field by hemipenes eversion, as we seek equal numbers of males and females for each species.

A subset of individuals captured are only required for DNA analysis, and will be tail-tipped in the field.The remainder will be transported to the laboratory, where all but four individuals will be live blood sampled for chromosome analysis (karyotyping), and live tail-tipped for genotyping, and released back to their site of capture.
 
Four individuals of C. ocellatus will be euthanised for brain and gonad tissue sampling for transcriptome sequencing, and subsequently blood and tail-tip sampled as per the other individuals.

BRAIN DISSECTION
Because the CNS of reptiles and amphibians is tolerant to hypoxic and hypotensive conditions, decapitation must be followed by pithing or another method of destroying brain tissue. Because we want to sample brain tissue intact, we seek to use pentobarbital (intracoelomic) euthanasia followed by brain dissection. Given the small number of individuals (n=4) and the nature of the procedure, we will request this euthanasia procedure to be undertaken by a Veterinarian.

TAIL TIPPING (field and laboratory)
DNA sampling is possible through taking a small tissue sample from the end of skinks’ tails. These species naturally lose their tails and regenerate them in natural situations – there is negligible blood loss (usually none) as the procedure utilises their natural response to autotomise their tail which is preceded by blood retraction to the body and shutting down of the nerves. Thus tail-tip samples are the least invasive way of obtaining tissues. DNA sampling is crucial to meeting several aims of the study.

TRANSPORT & HOUSING
Animals will be transported to the laboratory. During transport here and for all other periods of transportation, lizards will be held in cloth bags and kept cool and moist to minimise distress. The laboratory is thermally controlled (16.5˚C) with a 4.6kW reverse cycle air conditioner. Bright overhead lights, including UV lights, will be provided to the room during normal daylight hours (usually around 14 hours per day). Individuals will be housed singly in terraria (approx. 40 x 20 x 20 cm) with available cover (blocks of wood and paper pellets) and a basking rock, and fed a variety of live food (including mealworms, crickets and baby food with calcium powder). Water will be available ad libitum. Individuals will be checked twice daily. Individuals who do not appear to be consuming food will be monitored for weight loss and lethargy and returned to their site of capture.

BLOOD SAMPLING FOR CYTOGENETICS
Blood will be sampled (75 microlitres) from the sub - orbital sinus located in the corner of the upper jaw by first cleaning the area with a sterile swap, puncturing the sinus using a fine-gauge needle, and collecting blood using a heparinised capillary tube and immediately transferring to cell culture media. According to Heard et al. (2004), a safe sample is 10% of total blood volume in the healthy reptile. Reptile blood volume equates to around 5–8% of bodyweight;  therefore, 0.5–0.8% of bodyweight (g) can be taken. Adult skinks weigh 20-30 grams, and therefore 75 μL = 0.075 g = 0.00375% body weight. The animal will not be sedated for this procedure. Blood will be taken in captivity. Proximity to the laboratory is essential, as blood needs to be immediately transferred to cell culture media and incubated under laboratory conditions, hence the need for transfer of individuals into captivity.

EMERGENCY EUTHANASIA OF SMALL LIZARDS
If for any reason emergency euthanasia is required, we will follow cervical dislocation and pithing.

Fate of animals:
All individuals will be returned to their site of capture with the exception of the four Carinascincus ocellatus that will be euthanised for sampling or brain tissue.

Likely impact on species involved (including any by-catch):
No impact on any species with the exception of the 4 individuals of Carinascincus ocellatus to be sacrificed.

Contact

Scientific Research Permits
Natural and Cultural Heritage
Level 5, 134 Macquarie Street
Hobart TAS 7000
Email: Scientific.Permits@dpipwe.tas.gov.au

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