Pseudemoia Entrecasteauxii Research Articles

Comparing the potential for maternal-fetal signalling in oviparous and viviparous lizards.

The evolution of a placenta requires several steps including changing the timing of reproductive events, facilitating nutrient exchange, and the capacity for maternal-fetal communication. To understand the evolution of maternal-fetal communication, we used ligand-receptor gene expression as a proxy for the potential for cross-talk in a live-bearing lizard (Pseudemoia entrecasteauxii) and homologous tissues in a related egg-laying lizard (Lampropholis guichenoti). Approximately 70% of expressed ligand/receptor genes were shared by both species. Gene ontology (GO) analysis showed that there was no GO-enrichment in the fetal membranes of the egg-laying species, but live-bearing fetal tissues were significantly enriched for 50 GO-terms. Differences in enrichment suggest that the evolution of viviparity involved reinforcing specific signalling pathways, perhaps to support fetal control of placentation. One identified change was in transforming growth factor beta signalling. Using immunohistochemistry, we show the production of the signalling molecule inhibin beta B (INHBB) occurs in viviparous fetal membranes but was absent in closely related egg-laying tissues, suggesting that the evolution of viviparity may have involved changes to signalling via this pathway. We argue that maternal-fetal signalling evolved through co-opting expressed signalling molecules and recruiting new signalling molecules to support the complex developmental changes required to support a fetus in utero. This article is part of the theme issue 'Extraembryonic tissues: exploring concepts, definitions and functions across the animal kingdom'.

Corrigenda: The regulation of uterine proinflammatory gene expression during pregnancy in the live-bearing lizard, Pseudemoia entrecasteauxii.

Corrigenda: The regulation of uterine proinflammatory gene expression during pregnancy in the live-bearing lizard, Pseudemoia entrecasteauxii.

The Regulation of Uterine Proinflammatory Gene Expression during Pregnancy in the Live-Bearing Lizard, Pseudemoia entrecasteauxii.

The evolutionary transition from egg-laying to live-bearing in amniote vertebrates (reptiles and mammals) requires the development of a closer association between the maternal and embryonic tissue to facilitate gas and nutrient exchange with the embryo. Because the embryo is an allograft to the father and mother, it could be considered foreign by the maternal immune system and thus be immunologically rejected during pregnancy. In eutherian ("placental") mammals, the proinflammatory genes interleukin 1B (IL1B), tumor necrosis factor (TNF) and tumor necrosis factor receptor superfamily 1A (TNFRSF1A) are tightly regulated in the pregnant uterus to prevent embryonic rejection. We tested whether inflammation is similarly regulated in pregnant viviparous reptiles by comparing the expression of IL1B, TNF, and TNFRSF1A in the pregnant and nonpregnant uterus of the viviparous lizard, Pseudemoia entrecasteauxii. We found statistically significant support for the downregulation of pregnant uterine TNF mRNA expression in P. entrecasteauxii, but no statistically significant changes in mRNA expression of TNFRSF1A or IL1B between pregnant and nonpregnant uteri. Although these genes are apparently not regulated at the transcriptional level, our immunofluorescence microscopy analyses nonetheless demonstrate that the IL1B proteins are stored intracellularly during pregnancy, possibly resulting in inhibition of inflammatory response. We therefore conclude that processes of both transcriptional (TNF) and posttranslational (IL1B) gene regulation may reduce inflammation in the pregnant uterus of this viviparous reptile. Our study is important because it demonstrates that regulating the maternal immune system to prevent embryonic rejection may be important in reptilian pregnancy as it is in mammalian pregnancy.

Reptile Pregnancy Is Underpinned by Complex Changes in Uterine Gene Expression: A Comparative Analysis of the Uterine Transcriptome in Viviparous and Oviparous Lizards.

The evolution of new organs is difficult to study because most vertebrate organs evolved only once, more than 500 million years ago. An ideal model for understanding complex organ evolution is the placenta, a structure that is present in live bearing reptiles and mammals (amniotes), which has evolved independently more than 115 times. Using transcriptomics, we characterized the uterine gene expression patterns through the reproductive cycle of a viviparous skink lizard, Pseudemoia entrecasteauxii. Then we compare these patterns with the patterns of gene expression from two oviparous skinks Lampropholis guichenoti and Lerista bougainvillii. While thousands of genes are differentially expressed between pregnant and non-pregnant uterine tissue in the viviparous skink, few differentially expressed genes were identified between gravid and non-gravid oviparous skinks. This finding suggests that in P. entrecasteauxii, a pregnant-specific gene expression profile has evolved, allowing for the evolution of pregnancy-specific innovations in the uterus. We find substantial gene expression differences between the uterus of the chorioallantoic and the yolk sac placenta in P. entrecasteauxii, suggesting these placental regions are specialized for different placental functions. In particular, the chorioallantoic placenta is likely a major site of nutrient transport by membrane-bound transport proteins, while the yolk sac placenta also likely transports nutrients but via apocrine secretions. We discuss how the evolution of transcription factor networks is likely to underpin the evolution of the new transcriptional states in the uterine tissue of viviparous reptiles.

Comparative genomics of hormonal signaling in the chorioallantoic membrane of oviparous and viviparous amniotes

In oviparous amniotes (reptiles, birds, and mammals) the chorioallantoic membrane (CAM) lines the inside of the egg and acts as the living point of contact between the embryo and the outside world. In livebearing (viviparous) amniotes, communication during embryonic development occurs across placental tissues, which form between the uterine tissue of the mother and the CAM of the embryo. In both oviparous and viviparous taxa, the CAM is at the interface of the embryo and the external environment and can transfer signals from there to the embryo proper. To understand the evolution of placental hormone production in amniotes, we examined the expression of genes involved in hormone synthesis, metabolism, and hormone receptivity in the CAM of species across the amniote phylogeny. We collected transcriptome data for the chorioallantoic membranes of the chicken (oviparous), the lizards Lerista bougainvillii (both oviparous and viviparous populations) and Pseudemoia entrecasteauxii (viviparous), and the horse Equus caballus (viviparous). The viviparous taxa differ in their mechanisms of nutrient provisioning: L. bougainvillii is lecithotrophic (embryonic nourishment is provided via the yolk only), but P. entrecasteauxii and the horse are placentotrophic (embryos are nourished via placental transport). Of the 423 hormone-related genes that we examined, 91 genes are expressed in all studied species, suggesting that the chorioallantoic membrane ancestrally had an endocrine function. Therefore, the chorioallantoic membrane appears to be a highly hormonally active organ in all amniotes. No genes are expressed only in viviparous species, suggesting that the evolution of viviparity has not required the recruitment of any specific hormone-related genes. Our data suggest that the endocrine function of the CAM as a placental tissue evolved in part through co-option of ancestral gene expression patterns.

Allelic expression of mammalian imprinted genes in a matrotrophic lizard, Pseudemoia entrecasteauxii.

Genomic imprinting is a process that results in the differential expression of genes depending on their parent of origin. It occurs in both plants and live-bearing mammals, with imprinted genes typically regulating the ability of an embryo to manipulate the maternal provision of nutrients. Genomic imprinting increases the potential for selection to act separately on paternally and maternally expressed genes, which increases the number of opportunities that selection can facilitate embryonic control over maternal nutrient provision. By looking for imprinting in an independent matrotrophic lineage, the viviparous lizard Pseudemoia entrecasteauxii (Scincidae), we test the hypothesis that genomic imprinting facilitates the evolution of substantial placental nutrient transport to embryos (matrotrophy). We sequenced transcriptomes from the embryonic component of lizard placentae to determine whether there are parent-of-origin differences in expression of genes that are imprinted in mammals. Of these genes, 19 had sufficiently high expression in the lizard to identify polymorphisms in transcribed sequences. We identified bi-allelic expression in 17 genes (including insulin-like growth factor 2), indicating that neither allele was imprinted. These data suggest that either genomic imprinting has not evolved in this matrotrophic skink or, if it has, it has evolved in different genes to mammals. We outline how these hypotheses can be tested. This study highlights important differences between mammalian and reptile pregnancy and the absence of any shared imprinting genes reflects fundamental differences in the way that pregnancy has evolved in these two lineages.

Carbonic anhydrase II is found in the placenta of a viviparous, matrotrophic lizard and likely facilitates embryo-maternal CO2 transport.

The evolution of viviparity requires the development of mechanisms that facilitate transport of respiratory gases between mother and developing embryo. Of particular importance is maternal excretion of embryonic carbon dioxide (CO2 ), which increases as the embryo grows in size during development. The carbonic anhydrases are a family of enzymes that convert CO2 to bicarbonate for transport throughout the cardiovascular system and which may also be important for CO2 transport from embryo to mother. We used immunohistochemistry to localize carbonic anhydrase II in the placental tissues of a viviparous and highly placentotrophic lizard, Pseudemoia entrecasteauxii. Carbonic anhydrase II is localized in the uterine component of the paraplacentome, presumably to facilitate transport of embryonic CO2 to the mother. Carbonic anhydrase II is also localized in both the uterine and embryonic components of the placentome, a region heavily involved in placental nutrient transport rather than respiratory gas exchange. In contrast, carbonic anhydrase II is not present in the uterine or embryonic components of the omphaloplacenta, another region responsible for nutrient transport. While carbonic anhydrase II in the paraplacentomal uterus is likely to be responsible for embryo-maternal CO2 transport, the distribution of carbonic anhydrase II throughout the placentome indicates a different function. Instead of transporting embryonic CO2 , placentomal carbonic anhydrase II appears to be responsible for transporting CO2 produced by energetically expensive nutrient transport mechanisms in both the uterus and the embryo, which implies that the mechanisms of nutrient transport in the omphaloplacenta may not be as energetically expensive.

Phylogenetic evidence of historic mitochondrial introgression and cryptic diversity in the genus Pseudemoia (Squamata: Scincidae)

The Australian scincid genus Pseudemoia comprises six morphologically similar species restricted to temperate south-eastern Australia. Due to the high degree of morphological conservatism, phylogenetic relationships and taxonomic status within the Pseudemoia entrecasteauxii complex (comprising the nominal species P. entrecasteauxii, P. cryodroma, and P. pagenstecheri) remains unresolved. To further investigate the phylogenetic relationships and taxonomic status of Pseudemoia spp., and to test the hypothesis that P. cryodroma evolved from hybridization between P. entrecasteauxii and P. pagenstecheri, we sequenced one mitochondrial locus (ND4) and five nuclear loci (β-globin, LGMN, PRLR, Rhodopsin, RPS8). While we find strong support for the monophyly of the P. entrecasteauxii complex, there exists marked incongruence between the mitochondrial and nuclear markers, particularly in regards to the high altitude specialist, P. cryodroma. The most parsimonious explanation of this discordance is historic mitochondrial introgression, although a hybrid origin for P. cryodroma cannot be completely rejected. Within P. pagenstecheri sensu lato, we identified a strongly supported, highly divergent yet morphologically cryptic lineage restricted to northern New South Wales. Although more weakly supported by the nuDNA, we also identified a second geographically distinct lineage of P. pagenstecheri s.l., which may warrant separate conservation management. Our study reveals a more complex evolutionary history of the genus Pseudemoia than previously appreciated and contributes to our understanding of the biogeography and evolution of Australian mesic zone fauna.

High food abundance permits the evolution of placentotrophy: evidence from a placental lizard, Pseudemoia entrecasteauxii.

Mechanisms of reproductive allocation are major determinants of fitness because embryos cannot complete development without receiving sufficient nutrition from their parents. The nourishment of offspring via placentas (placentotrophy) has evolved repeatedly in vertebrates, including multiple times in squamate reptiles (lizards and snakes). Placentotrophy has been suggested to evolve only if food is sufficiently abundant throughout gestation to allow successful embryogenesis. If scarcity of food prevents successful embryogenesis, females should recoup nutrients allocated to embryos via abortion, reabsorption, and/or cannibalism. We tested these hypotheses in the placentotrophic southern grass skink Pseudemoia entrecasteauxii. We fed females one of four diets (high constant, high variable, low constant, and low variable) during gestation and tested the effects of both food amount and schedule of feeding on developmental success, cannibalism rate, placental nutrient transport, offspring size, and maternal growth and body condition. Low food availability reduced developmental success, placental nutrient transport, offspring size, and maternal growth and body condition. Cannibalism of offspring also increased when food was scarce. Schedule of feeding did not affect offspring or mothers. We suggest that high food abundance and ability to abort and cannibalize poor-quality offspring are permissive factors necessary for placentotrophy to be a viable strategy of reproductive allocation.

Placental lipoprotein lipase (LPL) gene expression in a placentotrophic lizard,Pseudemoia entrecasteauxii

Viviparity (live birth) relies on a functional placenta, which is formed by cooperating maternal and embryonic tissues. In some viviparous lineages, mothers use this placenta to transport nutrients to feed developing embryos through pregnancy (placentotrophy). The Australian lizard, Pseudemoia entrecasteauxii, provides approximately 60% of the lipid for embryonic growth and metabolism to embryos across the placenta. Lipoprotein lipase (LPL) is an important enzyme in lipid transport in vertebrates. We examined patterns of LPL gene expression to identify its role in the uterus of pregnant P. entrecasteauxii. We used reverse transcription quantitative real time PCR to measure the expression of the LPL gene in the uterine tissue throughout reproduction and compared uterine LPL expression in chorioallantoic and yolk-sac placentae. Expression of the LPL gene is significantly higher in the uterus of late pregnant compared to non-pregnant and early pregnant P. entrecasteauxii, indicating a greater capacity for lipid transport towards the end of pregnancy. The period of high LPL gene expression correlates with the time that developing embryos are undergoing the greatest growth and have the highest metabolic rate. LPL gene expression is significantly higher in the uterine tissue of the yolk-sac placenta than the chorioallantoic placenta, providing the first molecular evidence that the yolk-sac placenta is the major site of lipid transport in pregnant P. entrecasteauxii.

No implantation in an extra-uterine pregnancy of a placentotrophic reptile

Placentation is a common feature of live-bearing reptiles and mammals. Placentae are variable between species and can be classified by the extent that embryonic tissue breaches (invades) the uterus. Non-invasive placentation in eutherians is maternally imposed as extra-uterine embryos of species with epitheliochorial placentation will readily invade non-uterine tissues. This study documents the first observation of an extra-uterine pregnancy in a reptile; Pseudemoia entrecasteauxii, which in-utero exhibits non-invasive epitheliochorial placentation. The extra-uterine embryo did not invade maternal tissue suggesting fundamental differences between the nature and evolution of placentation in P. entrecasteauxii and eutherian mammals.

Interpopulational variation in costs of reproduction related to pregnancy in a viviparous lizard

Interpopulational variation in reproductive costs may affect variation in life history traits including reproductive investment (i.e. clutch mass relative to either maternal body mass or length). While the relationships between reproductive investment and costs of reproduction, especially costs to mobility, have been well studied in squamate reptiles, how these costs relate to investment and explain patterns within and between populations is not always straightforward. In the present study, we examined the relationship between reproductive investment and costs of reproduction (gravid and postpartum sprint speeds and maternal postpartum body condition) in two populations of a viviparous skink, Pseudemoia entrecasteauxii living in different habitat types. We found that costs of reproduction (i.e. impact on gravid and postpartum sprint speeds) depended on the interaction between relative reproductive burden (RRB) and population. There was no link between relative clutch mass (RCM) and maternal sprint speeds. Maternal postpartum body condition was not related to either RRB or RCM for either population. Gravid females living in the open habitat population showed significantly slower sprint speed compared with the same females immediately postparturition, and other gravid females living in a closed habitat population. Such females are likely to experience a higher cost of reproduction in terms of changes in sprint speed as well as exposure to predators and may show a behavioural shift to crypsis in order to compensate for locomotor impairment and to reduce the risk of predation. We suggest that factors which relate to costs of reproduction (i.e. sprint speeds) are complex and may involve multiple factors such as reproductive investment and habitat characteristics.

Effects of Maternal Basking and Food Quantity during Gestation Provide Evidence for the Selective Advantage of Matrotrophy in a Viviparous Lizard

The evolution of matrotrophy (i.e., direct supply of nutrients by the mother during gestation) may be associated with high maternal energy availability during gestation. However, we lack knowledge about the selective advantages of matrotrophic viviparity (live-bearing) in reptiles. In reptiles, the interaction between body temperature and food intake affect maternal net energy gain. In the present study, we examined the effects of basking and food availability (2 by 2 factorial design) during gestation on offspring phenotype in a matrotrophic viviparous lizard (Pseudemoia entrecasteauxii). Subsequently, we investigated if the maternal effects were context-dependent using offspring growth rate as an indicator of the adaptive significance of matrotrophy. Offspring were exposed either to the same thermal conditions as their mothers experienced or to thermal conditions different from those experienced by their mothers. We provide the first evidence that an interaction between maternal thermal and maternal food conditions during gestation strongly affects offspring phenotype, including date of birth, body size and performance ability, which affect offspring fitness. Offspring growth rate was dependent on offspring thermal conditions, but was not influenced by maternal effects or offspring sex. Matrotrophic viviparity provided gravid females with the means to enhance offspring fitness through greater energetic input to offspring when conditions allowed it (i.e., extended basking opportunity with high food availability). Therefore, we suggest that selective advantages of matrotrophic viviparity in P. entrecasteauxii may be associated with high maternal energy availability during gestation.

A Novel Pattern of Placental Leucine Transfer During Mid to Late Gestation in a Highly Placentotrophic Viviparous Lizard

Placentotrophy is the nourishment of embryos by resources provided via the placenta during gestation. The magnitude and timing of placental nutrient support during pregnancy are important for embryonic growth, especially in highly placentotrophic animals such as mammals. However, no study has yet investigated how placental organic nutrient support may change during pregnancy in highly placentotrophic viviparous reptiles. Amino acids are essential nutrients for embryonic growth and leucine is a common amino acid. The magnitude and timing of placental leucine transfer may affect embryonic growth and mass and, therefore, offspring phenotype. In this study, female Pseudemoia entrecasteauxii, a highly placentotrophic viviparous skink, were collected throughout gestation. We injected (3)H-leucine into these gravid females and assessed the transfer of (3)H-leucine into maternal compartments (i.e., the blood and the liver), and into embryonic compartments (i.e., the embryo, the yolk, and the amniotic fluid). At either 60 or 120 min post-injection, the radioactivity in each sample was extracted and then counted, and the transfer ratio was calculated. Our results provide direct evidence that circulating maternal leucine passes through the placenta into the embryos in this species. The relative rate of placental leucine transfer did not alter during mid to late gestation. This suggests the steady somatic growth of the embryos during mid-late pregnancy is dependent upon the placental transfer of nutrients rather than yolk stores. This pattern of placental nutrient support may determine offspring body size at birth and, therefore, offspring fitness in P. entrecasteauxii.

Do Gravid Females Become Selfish? Female Allocation of Energy during Gestation

Net energy availability depends on plasma corticosterone concentrations, food availability, and their interaction. Limited net energy availability requires energy trade-offs between self-maintenance and reproduction. This is important in matrotrophic viviparous animals because they provide large amounts of energy for embryos, as well as self-maintenance, for the extended period of time during gestation. In addition, gravid females may transmit environmental information to the embryos in order to adjust offspring phenotype. We investigated effects of variation in maternal plasma corticosterone concentration and maternal food availability (2 × 2 factorial design) during gestation on offspring phenotype in a matrotrophic viviparous lizard (Pseudemoia entrecasteauxii). Subsequently, we tested preadaptation of offspring phenotype to their postnatal environment by measuring risk-averse behavior and growth rate using reciprocal transplant experiments. We found that maternal net energy availability affected postpartum maternal body condition, offspring snout-vent length, offspring mass, offspring performance ability, and offspring fat reserves. Females treated with corticosterone allocated large amounts of energy to their own body condition, and their embryos allocated more energy to energy reserves than somatic growth. Further, offspring from females in high plasma corticosterone concentration showed compensatory growth. These findings suggest that while females may be selfish when gestation conditions are stressful, the embryos may adjust their phenotype to cope with the postnatal environment.

Uterine Epithelial Morphology and Progesterone Receptors in a Mifepristone‐treated Viviparous Lizard Pseudemoia entrecasteauxii (Squamata: Scincidae) During Gestation

Structural and functional changes to the uterus associated with maintenance of pregnancy are controlled primarily by steroid hormones such as progesterone. We tested the hypothesis that progesterone regulates uterine structural changes during pregnancy in the viviparous skink, Pseudemoia entrecasteauxii, by treating pregnant females with the progesterone receptor antagonist mifepristone at different stages of pregnancy. Expression and distribution of progesterone receptor was determined using Western blot and immunohistochemistry. During early pregnancy, mifepristone treatment resulted in altered uterine epithelial cell surface morphology and high embryo mortality, but did not affect females at mid and late stages of pregnancy. Females treated with mifepristone in early pregnancy exhibited abnormal uterine epithelial cell morphology such as lateral blebbing and presence of wide gaps between cells indicating loss of intercellular attachment. Chorioallantoic membranes of the embryo were not affected by mifepristone treatment. Two isoforms (55 kDa and 100 kDa) of progesterone receptor were identified using immunoblots and both isoforms were localized to the nucleus of uterine epithelial cells. The 55 kDa isoform was expressed throughout pregnancy, whereas the 100 kDa isoform was expressed during mid and especially late pregnancy. In P. entrecasteauxii, mifepristone may prevent successful embryo attachment in early pregnancy through its effects on uterine epithelial cells but may have little effect on pregnancy once the maternal-embryo structural relationship is established.

Effects of variation in maternal carotenoid intake during gestation on offspring innate immune response in a matrotrophic viviparous reptile

Summary1. The evolution of matrotrophy may be related to high resource availability during gestation and consequently increased opportunity for reproductive investment via the placenta. Thus, matrotrophic species have smaller yolk volume than lecithotrophic species. Innate immune response of offspring at birth, which is important in protection against disease and parasites, may be influenced by the degree of reproductive investment of carotenoids, such as β‐carotene. However, to date, no study has investigated the effects of maternal carotenoid intake during reproduction on the innate immune responses of offspring in reptiles.2. Some matrotrophic viviparous (live‐bearing) reptiles provide significant support for embryogenesis through both yolk and placenta. These species are excellent models for research into the evolutionary transitions from lecithotrophic reproduction (i.e. supply of resources from the yolks, e.g. birds) to highly matrotrophic reproduction (i.e. direct supply of most resources from the mother, e.g. mammals).3. We used high‐performance liquid chromatography (HPLC) to examine carotenoid concentration in newly ovulated eggs of a matrotrophic viviparous lizard (Pseudemoia entrecasteauxii). Most eggs did not contain any carotenoids, including β‐carotene.4. We then experimentally manipulated maternal β‐carotene availability (i.e. supplementation of β‐carotene in the diet or no supplementation) during gestation and investigated innate immune response of offspring (i.e. tissue swelling and proportion of monocytes in response to antigenic stimulation) at 2 days after birth. Subsequently, we investigated the effects of post‐natal β‐carotene intake on innate immune response of offspring using reciprocal transplant experiments in which we manipulated offspring β‐carotene availability for 2 weeks after birth. This experimental design also allowed us to assess the persistence of effects of maternal β‐carotene effects on innate immune response in offspring. We found that the immune response as measured by the proportion of monocytes was enhanced by high maternal β‐carotene intake during gestation, and this effect persisted for 2 weeks after birth. Furthermore, post‐natal β‐carotene intake enhanced innate immune response.5. The maternally transferred ß‐carotene via the placenta during embryogenesis may be an important physiological adaptation to counter the costs of a reduction in organic substances supplied in the yolk that occurs as matrotrophy evolves.

Evidence for placental transfer of maternal corticosterone in a viviparous lizard

In mammals, there is experimental evidence that circulating maternal cortisol is transferred to the embryos across the placenta during gestation. Direct effects of this maternal cortisol may allow embryos to display phenotypic plasticity to cope with postnatal environments (i.e., pre-programming). The potential for maternal hormone induced-adaptation may be of considerable evolutionary significance in viviparous animals. However, to date, there is no such direct evidence that circulating maternal corticosterone passes through the placenta and into the embryos of viviparous reptiles. In this study, we assessed the transfer of 3H-corticosterone injected into females of the lizard Pseudemoia entrecasteauxii into maternal blood, maternal liver, the embryo, the yolk and the amniotic fluid during mid-to-late gestation. We provide direct evidence that circulating maternal corticosterone passes through the placenta into the embryos in this species. Transfer of maternal corticosterone into the embryos significantly decreased at the end of embryonic development. We discuss these results in terms of the relationships between the degree of corticosterone transfer and embryonic stage. These results demonstrate the potential for direct effects of maternal corticosterone, including endocrine pre-programming, upon the developing embryos in viviparous lizards.

Transient Receptor Potential Ion Channels Control Thermoregulatory Behaviour in Reptiles

Biological functions are governed by thermodynamics, and animals regulate their body temperature to optimise cellular performance and to avoid harmful extremes. The capacity to sense environmental and internal temperatures is a prerequisite for the evolution of thermoregulation. However, the mechanisms that enable ectothermic vertebrates to sense heat remain unknown. The recently discovered thermal characteristics of transient receptor potential ion channels (TRP) render these proteins suitable to act as temperature sensors. Here we test the hypothesis that TRPs are present in reptiles and function to control thermoregulatory behaviour. We show that the hot-sensing TRPV1 is expressed in a crocodile (Crocodylus porosus), an agamid (Amphibolurus muricatus) and a scincid (Pseudemoia entrecasteauxii) lizard, as well as in the quail and zebrafinch (Coturnix chinensis and Poephila guttata). The TRPV1 genes from all reptiles form a unique clade that is delineated from the mammalian and the ancestral Xenopus sequences by an insertion of two amino acids. TRPV1 and the cool-sensing TRPM8 are expressed in liver, muscle (transversospinalis complex), and heart tissues of the crocodile, and have the potential to act as internal thermometer and as external temperatures sensors. Inhibition of TRPV1 and TRPM8 in C. porosus abolishes the typically reptilian shuttling behaviour between cooling and heating environments, and leads to significantly altered body temperature patterns. Our results provide the proximate mechanism of thermal selection in terrestrial ectotherms, which heralds a fundamental change in interpretation, because TRPs provide the mechanism for a tissue-specific input into the animals' thermoregulatory response.

Reproductive Seasonality of Three Cold-temperate Viviparous Skinks from Southeastern Australia

Reproductive cycles and patterns of sperm storage were examined for three regionally sympatric species of cold-temperate viviparous Australian skinks previously reported to have Type II reproductive cycles (autumn spermatogenesis and mating, with oviductal sperm storage (OSS) and spring ovulation). Histological examination of oviducts, testes and epididymides of winter- and spring-collected skinks were combined with data on monthly variation in gonad size (ovarian follicle diameter and testis length) obtained from museum specimens. Pseudemoia entrecasteauxii exhibited autumn spermatogenesis and mating, OSS over winter, and spring vitellogenesis and ovulation, concurring with previous studies for more southern populations. In contrast to previous studies from more southern populations, Niveoscincus coventryi and Hemiergis decresiensis displayed autumn spermatogenesis with epididymal sperm storage (ESS) over winter and spring vitellogenesis, mating and ovulation. Timing of mating and sperm storage patterns may vary geographically or possibly be plastic in response to local or annual variation in environmental factors. Autumn-onset vitellogenesis with OSS is hypothesized to be the optimal cycle for producing offspring earlier in the breeding season.