Non-invasive genetic sexing technique for analysis of short-beaked echidna (Tachyglossus aculeatus) populations
Tahlia Perry A * , Deborah Toledo-Flores A * , Wan X. Kang A , Arthur Ferguson B , Belinda Laming B , Enkhjargal Tsend-Ayush A , Shu L. Lim A and Frank Grützner
A C
+ Author Affiliations
- Author Affiliations
A The Environment Institute, School of Biological Sciences, The University of Adelaide, Adelaide, SA 5005, Australia.
B Perth Zoo, 20 Labouchere Road, South Perth, WA 6151, Australia.
C Corresponding author. Email: frank.grutzner@adelaide.edu.au
Reproduction, Fertility and Development 31(7) 1289-1295 https://doi.org/10.1071/RD18142
Submitted: 18 April 2018 Accepted: 4 May 2019 Published: 4 June 2019
Abstract
Identifying male and female echidnas is challenging due to the lack of external genitalia or any other differing morphological features. This limits studies of wild populations and is a major problem for echidna captive management and breeding. Non-invasive genetic approaches to determine sex minimise the need for handling animals and are used extensively in other mammals. However, currently available approaches cannot be applied to monotremes because their sex chromosomes share no homology with sex chromosomes in other mammals. In this study we used recently identified X and Y chromosome-specific sequences to establish a non-invasive polymerase chain reaction-based technique to determine the sex of echidnas. Genomic DNA was extracted from echidna hair follicles followed by amplification of two Y chromosome (male-specific) genes (mediator complex subunit 26 Y-gametolog (CRSPY) and anti-Müllerian hormone Y-gametolog (AMHY)) and the X chromosome gene (anti-Müllerian hormone X-gametolog (AMHX)). Using this technique, we identified the sex of 10 juvenile echidnas born at Perth Zoo, revealing that eight of the 10 echidnas were female. Future use of the genetic sexing technique in echidnas will inform captive management, continue breeding success and can be used to investigate sex ratios and population dynamics in wild populations.
Additional keywords: blood sample, captive breeding, echidna sexing, fluorescence in situ hybridization, hair sample, sex-specific polymerase chain reaction (PCR).
References
Augee, M. L., Gooden, B., and Musser, A. (2006). ‘Echidna: Extraordinary Egg-Laying Mammal.’ (CSIRO Publishing: Melbourne.)Cortez, D., Marin, R., Toledo-Flores, D., Froidevaux, L., Liechti, A., Waters, P. D., Grützner, F., and Kaessmann, H. (2014). Origins and functional evolution of Y chromosomes across mammals. Nature 508, 488–493.
| Origins and functional evolution of Y chromosomes across mammals.Crossref | GoogleScholarGoogle Scholar | 24759410PubMed |
Dallas, J. F., Carss, D. N., Marshall, F., Koeogli, K.-P., Kruuk, H., Piertney, S. B., and Bacon, P. J. (2000). Sex identification of the Eurasian otter Lutra lutra by PCR typing of spraints. Conserv. Genet. 1, 181–183.
| Sex identification of the Eurasian otter Lutra lutra by PCR typing of spraints.Crossref | GoogleScholarGoogle Scholar |
Dohm, J. C., Tsend-Ayush, E., Reinhardt, R., Grützner, F., and Himmelbauer, H. (2007). Disruption and pseudoautosomal localization of the major histocompatibility complex in monotremes. Genome Biol. 8, R175.
| Disruption and pseudoautosomal localization of the major histocompatibility complex in monotremes.Crossref | GoogleScholarGoogle Scholar | 17727704PubMed |
Durnin, M. E., Palsbøll, P. J., Ryder, O. A., and McCullough, D. R. (2007). A reliable genetic technique for sex determination of giant panda (Ailuropoda melanoleuca) from non-invasively collected hair samples. Conserv. Genet. 8, 715–720.
| A reliable genetic technique for sex determination of giant panda (Ailuropoda melanoleuca) from non-invasively collected hair samples.Crossref | GoogleScholarGoogle Scholar |
Faust, L. J., and Thompson, S. D. (2000). Birth sex ratio in captive mammals: patterns, biases, and the implications for management and conservation. Zoo Biol. 19, 11–25.
| Birth sex ratio in captive mammals: patterns, biases, and the implications for management and conservation.Crossref | GoogleScholarGoogle Scholar |
Ferguson, A., and Turner, B. (2013). Reproductive parameters and behaviour of captive short-beaked echidna (Tachyglossus aculeatus acanthion) at Perth Zoo. Aust. Mammal. 35, 84–92.
| Reproductive parameters and behaviour of captive short-beaked echidna (Tachyglossus aculeatus acanthion) at Perth Zoo.Crossref | GoogleScholarGoogle Scholar |
Glatston, A. R. (1997). Sex ratio research in zoos and its implications for captive management. Appl. Anim. Behav. Sci. 51, 209–216.
| Sex ratio research in zoos and its implications for captive management.Crossref | GoogleScholarGoogle Scholar |
Grant, T. R. (2004). Captures, capture mortality, age and sex ratios of platypuses, Ornithorhynchus anatinus, during studies over 30 years in the Upper Shoalhaven River in New South Wales. Proc. Linn. Soc. N. S. W. 125, 217–226.
Grant, T. R., and Temple-Smith, P. D. (1998). Field biology of the platypus (Ornithorhynchus anatinus): historical and current perspectives. Philos. Trans. R. Soc. Lond. B Biol. Sci. 353, 1081–1091.
| Field biology of the platypus (Ornithorhynchus anatinus): historical and current perspectives.Crossref | GoogleScholarGoogle Scholar | 9720106PubMed |
Griffiths, M. (1978). ‘The Biology of the Monotremes.’ (Academic Press: New York.)
Griffiths, M. (1989). Tachyglossidae. In ‘Fauna of Australia’. (Eds D. W. Walton and B. J. Richardson) pp. 407–435. (AGPS: Canberra.)
Grützner, F., Rens, W., Tsend-Ayush, E., El-Mogharbel, N., O’Brien, P. C., Jones, R. C., Ferguson-Smith, M. A., and Marshall Graves, J. A. (2004). In the platypus a meiotic chain of ten sex chromosomes shares genes with the bird Z and mammal X chromosomes. Nature 432, 913–917.
| In the platypus a meiotic chain of ten sex chromosomes shares genes with the bird Z and mammal X chromosomes.Crossref | GoogleScholarGoogle Scholar | 15502814PubMed |
Jackson, S. M. (2003). ‘Australian Mammals: Biology and Captive Management.’ (CSIRO Publishing: Melbourne.)
Johnston, S. D., Nicolson, V., Madden, C., Logie, S., Pyne, M., Roser, A., Lisle, A. T., and D’Occhio, M. (2007). Assessment of reproductive status in male echidnas. Anim. Reprod. Sci. 97, 114–127.
| Assessment of reproductive status in male echidnas.Crossref | GoogleScholarGoogle Scholar | 16476529PubMed |
Li, B., Zhang, W., Shi, X., and Zhou, X. (2013). Molecular sex identification of sable and stone marten. Russ. J. Ecol. 44, 137–141.
| Molecular sex identification of sable and stone marten.Crossref | GoogleScholarGoogle Scholar |
Lindsay, A. R., and Belant, J. L. (2008). A simple and improved PCR-based technique for white-tailed deer (Odocoileus virginianus) sex identification. Conserv. Genet. 9, 443–447.
| A simple and improved PCR-based technique for white-tailed deer (Odocoileus virginianus) sex identification.Crossref | GoogleScholarGoogle Scholar |
Lucchini, V., Fabbri, E., Marucco, F., Ricci, S., Boitani, L., and Randi, E. (2002). Noninvasive molecular tracking of colonizing wolf (Canis lupus) packs in the western Italian Alps. Mol. Ecol. 11, 857–868.
| Noninvasive molecular tracking of colonizing wolf (Canis lupus) packs in the western Italian Alps.Crossref | GoogleScholarGoogle Scholar | 11975702PubMed |
Lynch, Á. B., and Brown, M. J. F. (2006). Molecular sexing of pine marten (Martes martes): how many replicates? Mol. Ecol. Notes 6, 631–633.
| Molecular sexing of pine marten (Martes martes): how many replicates?Crossref | GoogleScholarGoogle Scholar |
Morrow, G. E. (2013). Optimizing reproduction in the Tasmanian echidna Tachyglossus aculeatus setosus: the influence of an obligatory hibernation period and intense sexual conflict. Ph.D. Thesis, University of Tasmania, Hobart.
Morrow, G. E., and Nicol, S. C. (2012). Maternal care in the Tasmanian echidna (Tachyglossus aculeatus setosus). Aust. J. Zool. 60, 289–298.
| Maternal care in the Tasmanian echidna (Tachyglossus aculeatus setosus).Crossref | GoogleScholarGoogle Scholar |
Morrow, G., Andersen, N. A., and Nicol, S. C. (2009). Reproductive strategies of the short-beaked echidna – a review with new data from a long-term study on the Tasmanian subspecies (Tachyglossus aculeatus setosus). Aust. J. Zool. 57, 275–282.
| Reproductive strategies of the short-beaked echidna – a review with new data from a long-term study on the Tasmanian subspecies (Tachyglossus aculeatus setosus).Crossref | GoogleScholarGoogle Scholar |
Nicol, S., and Andersen, N. A. (2006). Body temperature as an indicator of egg-laying in the echidna, Tachyglossus aculeatus. J. Therm. Biol. 31, 483–490.
| Body temperature as an indicator of egg-laying in the echidna, Tachyglossus aculeatus.Crossref | GoogleScholarGoogle Scholar |
Nicol, S., and Andersen, N. A. (2007). The life history of an egg-laying mammal, the echidna (Tachyglossus aculeatus). Ecoscience 14, 275–285.
| The life history of an egg-laying mammal, the echidna (Tachyglossus aculeatus).Crossref | GoogleScholarGoogle Scholar |
Nicol, S. C., and Morrow, G. E. (2012) Sex and seasonality: reproduction in the echidna (Tachyglossus aculeatus). In ‘Living in a Seasonal World’. (Eds T. Ruf, C. Bieber, W. Arnold and E. Millesi.) pp. 143–153. (Springer: Berlin Heidelberg.)
Rens, W., Grützner, F., O’Brien, P. C. M., Fairclough, H., Graves, J. A., and Ferguson-Smith, M. A. (2004). Resolution and evolution of the duck-billed platypus karyotype with an X1Y1X2Y2X3Y3X4Y4X5Y5 male sex chromosome constitution. Proc. Natl Acad. Sci. USA 101, 16257–16261.
| Resolution and evolution of the duck-billed platypus karyotype with an X1Y1X2Y2X3Y3X4Y4X5Y5 male sex chromosome constitution.Crossref | GoogleScholarGoogle Scholar | 15534209PubMed |
Rens, W., O’Brien, P. C., Grützner, F., Clarke, O., Graphodatskaya, D., Tsend-Ayush, E., Trifonov, V. A., Skelton, H., Wallis, M. C., Johnston, S., Veyrunes, F., Graves, J. A., and Ferguson-Smith, M. A. (2007). The multiple sex chromosomes of platypus and echidna are not completely identical and several share homology with the avian Z. Genome Biol. 8, R243.
| The multiple sex chromosomes of platypus and echidna are not completely identical and several share homology with the avian Z.Crossref | GoogleScholarGoogle Scholar | 18021405PubMed |
Rismiller, P. (1992). Field observations on Kangaroo Island echidnas (Tachyglossus aculeatus multiaculeatus) during the breeding season. In ‘Platypus and Echidna’. (Ed. M. L. Augee.) pp. 101–105. (Royal Zoological Society of New South Wales: Mosman, N.S.W.)
Rismiller, P. D., and McKelvey, M. W. (2000). Frequency of breeding and recruitment in the short-beaked echidna, Tachyglossus Aculeatus. J. Mammal. 81, 1–17.
| Frequency of breeding and recruitment in the short-beaked echidna, Tachyglossus Aculeatus.Crossref | GoogleScholarGoogle Scholar |
Rismiller, P. D., and McKelvey, M. W. (2003). Body mass, age and sexual maturity in short-beaked echidnas, Tachyglossus aculeatus. Comp. Biochem. Physiol. A Mol. Integr. Physiol. 136, 851–865.
| Body mass, age and sexual maturity in short-beaked echidnas, Tachyglossus aculeatus.Crossref | GoogleScholarGoogle Scholar | 14667849PubMed |
Stannard, H. J., Bekkers, J. M., Old, J. M., McAllan, B. M., and Shaw, M. E. (2017). Digestibility of a new diet for captive short-beaked echidnas (Tachyglossus aculeatus). Zoo Biol. 36, 56–61.
| Digestibility of a new diet for captive short-beaked echidnas (Tachyglossus aculeatus).Crossref | GoogleScholarGoogle Scholar | 28111821PubMed |
Taberlet, P., Mattock, H., Dubois‐Paganon, C., and Bouvet, J. (1993). Sexing free-ranging brown bears Ursus arctos using hairs found in the field. Mol. Ecol. 2, 399–403.
| Sexing free-ranging brown bears Ursus arctos using hairs found in the field.Crossref | GoogleScholarGoogle Scholar | 8162229PubMed |
Temple-Smith, P., and Grant, T. (2001). Uncertain breeding: a short history of reproduction in monotremes. Reprod. Fertil. Dev. 13, 487–497.
| Uncertain breeding: a short history of reproduction in monotremes.Crossref | GoogleScholarGoogle Scholar | 11999298PubMed |
Tsend-Ayush, E., Dodge, N., Mohr, J., Casey, A., Himmelbauer, H., Kremitzki, C. L., Schatzkamer, K., Graves, T., Warren, W. C., and Grützner, F. (2009). Higher-order genome organization in platypus and chicken sperm and repositioning of sex chromosomes during mammalian evolution. Chromosoma 118, 53–69.
| Higher-order genome organization in platypus and chicken sperm and repositioning of sex chromosomes during mammalian evolution.Crossref | GoogleScholarGoogle Scholar | 18726609PubMed |
Tsend-Ayush, E., Kortschak, R. D., Bernard, P., Lim, S. L., Ryan, J., Rosenkranz, R., Borodina, T., Dohm, J. C., Himmelbauer, H., and Harley, V. R. (2012). Identification of mediator complex 26 (Crsp7) gametologs on platypus X1 and Y5 sex chromosomes: a candidate testis-determining gene in monotremes? Chromosome Res. 20, 127–138.
| Identification of mediator complex 26 (Crsp7) gametologs on platypus X1 and Y5 sex chromosomes: a candidate testis-determining gene in monotremes?Crossref | GoogleScholarGoogle Scholar | 22215486PubMed |
Veyrunes, F., Waters, P. D., Miethke, P., Rens, W., McMillan, D., Alsop, A. E., Grützner, F., Deakin, J. E., Whittington, C. M., Schatzkamer, K., Kremitzki, C. L., Graves, T., Ferguson-Smith, M. A., Warren, W., and Marshall Graves, J. A. (2008). Bird-like sex chromosomes of platypus imply recent origin of mammal sex chromosomes. Genome Res. 18, 965–973.
| Bird-like sex chromosomes of platypus imply recent origin of mammal sex chromosomes.Crossref | GoogleScholarGoogle Scholar | 18463302PubMed |
Vigilant, L. (1999). An evaluation of techniques for the extraction and amplification of DNA from naturally shed hairs. Biol. Chem. 380, 1329–1331.
| An evaluation of techniques for the extraction and amplification of DNA from naturally shed hairs.Crossref | GoogleScholarGoogle Scholar | 10614826PubMed |
Wallage, A., Clarke, L., Thomas, L., Pyne, M., Beard, L., Ferguson, A., Lisle, A., and Johnston, S. (2015). Advances in the captive breeding and reproductive biology of the short-beaked echidna (Tachyglossus aculeatus). Aust. J. Zool. 63, 181–191.
| Advances in the captive breeding and reproductive biology of the short-beaked echidna (Tachyglossus aculeatus).Crossref | GoogleScholarGoogle Scholar |
Wallis, M. C., Waters, P. D., Delbridge, M. L., Kirby, P. J., Pask, A. J., Grützner, F., Rens, W., Ferguson-Smith, M. A., and Graves, J. A. (2007). Sex determination in platypus and echidna: autosomal location of SOX3 confirms the absence of SRY from monotremes. Chromosome Res. 15, 949.
| Sex determination in platypus and echidna: autosomal location of SOX3 confirms the absence of SRY from monotremes.Crossref | GoogleScholarGoogle Scholar | 18185981PubMed |
Warren, W. C., Hillier, L. W., Marshall Graves, J. A., Birney, E., Ponting, C. P., Grützner, F., Belov, K., Miller, W., Clarke, L., Chinwalla, A. T., et al. (2008). Genome analysis of the platypus reveals unique signatures of evolution. Nature 453, 175–183.
| Genome analysis of the platypus reveals unique signatures of evolution.Crossref | GoogleScholarGoogle Scholar | 18464734PubMed |
Woods, J. G., Paetkau, D., Lewis, D., McLellan, B. N., Proctor, M., and Strobeck, C. (1999). Genetic tagging of free-ranging black and brown bears. Wildl Soc. Bull. 27, 616–627.
