Molecular sexing of threatened Gyps vultures: an important strategy for conservation breeding and ecological studies.pdf
RESEARCH Open Access Molecular sexing of threatened Gyps vultures: an important strategy for conservation breeding and ecological studies Prabhakar B Ghorpade1, Praveen K Gupta2, Vibhu Prakash3, Richard J Cuthbert4, Mandar Kulkarni3, Nikita Prakash3, Asit Das1, Anil K Sharma1 and Mohini Saini1* Abstract During the last two decades populations of three resident species of Gyps vulture have declined dramatically and are now threatened with extinction in South Asia. Sex identification of vultures is of key importance for the purpose of conservation breeding as it is desirable to have an equal sex ratio in these monogamous species which are housed together in large colony aviaries. Because vultures are monomorphic, with no differences in external morphology or plumage colour between the sexes, other methods are required for sex identification. Molecular methods for sex identification in birds rely on allelic length or nucleotide sequence discrimination of the chromohelicase-DNA binding (CHD) gene located on male and female chromosomes ZZ and ZW, respectively. We characterized the partial sequences of CHD alleles from Gyps indicus, Gyps bengalensis, Gyps himalayensis and Aegypius monachus and analysed the applicability of five molecular methods of sex identification of 46 individual vultures including 26 known-sex G. bengalensis and G. indicus. The results revealed that W-specific PCR in combination with ZW-common PCR is a quick, accurate and simple method, and is ideal for sex identification of vultures. The method is also suitable to augment ecological studies for identifying sex of these endangered birds during necropsy examinations especially when gonads are not apparent, possibly due to regression during non-breeding seasons. Keywords: Molecular sex identification, Gyps vulture, Cinereous vulture, Vulture conservation, Captive breeding Background Nine species of vultures in the family Accipitridae are found in India, three of which are endemic to South and South-East Asia (the Oriental white-backed vulture (Gyps bengalensis), long-billed (G. indicus) and slender- billed vulture (G. tenuirostris) and are classified as Critically Endangered by the International Union for Conservation of Nature and Natural resources and are at high risk of extinction in the wild (IUCN, 2011). In India, populations of G. bengalensis have declined by more than 99.9% while those of G. indicus and G. tenuirostris have declined by around 97% between the early 1990s and 2007 (Prakash et al. 2007). Similar reductions in vulture populations have been recorded in Pakistan and Nepal (Pain@ et al. 2008). Although the Himalayan griffon (G. himalayensis) is not considered threatened (under category Least Concern) (IUCN, 2011), its population de- cline has been recorded in Nepal (Acharya et al. 2009). The status of another species, the Cinereous Vulture (Aegypius monachus), is classified as Near Threatened as per IUCN (IUCN, 2011). Veterinary use of non-steroidal anti-inflam- matory drugs (NSAIDs) such as diclofenac and ketoprofen have been shown to be toxic to Gyps vultures and are responsible for the decline of these species (Oaks et al. 2004; Green et al. 2006, 2007; Swan et al. 2006; Cuthbert et al. 2009; Naidoo et al. 2009, 2010; Das et al. 2011). In contrast the NSAID meloxicam has been demonstrated to be a safe and effective alternative drug for veterinary use (Swan et al. 2006; Swarup et al. 2007). Although the veter- inary use of diclofenac has been banned in India, Pakistan and Nepal (Kumar 2006; Singh 2008), it’s illegal use is still * Correspondence: mohini@ivri.res.in 1Centre for Wildlife Conservation, Management & Disease Surveillance, Indian Veterinary Research Institute, Izatnagar 243 122, India Full list of author information is available at the end of the article a SpringerOpen Journal © 2012 Ghorpade et al.; licensee Springer. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Ghorpade et al. SpringerPlus 2012, 1:62 http://www.springerplus.com/content/1/1/62 apparent, as diclofenac residues are still prevalent in cattle carcasses across India at concentrations sufficient to cause declines in vulture populations (Cuthbert et al. 2011a, 2011b; Saini et al. 2012). Due to the massive scale of the population declines and the continued use of diclofenac, populations of the three Critically Endangered resident Gyps species are being bred in captivity in India, Nepal and Pakistan, with the aim that their progeny will be introduced back in to the wild after ensuring that the environment is safe and diclofenac free (MoEF 2006; Bowden 2009). Vultures are monomorphic monogamous species and hence without knowing the sex of birds it is difficult to maintain the correct sex ratios in aviaries at conservation breeding centres in order to maximise the chances of successful breeding. As well as the key importance of iden- tifying gender for conservation breeding programmes, knowledge of sex is also important to complement forensic studies (An et al. 2007) and investigations on evolution and ecology (Griffiths and Tiwari 1995; Costantini 2008; Fukui et al. 2008). Various techniques have been employed for sex deter- mination of monomorphic birds such as laparotomy (Risser 1971), laparoscopy (Richner 1989), flow cytometry (Nakamura et al. 1990), karyotyping (Hatzofe and Getreide 1990) and Raman spectroscopy (Harz et al. 2008) but mo- lecular methods based on DNA analysis are most prevalent (Fridolfsson and Ellegren 1999). Except for the ratites, that have undifferentiated sex chromosomes, all male birds are homogametic with ZZ sex chromosomes and females are heterogametic with ZW sex chromosomes (Ellegren 1996; Griffiths et al. 1996). The most frequently exploited gene for sex identification is the Chromohelicase DNA binding (CHD) gene that is found conserved on both W and Z chromosomes (Griffiths 2000). Intronic length variation in CHD-Z and CHD-W allelles amplified by Griffiths univer- sal CHD primer pair P2/P8 has formed the basis of gender identification in most avian species (Griffiths et al. 1998; Fridolfsson and Ellegren 1999). However, in certain species of Accipitridae there is an extremely short difference in in- tronic length between CHD-Z and CHD-W P2/P8 ampli- con which makes sex identification more difficult and inaccurate (Ito et al. 2003; Chang et al. 2008). Hence, in order to circumvent the limitation of conventional PCR (Fridolfsson and Ellegren 1999), different approaches detecting small variation in nucleotides like Amplification Refractory Mutation System (ARMS) (Ito et al. 2003), Re- striction Fragment Length Polymorphism (RFLP) (Sacchi et al. 2004), Single strand conformation polymorphism (SSCP) (Ramos et al. 2009), Melting Curve analysis (Chang et al. 2008a), ZW common and W-specific PCR (Chang et al. 2008b), TaqMan Probe-based real time PCR (Chang et al. 2008c; Chou et al. 2010) have been suggested in order to identify gender in these species. Old World vultures along with other birds of prey be- long to the taxonomic order Falconiformes, family Acci- pitridae, and subfamily Accipitrinae (Chang et al. 2008b, 2008c). Due to their position within the Accipitridae it was observed that intronic length variation of CHD-Z and CHD-W amplicon in Griffiths universal CHD primer pair P2/P8 based PCR is unlikely to be suitable for sex discrimination in G. indicus or G. bengalensis (Reddy et al. 2007). However, a similar approach (Kahn et al. 1998) using denaturing polyacrylamide gel electrophor- esis combined with autoradiography has been reported to sex nestlings of these two species (Arshad et al. 2009). In the present study, based upon the chromohelicase gene sequences in male (ZZ) and female birds (ZW), the accuracy and reliability of five different approaches are compared for molecular gender identification in three vul- ture species (G. indicus, G. bengalensis, G. himalayensis) in order to identify an accurate and simple test to support the captive breeding programmes. Results Sequence characterization of CHD-Z and CHD-W sequences The CHD-Z and CHD-W sequences from the four vulture species used in this study were amplified and the sequences were determined. These sequences were sub- mitted to GenBank and accession numbers obtained were HQ236387, HQ236386 (G. indicus); HQ236388, HQ236385 (G. bengalensis); HQ236384, HQ236383 (G. himalayensis); HQ236382 (A. monachus). Independent alignment reports for CHD-Z and CHD-W sequences were prepared (Figure 1A and B), where primer binding regions for P2, P8, NP, MP; ZW-Common and W-specific primers and probes as well as restriction site for BamHI and RsaI were located. The primer binding region for MP and W-specific primers were found in all CHD-W but not in CHD-Z sequences. The recognition sequence for BamHI was found on CHD-Z but was absent on the CHD-W sequence, whereas the RsaI restriction site was located at different positions in the CHD-Z and CHD-W sequences. Based on these identified sequences the applicability and accuracy of PCR-RFLP, ARMS-PCR, W-specific PCR and TaqMan probe based real-time PCR methods for sex iden- tification was tested for all four species of vultures (Table 1). Standardization of PCR-based molecular methods for sex identification i) Conventional PCR-RFLP For standardization of conventional PCR-RFLP, known sex samples from G. bengalensis and G. indicus, G. hima- layensis and A. monachus were used. It was evident from Ghorpade et al. SpringerPlus 2012, 1:62 Page 2 of 12 http://www.springerplus.com/content/1/1/62 Restriction endonuclease RsaI and BamHI sites were selected for sex identification in PCR-RFLP analysis. Standardization of PCR-based molecular methods for sex identification i) Conventional PCR-RFLP Using Griffiths universal CHD primer pair P2/P8, the amplified PCR products were analysed using restriction endonuclease digestion with RsaI and BamHI and sex was identified. The restriction digestion was performed in a 30 μl reaction volume containing 5 μl of amplified PCR product and 2 U of restriction enzymes (RsaI or BamHI) and was incubated at 37°C overnight. The digested products were separated on 3% agarose gel along with 100 bp DNA ladder and analysed. ii) ARMS-PCR ARMS-PCR based on 3’-terminal mismatch primer (MP primer) point mutation conserved among Falconiformes CHD-W and CHD-Z sequences previously reported (Ito et al. 2003) was performed to identify sex in vultures with some modifications. Briefly, PCR was done in a 25 μl reaction volume containing 0.4 μM each of Griffiths univer- sal CHD primer P2 forward primer, another forward primer MP (5’-AGTCACTATCAGATCCGGAA-3’) and reverse primer NP (5’-GAGAAACTGTGCAAAACAG -3’), 100 ng genomic DNA, 0.2 mM each dNTP and 1U of Taq DNA Polymerase (Bangalore Genei, India). PCR amplification cycle involved initial denaturation at 94°C for 90 sec followed by 35 cycles of 94°C for 30 sec, 50°C for 45 sec, 72°C for 30 sec and final extention at 72°C for 5 min. The amplified PCR products were separated on 3% agarose gel along with 100 bp DNA ladder and analysed. iii) W-specific PCR An alternative W-specific sex identification method suggested for Crested Serpent Eagle (Spilornis cheela hoya) (Chang et al. 2008b) was also used in this study, where Griffith’s universal CHD primer P2 was used as a forward primer and CHD-W primer as a reverse primer which anneals to only the CHD-W allele sequence, or ZW-common primer which anneals to both CHD-Z and CHD-W allele sequences. The PCR reaction was performed in a 25 μl volume consisting of 0.4 μM each of Griffith’s universal CHD primer P2 and reverse primer CHD-ZW-common (5’-GATCAGCTTTAATGGA AGTGAAG-3’) or CHD-W specific (5’-GGTTTTCACAC ATGGCACA-3’), 100 ng genomic DNA, 0.2 mM each dNTP, 1.5 μl DMSO and 1U Taq DNA Polymerase (Bangalore Genei, India). The PCR cycling condition employed was an initial denaturation at 94°C for 3 min, followed by 45 repeated cycles of 94°C for 30 sec, 56°C for 30 sec, 72°C for 20 sec, and final extension at 72°C for 5 min. The amplified PCR products were resolved on 3% agarose gel along with 100 bp DNA ladder and analysed for presence (indicating female) or absence (indicating male) of 263 bp W-specific product. iv) TaqMan probe based real-time PCR The TaqMan based qualitative real-time PCR (qPCR) based on allele discrimination option for sex identifica- tion reported earlier for S. cheela hoya (Chang et al. 2008c) was used. This test utilises the considerable difference in composition of the CHD-W and CHD-Z sequences in vultures, with the W-specific probe (5’-FAM-TGTGCCATGTGTGAAAACCACCCA-TAMR A) recognising only the CHD-W region whereas the ZW common probe (5’-HEX-CCCTTCACTTCCAT TAAAGCTGATCTGG-TAMRA) recognises both the Z and W CHD chromosome regions. The PCR reaction mixture in a 20 μl volume consisted of 0.4 μM each of Griffith’s universal CHD primer pair P2/P8, 50–100 ng genomic DNA, 0.2 mM of each dNTP, 20nM each of W-specific and ZW common probes and 1 U of Taq DNA polymerase (Bangalore Genei, India). The DNA template was excluded from no template control (NTC), whereas the probe was excluded from no probe control (NPC). In addition, positive controls (with known male and female DNA samples) were also included in each test. Two steps PCR condition was employed with initial denaturation at 94°C for 4 min, followed by 50 repeated cycles of 92°C for 15 sec, 60°C for 1 min in Mx3005P real-time PCR machine (Agilent, USA). The results were recorded as an amplification plot, with text report and alleles discrimination made using MxPro™QPCR soft- ware (Agilent, USA) and compared with female and male positive controls. Application of the molecular methods for sex identification All molecular methods were employed for sex identifica- tion of vultures using tissue samples obtained during necropsy (n = 17) and blood samples obtained from live birds (n = 9) for which the sex was known. These tests were then employed for analysing eight blood samples and 12 necropsy tissues from unknown-sex vultures. Abbreviations CHD: Chromohelicase-DNA binding gene; IUCN: International Union for Conservation of Nature; NSAIDs: Non-steroidal antiinflammatory drugs; RFLP: Restriction Fragment Length Polymorphism; ARMS: Amplification Refractory Mutation System; SSCP: Single strand conformation polymorphism; qPCR: Qualitative real-time PCR; VCBC: Vulture Conservation Breeding Centre. Ghorpade et al. SpringerPlus 2012, 1:62 Page 10 of 12 http://www.springerplus.com/content/1/1/62 Competing interests The authors declare that they have no competing interests. Authors’ contributions PBG carried out cloning and characterization of sequences, performing tests, preparation of the draft and revision of the manuscript. PKG participated in conceiving the design of the study, performed sequence analysis and interpretations and helped in drafting and revising the manuscript. VP conceived the problem, coordinated the collection of samples from field post-mortems and breeding centres and helped to draft the manuscript. RJC participated in coordination of the study, preparing draft and critically revising the manuscript. MK and NP collected the tissue and blood samples from vultures. AD and AKS participated in collection of one tissue sample from Gyps himalayensis from necropsy examination, design of the study and helped to draft the manuscript. MS contributed in conception of the study, execution of the experiments, analysis and interpretation of data, drafting and revising the manuscript. All authors read and approved the final manuscript. Acknowledgements We thank the Director and Joint Director (Research), IVRI, Izatnagar and the Director, BNHS, Mumbai for providing the necessary facilities and funding to carry out this work. We acknowledge laboratory assistance provided by Mr Mohan Bhat, IVRI. The vulture conservation breeding centres in India are run by the Bombay Natural History Society (BNHS), India in collaboration with the state forest departments and Ministry of Environment and Forests, Government of India and supported by UK based organizations Royal Society for Protection of Birds (RSPB), Darwin Initiative for the survival of species, Zoological Society of London and National Birds of Prey Trust. Author details 1Centre for Wildlife Conservation, Management & Disease Surveillance, Indian Veterinary Research Institute, Izatnagar 243 122, India. 2Division of Veterinary Biotechnology, Indian Veterinary Research Institute, Izatnagar 243 122, India. 3Bombay Natural History Society, Hornbill House, S.B. Singh Road, Mumbai 400 001, India. 4Royal Society for the Protection of Birds, The Lodge, Sandy, Bedfordshire, UK. 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Available at http://www. cza.nic.in doi:10.1186/2193-1801-1-62 Cite this article as: Ghorpade et al.: Molecular sexing of threatened Gyps vultures: an important strategy for conservation breeding and ecological studies. SpringerPlus 2012 1:62. Submit your manuscript to a  journal and beneﬁ t from: 7 Convenient online submission 7 Rigorous peer review 7 Immediate publication on acceptance 7 Open access: articles freely available online 7 High visibility within the ﬁ eld 7 Retaining the copyright to your article     Submit your next manuscript at 7 springeropen.com Ghorpade et al. SpringerPlus 2012, 1:62 Page 12 of 12 http://www.springerplus.com/content/1/1/62
Title: Molecular sexing of threatened Gyps vultures: an important strategy for conservation breeding and ecological studies ​
Authors: Prabhakar B Ghorpade, Praveen K Gupta, Vibhu Prakash, Richard J Cuthbert, Mandar Kulkarni, Nikita Prakash, Asit Das, Anil K Sharma, Mohini Saini ​
Publisher: SpringerPlus
Date: December 12, 2012 ​
Abstract: During the last two decades populations of three resident species of Gyps vulture have declined dramatically and are now threatened with extinction in South Asia. ​ Sex identification of vultures is of key importance for the purpose of conservation breeding as it is desirable to have an equal sex ratio in these monogamous species which are housed together in large colony aviaries. ​ Because vultures are monomorphic, with no differences in external morphology or plumage colour between the sexes, other methods are required for sex identification. ​ Molecular methods for sex identification in birds rely on allelic length or nucleotide sequence discrimination of the chromohelicase-DNA binding (CHD) gene located on male and female chromosomes ZZ and ZW, respectively. ​ We characterized the partial sequences of CHD alleles from Gyps indicus, Gyps bengalensis, Gyps himalayensis and Aegypius monachus and analysed the applicability of five molecular methods of sex identification of 46 individual vultures including 26 known-sex G. bengalensis and G. indicus. ​ The results revealed that W-specific PCR in combination with ZW-common PCR is a quick, accurate and simple method, and is ideal for sex identification of vultures. ​ The method is also suitable to augment ecological studies for identifying sex of these endangered birds during necropsy examinations especially when gonads are not apparent, possibly due to regression during non-breeding seasons. ​