Molecular sex identification of Malaysian White‐Nest Swiftlet (Aerodramus fuciphagus Thunberg, 1812)

Abstract The difficulty in differentiating the sex of monomorphic bird species has made molecular sexing an important tool in addressing this problem. This method uses noninvasively collected materials such as feathers and may be advantageous for sexing endangered as well as commercialized bird species. In this study, seven primer sets for sexing birds were screened in Aerodramus fuciphagus using a total of 13 feather samples that were randomly selected from the state of Perak, Malaysia. From the screening analysis, only one primer set (P8/WZ/W) successfully differentiated the sex of A. fuciphagus. PCR amplification produced a single 255‐bp DNA fragment for males which was derived from CHD‐Z (CHD gene region in the sex chromosome Z), while for the females it produced two fragments (144 and 255 bp). The 144‐bp fragment was from CHD‐W (CHD gene region in the sex chromosome W). Results from sequencing showed no variations in the base sequences of the CHD‐W and CHD‐Z amplified fragments within the same sexes, except for one male sample (A23) where at position 166, a base substitution occurred (G → A). Phylogenetic analysis of CHD‐W showed that four (Apodiformes; Gruiformes; Passeriformes; and Pelecaniformes) out of the five orders investigated had formed four clear clusters within their orders, including the studied order: Apodiformes. Whereas in CHD‐Z, four (Accipitriformes; Columbiformes; Galliformes; and Passeriformes) out of five orders investigated formed four clear clusters within their orders, excluding the studied order. In addition, A. fuciphagus and Apus apus (both Apodiformes) showed less divergence in CHD‐W than CHD‐Z (0% c.f. 9%). The result suggests that in A. fuciphagus, CHD gene evolution occurred at a higher rate in males (CHD‐Z) compared to females (CHD‐W). This finding may be useful for further studies on sex ratio and breeding management of A. fuciphagus.


| INTRODUC TI ON
Individuals' sex information is important in the field of evolutionary biology, breeding, and conservation (Fridolfsson & Ellegren, 1999).
Animals may be sexed using morphological, surgical, cytological, and molecular methods. Among these methods, molecular sexing is considered as the most accurate method (Dubiec & Zalagska-Neubauer, 2006), especially for sexing animals that lack sexual dimorphism, such as most birds. Molecular sexing in birds is based on PCR amplification of alleles on Z and W chromosomes using specific primers designed to screen variations in length and nucleotide sequence (Morinha, Cabral, & Bastos, 2012). Female birds are heterogametic (ZW) while males are homogametic (ZZ). Over the years, numerous molecular sexing markers have been designed to accurately sex birds. However, due to the diverse species of birds investigated, only a few molecular sexing markers were able to successfully differentiate the sex of these birds.
Molecular sexing in birds was initiated by Griffiths and Tiwari (1995). They discovered the W-linked gene that was later identified for encoding a chromo-helicase-DNA-binding protein (CHD) which found to present in most birds (Ellegren, 1996;Griffiths, Daan, & Dijkstra, 1996), and related to human CHD1 (Woodage, Basrai, Baxevanis, Hieter, & Collins, 1997). A related Z-linked gene, CHD-Z, had been reported by Griffiths and Korn (1997). Griffiths et al. (1996) proved that this gene is remarkably conserved, and a single set of PCR primer may be used for molecular sexing in birds throughout the class Aves, with the exception of ratites. This primer simultaneously amplifies the homologous parts of CHD-W and CHD-Z. The two amplified CHD products were of the same size but may be differentiated using restriction enzyme which selectively cuts the CHD-Z DNA fragment, generating two electrophoretic bands for the females and only one for the males. To overcome the problem of the same PCR product sizes for CHD-W and CHD-Z, Griffiths, Double, Orr, and Dawson (1998) proposed a protocol for sexing of birds using two PCR primers which anneal at the conserved exonic region and amplified across an intron, which being noncoding and less conserved, varying in length between CHD-W and CHD-Z. Fridolfsson and Ellegren (1999), however, claimed that the primers suggested by Griffiths et al. (1998) were not always successful in differentiating CHD-W and CHD-Z fragments by using standard agarose electrophoresis, and therefore, proposed another set of primer which consistently amplified different sizes of DNA fragments for CHD-W and CHD-Z in the majority of bird species. This particular pair of primer (2550F and 2718R) was designed based on the constant size differences between CHD1W and CHD1Z introns. Using highly conserved pair of primer flanking the intron, PCR amplification, and agarose gel electrophoresis, female birds displayed either one (CHD1W) or two fragments (CHD1W and CHD1Z), while males showed a single fragment (CHD1Z) with a clear difference in size from the female-specific CHD1W fragment. From the 50 bird species studied, 47 species successfully amplified the CHD1W fragment between 400 and 450 bp in size and CHD1Z fragment between 600 and 650 bp. Later, an almost similar method was designed by Kahn, St. John, and Quinn (1998) which based on two highly conserved regions that flanked the intervening introns, common to both Z-linked and W-linked CHD genes. From it, a pair of primer was synthesized (1237L and 1272H). The sizes of amplification products for this pair of primer differ over a range of 210-285 bp for each bird species (total of 17 bird species investigated), and the size differences between the W-and Z-specific copies of this amplification also vary among those bird species. Thus, some of the bird species showed a very small size difference such as found in Red-tailed Hawk (Buteo jamaicensis) and Great Horned Owl (Bubo virginiasus). Furthermore, in some other bird species, the size differences cannot be resolved on standard agarose gel electrophoresis and needed the use of non-denaturing polyacrylamide gel electrophoresis method.
The most widely used molecular sexing method for birds usually involves the exploitation on size differences between introns of the CHD gene on the Z and W sex chromosomes (Fridolfsson & Ellegren, 1999;Kahn et al., 1998). Heterogametic (ZW) females are expected to have two different sized introns while homogametic (ZZ) males should only have one intron size. However, the existence of polymorphism in the introns of CHD-Z in several species has complicated the identification of sex because heterozygote (e.g., ZZ') males will also have two different sized introns (Dawson et al., 2001;Lee, Brain, Forman, Bradbury, & Griffiths, 2002;Robertson & Gemmell, 2006). Due to this concern, another molecular sexing primer set to improve the reliability in birds sexing had been introduced by amplifying an additional W chromosome-specific DNA fragment of a different size than that produced by the pre-existing sexing primer pairs to independently confirm the existence of the W chromosome. Therefore, Shizuka and Lyon (2008) developed a reverse primer (GWR2) designed to sit within the intron of the W chromosome and amplify a small DNA fragment that served as a W-specific marker. This primer combines with the primer pair (1237L and 1272H) developed by Kahn et al. (1998) and subsequently used to amplify the genes of the individual birds from American coots' species (Fulica americana). It was essentially a multiplex reaction of two primer pairs, the 1237L/1272H that amplified the entire CHD1 intron of both Z and W chromosomes, and 1237L/GWR2 pair that amplified a short fragment when the W chromosome was present.
The other recent molecular sexing in bird species was conducted by Hagadorn, Tell, Drazenovich, and Ernest (2016)which had evaluated 11 primer pairs from CHD region on sex chromosomes in hummingbird and obtained only two primer sets (P8/P2 and 1237L/1272H) resulted in reliable DNA amplification to determine sex in this species of bird. Therefore, molecular sexing for determining sex in avian species is important to accurately identify sex for sustainable species management (e.g., breeding) and provides vital data for comparative genomic studies such as sex evolutionary.
Though many studies on molecular sexing in birds had been conducted, but no study for Aerodramus fuciphagus (White-nest swiftlet; Figure 1) which is a bird with lack of sexual dimorphism has been done. This bird species showed no distinguishable external feature or behavior between male and female which make it hard to differentiate their sex using morphological method. It is important to be able to differentiate males from females of A. fuciphagus for sustainable breeding, to ensure population viability and growth of this economically important swiftlet species which produces nests that are edible (Lim, 2011). The nests have high commercial values because they are made from pure saliva of the birds (Kang & Lee, 1991) and contain ~8.6% sialic acid (a major component of glycoprotein) which is considered of high medicinal and nutrient benefits (Chau et al., 2003;Kathan & Weeks, 1969;Vimala, Hussain, & Nazaimoon, 2012). Nutrients in the nests have been claimed to be beneficial for reducing the risk of cardiometabolic conditions associated with estrogen deficiency such as diabetes and cardiovascular disease (Hou, Imam, Ismail, Ooi, et al., 2015). EBN may also possess anti-inflammatory (Vimala et al., 2012) and antioxidant properties (Hou, Imam, Ismail, Azmi, et al., 2015). Furthermore, EBN has been suggested to be effective for the improvement of bone loss (Matsukawa et al., 2011) and it may be a potential antidegenerative agent for treating osteoarthritis (Chua et al., 2013). Therefore, this study aims to identify molecular sexing markers for A. fuciphagus and compare the amplified CHD-W and CHD-Z sequences with other bird species.

| Sample collection and PCR protocol
In an attempt to obtain samples of male and female swiftlets, a postmortem was conducted on 13 randomly selected A. fuciphagus carcasses from Northern Region (Perak) swiftlet houses to positively confirm the sex of each individual via gross visual identification of the sex gonads. From the 13 individual carcasses dissected, the sex gonads could be identified in only three of the carcasses. Based on the distinct testes identified, these three carcasses were confirmed as male A. fuciphagus ( Figure 2) and were used as control (M1, M2, and M3) in the subsequent investigation for molecular sexing. On the other hand, feather samples collected from 10 unknown sex of dead chicks or chicks that had dropped from their nests at the breeding colonies were used as candidate samples to screen for the sexing markers. Hence, the electrophoresis banding patterns for the 10 candidate samples (unknown sex) were compared to those of the controls. Samples showed similar patterns as the confirmed males were identified as male individuals, and samples showed banding patterns that differed from the controls were assumed to be female individuals.
All thirteen A. fuciphagus individuals including three males (determine by postmortem) and 10 unknown sex were used as samples in this molecular sex identification study. All samples were taken from the calamus tip of a primary feather and extracted for DNA using DNeasy ® Blood & Tissue Kit (Qiagen, Hilden, Germany) by following the manufacturer's protocol. A total of seven different molecular sexing primers were used in this study (Table 1). Primers P8/P2/P0, P8/WZ/W, and 1237L/1272H/GW2 used the multiplex PCR method. PCR was carried out in a total volume of 25 μl containing 5 µl of 5 × PCR buffer, 50 ng/µl of genomic DNA, 1 unit of Taq polymerase (Promega Corporation), 1.5 mM MgCl2, 1 µM of each primer, 0.2 mM of dNTPs, and distilled water. Touchdown PCR protocol for all samples using primer set of P8/WZ/W was as follows: initial denaturation at 95°C for 5 min, followed by 10 cycles of 95°C denaturation for 30 s, 60°C to 50°C (decrease temperature by 1°C in

| Nucleotide sequence analysis
PCR products that showed variations in genotypic patterns were chosen for further investigation. The PCR products were purified and sent for sequencing to First Base Laboratories Sdn Bhd, Selangor. One male control (sex determine by post-mortem) together with two expected male and two expected female samples (identified as such from banding patterns) were selected for sequencing (a total of five samples). The two expected male and one male control samples displayed only a single band pattern on electrophoresis, and therefore, the PCR products were purified using PCR purification method. Meanwhile, the two expected female samples showed two band patterns on electrophoresis, the PCR product bands of interest (144-bp band) were visualized with the help of UV transilluminator and excised from the agarose gel using a sterile scalpel and transferred into a 1.5-ml microcentrifuge tube and weighed before purified using gel purification method. All selected samples were purified using Geneall ® ExpinTM Combo GP (Geneall Biotechnology, Korea) by following the manufacturer's protocol.

| Data analysis
The nucleotide sequences of representative males and females of A.

| RE SULTS
In this study, seven avian sexing marker sets based from CHD region on sex chromosome in birds had been screened and results showed that only one sexing marker set (P8/WZ/W) could successfully differentiate males from females A. fuciphagus (Figure 3). The results from this study showed the presence of CHD-Z gene (255 bp;  genes also showed that there were sex evolutionary patterns in CHD region on sex chromosomes among bird species (Figures 4 and 5). In this study, only one sexing marker set (P8/WZ/W) from seven avian sexing marker sets based from CHD region on sex chromosome in birds could successfully differentiated males from females A. fuciphagus (Figure 3). This may attribute to the fact that  and CHD-W (140 bp) genes (Wang et al., 2011). Wang et al. (2011) reported that there were some advantages in this method compared with the use of P2/P8 primer-based technique. First of all is the fact