Eggshell membrane for DNA sexing of the endangered Maleo (Macrocephalon maleo)

Introduction

Studies on molecular ecology have a great impact on our knowledge on ecology and evolution of animals, i.e. the phylogenetic relationships and systematics of organisms, population genetics, mating systems, micro-evolutionary processes and host-parasite interactions^1–4. Oftentimes a necessary prerequisite for answering evolutionary or ecological questions is access to a good DNA sample. Birds’ blood contains nucleated red blood cells with abundant DNA, making it a preferred source of DNA⁵. However, obtaining blood requires the capture of a bird, which can provoke an increased level of stress and might results in unusual behavior or nest desertion. For example, blood sampling has been reported to reduce annual survival in Cliff Swallows (Petrochelidon pyrrhonota)⁶, although the effects of blood collection in free-living adult and immature birds is not thought to have major negative effects on adult survival, reproductive success, body condition, or behavior⁷.

As an alternative to invasive sampling, researchers have adopted noninvasive sampling methods such as DNA capture from molted feathers^8,9, feces^10,11, and egg shell membrane^12,13. In addition the application of moderately invasive sampling such as buccal swabs^14,15 has also increased. Megapodes (family Megapodiidae) are a galliform clade, centered in Australasia¹⁶, that are known for their unique super-precocial behavior¹⁷. Megapodes are also renowned as the only bird species that use environmental heat rather than body temperature (brood) to incubate their eggs¹⁸. Their ground-living habits, large body size and large egg size make them particularly vulnerable to human persecution, habitat destruction and habitat loss: 11 out of 21 species are now considered endangered or threatened in some form 19. Given this precarious conservation situation, the application of noninvasive DNA sampling techniques is crucial for megapode birds. Yet previous genetic studies on this family have used either blood²⁰ or fresh tissue²¹.

Previously, all megapodes were assumed to be monogamous. The mating system is considered to correlate with sexual selection, with sexually dimorphic birds are non-monogamous and monomorphic birds are monogamous. The evolution of non-monogamous systems in birds was believed to be an adaptive solution to an unbalanced sex ratio²². The sex-ratio in Maleo (Macrocephalon maleo) is unknown, but based on previous assumptions, it is expected that the Maleo has an evenly balanced sex ratio. Even though Maleo are slightly sexual dimorphic, the available population data only report total population size and never mention sex ratio. A study on the correlation of incubation temperature and sex ratio of chicks has been carried out in the Australian brush-turkey (Alectura lathami), which revealed that at average temperature the hatched chicks in the proportion of 1:1 of male and female chicks²³.

The purpose of our study was to determine whether the eggshell membrane of the endangered Maleo, a monotypic genus within the megapodes, could be successfully extracted and amplified for DNA sexing. Adult male and female Maleo are morphologically slightly different, but the chicks are not. To determine the sex in Maleo chicks, vent sexing has been conducted. Base on cloaca size and shape, a one-day-old male Maleo chick cloaca is bigger (3.96 ± 0.11 cm) and rounded, than the female cloaca (3.20 ± 0.10 cm), which is more oval in shape. The concentration of estrogen in female birds was also higher²³. Until recently, no molecular technique has been applied for sex determination of Maleo. As in all non-ratite birds, determination of sex in Maleo are based on heteromorphic Z and W chromosomes. Female birds are heterogametic sex ZW, meanwhile males are homogametic sex ZZ^24,25. Maleo are endemic to Sulawesi, Indonesia^16,26. The bird is a burrow-nesting megapode that incubates its eggs in communal nesting sites on beaches (coastal nesting grounds) and in soil heated by volcanic activity mostly at inland localities. Due to its small, severely fragmented population and continued rapid decline, the International Union for Conservation of Nature has classified Maleo as an endangered species²⁷. Among the major threats are the over-exploitation of eggs and loss of connectivity between forest and nesting grounds²⁸. To minimize these threats at some nesting grounds, conservation programs are currently removing eggs and hatching them in safer, semi-natural hatcheries, built close to the nesting grounds. These facilities provide an opportunity to collect non-invasive DNA samples from the eggshell membrane left in the soil or brought to the soil surface by the hatched Maleo. The DNA of the chicks can be obtained from the extraembryonic membranes that remain attached to eggshell membranes of post-hatched eggs. The extraembryonic membranes consist of the amnion, allantois and chorion, and form to support the life and growth of the bird embryo²⁹. During the embryonic development stage, the allantois sac expands, causing the inner shell membrane and chorion being combined and forming a chorioallantois membrane (CAM), which contains lots of blood capillaries^29,30.

Methods

Study sites and genetic sampling

Post-hatched egg-shell membranes were collected from semi-natural hatcheries of Maleo at two different nesting grounds: an inland geothermal heated nesting ground at Tambun (Bogani Nani Wartabone National Parks and a sun-heated sand beach nesting ground at Tanjung Binerean, North Sulawesi, Indonesia (Figure 1). All samples were collected from 4^th April until 1^st May 2018. Samples were egg-shell membranes from maleo chicks that hatched less than 24 hours earlier based on records of staff from semi-natural hatcheries. The samples were collected from the surface and inside the substrate (soil and sand) where the eggs are laid. To prevent post-sampling contamination, each sample was placed separately in a zip-lock plastic bag and stored in silica gel for delivery to laboratory. The samples were stored at -40°C until DNA extraction were conducted.

Figure 1. Sampling sites of eggshell membrane of Maleo (Macrochepalon maleo) in North Sulawesi, Indonesia.

Results

All eggshell membranes were successfully extracted, with mean DNA concentrations around 267.5 ng/µl (range 47–510.5 ng/µl). The average DNA concentration extracted from eggshell membrane collected from coastal nesting grounds (Tanjung Binerean: 213±179 ng/µl,) was significantly lesser than of that of inland nesting grounds (Tambun: 322±153 ng/µl, p=0.004; Data Supp.1). These results demonstrate that all samples were adequate for further PCR based analysis.

The 260/280 nm absorbance ratio of all samples ranged from 1.81 to 1.89, with an average of 1.85 (±0.03). Meanwhile the average for Tambun and Tanjung Binerean samples were, respectively, 1.85 (±0.03) and 1.84 (±0.01); Table 1). This result suggested good purity of DNA extracted from eggshell samples. However. gel visualization of extracted DNA showed smears in all samples (Figure 2), pointing to some DNA degradation.

Figure 2. Gel visualizing DNA degeneration of eggshell membrane samples.

Above: Tambun; Below: Tanjung Binereaan; LD: 100 bp DNA Ladder (SMOBiO); AK01: domestic chicken eggshell membrane.

Sex determination

Out of 24 samples in which extracted DNA was used as a template, one sample (MB09) was not amplified. Meanwhile all samples based on lysate were successfully amplified. There was complete agreement in gender determination across all Maleo samples that were run with different DNA template (Figure 3). Females showed two bands (545 bp and 395 bp), whereas males exhibited one band (545 bp). Three sequences of CHD1 genes have been deposited in GenBank (accession numbers MT074328, MT074329 and MT074330). Sequence similarity searches on the upper band revealed a match with CHD-Z genes of other bird species (i.e. Anser cygnoides, Anser reevesii, Anas penelope). Meanwhile the lower band matched CHD-W submissions of other birds (i.e. Gallus gallus, Crossoptilon mantchuricum, Syrmaticus reevesii).

Figure 3. Gel visualizing of CHD-genes amplification of Maleo using extracted DNA (left) and Lysate (right) for molecular sexing of Tambun (above) and Tanjung Binerean (below) samples.

AK1, positive control, female chicken; K, negative control, no template.

In total 9 samples were identified as females and 15 were males (Table 2). Four out of five samples with no allantois blood vessels were identified as females. Based on this limited sample, the sex ration of Maleo’s chicks in Tambun and Tanjung Binerean is biased towards males. The sex ratio males to females was 1.6. Care should be taken using eggs to identify the sex of the chicks. The use of freshly laid eggs resulted in inaccurate estimates of the primary ratio, due to contaminants from the DNA of the hen³⁴. In the other hand, using DNA isolated from CAM were reliable to sex the newly hatched Denizli chicken. The results of DNA sexing of chicks in all samples matched with that determined by morphological appearance of gonads³⁵.

Table 2. Identified sex of Maleo (Macrocepalon maleo) based on molecular sexing with different templates.

No.	Sample Code	Template
		Extracted DNA	Lysate
1	MT02	Male	Male
2	MT03	Male	Male
3	MT04	Male	Male
4	MT06	Male	Male
5	MT07	Male	Male
6	MT10	Female	Female
7	MT12	Female	Female
8	MT15	Female	Female
9	MT17	Male	Male
10	MT18	Male	Male
11	MT19	Male	Male
12	MT20	Male	Male
13	MB01	Female	Female
14	MB02	Male	Male
15	MB03	Male	Male
16	MB04	Female	Female
17	MB05	Male	Male
18	MB06	Male	Male
19	MB07	Male	Male
20	MB08	Female	Female
21	MB09	-	Female
22	MB10	Female	Female
23	MB11	Male	Male
24	MB12	Female	Female

Discussion

This study has demonstrated the first successful DNA isolation from eggshell membranes of a megapode bird. Our success rate (100%) compares favorably to that of previous avian eggshell membrane studies of Black-tailed Godwits (Limosa limosa), which also successfully extracted DNA from all 47 eggshell membranes¹³. The freshness of the samples might be one of the determinant factors of DNA extraction success. Our samples were relatively fresh, extracted 5–15 days after collection and kept in the freezers until extraction, with no concomitant extraction failure and high purity (1.85±0.03) and concentration (267.5 ng/µl) of DNA.

PCR amplification of CHD genes succeeded in 96% of the eggshell membrane samples, with only one eggshell membrane isolate out of 24 failing to amplify. The quantity of the DNA sample (MB09; 47 ng/µl) might be the cause of the failure, since its quality was relatively good (1.88). However, using lysate as DNA template for PCR resulted in 100% amplifications across 24 samples. This result show that eggshell membrane isolates yielded DNA with little amplification problems. Compared to blood DNA isolates, eggshell membrane DNA isolates of Black-tailed Godwits (Limosa limosa) also yielded fewer amplification problems¹³. One eggshell membrane DNA isolate out of 21 and 3 samples of Black-tailed Godwit (Limosa limosa) did not amplify for 2 and 5 of the 11 microsatellite loci. The amplification success rates was 99.1%¹³. Meanwhile the success rate of eggshell membrane of Sage Grouse (Centrocercus uropihasianus) for DNA sexing was only 55.6%¹².

This study demonstrates that hatched eggshell membrane provides useful noninvasive DNA material as an alternative to invasive sampling in sex determination studies of Maleo. Information of the sex of the hatched eggs are important to understand demographic issues, such as the demographic consequences of offspring sex ratio bias or whether there is any sex-specific mortality or dispersal. Furthermore, this information is very important for translocation programs of endangered species, including Maleo.

This study provides additional evidence that noninvasive DNA samples yield reliable results and eliminating the need for capture and invasive sampling. Collection of post-hatched eggshell membrane of Maleo, and other megapodes does not require specific skills. This noninvasive DNA sampling also open the possibility to build participation of local community or local conservation area staff on DNA collections over large spatial scales. Furthermore, the collected samples provide sufficient samples required for population and other ecological and evolutionary study of endangered bird species.

Data availability