The Bali Starling/Mynah (Leucopsar rothschildi) endemic to Bali, is an endangered Balinese bird in Indonesia with its natural habitat in West Bali National Park (Taman Nasional Bali Barat, TNBB). These exotic starlings are increasingly in demand to be raised as pets; many are stolen or poached from their natural habitat. Conservation has been performed by sending and breeding the birds overseas to American and England zoos since the 1950's. After an overseas breeding process, the birds were returned to Bali Barat National Park and released into their natural habitats or either kept in captivity after passing habituation. These birds are expected to reproduce naturally to increase their natural population. For half a century, breeding of birds occurs in different environments overseas, but their genetic characteristics are not known. Therefore, the aim of this study is to investigate the genetic diversity of this Balinese starling through DNA analysis. Blood and tissue samples preserved in formaldehyde were collected from Bali Barat National Park, birds bred overseas, and from semi-range habitat on the island of Nusa Penida. The result showed that genetic diversity of the indigenous Balinese starling was no different after many generations. However, acknowledging that DNA samples can be analysed from deceased bird tissues preserved in formaldehyde is a novelty. This result can be used as a reference of technical DNA analysis, particularly in animal samples that are exceptionally rare as in Balinese Starling.
Key words: Bali Starling (Leucopsar rothschildi), Bali Barat National Park, DNA
Image right: Bali Starling, Nusa Penida (FNPF, 2009)
Leucopsar rothschildi or Bali Mynah is a critically endangered Balinese endemic bird species. Van Bale et al. (2000)  stated that the species was highly endangered due to its rather small population, the fact that it is restricted to small areas, illegal poaching and failing suitable habitat within its natural range.
This land bird is distinctive by its bluish body colour, almost completely covered with white plumage, except around the eyes, similar to its skin, giving the bird its gorgeous looks. These characteristics distinguish them from their Sturnus relatives, although impressively distributed throughout Eurasia, Africa, and the eastern Pacific, are restricted to the old world [2-3].
Many studies have been carried out to evaluate the population of the bird. Strategies have been improved to maintain a captive population size to meet the educational and conservational programmes using the so-called technique of the Bali Mynah Species Survival Plan (SSPs), an association of Zoos and Aquariums (AZA) on corporative breeding and management program (http://www.aza.org/ConScience/ConScienceSSPFact/index.html; ). As summarized by Earnhardt (2009) , most of the captive populations show a small loss of genetic diversity, interactions of population structure, stochastic and genetic diversity. SSPs actively manage captive populations to minimize the loss of genetic diversity  and maintain the population size and structure. In the early 1980's, the population size and composition of Bali Mynah SSPs has met the standard of AZA's genetic and demographic objectives (Long, 2005) in Earnhardt (2009)  in spite of SSP participants concerned with the ability to sustain its future population .
The fact that there was a high demand for the birds as pets and for other purposes has a negative impact on the Bali Mynah population, and as a result the bird population decreased. Although the population of L. rothschildi in overseas captivity increased and was considered a self-sustaining population, based on census conducted in October 1997, however, only 12-17 individuals birds were recorded in the entire wild population of Prapat Agung in Bali Barat National Park. After a temporary recovery, in 1998 the number of individuals of L. rothschildi was at its lowest level: fewer than 15 individuals restricted to the Bali Barat National Park[1, 5]. A survey conducted in March 2005, recorded that 1.000 individuals were in captivity, but only 24 were found in the wild (Bird Life International Indonesian Program 2007, Bogor).
The declining number of birds in the wild is thought to be affected by extrinsic anthropogenic factors, primarily poaching for pet trade and due to the contribution of habitat loss[5, 11]. Furthermore, Earnhardt  states that the decreasing number of the bird population has also affected by the intrinsic population factors (e.g. stochasticity, loss of genetic diversity) that was contributed to the large fluctuations in population size.
In order to maintain the populations of the Balinese Mynah in the wild, particularly in Bali Barat National Park, during late 1980's the SSP transferred the offspring to Bali. However, the supplementation had no impact on the persistence of the wild population, possibly because poaching pressure was not mitigated . The SSPs anticipate for poaching to be eliminated or greatly reduced, for they will act to reintroduce the Bali mynah birds. This action necessitates the SSPs to self-sustain birds that are demographically, behaviourally and genetically appropriate for reintroduction. Reintroduction of the Mynah in Bali Barat National Park may have an impact to the genetic diversity of the Bali Mynah; therefore this study was conducted to find out the genetic variability of the Balinese mynah native to West Bali National Park and from birds sent to Bali through the SSPs program. With regard to the conservationists and the present number of L. rothschildi in West Bali National Park, samples (blood and preserved muscle tissue from deceased birds) were collected in a manner to minimize the damage of the birds.
Materials and methods
Samples were collected from captive bird in Bali Barat National Park and Nusa Penida either alive or deceased and are preserved in formaldehyde. Blood samples were taken from living birds and muscle tissue samples from preserved deceased birds.
Blood samples were collected by a 1ml needle in the wing or toes of the bird's vein, 150-200µl for each of 6 birds collected. Samples were then placed in a 1.5 ml ependorf tube with 150µl cell buffer lyses. Muscle tissue samples were collected from 10 deceased birds preserved in formaldehyde.
DNA was extracted with the Phenol-Chloroform method and precipitated in ethanol following Sambrook and Russel (2001)  with modifications. Mixed blood sample and lyses buffer was added with 40µl SDS 10%, 40 µl 5M NaCl, 400µl – phenol pH 8.0, and 400 µl CIAA. Mixed samples were homogenised for two hours at room temperature. Samples were then centrifuged with the speed of 5000rpm for 5 minutes. Aqueous phase DNA was moved to a 1.5ml ependorf tube, then added with ethanol twice as much as the volume of the sample. The sample was frozen for two hours up to one night. Frozen samples were centrifuged with the speed of 10.000rpm for 15 minutes, supernatant was removed, but the pellet was resuspended with 900µl 700% ethanol in TE solution, centrifuged at a speed of 10.000rpm for 15 minutes. Supernatant was removed and the pellet was dried out at room temperature or dried in a vacuum drier for 30 min. Pellets then resuspended with 50µl 80% TE and DNA sample can be used directly for DNA analyses or kept in a refrigerator before analysis.
Before the extraction of samples, the muscle tissues preserved in formaldehyde were cut into small pieces, washed with lyses buffer many times until the formaldehyde was removed from the sample. The next procedure was similar to the blood sample.
Extracted DNA samples were amplified in a PCR machine with 6 pairs microsatellite primers: TH3, TH6, TH12, TH15, WB2 and WB5. The amplification was conducted at a volume of 12.5µl, containing 10.5µl PCR platinum super mix (Invitrogen), 1µl primer mix and 1µl DNA template (sample). Amplification was carried out in the PCR, which was set up in 94oC denaturation for 45 minutes, annealing at 50-60oC for 1.30 minutes, and elongation processes at 72oC for 2.15 minutes in 30 cycles. Amplification results and DNA standard of 100bp were electrophoreses in 6% polyacrilamide gel (PAGE) for 60 minutes. In order to visualize the DNA bands, the gel was stained in silver nitrate. DNA typing was performed by comparing the distance of DNA migration and DNA standard.
Results and discussion
The results of DNA extractions in 1% agarose gel stained in Ethidium Bromide is presented in Figure 1. Bands of DNAs migrated at similar distances from the first loading samples. Figure 1 indicates that the sample in the 8th column was not visible. The DNA band in the 7th column, which was taken from muscle tissue preserved in formaldehyde was barely visible. Less visibility of DNA bands in the gel could be the result of poor quality extraction, or in the case of the preserved muscle tissue, the cells may yet been bound in formaldehyde, although it has been washed for many times. However, findings that the preserved tissues can be extracted for DNA analyses is novel information.
Figure 1 (left): Bands of DNA on agarose gel show the speed/length of DNA migration. Samples were collected from Taman National Bali Barat (TNBB); Bali Bird Park (BBP); Nusa Penida (NS). The name of the samples (columns from left to right) are: TNBB DR 01(England), TNBB DR 04 (Yokohama), TNBB DR 05 (Yokohama), TNBB DR 06 (Surabaya Zoo), DNA Marker, TNBB DG 06 (TNBB), BBP DR 10 (America), BBP DR 12 (BBP), NS 01(NS), NS 02 (NS), NS 03 (NS). (DR = Blood; DG = Muscle)
Out of six pairs of primers, TH3, TH6, TH12, TH15, WB2 dan WB5, only two primers (TH6 and TH15) were amplified. The electrophoresis of 6% polyacrylamide gel showed that only four of 18 samples could be amplified using the two primers. This poor result could be affected not only by technical problems such as the absence of DNA due to failure during extraction, but also the failure of DNA attachment during DNA amplification. The amplification failure could be due to mutation or the changed primary sites, result in failure of primer attachment during DNA amplifications. This may be the case because during DNA extraction, there was a quantity of DNA of good quality observed and presented on agarose gel without smear.
The results also show the length of the alleles of birds endemic to Bali Barat National Park and bird from overseas breeding (England) was not different. This means that they are a monotype genus as found by van Balen  although they have been apart and bred in different condition and environment (see  for further historical distribution). This may also indicates that overseas breeding has similar response genetically in terms of demographic and behaviour, therefore the SSPs program on reintroducing birds bred overseas has no major impact to the genetic population of the birds in the wild. However, it is important to note that our results were only based on two alleles, therefore precaution should be taken when drawing conclusion.
The distance migration of DNA bands compared to 100bp DNA ladder, as a DNA standard, showed that each allele has similar 156bp for the TH6 primer. This means that TH6 locus only has one allele (156 pb). The TH15 locus, however, has two alleles. One of them was allele 153bp, which was found in TNBB DR. 01 and TNBB DR.02. Although the bird samples were from different sources, the TNBB DR.01 from England and TNBB DR.02 from West Bali National Park share similar alleles. However, it should be noted that the parents of the birds from England might have originated from Bali Barat National Park. The same results were found on muscle tissues from preserved deceased birds in formaldehyde.
Figure 2. Bands of DNA in polyacrilamide gell. The bands shown in TH6 'primer' are visible in column 1 (TNBB DR 01/England), 2 (TNBB DR 02/TNBB), 9 (TNBB DG 04/TNBB); and TH 15 'primer' are visible in column 11 (TNBB DR 01/England), 12 (TNBB DR 02/TNBB) and 20 (TNBB DG 04/TNBB). Bands in column 10 is a DNA ladder of 100bp.
The other allele of individual chicks bred at Bali Barat National Park also has variations shown in samples: TNBB DR.01, TNBB DR02, and TNBB DG.04. This result, however, needs further investigation. Blood samples from other places (captive birds in Nusa Penida; Image 2; Column 18 & 19), whose parents are from the UK, cannot confirm these findings because DNA cannot be verified.
In spite of the fact that this study only shows minimal results, yet there is tissue of the bird brains conserved in formaldehyde to be used for future DNA analysis.
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We are indebted for their expertise to the following: Drs. P. Bambang Darmadja, MS, The Head of Taman National Bali Barat; Drh I Gede Nyoman Bayu Wirayudha, The Director FNPF; and Drs. I.N. Nuyana SSi., The Curator of Bali Bird Park.