Non-invasive sampling technique for DNA extraction from captive Japanese Crested Ibis on Sado Island

INTRODUCTION

The Japanese Crested Ibis Nipponia nippon is a globally threatened bird (Collar et al. 1994) that was extinct in the wild in Japan. After the extinction in Japan, five individuals were gifted by the Chinese government. The population size has now increased to about 120 captive individuals because of great conservation efforts for captive-breeding programs implemented at the Sado Japanese Crested Ibis Conservation Center, Niigata, Japan. However, molecular genetics studies on genetic diversity are clearly needed to perform further effective management of conservation of the Japanese Crested Ibis.

Invasive samples, blood and plucked feathers, have been used as DNA sources for molecular genetics studies (e.g. Taberlet & Bouvet 1991; Petitte et al. 1994). However, these sampling methods seem to be unsatisfactory, because disturbance associated with trapping and handling compromises project goals of conservation of threatened species (Hoysak & Weatherhead 1991; Lanctot 1994; Lessells et al. 1996). The vascularized chorioallantois membrane from the inside of post-hatch eggs has been reported to be useful as a non-invasive DNA source in two bird species, the Spectacled Eider Somateria fischeri and the Brown-headed Cowbird Molothrus ater (Pearce et al. 1997; Strausberger & Ashley 2001). The post-hatch eggs of captive Japanese Crested Ibis have been stored at room temperature for the long-term in the Sado Japanese Crested Ibis Conservation Center.

In this study, we investigated whether the vascularized chorioallantois membrane from the inside of post-hatch eggs stored at room temperature for the long-term provides a reliable DNA source of offspring that had hatched from the egg possessing the membrane, in captive Japanese Crested Ibis on Sado Island.

MATERIALS AND METHODS

The post-hatch eggs of captive Japanese Crested Ibis were stored in plastic bags at room temperature for the long-term at the Sado Japanese Crested Ibis Conservation Center (Niigata, Japan), and the longest storage period was 10 years. The eggs (n = 21) used in this study had an average storage period of 4.9 years, ranging from 1 to 9 years. The vascularized chorioallantois membrane was excised from the eggs with forceps and a scalpel.Peripheral blood samples were drawn by wing venipuncture from offspring that had hatched from the eggs or its parents. DNA was extracted using the DNeasy Blood & Tissue Kit (QIAGEN, Hilden, Germany).

We used microsatellite loci, Nn01, Nn04, Nn12, Nn18, Nn21, Nn25, Nn26 and NnNF5, developed for the Japanese Crested Ibis (Ji et al. 2004; He et al. 2006). With these eight loci, one primer of each pair was end-labeled with a fluorescent dye, 6-FAM. PCR amplifications were carried out in a 10-µL volume using GeneAmp PCR System 9700 (Applied Biosystems, Foster City, CA, USA), as described by He et al. (2006) for Nn01, Nn04, Nn12, Nn18, Nn21, Nn25 and Nn26, and by Ji et al. (2004) for NnNF5. For allele scoring, a portion of PCR products (0.2 µL) from each locus was combined with 10 µL of formamide loading dye that contained the fluorescent labeled GeneScan-500LIZ size standard (Applied Biosystems), and assayed using a 3730xl DNA analyzer (Applied Biosystems). We used GeneMapper software version 4.0 (Applied Biosystems) to create electropherograms and to score PCR products.

To examine whether the vascularized chorioallantois membrane from the inside of post-hatch eggs stored at room temperature for the long-term could be regarded as a reliable DNA source of offspring that had hatched from the egg, we performed three experiments. In experiment 1, we compared genotypes obtained from five different sites of the identical membrane. In experiment 2, the genotype derived from the membrane was compared to that of the blood of parents of offspring that had hatched from the egg possessing the membrane. In experiment 3, we compared genotypes obtained between the membrane and the blood of offspring that had hatched from the egg possessing the membrane.

RESULTS AND DISCUSSION

We used the vascularized chorioallantois membranes from seven birds in experiment 1. We genotyped two loci, Nn01 and Nn21, for DNA prepared from five different sites on each of the bird membranes. The five different sites had the same genotypes for all of the samples (Table 1). Occasionally, we were unable to amplify either of the two loci, possibly due to low DNA yields. The low DNA yields are likely to be caused by a small quantity of blood residue present in the vascularized chorioallantois membrane. Thus, we concluded that the vascularized chorioallantois membrane stored at room temperature for the long-term can be regarded as a stable DNA source, if the membrane is used in its entirety or a portion of the membrane possessing a large quantity of blood residue is used.

Using DNA prepared from the vascularized chorioallantois membranes from eight full-sibling birds and the blood from their parents, eight loci, Nn01, Nn04, Nn12, Nn18, Nn21, Nn25, Nn26 and NnNF5, were genotyped in experiment 2. We obtained genotypes from the blood and membrane samples that were consistent with Mendelian parent–offspring relationships (Table 2). This result suggested that DNA in the membrane is that of offspring that had hatched from the egg possessing the membrane, and that the DNA is not contaminated by maternal DNA.

We used the vascularized chorioallantois membranes from six birds and the blood from offspring that had hatched from the eggs possessing the membranes in experiment 3. We genotyped eight loci, Nn01, Nn04, Nn12, Nn18, Nn21, Nn25, Nn26 and NnNF5, for DNA prepared from each of six birds. We confirmed that the DNA prepared from the membrane matched that of offspring that had hatched from the egg possessing the membrane by comparing the genotypes obtained from each (Table 3). Therefore, we concluded that the vascularized chorioallantois membrane from the inside of a post-hatch egg stored at room temperature for the long-term can be used as a reliable DNA source of offspring that had hatched from the egg in captive Japanese Crested Ibis on Sado Island.

Our results extended the suitability of the vascularized chorioallantois membrane as a source of offspring DNA to the Japanese Crested Ibis species, in addition to two bird species, the Spectacled Eider (Pearce et al. 1997) and the Brown-headed Cowbird (Strausberger & Ashley 2001). Furthermore, Pearce et al. (1997) and Strausberger and Ashley (2001) used fresh vascularized chorioallantois membrane for offspring DNA extraction. Our results supported offspring DNA extraction from the membrane stored at room temperature for a longer period of 9 years. However, it is worth noting that the membrane should be stored under dry conditions as far as possible, to prevent mold growth.

Application of DNA extraction from the vascularized chorioallantois membrane stored at room temperature for the long-term, together with the utilization of microsatellite loci developed for the Japanese Crested Ibis (Ji et al. 2004; He et al. 2006) and species related to the Crested Ibis (Tomasulo-Seccomandi et al. 2003 for the Wood Stork Mycteria Americana; Santos et al. 2006 for the Scarlet Ibis Eudocimus ruber; Sawyer & Benjamin 2006 for the Roseate Spoonbill Ajaia ajaja; and Yeung et al. 2009 for the Black-faced Spoonbill Platalea minor), will promote a molecular genetics study on genetic diversity, relatedness and kinship of the current captive Japanese Crested Ibis population on Sado Island. This study greatly contributes to effective management of conservation of this bird species.