Non-destructive sampling of maternal DNA from the external shell of bird eggs

Abstract

The use of non-destructive sampling methods to collect genetic material from wildlife allows researchers to minimize disturbance. Most avian studies employ capturing and handling of young and parents to draw blood for DNA analysis. In some cases adult female birds are difficult to catch, so maternal genotyping has required collection of contour feathers from nests, or destructive sampling of eggs. Many species do not leave contour feathers in the nest, and destructive sampling has been unreliable due to contamination with embryonic DNA. Alternative field sampling techniques for collection of maternal DNA from birds are therefore desirable. Here we demonstrate that avian maternal DNA can be isolated in a non-invasive and non-destructive way from the external surface of eggs. We used cotton swabs to collect maternal DNA from the external shells of herring gull (Larus argentatus) and Caspian tern (Sterna caspia) eggs. DNA was then amplified by the polymerase chain reaction (PCR) for microsatellite genotyping. We verified that the DNA samples were maternal by comparing microsatellite profiles to those obtained from adults and chicks from the same nests. In 100% of Caspian tern (n=16) and herring gull families (n=12), the egg swabs that amplified matched the maternal microsatellite genotype. In a screening of many nests of both species, we successfully amplified microsatellite markers from 101/115 (88%) egg swabs. Swabs from eggs with blood stains on the shell were more likely to amplify successfully than those from clean eggs. The advantages of this new method include increased parentage assignment/exclusion power, and increased availability of maternal DNA for genotyping of species that do not deposit contour feathers in nests.

Using eggshell membranes as a DNA source for population genetic research

“…In precocial species where the chicks leave the nesting area as soon as they can walk, eggshell remains can usually be collected after hatch. Egg-shells might be contaminated with parental and sibling DNA (Schmaltz et al 2006;Strausberger and Ashley 2001;Taberlet and Fumagalli 1996;Taberlet and Waits 1998). However, if eggshells would still yield high quality DNA, the collection of eggshells would ease the collection of material for genetic population structure.…”

Living with strangers: direct benefits favour non-kin cooperation in a communally nesting bird

“…(b) Genetic determination of egg and nestling maternity In order to determine which female laid each egg in the communal clutch, maternal DNA was isolated from blood stains and shed cells on the external surface of the freshly laid egg [23]. Samples were then genotyped with a set of 12 highly variable microsatellite markers developed for the greater ani [24].…”

Evaluation of methods for molecular sex-typing of three heron species from different DNA sources

“…The value of bird ringing data can increase if the data for the ringed individuals are coupled with sex information. Noninvasive genetic analyses can provide information about the birds' sex, population demography, and behavioral ecology (Horváth et al, 2005;Schmaltz et al, 2006;Hogan et al, 2008;Rudnick et al, 2009). Use of molecular markers for sex can help with tracking and predicting population status and growth in birds.…”

“…From the eggshell, a piece of vascularized membrane was excised and cut into smaller fragments. From the same eggshells, the external surface was swabbed as described by Schmaltz et al (2006) by using cotton tips. To avoid cross-contamination with nestling DNA, swabs were taken only from eggshells that did not have blood smears (which could have come from the mother) on the surface.…”