Nondestructive sampling of insect DNA from defensive secretion

Abstract

Nondestructive techniques to obtain DNA from organisms can further genetic analyses such as estimating genetic diversity, dispersal and lifetime fitness, without permanently removing individuals from the population or removing body parts. Possible DNA sources for insects include frass, exuviae, and wing and leg clippings. However, these are not feasible approaches for organisms that cannot be removed from their natural environment for long periods or when adverse effects of tissue removal must be avoided. This study evaluated the impacts and efficacy of extracting haemolymph from a defensive secretion to obtain DNA for amplification of microsatellites using a nondestructive technique. A secretion containing haemolymph was obtained from Bolitotherus cornutus (the forked fungus beetle) by perturbation of the defensive gland with a capillary tube. A laboratory experiment demonstrated that the sampling methodology had no impact on mortality, reproductive success or gland expression. To evaluate the quality of DNA obtained in natural samples, haemolymph was collected from 187 individuals in the field and successfully genotyped at nine microsatellite loci for 95.7% of samples. These results indicate that haemolymph-rich defensive secretions contain DNA and can be sampled without negative impacts on the health or fitness of individual insects.

Using Noninvasive Genetic Sampling to Survey Rare Butterfly Populations

“…Similarly, the use of residual egg debris has advantages over other documented noninvasive genetic sampling methods involving larval exuviae, frass, or the more traditional removal of tissue samples (e.g., palps, wing clippings, legs) from adult organisms. These can be problematic logistically as they often require longer organism holding times, necessitate organism removal from the environment, or may result in a limited number of viable available samples [18,19,20,21,22]. For example, Saarinen et al [13] demonstrated that wing clips represent a viable, non-lethal method of obtaining DNA from a federally endangered butterfly.…”

“…Despite being nonlethal, such methods still require capture and handling of the target organism which can inflict significant stress, inadvertent damage, or death, and may not be feasible for low density or at-risk populations. Alternatively, researchers have also successfully secured DNA from frass, exuviae, and hemolymph in defensive secretions [18,19,20,21,22]. However, these sources may not be broadly available for many taxa and certainly offer significant challenges for reliable collection in the field.…”

No Effect of Host Species on Phenoloxidase Activity in a Mycophagous Beetle

“…Animals were not fed during capture to avoid altering their diet. Samples of hemolymph were collected within 24 hours of capture following the procedure described in [ 32 ]. Briefly, individuals present a tenebrio-type defensive gland from their anal sternite when exposed to human breath [ 33 ].…”

Surprisingly little population genetic structure in a fungus‐associated beetle despite its exploitation of multiple hosts

“…Each individual received a unique ID, which was painted on its elytra using Testors Gloss Enamel in earth tone colors. We collected 0.2–5 μL of hemolymph from the defensive glands of all beetles using the method described in Donald et al (2012), which did not affect either survival or reproduction in laboratory trials. Hemolymph was stored in prepared lysis buffer (Promega DNA IQ system).…”

“…Fragment analysis was completed by GeneWiz Inc. (South Plainfield, NJ) using Applied Biosystems 3730xl DNA Analyzers. All individuals' genotypes at nine microsatellite loci (Donald et al 2012) were scored using GeneMarker (SoftGenetics, State College, PA).…”