Development of an easy and cost-effective method for non-invasive genotyping of insects

Abstract

Non-invasive genotyping methods provide valuable information on insect populations. However, poor DNA amplification and time-consuming sampling procedures limit these methods, especially for small insects. An efficient and convenient method was developed for non-invasive, non-lethal genotyping of a large insect, Mythimna separata , and a small insect, Drosophila melanogaster , by amplification of endogenous and exogenous, nuclear and mitochondrial genes from insect frass, exuviae, and food waste. For M . separata , the chitin synthesis gene MsCHSB and the COI gene were successfully detected by PCR from exuviae DNA. However, a COI fragment could not be detected directly by PCR from frass, probably due to DNA degradation. To improve the detection rate, DNA from frass was first amplified by Multiple Displacement Amplification with phi29 DNA polymerase, after which the COI fragment was detected from all samples by PCR. For D . melanogaster , second instar larvae were reared individually for three days and then DNA was extracted from food waste of each individual. The endogenous fragment serendipity α (sryα), exogenous transgene ΦC31 integrase, and the kl-5 gene, a Y-chromosome-located male-specific marker gene were successfully detected from most samples. We developed a simple, non-invasive, non-lethal method to determine gender and identify transgenic individuals early in the larval stage. This universal method is applicable to most insects and has potential application in genetic and ecological studies of insects and other arthropods.

Non-invasive genetic monitoring for the threatened valley elderberry longhorn beetle

“…Finally, some mitochondrial loci might be more challenging to amplify from frass than others. Similar to another publication [40], we were unsuccessful in amplifying COI in our beetle frass samples, although these primers successfully amplified a subset of museum specimens (S3 Table).…”

“…Frass has also been utilized for molecular species identification, which can be indispensable in distinguishing closely related species [30]. In addition, frass genetic methods can reveal gender and identify transgenic individuals, even in visually similar early larval stage insects [40]. A PCR-based approach has been documented for invasive wood-boring insects [41] but there is also potential for non-lethal genetic sampling of rare and protected organisms.…”

Non-Destructive Genotyping of Honeybee Queens to Support Selection and Breeding

“…Of course, the success of the non-destructive approach is not guaranteed on all occasions and purposes. Obtaining useful genetic information from samples, collected in a non-destructive way, seem to have a wide range of success rate among different insect taxa and for different purposes [ 14 , 15 , 17 , 21 , 48 ]. These results hint that any non-destructive approach should not be considered universal but rather decided upon after preliminary screening.…”

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.…”