GENETICS: ARTHROPODS
ARACHNIDS
Longhorn S. J., Nicholas M., Chuter J., Vogler A. P. (2007): The utility of molecular markers from non-lethal DNA samples of the CITES II protected “tarantula” Brachypelma vagans (Araneae, Theraphosidae). The Journal of Arachnology 35: 278-292.
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Tarantula spiders of the genus Brachypelma Simon 1891 are the only complete genus of arachnids protected from international trade under CITES law. To better understand the genetic cohesion of spiders within this genus, we evaluated multiple genetic fragments (totalling about 2200 bp) for their ability to recover population sub-structure among wild-caught Brachypelma vagans (Ausserer 1875) from Belize. We used a novel non-lethal method of tissue sampling, by inducing autospasy of the medial leg. This method allowed us to release wild-caught individuals of this protected species after DNA sampling. We used arachnid specific PCR primers to amplify targeted regions of B. vagans DNA, testing various combinations for consistency. We compared mitochondrial fragments from two populations of B. vagans (∼50 km apart) for variation in mitochondrial 16S lrRNA (plus 5′ ND1), CO1, and the nuclear ITS-2 spacer. Both lrRNA-ND1 and CO1 provided congruent estimates of population subdivision, and indicated that lrRNA-ND1 contained the greatest variation. The nuclear ITS-2 was surprisingly short (193 bp) and relatively invariant across B. vagans. While both mitochondrial fragments appear suitable to elucidate population subdivision and historical processes in B. vagans, we suggest that mitochondrial markers may overestimate population division in B. vagans. We conclude that along with valuable inferences from mitochondrial regions, the characterization of population sub-structure in tarantula spiders will be enhanced by other estimates from alternate nuclear fragments.
Petersen S. D., Mason T., Akber S., West R., White B., Wilson P. (2007): Species identification of tarantulas using exuviae for international wildlife law enforcement. Conservation Genetics 8: 497-502.
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This paper outlines a novel, non-invasive procedure to obtain DNA from Mexican tarantulas (Brachypelma spp.) using exuvia. These species are important in the pet trade and species identification is important for international wildlife law enforcement. Mitochondrial DNA sequence from the cytochrome c oxidase subunit I gene was used to investigate the relationship between various Brachypelma spp. This phylogeny was used as a framework to assign unknown specimens and spiderlings to species. The benefits to conservation, research, and international wildlife law enforcement that are gained by the ability to accurately identify species without the death of the specimen are explored.
Sint D., Thurner I. Kaufmann R., Traugott M. (2015): Sparing spiders: faeces as a non-invasive source of DNA. Frontiers in Zoology 12: 3.
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Spiders are important arthropod predators in many terrestrial ecosystems, and molecular tools have boosted our ability to investigate this taxon, which can be difficult to study with conventional methods. Nonetheless, it has typically been necessary to kill spiders to obtain their DNA for molecular applications, especially when studying their diet. We successfully tested the novel approach of employing spider faeces as a non-invasive source of DNA for species identification and diet analysis. Although the overall concentration of DNA in the samples was very low, consumer DNA, suitable for species identification, was amplified from 84% of the faecal pellets collected from lycosid spiders. Moreover, the most important prey types detected in the gut content of the lycosids were also amplified from the faecal samples. The ability to amplify DNA from spider faeces with specific and general primers suggests that this sample type can be used for diagnostic PCR and sequence-based species and prey identification such as DNA barcoding and next generation sequencing, respectively. These findings demonstrate that faeces provide a non-invasive alternative to full-body DNA extracts for molecular studies on spiders when killing or injuring the animal is not an option.
Xu C. C. Y., Yen I. J., Bowman D., Turner C. R. (2015): Spider web DNA: a new spin on noninvasive genetics of predator and prey. Plos One 10: e0142503.
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Noninvasive genetic sampling enables biomonitoring without the need to directly observe or disturb target organisms. This paper describes a novel and promising source of noninvasive spider and insect DNA from spider webs. Using black widow spiders (Latrodectus spp.) fed with house crickets (Acheta domesticus), we successfully extracted, amplified, and sequenced mitochondrial DNA from spider web samples that identified both spider and prey to species. Detectability of spider DNA did not differ between assays with amplicon sizes from 135 to 497 base pairs. Spider and prey DNA remained detectable at least 88 days after living organisms were no longer present on the web. Spider web DNA as a proof-of-concept may open doors to other practical applications in conservation research, pest management, biogeography studies, and biodiversity assessments.
Blake M., McKeown N. J., Bushell M. L. T., Shaw P. W. (2016): DNA extraction from spider webs. Conservation Genetics Resources 8: 219–221.
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Many spider species produce webs that represent a potential non-invasive source of DNA for conservation genetic analysis. Reported here is the successful isolation of target DNA from members of two families (Theraphosidae and Pholcidae) using a standard CTAB phenol–chloroform–isoamyl protocol. The isolated DNA was of sufficient quality to permit routine PCR amplification and sequencing of mtDNA COI fragments of various sizes (maximum 710 bp attempted). This adds to other studies in demonstrating that webbing offers an excellent resource for genetic studies of spiders across families. Applications of the technique include species identification and monitoring, faunistic surveys, population connectivity, subpopulation structuring, and ex situ breeding programs.
INSECTS
Feinstein J. (2004): DNA sequence from butterfly frass and exuviae. Conservation Genetics 5: 103-104.
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Two species of butterfly were investigated in this study: Vanessa cardui L., the “Painted Lady”, and Pieris rapae L., the “Cabbage Butterfly”. The last larval skin was collected from four individuals at pupation. In addition to exuviae, a combined frass sample for the cohort was collected from the communal cage to test the possibility of PCR amplification of DNA from frass of this species.
Fumanal B., Martin J. F., Bon M. C. (2005): High through-put characterization of insect morphocryptic entities by a non-invasive method using direct-PCR of fecal DNA. Journal of Biotechnology 119: 15-19.
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The development of non-invasive molecular techniques is currently increasing, particularly in the fields of behavioural ecology and conservation genetics of mammals. Surprisingly, genetic studies of Arthropods and particularly the insects have not benefited yet from the contributions that non-invasive methods have made. Here, we outline a strategy for identifying phytophagous insect genetic entities based on direct-PCR of fecal DNA combined with double strand conformation polymorphism (DSCP) typing. This allows the differentiation of morphocryptic entities within the species Ceutorhynchus assimilis (Coleoptera: Curculionidae), a candidate biocontrol agent of a noxious weed. The results obtained clearly demonstrate the potential for this method to provide a valuable means for genetic and ecological studies of Arthropods.
Watts P. C., Thompson D. J., Daguet C., Kemp S. J. (2005): Exuviae as a reliable source of DNA for population-genetic analysis of odonates. Odonatologica 34: 183-187.
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Genetic analyses are widely used for a variety of ecological research scenarios, especially to aid species’ conservation programs. Where genetic material is required from rare or endangered spp. it is essential that the samples be collected non-destructively, the ultimate goal should be to develop reliable DNA extraction protocols that may be used with non-invasively collected samples. In this paper 3 methods of DNA extraction (DNeasy tissue kit, proteinase-K/TNES and Chelex-100) that use odonate (Coenagrion mercuriale) exuviae as a non-invasive source of genetic material are described and compared. DNA extracted from exuviae produced consistent genotypes at 5 polymorphic microsatellite loci for all of the samples processed using the DNeasy tissue kit and proteinase-K/TNES methods and 4 out of the 6 exuviae treated with Chelex-100. Exuviae offer an effective source of genetic material from endangered odonates and also highly mobile spp. that are too difficult to catch in significant numbers. As such, it is expected DNA extracted from exuviae to be widely applied to odonatological genetic research.
Dhananjeyan K. J., Paramasivan R., Tewari S. C., Rajendran R., Thenmozhi V., Leo S. V., Venkatesh A., Tyagi B. K. (2010): Molecular identification of mosquito vectors using genomic DNA isolated from eggshells, larval and pupal exuvium. Tropical Biomedicine 27: 47-53.
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Correct and precise identification of mosquito vectors is important in many respects including development of vector control strategies. Conventional identification methods have limitations for sibling and closely related species of mosquitoes, stage and quality of the specimen used and this could be overcome by DNA-based identification methods using molecular markers such as nuclear ribosomal internal transcribed spacer (ITS) which do not demand intact or undamaged specimen. Genomic DNA is usually isolated from whole mosquito, legs, wings etc. Alternate sources for genomic DNA isolation such as eggshells, larval and pupal exuviae were explored in this study by amplifying the ITS markers. Standardization of genomic DNA extraction and ITS amplification were carried out with laboratory specimens. The same was applied to specimens collected from the field. The results show that PCR amenable genomic DNA could be isolated from fresh exuviae collected in the laboratory and not from older and/or field specimens. But exuviae of larvae and/or pupae collected in the field reared to adulthood in the laboratory yielded PCR amenable genomic DNA. The results also revealed that the ITS2 marker could very well differentiate Aedes aegypti and Aedes albopictus by producing amplicons of ~330 bp and ~520 bp, respectively. The genomic DNA from these alternate sources also supported the speciesspecific PCR to distinguish the Culex vishnui subgroup mosquitoes.
Hamm C. A., Aggarwal D., Landis D. A. (2010): Evaluating the impact of non-lethal DNA sampling on two butterflies, Vanessa cardui and Satyrodes eurydice. Journal of Insect Conservation 14: 11-18
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Genetic sampling of endangered species can inform conservation management and potentially aid the long-term survival of a species. However, when dealing with very small populations of rare species, the sacrifice of whole animals may not be desirable or permitted. We set out to develop a demonstrably non-lethal method of obtaining DNA from the federally-endangered Mitchell’s satyr butterfly, Neonympha mitchellii mitchellii. Because of its endangered status we developed our methods on related species. In greenhouse and fields trials, we demonstrate that removal of small amounts of hind wing (2–3 mm2) has no significant impact on the behavior or survival of Vanessa cardui and Satyrodes eurydice. Based on these studies we were successful in obtaining a permit from the US Fish and Wildlife Service to sample DNA from N. m. mitchellii populations. We suggest that our results can be extended to the sampling of other rare butterfly species.
Keller D., Brodbeck S., Flöss I., Vonwil G., Holderegger R. (2010): Ecological and genetic measurements of dispersal in a threatened dragonfly. Biological Conservation 143: 2658-2663.
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Leucorrhinia caudalis is a rare dragonfly, threatened throughout its European distribution. The species was formerly widespread in the Swiss lowlands, but only a single population remained in the 1980s. However, a spread has recently been observed, with additional ponds being colonised, sometimes at considerable distance. Despite this evidence of recent long-distance dispersal, it is unknown whether L. caudalis regularly moves among ponds or whether this is a rather rare event. A combination of an ecological mark-resight and a population genetic study was applied to investigate contemporary dispersal and the genetic footprint of the recent population history of L. caudalis in Switzerland. DNA for genetic microsatellite analysis was extracted from exuviae. The mark-resight study and the genetic analysis gave congruent results. They showed that L. caudalis is mostly a sedentary species, with only a few contemporary dispersal events over distances up to 5 km being observed. The genetic analysis was in agreement with the recent population history of the Swiss populations. The oldest and largest population showed large genetic diversity and acted as source population for the recent spread of L. caudalis in Switzerland. Recurrent gene flow among this source population and close populations caused substantial local genetic variation in the latter, as well as low population differentiation. The two recently founded distant populations (⩾30 km distance) were genetically less diverse and highly differentiated. These distant populations and another recently colonised population also expressed signatures of genetic bottlenecks.
Donald H. M., Wood C. W., Benowitz K. M., Johnson R. A., Brodie III, E. D., Formica V. A. (2012): Nondestructive sampling of insect DNA from defensive secretion. Molecular Ecology Resources 12: 856-860.
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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.
Lefort M. C., Boyer S., Worner S. P., Armstrong K. (2012): Noninvasive molecular methods to identify live scarab larvae: an example of sympatric pest and nonpest species in New Zealand. Molecular Ecology Resources 12: 389-395.
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Despite the negative impact that many scarab larvae have on agro‐ecosystems, very little attention has been paid to their taxonomy. Their often extremely similar morphological characteristics have probably contributed to this impediment, which has also meant that they are very difficult to identify in the field. Molecular methods can overcome this challenge and are particularly useful for the identification of larvae to enable management of pest species occurring sympatrically with nonpest species. However, the invasive collection of DNA samples for such molecular methods is not compatible with subsequent behavioural, developmental or fitness studies. Two noninvasive DNA sampling and DNA analysis methods suitable for the identification of larvae from closely related scarab species were developed here. Using the frass and larval exuviae as sources of DNA, field‐collected larvae of Costelytra zealandica (White) and Costelytra brunneum (Broun) (Scarabaeidae: Melolonthinae) were identified by multiplex PCR based on the difference in size of the resulting PCR products. This study also showed that small quantities of frass can be used reliably even 7 days after excretion. This stability of the DNA is of major importance in ecological studies where timeframes rarely allow daily monitoring. The approach developed here is readily transferable to the study of any holometabolous insect species for which morphological identification of larval stages is difficult.
Suzuki G., Inoda T., Kubota S. (2012): Nonlethal sampling of DNA from critically endangered diving beetles (Coleoptera: Dytiscidae) using a single antenna. Entomological Science 15: 352-356.
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Nonlethal DNA sampling is highly desirable in molecular genetic studies of protected and endangered species. To develop a demonstrably nonlethal method of obtaining DNA from endangered diving beetles (Dytiscus sharpi sharpi Wehncke, Cybister lewisianus Sharp and Cybister brevis Aubé), we amputated the antennae of these endangered diving beetles and investigated the impact of the amputation on reproductive behaviors, egg‐laying and lifespan. Diving beetles with either one or no antennae copulated without delay and laid eggs, comparable to the pairs of intact beetles under breeding conditions. The lifespan of antennae‐amputated D. sharpi sharpi was the same as that of the intact beetles. A single antenna was sufficient to allow polymerase chain reaction (PCR) detection of a mitochondrial DNA gene, cytochrome‐c oxidase subunit I (COI), and the sequence of the COI gene could be determined directly. The PCR‐ready genomic DNA was available both in fresh antennae isolated from living beetles and in old antennae from whole beetles preserved for at least 5–6 years in pure ethanol. These results suggest that an antenna is a good sampling site for isolating genomic DNA from endangered diving beetles without sacrificing and disturbing reproductive behaviors such as mating and egg‐laying, or lifespan.
Wong J., Chu Y. Y., Stoddard S. T., Lee Y., Morrison A. C., Scott T. W. (2012): Microsatellite-based parentage analysis of Aedes aegypti (Diptera: Culicidae) using nonlethal DNA sampling. Journal of Medical Entomology 49: 85-93.
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To track Aedes aegypti (L.) egg-laying behavior in the field in Iquitos, Peru, we developed methods for 1) sampling DNA from live mosquitoes and 2) high through-put parentage analysis using microsatellite markers. We were able to amplify DNA extracted from a single hind leg, but not from the pupal exuvia. Removal of a leg from teneral females caused no significant changes in female behavioral or life history traits (e.g., longevity, blood feeding frequency, fecundity, egg hatch rate, gonotrophic cycle length, or oviposition behavior). Using a panel of nine microsatellite markers and an exclusion-based software program, we matched offspring to parental pairs in 10 Ae. aegypti test families in which parents originated from natural development sites in Iquitos. By mating known individuals in the laboratory, retaining the male, sampling the female’s DNA before release, and collecting offspring in the field, the technique we developed can be used to genotype large numbers of Ae. aegypti, reconstruct family relationships, and track the egg-laying behavior of individual Ae. aegypti in nature.
Oi C. A., López-Uribe M. M., Cervini M., Del Lama M. A. (2013): Non-lethal method of DNA sampling in euglossine bees supported by mark–recapture experiments and microsatellite genotyping. Journal of Insect Conservation 17: 1071-1079.
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Non-lethal sampling methods are of great interest for conservation genetic studies to prevent the death of individuals in populations that are threatened or in decline. With this aim, we tested a non-lethal method of partial antennae removal for DNA sampling in two euglossine bee species: Euglossa cordata and Eulaema nigrita. We validated the survival of the individuals through mark–recapture experiments during 16 months. The quality and quantity of the tissue for DNA analysis was verified through amplification and genotyping of nine and eleven microsatellite loci, respectively. Our results from the mark–recapture experiments showed equal recapture rates of individuals with intact and removed antennae (E. cordata χ2 = 2.492, df = 1, p = 0.114; E. nigrita χ2 = 1.683, df = 1, p = 0.194). Microsatellite loci were successfully genotyped in 97.1 and 97.6 % of the E. cordata and E. nigrita individuals, respectively. Our results validate the feasibility of using antennae tissue for DNA genetic analysis without compromising the survival of individual bees.
Scriven J. J., Woodall L. C., Goulson D. (2013): Nondestructive DNA sampling from bumblebee faeces. Molecular Ecology Resources 13: 225-229.
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Genetic studies provide valuable data to inform conservation strategies for species with small or declining populations. In these circumstances, obtaining DNA samples without harming the study organisms is highly desirable. Excrements are increasingly being used as a source of DNA in such studies, but such approaches have rarely been applied to arthropods. Bumblebees are ecologically and economically important as pollinators; however, some species have recently suffered severe declines and range contractions across much of Western Europe and North America. We investigated whether bumblebee faeces could be used for the extraction of DNA suitable for genotyping using microsatellite markers. We found that DNA could be extracted using a Chelex method from faecal samples collected either in microcapillary tubes or on filter paper, directly from captured individuals. Our results show that genotypes scored from faecal samples are identical to those from tissue samples. This study describes a reliable, consistent and efficient noninvasive method of obtaining DNA from bumblebees for use in population genetic studies. This approach should prove particularly useful in breeding and conservation programs for bumblebees and may be broadly applicable across insect taxa.
Inoda T., Miyazaki Y., Kitano T., Kubota S. (2015): Noninvasive sampling of DNA from larval exuvia in diving beetles of the genera Cybister and Dytiscus. Entomological Science 18: 403-406.
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Nonlethal DNA sampling is a highly recommendable method in molecular genetic studies of protected and endangered species. To develop a demonstrably nonlethal method of obtaining DNA from larvae of endangered diving beetles (Cybister brevis, C. lewisianus, C. limbatus, C. rugosus, Dytiscus sharpi sharpi and D. sharpi validus), we obtained the larval exuvia (molted skin) of these endangered diving beetles under laboratory conditions. A single exuvia 24 h after molting was sufficient to allow polymerase chain reaction (PCR) detection of a mitochondrial DNA gene, cytochrome‐c oxidase subunit I (COI), and the sequence of the COI gene could be determined directly. Sequences obtained from the exuvial samples were used to further find similarities within DDBJ / EMBL / GenBank. Genomic DNA from the samples was successfully isolated, and we identified the species. This process suggests that exuvia provides a good sample for extracting DNA from endangered diving beetle larvae without killing them.
Kranzfelder P., Ekrem T., Stur E. (2016): Trace DNA from insect skins: a comparison of five extraction protocols and direct PCR on chironomid pupal exuviae. Molecular Ecology Resources 16: 353-363.
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Insect skins (exuviae) are of extracellular origin and shed during moulting. The skins do not contain cells or DNA themselves, but epithelial cells and other cell‐based structures might accidentally attach as they are shed. This source of trace DNA can be sufficient for PCR amplification and sequencing of target genes and aid in species identification through DNA barcoding or association of unknown life stages. Species identification is essential for biomonitoring programs, as species vary in sensitivities to environmental factors. However, it requires a DNA isolation protocol that optimizes the output of target DNA. Here, we compare the relative effectiveness of five different DNA extraction protocols and direct PCR in isolation of DNA from chironomid pupal exuviae. Chironomidae (Diptera) is a species‐rich group of aquatic macroinvertebrates widely distributed in freshwater environments and considered a valuable bioindicator of water quality. Genomic DNA was extracted from 61.2% of 570 sampled pupal exuviae. There were significant differences in the methods with regard to cost, handling time, DNA quantity, PCR success, sequence success and the ability to sequence target taxa. The NucleoSpin® Tissue XS Kit, DNeasy® Blood and Tissue kit, and QuickExtract™ DNA Extraction Solution provided the best results in isolating DNA from single pupal exuviae. Direct PCR and DTAB/CTAB methods gave poor results. While the observed differences in DNA isolation methods on trace DNA will be relevant to research that focuses on aquatic macroinvertebrate ecology, taxonomy and systematics, they should also be of interest for studies using environmental barcoding and metabarcoding of aquatic environments.
Nguyen H. Q., Kim Y. I., Borzée A., Jang Y. (2017): Efficient isolation method for high‐quality genomic DNA from cicada exuviae. Ecology and Evolution 7: 8161-8169.
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In recent years, animal ethics issues have led researchers to explore nondestructive methods to access materials for genetic studies. Cicada exuviae are among those materials because they are cast skins that individuals left after molt and are easily collected. In this study, we aim to identify the most efficient extraction method to obtain high quantity and quality of DNA from cicada exuviae. We compared relative DNA yield and purity of six extraction protocols, including both manual protocols and available commercial kits, extracting from four different exoskeleton parts. Furthermore, amplification and sequencing of genomic DNA were evaluated in terms of availability of sequencing sequence at the expected genomic size. Both the choice of protocol and exuvia part significantly affected DNA yield and purity. Only samples that were extracted using the PowerSoil DNA Isolation kit generated gel bands of expected size as well as successful sequencing results. The failed attempts to extract DNA using other protocols could be partially explained by a low DNA yield from cicada exuviae and partly by contamination with humic acids that exist in the soil where cicada nymphs reside before emergence, as shown by spectroscopic measurements. Genomic DNA extracted from cicada exuviae could provide valuable information for species identification, allowing the investigation of genetic diversity across consecutive broods, or spatiotemporal variation among various populations. Consequently, we hope to provide a simple method to acquire pure genomic DNA applicable for multiple research purposes.
Ali B., Zhou Y., Zhang Q., Niu C., Zhu Z. (2019): Development of an easy and cost-effective method for non-invasive genotyping of insects. Plos One 14: e0216998.
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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.
Storer C., Daniels J., Xiao L., Rossetti K. (2019): Using noninvasive genetic sampling to survey rare butterfly populations. Insects 10: 311.
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Advances in nondestructive genetic sampling techniques continue to offer new opportunities for studying organisms, particularly those of conservation concern where more traditional invasive sampling methods are often not available. As part of a proof-of-concept, we investigated the effectiveness of using the chorion from residual butterfly egg debris as a source of viable genetic material for analysis. Laboratory material from a captive breeding population of the federally endangered Miami blue butterfly (Cyclargus thomasi bethunebakeri) was used to test efficacy and refine the methodology. The resulting best practices were subsequently evaluated using field-collected material from extant north Florida populations of the at-risk frosted elfin butterfly (Callophyrs irus). Our results demonstrated that it is possible to extract DNA of sufficiently high quantity and quality for successful gene sequencing. We additionally describe a simple, low-cost, and reliable method of collecting and storing egg debris samples that can be consistently adopted for field or laboratory work as well as deployed with projects that have a larger geographic scope and/or involve citizen scientists. Potential limitations related to field sample collection are discussed as well as needs for future evaluation.
Bubnič J., Mole K., Prešern J., Moškrič A. (2020): Non-destructive genotyping of honeybee queens to support selection and breeding. Insects 11: 896.
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In traditional bee breeding, the honeybee queen is chosen for breeding based on the performance of the colony produced by its mother. However, we cannot be entirely certain that a specific queen will produce offspring with desirable traits until we observe the young queen’s new colony. Collecting the queen’s genetic material enables quick and reliable determination of the relevant information. We sampled exuviae, feces, and wingtips for DNA extraction to avoid fatally injuring the queen when using tissue samples. Quantity and purity of extracted DNA were measured. Two mitochondrial markers were used to determine the lineage affiliation and exclude possible contamination of DNA extracts with non-honeybee DNA. dCAPS (derived Cleaved Amplified Polymorphic Sequences) markers allowed detection of single nucleotide polymorphisms (SNPs) in nuclear DNA regions presumably associated with Varroa sensitive hygiene and set the example of successful development of genotyping protocol from non-destructive DNA sources. One of the logical future steps in honeybee breeding is introducing genomic selection and non-destructive sampling methods of genetic material may be the prerequisite for successful genotyping. Our results demonstrate that the extraction of DNA from feces and exuviae can be introduced into practice. The advantage of these two sources over wingtips is reducing the time window for processing the samples, thus enabling genotyping directly after the queen’s emergence.
Yumoto K., Kanbe T., Saito Y., Kaneko S., Tsuda Y. (2021): Efficient PCR amplification protocol of nuclear microsatellites for exuviae-derived DNA of cicada, Yezoterpnosia nigricosta. Frontiers in Insect Science 1: 696886.
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Although insect exuviae-based genetics is challenging, it can be a valuable method for obtaining reliable DNA resources by non-invasive sampling. This approach is especially effective when the target species is endangered/endemic or when sampling the adult is difficult. One example is cicadas, which during molt leave their exoskeletons on tree trunks, making them easily collectable. While cicada exuviae-derived DNA has previously been employed for mitochondrial DNA sequencing, this study aimed to develop a reliable method for the PCR amplification of nuclear microsatellite loci from cicada exuviae derived DNA for application in molecular ecology, conservation and population genetics. Five different PCR amplification protocols were performed, and the fragment patterns compared with those obtained using DNA extracted from adult individuals. Moreover, the relationship between the freshness of the exuviae and genotyping success was evaluated. TaKaRa LA Taq provided the best performance in the PCR amplification of DNA isolated from cicada exuviae and the electropherogram showed a clear fragment pattern that was equivalent to that obtained from the DNA extracted from the adult individual. This result suggests that cicada exuviae-derived DNA can be amplified by PCR and that multiple independent loci of nuclear DNA microsatellite markers can be easily genotyped. This study demonstrates that fresh cicada exuviae provide high quality DNA, which can be used for microsatellite genotyping. The methods developed in this study are applicable not only for cicada but other insect species for which exuviae are available. Thus, this study can make a significant contribution to insect sciences.
Wang X. X., Li J., Wang T. X., Yang Y. N., Zhang H. K., Zhou M., Kang L., Wei L. Y. (2022): A novel non‐invasive identification of genome editing mutants from insect exuviae. Insect Science 29: 21-32.
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With the wide application of genome editing in insects, a simple and efficient identification method is urgently needed to meet the increasing demand for mutation detection. Here, taking migratory locusts as a model system, we developed a non-invasive method to accurately identify genome-edited mutants by using DNA from insect exuviae. We compared the quantity and quality of genomic DNA from exuviae in five instar hoppers and found that the 1st instar exuviae had the highest DNA yield and content, while the 3rd instar exuviae had the best quality. Consensus genotypes were identified from genomic DNA of hoppers at different developmental stages in the same individuals. Moreover, we demonstrated that the amplification products from DNA extracted from locust exuviae are the consensus sequences with those from the hemolymph and foreleg pre-tarsus. Therefore, non-invasive samples provide the same genotyping results as minimally invasive and invasive samples of the same individuals. Furthermore, this identification method that uses genomic DNA from exuviae can be used for early screening of positive genome-edited individuals in each generation for adult crossing. In our study, the non-invasive identification method was not only simpler and provided results earlier than existing methods, but also had a better reproducibility and accuracy. This non-invasive identification approach using genomic DNA from exuviae can be adapted to meet the growing demand for genetic analysis and will find wide application in insect genome editing research.
Sittenthaler M., Fischer I., Chovanec A., Koblmüller S., Macek O., Sattmann H., Szucsich N., Zangl L., Haring E. (2023): DNA barcoding of exuviae for species identification of Central European damselflies and dragonflies (Insecta: Odonata). Journal of Insect Conservation 27: 435-450.
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Monitoring of odonates has become an important instrument for ecological status assessment of (semi-)aquatic habitats. Besides information on presence and abundance, knowledge about a species´ autochthony at the surveyed waterbody is a significant information within the assessment process. Here, the finding of exuviae represents the ultimate proof of successful reproduction. Although feasible for most odonate species, morphological identification of exuviae is often time consuming, as it relies on small, fragile structures. To facilitate species identification of exuviae, a DNA barcoding approach was developed, including (1) non-destructive extraction of DNA using whole exuviae or their tracheal tubes, and (2) primer systems for long (< 600 bp) and short (< 200 bp) CO1 fragments. A total of 85 exuviae from 33 species were analysed and compared to results of morphological identification. Additionally, factors potentially influencing DNA quality and quantity, as well as PCR and sequencing success were investigated. Eighty-two exuviae matched the morphologically identified genus, and 60 matched at species level. Of the 33 species present in the data set, 82% could be identified to species level via DNA barcoding. The results show how DNA-based approaches can support fast and accurate species identification and therefore enhance monitoring of an ecologically important taxonomic group, with high relevance for conservation and habitat restoration. Moreover, the use of exuviae as DNA resource once more shows that non-invasive sampling offers great potential for molecular species identification, which is essential when studying rare and endangered species.