GENETICS: MOLLUSCS
BIVALVES
Henley W. F., Grobler P. J., Neves R. J. (2006): Non-invasive method to obtain DNA from freshwater mussels (Bivalvia: Unionidae). Journal of Shellfish Research 25: 975-977.
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To determine whether DNA could be isolated from tissues obtained by brush-swabbing the mantle, viscera and foot, mantle-clips and swabbed cells were obtained from eight Quadrula pustulosa (Lea, 1831). DNA yields from clips and swabbings were 447.0 and 975.3 ηg/μL, respectively. Furthermore, comparisons of sequences from the ND-1 mitochondrial gene region showed a 100% sequence agreement of DNA from cells obtained by clips and swabs. To determine the number of swabs needed to obtain adequate yields of DNA for analyses, the visceras and feet of 5 Q. pustulosa each were successively swabbed 2, 4 and 6 times. DNA yields from the 2, 4 and 6 swabbed mussel groups were 399.4, 833.8 and 852.6 ng/μL, respectively. ND-1 sequences from the lowest yield still provided 846–901 bp for the ND-1 region. Nevertheless, to ensure adequate DNA yield from cell samples obtained by swabbing, we recommend that 4 swab-strokes of the viscera and foot be obtained. The use of integumental swabbing for collection of cells for determination of genetic relationships among freshwater mussels is noninvasive, when compared with tissue collection by mantle-clipping. Therefore, its use is recommended for freshwater mussels, especially state-protected or federally listed mussel species.
Karlsson S., et al. (2013): Four methods of nondestructive DNA sampling from freshwater pearl mussels Margaritifera margaritifera L. (Bivalvia: Unionoida). Freshwater Science 32: 525-530.
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Nondestructive tissue sampling is desirable for genetic or physiological studies of endangered freshwater mussels. We used the freshwater pearl mussel (Margaritifera margaritifera) to evaluate 4 sampling methods (haemolymph extraction, foot scraping, mantle biopsy, and viscera swabbing) with regard to their effectiveness for deoxyribonucleic acid (DNA) analyses and their effects on sampled mussels. One hundred twenty-eight days subsequent to tissue sampling (1 June–7 October), all sampled individuals were alive, and average growth of sampled individuals was not significantly different from unsampled, control individuals, except that the viscera-swabbing group had lower growth. The magnitude of decreased growth in viscera-swabbed individuals was small (∼0.5 mm less than controls), and the biological significance of this result is unclear. DNA yields from haemolymph extraction and foot scraping were significantly lower and more variable than yields from the other methods. Genotyping success was lowest for haemolymph extraction and mantle biopsy, but was high for the other methods. Viscera-swab samples stored in lysis buffer at room temperature prior to DNA extraction had higher DNA yield than samples stored in buffer at 4°C or samples stored dry, but genotyping success was equivalent among storage methods. On the basis of these results, we recommend use of the noninvasive viscera-swabbing method.
Holman L. E., Hollenbeck C. M., Ashton T. J., Johnston I. A. (2019): Demonstration of the use of environmental DNA (eDNA) for the non-invasive genotyping of a bivalve mollusc, the European Flat Oyster (Ostrea edulis). Frontiers in Genetics 10: 1159.
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Accurate SNP (single nucleotide polymorphism) genotype information is critical for a wide range of selective breeding applications in aquaculture, including parentage assignment, marker-assisted, and genomic selection. However, the sampling of tissue for genetic analysis can be invasive for juvenile animals or taxa where sampling tissue is difficult or may cause mortality (e.g. bivalve mollusks). Here, we demonstrate a novel, non-invasive technique for sampling DNA based on the collection of environmental DNA using European Flat Oysters (Ostrea edulis) as an example. The live animals are placed in individual containers until sufficient genetic material is released into the seawater which is then recovered by filtration. We compared the results of tissue and eDNA derived SNP genotype calls using a PCR based genotyping platform. We found that 100% accurate genotype calls from eDNA are possible, but depend on appropriate filtration and the dilution of the sample throughout the workflow. We also developed an additional low-cost DNA extraction technique which provided >99% correct SNP genotype calls in comparison to tissue. It was concluded that eDNA sampling can be used in hatchery and selective breeding programs applicable to any aquatic organism for which direct tissue sampling may result in animal welfare concerns or mortality.
Garrison N. L., Johnson P. D., Whelan N. V. (2021): Conservation genomics reveals low genetic diversity and multiple parentage in the threatened freshwater mussel, Margaritifera hembeli. Conservation Genetics 22: 217-231.
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Margaritifera hembeli is a federally threatened freshwater mussel species restricted to three central Louisiana drainages. Currently, management efforts are being formulated without an understanding of population-level genetic patterns, which could result in sub-optimal conservation outcomes. In particular, information about riverscape genetic patterns is needed to design effective propagation and reintroduction plans. We apply a genomic approach (RADseq) to assess genetic diversity and structure among four wild populations sampled from across the species range. We also assess the genetic diversity of a captively reared cohort produced from a single female. We recovered population differentiation between individuals sampled to the north and south of the Red River. All sites had similarly low levels of heterogeneity and other measures of genetic diversity. The captive cohort displayed higher levels of genetic diversity than expected and likely represents a case of multiple paternity. Future propagation efforts will likely be able to produce genetically diverse cohorts from a small number of wild-caught females, and we recommend future reintroduction efforts utilize brooders within the sub-drainage closest to the reintroduction effort.
Massault C., Jeffrey C., Jones D. B., Barnard R., Strugnell J. M., Zenger K. R., Jerry D. R. (2022): Non-invasive DNA collection for parentage analysis for bivalves: A case study from the silver-lipped pearl oyster (Pinctada maxima). Aquaculture 552: 738036.
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Due to the high revenues generated by the pearl industry, interest in developing breeding programs is increasing. The traditional approach for collecting DNA for pedigree analyses in breeding programs for the silver-lipped pearl oyster (Pinctada maxima) involves tissue excision from the muscular foot and/or mantle. Tissue excision causes injury and stress to the animal, and can lead to reduced growth efficiency, bacterial infection and subsequent mortality when oysters are returned to the non-sterile marine environment. The approach of swabbing tissue to collect cells for DNA analyses offers a potential non-invasive and rapid method but has not been reported previously for pearl oysters; particularly where DNA is destined for a high-throughput genotype-by-sequencing (GBS) single nucleotide polymorphism (SNP) genotyping platform. To evaluate the potential of swabbing as a method to collect DNA for downstream GBS SNP genotyping under commercial farming conditions, 29 broodstock pearl oysters were swabbed with two different swab brushes (a stiff and soft brush). Concentration of the extracted DNA from swabs was found to meet requirements for genotyping (44 to 59 ng/μL) and 260/280 ratios were in the acceptable range for DNA purity (1.9–2.2). SNP genotype calls derived from swab samples were highly correlated with those derived from tissue excised from the same oyster (mean of 0.97 ± 0.04 for soft bristle – mean of 0.98 ± 0.02 for stiff bristle). Finally, we evaluated the performance of swabbing under a commercial breeding program scenario, whereby 923 spat were genotyped and 18 months later 1927 pearl oysters from the same commercial cohort were swabbed to determine parental contributions for the cohort at the two time periods. Results demonstrated that the swab method is reliable for collecting sufficient quantity and quality of DNA for genome-wide SNP genotyping enabling accurate pedigree reconstruction. This study validated the efficiency of the use of the swab technique to collect DNA for routine parentage assignment, while limiting the adverse effects of destructive tissue sampling for commercial use and/or for the development of breeding programs.
CEPHALOPODS
Hollenbeck N., Scheel D., Gravley M. C., Sage G. K., Toussaint R., Talbot S. L. (2017): Use of swabs for sampling epithelial cells for molecular genetics analyses in Enteroctopus. American Malacological Bulletin 35: 145-157.
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We evaluated the efficacy of using swabs to collect cells from the epidermis of octopus as a non-invasive DNA source for classical genetic studies, and demonstrated value of the technique by incorporating it into an effort to determine, within a day, the lineage of captured, live Enteroctopus (E. dofleini or a cryptic lineage). The cryptic lineage was targeted for captive behavioral and morphological studies, while once genetically identified, the non-target lineage could be more rapidly released back to the wild. We used commercially available sterile foam tipped swabs and a high-salt preservation buffer to collect and store paired swab and muscle (arm tip) tissue sampled from live Enteroctopus collected from Prince William Sound, Alaska. We performed a one-day extraction of DNA from epithelial swab samples and amplification of two diagnostic microsatellite loci to determine the lineage of each of the 21 individuals. Following this rapid lineage assessment, which allowed us to release non-target individuals within a day of laboratory work, we compared paired swab and muscle tissue samples from each individual to assess quantity of DNA yields and consistency of genotyping results, followed by assessment of locus-by-locus reliability of DNA extracts from swabs. Epithelial swabs yielded, on average, lower quantities of DNA (170.32 ± 74.72 (SD) ng/μL) relative to DNA obtained from tissues collected using invasive or destructive techniques (310.95 ± 147.37 (SD) ng/μL. We observed some decrease in yields of DNA from extractions of swab samples conducted 19 and 31 months after initial extractions when samples were stored at room temperature in lysis buffer. All extractions yielded quantities of DNA sufficient to amplify and score all loci, which included fragment data from 10 microsatellite loci (nine polymorphic loci and monomorphic locus EdoμA106), and nucleotide sequence data from a 528 base pair portion of the nuclear octopine dehydrogenase gene. All results from genotyping and sequencing using paired swab and muscle tissue extracts were concordant, and experimental reliability levels for multilocus genotypes generated from swab samples exceeded 97%. This technique is useful for studies in which invasive sampling is not optimal, and in remote field situations since samples can be stored at ambient temperatures for at least 31 months. The use of epithelial swabs is thus a noninvasive technique appropriate for sampling genetic material from live octopuses for use in classical genetic studies as well as supporting experimental and behavioral studies.
Sykes A. V., Alves A., Capaz J. C., Madeira C., Couto A. T., Gonçalves R. A., Frias P. A., Leal I., Andrade J.P. (2017): Refining tools for studying cuttlefish (Sepia officinalis) reproduction in captivity: in vivo sexual determination, tagging and DNA collection. Aquaculture 479: 13-16.
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To overcome the bottleneck of reproduction in cuttlefish, Sepia officinalis, aquaculture development, there is need for new tools or refinement of methods. While attaining low welfare impact on individuals, procedures to allow for in vivo individual sexual and maturity determination, tagging and collection of samples for high quality DNA are needed. The present study evaluated the use of an endoscope, visual implant elastomer and swabbing for each, respectively, in a set of 3 experiments that registered its effects on growth, mortality and reproduction. Results showed that all the methodologies tested herein were of easy application, did not promote injuries nor poor growth, abnormal reproduction or increased mortality. This indicates that cuttlefish would not be experiencing pain, suffering, distress and lasting harm (PSDLH), resulting from the application of these procedures and that these are therefore recommended as new standards for reproduction studies in the species.
GASTROPODS
Kawai K., Shimizu M., Hughes R. N., Takenaka O. (2004): A non-invasive technique for obtaining DNA from marine intertidal snails. Journal of the Marine Biological Association of the United Kingdom 84: 773-774.
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DNA was extracted from mucus secreted by snails that had been allowed to crawl over glass microscope slides. The mucus contained many epithelial cells and a few blood cells. Microsatellite DNA regions were amplified using template DNA from the mucus and clear bands obtained showing the same positions as when using template DNA from the foot. Pedal mucus is therefore a reliable source of DNA, which can be extracted by a simple methodology that is readily applied in the field. The technique has considerable potential for conservation- and behavioural ecology.
Armbruster G. F. J., Koller B., Baur B. (2005): Foot mucus and periostracum fraction as non-destructive source of DNA in the land snail Arianta arbustorum, and the development of new microsatellite loci. Conservation Genetics 6: 313-316.
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We collected mucus samples by smoothly rubbing a 1-ml plastic tip on the foot of living A. arbustorum.
Régnier C., Gargominy O., Falkner G., Puillandre N. (2011): Foot mucus stored on FTA® cards is a reliable and non-invasive source of DNA for genetics studies in molluscs. Conservation Genetics Resources 3: 377-382.
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In this study, a non-invasive method for DNA sampling of terrestrial molluscs using mucus stored on FTA cards is described. DNA was isolated from FTA cards impregnated with terrestrial mollusc mucus, which were collected from four mollusc families. DNA was stored for approximately 2 months at room temperature. For two mollusc families, DNA was extracted from foot tissue as a cross-check against the DNA obtained from mucus. Amplification of mitochondrial and nuclear DNA was successful for all mucus samples and in all cases the sequences matched those obtained from tissue samples, but also sequences from the same species, genus or family (depending on their availability) from GenBank. These results show that isolating mucus on FTA cards is a very efficient alternative to sampling tissue for genetic studies of terrestrial molluscs, and particularly for conservation genetic studies. This non-invasive method is rapid, reliable and very easy to put in practice in field situations.
Morinha F., Travassos P., Carvalho D., Magalhães P., Cabral J. A., Bastos E. (2014): DNA sampling from body swabs of terrestrial slugs (Gastropoda: Pulmonata): a simple and non-invasive method for molecular genetics approaches. Journal of Molluscan Studies 80: 99-101.
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Body swabs of Geomalacus maculosus (n = 12) and Arion spp. (n = 12) were collected in Vila Real (Northern Portugal) in their natural habitats. This procedure was accomplished by carefully scraping a sterile cotton swab against each individuals’ body 10 times. Swabs were directly placed in sterile 1.5 ml eppendorf tubes and stored at −20°C until DNA extraction.
Ashton T. J., Kayoueche-Reeve M., Blight A. J., Moore J., Paterson D. M. (2016). Duplex DNA barcoding allows accurate species determination of morphologically similar limpets (Patella spp.) from non-destructive sampling. Journal of the Marine Biological Association of the United Kingdom 97: 1479–1482.
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Accurate discrimination of two morphologically similar species of Patella limpets has been facilitated by using qPCR amplification of species-specific mitochondrial genomic regions. Cost-effective and non-destructive sampling is achieved using a mucus swab and simple sample lysis and dilution to create a PCR template. Results show 100% concurrence with dissection and microscopic analysis, and the technique has been employed successfully in field studies. The use of highly sensitive DNA barcoding techniques such as this hold great potential for improving previously challenging field assessments of species abundance.
Cha D., Kim J. Y., Kim K. S., Kim Y.-J. (2023): Species identification method by a new non-invasive technique in Korean endangered terrestrial snail, Koreanohadra koreana (Gastropoda: Mollusca). Conservation Genetics Resources.
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Koreanohadra koreana (K. koreana) is an endemic species in South Korea that is listed as endangered. While the ecology and phylogenetics of K. koreana have been studied, its morphological similarity to the related species Koreanohadra kurodana (K. kurodana), can make species identification difficult. Furthermore, this has led to confusion when determining essential habitat information for the conservation of K. koreana. To bypass this issue, we have developed a non-invasive species identification method that can genetically differentiate between them. While there are already various non-invasive genomic DNA (gDNA) extraction methods that utilize the mucus from mollusks, they are limited as they require the target species to be physically located. To address this, in this investigation a method of extracting gDNA from the feces of snails was developed. The method utilized a primer set to amplify a cytochrome b fragment from K. koreana but not K. kurodana or other terrestrial snails. The feces of terrestrial snails could thus be used to obtain gDNA to a genetically usable level if collected within 5 days of excretion. This non-invasive species identification method using feces will help to facilitate genetic research without harming the endangered species and if the target species is not physically in the habitat. Moreover, K. koreana and K. kurodana could perhaps be further distinguished, using their habitat information to help facilitate essential conservation measures.
Leung K. (2023): A size and taxonomic assessment of non-lethal DNA sampling of gastropods using Flinders Technology Associates (FTA) cards. Conservation Genetics Resources 16: 45-61.
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Sampling the DNA of rare animal species should have minimal impacts on individual health. This can be accomplished through non-lethal/non-invasive sampling. Few of these methods have been developed for invertebrates, including the Mollusca, which are in global decline. Tissue clipping the foot is a common non-lethal method for gastropods. However, it causes permanent damage and is inappropriate for smaller snails. This study used Flinders Technology Associates (FTA) cards to sample DNA from snail mucus for species of different sizes and habitat types, and across evolutionarily distant lineages. In a survival assay, the death rate of individuals sampled with FTA cards (12.1%) was greater than in the controls (3.7%), but the difference was not significant. Of 224 individuals representing 27 snail species (17 Hawaiian native, ten non-native) sampled using both FTA cards and tissue clipping, 80.4% of FTA samples and 91.6% of tissue samples amplified for COI, a significant difference. COI sequencing success did not differ significantly between the two methods. For individuals that failed to produce a COI sequence, an attempt was made to sequence 16S. For 16S, amplification and sequencing rates did not differ significantly between FTA and tissue samples. Habitat type and shell size did not affect FTA sampling success. Phylogenetically basal taxa exhibited lower success rates, but this may have been because of difficulty in sampling operculate taxa, and not because of identity. These results indicate that the FTA sampling is a viable non-lethal alternative to tissue clipping and can be used for diverse gastropods.
OTHER MOLLUSCS
Palmer A. N., Styan C. A., Shearman D. C. (2008): Foot mucus is a good source for non-destructive genetic sampling in Polyplacophora. Conservation Genetics 9: 229-231.
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A non-destructive method for collecting samples for DNA analysis from the mucus of molluscs was successfully adapted for use with the genus Ischnochiton. DNA was extracted using a Chelex-based method and the COI subunit of the mtDNA was amplified and sequenced. Sequences from the mucus were crosschecked against sequences from the foot tissue of the same animal and were found to be identical. This method provides a non-destructive way of carrying out larger studies of the genetics of rare organisms and may be of general use for genetic-based field studies of molluscs.