To pluck or not to pluck: the hidden ethical and scientific costs of relying on feathers as a primary source of DNA

This article responds to the recent prominence of ornithological literature advocating the plucking or clipping of feathers to obtain DNA in avian studies. We argue that the practise of feather plucking or clipping should be strongly discouraged on both scientific and ethical grounds in the avian literature. Currently, despite claims to the contrary, it is not clear that feather sampling as a source of DNA has lower ethical impacts on birds than blood sampling. In addition, feather samples provide a smaller and less reliable biological resource, significantly jeopardising the short and long‐term outcomes that can be gained by the sampling. In contrast, blood collection has been experimentally demonstrated to be relatively safe, subject to operators being skilled and following published guidelines, providing large yields of high quality DNA that facilitates archival storage of samples in a manner that the destructive sampling of feathers cannot.     

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

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.     

To pluck or not to pluck: scientific methodologies should be carefully chosen,not ‘one size fits all’

McDonald and Griffith (2011) raise important points in their critique of reliance on feathers as a source of DNA for scientific research. Although those authors are right about many details, their one‐size‐fits all approach (i.e. prescribing blood draws for avian DNA analyses) obscures bigger picture issues that are of extraordinary relevance to avian biology. We introduce four points to provide alternative perspectives on their commentary. In particular, we feel that a) scientific goals should determine methodologies; b) stress to animals is context specific and blood sampling is not always less stressful to birds than feather plucking; c) feather DNA is too valuable to be ignored, especially when coupled with other analyses that require feathers; and d) logistical and other concerns often preclude blood sampling. A one size fits all approach to science is generally short‐sighted, be it in regard to the collection of genetic or other samples from birds, or to a suite of other research problems.     

A Simple Method of Genomic DNA Extraction from Human Samples for PCR-RFLP Analysis

Isolation of DNA from blood and buccal swabs in adequate quantities is an integral part of forensic research and analysis. The present study was performed to determine the quality and the quantity of DNA extracted from four commonly available samples and to estimate the time duration of the ensuing PCR amplification. Here, we demonstrate that hair and urine samples can also become an alternate source for reliably obtaining a small quantity of PCR-ready DNA. We developed a rapid, cost-effective, and noninvasive method of sample collection and simple DNA extraction from buccal swabs, urine, and hair using the phenol-chloroform method. Buccal samples were subjected to DNA extraction, immediately or after refrigeration (4–6°C) for 3 days. The purity and the concentration of the extracted DNA were determined spectrophotometerically, and the adequacy of DNA extracts for the PCR-based assay was assessed by amplifying a 1030-bp region of the mitochondrial D-loop. Although DNA from all the samples was suitable for PCR, the blood and hair samples provided a good quality DNA for restriction analysis of the PCR product compared with the buccal swab and urine samples. In the present study, hair samples proved to be a good source of genomic DNA for PCR-based methods. Hence, DNA of hair samples can also be used for the genomic disorder analysis in addition to the forensic analysis as a result of the ease of sample collection in a noninvasive manner, lower sample volume requirements, and good storage capability.     

Utilizing field collected insects for next generation sequencing: Effects of sampling,storage, and DNA extraction methods

DNA sequencing technologies continue to advance the biological sciences, expanding opportunities for genomic studies of non‐model organisms for basic and applied questions. Despite these opportunities, many next generation sequencing protocols have been developed assuming a substantial quantity of high molecular weight DNA (>100 ng), which can be difficult to obtain for many study systems. In particular, the ability to sequence field‐collected specimens that exhibit varying levels of DNA degradation remains largely unexplored. In this study we investigate the influence of five traditional insect capture and curation methods on Double‐Digest Restriction Enzyme Associated DNA (ddRAD) sequencing success for three wild bee species. We sequenced a total of 105 specimens (between 7–13 specimens per species and treatment). We additionally investigated how different DNA quality metrics (including pre‐sequence concentration and contamination) predicted downstream sequencing success, and also compared two DNA extraction methods. We report successful library preparation for all specimens, with all treatments and extraction methods producing enough highly reliable loci for population genetic analyses. Although results varied between species, we found that specimens collected by net sampling directly into 100% EtOH, or by passive trapping followed by 100% EtOH storage before pinning tended to produce higher quality ddRAD assemblies, likely as a result of rapid specimen desiccation. Surprisingly, we found that specimens preserved in propylene glycol during field sampling exhibited lower‐quality assemblies. We provide recommendations for each treatment, extraction method, and DNA quality assessment, and further encourage researchers to consider utilizing a wider variety of specimens for genomic analyses.     

A comparison of plucked feathers versus blood samples as DNA sources for molecular sexing

ABSTRACT.   Feathers are increasingly collected as a nondestructive source of DNA for avian genetic research. Although feather samples are not optimal in some important ways than more robust blood or tissue samples, feather sampling requires less training for field workers, results in shorter handling times for the organism, generates no hazardous wastes, and requires simpler storage procedures. Along with these largely positive attributes comes a set of challenges, particularly the relatively low copy number of DNA present in feather samples. We compared the utility and reliability of feathers to the more traditional blood samples as sources of DNA for polymerase chain reaction (PCR)-based molecular sexing of Black-capped Chickadees ( Poecile atricapilla ). DNA from 102 individuals was extracted separately from both single rectrices and from blood samples, and the sex of each bird was then determined using standard PCR-based methods. We found complete agreement between sex determinations based on feather versus blood DNA extractions. Slight variations in lab protocols were necessary to obtain consistent results from these two DNA sources; and we briefly discuss other sources of error that could occur in feather-based molecular sexing studies. This controlled comparison of feather versus blood samples demonstrates that plucked rectrices provide a highly reliable source of DNA for molecular sexing of wild birds.     

Reliable genotyping of the koala (Phascolarctos cinereus) using DNA isolated from a single faecal pellet

The koala, an Australian icon, has been added to the threatened species list. Rationale for the listing includes proposed declines in population size, threats to populations (e.g. disease) and loss and fragmentation of habitat. There is now an urgent need to obtain accurate data to assess the status of koala populations in Australia, to ensure the long‐term viability of this species. Advances in genetic techniques have enabled DNA analysis to study and inform the management of wild populations; however, sampling of individual koalas is difficult in tall, often remote, eucalypt forest. The collection of faecal pellets (scats) from the forest floor presents an opportunistic sampling strategy, where DNA can be collected without capturing or even sighting an individual. Obtaining DNA via noninvasive sampling can be used to rapidly sample a large proportion of a population; however, DNA from noninvasively collected samples is often degraded. Factors influencing DNA quality and quantity include environmental exposure, diet and methods of sample collection, storage and DNA isolation. Reduced DNA quality and quantity can introduce genotyping errors and provide inaccurate DNA profiles, reducing confidence in the ability of such data to inform management/conservation strategies. Here, we present a protocol that produces a reliable individual koala genotype from a single faecal pellet and highlight the importance of optimizing DNA isolation and analysis for the species of interest. This method could readily be adapted for genetic studies of mammals other than koalas, particularly those whose diet contains high proportions of volatile materials that are likely to induce DNA damage.     

Major histocompatibility complex class II polymorphisms in Macaca mulatta: factors influencing comprehensive genotyping of Macaca mulatta (Mamu)-DQA1 alleles by PCR-RFLP in archival samples.

In the last 5 years, HLA class II genotyping methods have been adapted for genotyping of class II loci in rhesus macaques. Since previously published typing protocols were used on samples that were collected and stored under ideal conditions, it was of interest to determine if these methods were adequate for genotyping a large collection of archival samples from which DNA had been isolated and stored under various conditions. Established macaque DQA1 typing protocols were modified to optimize the typing procedure and enhance the ability to successfully genotype DNA from samples that were of poor quality and/or quantity. Long-term storage of whole-blood buffy coats or stored DNA extracted from whole-blood buffy coats did not affect typing success; however, amplification and typing of DNA extracted from archival samples of plasma were difficult and resulted in a low success rate. This suggests that amplification and DQA1-genotyping of archival samples is possible with a modified protocol, but is influenced by the age and source of the sample, and to a lesser extent, the method used to extract DNA from sample substrates.     

Methodological confounds of measuring urinary oxidative stress in wild animals

Biomarkers of oxidative stress (OS) are useful in addressing a wide range of research questions, but thus far, they have had limited application to wild mammal populations due to a reliance on blood or tissue sampling. A shift toward non‐invasive measurement of OS would allow field ecologists and conservationists to apply this method more readily. However, the impact of methodological confounds on urinary OS measurement under field conditions has never been explicitly investigated. We combined a cross‐sectional analysis with a field experiment to assess the impact of four potential methodological confounds on OS measurements: (1) time of sampling, (2) environmental contamination from foliage; (3) delay between sample collection and flash‐freezing in liquid nitrogen; and (4) sample storage of up to 15 months below −80°C. We measured DNA oxidative damage (8‐hydroxy‐2′‐deoxyguanosine, 8‐OHdG), lipid peroxidation (malondialdehyde, MDA), total antioxidant capacity (TAC), and uric acid (UA) in 167 urine samples collected from wild Zanzibar red colobus (Piliocolobus kirkii). We found that MDA was higher in samples collected in the morning than in the afternoon but there were no diurnal patterns in any of the other markers. Contamination of samples from foliage and length of time frozen at −80°C for up to 15 months did not affect OS marker concentrations. Freezing delay did not affect OS levels cross‐sectionally, but OS values from individual samples showed only moderate‐to‐good consistency and substantial rank‐order reversals when exposed to different freezing delays. We recommend that diurnal patterns of OS markers and the impact of storage time before and after freezing on OS marker concentrations be considered when designing sampling protocols. However, given the high stability we observed for four OS markers subject to a variety of putative methodological confounds, we suggest that urinary OS markers provide a valuable addition to the toolkit of field ecologists and conservationists within reasonable methodological constraints.     

Isolation of high-molecular-weight DNA from small samples of blood having nucleated erythrocytes,collected, transported,and stored at room temperature

Blood samples collected in the field for isolating DNA suitable for molecular analysis need special care in their storage and handling. In this article, we describe a simple method for the isolation of good-quality high-molecular-weight DNA that does not require low temperature conditions during collection, storage, and/or transportation of blood samples. This method involves smearing small aliquots of blood onto clean slides and air drying them at room temperature. The slides with blood smears can then be transported or stored at room temperature and still serve as a very good source of high-molecular-weight DNA. Genomic DNA from these samples can be extracted by organic phase separation (phenol-chloroform extraction) after lysis. The DNA thus obtained is of high quality and yields DNA fingerprints qualitatively similar to those prepared from corresponding control DNA isolated from frozen blood samples. Needing minimal facilities at field sites, the method is very convenient for conducting RFLP analysis of wild/field populations for demographic, behavioral, and ecologic studies.     

Effects of storage type and time on DNA amplification success in tropical ungulate faeces

The present study compares the effect of three storage media (silica, RNAlater®, ethanol) and time to extraction (1 week, 1 month and 3 months) on mitochondrial and nuclear marker amplification success in faecal DNA extracts from a sympatric community of small to medium‐sized Central African forest ungulates (genera Cephalophus, Tragelaphus, Hyemoschus). The effect of storage type and time on nuclear DNA concentrations, genotyping errors and percentage recovery of consensus genotypes was also examined. Regardless of storage method, mitochondrial and nuclear amplification success was high in DNA extracted within the first week after collection. Over longer storage periods, RNAlater yielded better amplification success rates in the mitochondrial assay. However, samples stored on silica showed (i) highest nuclear DNA concentrations, (ii) best microsatellite genotyping success, (iii) lowest genotyping errors, and (iv) greatest percentage recovery of the consensus genotype. The quantity of nuclear DNA was generally a good predictor of microsatellite performance with 83% amplification success or greater achieved with sample DNA concentrations of ≥ 50 pg/µL. If faecal DNA samples are to be used for nuclear microsatellite analyses, we recommend silica as the best storage method. However, for maximum mitochondrial amplification success, RNAlater appears to be the best storage medium. In contrast, ethanol appeared inferior to the other two methods examined here and should not be used to store tropical ungulate faeces. Regardless of storage method, samples should be extracted as soon as possible after collection to ensure optimal recovery of DNA.     

Noninvasive genetic analysis in birds: testing reliability of feather samples

Noninvasive samples are useful for molecular genetic analysis of free‐ranging animals. I tested whether moulted feathers collected in the field are a reliable source of DNA for genotyping microsatellite loci. I prescreened extracts for DNA quantity and, using only samples with higher amounts of DNA, obtained reliable genotyping results. Polymerase chain reaction (PCR) amplification success was higher from extracts of plucked feathers than moulted feathers. DNA quantity in larger feathers was higher than that in smaller feathers. This study clearly demonstrates that moulted feathers could be used for genetic studies in birds.     

Effects of blood collection on wild birds: an update

Blood sampling is often critical for answering a variety of questions about wild birds. However, it is important to assess the impacts, if any, of blood collection on wild birds. Here, we examined the effects of blood sampling on adults or nestlings in three species of free-living birds. First, we examined the effects of blood collection on annual survival and reproductive success in adult buff-breasted wrens Thryothorus leucotis in Panama. In adult wrens, blood collection from the brachial vein during the breeding season had no effect on annual survival or reproductive success. Second, we examined whether blood collection influenced mass gain in developing smooth-billed anis Crotophaga ani in Puerto Rico. In developing anis, blood collection from the femoral or jugular veins had no effect on mass gain of nestlings. Third, in developing European starlings Sturnus vulgaris in British Columbia, Canada, blood collection from the brachial vein had no effect of body condition. Blood collection from the jugular vein had a transient effect on body condition during the first week post-hatch, but this effect disappeared by the second week of age. Lastly, we present an extensive up-to-date review of the literature on the effects of blood collection on free-living avian species. Taken together, these data show that blood collection has no major negative effects on developing or adult birds in the wild.     

A New Method for Noninvasive Genetic Sampling of Saliva in Ecological Research

Noninvasive samples for genetic analyses have become essential to address ecological questions. Popular noninvasive samples such as faeces contain degraded DNA which may compromise genotyping success. Saliva is an excellent alternative DNA source but scarcity of suitable collection methods makes its use anecdotal in field ecological studies. We develop a noninvasive method of collection that combines baits and porous materials able to capture saliva. We report its potential in optimal conditions, using confined dogs and collecting saliva early after deposition. DNA concentration in saliva extracts was generally high (mean 14 ng μl^-1). We correctly identified individuals in 78% of samples conservatively using ten microsatellite loci, and 90% of samples using only eight loci. Consensus genotypes closely matched reference genotypes obtained from hair DNA (99% of identification successes and 91% of failures). Mean genotyping effort needed for identification using ten loci was 2.2 replicates. Genotyping errors occurred at a very low frequency (allelic dropout: 2.3%; false alleles: 1.5%). Individual identification success increased with duration of substrate handling inside dog’s mouth and the volume of saliva collected. Low identification success was associated with baits rich in DNA-oxidant polyphenols and DNA concentrations <1 ng μl^-1. The procedure performed at least as well as other noninvasive methods, and could advantageously allow detection of socially low-ranked individuals underrepresented in sources of DNA that are involved in marking behaviour (faeces or urine). Once adapted and refined, there is promise for this technique to allow potentially high rates of individual identification in ecological field studies requiring noninvasive sampling of wild vertebrates.     

An overlooked DNA source for non-invasive genetic analysis in birds

Non-invasive sampling is a useful tool for genetic analyses of endangered and/or elusive species, but it is often inapplicable due to the low quality and quantity of the DNA obtained. In this study we show that the blood clot located in the superior umbilicus of the feather shaft is a better source of DNA than the previously used tip samples from moulted feathers. We found that feather clots from museum specimens provided results nearly as good as footpad and better than those from the more commonly used museum skin snips. Feather clots proved to be a good source of DNA for genetic analysis that will significantly facilitate genetic monitoring of wild bird populations.     

一种从鸟类剥制标本提取DNA的改进方法

应用非损伤性取样的方法,收集鸟类剥制标本的皮肤组织和羽毛,用无水乙醇、浸泡液预处理的方法抽提DNA,结果两者都可提取DNA供PCR扩增。将PCR产物经序列测定和比对分析,证明提取的DNA为目的DNA,表明本试验方法可行。鸟类剥制标本的皮肤组织和羽毛可以作为研究种群遗传学的资源。     

Single-tube HotSHOT technique for the collection, preservation and PCR-ready DNA preparation of faecal samples: the threatened Cabrera's vole as a model

A cost-effective, reliable and efficient method of obtaining DNA samples is essential in large-scale genetic analyses. This study examines the possibility of using a threatened vole species, Microtus cabrerae, as a model for the collection and preservation of faecal samples for subsequent DNA extraction with a protocol based on the HotSHOT technique. Through the examination of the probability of multi-copies (mitochondrial) and single copy (microsatellite) loci amplification (including the genotype error) and of the DNA yield (estimated by real-time qPCR), the new protocol was compared with both the frequently employed methods that successfully use ethanol to preserve faecal samples and with commercial kit-based DNA extraction. The single-tube HotSHOT-based protocol is a user-friendly, non-polluting, time-saving and inexpensive method of faeces sample collection, preservation and PCR-quality gDNA preparation. This technique therefore provides researchers with a new approach that can be employed in high-throughput, noninvasive genetic analyses of wild animal populations.     

Optimization of conditions to extract high quality DNA for PCR analysis from whole blood using SDS-proteinase K method

In case of studies associated with human genetics, genomics, and pharmacogenetics the genomic DNA is extracted from the buccal cells, whole blood etc. Several methods are exploited by the researchers to extract DNA from the whole blood. One of these methods, which utilizes cell lysis and proteolytic properties of sodium dodecyl sulfate (SDS) and proteinase K respectively, might also be called SDS-PK method. It does not include any hazardous chemicals such as phenol or chloroform and is inexpensive. However, several researchers report the same method with different formulas and conditions. During our experiments with whole blood DNA extraction we experienced problems such as protein contamination, DNA purity and yield when followed some SDS-PK protocols reported elsewhere. A260/A280 and A260/A230 ratios along with PCR amplification give a clear idea about the procedure that was followed to extract the DNA. In an effort to increase the DNA purity from human whole blood, we pointed out some steps of the protocol that play a crucial role in determining the extraction of high quality DNA.     

Salt drying: a low‐cost,simple and efficient method for storing plants in the field and preserving biological repositories for DNA diversity research

Although a variety of methods have been optimized for the collection and storage of plant specimens, most of these are not suited for field expeditions for a variety of logistic reasons. Drying specimens with silica gel in polyethylene bags is currently the standard for field‐sampling methods that are suitable for subsequent DNA extraction. However, silica‐gel repositories are not readily available in remote areas, and its use is not very cost‐effective for the long‐term storage of collections or in developing countries with limited research budgets. Salting is an ancient and traditional drying process that preserves food samples by dehydrating tissues and inhibiting water‐dependent cellular metabolism. We compared salt and silica‐gel drying methods with respect to dehydration rates overtime, DNA quality and polymerase chain reaction(PCR) success to assess whether dry salting can be used as an effective plant preservation method for DNA analysis. Specimens from eleven plant species covering a variety of leaf structures, leaf thicknesses and water contents were analysed. Experimental work indicated that (i) levels of dehydration in sodium chloride were usually comparable to those obtained when silica gel was used, (ii) no spoilage, fungal or bacterial growth was observed for any of the species with all drying treatments and (iii) good yields of quality genomic DNA suitable for PCR applications were obtained in the salt‐drying treatments. The preservation of plant tissues in commercial table salt appears to be a satisfactory, and versatile method that may be suitable in remote areas where cryogenic resources and silica repositories are not available.     

Quantitative polymerase chain reaction analysis of DNA from noninvasive samples for accurate microsatellite genotyping of wild chimpanzees (Pan troglodytes verus)

Noninvasive samples are useful for molecular genetic analyses of wild animal populations. However, the low DNA content of such samples makes DNA amplification difficult, and there is the potential for erroneous results when one of two alleles at heterozygous microsatellite loci fails to be amplified. In this study we describe an assay designed to measure the amount of amplifiable nuclear DNA in low DNA concentration extracts from noninvasive samples. We describe the range of DNA amounts obtained from chimpanzee faeces and shed hair samples and formulate a new efficient approach for accurate microsatellite genotyping. Prescreening of extracts for DNA quantity is recommended for sorting of samples for likely success and reliability. Repetition of results remains extensive for analysis of microsatellite amplifications beginning from low starting amounts of DNA, but is reduced for those with higher DNA content.