Noninvasive collection of fresh hairs from free‐ranging howler monkeys for DNA extraction

The use of noninvasive collected samples as source of DNA in studies of wild primate populations has increased in recent years. Fresh‐plucked hairs represent an important source of DNA, with relatively high quality and concentration. In this study, we describe a low‐cost noninvasive technique for collecting fresh‐plucked hairs used to obtain DNA samples from free‐ranging black howler monkey populations (Alouatta pigra). We designed and manufactured darts made of wooden dowels, with the anterior part smeared with glue, which were projected with blowpipes to trap howler monkey hairs. All of the materials to make the darts are inexpensive and are available locally. We collected 89 samples from 76 individuals residing in 15 troops, and the total number of hairs obtained was 754. We found no differences in the number of hairs collected among sex–age classes or among localities but the percentage of darts recovered with sample varied among localities. Preliminary results indicate that over 96% of samples yielded DNA suitable for polymerase chain reaction‐based microsatellite marker analysis. The technique proved successful for collecting fresh‐plucked hairs of free‐ranging black howler monkeys without any trauma to the animals and can be easily adapted to obtain samples from other wild primate and mammal species. Am. J. Primatol. 71:359–363, 2009. © 2009 Wiley‐Liss, Inc.     

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.     

Wild chimpanzee infant urine and saliva sampled noninvasively usable for DNA analyses

In many genetic studies on the great apes, fecal or hair samples have been used as sources of DNA. However, feces and hairs are difficult to collect from chimpanzee infants under 3 years of age. As alternative DNA sources, we investigated the efficiency of collecting urine samples from infants compared with fecal samples, as well as the validity of the DNA extracted from urine and saliva samples of well-habituated M group chimpanzees (Pan troglodytes schweinfurthii) in the Mahale Mountains National Park, Tanzania. We collected 40 urine and 3 fecal samples from 10 infants under 3 years. Compared with feces, the urine samples were relatively easy to collect. The saliva of infants, which remained on the twigs sucked by them, was collected using cotton swabs. The average amounts of DNA extracted from the 40 urine and 6 saliva samples were 3,920 and 458 pg/μl, respectively. The rate of positive PCR was low and the allelic dropout rate was high when using less than 25 pg of template DNA in the PCR mixtures. Based on the amounts of DNA, 50% of the urine samples and 100% of the saliva samples were judged usable for accurate microsatellite genotyping. For infant chimpanzees in particular, collecting urine and saliva as an alternative to fecal and hair samples can reduce the effort invested in collection in the field.     

Primates and parasites: A case for a multidisciplinary approach

Examinations of primate parasitic infections can inform both primatologists and parasitologists about evolutionary and ecological relationships. Interspecific, intraspecific, and interindividual variation in parasitic infections may correlate with environmental, demographic, behavioral, and human variables. Understanding these relationships is particularly important for conservation management issues for endangered species. We describe techniques for the noninvasive collection and preservation of fecal samples from wild primates and the salvaging of parasitological information from primate hosts in the field.     

Multiplex pre-amplification for noninvasive genetic sampling: is the extra effort worth it?

Microsatellite genotyping of hair and faeces using standard polymerase chain reaction (PCR) resulted in low success rates and high error rates in a 2003–2004 pilot study using noninvasive genetic sampling for the brown bear (Ursus arctos) in the Italian Alps. Thus, we evaluated the performance of multiplex pre-amplification for improving microsatellite genotyping results. Brown bear faecal DNA extracts of varying quality (n = 33) and hair DNA extracts of poor (n = 32) and good (n = 34) quality were used to compare standard PCR and pre-amplification. In contrast to previous studies, there was no significant difference between methods for individual locus amplification success, genotyping error and genotyping success rates for scat and hair samples. The use of pre-amplification requires an additional investment of time and resources, and our results raise questions about the universal value of pre-amplification approaches. We suggest that researchers carefully evaluate the performance of pre-amplification compared to standard PCR using field-collected samples from the study area of interest before engaging in large-scale noninvasive genetic analyses.     

Two-step multiplex polymerase chain reaction improves the speed and accuracy of genotyping using DNA from noninvasive and museum samples

Many studies in molecular ecology rely upon the genotyping of large numbers of low‐quantity DNA extracts derived from noninvasive or museum specimens. To overcome low amplification success rates and avoid genotyping errors such as allelic dropout and false alleles, multiple polymerase chain reaction (PCR) replicates for each sample are typically used. Recently, two‐step multiplex procedures have been introduced which drastically increase the success rate and efficiency of genotyping. However, controversy still exists concerning the amount of replication needed for suitable control of error. Here we describe the use of a two‐step multiplex PCR procedure that allows rapid genotyping using at least 19 different microsatellite loci. We applied this approach to quantified amounts of noninvasive DNAs from western chimpanzee, western gorilla, mountain gorilla and black and white colobus faecal samples, as well as to DNA from ~100‐year‐old gorilla teeth from museums. Analysis of over 45 000 PCRs revealed average success rates of > 90% using faecal DNAs and 74% using museum specimen DNAs. Average allelic dropout rates were substantially reduced compared to those obtained using conventional singleplex PCR protocols, and reliable genotyping using low (< 25 pg) amounts of template DNA was possible. However, four to five replicates of apparently homozygous results are needed to avoid allelic dropout when using the lowest concentration DNAs (< 50 pg/reaction), suggesting that use of protocols allowing routine acceptance of homozygous genotypes after as few as three replicates may lead to unanticipated errors when applied to low‐concentration DNAs.     

Genotyping of celiac disease-related-risk haplotypes using a closed-tube polymerase chain reaction analysis of dried blood and saliva disk samples.

Expansion of molecular diagnostics more widely into clinical routines requires simplified methods allowing automation. We developed a homogeneous, multilabel polymerase chain reaction (PCR) method based on time-resolved fluorometry, and studied the use of dried disk samples in PCR. Celiac disease-related HLA-DQA1*05, HLA-DQB1*02, and HLA-DQB1*0302 genotyping was used to verify the method with blood and saliva samples dried on S&S 903 and IsoCode sample collection papers. Three sample preparation procedures, including manufacturer’s manual elution, an automated elution, and direct use of disk samples, were compared using dried disk samples. The three procedures gave successful amplification and correct genotyping results. Owing to the simplicity of the direct use of disk samples in PCR, this method was chosen for the subsequent homogeneous analysis of blood (n = 194) and saliva (n = 30) disk samples on S&S 903 paper. The results revealed that, in addition to DNA samples (n = 29), both blood and saliva disk samples were successfully amplified and genotyped using the homogeneous PCR assays for HLA-DQA1 and HLA-DQB1. The homogeneous PCR assays developed provide a useful tool to genotype celiac disease-related HLA-DQA1*05, HLA-DQB1*02, and HLA-DQB1*0302 alleles. Furthermore, the method provides a direct way to perform a closed-tube PCR analysis of dried blood and saliva disk samples enabling simple automation.     

Saliva samples are a viable alternative to blood samples as a source of DNA for high throughput genotyping

ABSTRACT: BACKGROUND: The increasing trend for incorporation of biological sample collection within clinical trials requires sample collection procedures which are convenient and acceptable for both patients and clinicians. This study investigated the feasibility of using saliva-extracted DNA in comparison to blood-derived DNA, across two genotyping platforms: Applied Biosystems Taqman TM and Illumina Beadchip TM genome-wide arrays. METHOD: Patients were recruited from the Pharmacogenetics of Breast Cancer Chemotherapy (PGSNPS) study. Paired blood and saliva samples were collected from 79 study participants. The Oragene DNA Self-Collection kit (DNAgenotek(R)) was used to collect and extract DNA from saliva. DNA from EDTA blood samples (median volume 8 ml) was extracted by GenProbe, Livingstone, UK. DNA yields, standard measures of DNA quality, genotype call rates and genotype concordance between paired, duplicated samples were assessed. RESULTS: Total DNA yields were lower from saliva (mean 24 ug, range 0.2-52 ug) than from blood (mean 210 ug, range 58-577 ug) and a 2-fold difference remained after adjusting for the volume of biological material collected. Protein contamination and DNA fragmentation measures were greater in saliva DNA. 78/79 saliva samples yielded sufficient DNA for use on Illumina Beadchip arrays and using Taqman assays. Four samples were randomly selected for genotyping in duplicate on the Illumina Beadchip arrays. All samples were genotyped using Taqman assays. DNA quality, as assessed by genotype call rates and genotype concordance between matched pairs of DNA was high (>97%) for each measure in both blood and saliva-derived DNA. CONCLUSION: We conclude that DNA from saliva and blood samples is comparable when genotyping using either Taqman assays or genome-wide chip arrays. Saliva sampling has the potential to increase participant recruitment within clinical trials, as well as reducing the resources and organisation required for multicentre sample collection.     

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.     

Techniques for noninvasive genetic monitoring of Alpine Ibex <Emphasis Type="Italic">Capra ibex</Emphasis>

Noninvasive sampling is of increasing importance for the molecular genetic monitoring of wild animal populations, although reduced quality and quantity of such samples’ DNA can affect genetic data and their subsequent interpretation. Consequently, we performed a pilot study to establish a feasible approach for the genetic investigation of free-ranging Alpine ibex Capra ibex Linnaeus, 1758 populations. Establishing an ibex-specific PCR-RFLP based on Cytochrome b gene differences allowed the discrimination of noninvasive ibex samples from those of other sympatric ungulates. In addition, we established a quantitative PCR for ibex samples. The quantification of 35 faecal samples clearly exhibited a strong variability of DNA contents among samples and individuals. Furthermore, we performed threefold genotyping experiments on six microsatellite loci to determine the extent of genotyping errors in reference to blood samples of the respective individuals. The analyses exhibited a strong dependence of erroneous microsatellite genotypes on the starting amount of template DNA. Variability in reliability was observed between individual loci, resulting in a mandatory high DNA concentration necessary for consistent genotyping. This study serves as basis for further ibex research and we propose the application of DNA quantification of faecal samples to focus genotyping efforts solely on suitable samples.     

Reliable noninvasive genotyping: fantasy or reality?

Noninvasive genotyping has not gained wide application, due to the notion that it is unreliable, and also because remedial measures are time consuming and expensive. Of the wide variety of noninvasive DNA sources, dung is the most universal and most widely used in studies. We have developed collection, extraction, and amplification protocols that are inexpensive and provide a high level of success in amplifying both mitochondrial and nuclear DNA from dung. Here we demonstrate the reliability of genotyping from elephant dung using these protocols by comparing results from dung-extracted DNA to results from blood-extracted DNA. The level of error from dung extractions was only slightly higher than from blood extractions, and conducting two extractions from each sample and a single amplification from each extraction was sufficient to eliminate error. Di-, tri-, and tetranucleotide loci were equally reliable, and low DNA quantity and quality and PCR inhibitors were not a major problem in genotyping from dung. We discuss the possible causes of error in genotyping with particular reference to noninvasive samples and suggest methods of reducing such error.     

A test of the efficacy of whole‐genome amplification on DNA obtained from low‐yield samples

Conservation and population genetic studies are sometimes hampered by insufficient quantities of high quality DNA. One potential way to overcome this problem is through the use of whole genome amplification (WGA) kits. We performed rolling circle WGA on DNA obtained from matched hair and tissue samples of North American red squirrels (Tamiasciurus hudsonicus). Following polymerase chain reaction (PCR) at four microsatellite loci, we compared genotyping success for DNA from different source tissues, both pre‐ and post‐WGA. Genotypes obtained with tissue were robust, whether or not DNA had been subjected to WGA. DNA extracted from hair produced results that were largely concordant with matched tissue samples, although amplification success was reduced and some allelic dropout was observed. WGA of hair samples resulted in a low genotyping success rate and an unacceptably high rate of allelic dropout and genotyping error. The problem was not rectified by conducting PCR of WGA hair samples in triplicate. Therefore, we conclude that WGA is only an effective method of enhancing template DNA quantity when the initial sample is from high‐yield material.     

False alleles derived from microbial DNA pose a potential source of error in microsatellite genotyping of DNA from faeces

Microsatellite genotyping of wild animals using DNA extracted from noninvasive samples such as faeces is a powerful means to identify individuals within a population and examine aspects of genetic social structure, such as relatedness and paternity. However, the use of the low quantities of poor quality DNA typically obtained from noninvasive samples can result in genotyping errors. Here we report the first instance of artefactural ‘alleles’ resulting from specific co‐amplification of microorganismal DNA present in the total DNA derived from faeces.     

Noninvasive recovery and detection of possum Trichosurus vulpecula DNA from bitten bait interference devices (WaxTags)

The brushtail possum is a major agricultural and ecological pest in New Zealand. A novel noninvasive DNA sampling tool for detecting its presence (WaxTags, or WT) was tested. DNA was recovered from saliva left on WT, and two lengths (407 bp and 648 bp) of the cytochrome c oxidase I (COI) barcoding region were amplified by polymerase chain reaction (PCR). PCR products were considered (+) when a DNA band was clearly visible by electrophoresis. Different factors that might affect PCR (+) were investigated with captive possums: (i) both extraction protocols of the QIAGEN DNeasy Blood and Tissue Kit, (ii) effect of an overnight or longer delay of up to 3 weeks before DNA extraction on both COI amplicons, and (iii) effect of the individual, order and magnitude of the bite. Extraction protocols were not significantly different. The effect of the overnight delay was not significant, and amplification of the short amplicon was significantly higher (100%) than for the long fragment (48%). After a two or 3‐week delay, the short amplicon had 94% and 56% PCR (+), success rates, respectively. Individual, order and magnitude of a bite had no significant effect. The delay trial was repeated with WT from the wild, for which PCR (+) rate of the short amplicon was 63%, regardless of freshness. Four microsatellites were amplified from captive WT samples. We conclude that DNA from saliva traces can be recovered from WT, a potential new tool for noninvasive monitoring of possums and other wildlife.     

Evaluation of saliva as a source of human DNA for population and association studies

A simple noninvasive procedure for saliva sample collection and DNA extraction was developed. On average, the amount of human DNA (as measured by a TaqMan-based assay) was about 11.4 microg/mL saliva, which is more than can be obtained from other noninvasive samples such as cheek swabs. However, the presence of large amounts of nonhuman DNA (up to 90% of the total extracted DNA) in saliva samples does necessitate DNA quantitation methods that are specific for human DNA. We were able to reliably and accurately type different genetic markers (mDNA sequences, Y-chromosomal single-nucleotide polymorphisms, and autosomal microsatellite loci) from saliva samples stored for up to 30 days at 37 degrees C, making this method well-suited for field conditions and convenient transportation of samples back to the laboratory. Thus, saliva can be considered a reliable source of DNA for a wide variety of genetic studies.     

Highly accurate SNP genotyping from historical and low‐quality samples

Historical and other poor‐quality samples are often necessary for population genetics, conservation, and forensics studies. Although there is a long history of using mtDNA from such samples, obtaining and genotyping nuclear loci have been considered difficult and error‐prone at best, and impossible at worst. The primary issues are the amount of nuclear DNA available for genotyping, and the degradation of the DNA into small fragments. Single nucleotide polymorphisms offer potential advantages for assaying nuclear variation in historical and poor‐quality samples, because the amplified fragments can be very small, varying little or not at all in size between alleles, and can be amplified efficiently by polymerase chain reaction (PCR). We present a method for highly multiplexed PCR of SNP loci, followed by dual‐fluorescence genotyping that is very effective for genotyping poor‐quality samples, and can potentially use very little template DNA, regardless of the number of loci to be genotyped. We genotyped 19 SNP loci from DNA extracted from modern and historical bowhead whale tissue, bone and baleen samples. The PCR failure rate was < 1.5%, and the genotyping error rate was 0.1% when DNA samples contained > 10 copies/µL of a 51‐bp nuclear sequence. Among samples with ≤ 10 copies/µL DNA, samples could still be genotyped confidently with appropriate levels of replication from independent multiplex PCRs.     

Urine — a source for noninvasive genetic monitoring in wildlife

Noninvasive samples are of increasing importance to study wild populations. In this study, we investigate the applicability of urine samples as the sole source of DNA for routine noninvasive genetic monitoring of wildlife using wolves (Canis lupus) as an example. Within the scope of a long‐term wolf population survey, we collected during winter snow tracking in Bieszczady Mountains, Poland 41 urine samples considered as utilizable for genetic analyses. DNA concentration was determined by quantitative real‐time polymerase chain reaction (qPCR) and six microsatellite loci were genotyped in threefold repeated genotyping experiments to assess the reliability of genetic analyses of urine. DNA concentration of 33 urine samples was successfully quantified and of 14 samples, we obtained congruent results for all analysed loci and all repeated genotyping experiments. The gender of urine samples was identified with a Y‐chromosome‐linked marker. Considering the high discovery rate of urine in conjunction with its genotype reliability, our study confirms that urine is a valuable source in noninvasive genetic monitoring. Additionally, preselection of samples via qPCR proved to be a powerful tool contributing to a beneficial cost‐value ratio of genetic analyses by minimizing genotyping errors.     

Noninvasive methodology for the sampling and extraction of DNA from free‐ranging Atlantic spotted dolphins (Stenella frontalis)

Genetic sampling and molecular investigations are important parts of studying wild populations. However, collecting tissues from free‐ranging animals can be difficult or impractical. This study develops a sampling and extraction protocol for template DNA from faecal material collected in a marine environment from small cetaceans. DNA was extracted from faecal material of free‐ranging Atlantic spotted dolphins (Stenella frontalis) and subsequently tested for its suitability in molecular investigations by amplifying both mitochondrial and nuclear DNA. The resulting mitochondrial sequences were found to closely match known S. frontalis haplotypes. Three microsatellite loci were amplified and fall within the expected size range for cetaceans. Mother and calf families previously assigned by observation were genetically confirmed using both mitochondrial haplotype and allele sharing between the mother and offspring. The protocol effectively collects and extracts dolphin DNA from faecal samples and enables species identification as well as confirmation of genetic relatedness and should be considered as a noninvasive alternative to current protocols.     

Agroecosystems and Primate Conservation in The Tropics: A Review

Agroecosystems cover more than one quarter of the global land area (ca. 50 million km²) as highly simplified (e.g. pasturelands) or more complex systems (e.g. polycultures and agroforestry systems) with the capacity to support higher biodiversity. Increasingly more information has been published about primates in agroecosystems but a general synthesis of the diversity of agroecosystems that primates use or which primate taxa are able to persist in these anthropogenic components of the landscapes is still lacking. Because of the continued extensive transformation of primate habitat into human‐modified landscapes, it is important to explore the extent to which agroecosystems are used by primates. In this article, we reviewed published information on the use of agroecosystems by primates in habitat countries and also discuss the potential costs and benefits to human and nonhuman primates of primate use of agroecosystems. The review showed that 57 primate taxa from four regions: Mesoamerica, South America, Sub‐Saharan Africa (including Madagascar), and South East Asia, used 38 types of agroecosystems as temporary or permanent habitats. Fifty‐one percent of the taxa recorded in agroecosystems were classified as least concern in the IUCN Red List, but the rest were classified as endangered (20%), vulnerable (18%), near threatened (9%), or critically endangered (2%). The large proportion of threatened primates in agroecosystems suggests that agroecosystems may play an important role in landscape approaches to primate conservation. We conclude by discussing the value of agroecosystems for primate conservation at a broad scale and highlight priorities for future research. Am. J. Primatol. 74:696‐711, 2012. © 2012 Wiley Periodicals, Inc.     

Nutritional status of free‐ranging Mexican howler monkeys (Alouatta palliata mexicana) in Veracruz,Mexico: Serum chemistry; lipoprotein profile; vitamins D,A, and E; carotenoids; and minerals

The purpose of this work was to measure important nutritional status parameters for a group of free‐ranging Mexican mantled howler monkeys (A. palliata mexicana) and compare those data to published data for primates. The nutritional status of six free‐ranging Mexican mantled howler monkeys was examined using biochemical analysis. Blood samples were analyzed for serum chemistry; lipids; vitamins D, A, and E; carotenoids; and minerals. Serum chemistries were somewhat different from published values, but did not indicate clear abnormalities. Circulating lipids were not different from those in captive primates. Circulating vitamin D metabolites (83±16.3 for 25(OH)D ng/mL; 563±53.8 for 1,25(OH)₂D pg/mL) were similar to those in wild‐caught tamarins (Saguinus oedipus), lower than some published data for captive Cebidae and Callitrichidae, and higher than for Old World primates. Serum concentrations of retinol (16.5±1.64 μg/dl) were similar to those in captive spider monkeys (Ateles geoffroyi). Retinyl palmitate and retinyl stearate was present in howler samples and may have reflected recent dietary intake. Circulating α‐tocopherol (997±97.6 μg/dl) was similar to published values for other primates. Carotenoid levels in howlers were within the ranges reported for many primates. A significant finding was the presence of cadmium in samples that should be further studied. The number of individuals sampled was limited, and further investigation into the effects of seasonality is needed. However, this information provides new data for howler monkeys and for free‐ranging primates in general. Zoo Biol 22:239–251, 2003. © 2003 Wiley‐Liss, Inc.