Carrion fly‐derived DNA as a tool for comprehensive and cost‐effective assessment of mammalian biodiversity

Large‐scale monitoring schemes are essential in assessing global mammalian biodiversity, and in this framework, leeches have recently been promoted as an indirect source of DNA from terrestrial mammal species. Carrion feeding flies are ubiquitous and can be expected to feed on many vertebrate carcasses. Hence, we tested whether fly‐derived DNA analysis may also serve as a novel tool for mammalian diversity surveys. We screened DNA extracted from 201 carrion flies collected in tropical habitats of Côte d'Ivoire and Madagascar for mammal DNA using multiple PCR systems and retrieved DNA sequences from a diverse set of species (22 in Côte d'Ivoire, four in Madagascar) exploiting distinct forest strata and displaying a broad range of body sizes. Deep sequencing of amplicons generated from pools of flies performed equally well as individual sequencing approaches. We conclude that the analysis of fly‐derived DNA can be implemented in a very rapid and cost‐effective manner and will give a relatively unbiased picture of local mammal diversity. Carrion flies therefore represent an extraordinary and thus far unexploited resource of mammal DNA, which will probably prove useful for future inventories of wild mammal communities.     

Carrion fly‐derived DNA metabarcoding is an effective tool for mammal surveys: Evidence from a known tropical mammal community

Metabarcoding of vertebrate DNA derived from carrion flies has been proposed as a promising tool for biodiversity monitoring. To evaluate its efficacy, we conducted metabarcoding surveys of carrion flies on Barro Colorado Island (BCI), Panama, which has a well‐known mammal community, and compared our results against diurnal transect counts and camera trapping. We collected 1,084 flies in 29 sampling days, conducted metabarcoding with mammal‐specific (16S) and vertebrate‐specific (12S) primers, and sequenced amplicons on Illumina MiSeq. For taxonomic assignment, we compared blast with the new program protax , and we found that protax improved species identifications. We detected 20 mammal, four bird, and one lizard species from carrion fly metabarcoding, all but one of which are known from BCI. Fly metabarcoding detected more mammal species than concurrent transect counts (29 sampling days, 13 species) and concurrent camera trapping (84 sampling days, 17 species), and detected 67% of the number of mammal species documented by 8 years of transect counts and camera trapping combined, although fly metabarcoding missed several abundant species. This study demonstrates that carrion fly metabarcoding is a powerful tool for mammal biodiversity surveys and has the potential to detect a broader range of species than more commonly used methods.     

Tropical rainforest flies carrying pathogens form stable associations with social nonhuman primates

Living in groups provides benefits but also incurs costs such as attracting disease vectors. For example, synanthropic flies associate with human settlements, and higher fly densities increase pathogen transmission. We investigated whether such associations also exist in highly mobile nonhuman primate (NHP) Groups. We studied flies in a group of wild sooty mangabeys (Cercocebus atys atys) and three communities of wild chimpanzees (Pan troglodytes verus) in Taï National Park, Côte d'Ivoire. We observed markedly higher fly densities within both mangabey and chimpanzee groups. Using a mark–recapture experiment, we showed that flies stayed with the sooty mangabey group for up to 12 days and for up to 1.3 km. We also tested mangabey‐associated flies for pathogens infecting mangabeys in this ecosystem, Bacillus cereus biovar anthracis (Bcbva), causing sylvatic anthrax, and Treponema pallidum pertenue, causing yaws. Flies contained treponemal (6/103) and Bcbva (7/103) DNA. We cultured Bcbva from all PCR‐positive flies, confirming bacterial viability and suggesting that this bacterium might be transmitted and disseminated by flies. Whole genome sequences of Bcbva isolates revealed a diversity of Bcbva, probably derived from several sources. We conclude that flies actively track mangabeys and carry infectious bacterial pathogens; these associations represent an understudied cost of sociality and potentially expose many social animals to a diversity of pathogens.     

Back to basics: an evaluation of NaOH and alternative rapid DNA extraction protocols for DNA barcoding,genotyping, and disease diagnostics from fungal and oomycete samples

The ubiquity, high diversity and often‐cryptic manifestations of fungi and oomycetes frequently necessitate molecular tools for detecting and identifying them in the environment. In applications including DNA barcoding, pathogen detection from plant samples, and genotyping for population genetics and epidemiology, rapid and dependable DNA extraction methods scalable from one to hundreds of samples are desirable. We evaluated several rapid extraction methods (NaOH, Rapid one‐step extraction (ROSE), Chelex 100, proteinase K) for their ability to obtain DNA of quantity and quality suitable for the following applications: PCR amplification of the multicopy barcoding locus ITS1/5.8S/ITS2 from various fungal cultures and sporocarps; single‐copy microsatellite amplification from cultures of the phytopathogenic oomycete Phytophthora ramorum; probe‐based P. ramorum detection from leaves. Several methods were effective for most of the applications, with NaOH extraction favored in terms of success rate, cost, speed and simplicity. Frozen dilutions of ROSE and NaOH extracts maintained PCR viability for over 32 months. DNA from rapid extractions performed poorly compared to CTAB/phenol‐chloroform extracts for TaqMan diagnostics from tanoak leaves, suggesting that incomplete removal of PCR inhibitors is an issue for sensitive diagnostic procedures, especially from plants with recalcitrant leaf chemistry. NaOH extracts exhibited lower yield and size than CTAB/phenol‐chloroform extracts; however, NaOH extraction facilitated obtaining clean sequence data from sporocarps contaminated by other fungi, perhaps due to dilution resulting from low DNA yield. We conclude that conventional extractions are often unnecessary for routine DNA sequencing or genotyping of fungi and oomycetes, and recommend simpler strategies where source materials and intended applications warrant such use.     

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.     

Bimodal cue complex signifies suitable oviposition sites to gravid females of the common green bottle fly

Gravid females of the common green bottle fly, Lucilia sericata Meigen (Diptera: Calliphoridae), readily locate recently deceased vertebrates as oviposition sites, particularly when these animals have been injured. We investigated semiochemical and visual cues that mediate attraction of gravid females to fresh rat carrion. Female flies were more strongly attracted to incised rat carrion than to intact carrion. They were also attracted to Porapak Q headspace volatile (HSV) extract of incised rat carrion. Analyzing aliquots of Porapak Q HSV extract by gas chromatographic‐electroantennographic detection revealed nine components [phenol, para‐ and/or meta‐cresol (could not be separated), guaiacol, dimethyl trisulfide (DMTS), phenylacetaldehyde, (E)‐2‐octenal, nonanal, and tetramethyl pyrazine] that consistently elicited responses from blow fly antennae. In laboratory experiments, a synthetic blend of these nine components was as attractive to gravid females as Porapak Q HSV extract, but blend attractiveness was due entirely to DMTS. In both laboratory and field experiments, increasing doses of DMTS attracted increasingly more flies. Coupled with DMTS, carrion‐type color cues (dark red, black) were more effective than bright color cues (white, yellow) in attracting flies. In field experiments, dark traps baited with DMTS captured a total of 214 calliphorid flies (200 L. sericata, 10 Lucilia illustris Meigen, three Calliphora vicina Robineau‐Desvoidy, one Calliphora vomitoria L.), all of which were gravid females. These results support the conclusion that DMTS and dark color represent a bimodal cue complex that signifies suitable oviposition sites to gravid calliphorid females, particularly L. sericata.     

Quantitative PCR assessment of microsatellite and SNP genotyping with variable quality DNA extracts

In this study we developed eight quantitative PCR (qPCR) assays to evaluate the starting copy number of nuclear and mitochondrial DNA fragments ranging from 75 to 350 base-pairs in DNA extracts from Chinook salmon tissues with varying quality. Samples were genotyped with 13 microsatellite and 29 SNP assays and average genotyping success for good, intermediate, and poor quality samples was 96%, 24%, and 24% for microsatellite loci, and 98%, 97%, and 79% for SNPs, respectively. As measured by qPCR, good quality samples had a consistently high number of starting copies across all fragment sizes with little change between the smallest and largest size. In contrast, the intermediate and poor quality samples displayed decreases in starting copy number as fragment size increased, and was most pronounced with poor samples. Logistic regression of genotyping success by starting copy number indicated that in order to achieve at least 90% genotyping success, approximately 1,000 starting copies of nuclear DNA are necessary for microsatellite loci, and as few as 14 starting copies for SNP assays (but we recommend at least 50 copies to reduce genotyping error). While these guidelines apply specifically to Chinook salmon and the genetic markers included in this study, the principles are transferable to other species and markers due to the underlying process associated with template quantity and PCR amplification.     

Evaluating DNA degradation rates in faecal pellets of the endangered pygmy rabbit

Noninvasive genetic sampling of faecal pellets can be a valuable method for monitoring rare and cryptic wildlife populations, like the pygmy rabbit (Brachylagus idahoensis). To investigate this method's efficiency for pygmy rabbit monitoring, we evaluated the effect of sample age on DNA degradation in faecal pellets under summer field conditions. We placed 275 samples from known individuals in natural field conditions for 1–60 days and assessed DNA quality by amplifying a 294‐base‐pair (bp) mitochondrial DNA (mtDNA) locus and five nuclear DNA (nDNA) microsatellite loci (111–221 bp). DNA degradation was influenced by sample age, DNA type, locus length and rabbit sex. Both mtDNA and nDNA exhibited high PCR success rates (94.4%) in samples <1 day old. Success rates for microsatellite loci declined rapidly from 80.0% to 42.7% between days 5 and 7, likely due to increased environmental temperature. Success rates for mtDNA amplification remained higher than nDNA over time, with moderate success (66.7%) at 21 days. Allelic dropout rates were relatively high (17.6% at <1 day) and increased to 100% at 60 days. False allele rates ranged from 0 to 30.0% and increased gradually over time. We recommend collecting samples as fresh as possible for individual identification during summer field conditions. Our study suggests that this method can be useful for future monitoring efforts, including occupancy surveys, individual identification, population estimation, parentage analysis and monitoring of genetic diversity both of a re‐introduced population in central Washington and across their range.     

Availability of short microsatellite markers from butterfly museums and private specimens

Knowledge of the temporal changes in genetic diversity and structure is important for identifying factors causing a decline in threatened insect species, and for establishing conservation programs for these species. Thus, there is recently an increasing interest in the restoration of genetic diversity in conservation programs using DNA data from historical museum specimens. For butterfly specimens, we measured the yields and fragment sizes of the extracted DNA and investigated the genotyping success probability of nine short microsatellite markers (allele size 73–191 bp). We used leg samples of specimens of a medium‐sized butterfly species, Melitaea ambigua (Lepidoptera; Nymphalidae), collected from the 1960s to the 2010s. The yields of specimen‐extracted DNA longer than 150 bp decreased with increasing specimen age. There were negative correlations between the genotyping success probability and specimen age for each of all microsatellite markers. A negative correlation was also observed between the genotyping success probability and allele size of each microsatellite marker. We conclude that short microsatellite markers and analysis of recently obtained specimens are particularly suitable for microsatellite analysis of butterfly specimens.     

A test of the multiplex pre-amplification approach in microsatellite genotyping of wolverine faecal DNA

Recently, a two-step PCR approach, referred to as multiplex pre-amplification, was proposed to improve microsatellite amplification from non-invasive samples such as faecal DNA. Here, we compare this new approach to standard PCR with respect to amplification success and genotyping error rates in microsatellite analysis (18 markers) of wolverine faecal DNA (48 extracts initially shown to contain amplifiable DNA). The multiplex pre-amplification approach was clearly advantageous both in terms of successful PCR amplifications (91% vs. 80%) and allelic dropout rate (2.4% vs. 12.5%). However, dropouts were to a high extent repeated in all second-step amplifications following multiplex pre-amplification, indicative of being generated during the initial PCR. Analysing more than one PCR from the initial multiplex PCR product may thus be of limited value. We instead suggest to perform two initial multiplex PCRs and to analyse a single second-step PCR from each of them. This was tested for 22 extracts at 18 loci and proved to be an effective way to obtaining a correct genotype.     

Invasion genetics of American cherry fruit fly in Europe and signals of hybridization with the European cherry fruit fly

The American cherry fruit fly is an invasive pest species in Europe, of serious concern in tart cherry production as well as for the potential to hybridize with the European cherry fruit fly, Rhagoletis cerasi L. (Diptera: Tephritidae), which might induce new pest dynamics. In the first European reports, the question arose whether only the eastern American cherry fruit fly, Rhagoletis cingulata (Loew) (Diptera: Tephritidae), is present, or also the closely related western American cherry fruit fly, Rhagoletis indifferens Curran. In this study, we investigate the species status of European populations by comparing these with populations of both American species from their native ranges, the invasion dynamics in German (first report in 1993) and Hungarian (first report in 2006) populations, and we test for signals of hybridization with the European cherry fruit fly. Although mtDNA sequence genealogy could not separate the two American species, cross‐species amplification of 14 microsatellite loci separated them with high probabilities (0.99–1.0) and provided evidence for R. cingulata in Europe. German and Hungarian R. cingulata populations differed significantly in microsatellite allele frequencies, mtDNA haplotype and wing pattern distributions, and both were genetically depauperate relative to North American populations. The diversity suggests independent founding events in Germany and Hungary. Within each country, R. cingulata displayed little or no structure in any trait, which agrees with rapid local range expansions. In cross‐species amplifications, signals of hybridization between R. cerasi and R. cingulata were found in 2% of R. cingulata individuals and in 3% of R. cerasi. All putative hybrids had R. cerasi mtDNA indicating that the original between‐species mating involved R. cerasi females and R. cingulata males.     

Improved noninvasive genotyping method: application to brown bear (Ursus arctos) faeces

We redesigned new microsatellite primers and one sex‐specific primer for amplification of faecal DNA from brown bears (Ursus arctos). We also combined a semi‐nested polymerase chain reaction (PCR) with a newly developed multiplex preamplification method in order to increase the quality of the amplified DNA fragments. In comparison with a conventional PCR approach, the genotyping error rate was substantially reduced and the amplification rate was increased. This new approach could be transposed to other species where conventional PCR methods experience low success due to limited DNA concentration and/or quality.     

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.     

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.     

Development of novel real‐time TaqMan® PCR assays for the species and sex identification of otter (Lutra lutra) and their application to noninvasive genetic monitoring

Developing strategies to maintain biodiversity requires baseline information on the current status of each individual species. The development of genetic techniques and their application to noninvasively collected samples have the potential to yield information on the structure of elusive animal populations and so are important tools in conservation management. Using DNA isolated from faecal samples can be challenging owing to low quantity and quality. This study, however, presents the development of novel real‐time polymerase chain reaction assays using fluorescently labelled TaqMan^® MGB probes enabling species and sex identification of Eurasian otter (Lutra lutra) spraints (faeces). These assays can also be used in determining an optimum microsatellite panel and can be employed as cost‐saving screening tools for downstream genetic testing including microsatellite genotyping and haplotype analysis. The techniques are shown to work efficiently with L. lutra DNA isolated from tissue, hair, spraint, blood and anal jelly samples.     

Two highly validated SSR multiplexes (8‐plex) for Euphrates' poplar,Populus euphratica (Salicaceae)

Multiplex PCR amplification of microsatellites has significantly increased the throughput and decreased the costs of genotyping. We have developed two highly polymorphic microsatellite multiplexes for Populus euphratica, the only tree species found in desert regions of Western China and adjacent Central Asian countries. The first of these multiplex kits comprises an eight‐Plex of genomic SSRs (gSSRs) obtained from published databases. The second comprises an eight‐plex of newly designed EST‐SSRs (eSSRs) based on expressed sequence tags for P. euphratica. Both kits were tested on a sample of 170 individuals from four populations. The gSSRs exhibited slightly more polymorphism than the eSSRs. The new multiplex protocols yielded consistent results in the hands of multiple researchers, demonstrating their robustness. The 16 loci used in the kits exhibited a high transferability rate (82.0%) in eight other poplar species belonging to five different sections of the genus. Both kits should therefore be useful for further investigations of population genetics in P. euphratica and related species. Our results indicate that it is essential to follow recently established recommendations when developing microsatellite markers, including verifying the amplification efficiency, detecting null alleles and carefully measuring error rates.     

Comparative mitochondrial and nuclear quantitative PCR of historical marine mammal tissue,bone, baleen,and tooth samples

The use of historical and ancient tissue samples for genetic analysis is increasing, with ever greater numbers of samples proving to contain sufficient mitochondrial and even nuclear DNA for multilocus analysis. DNA yield, however, remains highly variable and largely unpredictable based solely on sample morphology or age. Quantification of DNA from historical and degraded samples can greatly improve efficiency of screening DNA extracts prior to attempting sequencing or genotyping, but requires sequence‐specific quantitative polymerase chain reaction (qPCR) based assays to detect such minute quantities of degraded DNA. We present two qPCR assays for marine mammal DNA quantification, and results from analysis of DNA extracted from preserved soft tissues, bone, baleen, and tooth from several cetacean species. These two assays have been shown to amplify DNA from 26 marine mammal species representing 12 families of pinnipeds and cetaceans. Our results indicate that different tissues retain different ratios of mitochondrial to nuclear DNA, and may be more or less suitable for analysis of nuclear loci. Specifically, historical bone and tooth samples average 60‐fold higher ratio of mitochondrial DNA to nuclear DNA than preserved fresh soft tissue, and the ratio is almost 8000‐fold higher in baleen.     

An efficient method for screening faecal DNA genotypes and detecting new individuals and hybrids in the red wolf (<Emphasis Type="Italic">Canis rufus</Emphasis>) experimental population area

Previously, sequencing of mitochondrial DNA (mtDNA) from non-invasively collected faecal material (scat) has been used to help manage hybridization in the wild red wolf (Canis rufus) population. This method is limited by the maternal inheritance of mtDNA and the inability to obtain individual identification. Here, we optimize the use of nuclear DNA microsatellite markers on red wolf scat DNA to distinguish between individuals and detect hybrids. We develop a data filtering method in which scat genotypes are compared to known blood genotypes to reduce the number of PCR amplifications needed. We apply our data filtering method and the more conservative maximum likelihood ratio method (MLR) of Miller et al. (2002 Genetics 160:357–366) to a scat dataset previously screened for hybrids by sequencing of mtDNA. Using seven microsatellite loci, we obtained genotypes for 105 scats, which were matched to 17 individuals. The PCR amplification success rate was 50% and genotyping error rates ranged from 6.6% to 52.1% per locus. Our data filtering method produced comparable results to the MLR method, and decreased the time and cost of analysis by 25%. Analysis of this dataset using our data filtering method verified that no hybrid individuals were present in the Alligator River National Wildlife Refuge, North Carolina in 2000. Our results demonstrate that nuclear DNA microsatellite analysis of red wolf scats provides an efficient and accurate approach to screen for new individuals and hybrids.     

High‐throughput microsatellite genotyping in ecology: improved accuracy,efficiency, standardization and success with low‐quantity and degraded DNA

Microsatellite markers have played a major role in ecological, evolutionary and conservation research during the past 20 years. However, technical constrains related to the use of capillary electrophoresis and a recent technological revolution that has impacted other marker types have brought to question the continued use of microsatellites for certain applications. We present a study for improving microsatellite genotyping in ecology using high‐throughput sequencing (HTS). This approach entails selection of short markers suitable for HTS, sequencing PCR‐amplified microsatellites on an Illumina platform and bioinformatic treatment of the sequence data to obtain multilocus genotypes. It takes advantage of the fact that HTS gives direct access to microsatellite sequences, allowing unambiguous allele identification and enabling automation of the genotyping process through bioinformatics. In addition, the massive parallel sequencing abilities expand the information content of single experimental runs far beyond capillary electrophoresis. We illustrated the method by genotyping brown bear samples amplified with a multiplex PCR of 13 new microsatellite markers and a sex marker. HTS of microsatellites provided accurate individual identification and parentage assignment and resulted in a significant improvement of genotyping success (84%) of faecal degraded DNA and costs reduction compared to capillary electrophoresis. The HTS approach holds vast potential for improving success, accuracy, efficiency and standardization of microsatellite genotyping in ecological and conservation applications, especially those that rely on profiling of low‐quantity/quality DNA and on the construction of genetic databases. We discuss and give perspectives for the implementation of the method in the light of the challenges encountered in wildlife studies.     

A real‐time PCR toolbox for accurate identification of invasive fruit fly species

Significant plant pests such as fruit flies that travel with fresh produce between countries as eggs or larvae pose a great economic threat to the agriculture and fruit industry worldwide. Time‐limited and expensive quarantine decisions require accurate identification of such pests. Immature stages are often impossible to identify, making them a serious concern for biosecurity agencies. Use of COI barcoding PCR, often the only molecular identification resource, is time‐consuming. We assess the suitability of the COI barcoding region for real‐time PCR assays to identify four pest fruit fly species (Family: Tephritidae), in a diagnostic framework. These species, namely Mediterranean fruit fly (Ceratitis capitata), Queensland fruit fly (Bactrocera tryoni), African invader fly (Bactrocera invadens) and Island fly (Dirioxa pornia) each provide a different set of genetic species delimitation problems. We discuss the benefits and limitations of using a single‐gene TaqMan^? real‐time approach for such species. Our results indicate that COI‐based TaqMan^? real‐time PCR assays, in particular for genetically distinct species, provide an accurate, sensitive and rapid diagnostic tool.