Genetic evidence for seasonal consumption of monkfish (Lophius spp.) and salmonids (Salmo spp.) by gray seals

Recoveries of gray seal (Halichoerus grypus) populations across their eastern Atlantic distribution have led to a steady increase in seal-fishery interactions. Fishers have estimated depredation of salmonids (Salmo spp.) and monkfish (Lophius spp.) as high as 40% and 59% respectively in Ireland. However, empirical evidence for the consumption of these species has been extremely limited due to diagnostic hard part remains not being found in scats or stomach samples. We applied species-specific primers and tested for the presence of monkfish and salmonids in gray seal diet genetically using quantitative polymerase chain reaction (qPCR) on scats. Monkfish occurred in 29.7% of sampled scats, while salmonids occurred in 12.7%. Seasonal and regional variability in occurrence were noted for both species, likely related to the migratory behavior of the prey species and proximity of seal haul-outs to aquaculture sites. Traditional hard part analysis of scats, including scats that tested positive for monkfish and salmonid DNA, failed to find any evidence of either species. This study provides important empirical evidence for the consumption of these species in Ireland that can inform management.     

大熊猫和小熊猫粪便DNA提取的简易方法

采集了大熊猫和小熊猫的新鲜粪便样品 ,使用 1 0 0 %乙醇保存。通过重复离心富集研究动物的肠道脱落细胞 ,并使用乙醇和双蒸水洗涤以除去抑制物。用 1 %的SDS快速裂解细胞 ,离心除去残渣后 ,向裂解液中加入蛋白酶进行消化。消化结束后使用等体积的酚 /氯仿抽提 ,乙醇沉淀DNA。用双蒸水溶解粪便DNA后 ,使用PCR产物纯化试剂盒对粪便DNA进行纯化。电泳检测结果显示 ,从乙醇保存的大、小熊猫粪便样品中抽提到高质量的粪便DNA。对线粒体控制区、细胞色素b基因、 1 2SrRNA基因的PCR扩增反应以及测序结果也证实了样品保存方法和DNA抽提方法可靠而高效。此方法使用实验室内常用的分子生物学试剂 ,不仅克服了分子粪便学研究中常见的抑制物粪便DNA微量降解严重等障碍 ,与商业化的粪便抽提试剂盒 (QIAampDNAStoolMiniKit,Qiagen)相比还是一种经济的试验方法 (抽提反应成本为试剂盒的 1 / 5 )。文中还对粪便DNA内细菌基因组等背景DNA可能对分子粪便学试验结果的影响进行了探讨。在基于PCR技术的遗传学研究中 ,对于植食性动物而言 ,粪便内的背景DNA对目标动物DNA片断的扩增和序列测定未见影响 ;但对于肉食性动物 ,则必须考虑被捕食者基因组对试验可能产生的影响 ,应谨慎对待     

粪便学在有蹄类动物数量调查研究中的应用与展望

随着与分子生物学的结合,传统的粪便学不仅拓宽了在种群生态学、行为生态学及保护遗传学等研究时取样的范围,而且能提供更多的有效信息,使得传统的粪便计数方法得以在新的领域里发展。本文对传统粪便学在有蹄类动物数量研究中的应用加以总结,并结合国内外研究现状对分子粪便学在这一领域内的可靠性、局限性及应用前景做了概述。     

Molecular prey identification in Central European piscivores

Diet analysis is an important aspect when investigating the ecology of fish‐eating animals and essential for assessing their functional role in food webs across aquatic and terrestrial ecosystems. The identification of fish remains in dietary samples, however, can be time‐consuming and unsatisfying using conventional morphological analysis of prey remains. Here, we present a two‐step multiplex PCR system, comprised of six assays, allowing for rapid, sensitive and specific detection of fish DNA in dietary samples. This approach encompasses 78 fish and lamprey species native to Central European freshwaters and enables the identification of 31 species, six genera, two families, two orders and two fish family clusters. All targeted taxa were successfully amplified from 25 template molecules, and each assay was specific when tested against a wide range of invertebrates and vertebrates inhabiting aquatic environments. The applicability of the multiplex PCR system was evaluated in a feeding trial, wherein it outperformed morphological prey analysis regarding species‐specific prey identification in faeces of Eurasian otters. Additionally, a wide spectrum of fish species was detected in field‐collected faecal samples and regurgitated pellets of Common Kingfishers and Great Cormorants, demonstrating the broad applicability of the approach. In conclusion, this multiplex PCR system provides an efficient, easy to use and cost‐effective tool for assessing the trophic ecology of piscivores in Central Europe. Furthermore, the multiplex PCRs and the primers described therein will be applicable wherever DNA of the targeted fish species needs to be detected at high sensitivity and specificity.     

Molecular methods for differentiating grey seal (Halichoerus grypus) and harbour seal (Phoca vitulina) scat

Pinniped dietary information is crucial for understanding marine ecosystems; however, in the North Atlantic, grey and harbour seals haulout concomitantly and their faeces are visually indistinguishable. Therefore, we developed molecular methods to differentiate the species' scat. PCR primers were created that amplify a portion of mitochondrial 16S ribosomal DNA in grey and harbour seals. The samples were amplified and the resulting products were digested using the restriction enzyme Tsp509I, producing diagnostic banding patterns. These tools provide a mechanism by which separate dietary analysis can be achieved for grey and harbour seals at mixed haulouts in New England.     

Reliable microsatellite genotyping of the Eurasian badger (Meles meles) using faecal DNA

The potential link between badgers and bovine tuberculosis has made it vital to develop accurate techniques to census badgers. Here we investigate the potential of using genetic profiles obtained from faecal DNA as a basis for population size estimation. After trialling several methods we obtained a high amplification success rate (89%) by storing faeces in 70% ethanol and using the guanidine thiocyanate/silica method for extraction. Using 70% ethanol as a storage agent had the advantage of it being an antiseptic. In order to obtain reliable genotypes with fewer amplification reactions than the standard multiple-tubes approach, we devised a comparative approach in which genetic profiles were compared and replication directed at similar, but not identical, genotypes. This modified method achieved a reduction in polymerase chain reactions comparable with the maximum-likelihood model when just using reliability criteria, and was slightly better when using reliability criteria with the additional proviso that alleles must be observed twice to be considered reliable. Our comparative approach would be best suited for studies that include multiple faeces from each individual. We utilized our approach in a well-studied population of badgers from which individuals had been sampled and reliable genotypes obtained. In a study of 53 faeces sampled from three social groups over 10 days, we found that direct enumeration could not be used to estimate population size, but that the application of mark-recapture models has the potential to provide more accurate results.     

Molecular identification of prey in predator diets

In many situations prey choice by predators in the field cannot be established or quantified using direct observation. The remains of some prey may be visually identified in the guts and faeces of predators but not all predators ingest such hard remains and even those that do consume them may also ingest soft-bodies prey that leave no recognizable remnants. The result is, at best, a biased picture of prey choice. A range of molecular techniques and applications are reviewed that allow prey remains to be identified, often to the species and even stage level. These techniques, all of which are still in use, include enzyme electrophoresis, a range of immunological approaches using polyclonal and monoclonal antibodies to detect protein epitopes, and recently developed polymerase chain reaction (PCR)-based methods for detecting prey DNA. Analyses may be postmortem, on invertebrate and vertebrate predators collected from the field, or noninvasive assays of the remains in regurgitated bird pellets or vertebrate faeces. It was concluded that although monoclonal antibodies are currently the most effective method in use today, PCR-based techniques have proved to be highly effective and versatile in recent laboratory trials and are likely to rapidly displace all other approaches.     

Empirical evaluation of preservation methods for faecal DNA

We evaluate the relative effectiveness of four methods for preserving faecal samples for DNA analysis. PCR assays of fresh faecal samples collected from free-ranging baboons showed that amplification success was dependent on preservation method, PCR-product size, and whether nuclear or mitochondrial DNA was assayed. Storage in a DMSO/EDTA/Tris/salt solution (DETs) was most effective for preserving nuclear DNA, but storage in 70% ethanol, freezing at –20°C and drying performed approximately equally well for mitochondrial DNA and short (<200 bp) nuclear DNA fragments. Because faecal DNA is diluted and degraded, repeated extractions from faeces may be necessary and short nuclear markers should be employed for genotyping. A review of molecular scatology studies further suggests that three to six faeces per individual should be collected.     

Food habits of harbor seals (Phoca vitulina richardii) as an indicator of invasive species in San Francisco Bay,California

The diet of harbor seals (Phoca vitulina richardii) in San Francisco Bay (SFB), California, was determined from July 2007 to July 2008 using prey hard parts recovered from 442 scats collected at five haul‐out sites. Twenty‐two species of fish and one species of crustacean were identified, but harbor seals primarily ate a nonnative invasive species, yellowfin goby (Acanthogobius flavimanus), which increased in dietary importance since the diet was last studied in 1991/1992. Additionally, another nonnative invasive fish species, chameleon goby (Tridentiger trigonocephalus), was found for the first time in the diet of harbor seals in SFB. Harbor seal diet was statistically different between years (1991/1992 and 2007/2008), between the pupping and nonpupping seasons, and between North SFB and South SFB haul‐out locations. The diet of harbor seals was significantly correlated with fish species caught in trawl surveys conducted by the California Department of Fish and Wildlife (CDFW) during the same time periods as this study (2007/2008). Harbor seals currently are influencing the health of the SFB ecosystem in a positive manner by consuming large quantities of nonnative invasive fish species.     

Genotyping faeces links individuals to their diet

The detection of individual variation in foraging behaviour within wild mammal populations requires large sample sizes and relies on the multifold re-sampling of individuals. However, limits for observational studies are posed by the rarity and nocturnal or otherwise elusive habits of many mammals. We propose that the detection of foraging variation within populations of mammals may be facilitated if conventional diet analysis from faeces is combined with DNA-based individual identification methods using "genetic fingerprinting" from faeces. We applied our approach to a coyote ( Canis latrans ) population, and showed how individuals may vary from one another in their diet profiles. Two main groups of coyotes were distinguished on the basis of their relative use of small mammals and "other vertebrates" as primary food sources, and these two groups were further subdivided on the basis of their relative use of "other vertebrates" and fruit as secondary food sources. We show that, unless a faecal sampling scheme is used that maximizes the number of different individuals included in a survey, individual foraging variation that is left unaccounted for may result in downwardly biased faecal diet diversity estimates. Our approach allows the re-sampling of individuals over time and space, and thus may be generally useful for the testing of optimal foraging theory hypotheses in mammals and also has conservation applications.