Satellite Tagging and Biopsy Sampling of Killer Whales at Subantarctic Marion Island: Effectiveness,Immediate Reactions and Long-Term Responses

Remote tissue biopsy sampling and satellite tagging are becoming widely used in large marine vertebrate studies because they allow the collection of a diverse suite of otherwise difficult-to-obtain data which are critical in understanding the ecology of these species and to their conservation and management. Researchers must carefully consider their methods not only from an animal welfare perspective, but also to ensure the scientific rigour and validity of their results. We report methods for shore-based, remote biopsy sampling and satellite tagging of killer whales Orcinus orca at Subantarctic Marion Island. The performance of these methods is critically assessed using 1) the attachment duration of low-impact minimally percutaneous satellite tags; 2) the immediate behavioural reactions of animals to biopsy sampling and satellite tagging; 3) the effect of researcher experience on biopsy sampling and satellite tagging; and 4) the mid- (1 month) and long- (24 month) term behavioural consequences. To study mid- and long-term behavioural changes we used multievent capture-recapture models that accommodate imperfect detection and individual heterogeneity. We made 72 biopsy sampling attempts (resulting in 32 tissue samples) and 37 satellite tagging attempts (deploying 19 tags). Biopsy sampling success rates were low (43%), but tagging rates were high with improved tag designs (86%). The improved tags remained attached for 26±14 days (mean ± SD). Individuals most often showed no reaction when attempts missed (66%) and a slight reaction–defined as a slight flinch, slight shake, short acceleration, or immediate dive–when hit (54%). Severe immediate reactions were never observed. Hit or miss and age-sex class were important predictors of the reaction, but the method (tag or biopsy) was unimportant. Multievent trap-dependence modelling revealed considerable variation in individual sighting patterns; however, there were no significant mid- or long-term changes following biopsy sampling or tagging.     

Molecular analysis of the efficiency of sloughed skin sampling in whale population genetics

Sloughed whale skin contains enough DNA for genetic analysis, and offers a nonintrusive method for collecting tissue. Here, we examine the efficiency of sloughed skin sampling using 1460 samples collected from free-ranging humpback whales. Samples were sexed and screened for up to 10 microsatellite markers. The vast majority of samples appear genetically compatible with field observations. About 1% of groups revealed more genotypes than whales, but we argue that this is more likely to be due to unobserved whales than to contamination. Sloughed skin sampling is particularly effective when applied to active groups and offers a viable alternative to biopsy darting in regions where darting is either not permitted or otherwise undesirable.     

Remote biopsy darting and marking of polar bears

Remote biopsy darting of polar bears (Ursus maritimus) is less invasive and time intensive than physical capture and is therefore useful when capture is challenging or unsafe. We worked with two manufacturers to develop a combination biopsy and marking dart for use on polar bears. We had an 80% success rate of collecting a tissue sample with a single biopsy dart and collected tissue samples from 143 polar bears on land, in water, and on sea ice. Dye marks ensured that 96% of the bears were not resampled during the same sampling period, and we recovered 96% of the darts fired. Biopsy heads with 5 mm diameters collected an average of 0.12 g of fur, tissue, and subcutaneous adipose tissue, while biopsy heads with 7 mm diameters collected an average of 0.32 g. Tissue samples were 99.3% successful (142 of 143 samples) in providing a genetic and sex identification of individuals. We had a 64% success rate collecting adipose tissue and we successfully examined fatty acid signatures in all adipose samples. Adipose lipid content values were lower compared to values from immobilized or harvested polar bears, indicating that our method was not suitable for quantifying adipose lipid content.     

Modeling Response of Target and Nontarget Dolphins to Biopsy Darting

Abstract: Remote biopsy sampling is a valuable technique to collect data on free-ranging cetaceans. Numerous researchers have evaluated effects of biopsy darting on the target animal, but biopsy sampling may also have unintentional effects on nearby nontarget animals. We evaluated reactions of target and nontarget animals during remote biopsy sampling conducted on bottlenose dolphins (Tursiops truncatus) in August 2004 off Folly Beach, South Carolina, and Jekyll Island, Georgia, USA, and in February-March and July-August 2005 off Hilton Head, South Carolina. We examined external factors that might influence response of the target animal as well as other individuals in the group. Target animals reacted to biopsy attempts 90.9% (n = 475) of the time. Of these, 97.5% (n = 432) of reactions were to biopsy hits, and 84.4% (n = 320) of all hits had reactions recorded as low. Nontarget animals reacted 21.9% (n = 1,158) of the time; interanimal distance from target animal, season, sea state, and depth affected reactions of nontarget animals. Overall, however, biopsy effects appear to be minimal. We present a statistical tool for identifying factors that elicit greater reactions. This model could be adapted for species or locations, as needed, to identify factors that could influence effects of biopsy sampling.     

Are most samples of animals systematically biased? Consistent individual trait differences bias samples despite random sampling

Sampling animals from the wild for study is something nearly every biologist has done, but despite our best efforts to obtain random samples of animals, ‘hidden’ trait biases may still exist. For example, consistent behavioral traits can affect trappability/catchability, independent of obvious factors such as size and gender, and these traits are often correlated with other repeatable physiological and/or life history traits. If so, systematic sampling bias may exist for any of these traits. The extent to which this is a problem, of course, depends on the magnitude of bias, which is presently unknown because the underlying trait distributions in populations are usually unknown, or unknowable. Indeed, our present knowledge about sampling bias comes from samples (not complete population censuses), which can possess bias to begin with. I had the unique opportunity to create naturalized populations of fish by seeding each of four small fishless lakes with equal densities of slow-, intermediate-, and fast-growing fish. Using sampling methods that are not size-selective, I observed that fast-growing fish were up to two-times more likely to be sampled than slower-growing fish. This indicates substantial and systematic bias with respect to an important life history trait (growth rate). If correlations between behavioral, physiological and life-history traits are as widespread as the literature suggests, then many animal samples may be systematically biased with respect to these traits (e.g., when collecting animals for laboratory use), and affect our inferences about population structure and abundance. I conclude with a discussion on ways to minimize sampling bias for particular physiological/behavioral/life-history types within animal populations.     

Immobilization of African buffaloes (Syncerus caffer coffer) in large numbers for veterinary research

Four hundred and eleven African buffaloes were immobilized with etorphine hydrochloride (M99, Reckitts) and sampled under difficult conditions in North Western Botswana. The buffaloes were darted from a helicopter in 36 days. 584 darts were fired giving a success rate of 70-5%. Various age groups in three separate buffalo populations were included. The technique and difficulties encountered when working from a helicopter are described. Mechanical failure of the darting equipment was negligible. A breakdown of the costs involved is given and a comparison made with similar work carried out solely from land vehicles.     

A CETACEAN BIOPSY SYSTEM USING LIGHTWEIGHT PNEUMATIC DARTS, AND ITS EFFECT ON THE BEHAVIOR OF KILLER WHALES

Lightweight untethered pneumatic darts were used to biopsy killer whales, Orcinus orca , for genetic and toxicological analysis. Samples of epidermal, dermal, and hypodermal tissue weighing approximately 0.5 g were obtained by 65% of the 91 darts fired during the study. Sufficient DNA for multiple analyses was extracted from the biopsies, which were also used for fatty acid and toxic contaminant analyses. Reactions such as momentary shakes or accelerations were observed after 81% of the dart hits and 53% of the misses. Aversion to the research vessel was assessed by reapproaching target whales after the sampling attempts. In 6% of the hits and 8% of the misses aversion to the research boat increased immediately following the attempt. No similar increases in aversion were seen when killer whales were reapproached one day to one year after being hit. The darts were also tested successfully on humpback whales, Megaptera novaeangliae . In view of the simplicity of the system, its effectiveness in acquiring multipurpose samples, and the apparently short-term disturbance it caused, it is recommended for future cetacean biopsy studies.     

BEHAVIORAL RESPONSE OF FOUR SPECIES OF BALAENOPTERID WHALES TO BIOPSY SAMPLING

Behavioral responses to biopsy sampling of four species of northwestern Atlantic balaenopterid whales summering in the estuary and Gulf of St. Lawrence, Quebec, from 1990 to 1995 were studied to determine if this technique was an important disturbance to the whales. A total of 447 biopsy samples were taken using a small punch-type biopsy tip fired from a crossbow. Biopsies were successfully taken from 91.2% of the whales approached. Whales displayed no reaction to 45.2% of the successful biopsy attempts. Whales that responded to biopsy sampling typically resumed their normal behavior immediately or within a few minutes. Most humpback whales displayed a hard tail flick, and the majority of fin and blue whales submerged following biopsy sampling. Significantly different frequencies and intensities of responses were found between whale species. Minke and humpback whales were found to be more sensitive to biopsy sampling than fin and blue whales. Response frequencies were similar between females and males for all species, with the exception of fin whales where females had a higher response frequency than males. Biopsy sample length, i. e., penetration depth, did not explain variations in response intensity but may influence response frequency to biopsy sampling. Group size, geographical region, and number of biopsies taken per whale were not factors that explained variation in behavioral responses. The biopsy technique was found to be an efficient method for obtaining high-quality whale skin and blubber samples with limited behavioral disturbance to balaenopterid whales.     

Comparative analysis and physiological impact of different tissue biopsy methodologies used for the genotyping of laboratory mice

Genotyping of genetically modified mice and control of authenticity of the genetic background of congenic or coisogenic strains by polymerase chain reaction (PCR) is a routine procedure that can be performed with different tissue biopsies causing variable grades of trauma. In this study, some invasive and non-invasive sampling methods were compared, with the main focus on the impact on animal physiology. We compared ear punch, tail biopsy, hair plugging, mouth and rectum swabs and the simple restraint of the animals, scoring for the impact on heart rate (HR), core body temperature (BT) and motor activity by telemetry, during biopsy and for the following 6 h. Furthermore, in order to correlate the physiological impact with the practicability and reliability of the genotyping results, we performed a PCR analysis of the biopsy samples obtained by using the same collection procedures analysed by telemetry. All sampling methods and restraint induced significant increase in HR and BT and a slight increase in motor activity for 1 h, independent of the invasiveness of the method used. Genotyping of all biopsies allowed the proper identification of transgenic animals, tail biopsies, ear punches and hair follicles giving clear signals, the last method being fast, but also susceptible to cross contaminations during sampling by large numbers of animals. Restraint and all biopsy methods provoked similar physiological changes, indicating that the handling of the animals is of major importance and that the sampling procedure does not strongly influence the physiological parameters.     

Darting terrestrial primates in the wild: A primer

While a goal of many field primatologists is to observe subjects in as undisturbed a setting as possible, it is often necessary to anesthetize animals for any of a variety of reasons. In this paper, we review techniques for anesthetizing wild primates, based on our experience with more than a thousand such procedures carried out on baboons in East Africa. We consider the following: 1) rationales for anesthetizing a wild primate; 2) systems for the delivery of anesthetic and choice of anesthetic; 3) the darting process itself and issues relevant to the period between darting and the safe removal of the animal; 4) handling of an anesthetized primate; 5) medical complications associated with darting; 6) when to reanesthetize an animal; 7) the process of recovery from anesthesia and release of an awake animal; 8) safety issues for humans. The range of information that can be obtained through field anesthetizations, when carried out successfully, as well as the potential benefit for the animals involved, can be enormous. However, this process is not trivial from the standpoint of the dangers involved to both the subjects and to humans and because of the potential disruption of social behavior to the group. As such, anesthetizations in the field should not be carried out without a strong rationale and without a tremendous priority given to the safety and care of all involved. Am. J. Primatol. 44:155–167, 1998. © 1998 Wiley-Liss, Inc.     

Temporally Adaptive Sampling: A Case Study in Rare Species Survey Design with Marbled Salamanders (Ambystoma opacum)

Improving detection rates for elusive species with clumped distributions is often accomplished through adaptive sampling designs. This approach can be extended to include species with temporally variable detection probabilities. By concentrating survey effort in years when the focal species are most abundant or visible, overall detection rates can be improved. This requires either long-term monitoring at a few locations where the species are known to occur or models capable of predicting population trends using climatic and demographic data. For marbled salamanders (Ambystoma opacum) in Massachusetts, we demonstrate that annual variation in detection probability of larvae is regionally correlated. In our data, the difference in survey success between years was far more important than the difference among the three survey methods we employed: diurnal surveys, nocturnal surveys, and dipnet surveys. Based on these data, we simulate future surveys to locate unknown populations under a temporally adaptive sampling framework. In the simulations, when pond dynamics are correlated over the focal region, the temporally adaptive design improved mean survey success by as much as 26% over a non-adaptive sampling design. Employing a temporally adaptive strategy costs very little, is simple, and has the potential to substantially improve the efficient use of scarce conservation funds.     

Sampling avian adipose tissue: assessing a nondestructive biopsy technique

ABSTRACT.   Adipose tissue samples can provide valuable information about the physiology, foraging ecology, and toxicology of birds. However, despite these varied applications, to date, no procedure for taking adipose samples from live birds has been described in detail, nor assessed for potential adverse effects. We describe a nonlethal method for collecting adipose tissue from adults and chicks of Black-legged Kittiwakes ( Rissa tridactyla ), Common Murres ( Uria aalge ), and Northern Fulmars ( Fulmarus glacialis ), and assess the short- and longer term effects of the procedure. Biopsies were carried out in the field using topical anesthetic and samples were taken from the synsacral region. Only two of 283 birds sampled (0.7%) had too little adipose tissue to be sampled successfully. Thirty-two kittiwakes were recaptured at varying intervals after the procedure (3–50 d) and the biopsy site inspected carefully. No signs of infection were observed and wounds healed completely within 6 d. Compared to birds captured for routine banding, biopsied kittiwakes showed neither greater weight loss nor reduced breeding success in the year of sampling. Similarly, recapture rates of biopsied birds in subsequent years were similar to those of individuals that had been blood sampled or banded. Our results suggest that collecting samples of adipose tissue by nondestructive biopsy has no more effect on birds than taking blood samples via syringe. Thus, we recommend nondestructive adipose tissue sampling via biopsy as an effective alternative to lethal methods in studies of wild birds.     

长江刀鲚不同生态型间的某些形态差异

摘要：为寻找和检验长江刀鲚（Coilia nasus）不同生态型间的形态差异,分析了江苏靖江、上海九段沙、浙江太湖和江西鄱阳湖4个种群906尾样本的脊椎骨数、臀鳍条数和上颌骨长。结果表明,即使是同一种群内的脊椎骨数和臀鳍条数变幅也较大,但均以靖江和九段沙种群的较多,众数分别为81、80和108、109;太湖和鄱阳湖种群...     

雄性短尾猴维持临时配偶关系的行为适应性

临时配偶关系指多雌多雄灵长类群体中的一只成年雄性连续跟随一只成年发情期/性接受期的雌性形成的异性关系,在季节性繁殖物种的交配季节表现的尤为明显,是雄性个体提高交配成功的策略之一。为了探讨能量消耗对这种行为的约束,本研究于2017年8月至2018年1月,以栖息于安徽黄山的短尾猴鱼鳞坑A1群（YA1群）的8~10只成年雄性为研究对象,采用目标动物取样法、行为取样法以及全事件记录法采集成年雄性自然发生的行为数据。通过分析移动时间、觅食时间和交配频次等行为指标,同时测定作为个体能量状态生理指标的尿液C-肽浓度（Urinary C-peptide , UCP）,从行为和生理两个方面研究雄性短尾猴维持临时配偶关系的适应性特征。研究期间,在临时配偶期内,雄性的移动时间无显著变化,但觅食时间和交配频率显著增加;在临时配偶期内,当存在雄性竞争者时,雄性的觅食时间显著减少;临时配偶关系对雄性的UCP水平无显著影响。结果表明,雄性短尾猴在临时配偶期内可能会根据能量消耗的情况以及守护雌性周围的社会环境对自身行为进行调整,以减少其在临时配偶期内的能量投资,提高自身维持临时配偶关系的行为适应性。     

Prevalence of Giardia spp. in beaver and muskrat populations in northeastern states and Minnesota: detection of intestinal trophozoites at necropsy provides greater sensitivity than detection of cysts in fecal samples.

Surveys of the prevalence of the intestinal protozoan Giardia spp. in animal populations have relied almost exclusively on the detection of cysts in fecal samples. We have determined the prevalence of Giardia spp. in beaver and muskrat populations in four northeastern states and Minnesota by using both the detection of trophozoites in mucosal scrapings from live-trapped animals at necropsy and the detection of cysts in fecal samples collected from kill-trapped animals. In muskrats the prevalence of Giardia infection was 36.6% by cyst detection in fecal samples (n = 790) from kill-trapped animals and 95.9% in live-trapped muskrats when the intestinal contents were analyzed for the presence of trophozoites (n = 219). Similarly, in beavers, Giardia infection was 9.2% by cyst detection in fecal samples (n = 662) from kill-trapped beavers and 13.7% in live-trapped animals examined for the presence of intestinal trophozoites (n = 302). The detection of trophozoites in mucosal scrapings from live-trapped animals consistently yielded a significantly higher prevalence for both muskrats and beavers than did the method based on detection of cysts in the fecal samples. The prevalence of Giardia infection in juvenile and adult live-trapped muskrats was similar (92.5 and 94.4%, respectively), but the prevalence in juvenile live-trapped beavers (23.2%) was significantly greater than that seen in the adult animals (12.6%). No difference in Giardia prevalence on the basis of sex was seen in either animal species. Regional variation, often statistically significant, was seen in the prevalence of Giardia in beavers in the northeastern states and Minnesota, but was not detected for muskrats.     

GENE DISPERSAL AND SPATIAL GENETIC STRUCTURE

Spatial autocorrelation statistics have been studied in theoretical population genetic models and widely used in experimental studies of spatial structure in many plant and animal populations. However, the statistical properties of spatial autocorrelation statistics have remained uncharacterized. Little is known about how values of spatial autocorrelation statistics in population samples depend on the level of dispersal and scheme of sampling. In this paper, we characterize the statistical properties of join-count spatial autocorrelation statistics for population genetic surveys under various conditions of dispersal and sampling. The results indicate generally high statistical power. These results can provide a method to estimate gene dispersal based on standing spatial patterns of genetic variation observed within populations.     

Application of Gibbs sampling for inference in a mixed major gene-polygenic inheritance model in animal populations

The application of Gibbs sampling is considered for inference in a mixed inheritance model in animal populations. Implementation of the Gibbs sampler on scalar components, as used for human populations, appeared not to be efficient, and an approach with blockwise sampling of genotypes was proposed for use in animal populations. The blockwise sampling of genotypes was proposed for use in animal populations. The blockwise sampling by which genotypes of a sire and its final progeny were sampled jointly was effective in improving mixing, although further improvements could be looked for. Posterior densities of parameters were visualised from Gibbs samples; from the former highly marginalised Bayesian point and interval estimates can be obtained.     

Prevalence of Giardia spp. in beaver and muskrat populations in northeastern states and Minnesota: detection of intestinal trophozoites at necropsy provides greater sensitivity than detection of cysts in fecal samples

Surveys of the prevalence of the intestinal protozoan Giardia spp. in animal populations have relied almost exclusively on the detection of cysts in fecal samples. We have determined the prevalence of Giardia spp. in beaver and muskrat populations in four northeastern states and Minnesota by using both the detection of trophozoites in mucosal scrapings from live-trapped animals at necropsy and the detection of cysts in fecal samples collected from kill-trapped animals. In muskrats the prevalence of Giardia infection was 36.6% by cyst detection in fecal samples (n = 790) from kill-trapped animals and 95.9% in live-trapped muskrats when the intestinal contents were analyzed for the presence of trophozoites (n = 219). Similarly, in beavers, Giardia infection was 9.2% by cyst detection in fecal samples (n = 662) from kill-trapped beavers and 13.7% in live-trapped animals examined for the presence of intestinal trophozoites (n = 302). The detection of trophozoites in mucosal scrapings from live-trapped animals consistently yielded a significantly higher prevalence for both muskrats and beavers than did the method based on detection of cysts in the fecal samples. The prevalence of Giardia infection in juvenile and adult live-trapped muskrats was similar (92.5 and 94.4%, respectively), but the prevalence in juvenile live-trapped beavers (23.2%) was significantly greater than that seen in the adult animals (12.6%). No difference in Giardia prevalence on the basis of sex was seen in either animal species. Regional variation, often statistically significant, was seen in the prevalence of Giardia in beavers in the northeastern states and Minnesota, but was not detected for muskrats.     

Review of cetacean biopsy techniques: Factors contributing to successful sample collection and physiological and behavioral impacts

Biopsy techniques have been developed to collect skin and blubber samples through non‐lethal methods. One sample can provide data on genetics, prey preferences, foraging ecology, contaminant loads, and physiological processes. The limited data available suggest that biopsy wounds heal quickly and that there are usually no discernable adverse health effects. Published accounts on factors contributing to the success of collecting biopsy samples and the behavioral impacts to cetaceans following biopsy sampling were standardized to permit statistical analysis. Several factors contribute to the success of acquiring samples; however, sampling rates do not differ significantly between delivery devices. Behavioral responses to biopsy sampling vary by species and other factors. The most predominant response for odontocetes is low, while low and moderate responses are equally prevalent for mysticetes. The use of retrieval lines may increase the occurrence of moderate and strong responses by mysticetes. These findings suggest that biopsy sampling is relatively benign, causing only minor and short‐lived responses. However, most researchers do not report sufficient data to assess short‐ and long‐term physiological and behavioral impacts. Finally, limited data suggest that biopsy sampling does not impact cetacean habitat use or distribution patterns. Yet these impacts are rarely investigated, so additional data are needed.     

Testing the reliability of noninvasive genetic sampling by comparing analyses of blood and fecal samples in Barbary macaques (Macaca sylvanus).

Genetic studies of wild animal populations are often hindered by difficulties in obtaining blood samples. Recent advances in molecular biology have allowed the use of noninvasive samples as sources of DNA (e.g., hair or feces), but such samples may provide low-quality DNA and prevent the determination of true genotypes in subsequent DNA analysis. We present a preliminary study aimed at assessing the reliability of using fecal samples for genotyping in Barbary macaques (Macaca sylvanus). The test was performed on samples of blood and feces from 11 captive animals, using three dinucleotide microsatellites. The CTAB DNA extraction method was found to be the most relevant for Barbary macaque feces, yielding successful amplification at all loci for 70% of PCRs. All the fecal samples tested gave correct genotypes at least once for each locus when referenced against blood-derived genotypes. An average of 18.3% of PCRs displayed spurious genotypes (false homozygous or false allele). The minimum theoretical probability required to obtain a 100% accurate genotype is 0.74, based on the criterion that a correct genotype is assessed only if it was observed at least twice. The observed probability of obtaining a correct genotype from three PCRs, based on our genotyping results, was greater (0.81 on average) than the minimum threshold. In conclusion, our comparison of blood and fecal samples showed that fecal sampling is a reliable tool for the further study of wild Barbary macaque populations.