Non-lethal Tissue Sampling Research Articles

Conservation genetics of the endangered California Freshwater Shrimp (Syncaris pacifica): watershed and stream networks define gene pool boundaries

Understanding genetic structure and diversity among remnant populations of rare species can inform conservation and recovery actions. We used a population genetic framework to spatially delineate gene pools and estimate gene flow and effective population sizes for the endangered California Freshwater Shrimp Syncaris pacifica. Tissues of 101 individuals were collected from 11 sites in 5 watersheds, using non-lethal tissue sampling. Single Nucleotide Polymorphism markers were developed de novo using ddRAD-seq methods, resulting in 433 unlinked loci scored with high confidence and low missing data. We found evidence for strong genetic structure across the species range. Two hierarchical levels of significant differentiation were observed: (i) five clusters (regional gene pools, FST = 0.38–0.75) isolated by low gene flow were associated with watershed limits and (ii) modest local structure among tributaries within a watershed that are not connected through direct downstream flow (local gene pools, FST = 0.06–0.10). Sampling sites connected with direct upstream-to-downstream water flow were not differentiated. Our analyses suggest that regional watersheds are isolated from one another, with very limited (possibly no) gene flow over recent generations. This isolation is paired with small effective population sizes across regional gene pools (Ne = 62.4–147.1). Genetic diversity was variable across sites and watersheds (He = 0.09–0.22). Those with the highest diversity may have been refugia and are now potential sources of genetic diversity for other populations. These findings highlight which portions of the species range may be most vulnerable to future habitat fragmentation and provide management consideration for maintaining local effective population sizes and genetic connectivity.

Analysis of Selenium in Fish Tissue: An Interlaboratory Study on Weight Constraints.

Environmental monitoring programs that target fish tissues for selenium (Se) analysis present unique sampling and analytical challenges. Se monitoring programs ideally focus on egg/ovary sampling but frequently sample multiple tissues with varying lipid content, often target small-bodied fish species due to their small home ranges, and require reporting in units of dry weight. Additionally, there is a growing impetus for non-lethal tissue sampling in fish monitoring. As a result, Se monitoring programs often generate low-weight tissue samples of varying lipid content, which challenges analytical laboratories to quantify tissue selenium concentrations accurately, precisely, and at desired detection limits. The objective of this study was to stress-test some conventional analytical techniques used by commercial laboratories in terms of their ability to maintain data quality objectives in the face of sample weight constraints. Four laboratories analyzed blind a suite of identical samples, and data were compared against a priori data quality objectives for accuracy, precision, and sensitivity. Data quality tended to decrease with decreasing sample weight, particularly when samples were less than the minimum weights requested by the participating laboratories; however, effects of sample weight on data quality were not consistent among laboratories or tissue types. This study has implications for accurately describing regulatory compliance in selenium monitoring programs, highlighting some important considerations for achieving high data quality from low-weight samples.

Snake scales record environmental metal(loid) contamination.

Wetland snakes, as top predators, are becoming globally recognised as bioindicators of wetland contamination. Livers are the traditional test organ for contaminant exposure in organisms, but research is moving towards a preference for non-lethal tissue sampling. Snake scales can be used as an indicator of exposure, as many metals bind to the keratin. We used laser ablation with inductively coupled plasma-atomic emission spectroscopy and mass spectrometry (LA-ICP-MS) to quantify the concentrations of 19 metals and metalloids (collectively referred to 'metals' hereafter) in Western tiger snake (Notechis scutatus occidentalis) scales from four wetlands along an urban gradient, and compared them to concentrations measured in captive tiger snake scales. We conducted repeat measures to determine the concentration accuracy of each metal using LA-ICP-MS. Concentrations in wild Western tiger snake scales were significantly higher than in reference tiger snake scales for most metals analysed, suggesting accumulation from environmental exposure. We compared the scale concentrations to sediment concentrations of sampled wetlands, and found inter-site differences between mean concentrations of metals in scales parallel patterns recorded from sediment. Four metals (Mn, As, Se, Sb) had strong positive correlations with liver tissue contents suggesting scale concentrations can be used to infer internal concentrations. By screening for a larger suite of metals than we could using traditional digestive methods, we identified additional metals (Ti, V, Sr, Cs, Tl, Th, U) that may be accumulating to levels of concern in tiger snakes in Perth, Western Australia. This research has progressed the use of LA-ICP-MS for quantifying a suite of metals available in snake scales, and highlights the significance of using wetland snake scales as a non-lethal indicator of environmental contamination.

Evaluation of Partial and Whole Maxillary Bone Excision as a Nonlethal Age Estimation Structure in Lake Trout

AbstractPartial excision, or clipping, of maxillary bones has been used for decades as a mark to assign year‐classes to hatchery fish stocked into the wild. Recent developments in the use of maxillary bones for age assignment purposes have raised the question of whether they could be used as age assignment structures that can be collected nonlethally. Such nonlethal tissue collections are of obvious importance to managers of low‐abundance and recovering populations or threatened and endangered species. We examined 120 Lake Trout Salvelinus namaycush from two cohorts in a hatchery setting for approximately 6 months to determine the feasibility of maxillary bone excision as a nonlethal sampling technique for age assignment purposes. Lake Trout were subjected to whole‐maxillary excision and partial‐maxillary excision and were evaluated for survival, efficiency of collection, and accuracy of age assignment. Survival in both the whole‐excision and partial‐excision groups was 97%, while untreated controls survived at a rate of 98% (not significantly different). There was no significant difference in sample collection time between groups. Age assignments from whole maxillary bones were more accurate than those from partial maxillary bones; assignments were more accurate when based on bones from younger fish than on bones from older fish. We conclude that for nonlethal collection purposes, excision of whole maxillary bones is advantageous over partial excision due to comparable efficiency, equal survival, and more accurate age estimation. While the value of maxillary bones as a reliable aging structure for Lake Trout has been noted, the use of maxillary bones for nonlethal tissue sampling in other studies, such as isotopic, trace element, and genetic analyses, may also be valuable.

Accumulation of coal combustion residues and their immunological effects in the yellow-bellied slider (Trachemys scripta scripta)

Anthropogenic activities such as industrial processes often produce copious amounts of contaminants that have the potential to negatively impact growth, survival, and reproduction of exposed wildlife. Coal combustion residues (CCRs) represent a major source of pollutants globally, resulting in the release of potentially harmful trace elements such as arsenic (As), cadmium (Cd), and selenium (Se) into the environment. In the United States, CCRs are typically stored in aquatic settling basins that may become attractive nuisances to wildlife. Trace element contaminants, such as CCRs, may pose a threat to biota yet little is known about their sublethal effects on reptiles. To assess the effects of CCR exposure in turtles, we sampled 81 yellow-bellied sliders (Trachemys scripta scripta) in 2014–2015 from CCR-contaminated and uncontaminated reference wetlands located on the Savannah River Site (Aiken, SC, USA). Specific aims were to (1) compare the accumulation of trace elements in T. s. scripta claw and blood samples between reference and CCR-contaminated site types, (2) evaluate potential immunological effects of CCRs via bacterial killing assays and phytohaemagglutinin (PHA) assays, and (3) quantify differences in hemogregarine parasite loads between site types. Claw As, Cd, copper (Cu), and Se (all p ≤ 0.001) and blood As, Cu, Se, and strontium (Sr; p ≤ 0.015) were significantly elevated in turtles from CCR-contaminated wetlands compared to turtles from reference wetlands. Turtles from reference wetlands exhibited lower bacterial killing (p = 0.015) abilities than individuals from contaminated sites but neither PHA responses (p = 0.566) nor parasite loads (p = 0.980) differed by site type. Despite relatively high CCR body burdens, sliders did not exhibit apparent impairment of immunological response or parasite load. In addition, the high correlation between claw and blood concentrations within individuals suggests that nonlethal tissue sampling may be useful for monitoring CCR exposure in turtles.

From tails to toes: developing nonlethal tissue indicators of mercury exposure in five amphibian species.

Exposure to environmental contaminants has been implicated as a factor in global amphibian decline. Mercury (Hg) is a particularly widespread contaminant that biomagnifies in amphibians and can cause a suite of deleterious effects. However, monitoring contaminant exposure in amphibian tissues may conflict with conservation goals if lethal take is required. Thus, there is a need to develop non-lethal tissue sampling techniques to quantify contaminant exposure in amphibians. Some minimally invasive sampling techniques, such as toe-clipping, are common in population-genetic research, but it is unclear if these methods can adequately characterize contaminant exposure. We examined the relationships between mercury (Hg) concentrations in non-lethally sampled tissues and paired whole-bodies in five amphibian species. Specifically, we examined the utility of three different tail-clip sections from four salamander species and toe-clips from one anuran species. Both tail and toe-clips accurately predicted whole-body THg concentrations, but the relationships differed among species and the specific tail-clip section or toe that was used. Tail-clips comprised of the distal 0-2 cm segment performed the best across all salamander species, explaining between 82 and 92% of the variation in paired whole-body THg concentrations. Toe-clips were less effective predictors of frog THg concentrations, but THg concentrations in outer rear toes accounted for up to 79% of the variability in frog whole-body THg concentrations. These findings suggest non-lethal sampling of tails and toes has potential applications for monitoring contaminant exposure and risk in amphibians, but care must be taken to ensure consistent collection and interpretation of samples.

Mating success and oviposition of a butterfly are not affected by non-lethal tissue sampling

Non-lethal methods of tissue sampling are increasingly used for genetic studies of insect species and the effects of this approach have long been assumed to be minimal. Tissue removal has the potential to influence insect reproductive behaviours such as mate recognition, courtship or oviposition but the effects of non-lethal sampling on reproductive success have not been widely and adequately tested. Here, we test potential effects of both wing-clipping and leg removal on reproductive behaviours of the cabbage white butterfly (Pieris rapae). We conducted a total of 93 male and 59 female mating trials, and found no significant differences in mating success between treated (i.e., tissue removed) and control individuals in either sex. We also monitored the number and location of eggs laid by 58 females. We found no significant differences in egg-laying behaviour among leg removed and control individuals. Power analysis indicated that we had sufficient statistical power to detect moderate effects of treatment on both mating and oviposition. Our study provides the most comprehensive examination to date of the effects of non-lethal sampling on reproductive behaviours in a butterfly/insect species, and supports the contention that tissue sampling is non-detrimental. To fully comprehend the general impacts of tissue sampling on butterfly reproductive behaviour however, additional similar studies need to be conducted on a variety of species with differing mating behaviours. Only through meta-analysis, may it be possible to detect more subtle effects of tissue removal which cannot be revealed within a single study due to sample size limitations.

Matching and mismatching stable isotope (δ13C and δ15N) ratios in fin and muscle tissue among fish species: a critical review

Using non-lethal tissue sampling for stable isotope analysis has become standard in many fields, but not for fishes, despite being desirable when species are rare or protected, when repeated sampling of individuals is required or where removal may bias other analyses. Here, we examine the utility of fish dorsal fin membrane as an alternative to muscle for analyzing δ13C and δ15N ratios in two reef fish species (blue cod Parapercis colias and spotty Notolabrus celidotus) that have differing feeding modes. Both species exhibited evidence of size-based feeding from fin δ15N values, but not from muscle. Blue cod fin δ15N increased steadily throughout the sampled size range (213–412 mm fork length), whereas spotty exhibited a distinct ontogenetic diet shift at approximately 120–140 mm fork length after which size-based feeding did not occur. Fin membrane was higher than muscle in δ13C in both species and in δ15N for blue cod, but fin δ15N was lower than muscle in spotty. The δ13C and δ15N fin–muscle offsets were constant in spotty regardless of size, while in blue cod, δ13C was constant with fish size, but δ15N offsets increased with increasing fish size. Non-lethal sampling utilizing fin tissue can be employed to estimate stable isotope values of muscle in fishes, but it is necessary to assess relationships among tissues and the effects of fish size on isotope values a priori for each species studied. Our data indicated that fin membrane may be a more sensitive tissue than muscle for detecting size-based feeding in some fish species using stable isotopes. A critical literature review revealed inconsistencies in tissue types tested, little understanding of tissue-specific trophic shift or turnover rates, and pseudo-replicated analyses leading to erroneous postulating of 1:1 relationships between tissues.

Effects of different methods of non-lethal tissue sampling on butterflies

1. We investigated the effects of two methods of non-lethal tissue sampling on post-release flight behaviour (short-term response) and survival (long-term response) of two butterflies, Pieris rapae and Coenonympha tullia, within the same natural habitat. We applied three treatments: control (no tissue removal), wing clipping, and leg removal. Our study is the first to directly compare the effects of these common sampling methods. 2. We monitored the flight behaviour of the butterflies by following individuals immediately after their release. In 99 behaviour trials of P. rapae and 101 of C. tullia we found no significant differences in proportion of time spent flying or displacement per unit time among treatment groups in either species. 3. We used standard mark–recapture techniques continuously throughout the flight season to compare the survival of individuals. We marked a total of 687 P. rapae and 490 C. tullia butterflies. We found no significant differences in survival among treatments in either species. 4. We detected differences between the sexes in survival in P. rapae and flight behaviour in C. tullia. In addition to indicating differences in ecology between the sexes, these results also suggest that our analyses were sufficiently powerful to detect a significant effect of tissue removal had such an effect existed. 5. Our work is an important addition to the accumulating evidence that these methods of non-lethal tissue sampling are generally not detrimental. These sampling techniques closely mimic conditions in the wild, as wing wear and leg losses occur naturally.

Field Evaluation of Mortality from Hemolymph Extraction as a Source of DNA, and Application to PCR-RFLP Identification of Threatened Freshwater Mussel Species

Evolutionary convergence and plasticity of shell characters creates great confusion in freshwater mussel systematics and complicates field census efforts. Genetic identification offers a powerful alternative. But methods involving DNA sequencing are expensive and require tissue sampling, whose effects on survival and health of animals from natural populations have seldom been assessed. We used hemolymph sampling as a nonlethal source of tissue for DNA extraction, and developed genetic identification methods using Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis as an alternative for field surveys. We focused on two morphologically similar endemic species in Lake Waccamaw, North Carolina: the abundant Elliptio waccamawensis and the less common Lampsilis fullerkati. These served as models for surveyors who are often faced with conducting accurate census of cryptic rare species that co-occur with more common forms. Hemolymph-sampled and control individuals of these two species and Leptodea ochracea were caged together in enclosures in Lake Waccamaw. Eight-week survival was 100% and we detected no significant effect of hemolymph extraction on shell growth. PCR-RFLP analysis of the 16s rRNA gene reliably identified species and detected cases of morphological misidentification, both in these collections and from belt transects in the lake. We also developed PCR-RFLP markers that distinguished pairs of cryptic taxa from surveys of streams in southeastern North Carolina. Our results show how nonlethal tissue sampling and PCR-RFLP assays designed for a regional fauna can be useful tools in freshwater mussel conservation programs.

Detection and quantitation of infectious pancreatic necrosis virus by real-time reverse transcriptase-polymerase chain reaction using lethal and non-lethal tissue sampling

Infectious pancreatic necrosis virus (IPNV) is a bisegmented double-stranded RNA virus belonging to the family Birnaviridae, genus Aquabirnavirus, which is a major viral pathogen of salmonid fish. The virus infects wild and cultured salmonids, causing high mortality in juvenile trout and salmon. A highly sensitive and specific real-time RT-PCR assay using the fluorogenic dye SYBR® Green I was developed for the detection and quantitation of IPNV in rainbow trout (Oncorhynchus mykiss). Rainbow trout were infected experimentally with IPNV in the laboratory by injection or immersion and then pectoral fin, spleen, and head kidney samples were collected for analysis. The corresponding cDNA was synthesized using DNase I-treated total RNA and then real-time RT-PCR was performed using primers based on the IPNV non-structural protein gene, designated as either NS or VP4. Rainbow trout β-actin and elongation factor 1α (EF-1α) genes were used as internal controls. Using real-time RT-PCR, the virus was successfully detected in pectoral fin, spleen, and head kidney tissue samples. The dissociation curves for each amplicon showed a single melting peak at 83, 81.5, and 84°C for IPNV NS, trout β-actin, and EF-1α genes, respectively. The amplicon size and nucleotide sequence was used to confirm the specificity of the products. Using a dilution series of in vitro transcribed RNA, IPNV was reliably detected down to 10 RNA copies and had a dynamic range up to 107 RNA copies. A time course assay, using immersion challenged samples, revealed that the virus could be detected in pectoral fin, spleen, and head kidney as early as 24h post-challenge. The average viral load in all three tissues increased over time, reaching its highest level at 21 days post-challenge, which was followed by a slight decrease at 28 days post-challenge. IPNV load in pectoral fin tissue was comparable to the viral load in spleen and head kidney tissues, indicating that pectoral fin could be used for the detection and quantification of IPNV. The development of a non-lethal detection method will be useful for the detection of IPNV and potentially other viruses of finfish in farmed and wild fish.

Assessing Organic Contaminants in Fish: Comparison of a Nonlethal Tissue Sampling Technique to Mobile and Stationary Passive Sampling Devices

As concerns mount over the human health risks associated with consumption of fish contaminated with persistent organic pollutants, there exists a need to better evaluate fish body burdens without lethally sampling many of the important commercial and sport species of interest. The aim of this study was to investigate two novel methods for estimating organic contaminants in fish that are a concern for both fish and human health. The removal of fish adipose fins, commonly done in mark-recapture studies with salmonid species, was evaluated as a nonlethal sampling technique to estimate concentrations of polychlorinated biphenyls (PCBs) and organochlorine pesticides (OCPs) in flathead catfish (Pylodictis olivaris), relative to those found in muscle fillets of the same fish. We also assessed the efficacy of using poly(dimethylsiloxane) (PDMS) as a mobile passive sampling device (PSD) attached directly to wild flathead catfish for assessing location-specific exposure of the fish to waterborne contaminants. The results of this study have demonstrated for the first time that organic contaminant concentrations in adipose fin were highly correlated (R2 = 0.87) with muscle fillet concentrations, indicating that the adipose fin of certain fishes may be used to accurately estimate tissue concentrations without the need for lethal sampling. Moreover, mobile PSDs attached directly to fish and used here for the first time accurately estimated ultratrace concentrations of waterborne PCBs and OCPs without any apparent harm to the fish, indicating that there are no practical or physical barriers to the use of mobile passive samplers attached to aquatic organisms. Among the many practical implications of this research, two potential priority items include the analysis of organic contaminants in farm-raised and sport fish intended for human consumption, without the economic and population losses associated with lethally sampling fish to obtain tissues, and identifying specific areas where fish may be accumulating large portions of their contaminant burden.