Lethal Sampling Research Articles

Ecological transcriptomics – a non‐lethal sampling approach for endangered fire salamanders

Summary The analysis of transcriptomes is well‐established and increasingly affordable in studies at the interface of ecology and evolution. Expression analysis of thousands of genes in parallel reveals functions and pathways involved in relevant phenotypic differentiation. The application of such methods typically involves the sacrifice of the analysed organisms, which is potentially subject to ethical and legal constraints. As an alternative to lethal sampling, transcriptome analyses can be performed using small biopsies of dispensable tissues. It has to be verified, however, to what extent such results are representative of the whole organism. Here, we use a custom microarray to compare transcriptomes of tail‐clip samples with those of the remaining whole body of fire salamander larvae (Salamandra salamandra). The microarray was calibrated using target RNA to validate the performance of each probe. We varied water temperature to test whether the thermal response in gene expression can be characterized in both types of sample. A large fraction (51%) of the differentially expressed genes showed parallel changes for both tail clips and whole bodies in response to temperature. While sets of differentially expressed were not identical, they largely belonged to the same functional categories. The gene functions thus revealed a common thermal response of larvae irrespective of the sampled tissue. This included an overexpression of mitochondrial transcripts, an expected thermal acclimatization response of ectotherms. Hence, ecological transcriptomics based on small biopsies represent an alternative to the analysis of lethally sampled tissues in situations where the sacrifice of individuals is not an option.

The effect of repeated, lethal sampling on wild bee abundance and diversity

Summary Bee pollinators provide a critical ecosystem service to wild and agricultural plants but are reported to be declining world‐wide due to anthropogenic change. Long‐term data on bee abundance and diversity are scarce, and the need for additional quantitative sampling using repeatable methods has been emphasized. Recently, monitoring programmes have begun using a standardized method that employs a combination of pan traps and sweep netting, resulting in lethal sampling of bees. This standardized method can remove a large number of bees from sites during each sampling day, raising concern that the sampling itself could have a negative effect on bee populations. We conducted an experiment to assess whether lethal sampling for bees using pan traps and netting affected bee abundance and diversity when done every two weeks throughout a season and over multiple years. We compared bee abundance, richness, evenness and functional group composition between sites that had been sampled every two weeks from 2009 to 2012 to similar sites not previously sampled. We found that the standardized method for sampling bees, with specimens from 132 morphospecies, did not affect bee communities in terms of abundance, rarefied richness, evenness, or functional group composition. Thus, our results indicate that the bee communities we sampled are robust to such sampling efforts, despite removing an average of 2862 bees per season. We discuss several explanations for why sampling did not affect bee abundance or community structure, including a density‐dependent response to reduced competition for resources. These results suggest that bee monitoring programmes sampling once every two weeks with pan traps and netting will not affect bee community structure. We urge researchers monitoring bees to utilize standardized protocols so that results can be compared across space and time.

Testing of candidate non-lethal sampling methods for detection of Renibacterium salmoninarum in juvenile Chinook salmon Oncorhynchus tshawytscha.

Non-lethal pathogen testing can be a useful tool for fish disease research and management. Our research objectives were to determine if (1) fin clips, gill snips, surface mucus scrapings, blood draws, or kidney biopsies could be obtained non-lethally from 3 to 15 g Chinook salmon Oncorhynchus tshawytscha, (2) non-lethal samples could accurately discriminate between fish exposed to the bacterial kidney disease agent Renibacterium salmoninarum and non-exposed fish, and (3) non-lethal samples could serve as proxies for lethal kidney samples to assess infection intensity. Blood draws and kidney biopsies caused ≥5% post-sampling mortality (Objective 1) and may be appropriate only for larger fish, but the other sample types were non-lethal. Sampling was performed over 21 wk following R. salmoninarum immersion challenge of fish from 2 stocks (Objectives 2 and 3), and nested PCR (nPCR) and real-time quantitative PCR (qPCR) results from candidate non-lethal samples were compared with kidney tissue analysis by nPCR, qPCR, bacteriological culture, enzyme-linked immunosorbent assay (ELISA), fluorescent antibody test (FAT) and histopathology/immunohistochemistry. R. salmoninarum was detected by PCR in >50% of fin, gill, and mucus samples from challenged fish. Mucus qPCR was the only non-lethal assay exhibiting both diagnostic sensitivity and specificity estimates>90% for distinguishing between R. salmoninarum-exposed and non-exposed fish and was the best candidate for use as an alternative to lethal kidney sample testing. Mucus qPCR R. salmoninarum quantity estimates reflected changes in kidney bacterial load estimates, as evidenced by significant positive correlations with kidney R. salmoninarum infection intensity scores at all sample times and in both fish stocks, and were not significantly impacted by environmental R. salmoninarum concentrations.

Non-lethal sampling of lake sturgeon for stable isotope analysis: Comparing pectoral fin-clip and dorsal muscle for use in trophic studies

When used to study the feeding ecology of fish, stable isotope analyses generally require sacrificing animals to collect tissue samples. Lethal sampling can be undesirable when studying a species at risk such as lake sturgeon (Acipenser fulvescens). We evaluated the feasibility of using pectoral fin clips as an alternative to dorsal muscle tissue in stable isotope studies for adult lake sturgeon. Because lipid content can affect stable isotope ratios, we also determined whether lipid extraction and mathematical normalization affected the relationship between fin-clip and muscle tissue signatures. Dorsal muscle plugs and fin clips were collected from adult lake sturgeon and analyzed for stable isotopes of carbon and nitrogen. Significant, positive relationships were observed between fin and muscle tissues for both δ15N (r2=0.43) and δ13C (r2=0.32). Lipid extraction significantly reduced the among-individual variation, and improved the δ13C relationship significantly (r2=0.74). While general non-linear mathematical lipid normalization did effectively reduce the among-individual variation, a consistent enrichment relative to chemical extraction was observed. An alternative mass balance correction method, however, yielded lake sturgeon specific parameter estimates that facilitated accurate lipid correction. The strong relationships between fin-clip and muscle tissue signatures demonstrate that fin-clips should be considered as good surrogates for muscle tissue, will allow trophic studies to accurately adjust for the effect of differential lipid accumulation and can effectively limit the need for invasive sampling for adult lake sturgeon.

Mysterious bio-duck sound attributed to the Antarctic minke whale (Balaenoptera bonaerensis).

For decades, the bio-duck sound has been recorded in the Southern Ocean, but the animal producing it has remained a mystery. Heard mainly during austral winter in the Southern Ocean, this ubiquitous sound has been recorded in Antarctic waters and contemporaneously off the Australian west coast. Here, we present conclusive evidence that the bio-duck sound is produced by Antarctic minke whales (Balaenoptera bonaerensis). We analysed data from multi-sensor acoustic recording tags that included intense bio-duck sounds as well as singular downsweeps that have previously been attributed to this species. This finding allows the interpretation of a wealth of long-term acoustic recordings for this previously acoustically concealed species, which will improve our understanding of the distribution, abundance and behaviour of Antarctic minke whales. This is critical information for a species that inhabits a difficult to access sea-ice environment that is changing rapidly in some regions and has been the subject of contentious lethal sampling efforts and ongoing international legal action.

Improved method for triacylglycerol-derived fatty acid profiling by various non-lethal and lethal sampling techniques in Atlantic salmon

AEI Aquaculture Environment Interactions Contact the journal Facebook Twitter RSS Mailing List Subscribe to our mailing list via Mailchimp HomeLatest VolumeAbout the JournalEditorsTheme Sections AEI 4:251-261 (2013) - DOI: https://doi.org/10.3354/aei00087 Improved method for triacylglycerol-derived fatty acid profiling by various non-lethal and lethal sampling techniques in Atlantic salmon Rolf Erik Olsen1,*, Geir Lasse Taranger2, Terje Svåsand2, Ove Tommy Skilbrei2 1Institute of Marine Research, Matre Research Station, 5984 Matredal, Norway 2Institute of Marine Research, PO Box 1870 Nordnes, 5817 Bergen, Norway *Email: rolf.erik.olsen@imr.no ABSTRACT: The current paper compares the use of adipose fin and muscle biopsies as non-lethal sampling procedures, and the suitability of triacylglycerol (TAG) versus total lipid (TOT) fatty acid composition (fatty acid methyl esters, FAME) to estimate dietary history in farmed and wild maturing Atlantic salmon Salmo salar. TAG FAME gives best resemblance to dietary history. Fatty acid composition differs between tissues, and sample series should preferably be from 1 tissue only. TOT FAME supplies informative data on dietary history in fatty tissues, but differs from TAG FAME when total lipid levels are low. The reason is a larger contribution of phospholipid FAME. In wild maturing salmon, lipid content in adipose fin and muscle is low. TAG provides good data on dietary history, while TOT is less informative. Escapees are well identified analysing 18:2n-6 and 18:3n-3 fatty acids, which are high in commercial feed. For estimating a wider range of dietary history after escape (switch to wild prey), or feed preferences in wild fish, other fatty acids have to be taken into account. This requires the use of multivariate methods, like principal component analysis. Adipose fin and muscle biopsies are good alternatives for non-lethal sampling using the TAG method. The minimum amounts of samples to be used are proposed to be 0.5 to 1 g of adipose fin and 60 to 100 mg of muscle. The method of storage (liquid nitrogen/dry ice) does not affect fatty acid compositions. Other tissues can also be used for fatty acid profiling. Snout is a good alternative, being lipid-rich even in wild fish with low muscle lipid. KEY WORDS: Fatty acid composition · Fatty acid profiling · Adipose fin · Triacylglycerol · Muscle · Snout · Escaped farmed salmon Full text in pdf format PreviousNextCite this article as: Olsen RE, Taranger GL, Svåsand T, Skilbrei OT (2013) Improved method for triacylglycerol-derived fatty acid profiling by various non-lethal and lethal sampling techniques in Atlantic salmon. Aquacult Environ Interact 4:251-261. https://doi.org/10.3354/aei00087 Export citation RSS - Facebook - Tweet - linkedIn Cited by Published in AEI Vol. 4, No. 3. Online publication date: November 14, 2013 Print ISSN: 1869-215X; Online ISSN: 1869-7534 Copyright © 2013 Inter-Research.

Non-lethal sampling of liver tissue for toxicologic evaluation of Florida cottonmouths snakes, Agkistrodon piscivorus conanti

Due to their longevity, strong site tenure, poikilothermic metabolism, and low-energy specializations, reptiles might serve as excellent environmental sentinels. Cottonmouth snakes are generalist predators and scavengers, and as such, may have higher exposure to persistent environmental contaminants as a result of bioaccumulation. Traditionally, assessment and monitoring of contaminant exposure in reptiles have involved lethal sampling techniques. In this paper, we describe a non-destructive technique for sampling liver tissue in live anesthetized Florida cottonmouths. Wild-caught snakes (n=21) were anesthetized with propofol, and a liver wedge biopsy was obtained by clamping the edge of the organ with two small hemostatic mosquito forceps via right-sided coeliotomy incision. A minimum required tissue sample weighing >100mg was harvested from all except one of the animals. No mortalities occurred during the procedures or recovery from anesthesia, and all snakes were released back into the field after the animal had consumed prey and defecated, usually within 2weeks following surgery. Hemorrhage was a minor complication in most snakes, especially those with friable discolored livers. The procedure appeared to have no short-term deleterious effects, and two biopsied individuals were captured after being released into the field and appeared to be normal and healthy. However, follow-up studies and recapture of more snakes are needed to assess long-term survivability. Our non-destructive liver sampling technique might be implemented in toxicological studies of other squamates and could help to minimize the lethal sampling of threatened species.

Assessing reproductive status in elasmobranch fishes using steroid hormones extracted from skeletal muscle tissue.

Elasmobranch fishes (sharks, skates, and rays) are particularly susceptible to anthropogenic threats, making a thorough understanding of their life history characteristics essential for proper management. Historically, elasmobranch reproductive data have been collected by lethal sampling, an approach that is problematic for threatened and endangered species. However, recent studies have demonstrated that non-lethal approaches can be as effective as lethal ones for assessment of the reproductive status of an animal. For example, plasma has been used to examine concentrations of steroid hormones. Additionally, skeletal muscle tissue, which can be obtained non-lethally and with minimal stress, can also be used to quantify concentrations of steroid hormones. Skeletal muscle progesterone, testosterone, and estradiol concentrations were determined to be statistically significant indicators of reproductive status in the oviparous Leucoraja erinacea, the yolk-dependent viviparous Squalus acanthias, and the yolk-sac placental viviparous Rhizoprionodon terraenovae. The results of the present study demonstrate that steroid hormones present in non-lethally harvested skeletal muscle tissue can be used as reliable indicators of reproductive status in elasmobranchs.

LOW DETECTION OF RANAVIRUS DNA IN WILD POSTMETAMORPHIC GREEN FROGS, RANA (LITHOBATES) CLAMITANS, DESPITE PREVIOUS OR CONCURRENT TADPOLE MORTALITY

Ranavirus (Iridoviridae) infection is a significant cause of mortality in amphibians. Detection of infected individuals, particularly carriers, is necessary to prevent and control outbreaks. Recently, the use of toe clips to detect ranavirus infection through PCR was proposed as an alternative to the more frequently used lethal liver sampling in green frogs (Rana [Lithobates] clamitans). We attempted reevaluate the use of toe clips, evaluate the potential use of blood onto filter paper and hepatic fine needle aspirates (FNAs) as further alternatives, and explore the adequacy of using green frogs as a target-sampling species when searching for ranavirus infection in the wild. Samples were obtained from 190 postmetamorphic (≥1-yr-old) green frogs from five ponds on Prince Edward Island (PEI), Canada. Three of the ponds had contemporary or recent tadpole mortalities due to Frog Virus 3 (FV3) ranavirus. PCR testing for ranavirus DNA was performed on 190 toe clips, 188 blood samples, 72 hepatic FNAs, and 72 liver tissue samples. Only two frogs were ranavirus-positive: liver and toe clip were positive in one, liver only was positive in the other; all blood and FNAs, including those from the two positive frogs, were negative. Results did not yield a definitive answer on the efficacy of testing each type of sample, but resemble what is found in salamanders infected with Ambystoma tigrinum (rana)virus. Findings indicate a low prevalence of FV3 in postmetamorphic green frogs on PEI (≤2.78%) and suggest that green frogs are poor reservoirs (carriers) for the virus.

Development of a non lethal sampling method to monitor immune response and disease progression in salmonid fish

Development of a non lethal sampling method to monitor immune response and disease progression in salmonid fish

Ovarian biopsy: a non-terminal method to determine reproductive status in giant kokopu, Galaxias argenteus (Gmelin 1789)

AIM: To establish a method of gonad biopsy for ovarian tissue collection in the declining giant kokopu Galaxias argenteus (Gmelin 1789) as an alternative to lethal sampling in order to understand the species' reproductive biology. METHODS: Six female giant kokopu weighing between 200 and 350 g were caught from the wild in early December of 2009 and transferred to a holding facility (Department of Zoology, University of Otago, Dunedin) where they were kept under a simulated natural photo-thermal regime for 10 months. Fish were repeatedly biopsied for ovarian tissue at near-monthly intervals (mean number of days between biopsies = 33) until ovulation. RESULTS AND CONCLUSIONS: Ovarian samples were successfully collected from giant kokopu by biopsy for use in downstream analyses. Among a total of 23 biopsy events, a single death occurred when a two-layered suturing approach was used, highlighting the value of this method for study of the reproductive biology of valuable fish. CLINICAL RELEVANCE: This biopsy method may have implications for veterinary research on fish physiology, pathology, conservation and development, when repeated tissue samples need to be collected over a prolonged period of time or for general surgical manipulations on fish when accessing the coelom. Furthermore, this approach allows the implementation of a more powerful experimental design, as repeated measures reduces the variability of estimates due to the removal of inherent stage differences among individuals.

Non-lethal dorsal fin sampling for stable isotope analysis in seahorses

Sampling collection for stable isotope analysis has traditionally involved the sacrifice of the animal. Seahorses (Hippocampus spp.) are listed as threatened by the Convention on International Trade in Endangered Species ( http://www.cites.org ) and consequently lethal sampling is undesirable. We evaluated the adequacy of dorsal fin tissue of adult seahorses Hippocampus guttulatus for stable isotope analysis as an alternative to lethal tissue sampling. Three seahorse tissues (dorsal fin, muscle, and liver) were analyzed for comparisons of δ15N and δ13C values. Similarities found between δ15N and δ13C values in dorsal fin and muscle tissue of H. guttulatus suggest that both tissues are adequate for stable isotope analysis to understand feeding ecology of seahorses. However, considering the threatened status of the species, dorsal fin tissue would be recommended in adult seahorses as a non-lethal sampling. The effect of lipid extraction on carbon and nitrogen stable isotope values was also evaluated in each seahorse tissues. Significant effects of lipids extraction did only occur for δ13C values in muscle and liver. It was found that lipid removal was not necessary to perform SIA in dorsal fin tissues. Due to the limited availability of fin tissue obtained from fin-clipping in seahorses, the relationship between the mass/surface of dorsal fin clip and stable isotope values was analyzed. δ15N and δ13C values in fin samples were found to be independent of the size of fin analyzed. According to our study, the use of fin-clipping sampling, with a minimum surface analyzed of 12.74 mm2, was found to be an adequate method for SIA in seahorses.

Reliability of non-lethal surveillance methods for detecting ranavirus infection

Ranaviruses have been identified as the etiologic agent in many amphibian die-offs across the globe. Polymerase chain reaction (PCR) is commonly used to detect ranavirus infection in amphibian hosts, but the test results may vary between tissue samples obtained by lethal and non-lethal procedures. Testing liver samples for infection is a common lethal sampling technique to estimate ranavirus prevalence because the pathogen often targets this organ and the liver is easy to identify and collect. However, tail clips or swabs may be more practicable for ranavirus surveillance programs compared with collecting and euthanizing animals, especially for uncommon species. Using PCR results from liver samples for comparison, we defined false-positive test results as occurrences when a non-lethal technique indicated positive but the liver sample was negative. Similarly, we defined false-negative test results as occurrences when a non-lethal technique was negative but the liver sample was positive. Using these decision rules, we estimated false-negative and false-positive rates for tail clips and swabs. Our study was conducted in a controlled facility using American bullfrog Lithobates catesbeianus tadpoles; false-positive and false-negative rates were estimated after different periods of time following exposure to ranavirus. False-negative and false-positive rates were 20 and 6%, respectively, for tail samples, and 22 and 12%, respectively, for swabs. False-negative rates were constant over time, but false-positive rates decreased with post-exposure duration. Our results suggest that non-lethal sampling techniques can be useful for ranavirus surveillance, although the prevalence of infection may be underestimated when compared to results obtained with liver samples.

Development of a sensitive assay for the detection of Pseudoloma neurophilia in laboratory ­populations of the zebrafish Danio rerio

The zebrafish Danio rerio is an increasingly important biological model in many areas of research. Due to the potential for non-protocol-induced variation, diseases of zebrafish, especially those resulting in chronic, sub-lethal infections, are of great concern. The microsporidium Pseudoloma neurophilia is a common parasite of laboratory zebrafish. Current methods for detection of this parasite require lethal sampling of fish, which is often undesirable with poorly spawning mutant lines and small populations. We present here an improved molecular-based diagnostic assay using real-time polymerase chain reaction (PCR), and including sonication treatment prior to DNA extraction. Comparisons of several DNA extraction methods were performed to determine the method providing the maximum sensitivity. Sonication was found to be the most effective method for disrupting spores. Compared to previously published data on PCR-based assay using a dilution experiment, sensitivity is increased. This shows that our assay, which includes sonication, is capable of detecting parasite DNA at 1 log higher dilution than the conventional PCR-based assay, which does not include sonication. Furthermore, we demonstrate the application of this method to testing of water, eggs, and sperm, providing a potential non-lethal method for detection of this parasite in zebrafish colonies with a sensitivity of 10 spores 1(-1) of water, 2 spores per spiked egg sample, and 10 spores microl(-1) of spiked sperm sample.

Sampling-Rate Calibration for Rapid and Nonlethal Monitoring of Organic Contaminants in Fish Muscle by Solid-Phase Microextraction

Solid-phase microextraction (SPME) is a promising technique for determining organic contaminants within biotic systems. Existing in vivo SPME-kinetic calibration (SPME-KC) approaches are unwieldy due to the necessity of predetermining a distribution coefficient for the analyte of interest in the tissue and the preloading of a calibrating compound to the fiber. In this study, a rapid and convenient SPME alternative calibration method for in vivo analysis, termed SPME-sampling rate (SPME-SR) calibration, was developed and validated under both laboratory and field conditions to eliminate such presampling requirements. Briefly, the SPME probe is inserted into tissue, in this study fish dorsal-epaxial muscle, for 20 min allowing the concentrations of target analytes in the fish muscle to be determined by the extracted amount of analyte and the predetermined sampling rates. Atrazine, carbamazepine, and fluoxetine were detected nonlethally in the low ppb levels within fish muscle, with both laboratory and field-derived results obtained by in vivo SPME-KC comparable (within a factor of 1.27) to those obtained by lethal sampling followed by tissue liquid extraction. The technique described in this study represents an important advance which broadens the application of SPME in vivo sampling technology.

Killing for conservation: the need for alternatives to lethal sampling of apex predatory sharks

ESR Endangered Species Research Contact the journal Facebook Twitter RSS Mailing List Subscribe to our mailing list via Mailchimp HomeLatest VolumeAbout the JournalEditorsSpecials ESR 14:135-140 (2011) - DOI: https://doi.org/10.3354/esr00354 AS WE SEE IT Killing for conservation: the need for alternatives to lethal sampling of apex predatory sharks Neil Hammerschlag1,2,3,*, James Sulikowski4 1Rosenstiel School of Marine and Atmospheric Science, University of Miami, 4600 Rickenbacker Causeway, Miami, Florida 33149, USA 2Leonard and Jayne Abess Center for Ecosystem Science and Policy, University of Miami, PO Box 248203, Coral Gables, Florida 33124, USA 3RJ Dunlap Marine Conservation Program, University of Miami, 4600 Rickenbacker Causeway, Miami, Florida 33149, USA 4University of New England, Marine Sciences Department, Marine Science Center, 11 Hills Beach Road, Biddeford, Maine 04005, USA *Email: nhammerschlag@rsmas.miami.edu ABSTRACT: Top oceanic predators, especially large predatory sharks (TOPS), appear to be experiencing varying degrees of population declines. Life history data (e.g. diet, reproductive status, age and growth, mortality) are critical for developing effective conservation strategies for TOPS. Presently, lethal sampling remains the most effective and accurate means of gathering these data. To meet such challenges, many scientists have utilized specimens obtained from recreational and commercial fisheries, but have needed to supplement those data with fishery-independent sampling. However, there is growing public and scientific debate as to whether lethal sampling of TOPS is justified for obtaining conservation data. Here we describe the development and use of non-lethal alternatives for collecting data on (1) trophodynamics; (2) maturity state and fecundity; and (3) growth and mortality rates necessary to enact conservation measures for threatened or even data-deficient TOPS. KEY WORDS: Conservation · Shark · Scientific sampling · Lethal sampling · Population status · LifeÊhistory · Fisheries Full text in pdf format PreviousNextCite this article as: Hammerschlag N, Sulikowski J (2011) Killing for conservation: the need for alternatives to lethal sampling of apex predatory sharks. Endang Species Res 14:135-140. https://doi.org/10.3354/esr00354 Export citation Mail this link - Contents Mailing Lists - RSS Facebook - Tweet - linkedIn Cited by Published in ESR Vol. 14, No. 2. Online publication date: June 22, 2011 Print ISSN: 1863-5407; Online ISSN: 1613-4796 Copyright © 2011 Inter-Research.

Evaluation of Calcein as a Mass Mark for Rainbow Trout Raised in Outdoor Hatchery Raceways

AbstractMass marks are useful for evaluating releases of hatchery fishes for management and research purposes. Calcein is a chemical that shows potential for producing a cost‐effective batch mark that can be easily applied to large numbers of small fish and can be detected externally by nonlethal means. However, calcein shows some drawbacks related to mark deterioration when exposed to direct sunlight. We evaluated calcein mark retention over time in rainbow trout Oncorhynchus mykiss by using both external (head, fins) and internal structures (otoliths), and we compared mark retention between fish reared in normal outdoor raceways under ambient light conditions and those reared indoors in shaded circular tanks. Calcein marks on rainbow trout that were marked as fry and reared in outdoor raceways deteriorated significantly within 8 d of marking and remained at low or undetectable levels throughout the study. Mark quality was much better for rainbow trout reared indoors but still degraded over the 7‐month evaluation period. Calcein marks were visible over the entire study period for otoliths of fish reared both indoors and outdoors, with better results being obtained for fish reared indoors. While calcein shows potential for generating a successful nonlethal batch mark, it may have limited use in situations where fish are exposed to sunlight, which can degrade the external mark. Marks in otoliths were retained well but required time‐consuming processing and lethal sampling. For use of otolith marks, we recommend combining calcein with some other external mark (e.g., adipose fin clip) to aid in identifying chemically marked individuals.

Comparison and Validation of Nonlethal Techniques for Estimating Condition in Juvenile Salmonids

AbstractFish proximate composition influences growth, reproduction, and survival and is commonly used to determine physiological condition; however, proximate composition is time‐consuming to measure and typically requires a lethal sample that cannot be analyzed in situ. As a result, researchers are increasingly interested in using nonlethal techniques that are quicker and less expensive to estimate proximate body components in fish. Bioelectrical impedance analysis (BIA) and microwave energy meters have been used with success on large‐bodied fishes, although no evaluation on small‐bodied Pacific salmon Oncorhynchus spp. has been conducted. We evaluated three nonlethal techniques (BIA, energy meter, and condition factor) as predictors of individual body composition (determined through lethal whole‐body proximate analysis) in juvenile hatchery steelhead O. mykiss (minimum fork length = 138 mm; minimum wet mass = 24.5 g). The BIA values were positively correlated with measured total body water (R2 = 0.77), protein (R2 = 0.74), and ash (R2 = 0.46) in steelhead; energy meter values were positively correlated with total body water (R2 = 0.83). Only condition factor was predictive of total body lipid, and both BIA and energy meter measures reflected somatic energy in muscle tissue rather than the total body energy reserves. Although modification of nonlethal methodologies is still required to generate accurate measures of total body energy reserves, BIA and energy meter technologies provide relatively quick and detailed data on a number of proximate body components without the need to kill the individual fish.

Characterization of the avian aryl hydrocarbon receptor 1 from blood using non-lethal sampling methods

The amino acid sequence of the aryl hydrocarbon receptor 1 ligand binding domain (AHR1 LBD) is an important determinant of sensitivity to dioxin-like compounds in avian species. We are interested in surveying AHR1 LBD sequences in a large number of birds as a means of identifying species that are particularly sensitive to dioxin-like compounds. Our original method for determining AHR1 LBD genotype used liver tissue and required lethal sampling. Here we present two alternate methods for determining AHR1 LBD genotype which use non-lethal sampling and are more appropriate for ecologically sensitive species. First, we establish that AHR1 LBD mRNA is expressed in avian blood and test a variety of blood collection and handling protocols in order to establish a method that is convenient for field collections. Our findings also identify which types of archival blood samples might be appropriate for AHR1 LBD sequence determination. Second, we present a method for obtaining AHR1 LBD coding sequences from DNA. A DNA-based method is advantageous because DNA can be isolated from many tissue types, is more stable than RNA, and requires less specific sample handling and preservation. This work extends applicability of a genetic screen for dioxin sensitivity to a larger number of species and sample types including endangered species and potentially museum specimens.

Non-lethal method to obtain stomach samples from a large marine predator and the use of DNA analysis to improve dietary information

Dietary information of apex predators is crucial to understanding community dynamics and ecosystem processes. However, as dietary studies traditionally involve lethal sampling, obtaining this essential information can have repercussions on predator populations and the structure and functioning of marine ecosystems. With stronger emphasis being placed on conservation of species that are vulnerable to overexploitation, the need for non-destructive methods of sampling is imperative, as is the requirement to maximize the information obtained from each sample. Stomach flushing (gastric lavage) and DNA analysis of stomach contents methods were tested on the broadnose sevengill shark Notorynchus cepedianus Peron 1807. Acoustic tracking and recaptures of sharks implied high survivorship post-fishing and stomach flushing. From 85 prey items collected, 36 (43%) could be identified to species level using morphological analysis. After DNA analysis, a further 35 items were identified to species level, doubling the information obtained from these stomachs. The number of N. cepedianus that were confirmed to have eaten gummy sharks Mustelus antarcticus Gunther 1870 also doubled after DNA analysis. Without DNA analysis (of stomach contents) the importance of M. antarcticus in the diets of N. cepedianus would have been substantially underestimated. In addition, the non-lethal approach provides an opportunity to obtain meaningful information from non-harvested, endangered or rare species or sampling of species within protected areas.