Non-lethal Sampling Research Articles

Validation of lipid extraction and correction methods for stable isotope analysis of freshwater food webs in southern Africa

Stable isotope analysis is ubiquitous as a method to investigate food-web dynamics at various scales in aquatic ecology. Most studies make use of dorsal muscle tissue, which involves lethal sampling of the fish. The sampling of muscle tissue is often followed by chemical lipid extraction pre-treatment before stable isotope analysis. In this study we tested whether stable light isotope results obtained from fin tissue were comparable to those from muscle, and we investigated whether lipid correction could be used as a substitute for lipid extraction. Various lipid correction equations were evaluated. Based on our results, we propose ethical and efficient methods of sample collection and preparation for stable isotope analysis of freshwater fish. We found that dorsal muscle and fin tissue samples could yield similar interpretations of freshwater food-web dynamics in South Africa, demonstrating that fin clippings might be more widely applied as a nonlethal sampling method for stable isotope studies. Existing lipid correction equations either over- or underestimated true lipid extracted δ13C values, therefore an amended lipid correction equation is proposed as it was successfully tested against a population of wild fish. The errors arising from existing lipid correction equations suggest that site-specific calibration should be employed.

Unintended Consequences? Lethal Specimen Collection Accelerates with Conservation Concern

Global declines of arthropods have garnered widespread attention because the loss of insect species threatens critical ecosystem services such as decomposition, pest control, and pollination (Sánchez-Bayo et al. 2019 and citations therein). Although the extent of the “insect apocalypse” is currently under debate (Didham et al. 2020), insect declines are well documented worldwide, with habitat loss, agricultural intensification, and climate change cited as primary drivers of losses in biomass and diversity (Raven and Wagner 2021). These losses have stimulated conservation action and research effort. However, lethal sampling continues as a common entomological practice, despite conservation concern and increasing research. Facing myriad extant pressures, vulnerable insect populations may be less resilient to traditional sampling norms than broadly assumed. We raise as an emerging concern the potentially damaging yet unknown impacts of contemporary specimen collection on wild insect populations. Reviewing the literature on temporal sampling trends in bumble bees as a case study, we highlight the value of non-lethal sampling alternatives and underscore the need for proactive, empirically informed sampling guidelines that reflect taxon-specific conservation needs.

Comparison of lethal and non-lethal age-based growth estimation methodologies to assess an endemic bay population of Atlantic cod (Gadus morhua)

The Gilbert Bay, Labrador, MPA (Marine Protected Area) was created in 2005 to protect a genomically distinct and locally adapted bay resident population of Atlantic cod (Gadus morhua) from commercial overfishing. Since then research showed that significant numbers of individuals migrate beyond MPA boundaries creating a concern that fishing continues to impact this endemic population. As part of research and MPA monitoring to assess the health of the protected cod population, lethal sampling of Gilbert Bay cod was conducted from 1998 to 2009 to gather length-at-age data from otoliths. Since then only non-lethal sampling methods were used so as to help conserve the protected population. This study was conducted to compare non-lethally (mark-recapture and length-frequency) obtained age and growth data with lethally (otolith-length-at-age) obtained data. The three methods produced similar results for the 1998 to 2009 period, and both of the non-lethal methods considered were consistent over the 20 year study, confirming their preference over lethal methods for this small endemic fish population.

Effect of body size, feeding ecology and maternal transfer on mercury accumulation of vulnerable silky shark Carcharhinus falciformis in the eastern tropical pacific

The silky shark Carcharhinus falciformis is a large pelagic species distributed in the global oceans and was recently listed as “Vulnerable” by the IUCN because of its decline in population due to overfishing. As an apex predator, the silky shark can accumulate elevated quantities of mercury (Hg), posing a potential risk to its remaining population. In this study, total Hg (THg) concentrations were determined in silky shark muscle, liver, dermis, red blood cells (RBC) and plasma sampled from the eastern tropical Pacific, and δ15N values were measured to explore the influence of feeding ecology on Hg accumulation. The highest THg concentrations were in muscle (7.81 ± 6.70 μg g−1 dry weight (dw) or 2.14 ± 1.83 μg g−1 wet weight (ww)) and liver (7.88 ± 10.22 μg g−1 dw or 4.66 ± 6.04 μg g−1 ww) rather than dermis, RBC and plasma. The THg concentrations in all tissue types were significantly correlated with fork length and showed faster accumulation rates after maturity. Maternal THg transfer was observed in silky sharks with embryos having 33.16% and 1.98% in muscle and liver compared with their respective mothers. The potentially harmful THg concentrations in silky shark tissues and embryos may lead to health problems of sharks and consumers. THg concentrations were negatively correlated with δ15N values for all tissues, indicating likely baseline variations in δ15N values that reflect changes in the foraging habitats or regions of silky sharks with size or age. Lastly, strong correlations were observed among THg concentrations of all tissue types, indicating that nonlethal sampling of muscle and dermis tissue can be used effectively to quantify THg concentration of other internal tissues.

Tissue Distribution of Mercury in the Bodies of Wild American Alligators (Alligator mississippiensis) from a Coastal Marsh in Louisiana (USA).

Total mercury (THg) concentrations were measured in wild alligators inhabiting a coastal marsh in southern Louisiana, to determine the tissue distribution of THg among various body organs and tissue compartments. Concentrations of THg in claws and dermal tail scutes were compared to those in blood, brain, gonad, heart, kidney, liver, and skeletal muscle to determine if the former tissues, commonly available by non-lethal sampling, could be used as measures of body burdens in various internal organs. Mercury was found in all body organs and tissue compartments. However, overall, THg concentrations measured in alligators were below the FDA action level for fish consumption and were comparable to previous data reported from southwestern Louisiana. Our results suggest consumption of meat from alligators found in this region may be of little public health concern. However, the extended period of time between sampling (in this study) and the present-day highlight the need for continuous, additional, and more recent sampling to ensure consumer safety. Total mercury concentrations were highest in the kidney (3.18 ± 0.69mg/kg dw) and liver (3.12 ± 0.76mg/kg dw). THg levels in non-lethal samples (blood, claws, and dermal tail scutes) were positively correlated with all tissue THg concentrations (blood: R2 = 0.513-0.988; claw: R2 = 0.347-0.637, scutes: R2 = 0.333-0.649). Because THg concentrations from blood, claws, and scutes were correlated with those of the internal organs, non-lethal sampling methods may be a viable method of estimating levels of THg in other body tissues.

Serum Protein Electrophoresis Reference Intervals for Six Species of Wild-Sampled Sharks in South Florida.

Serum proteins found in the blood have been used as diagnostic markers in both human and animal medicine. Evaluating proteins in the blood of sharks may be a valuable way to assess shark health. The purpose of this study was to perform serum protein electrophoresis from non-lethal blood samples of wild sharks and establish baseline values for six species off South Florida: blacknose (Carcharhinus acronotus), blacktip (C. limbatus), bull (C. leucas), lemon (Negaprion brevirostris), sandbar (C. plumbeus), and tiger shark (Galeocerdo cuvier). Two hundred and fifty-one serum samples were collected between 2015-2018 along with sex determination and morphometric measurements. Agarose gel analysis was performed using mammalian protein fraction delimits albumin, alpha 1-globulins, alpha 2-globulins, beta globulins, and gamma globulins alternatively defined as fractions 1-5. Reference electrophoretic profiles were established for each species, revealing the dominance of protein fractions 3 and 4. The fraction 3:4 ratio was determined for each shark and reference intervals for total solids, protein fractions, and fraction 3:4 ratio are reported for each species. There were no significant differences in total solids or protein fractions between males and females of any species. In both blacktip sharks and bull sharks, total solids were positively correlated with body size, but not in the other species evaluated. In blacknose sharks, the fraction 3:4 ratio was positively correlated with body condition. Results suggest that there may be differences in total solids values across dry and wet seasons for some species. Newly established reference intervals can now be utilized in future research to evaluate the health of captive sharks and to investigate the health status of individuals in the wild as well as relate health measures to environmental conditions.

Non-Lethal Sampling Supports Integrative Movement Research in Freshwater Fish.

Freshwater ecosystems and fishes are enormous resources for human uses and biodiversity worldwide. However, anthropogenic climate change and factors such as dams and environmental contaminants threaten these freshwater systems. One way that researchers can address conservation issues in freshwater fishes is via integrative non-lethal movement research. We review different methods for studying movement, such as with acoustic telemetry. Methods for connecting movement and physiology are then reviewed, by using non-lethal tissue biopsies to assay environmental contaminants, isotope composition, protein metabolism, and gene expression. Methods for connecting movement and genetics are reviewed as well, such as by using population genetics or quantitative genetics and genome-wide association studies. We present further considerations for collecting molecular data, the ethical foundations of non-lethal sampling, integrative approaches to research, and management decisions. Ultimately, we argue that non-lethal sampling is effective for conducting integrative, movement-oriented research in freshwater fishes. This research has the potential for addressing critical issues in freshwater systems in the future.

Tetrodotoxin levels in lab-reared Rough-Skinned Newts (Taricha granulosa) after 3 years and comparison to wild-caught juveniles

The origin and biogenesis of tetrodotoxin (TTX) is one of the most interesting and perplexing questions remaining for TTX researchers. Newts can possess extreme quantities of TTX and are one of the most well-studied of all TTX-bearing organisms, yet seemingly conflicting results between studies on closely related species continues to generate debate. In this study, eggs from 12 female newts (Taricha granulosa) were reared in captivity and the metamorphosed juveniles were fed a TTX-free diet for 3 years. Using a non-lethal sampling technique, we collected skin samples from each individual each year. Wild-caught juveniles from the same population were also sampled for TTX. In lab-reared juveniles, mass increased rapidly, and after only 2 years individuals approached adult body mass. TTX levels increased slowly during the first two years and then jumped considerably in year three when fed a diet free of TTX. However, wild-caught juvenile newts of unknown age were more toxic than their lab-reared counterparts. These results, coupled with additional data on the long-term production and synthesis of TTX in adult newts suggest that TTX is unlikely to come through dietary acquisition, but rather newts may be able to synthesize their own toxin or acquire it from symbiotic bacteria.

Non-Lethal Blood Sampling of Fish in the lab and Field With Methods for Dried Blood Plasma Spot Omic Analyses.

There is global acknowledgment that humane methods in animal research are a priority, but few environmental effects monitoring programs use nonlethal methods for fish. The goal of the present study was to determine the impacts of sampling small volumes of blood in larger-bodied fish on survival and healing. In addition to evaluating survival following blood sampling, we evaluated the utility of dried blood spots as an alternative for sample processing and storage in the field. In our approach, we housed 80 rainbow trout (Oncorhynchus mykiss) in our flow-through aquatic facility. We then anaesthetized using MS-222 and sampled 1 μl/g bw of blood via puncture of the caudal vasculature. We tested four different post-blood sampling treatments on the puncture wound: 1. application of liquid bandage; 2. a swab of betadine; 3. a swab of fish mucous; and 4. compared survival outcomes to a group where no post-treatment was performed (negative control). Overall, we observed 90% survival among all treatments, with the most effective approach being the negative control (100% survival). Based upon these results, we repeated the blood sampling with no-post treatment by housing 20 rainbow trout (not previously tested upon) in cages at a nearby creek and monitored survival for 2 weeks post sampling. The survival rate was 95% with full healing of the puncture site in all subjects. In addition to this, we tested the efficacy of dry blood spotting on proteomic, lipidomic and amino acid analysis as an alternative method for blood sample processing and storage. It was found that dried plasma spotting using parafilm in conjunction with a modified Bligh-Dyer extraction offered the best balance for good recovery of protein, lipid and amino acids relative to wet plasma and Noviplex dried plasma spot cards. In this article, we will present the detailed results of these combined studies and describe what we have determined to be the safest non-lethal blood sampling protocol.

Greater than the sum of your parts: Nonlethal stable isotope sampling methods in spiders

AbstractAs top consumers and generalist predators, spiders are ideal organisms to study food webs and complex ecological functions using stable isotopes. Most researchers use whole‐body samples to analyze stable isotope ratios in spiders. Spiders can regrow lost legs and produce multiple molts during a life cycle, and nonlethal sampling utilizing legs and molts may provide a useful alternative to whole‐body sampling especially in larger bodied or threatened species. Furthermore, removing spider abdomens and thus leftover prey in the gut contents may provide a more accurate isotopic value. We tested the hypothesis that the δ15N, δ13C, or δ2H isotopic values in spider legs are reliable proxies for spider prosomas, abdomens, or whole bodies. We used laboratory‐reared large‐bodied spiders (Pterinochilus murinus) and field‐collected Lycosidae to compare lethal and nonlethal tissue isotopic values. We found that nonlethal samples of spider legs and molts are acceptable alternatives to lethal whole‐body samples to determine δ13C and δ15N stable isotope signatures. Nonlethal samples are not suitable proxies for whole‐body samples to determine δ2H isotopic values. Using nonlethal leg or molts samples in stable isotope investigations of spiders will allow researchers to promote conservation efforts and study threatened species while ensuring accurate and repeatable results.

Season and species influence stable isotope ratios between lethally and non-lethally sampled tissues in freshwater fish.

The field of stable isotope ecology is moving away from lethal sampling (internal organs and muscle) towards non-lethal sampling (fins, scales and epidermal mucus). Lethally and non-lethally sampled tissues often differ in their stable isotope ratios due to differences in metabolic turnover rate and isotopic routing. If not accounted for when using non-lethal tissues, these differences may result in inaccurate estimates of resource use and trophic position derived from stable isotopes. To address this, the authors tested whether tissue type, season and their interaction influence the carbon and nitrogen stable isotope ratios of fishes and whether estimates of species trophic position and resource use are affected by tissue type, season and their interaction. This study developed linear conversion relationships between two fin types and dorsal muscle, accounting for seasonal variation. The authors focused on three common temperate freshwater fishes: northern pike Esox lucius, yellow perch Perca flavescens and lake whitefish Coregonus clupeaformis. They found that fins were enriched in 13 C and depleted in 15 N compared to muscle in all three species, but the effect of season and the interaction between tissue type and season were species and isotope dependent. The estimates of littoral resource use based on fin isotope ratios were between 13% and 36% greater than those based on muscle across species. Season affected this difference for some species, suggesting the potential importance of using season-specific conversions when working with non-lethal tissues. Fin and muscle stable isotopes produced similar estimates of trophic position for northern pike and yellow perch, but fin-based estimates were 0.2-0.4 trophic positions higher than muscle-based estimates for lake whitefish. The effect of season was negligible for estimates of trophic position in all species. Strong correlations existed between fin and muscle δ13 C and δ15 N values for all three species; thus, linear conversion relationships were developed. The results of this study support the use of non-lethal sampling in stable isotope studies of fishes. The authors suggest that researchers use tissue conversion relationships and account for seasonal variation in these relationships when differences between non-lethal tissues and muscle, and seasonal effects on those differences, are large relative to the scale of isotope values under investigation and/or the trophic discrimination factors under use.

Environmental DNA as Novel Technology: Lessons in Agenda Setting and Framing in News Media.

Simple SummaryIncreasing threats to wildlife have made assessing their populations, health, and adaptation to stressors ever more important. The use of environmental DNA to make these assessments is relatively new and offers many advantages, such as non-lethal sampling. How novel technologies such as these are framed in the news media is critically important because the general public gathers much of its information about scientific developments from the media, and public perceptions can impact use of technology, responses to data derived from its use, and ethical concerns. To date, media constructions of eDNA and perceptions among the general public have not been examined. The current paper begins to address this gap by undertaking an examination of media coverage of eDNA in Canada and the United States for the past approximately twenty years—likely a critical period in shaping understandings of and responses to eDNA. The findings indicate that eDNA is framed as a powerful tool, yet the social concerns that receive the most attention are those where there are financial interests at play, and these interests have to date eclipsed attending to relevant ethical considerations.Environmental DNA (eDNA) is an emerging technology used for understanding ecosystems, environmental change, and stressors. Cellular and extracellular DNA are collected from environmental samples instead of individual wildlife animals, and as such eDNA comes with associated logistical and ethical benefits. It is increasingly being used, yet to date public knowledge and perceptions of eDNA have not been explored. Given that most of the public gathers scientific information from news media sources, this is a logical first place to start. This paper reports on a framing and agenda-setting analysis of news media coverage of eDNA in Canada and the United States from 2000 to 2020. The findings indicate that eDNA is being framed as an emerging and powerful tool, although questions regarding its validity and reliability are raised vis-à-vis identifying the presence of invasive species. Less than half of the news articles analyzed address broader social or ethical issues in relation to eDNA, and the majority focus on the potential financial impacts of eDNA findings on development projects and business interests. The potential ethical advantages of non-lethal sampling methods used via eDNA sampling are not addressed, nor are the potential ethical issues raised by its potential use in bioprospecting, indicating that the current state of agenda setting regarding eDNA in these newspapers is focused on economic impacts, to the exclusion of potential ethical issues. This unfolding news coverage will likely be key to understanding public perceptions of this novel technology.

Fish environmental DNA in Tokyo Bay: A feasibility study on the availability of environmental DNA for fisheries

Environment DNA (eDNA) is a nonlethal sampling method that has potential for evaluating fisheries resources. To evaluate whether eDNA information is useful for fisheries, we collected and analysed eDNA samples from 14 sites in Tokyo Bay from April 2018 to March 2020. Using MiFish universal primers, fish mitochondrial 12S rRNAs were amplified from the eDNA by polymerase chain reaction (PCR) and then sequencing. A total of 535 eDNA samples yielded 44.5 million reads, with 197 of teleosts and 16 of elasmobranchs identified. Of those, 42 identified species were commercially important, with the most abundant being Engraulis japonicus (Japanese anchovy), followed by Konosirus punctatus (dotted gizzard shad) and Lateolabrax japonicusi (Japanese seabass). Mitochondrial cytochrome b copy numbers of K. punctatus and L. japonicus showed a seasonal trend, although catch per unit effort for L. japonicus was not correlated with copy number. Although eDNA is a powerful tool that can detect the presence of fish species from a water sample, caution should be used when interpreting fish biomass based on eDNA dynamics until the method has been better studied.

Body Remains Left by Bird Predators as a Reliable Source for Population Genetic Studies in the Great Capricorn Beetle Cerambyx cerdo, a Veteran Oak Specialist.

Simple SummaryMolecular genetic techniques can support species conservation by providing information about processes critical to population survival. Unfortunately, obtaining biological material for DNA extraction is often associated with an adverse impact on the animals under study. In legally protected or threatened species, non-invasive sampling (i.e., sampling without injuring or disturbing an animal) is preferred as it carries no risk to the population’s survival. Here, we tested the possibility of using body remains left by bird predators for microsatellite genotyping in Cerambyx cerdo, a veteran oak specialist. We compared results obtained from such remains samples with samples of fresh beetle tarsi (i.e., invasive and destructive but non-lethal samples). We found that the sample type had no significant effect on PCR amplification efficiency; instead, it was strongly affected by allele length and individual heterozygosity. Allele frequencies were perfectly correlated for both sample types (R2 = 0.94). Although point estimates of individual inbreeding were higher in remains than fresh samples (medians 0.08 vs. 0.02, respectively), both groups were not significantly different from each other and zero. Our study demonstrated that non-invasive remains samples could provide satisfactory data for population–genetic studies. However, we highlight the problem of potentially biased inbreeding estimates, which may result from allelic dropout.Obtaining biological material for DNA extraction is often challenging in organisms of conservation interest. Non-invasive sampling (i.e., sampling without injuring or disturbing an animal) is preferred as it carries no risk to the population’s survival. Here, we tested the possibility of using the body remains left by bird predators for microsatellite genotyping in Cerambyx cerdo, a veteran oak specialist. We compared results obtained from such potentially degraded samples with samples of fresh beetle tarsi (i.e., invasive and destructive but non-lethal samples). Using 10 SSR loci, we genotyped 28 fresh, and 28 remains samples. The analysis indicated that PCR amplification efficiency was not influenced by sample type but allele length and individual heterozygosity. Allele frequencies were perfectly correlated for both sample types (R2 = 0.94). Additionally, null allele frequencies and genotyping failure rates were not significantly different from zero. Although the point estimates of individual inbreeding rates (fi) were higher in remains than fresh samples (medians 0.08 vs. 0.02, respectively), both groups were not significantly different from each other and zero. Our study demonstrated that non-invasive remains samples could provide satisfactory data for population–genetic studies. However, we highlight the problem of biased inbreeding estimates, which may result from samples affected by allelic dropout.

The use of non-lethal sampling for transcriptomics to assess the physiological status of wild fishes.

Fishes respond to different abiotic and biotic stressors through changes in gene expression as a part of an integrated physiological response. Transcriptomics approaches have been used to quantify gene expression patterns as a reductionist approach to understand responses to environmental stressors in animal physiology and have become more commonly used to study wild fishes. We argue that non-lethal sampling for transcriptomics should become the norm for assessing the physiological status of wild fishes, especially when there are conservation implications. Processes at the level of the transcriptome provide a "snapshot" of the cellular conditions at a given time; however, by using a non-lethal sampling protocol, researchers can connect the transcriptome profile with fitness-relevant ecological endpoints such as reproduction, movement patterns and survival. Furthermore, telemetry is a widely used approach in fisheries to understand movement patterns in the wild, and when combined with transcriptional profiling, provides arguably the most powerful use of non-lethal sampling for transcriptomics in wild fishes. In this review, we discuss the different tissues that can be successfully incorporated into non-lethal sampling strategies, which is particularly useful in the context of the emerging field of conservation transcriptomics. We briefly describe different methods for transcriptional profiling in fishes from high-throughput qPCR to whole transcriptome approaches. Further, we discuss strategies and the limitations of using transcriptomics for non-lethally studying fishes. Lastly, as 'omics' technology continues to advance, transcriptomics paired with different omics approaches to study wild fishes will provide insight into the factors that regulate phenotypic variation and the physiological responses to changing environmental conditions in the future.

The presence of kelp Lessonia trabeculata drives isotopic niche segregation of redspotted catshark Schroederichthys chilensis

Kelp forests are associated with high biodiversity as they provide habitat and food source for many species. Redspotted catshark Schroederichthys chilensis is a coastal shark species associated with kelp forests of southern South America dominated by Lessonia trabeculata. Despite its common occurrence redspotted catshark the knowledge on its trophic ecology is rudimentary. This study aimed to assess the effects of presence/absence of kelp L. trabeculata on the isotopic niche of redspotted catshark through comparison of δ13C and δ15N in its blood and fin tissues (non-lethal samples). We found that males were characterized by higher trophic position and enriched carbon sources compared to females in locations where kelp was present. In contrast, males and females were characterized by similar isotopic niche in absence of kelp. These differences are most probably an effect of higher prey diversity associated with microhabitats generated by kelp forests that allow sexual segregation of isotopic niche. Therefore, extensive kelp harvest may cause significant effects in isotopic niche and nutritional status of adult redspotted catshark and may negatively affect its populations.

Variation of Total Mercury Concentrations in Different Tissues of Three Neotropical Caimans: Implications for Minimally Invasive Biomonitoring.

Mercury (Hg) is a global environmental contaminant that affects ecosystems. It is known to biomagnify through food webs and to bioaccumulate especially in the tissues of top predators. Large-scale comparisons between taxa and geographic areas are needed to reveal critical trends related to Hg contamination and its deleterious effects on wildlife. Yet, the large variety of tissues (keratinized tissues, internal organs, blood) as well as the variability in the units used to express Hg concentrations (either in wet- or dry-tissue weight) limits straightforward comparisons between studies. In the present study, we assessed the moisture content that could influence the total Hg (THg) concentrations measured in several tissues (claws, scutes, total blood, and red blood cells) of three caiman species. We evaluated the moisture content from the different tissues to provide information on THg concentrations in various matrices. Our results show a difference of THg concentrations between the tissues and intra- and interspecific variations of moisture content, with the highest THg values found in keratinized tissues (scute keratinized layers and claws). For the three species, we found positive relationships between body size and THg concentration in keratinized tissues. In the blood, the relationship between body size and THg concentration was species-dependent. Our results emphasize the need for a standardized evaluation of THg concentration and trace elements quantification based on dry weight analytical procedures. In addition, the use of both blood and keratinized tissues offers the possibility to quantify different time scales of THg exposure by non-lethal sampling.

Evidence for the Use of Mucus Swabs to Detect Renibacterium salmoninarum in Brook Trout.

Efforts to advance fish health diagnostics have been highlighted in many studies to improve the detection of pathogens in aquaculture facilities and wild fish populations. Typically, the detection of a pathogen has required sacrificing fish; however, many hatcheries have valuable and sometimes irreplaceable broodstocks, and lethal sampling is undesirable. Therefore, the development of non-lethal detection methods is a high priority. The goal of our study was to compare non-lethal sampling methods with standardized lethal kidney tissue sampling that is used to detect Renibacterium salmoninarum infections in salmonids. We collected anal, buccal, and mucus swabs (non-lethal qPCR) and kidney tissue samples (lethal DFAT) from 72 adult brook trout (Salvelinus fontinalis) reared at the Colorado Parks and Wildlife Pitkin Brood Unit and tested each sample to assess R. salmoninarum infections. Standard kidney tissue detected R. salmoninarum 1.59 times more often than mucus swabs, compared to 10.43 and 13.16 times more often than buccal or anal swabs, respectively, indicating mucus swabs were the most effective and may be a useful non-lethal method. Our study highlights the potential of non-lethal mucus swabs to sample for R. salmoninarum and suggests future studies are needed to refine this technique for use in aquaculture facilities and wild populations of inland salmonids.

Genetic diversity and population structure of orchid bees from the Brazilian savanna

Based on microsatellite markers and non-lethal sampling of orchid bee males, we assessed the genetic diversity and population structure of three species, Euglossa pleosticta, Euglossa imperialis, and Eulaema nigrita, collected from forest remnants in the Brazilian savanna biome. Among these species, El. nigrita has being acknowledged as more tolerant to savanic areas, since it is more frequently found in these habitats than the other two species. Thus, we hypothesized that species more associated with forest areas would show lower levels of genetic diversity and higher levels of population structure, since open areas could represent a barrier to the movement of these species. We observed lower levels of genetic diversity (HE ) in the species highly associated with forest areas (Eg. pleosticta, HE = 0.66; Eg. imperialis, HE = 0.44) compared to El. nigrita (HE = 0.81). Concerning the genetic structure, it was only remarkable among samples of Eg. pleosticta, a species strongly associated with the forest remnants. Despite the documented dispersal capacity of euglossine bees, maintenance of the connections among forest areas in this biome seems to be critical for their conservation in the Brazilian savanna.

Disease ecology of wild fish: opportunities and challenges for linking infection metrics with behaviour, condition, and survival

Surmounting evidence supports that infectious agents play a critical role in shaping fish physiology, behaviour, and survival. The exclusion of disease-causing agents from fisheries research has resulted in major knowledge gaps that may limit the predictive capacity of ecological models. A major barrier in wild fisheries epidemiology is the logistical constraints associated with observing disease and obtaining samples from free-ranging fish, restricting the vast majority of research to laboratory studies or aquaculture facilities. For fisheries ecologists, including infectious agents can provide greater insight into observed phenomena, particularly with respect to fish physiology (e.g., metabolism), movement (e.g., migration rates), behaviour (e.g., habitat selection), personality (e.g., bold versus shy), and survival. Here we provide a brief introduction to the current understanding of disease ecology in wild fish and describe technological advances in both epidemiology and fisheries and aquatic sciences that can be used in tandem to create comprehensive studies of disease ecology in wild fishes. Combining nonlethal sampling and molecular genetic-based identification methods with field studies creates vast opportunities for innovative study designs that have the potential to address the true complexity of aquatic ecosystems.