Non-lethal Methods Research Articles

Electron Spin Resonance (ESR) for the study of Reactive Oxygen Species (ROS) on the isolated frog skin (Pelophylax bergeri): A non-invasive method for environmental monitoring

BackgroundReactive oxygen species (ROS) in biological tissues of elected biosentinels represent an optimal biomarker for eco-monitoring of polluted areas. Electron spin resonance (ESR) is the most definitive method for detecting, quantifying and possibly identifying radicals in complex systems. ObjectiveA non-invasive method for monitoring polluted areas by the quantitative determination of ROS in frog skin biopsy is presented. MethodsWe assessed by ESR spectroscopy the ROS level in adult male of Pelophylax bergeri, specie not a risk of extinction, collected from the polluted Sarno River (SA, Italy) basin. The spin-trap ESR method was validated by immunohistochemical analysis of the well-assessed pollution biomarkers cytochrome P450 aromatase 1A (CYP1A) and glutathione S-transferase (GST), and by determining the poly(ADPribose) polymerase (PARP) and GST enzymatic activity. ResultsROS concentration in skin samples from frogs collected in the polluted area is significantly higher than that determined for the unpolluted reference area. Immunohistochemical analysis of CYP1A and GST supported the reliability of our approach, even in the absence of evident morphological and ultrastructural differences. PARP activity assay, connected to possible oxidative DNA damage, and the detoxification index by GST enzymatic assay give statistically significant evidence that higher levels of ROS are associated to alterations of the different biomarkers. ConclusionsROS concentration, measured by ESR on isolated frog skin, through the presented non-lethal method, is a reliable biomarker for toxicity screening and represents a useful basic datum for future modelling studies on environmental monitoring and biodiversity loss prevention.

Antimicrobial and Antibiofilm Activity of Unionid Mussels from the North of Portugal

The populations of freshwater mussels belonging to the family Unionidae have been facing drastic changes in terms of diversity and numbers caused by constant aggressions on their natural habitat. Nevertheless, bivalves are capable of developing strategies of defense to overcome potential aggressors. The present work aimed to assess the potential antibacterial capacity of different species of unionid mussels from the north of Portugal. For this purpose, circulating cells (hemocytes), fluids, and mucus were obtained by nonlethal methods from the species Anodonta anatina (Linnaeus), Anodonta cygnea (Linnaeus), Potomida littoralis (Cuvier), and Unio delphinus (Spengler), and tested against bacterial reference strains and multidrug-resistant isolates. The cellular fraction of A. anatina, A. cygnea, and P. littoralis showed antibacterial activity, detected by the agar disc diffusion method, against Bacillus subtilis ATCC 6683, Pseudomonas aeruginosa ATCC 27853 and Acinetobacter baumannii ATCC 19606. Circulating cells from P. littoralis and A. anatina also inhibited Listeria monocytogenes ATCC 19111 and A. cygnea has also inhibited a multidrug-resistant isolate of Pseudomonas putida. The plasma of all mentioned freshwater mussels, used directly or diluted, showed great ability to hamper or inhibit the biofilm formation of Staphylococcus aureus ATCC 25923, P. aeruginosa ATCC 27853, and Escherichia coli ATCC 25922. Anodonta cygnea hampered the biofilm formation by Enterococcus faecalis ATCC 29212 as well. Overall, these results showed that not only cells play a relevant role in the immune system of these species but also the plasma, which likely contains antibiofilm substances. Anodonta cygnea stood out by presenting the best antibacterial inhibition potential.

Use of a nursery area by cownose rays (Rhinopteridae) in southeastern Brazil

ABSTRACT Using non-lethal methods for data collection of individuals and participatory monitoring by fishermen, we provide the first empirical evidence of the use of a nursery area by neonate and young-of-the-year cownose rays Rhinoptera bonasus and R. brasiliensis in southeastern Brazil. Two methods were used to collect data: (1) information provided by fishermen (reports, pictures, and videos) and (2) field sampling by researchers. A total of 746 cownose rays were captured; 113 have been identified as R. bonasus, 15 as R. brasiliensis, and 618 were reported by fishermen and could not be identified to the species. Records of newborns were made only in late spring and summer in 2015, 2016, and 2017, which suggests an annual reproductive cycle, with birth in late spring, extending to summer. A repeated use of this area by R. bonasus suggests that it is potentially important to the reproduction of this species. However, R. brasiliensis requires more studies. Small increases in mortality, resulting from increased fishing or other anthropogenic stressors, can have a disproportionately large effect on population viability. Thus, management of areas used during critical stages of the life cycle of rays is crucial to their conservation.

Conflict and consensus in stakeholder views of seal management on Nantucket Island, MA, USA

With recovery of seal populations after their near extirpation from the coastal waters of Massachusetts, USA, controversy has emerged over seal-fishery interactions. To assess stakeholder attitudes toward management of seal-fishery conflicts and marine mammal protection, questionnaires were administered to the general public, on-site anglers, and tourists on Nantucket Island. All three groups agreed that the interests of the ecosystem should be the top management consideration and supported marine mammal protection. Opposition to lethal management was found among all groups, with tourists most opposed, followed by the public and on-site anglers. While non-lethal management received more support than lethal management, some support for leaving seals alone was found, particularly among tourists. No differences were found among stakeholder groups for non-lethal methods of seal management, with the exception of the scenario of using non-lethal methods to reduce population levels. These findings suggest that management of seal-fishery conflicts must respect ethical and ecological concerns to promote co-existence.

Exploring wildlife predation in the Limpopo Province of South Africa: A double‐hurdle approach

AbstractPredation is a well‐known problem in South Africa with large losses in the small and large livestock sectors. Predation in the wildlife ranching industry has also become more of a concern, as the financial losses due to predation on valuable antelope species are large. Predation data for small, large, and scarce/colour‐variant antelope species were collected using a structured questionnaire from wildlife ranchers in the Limpopo province, South Africa. We explore the factors that influence predation on these species by determining whether the perceptions of predation and consequent managerial decisions affect predation. The use of nonlethal control methods can be successfully employed to reduce the probability of predation occurrences, however, a combination of lethal and nonlethal control methods were used to reduce the level of predation. The type of antelope species will determine the type of predation control method to be employed. Therefore, the antelope species should be taken into account when making predation management decisions.

Efficacy of Otoliths and First Dorsal Spines for Preliminary Age and Growth Determination in Atlantic Tripletails

AbstractThe Atlantic Tripletail Lobotes surinamensis is a popular sport fish for which age and growth data are scarce in general and nonexistent for Georgia (GA), USA, waters. These data are necessary to ensure that management regulations are adequate to protect this species, especially given its popularity as a sport fish. We evaluated whether otoliths and spines were suitable for determining the estimated age (hereafter, “age”) and growth rates of Atlantic Tripletails, and we ascertained whether one method was more accurate than the other. Atlantic Tripletails were sampled by angling and trawling during March 30–August 10, 2009, and March 14–August 6, 2010, in nearshore GA waters of the Atlantic Ocean. During the study, 243 Atlantic Tripletails were captured and sampled for aging structures. Sagittal otoliths and the first dorsal spine were removed from each fish and used to estimate the age and growth rate. Mean differences in TL at age for spine and otolith data were evaluated with ANOVA. Estimated ages for males and females ranged from 1 to 5 years based on otoliths and spines. Both otolith and spine mean TLs at ages 1 and 2 were significantly different from each other as well as all other age‐classes, whereas mean TLs for ages 3–5 were not significantly different. Differences in Atlantic Tripletail TL among the otolith‐ and spine‐derived age‐classes were not significant. Each method used to age Atlantic Tripletails had advantages and disadvantages. Otoliths had higher initial reader agreement than spines, although agreement between the structures was 84.1%. However, otoliths require sacrifice of the fish, whereas a spine can be taken without sacrificing the fish. The lack of concrete life history data and population estimates suggests that when feasible, nonlethal aging methods would be preferred over lethal methods to ensure the survival of Atlantic Tripletail populations.

Killing wolves to prevent predation on livestock may protect one farm but harm neighbors.

Large carnivores, such as gray wolves, Canis lupus, are difficult to protect in mixed-use landscapes because some people perceive them as dangerous and because they sometimes threaten human property and safety. Governments may respond by killing carnivores in an effort to prevent repeated conflicts or threats, although the functional effectiveness of lethal methods has long been questioned. We evaluated two methods of government intervention following independent events of verified wolf predation on domestic animals (depredation) in the Upper Peninsula of Michigan, USA between 1998–2014, at three spatial scales. We evaluated two intervention methods using log-rank tests and conditional Cox recurrent event, gap time models based on retrospective analyses of the following quasi-experimental treatments: (1) selective killing of wolves by trapping near sites of verified depredation, and (2) advice to owners and haphazard use of non-lethal methods without wolf-killing. The government did not randomly assign treatments and used a pseudo-control (no removal of wolves was not a true control), but the federal permission to intervene lethally was granted and rescinded independent of events on the ground. Hazard ratios suggest lethal intervention was associated with an insignificant 27% lower risk of recurrence of events at trapping sites, but offset by an insignificant 22% increase in risk of recurrence at sites up to 5.42 km distant in the same year, compared to the non-lethal treatment. Our results do not support the hypothesis that Michigan’s use of lethal intervention after wolf depredations was effective for reducing the future risk of recurrence in the vicinities of trapping sites. Examining only the sites of intervention is incomplete because neighbors near trapping sites may suffer the recurrence of depredations. We propose two new hypotheses for perceived effectiveness of lethal methods: (a) killing predators may be perceived as effective because of the benefits to a small minority of farmers, and (b) if neighbors experience side-effects of lethal intervention such as displaced depredations, they may perceive the problem growing and then demand more lethal intervention rather than detecting problems spreading from the first trapping site. Ethical wildlife management guided by the “best scientific and commercial data available” would suggest suspending the standard method of trapping wolves in favor of non-lethal methods (livestock guarding dogs or fladry) that have been proven effective in preventing livestock losses in Michigan and elsewhere.

Why Do People Feed Free-roaming Cats? The Role of Anticipated Regret in an Extended Theory of Planned Behavior in Malaysia

ABSTRACTFree-roaming cats are common in residential and public areas in Malaysia and approach people for food. However, the psychological determinants of public feeding are unknown. This study investigated public perceptions of feeding free-roaming cats, based on an extended theory of planned behavior (TPB). It consisted of qualitative belief-elicitation interviews with 25 participants, followed by a quantitative survey of 167 participants, representative of the country’s population. The majority (87.2%) of the sample had fed free-roaming cats. The mean intention score (4.88 out of 7) indicated the public was likely, and would make an effort, to feed free-roaming cats in the future. The public’s benevolence toward animals largely explained the findings, based on generally positive attitudes and perceptions of moderate social credence and capability and confidence, underpinned by affective and cognitive beliefs. An important finding was the role of anticipated regret in predicting and explaining intentions, which contributed variance over and above that explained by the TPB constructs. The extended framework is explained by the influence of anticipated regret on the perceived evaluation of potential TPB outcomes, which in turn leads to the behavior becoming less volitional. Therefore, future TPB studies of people’s interactions with animals, such as free-roaming cats, should take account of affective and emotional antecedents of behavior, such as anticipated regret, to improve explanatory power. The study also has implications for managing public feeding of free-roaming cats, such as drawing on and strengthening the Malaysian public’s positive attitudes and emotional concern to redirect current feeding practices toward more constructive animal welfare initiatives. Such humane approaches align with the public’s sensitivity toward animal welfare and the historical development of cat population control from lethal methods to humane non-lethal methods to ensure adequate care.

Comparing biomarker responses during thermal acclimation: a lethal vs non-lethal approach in a tropical reef clownfish

Abstract Knowledge of thermal stress biology for most tropical fish species in reef ecosystems under climate change is still quite limited. Thus, the objective of this study was to measure the time-course changes of thermal stress biomarkers in the commercially exploited coral reef fish Amphiprion ocellaris , during a laboratory simulated event of increased temperature. Heat shock protein 70 kDa (Hsp70) and total ubiquitin (Ub) were determined in the muscle (lethal method) and in the fin (non-lethal alternative method) under two temperature treatments (control – 26 °C and elevated temperature – 30 °C) throughout one month with weekly samplings. Results suggest that biomarker basal levels are tissue-specific and influence the degree of response under temperature exposure. Responses were highly inducible in the muscle but not in fin tissue, indicating that the latter is not reliable for monitoring purposes. Thermal stress was observed in the muscle after one week of exposure (both biomarkers increased significantly) and Ub levels then decreased, suggesting the animals were able to acclimate by maintaining high levels of Hsp70 and through an effective protein turnover. In addition, the results show that mortality rates did not differ between treatments. This indicates that A. ocellaris is capable of displaying a plastic response to elevated temperature by adjusting the protein quality control system to protect cell functions, without decreasing survival. Thus, this coral reef fish species presents a significant acclimation potential under ocean warming scenarios of + 4 °C. Monitoring of thermal stress through a non-lethal method, fin-clipping, although desirable proved to be inadequate for this species.

Sunshine, Beaches, and Birds: Managing Raptor-Aircraft Collisions at Airports in Southern California

Wildlife-aircraft collisions (wildlife strikes) pose a serious safety risk to aircraft. Raptors (i.e., hawks and owls) are one of the most frequently struck guilds of birds within North America. Integrated wildlife damage management programs combine a variety of non-lethal and lethal management tools to reduce presence of raptors on airports. Live-capture and translocation away from an airport is a commonly used non-lethal method to reduce the risk of raptor-aircraft collisions. In southern California, USDA Wildlife Services airport biologists live-captured, marked with auxiliary markers (i.e., airport program-specific plastic leg band), and translocated approximately 1,232 raptors from seven airports and military bases located within the highly urbanized environment of the Los Angeles Basin during January of 2010-December of 2016. Ten different raptor species were marked and relocated during this effort. The composition of translocated raptors was red-tailed hawks (38.9%), Cooper’s hawks (27.5%), American kestrels (20.7%), barn owls (7.4%), and great horned owls (3.7%). Overall, the percentage of translocated raptors that returned to an airport was 11.1%. Although research is needed to better understand and increase the efficacy of such management efforts, this non-lethal method of reducing the presence of individual raptors at airports in southern California will be an important component of future wildlife management programs.

Could current fertility control methods be effective for landscape-scale management of populations of wild horses (Equus caballus) in Australia?

Context Fertility control is seen as an attractive alternative to lethal methods for control of population size and genetic diversity in managed animal populations. Immunocontraceptive vaccines have emerged as the most promising agents for inducing long-term infertility in individual animals. However, after over 20 years of scientific testing of immunocontraceptive vaccines in the horse, the scientific consensus is that their application as a sole management approach for reducing population size is not an effective strategy. Aims The purpose of this review is to evaluate currently available non-lethal fertility-control methods that have been tested for their contraceptive efficacy in Equidae, and to assess their suitability for effective management of wild (feral) horses in an Australian setting. Key results (1) Fertility-control agents, particularly injectable immunocontraceptive vaccines based on porcine zona pellucida (PZP) or gonadotrophin-releasing hormone (GnRH), can induce multi-year infertility (up to 3 years) in the horse. Some formulations require annual or biennial booster treatments. Remote dart delivery (on foot) to horses is possible, although the efficacy of this approach when applied to large numbers of animals is yet to be determined. (2) The proportion of females that must be treated with a fertility-control agent, as well as the frequency of treatment required to achieve defined management outcomes (i.e. halting population growth in the short term and reducing population size in the long term) is likely to be >50% per annum. In national parks, treatment of a large number of wild horses over such a broad area would be challenging and impractical. (3) Fertility control for wild horses could be beneficial, but only if employed in conjunction with other broad-scale population-control practices to achieve population reduction and to minimise environmental impacts. Conclusions In Australia, most populations of wild horses are large, dispersed over varied and difficult-to-access terrain, are timid to approach and open to immigration and introductions. These factors make accessing and effectively managing animals logistically difficult. If application of fertility control could be achieved in more than 50% of the females, it could be used to slow the rate of increase in a population to zero (2–5 years), but it will take more than 10–20 years before population size will begin to decline without further intervention. Thus, use of fertility control as the sole technique for halting population growth is not feasible in Australia.

Elevated exposure, uptake and accumulation of polycyclic aromatic hydrocarbons by nestling tree swallows (Tachycineta bicolor) through multiple exposure routes in active mining-related areas of the Athabasca oil sands region

In the Athabasca Oil Sands (OS) Region, the exposure (by air, water, diet), uptake and deposition of polycyclic aromatic compounds (PACs), including parent and alkylated hydrocarbons (PAHs) and dibenzothiophenes (DBTs), was assessed in nestling tree swallows (Tachycineta bicolor) at mining-related (OS1, OS2) and reference (REF) sites. The OS sites did not receive oil-sands processed waters (OSPW) and were ≥60km from the reference sites. Most of the 42 PACs (≤98%) were detected in all matrices. Swallows at the OS sites were exposed to higher air and water concentrations of individual PAC congeners, ΣPACs, Σparent-PAHs, Σalkyl-PAHs and ΣDBTs. Compared to reference nestlings (ΣPACs: 13–27ng/g wet weight (ww)), PACs were significantly higher in OS nestlings (31–106ng/gww) that also accumulated higher concentrations of major PAHs (i.e., naphthalene, C1-naphthalene, C2-naphthalene, C1-fluorenes, C2-fluorenes, C1-phenanthrenes) measured in 60% of nestlings. Uptake and deposition of PAHs in the birds' muscle was related to diet (δ15N: C1-naphthalenes, C2-naphthalenes, C1-fluorenes), water (C1-phenanthrenes), and air through inhalation and feather preening (C1-fluorenes), but fecal concentrations were not well explained by diet or environmental concentrations. While PAH concentrations were much higher in muscle than feces, they were highly correlated (p≤0.001 for all). Thus feces may represent a non-lethal method for characterizing PAH exposure of birds, with muscle characterizing accumulation and sources of PAH exposure. Tree swallows in the Athabasca OS Region are exposed to many PACs, accumulating higher concentrations when developing in close proximity to mining activity through diet, aerial deposition and mining-impacted freshwater sources (e.g., wetlands).

Identification of distinct haemocyte populations from the freshwater bivalves swan mussel (Anodontacygnea) and duck mussel (Anodontaanatina) using wheat-germ agglutinin

Haemocytes play a major role in molluscs immunity. Functional studies are, however, impaired by limited available experimental tools to identify and sort distinct haemocyte populations. Therefore, using nonlethal methods, we aimed at evaluating whether lectin staining combined with flow cytometry could be used to distinguish circulating haemocyte populations from two freshwater bivalves of the family Unionidae, the duck mussel (Anodonta anatina (L., 1758)) and the swan mussel (Anodonta cygnea (L., 1758)). Based on classical classification, haemocytes were distinguished as granulocytes and hyalinocytes and cytological features were visualized using transmission microscopy and staining techniques. Size, granularity, viability, and surface staining using lectins as specific probes were analysed by flow cytometry and fluorescence microscopy. The microscopic proportions of granulocytes and hyalinocytes significantly differed, being of 70% and 30% for A. cygnea and of 85% and 15% for A. anatina, respectively. Two haemocyte populations were sorted by flow cytometry based on size and granularity and confirmed as granulocytes and hyalinocytes. Interestingly, two different granulocyte populations could be further discriminated in A. cygnea according to their binding affinity to wheat-germ agglutinin (WGA), whereas granulocytes of A. anatina all stained similarly. Our results show that WGA labelling combined with flow cytometry can be used to better discriminate Anodonta haemocyte populations and obtain purified populations for functional studies.

Development of stereo camera methodologies to improve pelagic fish biomass estimates and inform ecosystem management in marine waters

Abstract To understand and manage marine ecosystems, long-term monitoring of fish biomass is needed. A challenge in estimating fish biomass using sampling nets is varying catchability with habitat, weather, or vessel traffic conditions. Underwater stereo cameras have shown promise in providing a non-lethal, efficient, and cost-effective method to observe and measure fish in areas that cannot be sampled otherwise. These methods, however, have yet to be demonstrated for mid-water pelagic or semi-pelagic fishes. We designed, built, and tested a stereo camera for its potential to augment survey assessments of pelagic fish biomass in areas where trawl net samples cannot be collected. In a pilot test, the stereo camera was used to identify fish species and to measure fish length, depth, tilt, and yaw. Five paired stereo camera deployments and pelagic midwater trawl hauls were compared during an acoustic survey in the Strait of Georgia, British Columbia. Fish sometimes moved in response to the stereo camera deployment, but acclimated quickly to its presence at depth. Pacific hake, Merluccius productus, and walleye pollock, Gadus chalcogrammus, were the two dominant species in both midwater trawl haul catches and stereo camera images, but fish sizes were significantly larger in most cases in stereo camera images compared to trawl haul catches. Fish length measurements were most accurate when yaw angles were

Natural Products as Platforms To Overcome Antibiotic Resistance.

Natural products have served as powerful therapeutics against pathogenic bacteria since the golden age of antibiotics of the mid-20th century. However, the increasing frequency of antibiotic-resistant infections clearly demonstrates that new antibiotics are critical for modern medicine. Because combinatorial approaches have not yielded effective drugs, we propose that the development of new antibiotics around proven natural scaffolds is the best short-term solution to the rising crisis of antibiotic resistance. We analyze herein synthetic approaches aiming to reengineer natural products into potent antibiotics. Furthermore, we discuss approaches in modulating quorum sensing and biofilm formation as a nonlethal method, as well as narrow-spectrum pathogen-specific antibiotics, which are of interest given new insights into the implications of disrupting the microbiome.

Optimal Control of Wild Horse Populations with Nonlethal Methods

Effective management of wild horse populations has proven difficult for land managers. This paper develops and evaluates dynamic optimization models of wild horse management for three nonlethal population control methods: removals, fertility control, and a hybrid method combining removals and fertility control. Two state variables are included in the models to account for horses on the range and horses in off-range holding facilities. The optimization problems are solved and dynamic simulations are generated for each method. The results indicate that a strategic approach to nonlethal wild horse population control has the potential to significantly improve management efficiency.

Morphological descriptions of the eggcases of skates (Rajidae) from the central-western Mediterranean, with notes on their distribution

Eggcases of eight rajiform skates (Dipturus nidarosiensis, D. oxyrinchus, Leucoraja melitensis, Raja asterias, R. brachyura, R. clavata, R. miraletus and R. polystigma) present in the central-western Mediterranean are described, based on specimens obtained from fishery surveys. Eggcase features such as dimensions, horns and apron lengths, and presence/absence of lateral keels were crucial to discriminate the eggcases of the various species. Morphological and morphometric data, confirmed by the multivariate analysis, indicated that the eggcase of R. miraletus and L. melitensis were distinct from those of the other species for being unkeeled. Within the species having keeled eggcases, those of the genus Dipturus and R. brachyura were discriminated from the remaining group by having the largest dimensions and aprons. Sandy bottoms (<100–150 m depth) were identified as egg-laying sites (i.e. sites with females bearing eggcases in uteri) for many species belonging to genus Raja Raja asterias, R. brachyura, R. miraletus and R. polystigma). The finding of R. asterias and R. miraletus carrying eggcases yearly on the same sites, seems to confirm the theory that many rajid species demonstrate site fidelity, returning to the same depositional area on an annual basis. Some remarks on reproductive biology of these skates are also provided. The eggcase identification key reported here represents the first for the Mediterranean and may be useful, in the future, to identify egg-laying grounds of skates with a nonlethal method.

Detection accuracy of Renibacterium salmoninarum in Chinook salmon, Oncorhynchus tshawytscha (Walbaum) from non-lethally collected samples: Effects of exposure route and disease severity

Bacterial kidney disease (BKD), caused by Renibacterium salmoninarum, threatens salmonid populations throughout the Northern hemisphere. Many fishery regulatory authorities require ongoing disease monitoring in hatcheries and spawning runs prior to gamete collection to prevent BKD outbreaks and spread. According to diagnostic protocols of the American Fisheries Society-Fish Health Section, monitoring for R. salmoninarum generally consists of lethal sampling of visceral organs from fish. However, non-lethal sampling would be preferable, especially for valuable broodstock or endangered species. In this study, non-lethal sampling methods were evaluated for their ability to detect R. salmoninarum in Chinook salmon (Oncorhynchus tshawytscha) that were experimentally infected via two different routes (e.g., intraperitoneal injection and waterborne immersion) to mimic acute and chronic disease courses. Non-lethal (e.g., blood, mucus, and a urine/feces mixture) and lethal (e.g., kidney and spleen homogenate) samples were collected from challenged and mock-challenged Chinook salmon and the presence of R. salmoninarum was assessed by culture on modified kidney disease medium, nested polymerase chain reaction (nPCR), and semi-quantitative enzyme-linked immunosorbent assay (ELISA). Sensitivity, specificity, and accuracy of lethal and non-lethal samples in detecting R. salmoninarum were calculated using receiver operating characteristic (ROC) analyses. For ROC analyses, true disease status was evaluated under two different assumptions: 1) that lethal samples represented the true disease status and 2) that all experimentally challenged fish were truly infected. We found that sensitivity and specificity of non-lethal samples depended upon time of sampling after experimental infection, sample type, and R. salmoninarum exposure route. Uro-fecal samples had the greatest potential as non-lethal samples compared to mucus and blood. In terms of future monitoring, combining lethal samples tested by ELISA assay with uro-fecal samples tested by nPCR could be the best strategy for detecting R. salmoninarum prevalence in a population as it reduces the overall number of fish required for sampling.

Trace element contamination in feather and tissue samples from Anna’s hummingbirds

Trace element contamination (17 elements; Be, V, Cr, Mn, Fe, Co, Ni, Cu, Zn, As, Se, Mo, Cd, Ba, Hg, Tl, and Pb) of live (feather samples only) and deceased (feather and tissue samples) Anna's hummingbirds (Calypte anna) was evaluated. Samples were analyzed using inductively coupled plasma-mass spectrometry (ICP-MS; 17 elements) and atomic absorption spectrophotometry (Hg only). Mean plus one standard deviation (SD) was considered the benchmark, and concentrations above the mean+1 SD were considered elevated above normal. Contour feathers were sampled from live birds of varying age, sex, and California locations. In order to reduce thermal impacts, minimal feathers were taken from live birds, therefore a novel method was developed for preparation of low mass feather samples for ICP-MS analysis. The study found that the novel feather preparation method enabled small mass feather samples to be analyzed for trace elements using ICP-MS. For feather samples from live birds, all trace elements, with the exception of beryllium, had concentrations above the mean+1 SD. Important risk factors for elevated trace element concentrations in feathers of live birds were age for iron, zinc, and arsenic, and location for iron, manganese, zinc, and selenium. For samples from deceased birds, ICP-MS results from body and tail feathers were correlated for Fe, Zn, and Pb, and feather concentrations were correlated with renal (Fe, Zn, Pb) or hepatic (Hg) tissue concentrations. Results for AA spectrophotometry analyzed samples from deceased birds further supported the ICP-MS findings where a strong correlation between mercury concentrations in feather and tissue (pectoral muscle) samples was found. These study results support that sampling feathers from live free-ranging hummingbirds might be a useful, non-lethal sampling method for evaluating trace element exposure and provides a sampling alternative since their small body size limits traditional sampling of blood and tissues. The results from this study provide a benchmark for the distribution of trace element concentrations in feather and tissue samples from hummingbirds and suggests a reference mark for exceeding normal. Lastly, pollinating avian species are minimally represented in the literature as bioindicators for environmental trace element contamination. Given that trace elements can move through food chains by a variety of routes, our study indicates that hummingbirds are possible bioindicators of environmental trace element contamination.

Post-Myocardial Infarction T-tubules Form Enlarged Branched Structures With Dysregulation of Junctophilin-2 and Bridging Integrator 1 (BIN-1).

BackgroundHeart failure is a common secondary complication following a myocardial infarction (MI), characterized by impaired cardiac contraction and t‐tubule (t‐t) loss. However, post‐MI nano‐scale morphological changes to the remaining t‐ts are poorly understood.Method and ResultsWe utilized a porcine model of MI, using a nonlethal microembolization method to generate controlled microinfarcts. Using serial block face scanning electron microscopy, we report that post‐MI, after mild left‐ventricular dysfunction has developed, t‐ts are not only lost in the peri‐infarct region, but also the remnant t‐ts form enlarged, highly branched disordered structures, containing a dense intricate inner membrane. Biochemical and proteomics analyses showed that the calcium release channel, ryanodine receptor 2 (RyR2), abundance is unchanged, but junctophilin‐2 (JP2), important for maintaining t‐t trajectory, is depressed (−0.5×) in keeping with the t‐ts being disorganized. However, immunolabeling shows that populations of RyR2 and JP2 remain associated with the remodeled t‐ts. The bridging integrator 1 protein (BIN‐1), a regulator of tubulogensis, is upregulated (+5.4×), consistent with an overdeveloped internal membrane system, a feature not present in control t‐ts. Importantly, we have determined that t‐ts, in the remote region, are narrowed and also contain dense membrane folds (BIN‐1 is up‐regulated +3.4×), whereas the t‐ts have a radial organization comparable to control JP2 is upregulated +1.7×.ConclusionsThis study reveals previously unidentified remodeling of the t‐t nano‐architecture in the post‐MI heart that extends to the remote region. Our findings highlight that targeting JP2 may be beneficial for preserving the orientation of the t‐ts, attenuating the development of hypocontractility post‐MI.