Non-lethal Methods Research Articles

Condensed tannins as a deterrent to crop depredation by white‐tailed deer: Effects of concentration and learning

ABSTRACTAgricultural producers lose millions of dollars annually to depredation caused by ungulates. Although multiple repellents exist, most are ineffective. Tannins are a plant‐defensive compound that have an astringent taste, reduce digestibility, reduce protein availability, and toxic to rumen microbes, resulting in the reduction of feeding by large herbivores. We hypothesized that chemical application of tannins to forages would provide a nonlethal method to reduce crop depredation in areas susceptible to ungulate damage. We designed an experiment to test the effectiveness of tannins at deterring feeding by captive white‐tailed deer (Odocoileus virginianus) on soybeans and other forages in 2010–2011 at the Wildlife Research Facility at South Dakota State University in Brookings, South Dakota, USA. First, using a cafeteria trial, we monitored weekly intake rates of captive deer (n = 10) offered a selected feed treated with varying concentrations of quebracho tannins (control, 3%, 5%, 10%, and 20%). Second, we manipulated food plots (n = 3) by treating 3 separate blocks with different concentrations (0% [control], 10%, and 20%) of tannins. We monitored feeding selection relative to tannin treatments within food plots 3 times/week, during summer (May–Jul) and autumn (Aug–Sep) using scan‐sampling. During cafeteria trials, intake rate of control feed was ≥3 times greater than all other feed treated with tannins. Similarly, in manipulated food plots, application of 10% and 20% tannins resulted in a 72% and 89% reduction in probability of feeding during the summer; this pattern of selection was intensified when trials occurred in consecutive days with the same deer. Strategic application of tannins to agricultural crops in areas prone to damage may provide a natural deterrent to crop depredation by ungulates. © 2019 The Wildlife Society.

Culling recolonizing mesopredators increases livestock losses: Evidence from the South African Karoo.

Populations of adaptable mesopredators are expanding globally where passive rewilding and natural recolonization are taking place, increasing the risk of conflict with remaining livestock farmers. We analysed data from two social surveys of farmers in the Karoo, South Africa, where black-backed jackals (Canis mesomelas) and caracals (Caracal caracal) have re-emerged as a threat to sheep farms in the context of falling agricultural employment and the expansion of natural areas. We show that irrespective of measurement approach, lethal control of mesopredators in this fragmented socio-economic landscape was associated with increased livestock losses the following year. Terrain ruggedness was positively, and number of farmworkers negatively, associated with livestock losses. Our study provides further evidence that lethal control of mesopredators in this context is probably counter-productive and supports calls to develop, share and financially support a range of non-lethal methods to protect livestock, especially where natural recolonization of mesopredators is occurring. A graphical abstract can be found in Electronic supplementary material.

Standardizing a Non-Lethal Method for Characterizing the Reproductive Status and Larval Development of Freshwater Mussels (Bivalvia: Unionida).

Actively monitoring the timing, development, and reproductive patterns of endangered species is critical when managing for population recovery. Freshwater mussels are among the most imperiled organisms in the world, but information about early larval (glochidial) development and brooding periods is still lacking for many species. Previous studies have focused on the complex life history stage when female mussels are ready to parasitize host fish, but few studies have focused on the brooding period and timing of larval development. The protocol described here allows researchers to non-lethally evaluate the state of gravidity for female mussels. The results of this study show that this method does not affect a female mussel's ability to stay gravid or become gravid again after sampling has been performed. The advantage of this method may permit its use on federally threatened or endangered species or other populations of high conservation concern. This protocol can be adapted for use on both preserved or live individuals and was tested on a variety of mussel species. The database provided is a repository for a breadth of information on timing of reproductive habits and will facilitate future freshwater mussel research, conservation, and recovery efforts.

Standardizing a Non-Lethal Method for Characterizing the Reproductive Status and Larval Development of Freshwater Mussels (Bivalvia: Unionida)

Standardizing a Non-Lethal Method for Characterizing the Reproductive Status and Larval Development of Freshwater Mussels (Bivalvia: Unionida)

Puma and livestock in central Patagonia (Argentina): from ranchers’ perceptions to predator management

ABSTRACTWe analyzed surveys from 383 ranchers to determine their perceptions of puma damage in Chubut province (Central Patagonia). Thirty-two percent of respondents had a negative perception, while 65% were neutral. Negative perceptions increased: (a) with the breeding of mixed livestock, (b) with the presence of neighboring inactive, and (c) in small and medium ranches. Our results showed that ranchers use both lethal control and non-lethal methods (regular patrol of the ranch areas, night and seasonal enclosures, guardian dogs) to reduce livestock predation. Lethal control has great acceptance among ranchers, but there is a general lack of evidence about its effectiveness. Considering that our results indicated that the puma may not be the major problem in livestock areas, it is necessary to review the current management policy. We suggest focusing efforts to reduce livestock depredation only on affected ranches, prioritizing the development of non-lethal methods.

Energetic status and bioelectrical impedance modeling of Arctic grayling Thymallus arcticus in interior Alaska Rivers

The energetic status of fishes represents energy stored as protein and lipids and reflects the ability of an individual to reproduce, migrate, and transition through life stages, ultimately influencing survival. However, traditional measurement methods, while highly accurate, are time consuming, expensive, and lethal, and nonlethal methods such as condition factor may not adequately characterize energetic status. We collected 161 Arctic grayling (Thymallus arcticus) from four interior Alaska river basins with varying hydrologic regimes during early summer and autumn seasons, and used multiple regression and model selection to evaluate the efficacy of bioelectrical impedance analysis (BIA), a nonlethal condition assessment method, to predict percent dry mass and percent lipid content estimated from proximate analysis. We found that Arctic grayling energetic status varied across seasons, by sex, and fish from sites with spawning runs of Pacific salmon had higher energy content than those from sites without salmon, potentially due to the influence of salmon-derived food subsidies. Electrical measurements explained 82% and 80% of the variability in percent dry mass and percent total lipids, respectively, and top models showed high predictive performance (observed vs. predicted root mean squared error ≤2.2%). Overall, we found the BIA approach to provide rapid, precise, and non-lethal estimates of Arctic grayling body condition. Such an approach may be useful for future work to characterize Arctic grayling bioenergetics and monitor fish condition under a rapidly changing Arctic environment.

F2-isoprostanes in Fish mucus: A new, non-invasive method for analyzing a biomarker of oxidative stress

F2-isoprostanes (F2-isoPs) are a reliable biomarker class for oxidative stress in vivo in animals. These compounds are traditionally measured in matrices like liver and plasma, however social and environmental pressures warrant the development of non-lethal and non-invasive methods to assess animal health. Therefore, this study aimed to develop a high-performance liquid chromatography tandem mass spectrometry (HPLC-ESI-MS/MS) method to separate and detect F2-isoPs in fish mucus. The method was developed and validated for four native F2-isoP isomers using Northern pike mucus (Esox lucius). Linearity was observed between 5 and 1000 pg/μL. The limits of detection of the four F2-IsoP isomers ranged from 0.63 to 2.0 ng/g. Recoveries ranged from 78 to 95%, and matrix effects were small (<10%). The between-day and within-day repeatability for all target analytes was lower than 20% RSD. Endogenous F2-isoPs were measured in the pike mucus (5.3–28.8 ng/g). A preliminary study of baseline F2-isoP concentrations in lake trout (Salvelinus namaycush) captured from five lakes at the IISD-Experimental Lakes Area in Northwestern Ontario, Canada, was also conducted to test the interspecies applicability of the method. Endogenous F2-isoPs were quantified in lake trout (6.3–132 ng/g). Lake trout samples displayed large variability within and between the different lakes, which suggests sampling methods may require adjustment for this species. This work developed a sensitive analytical method for measuring F2-isoPs in fish mucus, however several further studies are required to determine its ability to accurately measure oxidative stress in fish species.

In vivo investigation of pesticide residues in garlic using solid phase microextraction-gas chromatography-mass spectrometry

In this study, a novel multiwalled carbon nanotubes/polyaniline-polypyrrole@polydimethylsiloxane (MWCNTs/PANI-PPy@PDMS) fiber was prepared by a facile electrodeposition strategy followed by polymer surface modification, and used as the sorbent of in vivo solid phase microextraction (in vivo SPME). The custom-made fiber exhibits better enrichment capacity than three commercial SPME fibers. Ultra-high enrichment factor (438–2659), satisfactory thermal and mechanical stability, excellent matrix-compatibility and anti-biofouling ability render the fiber attractive as in vivo sampling probe. Combining in vivo SPME with gas chromatography-mass spectrometry (GC-MS), a convenient and sensitive method was successfully developed for the rapid determination of pesticides (hexachlorobenzene, chlorothalonil, fipronil, chlorfenapyr) in garlic. Under the optimal conditions, the proposed method displays relatively wide linear range (three or four orders of magnitude) with a coefficient of determination above 0.9944 both in the standard solution and spiked homogenized garlic samples, respectively. Low detection limits from 0.38 to 1.90 ng g−1 were obtained in homogenized garlic samples. Relative standard deviations (RSDs) less than 15.5% and recovery between 84.0% and 108.2% indicate satisfactory precision and accuracy of the method. In summary, a promising non-lethal method based on in vivo SPME-GC-MS is developed in this study, which provides a green, efficient, economic and rapid strategy for the determination of trace pesticide residues in edible plants.

Conditioned food aversion in domestic dogs induced by thiram.

The conflict between predators and humans for resources such as game species or livestock is an ancient issue, and it is especially sharp in the case of medium-large wild canids. In order to manage this conflict, lethal control methods are often used, which can sometimes be illegal, such as poisoning. As an alternative, conditioned food aversion (CFA) is a non-lethal method to reduce predation in which animals learn to avoid a given food due to the adverse effects caused by the ingestion of an undetectable chemical compound added to this food. The present study aimed to test thiram as a CFA agent in penned dogs as a first approach to use this substance for reducing the predation conflict associated with wild canids. Thiram, with or without an additional odor cue, produced CFA in penned dogs for more than 2 months. Moreover, thiram seemed to be undetectable and safe after the third ingestion of a 40-60 mg kg-1 dose. Desirable adverse effects, such as vomits, appeared around 1h after exposure. These characteristics make thiram optimal for its use in predation reduction through CFA. However, individual variability could prevent CFA acquisition by some animals. Thiram has the potential to be used as a CFA agent in wildlife management and conservation to reduce predation by wild canids. Since thiram produced CFA without the problems of detectability and toxicity caused by other substances, it may be an alternative to lethal control methods used to reduce predation on game, livestock and endangered species. © 2019 Society of Chemical Industry.

Characteristics of adolescents who visit the emergency department following suicide attempts: comparison study between adolescents and adults

BackgroundThe purpose of this study was to identify the demographic and clinical characteristics of suicide attempts in adolescents who visit the emergency department compared to those of adults.MethodsThis study included 149 children under the age of 18, and 1427 people in the age of 19–65 who came to the emergency department with suicide attempt from 2009 to 2015. We compare sociodemographic, clinical, and suicide attempt-related characteristics through Chi-square test and logistic regression analysis to evaluate the difference between two groups.ResultsIn adolescents, suicide attempters had more number of previous suicide attempt history than adults. Adolescents used more non-lethal method such as poisoning of over the counter drugs and had about 5 times higher odds ratio in suicide attempts with analgesics. The motivation of suicide attempt among adolescents was more related with interpersonal problems but less with financial or illness-related problems. The intention of suicide attempt in adolescents was less serious and lethal compared to adults.ConclusionSuicide attempts among adolescents had showed different from adults in method, motivation and intention. Considering the characteristics of suicide attempt among adolescent, it is necessary to keep close attention to adolescent’s suicide attempters and develop the customized intervention program to prevent the suicide attempt in this groups.

Mercury Concentrations in the Two-Toed Amphiuma (Amphiuma means) and the Lesser Siren (Siren intermedia): Validating Non-lethal Sampling Methods in Southeastern Aquatic Salamanders.

The global decline of amphibians is a major conservation issue. Many stressors are recognized for this decline including exposure to environmental contaminants. Mercury (Hg) is an environmental contaminant that bioaccumulates in wildlife and can cause a variety of negative impacts across taxa, including amphibians. Amphiuma and Siren spp. can comprise a large portion of biomass within their respective ecosystems, and thus, likely serve as important predators or prey in wetland communities. However, due to their cryptic nature, little is known about their ecology, diet, and accumulation potential. We sought to validate a nonlethal sampling method to quantify total mercury (THg) in two enigmatic species of aquatic salamanders: the two-toed amphiuma (Amphiuma means) and the lesser siren (Siren intermedia). We examined relationships between THg content in lethal (whole-body) and nonlethal (tail clip) samples. Tail clips were statistically significant predictors of whole-body THg (all p < 0.001), explaining 84-89% of variation in whole-body THg. Average whole-body THg (mg/kg) did not significantly differ between the two species (p = 0.97), and overall, they had similar whole-body THg content (S. intermedia = 0.330 ± 0.04, n = 18; A. means = 0.333 ± 0.07, n = 11). To our knowledge, these data represent the first reported Hg burdens in A. means and S. intermedia.

A non-destructive method for assessing the feeding habits of coastal fish

Studies on the feeding ecology of sympatric coastal species is important, especially in revealing different strategies to reduce competition. The aim of this study is to test the diet of littoral fish species with a non-destructive method, which does not necessitate the sacrifice of fish specimens. The clingfish Lepadogaster lepadogaster (Bonnaterre, 1788) was used to test this diet assessment method. Freshly caught specimens were delivered immediately to a specially designed box for collecting faecal pellets, supplied by an aerator. Clingfishes were left in the chambers for 24 hours to defecate. The pellets were carefully removed and fixed in 70% alcohol. The content of the pellets was analysed under stereomicroscope and prey items were determined and counted. The comparison with the existing studies showed similarity and consistency between their results, which proves the usefulness and applicability of the method for studying clingfish diet. The suitability of the proposed method was tested on related species L. candollei Risso, 1810, where all ingested prey items were also found and identified in faecal pellets, which confirms its effectiveness for diet analysis. A method of collecting faecal pellets immediately after defecation has proved to be more useful and effective than other nonlethal methods. After defecation, the specimens were released at the site where they were collected. Due to 100% survival as shown in this research, the proposed method is also suitable for studying the diet of rare and endangered fish species, and also coastal fish fauna in protected areas where traditional destructive sampling methods are not appropriate or allowed.

Nonlethal Detection of Asian Fish Tapeworm in the Federally Endangered Humpback Chub Using a Molecular Screening Tool

AbstractOptimal spawning habitat of federally endangered Humpback Chub Gila cypha exists within the Little Colorado River; however, temperatures in the Little Colorado River are also ideal for proliferation of the invasive pathogenic Asian fish tapeworm Schyzocotyle acheilognathi. The current standard for positive identification of the parasite is necropsy and visual examination of the gut via microscopy, a methodology undesirable for assessing infection in endangered fishes. A swab taken at the rectum of the fish and analyzed using a novel DNA primer targeting the cestode cytochrome oxidase I subunit mtDNA gene region offers a convenient nonlethal sampling tool. To validate the nonlethal methodology, primer sensitivity (51.9%) and specificity (79.5%) were calculated by exposing captive fish to the parasite and comparing results using the lethal standard method (i.e., visual examination). Subsequently, we utilized this nonlethal methodology to address prevalence of infection and infection frequency across a range of size‐classes in a wild population of Humpback Chub and addressed possible patterns and mechanisms of infection in the Little Colorado River. When wild Humpback Chub were screened for infection prevalence in spring 2015 (21.4%, n = 140) and fall 2015 (6.6%, n = 258), the relative frequency of infection was highest in juveniles and subadult fish (200–300 mm). Elevated levels of infection near the major spawning grounds of Humpback Chub in the Little Colorado River promote parasitic infection, which may continue to persist without treatment or actions to control infections. We demonstrated that rectal swabs, in conjunction with PCR and primer‐specific identification, offer a time‐efficient nonlethal method to detect infection of Asian fish tapeworm in an endangered fish.

Molecular composition of the paralyzing venom of three solitary wasps (Hymenoptera: Pompilidae) collected in southeast Mexico

The chemical and biological characterization of peptide and protein components of the paralyzing venom from three Pompilidae solitary spider wasps (Pepsis mexicana, Pepsis terminata, and Anoplius nigritus) is described for the first time. The molecular masses of the most abundant peptides were determined. The N-terminal sequences of two cysteine-rich peptides were obtained from Pepsis. Metalloproteinase and hyaluronidase activities were identified in the venom of P. mexicana. A novel non-lethal method to collect venom is described.

Development of a non-lethal hydrogen peroxide treatment for surveillance of Gyrodactylus salaris on trout farms and its application to testing wild salmon populations.

This study documents the development of a non‐lethal sampling method to recover gyrodactylid parasites from large numbers of fish that will underpin an improved surveillance strategy for Gyrodactylus salaris. A review of published literature identified over 80 compounds that have previously been tested against gyrodactylids or closely related parasite species. Five safe and relatively fast‐acting compounds were selected for testing to determine their efficiency in removing gyrodactylids from host fish in small‐scale aquaria trials using three‐spined stickleback infected with Gyrodactylus gasterostei as a model host–parasite system. The most effective compound was hydrogen peroxide; short‐duration exposure (3 min) achieved a parasite detection sensitivity of 80%–89%. The practicality of exposing farmed salmonids to hydrogen peroxide for G. salaris surveillance was tested in the field by conducting a parasite recovery trial using a brown trout stock endemically infected with G. truttae and G. derjavinoides and comparing this to the whole‐body examination procedure currently conducted by UK authorities. Significantly more parasites were recovered after exposing fish to hydrogen peroxide and filtering the treatment solution than by direct whole‐body examination of killed fish (mean: 225 vs. 138 parasites per fish). The gyrodactylid recovery rate of the two methods was 84.6% and 51.9%, respectively. A comparison of timings for the two methods indicated scope for significant time savings in adopting the chemical screening method. The study demonstrated that hydrogen peroxide bath treatment may be successfully applied to the surveillance of gyrodactylid parasites and established as a non‐lethal method for sampling farmed and wild fish. This approach has the potential to reduce resources required to collect and isolate parasites for diagnostic testing and improve the sensitivity and confidence of surveillance programmes designed to demonstrate freedom from disease, thus underpinning a robust and defensible surveillance strategy for G. salaris for the UK aquatic animal disease contingency plan.

Chloroform and desflurane immobilization with recovery of viable Drosophila larvae for confocal imaging

Imaging of living, intact Drosophila larvae is challenged if normal bodily function must be observed or when healthy larvae must be recovered for subsequent studies. Here, we describe a simple and short protocol that employs transient airborne chloroform or desflurane (1,2,2,2-tetrafluoroethyl difluoromethyl ether) to efficiently immobilize larvae without the use of manipulation devices, vaporizers or imaging chambers. This non-lethal method allows the use of anesthetics while allowing tracking of individual Drosophila into adulthood for follow-up experiments. At dosages sufficient to immobilize larvae, Desflurane, but not chloroform reduced the central nervous system response to auditory stimulus. Desflurane doses were sufficient to arrest the heart, however significant rapid recovery was observed. With our method, chloroform provided more rapid anesthesia but slower recovery than Desflurane. Without specialized hardware, this technique allows for repeated imaging of living Drosophila larvae.

Establishing a nonlethal and efficient mouse model of male gonadotoxicity by intraperitoneal busulfan injection

An ideal animal model of azoospermia would be a powerful tool for the evaluation of spermatogonial stem cell (SSC) transplantation. Busulfan has been commonly used to develop such a model, but 30%–87% of mice die when administered an intraperitoneal injection of 40 mg kg−1. In the present study, hematoxylin and eosin staining, Western blot, immunofluorescence, and quantitative real-time polymerase chain reaction were used to test the effects of busulfan exposure in a mouse model that received two intraperitoneal injections of busulfan at a 3-h interval at different doses (20, 30, and 40 mg kg−1) on day 36 or a dose of 40 mg kg−1 at different time points (0, 9, 18, 27, 36, and 63 days). The survival rate of the mice was 100%. When the mice were treated with 40 mg kg−1 busulfan, dramatic SSC depletion occurred 18 days later and all of the germ cells were cleared by day 36. In addition, the gene expressions of glial cell line-derived neurotrophic factor (GDNF), fibroblast growth factor 2 (FGF2), chemokine (C-X-C Motif) ligand 12 (CXCL12), and colony-stimulating factor 1 (CSF1) were moderately increased by day 36. A 63-day, long-term observation showed the rare restoration of endogenous germ cells in the testes, suggesting that the potential period for SSC transplantation was between day 36 and day 63. Our results demonstrate that the administration of two intraperitoneal injections of busulfan (40 mg kg−1 in total) at a 3-h interval to mice provided a nonlethal and efficient method for recipient preparation in SSC transplantation and could improve treatments for infertility and the understanding of chemotherapy-induced gonadotoxicity.

Development of an easy and cost-effective method for non-invasive genotyping of insects.

Non-invasive genotyping methods provide valuable information on insect populations. However, poor DNA amplification and time-consuming sampling procedures limit these methods, especially for small insects. An efficient and convenient method was developed for non-invasive, non-lethal genotyping of a large insect, Mythimna separata, and a small insect, Drosophila melanogaster, by amplification of endogenous and exogenous, nuclear and mitochondrial genes from insect frass, exuviae, and food waste. For M. separata, the chitin synthesis gene MsCHSB and the COI gene were successfully detected by PCR from exuviae DNA. However, a COI fragment could not be detected directly by PCR from frass, probably due to DNA degradation. To improve the detection rate, DNA from frass was first amplified by Multiple Displacement Amplification with phi29 DNA polymerase, after which the COI fragment was detected from all samples by PCR. For D. melanogaster, second instar larvae were reared individually for three days and then DNA was extracted from food waste of each individual. The endogenous fragment serendipity α (sryα), exogenous transgene ΦC31 integrase, and the kl-5 gene, a Y-chromosome-located male-specific marker gene were successfully detected from most samples. We developed a simple, non-invasive, non-lethal method to determine gender and identify transgenic individuals early in the larval stage. This universal method is applicable to most insects and has potential application in genetic and ecological studies of insects and other arthropods.

The Marin County Livestock Protection Program: 15 Years in Review

Author(s): Larson, Stephanie; McGranahan, Devan A.; Timm, Robert M. | Abstract: In 2001, Marin County, California, replaced its USDA Wildlife Services (WS) cooperative predator damage management program with a county-run program that emphasized non-lethal methods for preventing and controlling coyote predation on sheep. This new “Livestock Protection Program” cost-shared with livestock producers’ efforts to improve fencing, obtain and maintain guard animals, and other such non-lethal methods, and initially it compensated producers for documented losses to predators. We surveyed sheep producers in Marin County in an effort to review the program over the past 15 years, evaluating the program in relation to livestock production, economics, predation management, and other measures of producer satisfaction. Lack of standardized data collection during the current program complicates its evaluation; however, from available information, we conclude the number of sheep and lambs are being produced in Marin County has continued to decline; some producers left the sheep business and other who remain graze less acreage with smaller flocks; predation by coyotes remains a high concern to producers; and most producers are dissatisfied with the Livestock Protection Program.

Conditioned food aversion mediated by odour cue and microencapsulated levamisole to avoid predation by canids

Worldwide, predators and humans are in conflict for resources such as game species or livestock, especially in the case of wild canids. One non-lethal method to reduce predation is conditioned food aversion (CFA), in which animals learn to avoid a food due to the illness after ingestion, caused by the addition of an undetected chemical compound. CFA can be enhanced by adding an artificial odour cue, in a process known as taste-potentiated odour aversion (TPOA). We tested CFA and TPOA with three experimental groups of penned dogs. Food was offered with a combination of microencapsulated levamisole + vanilla odour (ODO), microencapsulated levamisole (LEV), or plain food as a control. The aims were (a) to test whether dogs detected the microencapsulated levamisole, (b) to analyse the strength and extinction time of CFA induced by microencapsulated levamisole, and (c) to analyse the strength and extinction time of TPOA. Two-choice tests were carried out during 11 post-conditioning months, and two reinforcements with microencapsulated levamisole were performed during the first post-conditioning month. In the first post-conditioning test, ODO and LEV groups ate significantly less untreated food than control group. After reinforcement, the dogs in LEV group resumed eating the food. Three of four dogs in ODO group showed long-lasting CFA until the 11th month. These results show that TPOA could be used to induce odour aversion on canids and that the odour cue overshadows the slight bitter taste of microencapsulated levamisole. These results show TPOA as a promising tool to reduce predation by wild canids.