Sentinel Species Research Articles

Assessing the Occurrence of Carbapenemase Producers Using Marine Animals as Sentinel Species

Carbapenemase-producing (CP) strains represent a substantial global threat, deactivating carbapenems and conferring resistance to β-lactam antibiotics. They can spread across various environments, yet data on their presence in marine animals are sparse. This study aimed to assess the occurrence of carbapenemase-producing strains in wild marine animals and to analyse their antimicrobial resistance (AMR) profiles to crucial antimicrobials and heavy metals frequently encountered in the marine environment due to anthropogenicactivity. A total of 28 samples were obtained from a fish auction in the Centre Region of Portugal. Non-fermenting bacilli (NFB) was isolated from the visceral content of wild marine animals. Identification of isolates was achieved through PCR-based amplification of the 16S rRNA gene, followed by sequencing. Antimicrobial susceptibility testing was conducted according to EUCAST guidelines, covering nine antimicrobials. Research of carbapenemases and metal tolerance genes was conducted by PCR, and statistical analysis utilized the Fisher’s exact test. Pseudomonas spp. and Aeromonas spp., among other isolates were identified (n=47/9/7, respectively). Susceptibility profiles showed 100% resistance or intermediate resistance to ticarcillin, piperacillin, piperacillin-tazobactam, ceftazidime, ciprofloxacin, and imipenem (n=63/63), while 27% were resistant to meropenem (n=17/63) and 13% to tobramycin (n=8/63). All of them exhibited susceptibility to amikacin and carried multidrug resistance (MDR) profiles, including heavy metal genes (merA and silA). None harboured the carbapenemase genes searched (blaKPC, blaGES, blaIMP, blaNDM or/and blaVIM). In this study, MDR profiles to clinically important antimicrobials were observed, including to carbapenems. However, no carbapenemase-producing strains were identified, suggesting the presence of other genes or alternative mechanisms of resistance. These findings underscore the importance of monitoring AMR in marine ecosystems, particularly given its close ties to the food chain.

Ecosystem Sentinels as Early-Warning Indicators in the Anthropocene

We are already experiencing the rapid pace of environmental perturbation in the Anthropocene, necessitating the development of new tools and techniques for measuring changes in ecosystem dynamics. Sentinel species, from birds to invertebrates, have been used to provide insights into ecosystem function, as leading indicators of risk to human health and as harbingers of future change, with implications for ecosystem structure and function. Here, we offer an update to previous research identifying marine top predators as indicators of ecosystem shifts and examine terrestrial sentinels and the latest research on sentinels of pollution and human health. Using ecosystem sentinels enables rapid response and adaptation to ecosystem variability and environmental change in part because they may be easier to observe and in part because they may serve as leading indicators of ecosystem disruption. While there may not be a given taxon that is best suited as sentinels, we highlight how to select the most effective sentinels, including examples of when sentinel species have been incorporated into management. Choosing a suite of appropriate sentinels both will give insight into ecosystem processes and can help manage changing ecosystems into the future.

Combined toxicity of perfluoroalkyl substances and microplastics on the sentinel species Daphnia magna: Implications for freshwater ecosystems

Persistent chemicals from industrial processes, particularly perfluoroalkyl substances (PFAS), have become pervasive in the environment due to their persistence, long half-lives, and bioaccumulative properties. Used globally for their thermal resistance and repellence to water and oil, PFAS have led to widespread environmental contamination. These compounds pose significant health risks with exposure through food, water, and dermal contact. Aquatic wildlife is particularly vulnerable as water bodies act as major transport and transformation mediums for PFAS. Their co-occurrence with microplastics may intensify the impact on aquatic species by influencing PFAS sorption and transport. Despite progress in understanding the occurrence and fate of PFAS and microplastics in aquatic ecosystems, the toxicity of PFAS mixtures and their co-occurrence with other high-concern compounds remains poorly understood, especially over organisms’ life cycles.Our study investigates the chronic toxicity of PFAS and microplastics on the sentinel species Daphnia, a species central to aquatic foodwebs and an ecotoxicology model. We examined the effects of perfluorooctane sulfonate (PFOS), perfluorooctanoic acid (PFOA), and polyethylene terephthalate microplastics (PET) both individually and in mixtures on Daphnia ecological endpoints. Unlike conventional studies, we used two Daphnia genotypes with distinct histories of chemical exposure. This approach revealed that PFAS and microplastics cause developmental failures, delayed sexual maturity and reduced somatic growth, with historical exposure to environmental pollution reducing tolerance to these persistent chemicals due to cumulative fitness costs. We also observed that the combined effect of the persistent chemicals analysed was 59% additive and 41% synergistic, whereas no antagonistic interactions were observed. The genotype-specific responses observed highlight the complex interplay between genetic background and pollutant exposure, emphasizing the importance of incorporating multiple genotypes in environmental risk assessments to more accurately predict the ecological impact of chemical pollutants.

Uptake, Efflux, and Sequestration of Mercury in the Asian Clam, Corbicula fluminea, at Environmentally Relevant Concentrations, and the Implications for Mercury Remediation

(1) Mercury (Hg) is a persistent, ubiquitous contaminant that readily biomagnifies into higher trophic level species in aquatic environments across the globe. It is crucial to understand the movement of environmentally relevant concentrations of Hg in impacted freshwater streams to minimize risks to ecological and human health. (2) The bioconcentration kinetics of aqueous Hg exposure (20, 100, and 200 ng/L) in the invasive Asian Clam, Corbicula fluminea, were measured. A toxicokinetic model, the first parameterized for Hg accumulation in freshwater clams, was developed to estimate uptake and efflux parameters and compared to previous parameter values estimated for other mollusk species. (3) Results demonstrated that even at low Hg concentrations, Corbicula record signals of contamination through bioconcentration, and both direct measurement and toxicokinetic models demonstrate large Hg bioconcentration factors (as high as 1.34 × 105 mL/g dry tissue), similar to partitioning coefficients seen in engineered Hg sorbents. (4) Our study found that Corbicula accumulated Hg at aqueous concentrations relevant to impacted streams, but well below regulatory drinking water limits, demonstrating their utility as a sensitive sentinel species and potential bioremediator.

Investigations on the Health Status and Infection Risk of Harbour Seals (Phoca vitulina) from Waters of the Lower Saxon Wadden Sea, Germany.

Harbour seals (Phoca vitulina) are the most common pinniped species in the Wadden Sea of Schleswig-Holstein, Hamburg and Lower Saxony, Germany. Their numbers have recovered after significant depletion due to viral outbreaks and effects of anthropogenic activities like pollution and habitat disturbance. Within the Wadden Sea National Park of Lower Saxony the harbour seal is protected. As a top predator in the Wadden Sea ecosystem, the harbour seal is a sentinel species for the state of the environment. Between 2015 and 2017, a total of 80 stranded dead harbour seals were collected along the coastline of Lower Saxony and submitted for pathological investigations. Of these, 70 seals were born in the same year (0-7 months, age group 1) and eight in the previous year (8-19 months, age group 2), due to high mortality rates in these age groups. However, two perennial animals were also available for examination during this period, one of which was in good nutritional condition. Many of the seals that had been mercy-killed and found dead were in poor nutritional status. Histopathological, microbiological, parasitological and virological examinations were conducted on 69 individuals (86% (69/80)) in a suitable state of preservation. Respiratory tract parasitosis, cachexia, and bronchopneumonia were the most common causes of death or disease. Overall, there was no evidence of a relapse of a viral disease outbreak. Macrowaste, such as plastic waste or fishery-related debris, were not found in any gastrointestinal tract of the animals examined. There was also no evidence of grey seal predation. Weakness and cachexia were prominent causes of disease and death in harbour seals found within a few weeks after birth, but bronchopneumonia and septicaemia also developed in slightly older animals. Frequently found microbial pathogens in seals from Lower Saxony were similar to those found in other studies on seals from the Wadden Sea region in Schleswig-Holstein, for example streptococci and Escherichia coli/v. haemolytica, Brucella spp. and Erysipelothrix rhusiopathiae, potentially human pathogenic germs. The results of the examinations of dead harbour seals from Lower Saxony show that pathological investigations on a representative number of animals deliver urgently needed information on the health status of the population. The results represent an important contribution to the state of the top predators of the Wadden Sea as part of the obligations within the Trilateral Wadden Sea Agreement, Oslo and Paris Convention for the Protection of the Marine Environment of the North-East Atlantic (OSPAR) and the Marine Framework Directive. The investigations should be continued as a matter of urgency and the stranding network should be expanded.

Determination of PCB and PAH tissue levels in bottlenose dolphins that stranded in the Mississippi sound before and after the unusual mortality event in 2019

Cetaceans are regarded as a sentinel species because their health and population changes can serve as indicators of effects on marine ecosystems. We characterized levels of 24 polycyclic aromatic hydrocarbons (PAHs) and 7 polychlorinated biphenyls (PCBs) in blubber, kidney, liver, and muscle of 138 bottlenose dolphins (Tursiops truncatus) that stranded in the Mississippi Sound (MSS) between 2010 and 2021. The samples were divided into four time periods: 2010–2018, 2019, 2020, and 2021, to assess whether there was a significant association between chemical levels in dolphin tissues and the 2019 unusual mortality event (UME) in the MSS resulting from the unprecedented freshwater incursion from 2 openings of the Bonnet Carré Spillway (BCS). We found 7 PCBs were readily detected in all tissues across time, and the major PAH was naphthalene, with detection of biphenyl and acenaphthene at lower levels and frequency. There was little change in tissue PCB levels over time; only naphthalene in the blubber was higher in tissues from dolphins that stranded before 2019. However, there were not significant changes in chemical levels in tissues from stranded bottlenose dolphins in the MSS before or after the UME in 2019.

Tracing troubles: Unveiling the hidden impact of inorganic contamination on juvenile green sea turtle

Human activities and climate change have negatively affected the world's oceans, leading to a decline of 30 to 60 % in coastal ecosystems' biodiversity and habitats. The projected increase in the human population to 9.7 billion by 2050 raises concerns about the sustainability of marine ecosystem conservation and exploitation. Marine turtles, as sentinel species, accumulate contaminants, including trace elements, due to their extensive migration and long-life span. However, there is a lack of data on the degree of contamination and their effects on marine turtles' health. This study focuses on assessing in-situ inorganic contamination in juvenile green sea turtles from La Réunion Island and its short-term impact on individual health, using conventional biomarkers and proteomics. The goals include examining contamination patterns in different tissues and identifying potential new biomarkers for long-term monitoring and conservation efforts. The study identified differential metal contamination between blood and scute samples, which could help illuminate temporal exposure to trace elements in turtle individuals. We also found that some conventional biomarkers were related to trace element exposure, while the proteome responded differently to various contaminant mixtures. Immune processes, cellular organization, and metabolism were impacted, indicating that contaminant mixtures in the wild would have an effect on turtle's health. Fifteen biomarker candidates associated with strong molecular responses of sea turtle to trace element contamination are proposed for future long-term monitoring. The findings emphasize the importance of using proteomic approaches to detect subtle physiological responses to contaminants in the wild and support the need for non-targeted analysis of trace elements in the biomonitoring of sea turtle health.

Agricultural pesticides in feathers of the burrowing owl (Athene cunicularia) and its relationship to land-use in the argentinean pampas

The pampas of South America represent one of the most productive lands for agriculture in the world, and consequently this activity has expanded throughout the region, especially in Argentina. In this context, native fauna faces various risks associated with agriculture, with exposure to pesticides being one of the most dangerous and deadly. Assessing the impact of pesticides on wildlife becomes fundamental and the use of sentinel species emerges as an important tool to monitor environmental health. In this study, we determined pesticide levels in the burrowing owl Athene cunicularia nesting in both rural and urban areas of the argentinean pampas. We used a multiple scale approach to evaluate the influence of land-uses (urban, crops, and grazing fields) at the nesting site on the contaminant load of A. cunicularia individuals, and assessed the potential use of this owl as a sentinel species. From March 2018 to January 2020, A. cunicularia feathers were collected at their nest sites in one urban and two rural areas. These samples were analyzed for the presence of contaminants, including chlorpyrifos and 18 organochlorine pesticides. All the compounds analyzed were detected in A. cunicularia samples. The chlorpyrifos showed the highest frequency of occurrence and concentration, thus denoting the exposure of owls to current-use pesticides. ∑DDTs showed the highest occurrence among organochlorines, followed by ∑HCHs > ∑Endosulfans > ∑Heptachlors > ∑Drins = ∑Chlordanes > methoxychlor. Principal Component Analysis showed that most of the compounds found in A. cunicularia samples showed a positive association with grazing fields, suggesting that exposure to contaminants does not depend on specific habitat use at the local scale, but probably the regional scale. Our results evidenced the exposure of A. cunicularia individuals to past and current-use pesticides in the pampas of Argentina, and indicate that this owl would be useful as a sentinel species.

Gross pathology and liver mercury concentrations in harbour porpoises, harbour seals and grey seals in Denmark, Northern Europe

Here we report the first investigation of gross pathology and mercury (Hg) in liver tissue from harbour porpoises, harbour seals and grey seals from Denmark, Northern Europe. Mercury concentrations ranged between 0.2 and 248 μg/g wet weight (ww) with highest concentrations found in grey seals and subadult harbour seals from the Baltic Sea, with no relationship to body condition. Necropsy findings across all three species decreasing in the following order: pneumonia (n = 60) > respiratory parasitism (n = 56) > wounds (n = 18) > GI-parasites (stomach nematodes and/or parasitic colitis) (n = 16) > ectoparasites (skin lice) (n = 12) > hepatic parasites/lesions (n = 8) > focal alopecia (n = 5) > nephropathy (n = 4) > middle ear complex parasites (n = 3) > nasal parasites (n = 2). Heart and/or lung worms were significantly highest in subadult harbour porpoises, GI parasites and nephropathy significantly lowest in subadult harbour seals and focal alopecia significantly highest in subadult harbour seals. Most cases of pneumonia were associated with respiratory parasites (68 %), while nine cases of wounds led to signs of septicaemia. Significant positive relationships were observed between Hg and the presence of respiratory parasites in subadult harbour porpoises and between Hg and the presence of focal alopecia, nephropathy, and gastrointestinal parasites in subadult harbour seals. Levels of Hg were in the categories for low risk (16–64 μg/g ww) in 18 %, moderate risk (64–83 μg/g ww) in 3 %, high risk (83–123 μg/g ww) in 2 % and severe risk (>123 μg/g ww) in 3 % of all individuals for health effects in marine mammals. In conclusion, using marine mammals as integrative sentinel species for Danish North Sea, Inner Danish Waters and the Baltic Sea ecosystems provides monitoring of ocean health in terms of multiple stressors such as anthropogenic contaminants and infectious diseases all being important in the context of global change.

Multiple “spaces”: using wildlife surveillance, climatic variables, and spatial statistics to identify and map a climatic niche for endemic plague in California, U.S.A.

Regional climatic features in endemic areas can help inform surveillance for plague, a bacterial disease typically transmitted by fleas and maintained in mammals. We use 7,954 coyotes (Canis latrans), a sentinel species for plague, screened for plague exposure by the California Department of Public Health - Vector-Borne Disease Section (CDPH-VBDS; 1983-2015) to identify and map plague-suitable local climates within California to empirically inform ongoing sampling and surveillance plans. Using spatial point processes, we compare the distributions of seropositive and seronegative coyotes within the “space” defined by the first two principal components of PRISM Climate Group 30-year average climate variables (primarily temperature and moisture). The approach identifies both regions consistent with CDPH-VBDS mapping of plague-positive rodent and other carnivore samples over the same period and additional plague-suitable areas with climate profiles similar to seropositive samples elsewhere but little or no historical sampling, providing new data-informed insight for prioritizing limited surveillance resources.

Low-level ionizing radiation-induced DNA responses in the Asian green mussel Perna viridis

Cesium-137 (Cs-137) is a radioactive isotope present in marine environments due to the operation of nuclear power plants and weapons testing. Radiocesium poses a potential risk to marine life due to its long half-life and bioaccumulation. This study evaluated the genotoxicity of low doses of Cs-137 in the Asian green mussel Perna viridis, a sentinel species for marine pollution monitoring, by performing the comet assay and micronucleus test on hemolymph samples. Genotoxicity was assessed after exposing mussels to Cs-137 at dose rates of 0, 5, 10, and 15 μGy/h for 48 h. Cs-137's organ-specific distribution was also determined using HPGe gamma spectrometry. Even at low radiation doses, Cs-137 was found to exert genotoxic effects. Significant increases in DNA strand breaks (%Tail DNA) and micronucleus formation (MNF) were observed at all tested dose rates compared with the levels in controls, with dose-dependent responses. Cs-137 predominantly accumulated in the soft tissues, specifically the gills and digestive gland. The findings support the recommended safety level of 10 μGy/h for aquatic organisms, suggesting its appropriateness as a fundamental criterion for developing the national marine water quality standard for Cs-137 in Thailand.

Does temperature influence on biomarker responses to copper exposure? The invasive bivalve Limnoperna fortunei (Dunker 1857) as a model

Biomarkers are useful tools for assessing the early warning effects of pollutants. However, their responses can be influenced by confounding factors. In this study, we investigated the influence of temperature on multiple biomarkers in the invasive freshwater bivalve Limnoperna fortunei exposed to copper (Cu). The mussels were exposed to low and high environmental Cu concentrations at two temperatures (15 °C and 25 °C). After 96 h, the oxidative stress, neurotoxicity, and metabolic parameters were assessed. Our results showed that temperature is a key factor influencing biomarker responses in mussels, with higher glutathione S-transferase activity and lower energy reserves at cold temperature. In addition, the effects of Cu were greater at the highest concentration at 15 °C (increased lipid peroxidation and cholinesterase activity). Overall, these findings suggest that cold stress increases the susceptibility of L. fortunei to metal effects and highlight the importance of including temperature in toxicity testing and biomonitoring. In addition, using the invasive bivalve L. fortunei as a model could prove valuable in its role as a sentinel species for other organisms.

Comparing three common nest survey methods, using double-crested cormorants as a proposed sentinel for monitoring plastic pollution in freshwater environments

Many bird species use plastic as nest-building material, and nest surveys represent a unique opportunity to monitor environmental plastic pollution. However, the current literature lacks consistent, repeatable methodologies, making comparison across studies challenging. This study evaluated three common nest survey methods to assess the use of nest debris: photographic assessment, visual assessment, and nest deconstruction. We applied these methods to double-crested cormorant nests (Nannopterum auritum) at two locations on Lake Ontario, Canada. We found that for this species, nest deconstruction yielded the greatest accuracy and detail for detecting debris abundance, type, and colour. Of the two non-invasive methods, visual surveys outperformed photographic surveys across all metrics assessed. In our mainland colony, 100% of nests contained debris based on visual assessment, while at our offshore colony only 37.4% of nests contained debris, suggesting that location influences nest debris for this species. We detected Personal Protective Equipment (PPE)-related debris in 20% of all nests across survey years at our mainland colony (2022 and 2023), representing the first time that PPE has been documented in the nests of this species and demonstrating that nest surveys can be a useful tool for capturing changes in an evolving pollution landscape. This study contributes evidence that double-crested cormorants may be a useful sentinel species for monitoring plastic pollution in understudied, freshwater environments. Our results also demonstrate that the chosen method can greatly impact the results of a nest survey, and careful consideration of methodology should be undertaken before implementing a nest survey to monitor plastic pollution.

Assessment of health risk of antibiotics resistance genes from human disturbed habitat to wild animals: Metagenomic insights into availability and functional changes of gut microbiome

Not all antibiotic resistance genes (ARGs) pose an ecological risk to their host animals. A standard should be developed to study which types of ARGs posed an ecological risk to wild animals under human disturbances (HDs). In this study, the golden snub-nosed monkeys (Rhinopithecus roxellana) were used as sentinel species. According to the animals-associated enrichment, mobility, and pathogenicity, the ARGs in habitat of sentinel species were divided into four levels. If the mobile and pathogenic ARGs that could be collinear with the metagenome-assembled genome (MAGs) in the gut of the sentinel species, the ARGs were defined as Rank I ARGs and they were considered to have ecological risk to sentinel species. Functional genes in the MAGs that collinear with the Rank I ARGs were used to predict the health risks of sentinel species. The ecological risk to sentinel species was present in 0.158 % of the ARGs-contigs in the habitat. Cultivation and villages, but not grazing, agriculture and ecotourism, increased the ecological risk of the ARGs to wild animals, The ability of gut microbiome to acquire mobile and pathogenic ARGs increased, as did the collinear functional genes, and the health risks of the wild animals also enhanced by the disturbances of cultivation and villages. Cultivation and villages increased the nutrient content of the soil, and they had a positive effect on the ecological risk of Rank I ARGs by affecting the mobile genetic elements (MGEs), microbiome and the resistant group in the habitat, which was why the cultivation and villages increased the health risks of wild animals. We proposed that cultivation and living should be controlled, while grazing, agriculture and ecotourism could be developed in nature reserves of wild animals, but the nutrients in the wild animals' habitat should be monitored.

Contaminant Exposure and Liver and Kidney Lesions in North American River Otters in the Indian River Lagoon, Florida.

The harmful algal bloom (HAB) liver toxin microcystin (MC) and trace element biomagnification were previously detected in organisms in the Indian River Lagoon (IRL), Florida. Since there are no routine screening programs for these contaminants, liver tissue from North American river otters (Lontra canadensis), an important sentinel species in the IRL, was screened for MC via enzyme-linked immunoassay (ELISA), followed by confirmatory analyses via liquid-chromatography/mass spectrometry methods (LC-MS/MS). Liver and kidney samples were evaluated for trace element (As, Cd, Co, Cu, Fe, Hg, Mn, Mo, Pb, Se, Tl, and Zn) bioaccumulation via inductively coupled plasma mass spectrometry (ICP-MS). Histopathologic evaluation of the liver and kidney was conducted to assess possible correlation with toxic insults. Forty-three river otters were evaluated (2016-2022). Microcystin was not detected in any river otter sample (n = 37). Of those tested for trace element bioaccumulation (n = 22), no sample measured above provided reference ranges or estimated toxic thresholds for this species. There were no statistically significant patterns observed based on season, year, or age class, but sex had a small influence on trace element levels in the kidney. One individual had a kidney Cu level (52 μg/g dry weight) higher than any previously reported for this species. Trace elements were detected at presumed background levels providing baselines for future monitoring. For otters with available histopathologic evaluation (n = 28), anomalies indicative of contaminant exposure (non-specific inflammation, necrosis, and/or lipidosis) were present in the liver and kidney of 18% and 4% of individuals, respectively. However, since these lesions were not linked to abnormal trace element bioaccumulation or MC exposure, other causes (e.g., infectious disease) should be considered.

Proteogenomic reconstruction of organ-specific metabolic networks in an environmental sentinel species, the amphipod Gammarus fossarum

Metabolic pathways are affected by the impacts of environmental contaminants underlying a large variability of toxic effects across different species. However, the systematic reconstruction of metabolic pathways remains limited in environmental sentinel species due to the lack of available genomic data in many taxa of animal diversity. In this study we used a multi-omics approach to reconstruct the most comprehensive map of metabolic pathways for a crustacean model in biomonitoring, the amphipod Gammarus fossarum in order to improve the knowledge of the metabolism of this sentinel species.We revisited the assembly of RNA-seq data by de novo approaches to reduce RNA contaminants and transcript redundancy. We also acquired extensive mass spectrometry shotgun proteomic data on several organs from a reference population of G. fossarum males and females to identify organ-specific metabolic profiles.The G. fossarum metabolic pathway reconstruction (available through the metabolic database GamfoCyc) was performed by adapting the genomic tool CycADS and we identified 377 pathways representing 7630 annotated enzymes, 2610 enzymatic reactions and the expression of 858 enzymes was experimentally validated by proteomics. To our knowledge, our analysis provides for the first time a systematic metabolic pathway reconstruction and the proteome profiles of these pathways at the organ level in this sentinel species. As an example, we show an elevated abundance in enzymes involved in ATP biosynthesis and fatty acid beta-oxidation indicative of the high-energy requirement of the gills, or the key anabolic and detoxification role of the hepatopancreatic caeca, as exemplified by the specific expression of the retinoid biosynthetic pathways and glutathione synthesis.In conclusion, the multi-omics data integration performed in this study provides new resources to investigate metabolic processes in crustacean amphipods and their role in mediating the effects of environmental contaminant exposures in sentinel species. SynopsisThis study provide the first evidence that it is possible to combine multiple omics data to exhaustively describe the metabolic network of a model species in ecotoxicology, Gammarus fossarum, for which a reference genome is not yet available.

Detection of Trypanosoma cruzi DNA in lizards: Using non-lethal sampling techniques in a sylvatic species with zoonotic reservoir potential in Chile

Several reptile species have been described as hosts of Trypanosoma cruzi, the causative agent of Chagas disease, and therefore, they have become vertebrates of epidemiological interest. In recent decades, there has been a growing interest in animal welfare, especially in populations with small numbers where lethal sampling could have catastrophic consequences, and non-lethal methodologies have been developed for detecting zoonotic parasites. In this study, we compared three non-lethal sampling methodologies for detecting T. cruzi DNA in 21 captured specimens of the native lizard Liolaemus monticola, collected from the semiarid Mediterranean ecosystem of Chile. Specimens were subjected to xenodiagnosis (XD), tail clipping, and living syringe sampling procedures to evaluate whether lizards could serve as sentinel species for T. cruzi in endemic regions. To detect the protozoan, real-time PCR (qPCR) was performed on the DNA extracted from the samples (intestinal contents, tail tissues, and blood from living syringes). Trypanosoma cruzi DNA was detected in 12 of 21 lizards, considering all three methodologies. By XD, 12 specimens showed infection (57.1 %), and both living syringe and tail sampling methodologies detected only one infected lizard (4.8 %). Therefore, T. cruzi can be detected in lizards by qPCR using the three methodologies but XD is by far the most effective non-lethal detection methodology. The use of tail and living syringe methodologies showed a large underestimation; however, they might be options for monitoring the presence of T. cruzi in lizard populations when large sample sizes are available.

Biotransformation of carbamazepine and nicotine in juvenile American alligator (Alligator mississippiensis) in vitro hepatic S9 vs. in situ perfused liver

American alligators (Alligator mississippiensis) are apex predators and sentinel species in the coastal wetland ecosystem along the Gulf of Mexico. There is concern for alligator exposure and susceptibility to chemical contaminants due to their high trophic level and lower metabolic capability. At present, their hepatic biotransformation capacity to metabolize or detoxify contaminants has not been comprehensively determined. In this study, the hepatic biotransformation capability of juvenile American alligators to metabolize two commonly found environmental pharmaceuticals: carbamazepine (CBZ) or nicotine (NCT) was evaluated. The formation of their respective primary metabolites, i.e., carbamazepine-10,11-epoxide (CBZ-E) and cotinine (CTN), was evaluated at 10 μM (within the human therapeutic range). The in vitro S9 and a novel in situ liver perfusion assays were used to characterize and compare metabolic ability in isolated hepatic enzymes vs. whole organ (liver). For CBZ, the perfused livers exhibited only 30% of intrinsic formation clearance (CLf,int) relative to the S9 assay. The metabolism of NCT was not detectable in the S9 assay and was only observed in the perfused liver assay. Compared to the corresponding rat models (S9 or perfused livers),alligators' CLf,int was 2060% for CBZ and 50% for NCT of rats. Additionally, NCT exposure increased lactate levels in perfused livers indicating metabolic stress. This study provides insight into the hepatic capability of alligators to metabolize CBZ and NCT using an established in vitro (S9) system and a newly developed in situ liver perfusion system.

Legacy effects of Phragmites australis and herbicide treatments can reduce survival but do not prevent native plant establishment

Introduced Phragmites australis represents a widespread threat to North American wetlands. Management agencies invest millions of dollars annually to manage the species, mostly by applying herbicides, to mitigate and prevent negative impacts. The often temporary reduction of P. australis rarely increases native diversity, and long‐term legacy effects of P. australis and repeat herbicide treatments are unknown. We used a coordinated management program targeting mostly small P. australis populations in the Adirondack Park in New York State, United States, to assess the potential for such legacy effects. We planted individuals of three native species as sentinels into treated and untreated, uninvaded reference wetlands after unassisted succession had occurred in treatment areas. Sentinel plants survived and grew in treated areas, suggesting legacy of P. australis and its management did not permanently limit establishment of native plants. However, responses were variable among sentinel species, with negative or neutral impacts on survival rates and neutral or positive effects on growth. Species‐specific responses and large variation in survival rates between sites and sentinels indicate that site factors are a dominant influence on survival and growth. Importantly, as treatment frequency increased, survival of one sentinel species decreased significantly, indicating a potential for long‐term negative impacts of repeated herbicide applications. Additional replanting after P. australis treatment and initial unassisted plant succession may enable more diverse plant communities to return, including species not able to recruit from local seed banks or seed sources. However, it remains unclear if active transplanting will enable more long‐term suppression of P. australis.

Active mussel biomonitoring for the health status assessment of the Western Mediterranean Sea

The Western Mediterranean coast is under the influence of anthropogenic pressures, including land use, increasing amounts of dangerous waste and habitat destruction. In 2021, the French RINBIO network (http://www.ifremer.fr/envlit/) originally dedicated to assess chemical contamination in the region, focused on biological effects produced by contaminants and the interaction with natural variability in mussels using an active caging strategy. Cell and tissue level biomarkers were applied for 17 sampling sites divided in three sub-regions categorized by different environmental conditions. Results provide critical information for ecosystem health assessment using mussels as sentinel species in the Western Mediterranean Sea. The influence of natural and confounding factors (trophic condition, reproductive cycle, caging strategy), on biological responses to mild chemical contamination, was discussed and discriminated for health status assessment. Results provide valuable data available as reference values for the assessment of biomarkers and histopathological alterations for large-scale active biomonitoring campaigns in the Western Mediterranean Sea.