Determination of residue levels of rodenticide in rodent livers offered novel diphacinone baits by liquid chromatography-tandem mass spectrometry.

A specific and sensitive liquid chromatography-tandem mass spectrometry method was developed and validated for the determination of the anticoagulant rodenticide diphacinone (DPN) in mouse and rat liver. Tissue samples were extracted with a mixture of water and acetonitrile containing ammonium hydroxide. The extracted sample was cleaned up with a combination of liquid-liquid partitioning and dispersive solid phase extraction. Chromatographic separation was achieved using a Waters X-Bridge BEH C-18 LC column (50 mm, 2.1 mm ID, 2.5 µm particle size) with detection on a triple quadrupole mass spectrometer in multiple reaction monitoring (MRM) mode. The monitored transition for DPN was m/z 339.0 → 167.0 for quantitation and 339.0 → 172.0 and 339.0 → 116.0 for confirmation. The linear range was 0.5 to 375 ng/mL. The average precision of DPN, represented by the relative standard deviation of the observed concentrations, was 7.2% (range = 0.97% - 20.4%) and the average accuracy, represented by the relative error, was 5.8% (range = 1.06% - 14.7%). The recovery of DPN fortified at 3 different levels averaged 106% in rat liver and 101% in mouse liver. The established method was successfully used to determine DPN residue levels in Polynesian rats (Rattus exulans) and mice (Mus musculus) fed two different formulated baits containing DPN. The observed residue levels were consistent with values observed in other rodent studies. However, the amount of bait consumed was lower for the novel baits evaluated in this study.

Evaluation of roost culling as a management strategy for reducing invasive rose-ringed parakeet (Psittacula krameri) populations.

Rose-ringed parakeets (Psittacula krameri) are one of the most widespread invasive avian species worldwide. This species was introduced to the island of Kaua'i, Hawai'i, USA, in the 1960s. The rapidly increasing population has caused substantial economic losses in the agricultural and tourism industries. We evaluated the efficacy of a roost culling program conducted by an independent contractor from March 2020 to March 2021. We estimated island-wide minimum abundance was 10,512 parakeets in January 2020 and 7,372 in April 2021. Over 30 nights of culling at four roost sites, approximately 6,030 parakeets were removed via air rifles with 4,415 (73%) confirmed via carcasses retrieval. An estimated average of 45 parakeets were removed per hour of shooter effort. The proportion of adult females removed in 2020 was 1.9 × greater when culled outside of the estimated nesting season. Of the four roosts where culling occurred, the parakeets fully abandoned three and partially abandoned one site. Of the three fully abandoned roosts, an estimated average of 29.6% of birds were culled prior to roost abandonment. The roost culling effort was conducted during the COVID-19 pandemic, when tourist numbers and foot traffic were greatly reduced. It is unknown how public perception of roost culling in public areas may impact future efforts. Findings suggest roost culling can be utilized for management of nonnative rose-ringed parakeet populations when roost size is small enough and staff size large enough to cull entire roosts in no greater than two consecutive nights (e.g., if two shooters are available for three hours per night, roost culling should only be attempted on a roost with ≤ 540 rose-ringed parakeets). Supplementary Information: The online version contains supplementary material available at 10.1007/s10530-022-02984-3.

Predation thresholds for reintroduction of native avifauna following suppression of invasive Brown Treesnakes on Guam.

The brown treesnake (BTS) (Boiga irregularis) invasion on Guåhan (in English, Guam) led to the extirpation of nearly all native forest birds. In recent years, methods have been developed to reduce BTS abundance on a landscape scale. To help assess the prospects for the successful reintroduction of native birds to Guåhan following BTS suppression, we modeled bird population persistence based on their life history characteristics and relative sensitivity to BTS predation. We constructed individual-based models and simulated BTS predation in hypothetical founding populations for each of seven candidate bird species. We represented BTS predation risk in two steps: risk of being encountered and risk of mortality if encountered. We link encounter risk from the bird's perspective to snake contact rates at camera traps with live animal lures, the most direct practical means of estimating BTS predation risk. Our simulations support the well-documented fact that Guåhan's birds cannot persist with an uncontrolled population of BTS but do indicate that bird persistence in Guåhan's forests is possible with suppression short of total eradication. We estimate threshold BTS contact rates would need to be below 0.0002-0.0006 snake contacts per bird per night for these birds to persist on the landscape, which translates to an annual encounter probability of 0.07-0.20. We simulated the effects of snake-proof nest boxes for Sihek (Todiramphus cinnamominus) and Såli (Aplonis opaca), but the benefits were small relative to the overall variation in contact rate thresholds among species. This variation among focal bird species in sustainable predation levels can be used to prioritize species for reintroduction in a BTS-suppressed landscape, but variation among these species is narrow relative to the required reduction from current BTS levels, which may be four orders of magnitude higher (>0.18). Our modeling indicates that the required predation thresholds may need to be lower than have yet been demonstrated with current BTS management. Our predation threshold metric provides an important management tool to help estimate target BTS suppression levels that can be used to determine when bird reintroduction campaigns might begin and serves as a model for other systems to match predator control with reintroduction efforts.

Locally abundant, endangered Mariana swiftlets impact the abundance, behavior, and body condition of an invasive predator.

Invasive predators are known to have negative consumptive and non-consumptive effects on native species, but few examples show how the abundance of native prey may influence an established invasive predator. We compared invasive brown treesnakes (Boiga irregularis; BTS) found in caves occupied by endangered Mariana swiftlets (Aerodramus bartschi) to snakes found in nearby forests and caves without birds to quantify how the abundance of native avian prey impacts BTS abundance and behavior on Guam. From 2011 to 2017 we removed 151 BTS in caves occupied by swiftlets and never observed BTS in caves without birds. Notable locations included snakes foraging near swiftlets and in holes that allowed cave access and escape from capture. Of 43 BTS with gut contents, 27 (63%) contained swiftlets. BTS in swiftlet-occupied caves had greater fat mass compared to forests, indicating access to swiftlets may increase body condition and promote reproduction. Number of ovarian follicles was significantly greater in female snakes from swiftlet-occupied caves compared to those from ravine, but not limestone forests; evidence of male BTS being more capable of reproduction was limited (i.e., fewer non-discernible but not significantly larger testes in snakes from caves). Assuming other limiting factors are considered, altering the functional response of predators through the modification of caves or interdiction lures to exclude or hinder the largest BTS could bolster swiftlet populations by increasing nesting refugia in currently-occupied caves and facilitate recolonization of historical caves.

Variation in Angiostrongylus cantonensis infection in definitive and intermediate hosts in Hawaii, a global hotspot of rat lungworm disease.

Angiostrongylus cantonensis (rat lungworm) is a tropical and subtropical parasitic nematode, with infections in humans causing angiostrongyliasis (rat lungworm disease), characterized by eosinophilic meningitis. Hawaii has been identified as a global hotspot of infection, with recent reports of high infection rates in humans, as well as rat definitive and snail intermediate hosts. This study investigated variation in A. cantonensis infection, both prevalence and intensity, in wild populations of two species of rats (Rattus exulans and R. rattus) and one species of snail (Parmarion martensi). An overall infection prevalence of 86.2% was observed in P. martensi and 63.8% in rats, with R. exulans (77.4%) greater than R. rattus (47.6%). We found infections to vary with environmental and host-related factors. Body mass was a strong predictor of infection in all three species, with different patterns seen between sexes and species of rats. Infection prevalence and intensity for R. exulans were high in May 2018 and again in February 2019, but generally lower and more variable during the intervening months. Information on sources of variability of infection in wild host populations will be a crucial component in predicting the effectiveness of future disease surveillance or targeted management strategies.

Occurrence of Rat Lungworm (Angiostrongylus cantonensis) in Invasive Coqui Frogs (Eleutherodactylus coqui) and Other Hosts in Hawaii, USA.

The rat lungworm (Angiostrongylus cantonensis) has emerged as an important human and animal health concern in Hawaii, US. Although the life cycle of the parasite requires both rat and gastropod hosts, other animals acting as paratenic hosts, such as frogs and centipedes, have been identified as sources of infection. We investigated the occurrence of rat lungworm infections in potential paratenic hosts in Hawaii to provide information on how they might be involved in transmission of angiostrongyliasis. We confirmed the presence of rat lungworm in 87% (21/24) of introduced Puerto Rican coqui frogs (Eleutherodactylus coqui) in Hilo, Hawaii, by real-time PCR. Additionally, four Cuban greenhouse frogs (Eleutherodactylus planirostris), two cane toads (Rhinella marina), and three centipedes (Scolopendra subspinipes) were found to be infected. In the frogs and toads, multiple tissue types were positive, including stomach and intestine, muscle, liver, heart, and brain, indicating larval migration. We identified rat lungworm infections in frogs, toads, and centipedes in Hawaii and highlighted the lack of knowledge of the role paratenic hosts may be playing in the transmission and life cycle maintenance of rat lungworm in Hawaii.

Validation of a death assay for Angiostrongylus cantonensis larvae (L3) using propidium iodide in a rat model (Rattus norvegicus).

Angiostrongylus cantonensis is a pathogenic nematode and the cause of neuroangiostrongyliasis, an eosinophilic meningitis more commonly known as rat lungworm disease. Transmission is thought to be primarily due to ingestion of infective third stage larvae (L3) in gastropods, on produce, or in contaminated water. The gold standard to determine the effects of physical and chemical treatments on the infectivity of A. cantonensis L3 larvae is to infect rodents with treated L3 larvae and monitor for infection, but animal studies are laborious and expensive and also raise ethical concerns. This study demonstrates propidium iodide (PI) to be a reliable marker of parasite death and loss of infective potential without adversely affecting the development and future reproduction of live A. cantonensis larvae. PI staining allows evaluation of the efficacy of test substances in vitro, an improvement upon the use of lack of motility as an indicator of death. Some potential applications of this assay include determining the effectiveness of various anthelmintics, vegetable washes, electromagnetic radiation and other treatments intended to kill larvae in the prevention and treatment of neuroangiostrongyliasis.

Behavioral differences following ingestion of large meals and consequences for management of a harmful invasive snake: A field experiment.

Many snakes are uniquely adapted to ingest large prey at infrequent intervals. Digestion of large prey is metabolically and aerobically costly, and large prey boluses can impair snake locomotion, increasing vulnerability to predation. Cessation of foraging and use of refugia with microclimates facilitating digestion are expected to be strategies employed by free-ranging snakes to cope with the demands of digestion while minimizing risk of predation. However, empirical observations of such submergent behavior from field experiments are limited. The brown treesnake (Serpentes: Colubridae: Boiga irregularis) is a nocturnal, arboreal, colubrid snake that was accidentally introduced to the island of Guam, with ecologically and economically costly consequences. Because tools for brown treesnake damage prevention generally rely on snakes being visible or responding to lures or baits while foraging, cessation of foraging activities after feeding would complicate management. We sought to characterize differences in brown treesnake activity, movement, habitat use, and detectability following feeding of large meals (rodents 33% of the snake's unfed body mass) via radio telemetry, trapping, and visual surveys. Compared to unfed snakes, snakes in the feeding treatment group showed drastic decreases in hourly and nightly activity rates, differences in refuge height and microhabitat type, and a marked decrease in detectability by trapping and visual surveys. Depression of activity lasted approximately 5-7 days, a period that corresponds to previous studies of brown treesnake digestion and cycles of detectability. Our results indicate that management strategies for invasive brown treesnakes need to account for cycles of unavailability and underscore the importance of preventing spread of brown treesnakes to new environments where large prey are abundant and periods of cryptic behavior are likely to be frequent. Characterization of postfeeding behavior changes provides a richer understanding of snake ecology and foraging models for species that consume large prey.

Quantile regression of microgeographic variation in population characteristics of an invasive vertebrate predator.

Localized ecological conditions have the potential to induce variation in population characteristics such as size distributions and body conditions. The ability to generalize the influence of ecological characteristics on such population traits may be particularly meaningful when those traits influence prospects for successful management interventions. To characterize variability in invasive Brown Treesnake population attributes within and among habitat types, we conducted systematic and seasonally-balanced surveys, collecting 100 snakes from each of 18 sites: three replicates within each of six major habitat types comprising 95% of Guam's geographic expanse. Our study constitutes one of the most comprehensive and controlled samplings of any published snake study. Quantile regression on snake size and body condition indicated significant ecological heterogeneity, with a general trend of relative consistency of size classes and body conditions within and among scrub and Leucaena forest habitat types and more heterogeneity among ravine forest, savanna, and urban residential sites. Larger and more robust snakes were found within some savanna and urban habitat replicates, likely due to relative availability of larger prey. Compared to more homogeneous samples in the wet season, variability in size distributions and body conditions was greater during the dry season. Although there is evidence of habitat influencing Brown Treesnake populations at localized scales (e.g., the higher prevalence of larger snakes-particularly males-in savanna and urban sites), the level of variability among sites within habitat types indicates little ability to make meaningful predictions about these traits at unsampled locations. Seasonal variability within sites and habitats indicates that localized population characterization should include sampling in both wet and dry seasons. Extreme values at single replicates occasionally influenced overall habitat patterns, while pooling replicates masked variability among sites. A full understanding of population characteristics should include an assessment of variability both at the site and habitat level.

Invaded Invaders: Infection of Invasive Brown Treesnakes on Guam by an Exotic Larval Cestode with a Life Cycle Comprised of Non-Native Hosts.

BACKGROUND: Multiple host introductions to the same non-native environment have the potential to complete life cycles of parasites incidentally transported with them. Our goal was to identify a recently detected parasitic flatworm in the invasive Brown Treesnake (Boiga irregularis) on the remote Pacific island of Guam. We considered possible factors influencing parasite transmission, and tested for correlations between infection status and potential indicators of host fitness. We used genetic data from the parasite and information about the native ranges of other possible non-native hosts to hypothesize how it arrived on Guam and how its life cycle may be currently supported. METHODS: We identified the parasite by comparing larval morphology and mtDNA sequences with other Pseudophyllid tapeworms. We assessed probability of infection in individual snakes using logistic regression and examined different factors influencing presence of parasites in hosts. RESULTS: We identified the parasite as the pseudophyllid cestode Spirometra erinaceieuropaei, with all sampled worms from multiple snakes sharing a single mtDNA haplotype. Infection appears to be limited to the only freshwater watershed on the island, where infection prevalence was high (77.5%). Larger snakes had a higher probability of being infected, consistent with the chronic nature of such infections. While infection status was positively correlated with body condition, infected snakes tended to have lower intra-peritoneal fat body mass, potentially indicating a negative effect on energy stores. CONCLUSIONS: We discovered that B. irregularis inhabiting a small area of forested habitat in a freshwater watershed on Guam are often infected by a novel parasite of Asian origin. While further work is needed, this species of Spirometra, itself a non-native species, likely depends on a suite of recently introduced hosts from different parts of the world to complete the life cycle. This baseline study provides little evidence of any effects on host fitness, but additional data are needed to more thoroughly explore the consequences of infection in this invasive snake population.