The role of ion homeostasis imbalance due to citrate accumulation in fluoroacetic acid (FAA) toxicity in Neurospora crassa.

Fluoroacetic acid (FAA) is a poison commonly used for the lethal control of invasive species in Australia and New Zealand. Despite its widespread use and long history as a pesticide, no effective treatment for accidental poisoning exists. Although it is known to inhibit the tricarboxylic acid (TCA) cycle, specific details of FAA toxicology have remained elusive, with hypocalcemia suggested to be involved in the neurological symptoms prior to death. Here, we study the effects of FAA on cell growth and mitochondrial function using the filamentous fungi Neurospora crassa as model organism. FAA toxicosis in N. crassa is characterized by an initial hyperpolarization and subsequent depolarization of the mitochondrial membranes, followed by a significant intracellular decrease in ATP and increase in Ca2+. The development of mycelium was markedly affected within 6 h, and growth impaired after 24 h of FAA exposure. Although the activity of mitochondrial complexes I, II and IV was impaired, the activity of citrate synthase was not affected. Supplementation with Ca2+ exacerbated the effects of FAA in cell growth and membrane potential. Our findings suggest that an imbalance created in the ratio of ions within the mitochondria may lead to conformational changes in ATP synthase dimers due to mitochondrial Ca2+ uptake, that ultimately result in the opening of the mitochondrial permeability transition pore (MPTP), a decrease in membrane potential, and cell death. Our findings suggest new approaches for the treatment research, as well as the possibility to use N. crassa as a high-throughput screening assay to evaluate a large number of FAA antidote candidates.

Monitoring methaemoglobinaemia in birds using 5 µL of whole blood.

Methaemoglobin (MetHb) forming compounds such as para-aminopropiophenone (PAPP) and sodium nitrite (NaNO2) have recently been adopted for the lethal control of a range of invasive carnivores and mustelids. Determining the relative hazard of these compounds to non-target bird species is an important component of ecological risks evaluation. Problematically, some potential non-target bird species may be as small as 10 g in body mass, thus placing limitations on blood volumes that can be routinely sampled. Accordingly, we developed methods to quantify markers of increasing methaemoglobinaemia at their point of collection that required only 5 µL of whole blood. A 3 µL blood aliquot is pipetted into a plastic micro-cuvette and placed in a custom made holder optically coupled to the Ocean Optics spectrometer, enabling absorbance for oxyhaemoglobin (HbO: 575 nm) and MetHb (630 nm) to be determined. Haemoglobin (HbFe2+), packed cell volume (PCV) and lactate (LAC) data were generated from the remaining 2 µL aliquot apportioned to biosensor strips for the Cera-Check® and Lactate Scout® point-of-care devices. After oral doses of PAPP, a methaemoglobinaemia absorbance index (MAI = absorbance 575 nm-absorbance 630 nm) was strongly and significantly associated with dose-dependent declines in HbFe2+ in 9 bird species. Quantifying dose-dependent responses to MetHb-forming agents at the point of sample collection avoids analytical and storage artifacts arising from sample degradation that appears to be a much greater problem in avian blood compared to mammalian blood.

Sensitivity of High Conservation Value Birds to Para-Aminopropiophenone (PAPP) Determined by Sub-Lethal Dose-Response Assay.

Para-aminopropiophenone (PAPP) is a methaemoglobin (MetHb) forming compound used for the lethal control of invasive carnivores and mustelids. By measuring the dose-dependent inhibition of O2 transport arising from the oxidation of haemoglobin (HbFe2+) to MetHb (HbFe3+), we determined the sensitivity of nine bird species to PAPP. A methaemoglobinaemia absorbance index (MAI) was validated in five common bird species to determine thresholds associated with a 99% probability of survival (ST99) and a 50% probability of mortality (LT50). Dose-response trials in high conservation value birds sought MAI values below the ST99 threshold, projecting the LT50 value and avoiding the need for lethal outcomes. Black-backed gull (LT50 = 1784.7) and eastern rosella (LT50 = 1074 mg kg-1) were the most tolerant species, while brown kiwi (LT50 = 8.4 mg kg-1) and weka (LT50 = 9.3 mg kg-1) were the most sensitive. Takahe were of intermediate acute sensitivity (LT50 = 51 mg kg-1), although protracted impacts on haemoglobin were observed in takahe up to 72 h later and associated with PAPP doses as low as 25.6 mg kg-1. In pukeko (LT50 = 138.4 mg kg-1), protracted declines in haemoglobin 72 h later occurred at doses as low as 29.5 mg kg-1, while at higher doses (253 and 112 mg kg-1), deaths resulted after 4-6 days. Based upon PAPP doses that caused acute and protracted responses, we provide estimates for the lowest observable adverse effect level (LOAEL) and no observable effects level (NOEL) for nine bird species.

Non-Lethal Dose-Response Models Replace Lethal Bioassays for Predicting the Hazard of Para-Aminopropiophenone to Australian Wildlife.

Para-aminopropiophenone (PAPP) is a potent methaemoglobin (MetHb) forming agent used for the lethal control of exotic carnivores and mustelids. To assess the sensitivity of Australian wildlife to PAPP we developed an in vivo assay that did not use death as an endpoint. Sub-lethal dose-response data were modelled to predict PAPP doses required to achieve an endpoint set at 80% MetHb (MetHb80). The comparative sensitivity of non-target mammals referenced to this endpoint was found to be highly variable, with southern brown bandicoots (Isoodon obesulus) the most sensitive species (MetHb80 = 6.3 mg kg-1) and bush rats (Rattus fuscipes) the most tolerant (MetHb80 = 1035 mg kg-1). Published LD50 estimates were highly correlated with PAPP doses modelled to achieve the MetHb80 endpoint (r2 = 0.99, p < 0.001). Most dose-response data for native mammals were collected in the field or in semi-natural enclosures, permitting PAPP and placebo dosed animals to be fitted with tracking transmitters and transponders and released at their point of capture. A protracted morbidity and mortality was observed only in Australian ravens (Corvus coronoides). The combination of sub-lethal dose-response assay and survival data collected in the field provided more relevant information about the actual hazard of pest control agents to non-target wildlife species than laboratory-based lethal-dose bioassays. We discuss the need to replace lethal-dose data with biologically meaningful insights able to define a continuum of toxicological hazards that better serve the needs of conservation and veterinary scientists and wildlife managers.

Trends in anecdotal fox sightings in Tasmania accounted for by psychological factors.

There has been little evaluation of anecdotal sightings as a means to confirm new incursions of invasive species. This paper explores the potential for equivocal information communicated by the media to account for patterns of anecdotal reports. In 2001, it was widely reported that red foxes (Vulpes vulpes) had been deliberately released in the island state of Tasmania (Australia), although this claim was later revealed to be baseless. Regardless, by 2013 a total of 3153 anecdotal fox sightings had been reported by members of the public, which implied their distribution was wide. For each month in 2001-2003, we defined a monthly media index (MMI) of fox-related media coverage, an index of their relative seasonal abundance (abundance), and a factor denoting claims of fox evidence (claimed evidence) regardless of its evidentiary quality. We fitted a generalized linear model with Poisson error for monthly totals of anecdotal sightings with factors of year and claimed evidence and covariates of MMI, abundance, and hours of darkness. The collective effect of psychological factors (MMI, claimed evidence, and year) relative to biophysical factors (photoperiod and abundance) was highly significant (χ2 = 122.1, df = 6, p < 0.0001), whereas anticipated changes in abundance had no significant influence on reported sightings (p = 0.15). An annual index of fox media from 2001 to 2010 was strongly associated with the yearly tally of anecdotal sightings (p = 0.018). The odds ratio of sightings ranked as reliable by the fox eradication program in any year decreased exponentially at a rate of 0.00643 as the total number of sightings increased (p < 0.0001) and was indicative of an observer-expectancy bias. Our results suggest anecdotal sightings are highly susceptible to cognitive biases and when used to qualify and quantify species presence can contribute to flawed risk assessments.

The dispersion and detection patterns of mtDNA-assigned red fox Vulpes vulpes scats in Tasmania are anomalous.

Models used for resource allocation in eradication programmes must be based on replicated data of known quality and have proven predictive accuracy, or they may provide a false indication of species presence and/or distribution. In the absence of data corroborating the presence of extant foxes Vulpes vulpes in Tasmania, a habitat-specific model based upon mtDNA data (Sarre et al. 2012. Journal Applied Ecology, 50, 459-468) implied that foxes were widespread. Overall, 61 of 9940 (0·6%) surveyed scats were assigned as mtDNA fox positive by the fox eradication programme (FEP). We investigated the spatiotemporal distribution of the 61 mtDNA-assigned fox scats and modelled the probability of replicating scat detection in independent surveys using detection dogs based upon empirically derived probabilities of scat detection success obtained by the FEP using imported fox scats. In a prior mainland study, fox genotypes were recurrently detected in a consecutive four-day pool of scats. In Tasmania, only three contemporaneously collected scat pairs of unknown genotype were detected by the FEP within an area corresponding to a conservatively large mainland fox home range (639 ha) in a decade. Nearest neighbour pairs were widely spaced (mean = 7·0 km; circular area = 153 km2) and generated after a mean of 281 days. The majority of assigned mtDNA positive scats were found in urban and peri-urban environments corresponding to small mainland fox home ranges (30-45 ha) that imply higher scat density and more certain replication. Using the lowest empirically determined scat detection success for dogs, the failure to replicate fox scat detection on 34 of 36 occasions in a large (639 ha) home range is highly improbable (P = 0·00001) and suggestive of Type I error. Synthesis and applications. Type I error, which may have various sources, should be considered when scat mtDNA data are few, accumulated over many years, uncorroborated by observations of extant specimens, inadequately replicated in independent surveys within an expected spatiotemporal scale and reported in geographically isolated environments unlikely to have been colonized.

Bait-delivered pimozide causes precocious embryo implantation in mink: a fertility control option for the exotic stoat?

Stoats (Mustela erminea), an exotic pest in New Zealand, threaten the conservation of several ground-nesting bird species and broad-scale methods for their control are sought. Females are seasonally monestrous, show a 9-month period of obligatory diapause and usually do not breed more than once in their lives. A bait-delivered agent that terminates diapause and results in a non-viable embryo may have a significant impact on their reproductive success. Prolactin (PRL) is hypothesised to be the only gonadotrophin required for renewal of luteal activity and blastocyst implantation in some mustelids. We investigated the effects of bait-delivered dopamine (DA) antagonists (which stimulate the release of PRL) using a mink model (Mustela vison), a species that maintains a short period of diapause. A bait dose of 0.8 mg kg(-1) of pimozide was more effective in elevating PRL levels than equivalent doses of fluphenazine, sulpiride (P < 0.01) or haloperidol (P < 0.05). Bait doses of 1.6 mg kg(-1) pimozide given at Days 0, 3, 9 and 11 after mating caused a significant reduction in the length of pregnancy compared with a positive control and placebo (46 days v. 51 days), indicating early termination of diapause (P < 0.01). Pimozide doses caused higher elevations in PRL concentration relative to the oral placebo by Day 12, but mean PRL levels fell below all other groups by Day 18. A borderline significant increase in progesterone (P4) secretion compared with the oral placebo was detected at Day 18. These results suggest that bait-delivered pimozide can elevate PRL outside of the normal breeding season and doses of 1.6 mg kg(-1) are effective in terminating embryonic diapause in mink. The implications and limitations of these data are discussed with reference to the use of bait-delivered DA antagonists as a possible means to affect the reproductive success of wild stoats.

Control of fertility in the red fox (Vulpes vulpes): effect of a single oral dose of cabergoline in early pregnancy.

Single oral doses of 100 microg kg(-1) cabergoline (CAB) were previously found to affect the reproductive success of silver fox (Vulpes vulpes) vixens from Day 28 post insemination (PI) onwards by causing abortions and postnatal mortality. The present trial investigated the effect of a single oral dose of 100 microg kg(-1) CAB given to farmed silver fox vixens at 7, 14, 21 or 28 days PI. Six vixens were dosed with CAB at each day PI and abortions, total post-partum litter loss and the combined effects of both were compared with a group of vixens that received a placebo and a control group of vixens that received no treatment. No significant difference existed between the placebo and control groups in the incidence of abortion, postnatal litter loss and litter size at 3 weeks post partum. Overall, a significantly higher frequency of abortions were detected in vixens dosed with CAB (6/24) compared with the combined placebo and control (0/41) groups (P<0.01). Cabergoline administration was significantly associated with complete litter loss (P<0.01) and the combined effects of abortion and litter loss (P<0.001). Oral CAB significantly reduced the reproductive success of vixens when given at Day 21 and Day 28 PI, but not at Day 7 and Day 14 PI.