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(A) Background, Problem Statement and Strategies for Mountain Chickens in Montserrat 1. Background The mountain chicken Leptodactylus fallax, a frog, is currently assessed as Critically Endangered by IUCN due to a severe population decline inferred from chytridiomycosis infection (‘chytrid’) and habitat loss through volcanic damage (Fa et al 2008). The species is now known only from two Eastern Caribbean Islands, Montserrat and Dominica. The causative agent of chytrid, the fungus Batrachochytrium dendrobatidis (Bd), was first recorded from Dominica in 2002 and is thought, within a single year, to have lead to an 80-90% decline in that island’s mountain chicken population, which has remained at these greatly reduced levels since. Symptoms of chytrid were first reported from Montserrat on 14th February 2009, when Montserrat Department of Environment (DOE) forestry officers found dead and dying mountain chickens in the island’s Centre Hills reserve. In response, the Durrell Wildlife Conservation Trust (Durrell) and DOE organized an immediate evacuation of 50 uninfected mountain chickens to biosecure (quarantined) captive breeding facilities in Europe. Since then, an apparent collapse of the wild population similar to that found in Dominica in 2002, has been reported. By May 2009, an international collaboration, the Mountain Chicken Recovery Programme (MCRP), was established between Durrell, The Zoological Society of London (ZSL), Chester Zoo (CZ), Parken Zoo (PZ) and the Government of Montserrat (GOM). The aim of this programme is to recover wild populations of the mountain chicken, focussing initially on Montserrat. This document details activities proposed for 2011 under the Darwin Initiative project Enabling Montserrat to save the Critically Endangered mountain chicken (project HYR1) lead by Durrell and DOE in partnership with ZSL’s Institute of Zoology (IOZ). It focuses on efforts to augment the mountain chicken population in Montserrat by reintroducing captive-bred frogs to their native range and to develop optimal strategies and protocols for future reintroductions. 2. Problem statement Chytrid causes severe long-term declines in mountain chicken populations, to the point where they may have little or no viability. Chytrid is highly infectious to mountain chickens and persists in the environment; it is also spread by other amphibian species infected by it but less severely impacted. Managing mountain chickens in a chytrid-infected environment is further hampered by other ongoing threats: non-native predators, habitat loss and hunting. The experience of trying to manage chytrid infection of the mountain chicken population on Dominica indicates the disease has a severe and long-lasting effect on this species. It seems likely, though not yet confirmed, that the mountain chickens population on Dominica is at densities now so low that they compromise breeding in those frogs. This now also appears to be the case in Montserrat. Chytrid is also infecting the other two amphibian species on Montserrat (the tree frog, Eleutherodactylus johnstonei, and the cane toad, Rhinella marina), albeit with less severe impacts on these populations. These species provide a reservoir for Bd – which can also survive in soil and water – and a vector for spreading it across the island and infecting mountain chickens. In 2010, it was confirmed that chytrid was present at the southern-most tip of Montserrat, in the island’s volcanic Exclusion Zone, uninhabited by people since 1997. It thus appears that the whole of Montserrat must now be considered infected with this disease. The fundamental problem facing efforts to re-establish a viable population of mountain chickens in Montserrat is the island-wide occurrence of chytrid and the severe impacts, normally fatal, of the disease on this species. Infected frogs can be treated to remove the infection (typically, in captivity). The reservoir of the fungus in the environment (including other amphibian species), however, means that the benefits of this treatment are only temporary: Frogs released back into their native range are re-exposed to the disease and can be re-infected. Secondary to the problem of chytrid infection once mountain chickens are reintroduced to Montserrat are the other threats also facing this species. A number of invasive alien species occur at high densities on Montserrat and are known to predate mountain chickens and/or damage their habitat. These are believed to be primarily rats (Rattus norvegicus and R. rattus) and feral pigs (Sus scrofa). Domestic and feral cats and dogs are other likely predators and other feral livestock. Loss of habitat in Montserrat due to the volcanic eruptions from 1995 to the present has been severe. Most of the mountain chicken habitat known from pre-1995 does, however, remain, primarily in Montserrat’s Centre Hills reserve. This is, nonetheless, a relatively small area of suitable mountain chicken habitat, and it is subject to periodic falls of acidic volcanic ash. Hunting mountain chickens for food is believed to have led to marked population declines on Montserrat before chytrid reached the island. At present, there seems to be little if any hunting whilst mountain chickens are at such low densities. It does, however, remain a potential threat if the population begins to recover. 3. Programme objectives The MCRP aims to recover populations of the mountain chicken to viable levels across its native range. We have restricted this document to covering the project activities for reintroducing mountain chickens to Montserrat in 2011 in support of this aim. 3.1. Project objectives for 2011 – reintroduction of mountain chickens to Montserrat. In 2011, Durrell, DOE and IOZ aim to investigate the factors that determine the success of a reintroduction. Montserrat’s Centre Hills mountain chicken population by the reintroduction of chytrid-free frogs bred in captivity in 2009-2010. There will be two reintroductions that will take the form of pilot studies to examine the effects of various factors on the success of reintroductions. These pilot studies will allow us to develop optimal strategies and protocols for future reintroductions to augment mountain chicken populations in their native range. The specific objectives of the 2011 reintroductions are to: 1. Determine survival of released frogs and causes of mortality over the next three years, but with a three-month intensive monitoring period immediately after release. 2. Determine the effect of age-at-release, season (wet or dry), and parentage on dispersal, survival of released frogs and causes of mortality 3. Determine if reintroduction leads to increased levels of chytrid in mountain chickens and other amphibian species on Montserrat which are part of the mountain chickens’ environment 4. Determine state of surviving frogs on Montserrat over the next three years, especially their infection and health status and, if possible the population distribution and trend in numbers The hypotheses that are to be tested to meet these objectives are: 1. A proportion of reintroduced frogs survive 2. (a) A higher proportion of older age-at-release and larger frogs will survive (b) A higher proportion of frogs released in the wet season will survive 3. Introducing frogs at a density of 8 frogs per hectare does not trigger mass mortality in the introduced population. 3. Reintroduction does not lead to increased levels of chytrid in other amphibian species or mountain chickens surviving at sites other than the reintroduction site. 4. Surviving (non-reintroduced) frogs remain chytrid-free (or infected but surviving) 5. The reintroduced population will breed within in the year 4. Ethical statement We believe that the long term survival of this species in its native range depends on finding ways to recover the wild population. We propose that a critical approach to this is to determine the factors affecting the survival of (a) the few mountain chickens still remaining in Montserrat and (b) captive bred mountain chickens reintroduced back into Montserrat. To do this we need to monitor surviving frogs and test reintroduction protocols. The welfare of mountain chickens will be paramount during these activities. Strict biosecurity protocols will mitigate the risk of transferring infections between frogs and between field sites. Frogs’ health status will be monitored intensively and humane euthanasia used for frogs whose health status is severely compromised by chytrid infection. We recognize that at least some chytrid-free mountain chickens reintroduced into Montserrat are likely to become infected with chytrid and that this infection may prove fatal. We recognize that at least some of the secondary threats to mountain chicken populations may also confront reintroduced frogs, primarily non-native predators. We also recognize, however, that some mountain chickens are still surviving in Montserrat since the arrival of, and (we believe) in the presence of, chytrid. They are also surviving in the face of any secondary threats. With no known method for removing the environmental reservoir of Bd on Montserrat, we believe the best course of action is to determine what affects the degree to which reintroductions are successful. We aim to identify which factors that are under our control to maximize the survival of reintroduced mountain chickens and their ability to reproduce. These factors include the season in which frogs are reintroduced; the age of frogs at reintroduction; the location of reintroductions on Montserrat and the density at which frogs are introduced. Without determining these basic parameters we believe we will never be able to move beyond a situation of a small population of frogs held in captivity indefinitely or until funds expire. Our aim is to re-establish viable mountain chicken populations in the wild in their native range. To do this we need determine why surviving frogs are surviving – albeit at apparently very low densities – and how we can ultimately augment their numbers. We believe we can only attempt to determine this if our captive-bred frogs confront the conditions now present in their native range. Released frogs will be very closely monitored to learn as much about their responses and fates as possible. A large sample of 34 will be individually tracked using radio transmitters and all frogs will be individually identifiable from PIT tags (‘microchips’). Their health status will be monitored throughout and detailed protocols are presented for the humane euthanasia of any frogs that do contact chytrid. At all stages of this work, robust biosecurity protocols are being implemented to prevent project workers from inadvertently transferring Bd between frogs (of the same species or between species) or between sites. It may be that the continued survival of mountain chickens on Montserrat is a result of them having been reduced to very low densities. Reintroduced frogs will not be released at any sites currently known, or believed, to support small numbers of surviving mountain chickens on Montserrat, so that we don’t increase the density of these survivors during our 2011 releases. The reintroduced frogs will be introduced at densities comparable to those known from the release site prior to chytrid reaching Montserrat. They will not all be released at a single point leading to inflated densities and hypothesized greater risk of chytrid infection. Candidate release sites have been assessed prior to the release to determine the presence of suitable habitat, the likely level of non-native predators and the risk of human foot traffic spreading chytrid between sites. The release site selected is known to have supported a healthy mountain chicken population prior to chytrid infecting Montserrat and is only rarely visited by people. Field monitoring programs have been designed with the welfare of individual animals in mind using information gained over many years on the reaction of frogs to handling and disturbance in captivity and in the wild. Given the location of the selected release site relatively far from human population, we believe that the secondary threat of predation by introduced predators will be minor. We have, however, developed protocols for rapidly implementing rat control at the release site if our monitoring of the reintroduced frogs reveals significant numbers of injuries from rats. Implantation of radio transmitters will be completed under anaesthesia and with a supervised recovery period of 10 days in captivity and under quarantine. Transport of frogs from Europe to Montserrat will follow the welfare in transport guidelines presented in this document and frogs will be held under supervision in secure captive conditions on arrival in Montserrat for a further four days prior to being released. Supplying food to the frogs during this period of captivity in Montserrat necessitates importing live insect food, with the danger that it could escape and contribute further to the diversity of invasive species already found on the island. Mitigative measures have been put in place to prevent this. Concurrent with the release, chytrid levels in the environment (i.e. in other, more numerous, amphibian species) will be monitored at the release site and at two control sites: one known to be still supporting surviving mountain chickens and one not known to be. We believe it is unlikely that chytrid levels at sites known to be still supporting surviving mountain chickens will be elevated as a result of our reintroduction of mountain chickens at a separate site. We have however, in 2009, demonstrated our ability to be able to rapidly evacuate frogs from an infected area to biosecure accommodation if deemed appropriate. We have been very successful in breeding the species in captivity. We believe that captive bred frogs have the maximum chance of surviving in the wild if they have not been maintained in captivity for an extended period of many years (>2yrs). Therefore we propose that an early release will improve their chances of surviving in the wild. 5. Strategy for reintroducing mountain chickens to Montserrat in 2011 5.1. Biosecurity and animal welfare Robust biosecurity protocols will be observed before during and after the captive-bred mountain chickens arrive in Montserrat. They will also be observed during all phases of the field work described below to mitigate the risk of transferring infections between frogs and between field sites. Frogs’ health status will be monitored intensively and humane euthanasia used for frogs whose health status is severely compromised by chytrid infection. Detailed protocols to ensure strict biosecurity throughout this work are presented in the biosecurity maunals. These were developed successfully for field work during the 2009 evacuation of mountain chickens from Montserrat to biosecure ex situ accommodation will be applied in all phases of the work. Our veterinary staff are providing a detailed diagnostic protocol for assessing health and infection status of mountain chickens in the wild that will be used by field staff to determine when humane euthanasia is indicated. All field staff will receive hands-on training in biosecurity and euthanasia protocols. 5.2. Reintroduction Chytrid-free mountain chickens bred in biosecure captivity are reintroduced to Montserrat in January 2011 (Montserrat’s dry season) and August 2011 (wet season). In January 2011, frogs are released at a single site known to have supported mountain chickens in the past, but not at present. The frogs in each release are in two cohorts: younger frogs bred in 2010 and older frogs breed in 2009. A sample of 34 released frogs are radio tracked to monitor their health status and determine their fates during the first three months after release. Uninfected frogs bred under strict quarantine will be released at a single site (for logistical reasons, primarily to make radio tracking feasible) in Montserrat. Prior to reintroduction a survey assessed candidate release sites (ghauts, as watercourses are known locally) was completed, to select a release site on (a) presence of suitable mountain chicken habitat; (b) ease of access to and within the site for radiotracking; and (c) low human usage (and, by implication, lower nonnative predator levels). Sweetwater Ghaut in the north east of Montserrat has been selected based on these criteria: It had a healthy mountain chicken population prior to chytrid arriving on Montserrat and we have a baseline population density estimate from mark-recapture data collected in 2005. It is relatively far from centres of human population on Montserrat and is believed to have relatively low human foot traffic visiting it. For these reasons, non-native predator levels are also believed to be relatively low. It is not a site from which mountain chicken calls have reportedly been heard in 2010. This is to minimize the risk of compromising the existing surviving mountain chickens on Montserrat for example by through increasing frog density and triggering a chytrid outbreak. All released frogs PIT tagged for individual recognition and a sample of 34 will be implanted with radio transmitters (and supervised for 10 days afterwards) in Europe prior to being transported to Montserrat. The radio transmitters will allow us to intensively monitor the status and fates of this sample over three months. Frogs will be transported from ex situ facilities under biosecure conditions and held for 4 days in Montserrat prior to reintroduction, to monitor general health post-transit, post-radio transmitter implantation. Frogs will be released along a ghaut to achieve mountain chicken densities equivalent to what was estimated for this site pre-chytrid (namely about 10 frogs for every 100m length of Sweetwater) One release will be carried out during the dry season, followed by a second one in the wet season to examine effect of season on survival. The protocols presented in this document have been developed, in the first instance, for the first January/February) release. They will be reviewed and adapted if necessary prior to the second (July/August) release. Each release cohort will contain frogs born in 2009 and 2010 to examine effect of age-atrelease on survival. Each release cohort will also contain frogs from multiple crosses (i.e. different parents) which may allow us to examine any effect of parentage on survival (though sample sizes may be too small to detect any such latter effect). As far as possible, given existing stock, sexes will be equally represented within the cohort of 34 radio tagged frogs. 5.3. Monitoring reintroduced frogs A sample of 34 radio tagged frogs will be located as frequently as possible (at least once every ten days per individual, with approximate locations taken daily) to monitor dispersal, health status and survival. Data on non-radio tagged frogs will also be collected opportunistically. Radio transmission and reception characteristics of the release site and surrounding sites will be assessed using a non-implanted transmitter Radio-tagged frogs will be relocated (but not captured) as frequently as possible to examine (a) dispersal, (b) habitat and microhabitat use. Habitat variables will be extracted from a GIS posttracking; a few simple microhabitat measures will be recorded in situ. We will also record the proximity of other mountain chickens and cane toads. Radio-tagged frogs will be captured at least once every ten days to (a) (to assess chytrid load) through swabbing, (b) record any visible signs of illness, (c) record any signs of injury (i.e. primarily from predators). Radio transmitters from which no signal has been obtained for a week or more will be searched for intensively to determine if loss of signal is due to (a) dispersal, (b) frog mortality, (c) frog location (e.g. in a burrow that attenuates radio transmission), (d) transmitter failure. This intensive monitoring is projected to last for three months (depending on frg and radio transmitter survival). During this period, a less intensive monitoring protocol will be developed to continue mountain chicken monitoring at the release site beyond radio tracking. This protocol will use automated call recording (‘frog loggers’) and intensive searches two days every week to find and record and sample the health status surviving mountain chickens… Individual PIT tags will allow us to follow the fates of individual frogs beyond the radio tracking period. Non-radio tagged frogs will be opportunistically mapped (GPS) and captured to sample chytrid/health/injuries. Habitat and microhabitat variables will also be recorded for these locations as above. From July onwards bi-weekly surveys at this site will also allow us to look for the presence of juveniles and/or signs of reproductive activity in adults indicating breeding by the frogs we introduced. 5.4. Monitoring surviving mountain chickens A sample of sites at which mountain chicken are believed to still survive on Montserrat will be monitored concurrently with the release monitoring. Where possible, surviving frogs will be captured to determine their health and infection status. Individual marking of these mountain chickens will be used to attempt a mark-recapture estimate of the abundance of these frogs. A sample of four sites (ghauts) from which mountain chickens are reported to have been heard calling from in 2010 will be sampled concurrently with the reintroduction and radiotracking work outlined above. Four ghauts will be selected and each will be monitored once a fortnight. These sites will be monitored using the transect monitoring protocol established in Montserrat in 1997, but traversing longer lengths of the ghaut (i.e. including but not limited to the 1997 transects). We will capture any mountain chickens located during these transects to sample chytrid load and assess health and breeding status. In addition, captures will be individually marked with PIT tags in an attempt to estimate population abundance from mark-recapture data. Location, microhabitat, and any injuries of captured frogs will be also be recorded as for radiotagged reintroduced frogs (above). We will also take DNA samples from buccal swabs (as, speculatively, it may be possible to identify genetic differences – potentially heritable resistance – in surviving frogs compared with DNA from frogs captured pre-chytrid). As with the radio tagged frogs, this intensive monitoring is projected to continue through the year. 5.5. Monitoring chytrid levels Chytrid load in mountain chickens and their environment (in this case, in tree frogs and cane toads and cane toads) may vary between sites or within sites over time. These variations may be an effect that explains survival of frogs at the release site or the sites where mountain chickens are believed to still survive. We also need to monitor whether the release appears to lead to an increase in chytrid load at the surviving frog sites, though we believe this to be unlikely as they are in different watersheds. Tree frogs and cane toads will be sampled and swabbed to determine chytrid load at (a) the mountain chicken release site plus (b) a control site from which mountain chickens are not known to survive, and (c) a control site at which small numbers of mountain chickens are believed to be surviving. We will attempt to obtain minimum samples of swabs from 60 tree frogs (and up to 300 tree frogs and cane toads if possible). This sampling will be done immediately prior to the first release of mountain chickens and then monthly thereafter for the remainder of the year. At the reintroduction site we will individually mark cane toads so we can monitor any progression of chytrid in a second amphibian species in the immediate vicinity of the introduced mountain chicken's. We will attempt to relate the chytrid loads in mountain chickens, and mountain chicken survival, to chytrid loads in the tree frogs and cane toads that make up part of their environment.