The Colorado Division of Wildlife has devoted significant resources toward researching the causes of boreal toad declines in the state, and exploring ways to recover the species. Results of these research activities can be found in the following reports:
Current research has focused on developing methodologies for reintroducing toads in historically occupied habitats, detecting the chytrid fungus Batrachochytrium dendrobatidis in the wild, and refining husbandry practices.
Repatriation of boreal toads on the Grand Mesa, Colorado
This study explores the efficacy of introducing various boreal toad life stages to establish new populations. The study site lies in the Kannah Creek drainage on the southern end of the Grand Mesa in Mesa County, Colorado. Approximately twelve thousand tadpoles were raised each year at the Native Aquatic Species Restoration Facility (NASRF) in Alamosa, CO. They were released unmarked around the margins of three ponds at the study site in late June 2003, 2004, and 2005. An additional 1200 were divided among six pens (two per pond), raised to metamorphosis, toe clipped for future identification, and released. Remaining tadpoles were kept at NASRF until approximately 3 weeks post metamorphosis. Nine hundred were then given a different toe clip and divided among the same three ponds in late August of each year. About 2500 tadpoles were released unmarked to a single pond in 2006, completing the initial reintroduction effort.
We had excellent success raising tadpoles to metamorphosis in the pens, with most having better than 80% surviving to metamorphosis. Of the mortalities, desiccation and predation were to blame, rather than inadequate husbandry practices. Metamorphosis was initiated the last week in July, and was completed by mid-August. Average water temperature in the pens over that time (release date to metamorphosis) was 17 C.
We did notice that pen raised toadlets captured in late August were only half as heavy as those released directly into the wild as tadpoles. The ability to behaviorally thermoregulate apparently was critical for maximizing growth in the wild release group, as their counterparts in the pens were fed daily. It was thought that this increase in mass would confer some survival advantage, suggesting that wild released tadpoles would be the most suitable life stage for subsequent repatriation efforts. Interestingly, this increased size going in to winter did not seem to affect survival to the following summer, as individuals from all release groups were recovered in proportion to numbers released the previous year. Future releases will likely all be wild-release tadpoles since they are much less labor intensive.
Detection of the chytrid fungus Batrachochytrium dendrobatidis
Since the late 1990s, researchers have discovered the chytrid fungus Batrachochytrium dendrobatidis (Bd) infecting frogs in areas experiencing amphibian population declines in Australia, Europe, Africa, and North, Central, and South America. In 1999, a decline in the Henderson/Urad boreal toad population in Colorado was attributed to this "frog chytrid". This chytrid fungus has now been identified in boreal toads from over a dozen Colorado populations and evidence suggests that this pathogen was responsible for the declines documented in the late 1970's and early 1980's. The chytrid life cycle begins with a motile zoospore, which is the infective stage of this pathogen. During the course of infection, chytrid zoospores enter skin cells on the amphibian. The fungus grows and develops asexually within the skin cells. Eventually, discharge tubes form, that extend to the surface of the cells. Mature zoospores emerge from the discharge tube and begin the life cycle again. Infections are restricted to the skin of the amphibian. Infected amphibians often slough skin more frequently than healthy amphibians. Scientists are currently exploring how this fungus kills (pdf) amphibians.
Until recently, the only way to positively diagnose Bd infection on boreal toads was through tedious histological examination of dead toads. With the cooperation of the University of Maine and the USGS Biological Research Division, the Colorado Division of Wildlife sponsored the development of a polymerase chain reaction (PCR) test to enable rapid detection of Bd DNA with a non-lethal approach (pdf).
Looking for reservoirs of Batrachochytrium dendrobatidis infection
An increasing range-wide interest in reintroducing boreal toads back in to historic habitats has spurred the need to develop a test for the presence of Bd in the wild. Repatriation efforts are time consuming and costly, and their success may hinge on the disease status of a potential site. As such, it is imperative that the disease status be considered when evaluating potential reintroduction efforts. This process is further complicated by the fact that many of our most promising sites currently have no resident amphibian species. Since Bd can persist at a location even in the absence of amphibian species, we suspect that amphibians may not be the primary host, and that infection can be maintained through other organisms. We have looked for infection in aquatic insects, small fish, snails, clams, algae, feathers, and keratin, from known Bd positive sites but have still not been able to detect the organism in a non-amphibian host. In 2007 we detected Bd in samples of mosquito larvae and pupae, but not consistently. Other researchers are developing water filtration strategies (pdf) for detecting Bd environmentally.
Sampling strategies for Batrachochytrium dendrobatidis in the wild
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| Figure 1: Prevalence of Bd infection in wild chorus frogs on the Grand Mesa from the third week in June through the first week in August. The ordinal score represents the intensity of infection on a scale of zero to four. |
Many potential boreal toad repatriation sites are home to other montane amphibians. In these situations, it is possible to test for the presence of Bd using an established protocol (pdf). Intensity and prevalence of the disease is seasonal however, so care must be taken when sampling for the disease to maximize ones chances of detecting Bd if it is present. Infection appears to be most common and intense early in the summer (Figure 1).
Hatchery studies
The Colorado Division of Wildlife's Native Aquatic Species Restoration Facility (NASRF) plays a critical role in State's efforts to restore boreal toads. The facility houses over 600 adult boreal toads, providing a key link in the production of these animals for repatriation efforts and research, as well as serving as a genetic bank for this state endangered species. Protocols used in maintaining boreal toads at NASRF can be found in the Boreal Toad Husbandry Manual (pdf). In addition, several other research projects have been completed at the facility:
The importance of diet on growth and survival to metamorphosis of boreal toads Bufo boreas in captivity
Refining husbandry practices at NASRF will ensure the vitality of boreal toad restoration programs. We initiated a study that considered four diets in an effort to improve growth and survival to metamorphosis on tadpoles raised at the hatchery. Each of twelve tanks received 35 boreal toad tadpoles (Gosner stage 23) then randomly assigned one of four treatments (diets). These diets included various combinations of Mazuri Feed (Mz; Mazuri® Purina Mills, St. Louis, MO), a mix of vegetables (mustard greens, collard greens, yellow squash, and zucchini) that were blended then frozen in to cubes (Vc), brine shrimp (Bs), and liquid vitamin B complex (B). Tadpoles were fed daily, and growth was monitored at roughly two-week intervals. Individual tadpoles were measured for total length, while an average mass for each individual was estimated by weighing all individuals in each tank. Mortalities were noted and removed daily. Timing of metamorphosis was also noted, as was metamorphic snout-vent length.
Interestingly, the commercially available tadpole food (Mz) was least effective in promoting growth and survival of the diets used. Weight gain was almost half what it was for our best diet (MzVcB), and length at metamorphosis was smaller as well. Perhaps more importantly, only a third as many tadpoles actually metamorphosed under the Mz diet as compared to the MzVcB group. Performance of the VcBs and MzVc diets was generally intermediate, though the VcBs group shared very high survival to metamorphosis with the MzVcB group. Interestingly, trace amounts of vitamin B were present in the Bs treatment, perhaps alluding to the importance of the role vitamin B plays in the developmental process. The variability in growth and survival attributed to these diets underscores the importance of continued work in this arena.
Tag retention in a captive population of boreal toads Bufo boreas using four marking schemes
The development of reliable marks to identify individuals or groups of boreal toads is critical to the success of research and captive breeding efforts on this species. Given the widespread use of various marking schemes, it is necessary to quantify the extent of the tag loss, to determine if it indeed poses a problem. The Colorado Division of Wildlife's Native Aquatic Species Restoration Facility (NASRF) in Alamosa lends itself to this sort of study, as large numbers of toads can be tagged and retention evaluated on a regular basis.
Over 300 captive boreal toads were used in this study. Adults received passive integrated transponders (PIT), visual implant elastomer (VIE), coded wire, and toe clips. Toadlets were subjected to the same treatment without the PIT tags. The PIT tags are uniquely coded and ideal for identifying specific individuals required for breeding records as well as robust schemes for population monitoring. The remaining marks are more suitable as batch marks used to identify a group of individuals. While it is true that the coded wire tags have a unique sequence of numbers on them, reading those numbers requires that the tag be removed from the body and inspected under a magnifying glass. As such, they were simply used as a batch mark in this study, and were readily detectable with an electromagnetic wand waved over the animal.
All toads were inspected at monthly intervals for marks, unless they were in hibernation. PIT and coded wire tags were scored as being either present or absent, while VIE and toe clips were scored on a scale ranging from 0-3. A VIE score of zero represented no detectable VIE tag even under scrutiny with a blue light and amber tinted glasses, while a three was clearly visible to the naked eye. A toe clip score of zero indicated complete regeneration of the toe without any deformity, while a three was assigned to toes with no evidence of regeneration and a clearly recognizable mark.
Though this study was only recently initiated, some interesting results have already come to light. Both VIE and toe clips worked well as batch marks for small toadlets (mean snout-vent length = 19.5 mm) with both displaying better than 99% retention over four months. The coded wire marks worked well initially, but loss has accumulated over time so that now 14% of the toadlets are without tags. For the adult toads, toe clipping, coded wire, and VIE were all excellent marks for identifying batches of individuals. Separation between methods in this portion of the study will presumably be more apparent after greater time has elapsed as most of these individuals are now in hibernation, and have not been evaluated in several months. Retention of the PIT tags was very disappointing, with 30% losing their tags in the first month, and 40% having lost their unique marks after two months. This degree of tag loss is unacceptable, and alternative methods for identifying individuals will need to be developed.
Using pattern recognition to identify individual boreal toads
Many researchers are investigating pattern recognition techniques to identify individual animals –everything from penguins to sharks and polar bears. Biologists in Switzerland have used unique abdominal patterns to recognize individual yellow-bellied toads. In the United States flank photographs have been used to identify Mountain Yellow-legged frogs. Boreal toads display similar unique marks on their bellies, analogous to a fingerprint in humans.
We have had a pilot computer program developed to digitally characterize these patterns so that all 600 adult boreal toads at the NASRF can be identified. We’ve completed photographing every toad at the NASRF facility and every individual has been loaded into the program. Now we are dealing with quality control – investigating how the photographs should be posed to ensure the best possible information, how to speed the recognition algorithm, and whether this program will eventually be useful in both hatchery and field situations. Even now, the presence of the physical photographs at our hatchery has proven useful in identifying individuals by comparing photos to toads within lots (fortunately, in the hatchery one would not have to compare a live toad with 600 photos in order to identify the toad, since they are kept separate by lots). This will ensure the integrity of our breeding program at the facility.