Chapter 16 Veterinary Participation in Puerto Rican Crested Toad Program
The crested toad, or sapo concho (Bufo [Peltophryne] lemur), is one of eight endemic West Indian bufonids and is now found only on Puerto Rico. It has prominent supraorbital crests and an upturned snout. The toads are sexually dimorphic; females are 80 to 120 mm long and ash gray–white to charcoal black; whereas the males are slightly smaller, 70 to 80 mm long, with a brighter, yellow-green coloration (Figure 16-1).
First described in 1868, the Puerto Rican crested toad (PRCT) was distributed over much of the coastal karst areas of the island 100 years ago, but climatic change and loss of habitat resulting from land alteration for agriculture and commercial development seriously reduced the numbers of toads and threatened to eliminate the species altogether. The introduction of the marine toad (Bufo marinus) and the small Indian mongoose (Herpestes auropunctatus) to the West Indies has also had an impact on the endemic toad. Mongoose predation was a significant factor identified by radiotracking captive-reared toads released in Puerto Rico.
The PRCT had not been seen for more than 35 years until its rediscovery in 1965.5 No record of captive animals exists before 1980. Puerto Rico is a mountainous island, and only two isolated toad populations were known to exist: a northern population, now believed to be extirpated, and a southern population believed to be as low as 1500 to 2000 in the 1980s.6 These populations have been isolated since the Pleistocene, and they are managed in captivity as two genetically distinct groups. Because of its fossorial nature, the species is difficult to inventory. During the day, toads live underground, usually entering secure, moist crevices or holes in the limestone karst. The largest known breeding population is found in Guanica Forest Reserve on the southwestern coast.
Typically, when the rainfall exceeds 18 cm over a 24-hour period, the adults emerge to breed at temporary ponds near the coastal beach. The breeding area is located adjacent to the sea, and inundation by seawater during hurricanes is a real threat. Before 1984 the breeding pond was drained to provide easier beach access. When this practice was stopped, it was discovered that toads were using this pond as a breeding site (Figure 16-2). The northern toad populations, found near Quebradillas, had bred in concrete walk-in cattle troughs together with B. marinus. Here, no more than 25 PRCTs had been seen at any one breeding episode. There has been no sighting of northern toads since 1992, and wild northern toads at this time are functionally extirpated. The remaining northern toads that now exist in zoos originate from a single breeding of the last pair of this race. In May 2006, tadpole descendants from these were released in newly constructed ponds near Arecibo, the first time in 15 years.
Fig 16-2 Breeding habitat in the Guanica Forest Reserve, Puerto Rico, in the dry season.
(Courtesy B. Johnson.)
For several years, censuses in the Guanica Forest showed a population at the breeding pond of fewer than 300 toads, but in 2004 more than 600 adult toads appeared at the pond in three separate breeding events.1 In 2005, four breeding events took place involving 2200 toads.
The PRCT species was considered suitable for a species survival plan (SSP) under the auspices of the Association of Zoos and Aquariums (AZA) in 1984 and was the first amphibian included in the SSP program. The 21 facilities currently holding 340 Bufo lemur manage approximately 400 captive animals as one population. Since the late 1980s, a captive breeding and release program has been undertaken to augment the wild population. Releases are only considered for suitable habitats beyond the range of the extant population. To date (2006), approximately 100,000 tadpoles hatched in North American zoos have been released in Puerto Rico.7 Additional breeding sites have been constructed, and in 2003, for the first time, adult toads grown up from captive-bred tadpoles were breeding at the new sites.
Veterinary involvement in the PRCT program has focused on reproduction, medical care, and pathology of captive animals and training of staff in the care of toads in Puerto Rico. Field projects are proposed, as described next.
As in other breeding programs for threatened species, captive populations of PRCT have been established (1) to provide a reservoir of animals against catastrophic loss of toad in the wild, (2) to reinforce wild populations, and (3) to provide animals to establish new populations. The recovery program is a joint effort of zoos, the Puerto Rican Department of Natural Resources, and the United States Fish and Wildlife Service in Puerto Rico. The University of Puerto Rico provides staff for field projects. In zoos, PRCTs are displayed in terraria equipped with rock piles or rock and cemented caves to mimic their natural karst habitat. They are behaviorally reclusive, and hiding places are provided. Full-spectrum (Vita-lite, Duro-test, Philadelphia) and black-light (Sylvania, Danvers, Mass) bulbs are suspended directly over the tanks on a 12:12 light/dark photoperiod. Off-exhibit breeding animals are held in plain tanks with hollow polyvinylchloride (PVC) plumbing tubes to provide the tight, secure environment they prefer. Shallow water provides the ability to rehydrate. Feed requirements are the same as those of other equivalent anurans and typically include domestic crickets and other insects and newborn mice.
As noted, PRCTs are generally sexually dimorphic, and the gender of mature males may be confirmed by the presence of black nuptial pads on the “thumbs.” Despite prebreeding conditioning with a period of cooling or drying, most pairs of PRCTs fail to reproduce spontaneously, and in almost all cases, exogenous hormones are used to promote reproductive behavior and success. Production also must be maximized and is timed to coincide with the rainy season in Puerto Rico when the tadpoles are released. Reproductive induction of anurans involves the use of human chorionic gonadotropin (hCG) or gonadotropin-releasing hormone/luteinizing hormone–releasing hormone (GnRH or LHRH).
Several chemical analogs of LHRH exist, and not all work well in each species. The analog most often used for anurans is des-Gly (d-Ala) LHRH ethylamide (catalog #L4513, Sigma, St. Louis). To achieve natural reproduction, it is necessary not only to induce gamete production and release, but also to promote behavioral amplexus for fertilization of released ova. Experiments have shown that hCG injected into PRCT males is somewhat more effective than LHRH at inducing amplexus, although they were similar at promoting spermiation. Other studies have examined the need for preconditioning, and results have suggested that, in males at least, preconditioning is not a prerequisite for successful gamete production. However, it apparently is not possible to induce ovulation in female PRCTs that have not been cooled or environmentally cycled, and hormone therapy will never be effective in the absence of mature ova in the ovary. In other anurans, ova maturation depends on an extended quiescent time and good nutritional status.
The timing of the injection is based on work that shows a peak in sperm production between 6 and 24 hours after injection. At the end of the cooling period for females, the toads are warmed back up to 28°C over 3 days. On day 2 the tanks are filled with 1 inch of dechlorinated, aged water. Audiotape toad calls are played throughout the day and into the night. As early as possible on the morning of day 3, males are injected with the LHRH analog ethylamide (Sigma), 0.1 μg/g subcutaneously (SC), or hCG (Chorulon, Intervet Canada, Whitby, Ontario), 4 IU/g.7
Female toads are also injected at this time with 0.1 μg/g LHRH. Eggs are usually laid overnight within 24 hours of amplexus or after hormonal injections. Females seem to be susceptible to fluid overload, and death from drowning may occur. The breeding period is a stressful time for toads, and deaths have occurred from infection, fluid overload, and glomerulopathy, perhaps because of intense mobilization of protein in this breeding period.
In some cases, pairs of toads will go into amplexus for variable periods but fail to deposit eggs. The use of hormones in toads that are not “ready” may result in internal laying in which ova are not collected by the oviducts and remain to decompose in the coelom, leading to toxic or septic coelomitis. I have also seen prolapse of the ovisac (oviduct) subsequent to egg laying, particularly if the eggs are not released completely.
Puerto Rican toads have been anesthetized with tricaine (MS-222, Argent, Redmond, Wash). It was found that 2 g/L buffered to a pH with the same dosage of sodium bicarbonate produces an adequate level in 8 minutes or less. Toads are placed in a shallow solution with care taken to keep the nostrils above the water level. Once toads have reached the required level, they are removed for the anesthetic solution. Toads anesthetized with topical tricaine have been kept for an hour or more at cool temperatures, without the need for additional drugs. It is generally not necessary and may be harmful to keep them in anesthetic solution for longer than induction. Local anesthesia is used for treatment of dermal lesions and microchipping (see following discussion).
Local anesthesia is used for inserting transponders (“PIT tags”). Toads are held, and the location for needle insertion is cleaned with sterile water, followed by the application of a small amount of lidocaine/prilocaine cream (EMLA, AstraZeneca, Wilmington, Del). The local anesthetic takes only a few minutes to be effective, and the chip is placed subcutaneously on the left side of the upper thorax beneath the overhanging parotoid gland. The opening in the skin is closed with tissue glue (e.g., Vetbond, 3M, St. Paul, Minn). The area is then rinsed with water to remove traces of the anesthetic, which is capable of causing general anesthesia in high doses in toads. In most cases the transponders remain at this location, although migration, even into the coelomic cavity, has been seen. Microchipping has been successful in toads as small as 30 g. Implanting with PIT tags means that all the toads may be housed together rather than in pairs, which reduces holding-space requirements. The ventral pattern may also be used to identify individuals.
Knowledge of the diseases of amphibians in general is increasing.10 There has been considerable research in the role of disease, and of chytridiomycosis in particular (see Chapter 17), in the decline of amphibian populations globally. There is no reason to believe that B. lemur is unique among amphibians from a disease point of view. However, the health of the wild population has never been studied systematically, a task made difficult by their highly secretive nature. PRCTs are typically only found during the breeding events. A health survey has been initiated, first encompassing the sympatric and more common B. marinus, to include screening for pathology, parasites, and pathogens such as chytrid fungus. The study will subsequently be extended to include B. lemur. In captive animals, most deaths have been sporadic, although several multiple die-offs have occurred in both adults and juveniles. These cases appear to have been caused by suboptimal husbandry rather than specific pathogens.
A review of 117 necropsies at the Toronto Zoo (TZ) from 1985 to 2002 revealed that the majority of toads had two or more histologic diagnoses. Nineteen cases (16%) of systemic infectious disease were noted. Most of these (67%) were diagnosed with single- or multiple-organism septicemia. Pseudomonas was the most frequently isolated bacterial organism from septic animals. Eight cases (7%) of musculoskeletal disease were noted, six of which (75%) were a myopathy of unknown etiology. Histologic lesions included myodegeneration, increased granularity, and hypereosinophilia of the sarcoplasm, with variable inflammation ranging from none to lymphocytic.
Twenty-two cases (21%) of enteric disease were observed. Many of these were the overeating/gastric dilation syndrome, for which no other cause of death could be attributed. Other cases of enteric disease included hepatocellular degeneration, hepatitis, unspecified endoparasitism, and mixed–inflammatory cell enteritis, as well as ceroid accumulation in the intestinal wall.
Seven cases (6%) of integumentary disease were diagnosed. Three of these were mycotic dermatitis, from which organisms such as Nigrosporum and Trichoderma were isolated. The remainder included a bacterial dermatitis concurrent with septicemia, suggesting that the skin was the portal for the pathogens. Three cases of respiratory disease were diagnosed, including one case of aspiration pneumonia and two of heterophilic pneumonia. Two cases of trauma and one case of ammonia toxicity (“tank die-off”) were noted.
Of the 117 cases, 29 (25%) were diagnosed with renal disease, principally glomerulonephropathy. Eight cases (7%) of interstitial nephritis and one case of cystitis were also diagnosed. The exact cause of glomerular disease is unclear. All except one of these cases were in adult animals. Many of the toads with glomerulopathies had other diseases that might have predisposed them to glomerular disease, the most common being myopathy, dermatitis, and sepsis. Immune stimulation associated with dermatitis or sepsis may ultimately cause renal immune complex disease.
Limb paralysis has been seen in several toads in the collection. Because many of these amphibians improve with treatment, necropsy has been sporadic. Lesions found in these animals include one case each of wallerian degeneration and peripheral axonopathy. In toads in which the forelimbs were principally affected, pathologic findings were minimal, and in retrospect these may have been cases of thiamine deficiency. Others with hind limb paralysis have shown spinal axonopathy. Possible causes of such a condition include dietary (hypovitaminosis E or B), infectious (neuritis, myositis, myelitis), autoimmune, or genetic processes. One affected toad had an osteomyelitis of the spine. Several cases of spindly leg syndrome were seen.
Several toads were diagnosed with anasarca and ascites of unknown origin. Some animals had been recently placed in breeding tanks and presumably had some alteration in normal osmoregulatory function. In addition, one adult animal was diagnosed with a systemic myeloproliferative disease of possible granulocytic origin.
No viruses have been identified to date in PRCTs. Die-offs in tadpoles have been investigated for the possibility of ranavirus infection but, to date, none has been detected by polymerase chain reaction (PCR).