168 A NONINVASIVE, TRANSDERMAL ABSORPTION APPROACH FOR EXOGENOUS HORMONE INDUCTION OF SPAWNING IN THE NORTHERN CRICKET FROG, ACRIS CREPITANS: A MODEL FOR SMALL ENDANGERED AMPHIBIANS

The World Conservation Union (IUCN) currently recognises Eleutherodactylus locustus from Puerto Rico and Plectrohyla acanthodes from Mexico and Guatemala as critically endangered, and they are recommended for ex situ breeding by several amphibian conservation organizations (e.g. AZA, ATAG, RCP, and AARK). Because of their very small and delicate bodies, conventional injectable methods developed for hormonally stimulating spawning in larger frogs and toads are undesirable, because this can result in trauma and injury. Recently, a protocol was developed for inducing spawning in frogs by co-injection of a gonadotropin-releasing hormone agonist (GnRHa) with metoclopramide-HCl (MET), a dopamine antagonist (Amphiplex; Trudeau et al. 2010 Rep. Biol. Endo. 8, 36). The goal of this study, therefore, was to determine the effectiveness of Amphiplex using 3 routes of exogenous treatments: (1) direct IP injection (0.4 µg of GnRHa + 10 µg of MET per gram of body weight), (2) injection into prey (1 µg of GnRHa + 25 µg of MET per insect) before feeding, and (3) a 60-min immersion in 10 mL of pond water containing 0.17 µg of GnRHa + 0.42 µg of MET per microliter. The northern cricket frog (Acris crepitans), a very common local species, was used as a model because it is also small, delicate, and difficult to handle. A total of 110 cricket frogs were caught locally either late or after their normal breeding season, sexed as males or females, and segregated into a control group that was housed similarly ex situ but not exposed to Amphiplex and the 3 Amphiplex experimental groups. The frogs were monitored daily for egg production and behavioural amplexus (potential spermiation). The results are summarised as follows (Table 1): of the frogs exposed to the Amphiplex-supplemented pond water, 84% of 38 females deposited eggs, and 6 pairs were observed in amplexus (indicating spermiation). Approximately 27% of more than 2242 eggs were found to develop cleavage divisions, and tadpoles were first observed in vitro after 4 days. In conclusion, to our knowledge this is the first report indicating that transdermal absorption of a reproductive hormone cocktail (e.g. Amphiplex) may serve in the application of assisted reproductive technology to increase the populations of critically endangered amphibians such as frogs and toads that may be too small or delicate for conventional methods of exogenous hormone treatments that require handling and injections. Table 1.Treated northern cricket frogs

Acoustic and morphological identification of the sympatric cricket frogs Acris crepitans and A. gryllus and the disappearance of A. gryllus near the edge of its range

Although the Northern Cricket Frog, Acris crepitans, and Blanchard’s Cricket Frog, A. c blanchardi or A. blanchardi, have declined in areas near the northern margins of their ranges in the eastern and central United States, no such declines have been reported for the Southern Cricket Frog, A. gryllus, at the northern margin of its range in the Southeast. However, the status of A. gryllus is obscured by its sympatry with A. crepitans and the difficulty of identifying the two species. To address these difficulties, we studied A. crepitans and A. gryllus from North Carolina in the field and in a large museum collection. We quantified distinct differences in the click components of the vocalizations of A. crepitans and A. gryllus and used these differences to identify calling males from field recordings. Based on field recording and collection, we developed a discriminant function to identify preserved specimens by the extent of hind-foot webbing and the diameter of anal tubercles. During surveys from 2004 to 2007, we found that Acris crepitans was more widespread than expected from published reports, as it was frequently found in the Coastal Plain. Acris gryllus was less widespread than expected, as we could not find it in the northern part of the upper Coastal Plain, where published reports indicated it should occur. By applying our discriminant function to Acris collected in 1962 and 1963, we confirmed that A. gryllus once occurred in this area. In 2008, we resurveyed sites where A. gryllus had once occurred alone or in syntopy with A. crepitans and confirmed that A. gryllus is now missing from three major river basins in the upper Coastal Plain of North Carolina. We conclude that while the range of A. crepitans is stable in North Carolina, A. gryllus has disappeared from a large area near the northern edge of its range.