Effects of ultraviolet light and methoprene on survival and development of <i>Rana pipiens</i>

Abstract Recently a suite of relatively specific hindlimb deformities have been observed in several anuran species in North America. These deformities include ectopic and supernumerary limbs and missing limbs, limb segments, or digits. The objective of this study was to assess two stressors hypothesized as responsible for limb malformations in amphibians: methoprene, an insect growth regulator that, through interaction with the retinoic acid signaling system, could possibly cause limb deformities, and ultraviolet (UV) light. Northern leopard frogs (Rana pipiens) were exposed to several different concentrations of methoprene both in the absence and presence of UV light designed to mimic the UV wavelength spectrum present in sunlight. Exposures were initiated at early embryonic stages (newly fertilized eggs) and continued through emergence of the forelimbs of the frogs. At the highest methoprene concentration tested, both in the absence and presence of UV light, severe developmental effects were observed, with all organisms dying within 12 to 16 d of test initiation. However, exposure to the pesticide did not cause limb malformations. Irrespective of methoprene treatment, a very high percentage (∼50%) of animals held under the UV light for 24 d developed hindlimb malformations. These malformations usually were bilateral and sometimes completely symmetrical, and consisted of missing limb segments and missing or reduced digits. A complete proximal to distal representation of the deficiencies occurred, ranging from missing or malformed femurs to the absence of single digits or digit segments. The developmental period of greatest sensitivity to UV light occurred during very early limb bud development, corresponding with formation of the apical ectodermal ridge. The significance of these findings in terms of deformed frogs in the field is uncertain. Although the deformity types observed (i.e., missing limb segments and digits) were similar to those seen in some field specimens, the UV light treatment did not cause the full range of malformations observed in animals from the field (e.g., supernumerary limbs, nonbilateral deformities). Furthermore, although the artificial light spectrum utilized mimicked the relative UV spectrum present in sunlight, it did not match full sunlight intensity, and did not accurately mimic visible wavelengths. Finally, the relationship of the UV light dose used in the laboratory to that actually experienced by amphibians in the field is uncertain. Despite these questions, our findings suggest that UV light should be further considered as a plausible factor contributing to amphibian malformations in field settings.