Underwater ultrasonography and blood sampling provide the first observations of reproductive biology in free-swimming whale sharks

We report on a non-invasive technique for observing the reproductive states of wild, free-swimming whale sharks Rhincodon typus for the first time. Female whale sharks (n = 22) were assessed using underwater ultrasonography and a novel blood-sampling technique at Darwin Island in the Galapagos Marine Reserve, Ecuador. Despite the widely held assumption among researchers that the post-pelvic distention of large females is indicative of pregnancy, ultrasound provided no evidence of embryos or egg cases. However, the presence of follicles (diameter: 28.5-83.6 mm) was confirmed in 2 female sharks of 11-12 m total length (TL). Additionally, 3 steroid hormones (estradiol, testosterone, and progesterone) were analyzed in blood plasma from 6 female sharks (11-12 m TL). Hormone levels were similar to, or lower than, those obtained from an immature female in the Okinawa Churaumi Aquarium. Based on these results, we infer that female whale sharks (TL >11 m) in this study were mature but not pregnant. The techniques used here for whale sharks can be successfully used to obtain non-lethal field data on the biology and reproductive anatomy of this globally endangered fish, and are adaptable for use in other large marine species.