In situ vital staining for chasing the galatheid crab Shinkaia crosnieri on deep-sea floor

Shinkaia crosnieri, a galatheid crab, has ectosymbiotic bacteria on its ventral setae, and forms very dense crowds in hydrothermally active regions and seep areas. They feed on the symbiotic bacteria and do not chase other animals for predation. To study how they move and behave in jostling crowds, we developed a vital staining to mark their individuals and trace them by using a camera on a remotely operated vehicle (ROV). Among the various dyes examined, Coomasie Brilliant Blue R250 (CBB) stained the galatheid crab the darkest, and its color lasted for more than 5 months in the laboratory at 4–5°C. The ventral setae were strongly stained, while the dorsal shell was weakly stained. The stained galatheid crab survived for more than 8 months. For the in situ staining of S. crosnieri at the Iheya North hydrothermal field in the Okinawa Trough, Japan, we applied a dye solution mixture (20 L) containing CBB and Acid Blue 161 to the galatheid crab population through a funnel equipped on the ROV Hyper-Dolphin. After staining for approximately 5 minutes, more than 18 individuals of S. crosnieri were dyed blue. They were disturbed by the staining process but seemed to be unharmed. The dyed galatheid crabs were identified by the ROV one and two days post staining. They seemed to remain at the place where they were stained. The present vital-staining marking method may present a new way to analyze the behavior and changing habitable range of deep-sea animals likeS. crosnieri, and may give us a deeper insight into how these animals behave in a very dense population and explore newer habitats.