The dual nature of haemocyanin in the establishment and persistence of the squid-vibrio symbiosis.

We identified and sequenced from the squid Euprymna scolopes two isoforms of haemocyanin that share the common structural/physiological characteristics of haemocyanin from a closely related cephalopod, Sepia officinalis, including a pronounced Bohr effect. We examined the potential roles for haemocyanin in the animal's symbiosis with the luminous bacterium Vibrio fischeri. Our data demonstrate that, as in other cephalopods, the haemocyanin is primarily synthesized in the gills. It transits through the general circulation into other tissues and is exported into crypt spaces that support the bacterial partner, which requires oxygen for its bioluminescence. We showed that the gradient of pH between the circulating haemolymph and the matrix of the crypt spaces in adult squid favours offloading of oxygen from the haemocyanin to the symbionts. Haemocyanin is also localized to the apical surfaces and associated mucus of a juvenile-specific epithelium on which the symbionts gather, and where their specificity is determined during the recruitment into the association. The haemocyanin has an antimicrobial activity, which may be involved in this enrichment of V. fischeri during symbiont initiation. Taken together, these data provide evidence that the haemocyanin plays a role in shaping two stages of the squid-vibrio partnership.