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

Abstract

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.

The chemistry of negotiation: Rhythmic, glycan-driven acidification in a symbiotic conversation

“…4B and Fig. S4C), consistent with the acidic pH measured in crypt contents at night (34). In contrast, symbionts released in the morning (3 hpd) showed no evidence of ATR induction, even though they were physiologically capable of inducing this response if they were exposed to acidic conditions before the assay (Fig.…”

“…This excretion of acetate during aerobic growth is consistent with a metabolic imbalance called the Crabtree effect (42). In the context of the light-organ environment, the Crabtree effect would lead to the excretion of SCFA like acetate during growth on COS, thereby acidifying the crypt lumen and enhancing the allocation of ambient oxygen toward bioluminescence (34).…”

“…At dusk, hemocytes (red) migrate into the light-organ crypt lumen, lyse, and release chitin oligosaccharides (COS, green). The COS are catabolized by symbionts to produce SCFA, which acidify the crypt lumen and promote oxygen release (34). Oxygen fuels the enzymatic production of light by symbiont luciferase, the functional basis of the squid-vibrio mutualism.…”

“…Light emission, which requires oxygen (32), is highest during the night (33). In the fully developed light organ, where the symbionts are oxygen limited (24,33), the diel bioluminescent rhythm is potentiated by an acidic crypt environment, which creates a Bohr effect that releases oxygen from the carrier protein, hemocyanin (34). Here, we demonstrate that, in this mature state of light-organ development, the cyclic provision of COS to symbionts, combined with their fermentation of this glycan, leads to the nightly acidification of the symbiont-containing extracellular crypts.…”

“…Hemolymph, which is the major constituent of the crypt environment (34), required the addition of ∼7.5 mM acetic acid to reach a pH of 5.5 (Fig. S4E).…”