Haemocyanin – the underdog of the squid-vibrio symbiosis.

Euprymna scolopes are known for their light organ symbiosis with the luminous bacterium Vibrio fischeri. The bobtail squid harvest planktonic symbiont cells from seawater each generation and juvenile squid’s light organ exposes two pairs of prominent ciliated epithelial appendages. These appendages secrete mucus, which creates currents that facilitates recruitment of symbionts.  Gram-negative bacteria all adhere to the cilia however after 3 hours V. fischeri are the only bacteria able to persist. V fischeri then travel down ducts and reach internal crypts where they multiply, which causes the light organ to undergo irreversible morphogenesis. The mechanisms behind the specificity of V. fischeri symbionts has been speculated and a study by Kremer et al. 2014, suggests the oxygen binding mechanism, haemocyanin may be involved in the establishment and persistence of the V. fischeri symbiosis.

Adult Hawaiian bobtail squid (E. scolopes) were collected from Oahu, Hawaii and maintained and bred in a recirculating seawater system. Juveniles were incubated overnight with either V. fischeri wild type, a light production defective mutant or with the absence of V. fischeri. Full length cDNA sequences of haemocyanin isoforms were obtained by rapid-amplification of cDNA ends (RACE) and aligned with sequences from a variety of mollusc species for phylogenetic reconstruction. Haemocyanin transcripts from both isoforms were then quantified by qRT-PCR. Affinity-purified polyclonal antibody of haemocyanin was produced and immunocytochemistry experiments were carried out to visualise and localise haemocyanin transcript and protein in the squid tissues. The functional properties, including oxygen affinity of the hemocyanins were measured with a diffusion chamber. Oxygen affinity was determined from whole-blood samples, collected either at night or in the day.  A pH sensitive fluorescent probe (SNARF) was used to identify the flow between haemolymph and the light organ crypts. Associated pH was measured at both of these sites using a pH- sensitive probe. Phenol oxidase and antimicrobial activities of haemocyanin were also determined.

Results showed Euprymna scolopes synthesises two distinct isoforms of haemocyanin. Localisation and expression of haemocyanin transcripts showed that both isoforms were expressed in similar levels in all tissues. The main site of synthesis of both haemocyanin isoforms was in the gills. Mantle and gut within the juveniles, eyes and central core of the light organ of adults, were also secondary sites of synthesis. Fine-scale distribution of haemocyanin protein also showed high levels in the gills, pore and epithelium regions of the light organ and within the mucus adjacent to these regions. Haemolymph passes from the vascular system into the crypt spaces, which has a lower pH than the haemolymph. This suggests high affinity haemocyanin takes up oxygen in the gills and circulates until it reaches the crypt space where the pH is significantly lower due to bacterial metabolism. V. fischeri luciferase has a higher affinity for the oxygen, which causes the oxygen to dissociate from the haemocyanin.  Therefore bacteria metabolism over a diel cycle in relation to their behaviour influences the delivery of oxygen by altering the pH of the crypt spaces and haemocyanin is an important component of this cycle. V. fischeri showed resistance to the phenol oxidase antimicrobial activity of the haemocyanin, whereas growth was inhibited in other marine gram negative and positive bacteria. This therefore infers the haemocyanin within the mucus may play a role in the specificity of symbiont selection.

I found this paper to be interesting as the specificity of V. fischeri in this symbiosis was briefly mentioned in a previous piece of literature I read, which speculated that the specificity was due to the high oxidative stress the bacteria have to endure within the crypt space. As this paper suggests the specificity is in fact determined before the symbiont enters the host, it provided an alternative and conflicting theory, which was interesting to study. Overall this study highlights the emerging importance of haemocyanin in the role of specificity in selecting V. fischeri for the symbiosis as well as their importance in the control and adjustment of the physiology involved in the maintenance of the squid-vibrio symbiosis. I believe this paper and its findings provide a good basis for future research regarding the antimicrobial properties of haemocyanin.

Kremer, N., Schwartzman, J., Augustin, R., Zhou, L., Ruby, E., Hourdez, S. and McFall-Ngai, M. (2014). The dual nature of haemocyanin in the establishment and persistence of the squid-vibrio symbiosis. Proceedings of the Royal Society B: Biological Sciences, 281(1785), pp.20140504-20140504.