The squid –Vibrio story continues... squid derived chitin is important in colonisation
Euprymna scolopes, the Hawaiian bobtail squid, has a well known symbiotic relationship with bioluminescent bacteria Vibrio fischeri (recently reclassified as Allivibrio fischeri), that colonise the light organ of the squid post hatch. However in a world saturated with an array of microorganisms how are the correct bacteria enticed and selected to colonise the juvenile squid light organ?
A ‘winnowing’ effect in recruitment has been observed: initially gram negative bacteria are selected, followed by A. fischeri then subsequently motile A. fischeri which form tight clusters at the entrance of the light organ pore. Aggregation of A. fisheri symbionts occurs in squid-derived mucus, induced by a two-component relay signalling mechanism in the bacteria. In this experiment, derivatives of wild type A. fischeri labelled with green fluorescent protein (GFP) could be seen aggregated in the mucus field just outside the pore within 2 hours of exposure.
The underlying mechanism of colonization of the light-organ and the related specificity challenges has been shown to be achieved by chemical communication. Using fluorescein isothiocyanate (FITC), a chitin-binding protein, and confocal microscopy, chitin was found to be present within the light organ ducts, indicating the presence of light-organ derived chitin. Chitin is thought to be a chemoattractact and important in symbiont colonization.
Using soft-agar swim plates inoculated with N-acetylglucosamime (GlcNAc or (GlcNAc)2, a mono and disaccharide of chitin respectively), a positive chemoattraction was observed in A. fischeri. However when added to water containing squid, the chemoattraction to squid-produced chitin was disrupted. Exogenous addition of the monosaccharide GlcNAc had no effect on squid colonization efficiency, however addition of disaccharide (GlcNAc)2 diluted the endogenous gradient produced by the squid, and resulted in <10% of squid becoming colonized (fig. 1).
Figure 1. (j) Efficiency of squid colonisation upon addition of chemoattractants at 0.625% (wt/vol) and (k) effect of dose of (GlcNAc)2.
Newly hatched squid were exposed to A. fischeri in combination with an inoculum of 0.625% (wt/vol) of either GlcNAc or (GlcNAc)2 (fig. 1), squid were anaesthetised at various intervals and examined under a microscope. By staining host tissues with molecular probes, and using GFP properties, the location and aggregation of bacteria relative to the light-organ, ducts and pores was observed. After 4 hours, bacterial aggregates in 90% of squid (GlcNAc treated) had begun travelling down ducts to the crypts of light organ, whereas in (GlcNAc)2 treated water, the effect was only apparent around the pore, with only 7% of bacterial aggregates entering the ducts. This suggests that chemotaxis to chitin oligosaccharides derived from the light-organ are important in inducing the bacteria to swim towards and into the light organ from the pore entrance. As an aside, it would be interesting to find out if A. fischeri use flick motility (see Stocker, 2012) to increase their efficiency in swimming against the host ciliary mucus current through the pores and into the light organ.
The authors suggest that squid-derived chitin oligosaccharides act as a synomone “a compound that is produced by one species and invokes a behavioural response in another that is beneficial to them both”. It is now hypothesized that encoded chitinases degrade chitin to chitin oligosaccharides that A. fischeri chemotactically use to colonise the crypts of the light organ. Whilst chitin oligosaccharides are ubiquitous in the marine environment and also found in the tissues of the host, the increased concentrations coming from the light organ induce the chemotactic response. This migration is dependent upon this chemical gradient which, if disrupted, impairs the colonisation of the squid.
By Marie Dale and Rachel Coppock