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
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