Population genomics of a bioluminescent symbiosis sheds light on symbiont transmission and specificity

All organisms depend on symbiotic associations with bacteria for their success, yet the processes by which specific symbioses are established and persist remain largely undescribed. To examine the ecological mechanisms involved in maintaining symbiont specificity over host generations, we examined the population genomics of a binary symbiosis involving the coral reef cardinalfish Siphamia tubifer and the luminous bacterium Photobacterium mandapamensis. Using restriction site-associated sequencing (RAD-Seq) methods we demonstrate that the facultative symbiont of S. tubifer exhibits genetic structure at spatial scales of tens of kilometers in Okinawa, Japan in the absence of physical dispersal barriers and in contrast to the host fish. These results suggest the host’s behavioral ecology help structure symbiont populations at a reef site by symbiont enrichment, consequently fostering symbiont specificity. This approach also revealed several symbiont genes that were divergent between host populations including genes known to play a role in other host-bacteria associations.

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.

Squid-Derived Chitin Oligosaccharides Are a Chemotactic Signal during Colonization by Vibrio fischeri

ABSTRACTChitin, a polymer ofN-acetylglucosamine (GlcNAc), is noted as the second most abundant biopolymer in nature. Chitin serves many functions for marine bacteria in the familyVibrionaceae(“vibrios”), in some instances providing a physical attachment site, inducing natural genetic competence, and serving as an attractant for chemotaxis. The marine luminous bacteriumVibrio fischeriis the specific symbiont in the light-emitting organ of the Hawaiian bobtail squid,Euprymna scolopes. The bacterium provides the squid with luminescence that the animal uses in an antipredatory defense, while the squid supports the symbiont's nutritional requirements.V. fischericells are harvested from seawater during each host generation, andV. fischeriis the only species that can complete this process in nature. Furthermore, chitin is located in squid hemocytes and plays a nutritional role in the symbiosis. We demonstrate here that chitin oligosaccharides produced by the squid host serve as a chemotactic signal for colonizing bacteria.V. fischeriuses the gradient of host chitin to enter the squid light organ duct and colonize the animal. We provide evidence that chitin serves a novel function in an animal-bacterial mutualism, as an animal-produced bacterium-attracting synomone.

Population Dynamics of Vibrio fischeri during Infection of Euprymna scolopes

ABSTRACT The luminous bacterium Vibrio fischeri colonizes a specialized light-emitting organ within its squid host, Euprymna scolopes. Newly hatched juvenile squid must acquire their symbiont from ambient seawater, where the bacteria are present at low concentrations. To understand the population dynamics of V. fischeri during colonization more fully, we used mini-Tn7 transposons to mark bacteria with antibiotic resistance so that the growth of their progeny could be monitored. When grown in culture, there was no detectable metabolic burden on V. fischeri cells carrying the transposon, which inserts in single copy in a specific intergenic region of the V. fischeri genome. Strains marked with mini-Tn7 also appeared to be equivalent to the wild type in their ability to infect and multiply within the host during coinoculation experiments. Studies of the early stages of colonization suggested that only a few bacteria became associated with symbiotic tissue when animals were exposed for a discrete period (3 h) to an inoculum of V. fischeri cells equivalent to natural population levels; nevertheless, all these hosts became infected. When three differentially marked strains of V. fischeri were coincubated with juvenile squid, the number of strains recovered from an individual symbiotic organ was directly dependent on the size of the inoculum. Further, these results indicated that, when exposed to low numbers of V. fischeri, the host may become colonized by only one or a few bacterial cells, suggesting that symbiotic infection is highly efficient.