A case study assessing the impact of mating frequency on the reproductive performance of the Hawaiian bobtail squid Euprymna scolopes.

BACKGROUND: The symbiosis between the Hawaiian bobtail squid Euprymna scolopes and bacterium Vibrio fischeri serves as a model for investigating the molecular mechanisms that promote the initial formation of animal-bacterial symbioses. Research with this system frequently depends on freshly hatched E. scolopes, but the husbandry factors that promote hatchling production in a mariculture facility remain underreported. Here we report on the reproductive performance of E. scolopes in response to decreased mating frequency. RESULTS: One animal cohort was maintained in a mariculture facility for 107 days, with females assigned to either a control group (mating once every 14 days) or an experimental group (mating once every 21 days). No differences between the groups were observed in survival, the number of egg clutches laid, or hatchling counts. Each group featured multiple females that were hyper-reproductive, i.e., they generated more than 8 egg clutches while in captivity. Examination of the distributions for daily hatchling counts of individual egg clutches revealed significant variation in the hatching patterns among clutches that was independent of mating frequency. Finally, an assessment of hatchling production showed that 93.5% of total hatchlings produced by the cohort were derived from egg clutches laid within the first 70 days. CONCLUSIONS: These results suggest a lower mating frequency does not impede hatchling production. Furthermore, the variation in hatchling production among egg clutches provides new insight into the reproductive performance of E. scolopes as a lab animal for microbiology research.

Quantification of the capacity of vibrio fischeri to establish symbiosis with Euprymna scolopes.

Most animals establish long-term symbiotic associations with bacteria that are critical for normal host physiology. The symbiosis that forms between the Hawaiian squid Euprymna scolopes and the bioluminescent bacterium Vibrio fischeri serves as an important model system for investigating the molecular mechanisms that promote animal-bacterial symbioses. E. scolopes hatch from their eggs uncolonized, which has led to the development of squid-colonization assays that are based on introducing culture-grown V. fischeri cells to freshly hatched juvenile squid. Recent studies have revealed that strains often exhibit large differences in how they establish symbiosis. Therefore, we sought to develop a simplified and reproducible protocol that permits researchers to determine appropriate inoculum levels and provides a platform to standardize the assay across different laboratories. In our protocol, we adapt a method commonly used for evaluating the infectivity of pathogens to quantify the symbiotic capacity of V. fischeri strains. The resulting metric, the symbiotic dose-50 (SD50), estimates the inoculum level that is necessary for a specific V. fischeri strain to establish a light-emitting symbiosis. Relative to other protocols, our method requires 2-5-fold fewer animals. Furthermore, the power analysis presented here suggests that the protocol can detect up to a 3-fold change in the SD50 between different strains.

Two enhancer binding proteins activate σ54-dependent transcription of a quorum regulatory RNA in a bacterial symbiont.

To colonize a host, bacteria depend on an ensemble of signaling systems to convert information about the various environments encountered within the host into specific cellular activities. How these signaling systems coordinate transitions between cellular states in vivo remains poorly understood. To address this knowledge gap, we investigated how the bacterial symbiont Vibrio fischeri initially colonizes the light organ of the Hawaiian bobtail squid Euprymna scolopes. Previous work has shown that the small RNA Qrr1, which is a regulatory component of the quorum-sensing system in V. fischeri, promotes host colonization. Here, we report that transcriptional activation of Qrr1 is inhibited by the sensor kinase BinK, which suppresses cellular aggregation by V. fischeri prior to light organ entry. We show that Qrr1 expression depends on the alternative sigma factor σ54 and the transcription factors LuxO and SypG, which function similar to an OR logic gate, thereby ensuring Qrr1 is expressed during colonization. Finally, we provide evidence that this regulatory mechanism is widespread throughout the Vibrionaceae family. Together, our work reveals how coordination between the signaling pathways underlying aggregation and quorum-sensing promotes host colonization, which provides insight into how integration among signaling systems facilitates complex processes in bacteria.

Quorum sensing facilitates interpopulation signaling by Vibrio fischeri within the light organ of Euprymna scolopes.

Quorum sensing is an intercellular signaling mechanism that enables bacterial cells to coordinate population-level behaviors. How quorum sensing functions in natural habitats remains poorly understood. Vibrio fischeri is a bacterial symbiont of the Hawaiian bobtail squid Euprymna scolopes and depends on LuxI/LuxR quorum sensing to produce the symbiotic trait of bioluminescence. A previous study demonstrated that animals emit light when co-colonized by a Δlux mutant, which lacks several genes within the lux operon that are necessary for bioluminescence production, and a LuxI- mutant, which cannot synthesize the quorum signaling molecule N-3-oxohexanoyl-homoserine lactone. Here, we build upon that observation and show that populations of LuxI- feature elevated promoter activity for the lux operon. We find that population structures comprising of Δlux and LuxI- are attenuated within the squid, but a wild-type strain enables the LuxI- strain type to be maintained in vivo. These experimental results support a model of interpopulation signaling, which provides basic insight into how quorum sensing functions within the natural habitats found within a host.

Impact of transit time on the reproductive capacity of Euprymna scolopes as a laboratory animal.

BACKGROUND: The Hawaiian bobtail squid Euprymna scolopes hosts various marine bacterial symbionts, and these symbioses have served as models for the animal-microbe relationships that are important for host health. Within a light organ, E. scolopes harbors populations of the bacterium Vibrio fischeri, which produce low levels of bioluminescence that the squid uses for camouflage. The symbiosis is initially established after a juvenile squid hatches from its egg and acquires bacterial symbionts from the ambient marine environment. The relative ease with which a cohort of wild-caught E. scolopes can be maintained in a mariculture facility has facilitated over 3 decades of research involving juvenile squid. However, because E. scolopes is native to the Hawaiian archipelago, their transport from Hawaii to research facilities often represents a stress that has the potential to impact their physiology. RESULTS: Here, we describe animal survival and reproductive capacity associated with a cohort of squid assembled from two shipments with markedly different transit times. We found that the lower juvenile squid counts generated by animals with the longer transit time were not due to the discrepancy in shipment but instead to fewer female squid that produced egg clutches at an elevated rate, which we term hyper-reproductivity. We find that hyper-reproductive females were responsible for 58% of the egg clutches laid. CONCLUSIONS: The significance of these findings for E. scolopes biology and husbandry is discussed, thereby providing a platform for future investigation and further development of this cephalopod as a valuable lab animal for microbiology research.

Vibrio fischeri imports and assimilates sulfate during symbiosis with Euprymna scolopes.

Sulfur is in cellular components of bacteria and is, therefore, an element necessary for growth. However, mechanisms by which bacteria satisfy their sulfur needs within a host are poorly understood. Vibrio fischeri is a bacterial symbiont that colonizes, grows, and produces bioluminescence within the light organ of the Hawaiian bobtail squid, which provides an experimental platform for investigating sulfur acquisition in vivo. Like other γ-proteobacteria, V. fischeri fuels sulfur-dependent anabolic processes with intracellular cysteine. Within the light organ, the abundance of a ΔcysK mutant, which cannot synthesize cysteine through sulfate assimilation, is attenuated, suggesting sulfate import is necessary for V. fischeri to establish symbiosis. Genes encoding sulfate-import systems of other bacteria that assimilate sulfate were not identified in the V. fischeri genome. A transposon mutagenesis screen implicated YfbS as a sulfate importer. YfbS is necessary for growth on sulfate and in the marine environment. During symbiosis, a ΔyfbS mutant is attenuated and strongly expresses sulfate-assimilation genes, which is a phenotype associated with sulfur-starved cells. Together, these results suggest V. fischeri imports sulfate via YfbS within the squid light organ, which provides insight into the molecular mechanisms by which bacteria harvest sulfur in vivo.

The Bacterial Enhancer Binding Protein VasH Promotes Expression of a Type VI Secretion System in Vibrio fischeri during Symbiosis.

Vibrio fischeri is a bacterial symbiont that colonizes the light organ of the Hawaiian bobtail squid, Euprymna scolopes Certain strains of V. fischeri express a type VI secretion system (T6SS), which delivers effectors into neighboring cells that result in their death. Strains that are susceptible to the T6SS fail to establish symbiosis with a T6SS-positive strain within the same location of the squid light organ, which is a phenomenon termed strain incompatibility. This study investigates the regulation of the T6SS in V. fischeri strain FQ-A001. Here, we report that the expression of Hcp, a necessary structural component of the T6SS, depends on the alternative sigma factor σ54 and the bacterial enhancer binding protein VasH. VasH is necessary for FQ-A001 to kill other strains, suggesting that VasH-dependent regulation is essential for the T6SS of V. fischeri to affect intercellular interactions. In addition, this study demonstrates VasH-dependent transcription of hcp within host-associated populations of FQ-A001, suggesting that the T6SS is expressed within the host environment. Together, these findings establish a model for transcriptional control of hcp in V. fischeri within the squid light organ, thereby increasing understanding of how the T6SS is regulated during symbiosis.IMPORTANCE Animals harbor bacterial symbionts with specific traits that promote host fitness. Mechanisms that facilitate intercellular interactions among bacterial symbionts impact which bacterial lineages ultimately establish symbiosis with the host. How these mechanisms are regulated is poorly characterized in nonhuman bacterial symbionts. This study establishes a model for the transcriptional regulation of a contact-dependent killing machine, thereby increasing understanding of mechanisms by which different strains compete while establishing symbiosis.

Incompatibility of Vibrio fischeri Strains during Symbiosis Establishment Depends on Two Functionally Redundant hcp Genes.

Bacteria that have the capacity to fill the same niche will compete with one another for the space and resources available within an ecosystem. Such competition is heightened among different strains of the same bacterial species. Nevertheless, different strains often inhabit the same host. The molecular mechanisms that impact competition between different strains within the same host are poorly understood. To address this knowledge gap, the type VI secretion system (T6SS), which is a mechanism for bacteria to kill neighboring cells, was examined in the marine bacterium Vibrio fischeri Different strains of V. fischeri naturally colonize the light organ of the bobtail squid Euprymna scolopes The genome of FQ-A001, a T6SS-positive strain, features two hcp genes that are predicted to encode identical subunits of the T6SS. Coincubation assays showed that either hcp gene is sufficient for FQ-A001 to kill another strain via the T6SS in vitro Additionally, induction of hcp expression is sufficient to induce killing activity in an FQ-A001 mutant lacking both hcp genes. Squid colonization assays involving inocula of FQ-A001-derived strains mixed with ES114 revealed that both hcp genes must be deleted for FQ-A001 and ES114 to occupy the same space within the light organ. These experimental results provide insight into the genetic factors necessary for the T6SS of V. fischeri to function in vivo, thereby increasing understanding of the molecular mechanisms that impact strain diversity within a host.IMPORTANCE Different bacterial strains compete to occupy the same niche. The outcome of such competition can be affected by the type VI secretion system (T6SS), an intercellular killing mechanism of bacteria. Here an animal-bacterial symbiosis is used as a platform for study of the genetic factors that promote the T6SS-mediated killing of one strain by another. Identification of the molecular determinants of T6SS function in vivo contributes to the understanding of how different strains interact within a host.

Draft Genome Sequences of Type VI Secretion System-Encoding Vibrio fischeri Strains FQ-A001 and ES401.

The type VI secretion system (T6SS) facilitates lethal competition between bacteria through direct contact. Comparative genomics has facilitated the study of these systems in Vibrio fischeri, which colonizes the squid host Euprymna scolopes Here, we report the draft genome sequences of two lethal V. fischeri strains that encode the T6SS, FQ-A001 and ES401.

Bacterial symbionts use a type VI secretion system to eliminate competitors in their natural host.

Intraspecific competition describes the negative interaction that occurs when different populations of the same species attempt to fill the same niche. Such competition is predicted to occur among host-associated bacteria but has been challenging to study in natural biological systems. Although many bioluminescent Vibrio fischeri strains exist in seawater, only a few strains are found in the light-organ crypts of an individual wild-caught Euprymna scolopes squid, suggesting a possible role for intraspecific competition during early colonization. Using a culture-based assay to investigate the interactions of different V. fischeri strains, we found "lethal" and "nonlethal" isolates that could kill or not kill the well-studied light-organ isolate ES114, respectively. The killing phenotype of these lethal strains required a type VI secretion system (T6SS) encoded in a 50-kb genomic island. Multiple lethal and nonlethal strains could be cultured from the light organs of individual wild-caught adult squid. Although lethal strains eliminate nonlethal strains in vitro, two lethal strains could coexist in interspersed microcolonies that formed in a T6SS-dependent manner. This coexistence was destabilized upon physical mixing, resulting in one lethal strain consistently eliminating the other. When juvenile squid were coinoculated with lethal and nonlethal strains, they occupied different crypts, yet they were observed to coexist within crypts when T6SS function was disrupted. These findings, using a combination of natural isolates and experimental approaches in vitro and in the animal host, reveal the importance of T6SS in spatially separating strains during the establishment of host colonization in a natural symbiosis.

Intraspecific Competition Impacts Vibrio fischeri Strain Diversity during Initial Colonization of the Squid Light Organ.

UNLABELLED: Animal development and physiology depend on beneficial interactions with microbial symbionts. In many cases, the microbial symbionts are horizontally transmitted among hosts, thereby making the acquisition of these microbes from the environment an important event within the life history of each host. The light organ symbiosis established between the Hawaiian squid Euprymna scolopes and the bioluminescent bacterium Vibrio fischeri is a model system for examining how hosts acquire horizontally transmitted microbial symbionts. Recent studies have revealed that the light organ of wild-caught E. scolopes squid contains polyclonal populations of V. fischeri bacteria; however, the function and development of such strain diversity in the symbiosis are unknown. Here, we report our phenotypic and phylogenetic characterizations of FQ-A001, which is a V. fischeri strain isolated directly from the light organ of an E. scolopes individual. Relative to the type strain ES114, FQ-A001 exhibits similar growth in rich medium but displays increased bioluminescence and decreased motility in soft agar. FQ-A001 outcompetes ES114 in colonizing the crypt spaces of the light organs. Remarkably, we find that animals cocolonized with FQ-A001 and ES114 harbor singly colonized crypts, in contrast to the cocolonized crypts observed from competition experiments involving single genotypes. The results with our two-strain system suggest that strain diversity within the squid light organ is a consequence of diversity in the single-strain colonization of individual crypt spaces. IMPORTANCE: The developmental programs and overall physiologies of most animals depend on diverse microbial symbionts that are acquired from the environment. However, the basic principles underlying how microbes colonize their hosts remain poorly understood. Here, we report our findings of bacterial strain competition within the coevolved animal-microbe symbiosis composed of the Hawaiian squid and bioluminescent bacterium Vibrio fischeri Using fluorescent proteins to differentially label two distinct V. fischeri strains, we find that the strains are unable to coexist in the same niche within the host. Our results suggest that strain competition for distinct colonization sites dictates the strain diversity associated with the host. Our study provides a platform for studying how strain diversity develops within a host.