Tracing the evolution of bioluminescent light organs across the deep-sea shrimp family Sergestidae using a genomic skimming and phylogenetic approach

Deep-sea shrimp of the family Sergestidae Dana, 1852 provide a unique system for studying the evolution of bioluminescence. Most species within the family possess autogenic bioluminescent photophores in one of three distinct forms: lensed photophores; non-lensed photophores; or internal organs of Pesta. This morphological diversity across the Sergestidae has resulted in recent major taxonomic revisions, dividing the two major genera (Sergia Stimpson, 1860 and Sergestes Milne Edwards, 1830) into 15. The present study capitalises on molecular data to construct an updated genus-level phylogeny of sergestid shrimp. DNA was successfully extracted from ~87 individuals belonging to 13 of the 15 newly proposed genera. A ‘genome skimming’ approach was implemented, allowing the capture of mitochondrial genomic data across 19 sergestid species. Additional individuals have been incorporated into the phylogeny through Sanger sequencing of both nuclear (H3 and NAK) and mitochondrial (16S and COI) genes. The resulting molecular phylogeny is compared with previous morphological trees with specific attention to genus-level relationships. The -sergestes group was rendered non-monophyletic and the -sergia group was recovered as monophyletic. Ancestral state reconstructions of light organ type indicate that organs of Pesta is the ancestral state for the family. Non-lensed photophores evolved once across the -sergia group, but were later lost in the deepest living genus, Sergia. Lensed photophores also evolved once within the genera Prehensilosergia Vereshchaka, Olesen & Lunina, 2014, Lucensosergia Vereshchaka, Olesen & Lunina, 2014 and Challengerosergia Vereshchaka, Olesen & Lunina, 2014. These findings identify preliminary patterns across light organ type and species’ depth distributions; however, future research that incorporates finer-scale depth data and more species is needed to confirm our findings.

Description of a new codling species of Physiculus from Taiwan (Gadiformes: Moridae)

A new species of codling Physiculus megastomus sp. nov. is described based on the holotype and a subadult paratype collected from northern and eastern Taiwan. The new species is classified in Physiculus by the presence of a ventral light organ on the abdomen and a chin barbel, and the absence of vomerine teeth. It is distinguished from congeners in having a large mouth with the posterior end of the maxilla extending well behind the level of the posterior margin of the orbit, its length 57.8‒60.7% in head length (HL) and the combination of the following characters: both jaws bearing caniniform teeth; snout, suborbital area, and gular region fully scaled; ventral light organ small, its length 5.5‒6.7% of distance from the interventral line to the origin of the anal fin (InV-af), located approximately at the mid-point of InV-af; five gill rakers on the upper limb of the first gill arch. DNA barcoding supported the establishment of the new species.

Bacterial Quorum-Sensing Regulation Induces Morphological Change in a Key Host Tissue during the Euprymna scolopes-Vibrio fischeri Symbiosis

Interbacterial signaling within a host-associated population can have profound effects on the behavior of the bacteria, for instance, in their production of virulence/colonization factors; in addition, such signaling can dictate the nature of the outcome for the host, in both pathogenic and beneficial associations. Using the monospecific squid-vibrio model of symbiosis, we examined how quorum-sensing regulation by the Vibrio fischeri population induces a biogeographic tissue phenotype that promotes the retention of this extracellular symbiont within the light organ of its host, Euprymna scolopes .

Host-Like Conditions Are Required for T6SS-Mediated Competition among Vibrio fischeri Light Organ Symbionts

Bacteria have evolved diverse strategies to compete for limited space and resources. Because these mechanisms can be costly to use, their expression and function are often restricted to specific environments where the benefits outweigh the costs.

A subcellular biochemical model for T6SS dynamics reveals winning competitive strategies

The Type VI secretion system (T6SS) is a broadly distributed interbacterial weapon that can be used to eliminate competing bacterial populations. Although unarmed target populations are typically used to study T6SS function, bacteria most likely encounter other T6SS-armed competitors in nature. The outcome of such battles is not well understood, neither is the connection between the outcomes with the subcellular details of the T6SS. Here, we incorporated new biological data derived from natural competitors of Vibrio fischeri light organ symbionts to build a biochemical model for T6SS function at the single cell level. The model accounts for activation of structure formation, structure assembly, and deployment. By developing an integrated agent-based model (IABM) that incorporates strain-specific T6SS parameters, we replicated outcomes of biological competitions, validating our approach. We used the IABM to isolate and manipulate strain-specific physiological differences between competitors, in a way that is not possible using biological samples, to identify winning strategies for T6SS-armed populations. We found that a tipping point exists where the cost of building more T6SS weapons outweighs their protective ability. Furthermore, we found that competitions between a T6SS-armed population and a unarmed target had different outcomes dependent on the geometry of the battlefield: target cells survived at the edges of a range expansion scenario where unlimited territory could be claimed, while competitions within a confined space, much like the light organ crypts where natural V. fischeri compete, resulted in the rapid elimination of the unarmed competitor.

Phylogenomics illuminates the evolution of bobtail and bottletail squid (order Sepiolida)

Bobtail and bottletail squid are small cephalopods with striking anti-predatory defensive mechanisms, bioluminescence, and complex morphology; that inhabit nektobenthic and pelagic environments around the world’s oceans. Yet, the evolution and diversification of these animals remain unclear. Here, we used shallow genome sequencing of thirty-two bobtail and bottletail squids to estimate their evolutionary relationships and divergence time. Our phylogenetic analyses show that each of Sepiadariidae, Sepiolidae, and the three subfamilies of the Sepiolidae are monophyletic. We found that the ancestor of the Sepiolinae very likely possessed a bilobed light organ with bacteriogenic luminescence. Sepiolinae forms a sister group to Rossinae and Heteroteuthinae, and split into Indo-Pacific and Atlantic-Mediterranean lineages. The origin of these lineages coincides with the end of the Tethys Sea and the separation of these regions during the Eocene and the beginning of the Oligocene. We demonstrated that sepiolids radiated after the Late Cretaceous and that major biogeographic events might have shaped their distribution and speciation.

Hybrid histidine kinase BinK represses Vibrio fischeri biofilm signaling at multiple developmental stages

The symbiosis between the Hawaiian bobtail squid, Euprymna scolopes, and its exclusive light-organ symbiont, Vibrio fischeri, provides a natural system in which to study host-microbe specificity and gene regulation during the establishment of a mutually-beneficial symbiosis. Colonization of the host relies on bacterial biofilm-like aggregation in the squid mucus field. Symbiotic biofilm formation is controlled by a two-component signaling (TCS) system consisting of regulators RscS-SypF-SypG, which together direct transcription of the Syp symbiotic polysaccharide. TCS systems are broadly important for bacteria to sense environmental cues and then direct changes in behavior. Previously, we identified hybrid histidine kinase BinK as a strong negative regulator of V. fischeri biofilm regulation, and here we further explore the function of BinK. To inhibit biofilm formation, BinK requires the predicted phosphorylation sites in both the histidine kinase (H362) and receiver (D794) domains. Furthermore, we show that RscS is not essential for host colonization when binK is deleted from strain ES114, and imaging of aggregate size revealed no benefit to the presence of RscS in a background lacking BinK. Strains lacking RscS still suffered in competition. Finally, we show that BinK functions to inhibit biofilm gene expression in the light organ crypts, providing evidence for biofilm gene regulation at later stages of host colonization. Overall, this study provides direct evidence for opposing activities of RscS and BinK and yields novel insights into biofilm regulation during the maturation of a beneficial symbiosis. IMPORTANCE Bacteria are often in a biofilm state, and transitions between planktonic and biofilm lifestyles are important for pathogenic, beneficial, and environmental microbes. The critical nature of biofilm formation during Vibrio fischeri colonization of the Hawaiian bobtail squid light organ provides an opportunity to study development of this process in vivo using a combination of genetic and imaging approaches. The current work refines the signaling circuitry of the biofilm pathway in V. fischeri, provides evidence that biofilm regulatory changes occur in the host, and identifies BinK as one of the regulators of that process. This study provides information about how bacteria regulate biofilm gene expression in an intact animal host.

MicroRNA-Mediated Regulation of Initial Host Responses in a Symbiotic Organ

ABSTRACT One of the most important events in an animal’s life history is the initial colonization by its microbial symbionts, yet little is known about this event’s immediate impacts on the extent of host gene expression or the molecular mechanisms controlling it. MicroRNAs (miRNAs) are short, noncoding RNAs that bind to target mRNAs, rapidly shaping gene expression by posttranscriptional control of mRNA translation and decay. Here, we show that, in the experimentally tractable binary squid-vibrio symbiosis, colonization of the light organ induces extensive changes in the miRNA transcriptome. Examination of the squid genome revealed the presence of evolutionarily conserved genes encoding elements essential for the production and processing of miRNAs. At 24 h postcolonization, 215 host miRNAs were detected in the light organ, 26 of which were differentially expressed in response to the symbionts. A functional enrichment analysis of genes potentially targeted by downregulation of certain miRNAs at the initiation of symbiosis revealed two major gene ontology (GO) term categories, neurodevelopment and tissue remodeling. This symbiont-induced downregulation is predicted to promote these activities in host tissues and is consistent with the well-described tissue remodeling that occurs at the onset of the association. Conversely, predicted targets of upregulated miRNAs, including the production of mucus, are consistent with attenuation of immune responses by symbiosis. Taken together, our data provide evidence that, at the onset of symbiosis, host miRNAs in the light organ drive alterations in gene expression that (i) orchestrate the symbiont-induced development of host tissues, and (ii) facilitate the partnership by dampening the immune response. IMPORTANCE Animals often acquire their microbiome from the environment at each generation, making the initial interaction of the partners a critical event in the establishment and development of a stable, healthy symbiosis. However, the molecular nature of these earliest interactions is generally difficult to study and poorly understood. We report that, during the initial 24 h of the squid-vibrio association, a differential expression of host miRNAs is triggered by the presence of the microbial partner. Predicted mRNA targets of these miRNAs were associated with regulatory networks that drive tissue remodeling and immune suppression, two major symbiosis-induced developmental outcomes in this and many other associations. These results implicate regulation by miRNAs as key to orchestrating the critical transcriptional responses that occur very early during the establishment of a symbiosis. Animals with more complex microbiota may have similar miRNA-driven responses as their association is initiated, supporting an evolutionary conservation of symbiosis-induced developmental mechanisms.

Reconstruction of Light Organ in Squid With The Histological Method of Electron Transmission Microscope

The light organ is an electronic device that can emit light. However, there are light organs in animals that can produce light naturally. Loligo duvaucelii is a species whose biolumenesence comes from fluorescent bacteria that live in symbiosis in its ink sacs. This study aims to determine in detail the construction of the squid light organ using the transmission electron microscopy (TEM) method. The results showed that this type of squid has a pair of light organs attached to the dorso-lateral ink sac. The light organ is spherical, some are found on the surface and some are embedded on the wall of the ink sac. It consists of a lens that is located on the outer surface of the ink sac, and a sac of light organs (embedded on the wall of the ink sac) with channels connecting the pocket to the mantle cavity. The wall of the sac of the light organ consists of three layers, namely the innermost layer which is multi-fold with microvilli on the cell surface and between the folds of the sac populated with bacteria, the dense layer that acts as a reflector, and the pigment layer. Cilia are observed on the surface of the duct connecting the sac with the mantle cavity. This study concluded that the construction of the squid light organ has a convex-shaped lens structure and is muscular. In the pockets of light organs, a dense population of bacteria is found. The reflector consists of many layers, and the pigment layer contains many granules.

Hybrid histidine kinase BinK represses Vibrio fischeri biofilm signaling at multiple developmental stages

ABSTRACTThe symbiosis between the Hawaiian bobtail squid, Euprymna scolopes, and its exclusive light-organ symbiont, Vibrio fischeri, provides a natural system in which to study host-microbe specificity and gene regulation during the establishment of a mutually-beneficial symbiosis. Colonization of the host relies on bacterial biofilm-like aggregation in the squid mucus field. Symbiotic biofilm formation is controlled by a two-component signaling (TCS) system consisting of regulators RscS-SypF-SypG, which together direct transcription of the Syp symbiotic polysaccharide. TCS systems are broadly important for bacteria to sense environmental cues and then direct changes in behavior. Previously, we identified hybrid histidine kinase BinK as a strong negative regulator of V. fischeri biofilm regulation, and here we further explore the function of BinK. To inhibit biofilm formation, BinK requires the predicted phosphorylation sites in both the histidine kinase (H362) and receiver (D794) domains. Furthermore, we show that a strain lacking BinK yields RscS non-essential for host colonization, and imaging of aggregate size revealed no benefit to the presence of RscS in a background lacking BinK. Strains lacking RscS still suffered in competition, suggesting another function for the protein. Finally, we show that BinK functions to inhibit biofilm gene expression in the light organ crypts, providing evidence for biofilm gene regulation at later stages of host colonization. Overall, this study provides direct evidence for opposing activities of RscS and BinK and yields novel insights into biofilm regulation during the maturation of a beneficial symbiosis.IMPORTANCEBacteria are often in a biofilm state, and transitions between planktonic and biofilm lifestyles are important for pathogenic, beneficial, and environmental microbes. The critical nature of biofilm formation during Vibrio fischeri colonization of the Hawaiian bobtail squid light organ provides an opportunity to study development of this process in vivo using a combination of genetic and imaging approaches. The current work refines the signaling circuitry of the biofilm pathway in V. fischeri, provides evidence that biofilm regulatory changes occur in the host, and identifies BinK as one of the regulators of that process. This study provides information about how bacteria regulate biofilm gene expression in an intact animal host.