Males conditionally inseminate at three female body locations according to female mating history and female maturity status in a squid.

In some squids, such as those in the family Loliginidae, upon copulation, females receive and store male-delivered sperm capsules, spermatangia, at two different body locations: the buccal membrane and the distal end of the oviduct. This insemination site dimorphism is associated with alternative reproductive strategies. However, in Loliolus sumatrensis, a species of Loliginidae, the females possess three insemination sites: buccal membrane (BM), basal left IV arm (ARM) and lateral head behind the left eye (EYE), therefore we studied such the unusual phenomena. We developed microsatellite markers and genotyped the paternity of each spermatangium on three sites. We found multiple paternity at every single site and simultaneous usage of all three sites by a few males. The seasonal dynamics of a population in the Seto Inland Sea revealed a set priority for the initial use of insemination sites as BM, followed by ARM and then EYE, whereas the maximum number of stored spermatangia was greater in EYE > ARM > BM. Female maturity status was correlated with the usage pattern of insemination sites but not with the number of stored spermatangia at any insemination site. These results suggest that a male squid inseminates at different locations according to female mating history and female maturity status.

Bacterial growth dynamics in a rhythmic symbiosis.

The symbiotic relationship between the bioluminescent bacterium Vibrio fischeri and the bobtail squid Euprymna scolopes serves as a valuable system to investigate bacterial growth and peptidoglycan (PG) synthesis within animal tissues. To better understand the growth dynamics of V. fischeri in the crypts of the light-emitting organ of its juvenile host, we showed that, after the daily dawn-triggered expulsion of most of the population, the remaining symbionts rapidly proliferate for ∼6 h. At that point the population enters a period of extremely slow growth that continues throughout the night until the next dawn. Further, we found that PG synthesis by the symbionts decreases as they enter the slow-growing stage. Surprisingly, in contrast to the most mature crypts (i.e., Crypt 1) of juvenile animals, most of the symbiont cells in the least mature crypts (i.e., Crypt 3) were not expelled and, instead, remained in the slow-growing state throughout the day, with almost no cell division. Consistent with this observation, the expression of the gene encoding the PG-remodeling enzyme, L,D-transpeptidase (LdtA), was greatest during the slowly growing stage of Crypt 1 but, in contrast, remained continuously high in Crypt 3. Finally, deletion of the ldtA gene resulted in a symbiont that grew and survived normally in culture, but was increasingly defective in competing against its parent strain in the crypts. This result suggests that remodeling of the PG to generate additional 3-3 linkages contributes to the bacterium's fitness in the symbiosis, possibly in response to stresses encountered during the very slow-growing stage.

Squid male alternative reproductive tactics are determined by birth date.

Alternative reproductive tactics (ARTs) are discontinuous phenotypes associated with reproduction, observed in males of many species. Typically, large males adopt a tactic of competing with rivals for mating, while small males adopt a tactic of stealing fertilization opportunities from the large males. The 'birth date hypothesis', proposing that the date of birth influences the determination of each male's reproductive tactic, has been tested only in teleost fish to date. Here, the birth date hypothesis was tested in ARTs of Japanese spear squid Heterololigo bleekeri (consort/sneaker) by analysing statolith growth increments. The birth date significantly differed between consorts (early-hatched) and sneakers (late-hatched). However, no differences were detected in growth history up to 100 days from hatching. Most immature males caught during the reproductive season were larger than sneakers, and their hatch date was similar to that of consorts, suggesting that these immature males had already been following a life-history pathway as a consort. These results indicate that ARTs of H. bleekeri are determined based on their hatch date in early life. This study firstly suggests that the birth date hypothesis applies to aquatic invertebrates, suggesting that the mechanism by which birth date determines the individual phenotype is a phenomenon more common than previously believed.

Modulations of ocean-atmosphere interactions on squid abundance over Southwest Atlantic.

Anthropogenic shifts in seas are reshaping fishing trends, with significant implications for aquatic food sources throughout this century. Examining a 21-year abundance dataset of Argentine shortfin squids Illex argentinus paired with a regional oceanic analysis, we noted strong correlations between squid annual abundance and sea surface temperature (SST) in January and February and eddy kinetic energy (EKE) from March to May in the Southwest Atlantic. A deeper analysis revealed combined ocean-atmosphere interactions, pinpointed as the primary mode in a rotated empirical orthogonal function analysis of SST. This pattern produced colder SST and amplified EKE in the surrounding seas, factors crucial for the unique life stages of squids. Future projections from the CMIP6 archive indicated that this ocean-atmosphere pattern, referred to as the Atlantic symmetric pattern, would persist in its cold SST phase, promoting increased squid abundance. However, rising SSTs due to global warming might counteract the abundance gains. Our findings uncover a previously unrecognized link between squids and specific environmental conditions governed by broader ocean-atmosphere interactions in the Southwest Atlantic. Integrating these insights with seasonal and decadal projections can offer invaluable information to stakeholders in squid fisheries and marine conservation under a changing climate.

Impact of modeled microgravity stress on innate immunity in a beneficial animal-microbe symbiosis.

The innate immune response is the first line of defense for all animals to not only detect invading microbes and toxins but also sense and interface with the environment. One such environment that can significantly affect innate immunity is spaceflight. In this study, we explored the impact of microgravity stress on key elements of the NFκB innate immune pathway. The symbiosis between the bobtail squid Euprymna scolopes and its beneficial symbiont Vibrio fischeri was used as a model system under a simulated microgravity environment. The expression of genes associated with the NFκB pathway was monitored over time as the symbiosis progressed. Results revealed that although the onset of the symbiosis was the major driver in the differential expression of NFκB signaling, the stress of simulated low-shear microgravity also caused a dysregulation of expression. Several genes were expressed at earlier time points suggesting that elements of the E. scolopes NFκB pathway are stress-inducible, whereas expression of other pathway components was delayed. The results provide new insights into the role of NFκB signaling in the squid-vibrio symbiosis, and how the stress of microgravity negatively impacts the host immune response. Together, these results provide a foundation to develop mitigation strategies to maintain host-microbe homeostasis during spaceflight.

Cephalopod learning and memory.

Cephalopod molluscs are renowned for their unique central nervous system - a donut-shaped brain organised around the oesophagus. This brain supports sophisticated learning and memory abilities. Between the 1950s and 1980s, these cognitive abilities were extensively studied in octopus (Figure 1A) - a now leading model for the study of memory and its neural substrates (approximately 200 papers during this period). The focus on octopus learning and memory was mainly due to their curious nature and the fact that they adapt to laboratory-controlled conditions, making them easy to test and maintain in captivity. Research on cephalopod cognition began to widen in the late 20th century, when scientists started focusing on other coleoid cephalopods (i.e., cuttlefish and squid) (Figure 1B,C), and not just on associative learning and memory per se, but other more complex aspects of cognition such as episodic-like memory (the ability to remember the what, where, and when of a past event), source memory (the retrieval of contextual details from a memory), and self-control (the ability to inhibit an action in the present to gain a more valuable future reward). Attention broadened further over the last two decades to focus on the shelled cephalopods - the nautiloids (Figure 1D). The nautiloids have relatively primitive brains compared to their soft-bodied cousins (octopus, cuttlefish, and squid) but research shows that they are still able to comparatively succeed in some cognitive tasks. In this primer, we will provide a general description of the types of memory studied in cephalopods, and discuss learning and memory experiments that address the main challenges cephalopods face during their daily lives: navigation, timing, and food selection. Determining the type of information cephalopods learn and remember and whether they use such information to overcome ecological challenges will highlight why these invertebrates evolved large and sophisticated brains.

Neural control of cephalopod camouflage.

In Die Another Day, James Bond receives an Aston Martin that can render itself invisible by dynamically reproducing the surroundings on the car's "polymer skin". In what is widely regarded as the worst Bond movie ever, the invisible car scene is cited as the moment the plot plunges into the truly absurd. But what if nature had actually invented such a technology, and did so hundreds of millions of years ago? The coleoid cephalopods - octopus, cuttlefish and squid - are living examples of dynamic camouflage. Their skin is covered with a high-resolution array of 'cellular pixels' (chromatophores) that are controlled by the brain. To disappear into their surroundings, cephalopods recreate an approximation of their environment on their skin by activating different combinations of colored chromatophores. However, unlike the fictional Bond car, whose surface is coated in tiny cameras to detect the environment, cephalopods don't see the world with their skin. Instead, the visual world is detected by the eyes, processed in the brain, and then used to activate motor commands that direct the skin's camouflage pattern. Thus, cephalopod skin patterns are an external manifestation of their internal perception of the world. How do cephalopods approximate the world with their skin? What can this teach us about how brains work? And which neurobiological tools will be needed to uncover the neural basis of camouflage?

The Inner Lives of Cephalopods.

The minds of cephalopods have captivated scientists for millennia, yet the extent that we can understand their subjective experiences remains contested. In this article, we consider the sum of our scientific progress towards understanding the inner lives of cephalopods. Here, we outline the behavioral responses to specific experimental paradigms that are helping us to reveal their subjective experiences. We consider evidence from three broad research categories, which help to illuminate whether soft-bodied cephalopods (octopus, cuttlefish, and squid) have an awareness of self, awareness of others, and an awareness of time. Where there are current gaps in the literature, we outline cephalopod behaviors that warrant experimental investigation. We argue that investigations, especially framed through the lens of comparative psychology, have the potential to extend our understanding of the inner lives of this extraordinary class of animals.

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.

Hydrodynamic Diversity of Jets Mediated by Giant and Non-Giant Axon Systems in Brief Squid.

Neural input is critical for establishing behavioral output, but understanding how neuromuscular signals give rise to behaviors remains a challenge. In squid, locomotion through jet propulsion underlies many key behaviors, and the jet is mediated by two parallel neural pathways, the giant and non-giant axon systems. Much work has been done on the impact of these two systems on jet kinematics, such as mantle muscle contraction and pressure-derived jet speed at the funnel aperture. However, little is known about any influence these neural pathways may have on the hydrodynamics of the jet after it leaves the squid and transfers momentum to the surrounding fluid for the animal to swim. To gain a more comprehensive view of squid jet propulsion, we made simultaneous measurements of neural activity, pressure inside the mantle cavity, and wake structure. By computing impulse and time-averaged forces from the wake structures of jets associated with giant or non-giant axon activity, we show that the influence of neural pathways on jet kinematics could extend to hydrodynamic impulse and force production. Specifically, the giant axon system produced jets with, on average, greater impulse magnitude than those of the non-giant system. However, non-giant impulse could exceed that of the giant system, evident by the graded range of its output in contrast to the stereotyped nature of the giant system. Our results suggest that the non-giant system offers flexibility in hydrodynamic output, while recruitment of giant axon activity can provide a reliable boost when necessary.

An Investigation into the Mechanism Mediating Counterillumination in Myctophid Fishes (Myctophidae).

AbstractCounterillumination is a camouflage strategy employed primarily by mesopelagic fishes, sharks, crustaceans, and squid, which use ventral bioluminescence to obscure their silhouettes when viewed from below. Although certain counterilluminating species have been shown to control the intensity of their ventral emissions to match the background downwelling light, the feedback mechanism mediating this ability is poorly understood. One proposed mechanism involves the presence and use of eye-facing photophores that would allow simultaneous detection and comparison of photophore emissions and downwelling solar light. Eye-facing photophores have been found in at least 34 species of counterilluminating stomiiform fishes and the myctophid Tarletonbeania crenularis. Here, we examined nine phylogenetically spaced myctophid species for eye-facing photophores to assess whether this mechanism is as prevalent in this group as it is in the Stomiiformes. First, microcomputed tomography imaging data were collected for each species, and three-dimensional reconstructions of the fishes were developed to identify potential eye-facing photophores. The fishes were then dissected under a stereomicroscope to confirm the presence of all identified photophores, probe for any photophores missed in the reconstruction analysis, and determine the orientation of the photophores' emissions. Although photophores were identified near the orbits of all species examined, none of the fishes' photophores directed light into their orbits, suggesting that myctophids may regulate bioluminescence through an alternative mechanism.

Sensory specializations drive octopus and squid behaviour.

The evolution of new traits enables expansion into new ecological and behavioural niches. Nonetheless, demonstrated connections between divergence in protein structure, function and lineage-specific behaviours remain rare. Here we show that both octopus and squid use cephalopod-specific chemotactile receptors (CRs) to sense their respective marine environments, but structural adaptations in these receptors support the sensation of specific molecules suited to distinct physiological roles. We find that squid express ancient CRs that more closely resemble related nicotinic acetylcholine receptors, whereas octopuses exhibit a more recent expansion in CRs consistent with their elaborated 'taste by touch' sensory system. Using a combination of genetic profiling, physiology and behavioural analyses, we identify the founding member of squid CRs that detects soluble bitter molecules that are relevant in ambush predation. We present the cryo-electron microscopy structure of a squid CR and compare this with octopus CRs1 and nicotinic receptors2. These analyses demonstrate an evolutionary transition from an ancestral aromatic 'cage' that coordinates soluble neurotransmitters or tastants to a more recent octopus CR hydrophobic binding pocket that traps insoluble molecules to mediate contact-dependent chemosensation. Thus, our study provides a foundation for understanding how adaptation of protein structure drives the diversification of organismal traits and behaviour.

Maturation state of colonization sites promotes symbiotic resiliency in the Euprymna scolopes-Vibrio fischeri partnership.

BACKGROUND: Many animals and plants acquire their coevolved symbiotic partners shortly post-embryonic development. Thus, during embryogenesis, cellular features must be developed that will promote both symbiont colonization of the appropriate tissues, as well as persistence at those sites. While variation in the degree of maturation occurs in newborn tissues, little is unknown about how this variation influences the establishment and persistence of host-microbe associations. RESULTS: The binary symbiosis model, the squid-vibrio (Euprymna scolopes-Vibrio fischeri) system, offers a way to study how an environmental gram-negative bacterium establishes a beneficial, persistent, extracellular colonization of an animal host. Here, we show that bacterial symbionts occupy six different colonization sites in the light-emitting organ of the host that have both distinct morphologies and responses to antibiotic treatment. Vibrio fischeri was most resilient to antibiotic disturbance when contained within the smallest and least mature colonization sites. We show that this variability in crypt development at the time of hatching allows the immature sites to act as a symbiont reservoir that has the potential to reseed the more mature sites in the host organ when they have been cleared by antibiotic treatment. This strategy may produce an ecologically significant resiliency to the association. CONCLUSIONS: The data presented here provide evidence that the evolution of the squid-vibrio association has been selected for a nascent organ with a range of host tissue maturity at the onset of symbiosis. The resulting variation in physical and chemical environments results in a spectrum of host-symbiont interactions, notably, variation in susceptibility to environmental disturbance. This "insurance policy" provides resiliency to the symbiosis during the critical period of its early development. While differences in tissue maturity at birth have been documented in other animals, such as along the infant gut tract of mammals, the impact of this variation on host-microbiome interactions has not been studied. Because a wide variety of symbiosis characters are highly conserved over animal evolution, studies of the squid-vibrio association have the promise of providing insights into basic strategies that ensure successful bacterial passage between hosts in horizontally transmitted symbioses. Video Abstract.

The mechanical properties of the mantle muscle of European cuttlefish (Sepia officinalis).

The circular muscles surrounding the mantle cavity of European cuttlefish (Sepia officinalis) generate the mechanical power to compress the cavity, forcing a jet of water out of the funnel, propelling the animal during jet propulsion swimming. During ontogeny, jetting frequency decreases in adults compared with juveniles, and this is expected to be reflected in the contractile properties of the locomotory muscles. To develop greater insight into how the locomotion of these animals is powered during ontogeny, we determined the mechanical properties of bundles of muscle fascicles during isometric, isotonic and cyclic length changes in vitro, at two life stages: juveniles and adults. The twitch kinetics were faster in juveniles than in adults (twitch rise time 257 ms compared with 371 ms; half-twitch relaxation 257 ms compared with 677 ms in juveniles and adults, respectively); however, twitch and tetanic stress, the maximum velocity of shortening and curvature of the force-velocity relationship did not differ. Under cyclic conditions, net power exhibited an inverted U-shaped relationship with cycle frequency in both juveniles and adults; the frequency at which maximum net power was achieved was shifted to lower cycle frequencies with increased maturity, which is consistent with the slower contraction and relaxation kinetics in adults compared with juveniles. The cycle frequency at which peak power was achieved during cyclical contractions in vitro was found to match that seen in vivo in juveniles, suggesting power is being maximised during jet propulsion swimming.

Quadrupedal Walking with the Skin: The Ambulatory Flaps in "Walking" Cuttlefish (Paintpot Cuttlefish, Metasepia tullbergi).

AbstractThe locomotion strategy of cephalopods is an important factor that influences their ability to exploit various oceanic environments. Particularly, Metasepia cuttlefish have a unique locomotion strategy; they prefer slow walking (ambling) on the seafloor over swimming. For this locomotion, they use their ventral arms as forelimbs and ambulatory flaps as hindlimbs. This locomotion is similar to the gait of quadruped vertebrates, where the forelimbs and hindlimbs on the left and right move alternately. The original description and some textbooks have considered these flaps to be muscular; however, this has not been proven. Here, we report the histological morphology of the ambulatory flaps of Metasepia tullbergi and their ambling locomotion. Histological observations indicated that the ambulatory flaps had a papillae structure comprising papillae musculature (dermal erector or retractor muscles) and connective tissue in the skin. Behavioral observations indicated that the ambulatory flaps changed their shape during ambling, which could explain the existence of the skin papillae. Our results suggest that ambulatory flaps are skin papillae, which can change shape by using their papillae musculature and connective tissue. This is a unique feature of Metasepia species that use the skin papillae for locomotion.

Temperature increases induce metabolic adjustments in the early developmental stages of bigfin reef squid (Sepioteuthis lessoniana).

Climate changes, such as extreme temperature shifts, can have a direct and significant impact on animals living in the ocean system. Ectothermic animals may undergo concerted metabolic shifts in response to ambient temperature changes. The physiological and molecular adaptations in cephalopods during their early life stages are largely unknown due to the challenge of rearing them outside of a natural marine environment. To overcome this obstacle, we established a pelagic bigfin reef squid (Sepioteuthis lessoniana) culture facility, which allowed us to monitor the effects of ambient thermal elevation and fluctuation on cephalopod embryos/larvae. By carefully observing embryonic development in the breeding facility, we defined 23 stages of bigfin reef squid embryonic development, beginning at stage 12 (blastocyst; 72 h post-egg laying) and continuing through hatching (~1 month post-egg laying). Since temperature recordings from the bigfin reef squid natural habitats have shown a steady rise over the past decade, we examined energy substrate utilization and cellular/metabolic responses in developing animals under different temperature conditions. As the ambient temperature increased by 7 °C, hatching larvae favored aerobic metabolism by about 2.3-fold. Short-term environmental warming stress inhibited oxygen consumption but did not affect ammonium excretion in stage (St.) 25 larvae. Meanwhile, an aerobic metabolism-related marker (CoxI) and a cellular stress-responsive marker (HSP70) were rapidly up-regulated upon acute warming treatments. In addition, our simulations of temperature oscillations mimicking natural daily rhythms did not result in significant changes in metabolic processes in St. 25 animals. As the ambient temperature increased by 7 °C, referred to as heatwave conditions, CoxI, HSP70, and antioxidant molecule (SOD) were stimulated, indicating the importance of cellular and metabolic adjustments. As with other aquatic species with high metabolic rates, squid larvae in the tropical/sub-tropical climate zone undergo adaptive metabolic shifts to maintain physiological functions and prevent excessive oxidative stress under environmental warming.

Multi-level control of adaptive camouflage by European cuttlefish.

To camouflage themselves on the seafloor, European cuttlefish Sepia officinalis control the expression of about 30 pattern components to produce a range of body patterns.1 If each component were under independent control, cuttlefish could produce at least 230 patterns. To examine how cuttlefish deploy this vast potential, we recorded cuttlefish on seven experimental backgrounds, each designed to resemble a pattern component, and then compared their responses to predictions of two models of sensory control of component expression. The body pattern model proposes that cuttlefish integrate low-level sensory cues to categorize the background and co-ordinate component expression to produce a small number of overall body patterns.2-4 The feature matching model proposes that each component is expressed in response to one (or more) local visual features, and the overall pattern depends upon the combination of features in the background. Consistent with the feature matching model, six of the backgrounds elicited a specific set of one to four components, whereas the seventh elicited eleven components typical of a disruptive body pattern. This evidence suggests that both modes of control are important, and we suggest how they can be implemented by a recent hierarchical model of the cuttlefish motor system.5,6.

Transitioning to confined spaces impacts bacterial swimming and escape response.

Symbiotic bacteria often navigate complex environments before colonizing privileged sites in their host organism. Chemical gradients are known to facilitate directional taxis of these bacteria, guiding them toward their eventual destination. However, less is known about the role of physical features in shaping the path the bacteria take and defining how they traverse a given space. The flagellated marine bacterium Vibrio fischeri, which forms a binary symbiosis with the Hawaiian bobtail squid, Euprymna scolopes, must navigate tight physical confinement during colonization, squeezing through a tissue bottleneck constricting to ∼2 µm in width on the way to its eventual home. Using microfluidic in vitro experiments, we discovered that V. fischeri cells alter their behavior upon entry into confined space, straightening their swimming paths and promoting escape from confinement. Using a computational model, we attributed this escape response to two factors: reduced directional fluctuation and a refractory period between reversals. Additional experiments in asymmetric capillary tubes confirmed that V. fischeri quickly escape from confined ends, even when drawn into the ends by chemoattraction. This avoidance was apparent down to a limit of confinement approaching the diameter of the cell itself, resulting in a balance between chemoattraction and evasion of physical confinement. Our findings demonstrate that nontrivial distributions of swimming bacteria can emerge from simple physical gradients in the level of confinement. Tight spaces may serve as an additional, crucial cue for bacteria while they navigate complex environments to enter specific habitats.

The effect of unexpected rewards on decision making in cuttlefish.

Despite numerous studies demonstrating the cognitive ability of cephalopods, there is currently no study showing an emotion-like behavior in this group of animals. To examine whether cuttlefish have different internal states, we developed a behavioral paradigm to assess if prior surprised events are able to alter the choice made by cuttlefish. By presenting unexpected food rewards to cuttlefish before the test, we investigated whether the reaction time of choosing between two shrimps, an intuitive response toward the prey without previous learning, at three different levels of discriminative tests (easy, difficult, and ambiguous), are different compared to the one without an unexpected reward. This behavioral paradigm serves to demonstrate whether cuttlefish are aware of ambiguous situations, and their choice outcome and reaction time are dependent of their internal states. The results show that the response latency was significantly shortened in the difficult and ambiguous tests when choosing from two shrimps that are either moderately different in size or similar sizes, respectively, when cuttlefish have received unexpected rewards before the test. These results were compared with tests during which the cuttlefish did not receive any reward in advance. Furthermore, this shortening of latency did not result in a difference in choice outcome during the difficult and ambiguous tests. Interestingly, even when cuttlefish have obtained the expected food rewards or simply made tentacular strike without prey capture each time before test, these prior experiences were sufficient to shorten the response latency in the difficult and ambiguous tests. However, different from the result of unexpected rewards, food consumption alone or prey capture failure did affect the choice outcome during the simple and difficult tests. Taken together, our findings suggest that pre-test treatments of unexpected and expected rewards or simply unsuccessful visual attack seem to induce cuttlefish to adopt different foraging behaviors. This context dependent decision making suggests that cuttlefish's foraging strategies are influenced by the previously surprised event and their internal states. It also shows a speed-accuracy tradeoff in difficult and ambiguous situations when foraging for prey. This observation may lead to a future investigation of the presence of emotional state in cephalopods.

Para-Aminobenzoic Acid, Calcium, and c-di-GMP Induce Formation of Cohesive, Syp-Polysaccharide-Dependent Biofilms in Vibrio fischeri.

The marine bacterium Vibrio fischeri efficiently colonizes its symbiotic squid host, Euprymna scolopes, by producing a transient biofilm dependent on the symbiosis polysaccharide (SYP). In vitro, however, wild-type strain ES114 fails to form SYP-dependent biofilms. Instead, genetically engineered strains, such as those lacking the negative regulator BinK, have been developed to study this phenomenon. Historically, V. fischeri has been grown using LBS, a complex medium containing tryptone and yeast extract; supplementation with calcium is required to induce biofilm formation by a binK mutant. Here, through our discovery that yeast extract inhibits biofilm formation, we uncover signals and underlying mechanisms that control V. fischeri biofilm formation. In contrast to its inability to form a biofilm on unsupplemented LBS, a binK mutant formed cohesive, SYP-dependent colony biofilms on tTBS, modified LBS that lacks yeast extract. Moreover, wild-type strain ES114 became proficient to form cohesive, SYP-dependent biofilms when grown in tTBS supplemented with both calcium and the vitamin para-aminobenzoic acid (pABA); neither molecule alone was sufficient, indicating that this phenotype relies on coordinating two cues. pABA/calcium supplementation also inhibited bacterial motility. Consistent with these phenotypes, cells grown in tTBS with pABA/calcium were enriched in transcripts for biofilm-related genes and predicted diguanylate cyclases, which produce the second messenger cyclic-di-GMP (c-di-GMP). They also exhibited elevated levels of c-di-GMP, which was required for the observed phenotypes, as phosphodiesterase overproduction abrogated biofilm formation and partially rescued motility. This work thus provides insight into conditions, signals, and processes that promote biofilm formation by V. fischeri. IMPORTANCE Bacteria integrate environmental signals to regulate gene expression and protein production to adapt to their surroundings. One such behavioral adaptation is the formation of a biofilm, which can promote adherence and colonization and provide protection against antimicrobials. Identifying signals that trigger biofilm formation and the underlying mechanism(s) of action remain important and challenging areas of investigation. Here, we determined that yeast extract, commonly used for growth of bacteria in laboratory culture, inhibits biofilm formation by Vibrio fischeri, a model bacterium used for investigating host-relevant biofilm formation. Omitting yeast extract from the growth medium led to the identification of an unusual signal, the vitamin para-aminobenzoic acid (pABA), that when added together with calcium could induce biofilm formation. pABA increased the concentrations of the second messenger, c-di-GMP, which was necessary but not sufficient to induce biofilm formation. This work thus advances our understanding of signals and signal integration controlling bacterial biofilm formation.