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.

A resonant squid-inspired robot unlocks biological propulsive efficiency.

Elasticity has been linked to the remarkable propulsive efficiency of pulse-jet animals such as the squid and jellyfish, but reports that quantify the underlying dynamics or demonstrate its application in robotic systems are rare. This work identifies the pulse-jet propulsion mode used by these animals as a coupled mass-spring-mass oscillator, enabling the design of a flexible self-propelled robot. We use this system to experimentally demonstrate that resonance greatly benefits pulse-jet swimming speed and efficiency, and the robot's optimal cost of transport is found to match that of the most efficient biological swimmers in nature, such as the jellyfish Aurelia aurita The robot also exhibits a preferred Strouhal number for efficient swimming, thereby bridging the gap between pulse-jet propulsion and established findings in efficient fish swimming. Extensions of the current robotic framework to larger amplitude oscillations could combine resonance effects with optimal vortex formation to further increase propulsive performance and potentially outperform biological swimmers altogether.

Transient pressure modeling in jetting animals.

There are many marine animals that employ a form of jet propulsion to move through the water, often creating the jets by expanding and collapsing internal fluid cavities. Due to the unsteady nature of this form of locomotion and complex body/nozzle geometries, standard modeling techniques prove insufficient at capturing internal pressure dynamics, and hence swimming forces. This issue has been resolved with a novel technique for predicting the pressure inside deformable jet producing cavities (M. Krieg and K. Mohseni, J. Fluid Mech., 769, 2015), which is derived from evolution of the surrounding fluid circulation. However, this model was only validated for an engineered jet thruster with simple geometry and relatively high Reynolds number (Re) jets. The purpose of this manuscript is twofold: (i) to demonstrate how the circulation based pressure model can be used to analyze different animal body motions as they relate to propulsive output, for multiple species of jetting animals, (ii) and to quantitatively validate the pressure modeling for biological jetting organisms (typically characterized by complicated cavity geometry and low/intermediate Re flows). Using jellyfish (Sarsia tubulosa) as an example, we show that the pressure model is insensitive to complex cavity geometry, and can be applied to lower Re swimming. By breaking down the swimming behavior of the jellyfish, as well as that of squid and dragonfly larvae, according to circulation generating mechanisms, we demonstrate that the body motions of Sarsia tubulosa are optimized for acceleration at the beginning of pulsation as a survival response. Whereas towards the end of jetting, the velar morphology is adjusted to decrease the energetic cost. Similarly, we show that mantle collapse rates in squid maximize propulsive efficiency. Finally, we observe that the hindgut geometry of dragonfly larvae minimizes the work required to refill the cavity. Date Received: 10-18-2019, Date Accepted: 99-99-9999 *kriegmw@hawaii.edu, UHM Ocean and Res Eng, 2540 Dole St, Honolulu, HI 96822.

An Expanded Transposon Mutant Library Reveals that Vibrio fischeri δ-Aminolevulinate Auxotrophs Can Colonize Euprymna scolopes.

Libraries of defined mutants are valuable research tools but necessarily lack gene knockouts that are lethal under the conditions used in library construction. In this study, we augmented a Vibrio fischeri mutant library generated on a rich medium (LBS, which contains [per liter] 10 g of tryptone, 5 g of yeast extract, 20 g of NaCl, and 50 mM Tris [pH 7.5]) by selecting transposon insertion mutants on supplemented LBS and screening for those unable to grow on LBS. We isolated strains with insertions in alr, glr (murI), glmS, several heme biosynthesis genes, and ftsA, as well as a mutant disrupted 14 bp upstream of ftsQ Mutants with insertions in ftsA or upstream of ftsQ were recovered by addition of Mg2+ to LBS, but their cell morphology and motility were affected. The ftsA mutant was more strongly affected and formed cells or chains of cells that appeared to wind back on themselves helically. Growth of mutants with insertions in glmS, alr, or glr was recovered with N-acetylglucosamine (NAG), d-alanine, or d-glutamate, respectively. We hypothesized that NAG, d-alanine, or d-glutamate might be available to V. fischeri in the Euprymna scolopes light organ; however, none of these mutants colonized the host effectively. In contrast, hemA and hemL mutants, which are auxotrophic for δ-aminolevulinate (ALA), colonized at wild-type levels, although mutants later in the heme biosynthetic pathway were severely impaired or unable to colonize. Our findings parallel observations that legume hosts provide Bradyrhizobium symbionts with ALA, but they contrast with virulence phenotypes of hemA mutants in some pathogens. The results further inform our understanding of the symbiotic light organ environment.IMPORTANCE By supplementing a rich yeast-based medium, we were able to recover V. fischeri mutants with insertions in conditionally essential genes, and further characterization of these mutants provided new insights into this bacterium's symbiotic environment. Most notably, we show evidence that the squid host can provide V. fischeri with enough ALA to support its growth in the light organ, paralleling the finding that legumes provide Bradyrhizobium ALA in symbiotic nodules. Taken together, our results show how a simple method of augmenting already rich media can expand the reach and utility of defined mutant libraries.

Loudness-dependent behavioral responses and habituation to sound by the longfin squid (Doryteuthis pealeii).

Sound is an abundant cue in the marine environment, yet we know little regarding the frequency range and levels which induce behavioral responses in ecologically key marine invertebrates. Here we address the range of sounds that elicit unconditioned behavioral responses in squid Doryteuthis pealeii, the types of responses generated, and how responses change over multiple sound exposures. A variety of response types were evoked, from inking and jetting to body pattern changes and fin movements. Squid responded to sounds from 80 to 1000 Hz, with response rates diminishing at the higher and lower ends of this frequency range. Animals responded to the lowest sound levels in the 200-400 Hz range. Inking, an escape response, was confined to the lower frequencies and highest sound levels; jetting was more widespread. Response latencies were variable but typically occurred after 0.36 s (mean) for jetting and 0.14 s for body pattern changes; pattern changes occurred significantly faster. These results demonstrate that squid can exhibit a range of behavioral responses to sound include fleeing, deimatic and protean behaviors, all of which are associated with predator evasion. Response types were frequency and sound level dependent, reflecting a relative loudness concept to sound perception in squid.

Dietary squid ink polysaccharide induces goblet cells to protect small intestine from chemotherapy induced injury.

Gastrointestinal mucositis induced by chemotherapy is associated with alterations of intestinal barrier function due to the potential damage induced by anti-cancer drugs on the epithelial cells. Goblet cells, an important epithelial lining in the intestine, contribute to innate immunity by secreting mucin glycoproteins. Employing a mouse model of chemotherapy induced intestinal mucosal immunity injury by cyclophosphamide, we demonstrated for the first time that polysaccharide from the ink of Ommastrephes bartramii (OBP) enhanced Cyto18, which is a mucin expression in goblet cells. The up-regulation of mucins by OBP relied on the augmented quantity of goblet cells, but not on the changes in the ultrastructure of endoplasmic reticulum (ER). Our results may have important implications for enhanced immunopotentiation function of functional OBP on intestinal mucosal immunity against intestinal disorders involving inflammation and infection.

Acoustically evoked potentials in two cephalopods inferred using the auditory brainstem response (ABR) approach.

It is still a matter of debate whether cephalopods can detect sound frequencies above 400 Hz. So far there is no proof for the detection of underwater sound above 400 Hz via a physiological approach. The controversy of whether cephalopods have a sound detection ability above 400 Hz was tested using the auditory brainstem response (ABR) approach, which has been successfully applied in fish, crustaceans, amphibians, reptiles and birds. Using ABR we found that auditory evoked potentials can be obtained in the frequency range 400 to 1500 Hz (Sepiotheutis lessoniana) and 400 to 1000 Hz (Octopus vulgaris), respectively. The thresholds of S. lessoniana were generally lower than those of O. vulgaris.

Cloning and functional characterization of squid voltage-dependent Ca2+ channel beta subunits: involvement of N-terminal sequences in differential modulation of the current.

cDNAs that encode beta subunits of voltage-dependent Ca(2+) channel were cloned from the optic lobe of the squid Loligo bleekeri. The subunits, LoCa(v)beta(1a) and LoCa(v)beta(1b) are 96% identical in amino acid sequence. The sole sequence differences are in the N-terminal region and in a five amino acid insertion in the central region of LoCa(v)beta(1b). RT-PCR revealed that LoCa(v)beta(1a) and LoCa(v)beta(1b) transcripts were expressed mainly in the optic lobe and stellate ganglion, and more weakly in mantle muscle, systemic heart, gill, branchial heart, stomach and liver. Coexpression of LoCa(v)beta(1a) or LoCa(v)beta(1b) with mammalian Ca(v)2.3 and alpha(2)/delta subunits in the Xenopus oocyte resulted in high-voltage-activated currents, and showed slow current inactivation and moderate steady-state inactivation. Comparison of the squid subunits with four mammalian beta subunits, beta(1b), beta(2a), beta(3) and beta(4), demonstrated that the modulatory effects of the beta subunits on steady-state inactivation kinetics were beta(3)

Direct visualization of particle velocity distribution by pseudostereoscopic viewing of time-lapsed sequential images: application to fast axonal transport.

We describe a simple method for direct visualization of the velocity distribution of particles moving against an immobile background. The technique involves pseudostereoscopic viewing of image pairs separated by an appropriate time interval in a sequential recording of the subject. Under these conditions, the positive or negative parallax arising from particle motion results in the binocular image of a particle being perceived as raised or lowered relative to an immobile background plane depending on its direction of movement, and with the degree of perceived elevation being proportional to its speed. In effect, the binocular optic axis becomes a velocity (speed) axis under these conditions. The technique is illustrated with examples of image pair sequences showing fast axonal transport in lobster and squid axons using video-enhanced differential interference contrast microscopy. However, the pseudostereoscopic method is quite generally applicable to both microscopic and macroscopic time-dependent phenomena. Particle speeds can be quantitated using standard procedures for measuring frame-to-frame particle displacements, or alternatively, by determination of parallax using stereogrammatic methods. It should be also readily adaptable for on-line monitoring of particle velocity distribution, particularly in video systems where frame buffers can be utilized to extract and present serial image pairs having any desired time separation from video-taped sequences.

The August Krogh Principle: "For many problems there is an animal on which it can be most conveniently studied".

The idea stated in the title, first clearly expressed by August Krogh, is illustrated by examples from animal biochemistry, physiology, general cell biology, experimental medicine, ethology and botany. General aspects of the concept are discussed.