Molecular characterization of cell types in the squid Loligo vulgaris.

Cephalopods are set apart from other mollusks by their advanced behavioral abilities and the complexity of their nervous systems. Because of the great evolutionary distance that separates vertebrates from cephalopods, it is evident that higher cognitive features have evolved separately in these clades despite the similarities that they share. Alongside their complex behavioral abilities, cephalopods have evolved specialized cells and tissues, such as the chromatophores for camouflage or suckers to grasp prey. Despite significant progress in genome and transcriptome sequencing, the molecular identities of cell types in cephalopods remain largely unknown. We here combine single-cell transcriptomics with in situ gene expression analysis to uncover cell type diversity in the European squid Loligo vulgaris. We describe cell types that are conserved with other phyla such as neurons, muscles, or connective tissues but also cephalopod-specific cells, such as chromatophores or sucker cells. Moreover, we investigate major components of the squid nervous system including progenitor and developing cells, differentiated cells of the brain and optic lobes, as well as sensory systems of the head. Our study provides a molecular assessment for conserved and novel cell types in cephalopods and a framework for mapping the nervous system of L. vulgaris.

The functional-morphological adaptive strategy of digestive organs of decapodiform cephalopods.

The digestive organs in decapodiform cephalopod species morphologically vary by individual lifestyle. We examined the following six species of adult decapodiformes cephalopods representing different habitats: Todarodes pacificus, Loligo bleekeri, Loligo edulis, Watasenia scintillans (pelagic), Sepia lycidas and Euprymna morsei (benthic). L. bleekeri and L. edulis possess a bursiform cecal sac connected to the cecum. Pelagic species have a single digestive gland smaller than in benthic species. T. pacificus has an oval digestive gland larger than that of L. bleekeri and L. edulis, which possess withered-looking and smaller digestive glands. In contrast, the digestive glands in benthic species are paired. S. lycidas and E. morsei have well-developed and larger digestive glands than those of the pelagic species. Well-developed digestive duct appendages are found in benthic species. In qualification of the mass of digestive organs, pelagic species have smaller stomachs, digestive glands and digestive ducts' appendages than benthic species. Because pelagic species need to swim, they may possess smaller stomachs and larger cecums for more rapid digestion. A smaller digestive gland may have the advantage of reducing the body weight in pelagic species for rapid swimming. In contrast, since benthic species require a longer time for digestion than pelagic species, they compact more food in their stomachs and absorb nutrients via more organs, such as the digestive grand and digestive duct appendages, in addition to cecum.

Ultrastructural damage of Loligo vulgaris and Illex coindetii statocysts after low frequency sound exposure.

There is a considerable lack of information concerning marine invertebrate sensitivity to sound exposure. However, recent findings on cuttlefish and octopi showed that exposure to artificial noise had a direct consequence on the functionality and physiology of the statocysts, sensory organs, which are responsible for their equilibrium and movements in the water column. Owing to a lack of available data on deep diving cephalopod species, we conducted a noise exposure comparative experiment on one Mediterranean squid, Illex coindetii, and on the European squid Loligo vulgaris. Scanning electron microscopy (SEM) revealed similar injuries in the inner structure of the statocysts, as those found in cuttlefish and octopi. In addition to the ultrastructural description of the lesions, we publish here the first images of the crista-cupula system and inner statocyst cavity of I. coindetii.

Orientation dependence of broadband acoustic backscattering from live squid.

A controlled laboratory experiment of broadband acoustic backscattering from live squid (Loligo pealeii) was conducted using linear chirp signals (60-103 kHz) with data collected over the full 360° of orientation in the lateral plane, in <1° increments. The acoustic measurements were compared with an analytical prolate spheroid model and a three-dimensional numerical model with randomized squid shape, both based on the distorted-wave Born approximation formulation. The data were consistent with the hypothesized fluid-like scattering properties of squid. The contributions from the front and back interfaces of the squid were found to dominate the scattering at normal incidence, while the arms had a significant effect at other angles. The three-dimensional numerical model predictions out-performed the prolate spheroid model over a wide range of orientations. The predictions were found to be sensitive to the shape parameters, including the arms and the fins. Accurate predictions require setting these shape parameters to best describe the most probable squid shape for different applications. The understanding developed here serves as a basis for the accurate interpretation of in situ acoustic scattering measurements of squid.

Why small males have big sperm: dimorphic squid sperm linked to alternative mating behaviours.

BACKGROUND: Sperm cells are the target of strong sexual selection that may drive changes in sperm structure and function to maximize fertilisation success. Sperm evolution is regarded to be one of the major consequences of sperm competition in polyandrous species, however it can also be driven by adaptation to the environmental conditions at the site of fertilization. Strong stabilizing selection limits intra-specific variation, and therefore polymorphism, among fertile sperm (eusperm). Here we analyzed reproductive morphology differences among males employing characteristic alternative mating behaviours, and so potentially different conditions of sperm competition and fertilization environment, in the squid Loligo bleekeri. RESULTS: Large consort males transfer smaller (average total length = 73 µm) sperm to a female's internal sperm storage location, inside the oviduct; whereas small sneaker males transfer larger (99 µm) sperm to an external location around the seminal receptacle near the mouth. No significant difference in swimming speed was observed between consort and sneaker sperm. Furthermore, sperm precedence in the seminal receptacle was not biased toward longer sperm, suggesting no evidence for large sperm being favoured in competition for space in the sperm storage organ among sneaker males. CONCLUSIONS: Here we report the first case, in the squid Loligo bleekeri, where distinctly dimorphic eusperm are produced by different sized males that employ alternative mating behaviours. Our results found no evidence that the distinct sperm dimorphism was driven by between- and within-tactic sperm competition. We propose that presence of alternative fertilization environments with distinct characteristics (i.e. internal or external), whether or not in combination with the effects of sperm competition, can drive the disruptive evolution of sperm size.

A highly distributed Bragg stack with unique geometry provides effective camouflage for Loliginid squid eyes.

Cephalopods possess a sophisticated array of mechanisms to achieve camouflage in dynamic underwater environments. While active mechanisms such as chromatophore patterning and body posturing are well known, passive mechanisms such as manipulating light with highly evolved reflectors may also play an important role. To explore the contribution of passive mechanisms to cephalopod camouflage, we investigated the optical and biochemical properties of the silver layer covering the eye of the California fishery squid, Loligo opalescens. We discovered a novel nested-spindle geometry whose correlated structure effectively emulates a randomly distributed Bragg reflector (DBR), with a range of spatial frequencies resulting in broadband visible reflectance, making it a nearly ideal passive camouflage material for the depth at which these animals live. We used the transfer-matrix method of optical modelling to investigate specular reflection from the spindle structures, demonstrating that a DBR with widely distributed thickness variations of high refractive index elements is sufficient to yield broadband reflectance over visible wavelengths, and that unlike DBRs with one or a few spatial frequencies, this broadband reflectance occurs from a wide range of viewing angles. The spindle shape of the cells may facilitate self-assembly of a random DBR to achieve smooth spatial distributions in refractive indices. This design lends itself to technological imitation to achieve a DBR with wide range of smoothly varying layer thicknesses in a facile, inexpensive manner.

D-Aspartic acid is a novel endogenous neurotransmitter.

D-aspartic acid (D-Asp) is present in invertebrate and vertebrate neuroendocrine tissues, where it carries out important physiological functions and is implicated in nervous system development. We show here that D-Asp is a novel endogenous neurotransmitter in two distantly related animals, a mammal (Rattus norvegicus) and a mollusk (Loligo vulgaris). Our main findings demonstrate that D-Asp is present in high concentrations in the synaptic vesicles of axon terminals; synthesis for this amino acid occurs in neurons by conversion of L-Asp to D-Asp via D-aspartate racemase; depolarization of nerve endings with K(+) ions evokes an immediate release of D-Asp in a Ca(2+) dependent manner; specific receptors for D-Asp occur at the postsynaptic membrane, as demonstrated by binding assays and by the expansion of squid skin chromatophores; D-aspartate oxidase, the specific enzyme that oxidizes D-Asp, is present in the postsynaptic membranes; and stimulation of nerve endings with D-Asp triggers signal transduction by increasing the second messenger cAMP. Taken together, these data demonstrate that D-Asp fulfills all criteria necessary to be considered a novel endogenous neurotransmitter. Given its known role in neurogenesis, learning, and neuropathologies, our results have important implications for biomedical and clinical research.

Sound detection by the longfin squid (Loligo pealeii) studied with auditory evoked potentials: sensitivity to low-frequency particle motion and not pressure.

Although hearing has been described for many underwater species, there is much debate regarding if and how cephalopods detect sound. Here we quantify the acoustic sensitivity of the longfin squid (Loligo pealeii) using near-field acoustic and shaker-generated acceleration stimuli. Sound field pressure and particle motion components were measured from 30 to 10,000 Hz and acceleration stimuli were measured from 20 to 1000 Hz. Responses were determined using auditory evoked potentials (AEPs) with electrodes placed near the statocysts. Evoked potentials were generated by both stimuli and consisted of two wave types: (1) rapid stimulus-following waves, and (2) slower, high-amplitude waves, similar to some fish AEPs. Responses were obtained between 30 and 500 Hz with lowest thresholds between 100 and 200 Hz. At the best frequencies, AEP amplitudes were often >20 µV. Evoked potentials were extinguished at all frequencies if (1) water temperatures were less than 8°C, (2) statocysts were ablated, or (3) recording electrodes were placed in locations other than near the statocysts. Both the AEP response characteristics and the range of responses suggest that squid detect sound similarly to most fish, with the statocyst acting as an accelerometer through which squid detect the particle motion component of a sound field. The modality and frequency range indicate that squid probably detect acoustic particle motion stimuli from both predators and prey as well as low-frequency environmental sound signatures that may aid navigation.

Immunohistochemical localization of a GnRH-like peptide in the brain of the cephalopod spear-squid, Loligo bleekeri.

We examined whether a gonadotropin-releasing hormone (GnRH)-like peptide exists in the brain of the cephalopod spear-squid, Loligo bleekeri, by performing a time-resolved fluoroimmunoassay and immunohistochemistry. The displacement curve obtained for serially diluted extracts of the spear-squid brain paralleled the chicken GnRH-II (cGnRH-II) standard curve, indicating the existence of a cGnRH-II-like peptide in the brain. For immunohistochemistry, a mouse monoclonal antibody raised against the common amino acid sequence of GnRH (LRH13) and a rabbit polyclonal antibody raised against cGnRH-II were used. GnRH-like-immunoreactive (ir) cell bodies (that reacted with LRH13) were mainly detected in the central part of the ventral magnocellular lobe (vmL), and a few cell bodies were also detected in the olfactory lobe and palliovisceral lobe (pvL). Bundles of GnRH-like-ir axons were observed running from the vmL to the internal brain regions. GnRH-like-ir fibers were widely distributed in almost all the brain regions. cGnRH-II-ir cell bodies were localized in the optic gland, outer region of the vmL, and pvL. Further, cGnRH-II-ir fibers were distributed in the wide areas of the brain. These results suggest that at least two forms of GnRH-like peptidergic neuronal systems exist in the spear-squid brain.

Partial phase synchronization of neural populations due to random Poisson inputs.

We show that populations of identical uncoupled neurons exhibit partial phase synchronization when stimulated with independent, random unidirectional current spikes with interspike time intervals drawn from a Poisson distribution. We characterize this partial synchronization using the phase distribution of the population, and consider analytical approximations and numerical simulations of phase-reduced models and the corresponding conductance-based models of typical Type I (Hindmarsh-Rose) and Type II (Hodgkin-Huxley) neurons, showing quantitatively how the extent of the partial phase synchronization depends on the magnitude and mean interspike frequency of the stimulus. Furthermore, we present several simple examples that disprove the notion that phase synchrony must be strongly related to spike synchrony. Instead, the importance of partial phase synchrony is shown to lie in its influence on the response of the population to stimulation, which we illustrate using first spike time histograms.

Spectral and spatial properties of polarized light reflections from the arms of squid (Loligo pealeii) and cuttlefish (Sepia officinalis L.).

On every arm of cuttlefish and squid there is a stripe of high-reflectance iridophores that reflects highly polarized light. Since cephalopods possess polarization vision, it has been hypothesized that these polarized stripes could serve an intraspecific communication function. We determined how polarization changes when these boneless arms move. By measuring the spectral and polarizing properties of the reflected light from samples at various angles of tilt and rotation, we found that the actual posture of the arm has little or no effect on partial polarization or the e-vector angle of the reflected light. However, when the illumination angle changed, the partial polarization of the reflected light also changed. The spectral reflections of the signals were also affected by the angle of illumination but not by the orientation of the sample. Electron microscope samples showed that these stripes are composed of several groups of multilayer platelets within the iridophores. The surface normal to each group is oriented at a different angle, which produces essentially constant reflection of polarized light over a range of viewing angles. These results demonstrate that cuttlefish and squid could send out reliable polarization signals to a receiver regardless of arm orientation.

Malleable skin coloration in cephalopods: selective reflectance, transmission and absorbance of light by chromatophores and iridophores.

Nature's best-known example of colorful, changeable, and diverse skin patterning is found in cephalopods. Color and pattern changes in squid skin are mediated by the action of thousands of pigmented chromatophore organs in combination with subjacent light-reflecting iridophore cells. Chromatophores (brown, red, yellow pigment) are innervated directly by the brain and can quickly expand and retract over underlying iridophore cells (red, orange, yellow, green, blue iridescence). Here, we present the first spectral account of the colors that are produced by the interaction between chromatophores and iridophores in squid (Loligo pealeii). Using a spectrometer, we have acquired highly focused reflectance measurements of chromatophores, iridophores, and the quality and quantity of light reflected when both interact. Results indicate that the light reflected from iridophores can be filtered by the chromatophores, enhancing their appearance. We have also measured polarization aspects of iridophores and chromatophores and show that, whereas structurally reflecting iridophores polarize light at certain angles, pigmentary chromatophores do not. We have further measured the reflectance change that iridophores undergo during physiological activity, from "off" to various degrees of "on", revealing specifically the way that colors shift from the longer end (infra-red and red) to the shorter (blue) end of the spectrum. By demonstrating that three color classes of pigments, combined with a single type of reflective cell, produce colors that envelop the whole of the visible spectrum, this study provides an insight into the optical mechanisms employed by the elaborate skin of cephalopods to give the extreme diversity that enables their dynamic camouflage and signaling.

Intense ultrasonic clicks from echolocating toothed whales do not elicit anti-predator responses or debilitate the squid Loligo pealeii.

Toothed whales use intense ultrasonic clicks to echolocate prey and it has been hypothesized that they also acoustically debilitate their prey with these intense sound pulses to facilitate capture. Cephalopods are an important food source for toothed whales, and there has probably been an evolutionary selection pressure on cephalopods to develop a mechanism for detecting and evading sound-emitting toothed whale predators. Ultrasonic detection has evolved in some insects to avoid echolocating bats, and it can be hypothesized that cephalopods might have evolved similar ultrasound detection as an anti-predation measure. We test this hypothesis in the squid Loligo pealeii in a playback experiment using intense echolocation clicks from two squid-eating toothed whale species. Twelve squid were exposed to clicks at two repetition rates (16 and 125 clicks per second) with received sound pressure levels of 199-226 dB re1 microPa (pp) mimicking the sound exposure from an echolocating toothed whale as it approaches and captures prey. We demonstrate that intense ultrasonic clicks do not elicit any detectable anti-predator behaviour in L. pealeii and that clicks with received levels up to 226 dB re1 microPa (pp) do not acoustically debilitate this cephalopod species.

Anatomical basis for camouflaged polarized light communication in squid.

Camouflage is a means to defeat visual detection by predators, whereas visual communication involves a signal that is conspicuous to a receiver (usually a conspecific). However, most intraspecific visual signals are also conspicuous to predators, so that signalling can lead to the serious consequence of predation. Could an animal achieve visual camouflage and simultaneously send a hidden visual message to a conspecific? Here, we present evidence that the polarized aspect of iridescent colour in squid skin is maintained after it passes through the overlying pigmented chromatophores, which produce the highly evolved--and dynamically changeable--camouflaged patterns in cephalopods. Since cephalopods are polarization sensitive, and can regulate polarization via skin iridescence, it is conceivable that they could send polarized signals to conspecifics while staying camouflaged to fish or mammalian predators, most of which are not polarization sensitive.

Acoustic detection and quantification of benthic egg beds of the squid Loligo opalescens in Monterey Bay, California.

The squid Loligo opalescens is a key species in the nearshore pelagic community of California, supporting the most valuable state marine fishery, yet the stock biomass is unknown. In southern Monterey Bay, extensive beds occur on a flat, sandy bottom, water depths 20-60 m, thus sidescan sonar is a prima-facie candidate for use in rapid, synoptic, and noninvasive surveying. The present study describes development of an acoustic method to detect, identify, and quantify squid egg beds by means of high-frequency sidescan-sonar imagery. Verification of the method has been undertaken with a video camera carried on a remotely operated vehicle. It has been established that sidescan sonar images can be used to predict the presence or absence of squid egg beds. The lower size limit of detectability of an isolated egg bed is about 0.5 m with a 400-kHz sidescan sonar used with a 50-m range when towed at 3 knots. It is possible to estimate the abundance of eggs in a region of interest by computing the cumulative area covered by the egg beds according to the sidescan sonar image. In a selected quadrat one arc second on each side, the estimated number of eggs was 36.5 million.

Discriminative responses of squid (Loligo pealeii) photoreceptors to polarized light.

Cephalopods behaviorally respond to polarized light. Electrophysiology experiments with the squid, Loligo pealeii, demonstrated that spike responses from individual photoreceptors are a cosine2 function of the e-vector orientation of a polarized stimulus. The discrimination limit to this polarization sensitivity depended upon the difference between the orientation of a polarized stimulus with a preferred e-vector. The limit ranged from 2 degrees to 9.2 degrees with a direct stimulus in the dark or 4.8 degrees -22.1 degrees with non-directed background illumination and the cells were least discriminative at the preferred orientations. This limit can be explained partly by the variability in anatomical alignment of microvilli in the photoreceptors around a dominant axis. A few light-sensitive retinal fibers showed no polarization sensitivity. The coding of polarization information suggests that light intensity is transformed into an average spike rate. This average results from silent periods interspersed between bursts of spikes, each burst possessing a consistent interspike interval. The variations in the length and frequency of silent periods depend upon the difference between the polarization e-vector and a preferred e-vector orientation. The minimal discriminated orientation of a squid photoreceptor agrees well with the minimum behavioral discrimination of polarized light by another cephalopod, the octopus.