Dynamic biophotonics: female squid exhibit sexually dimorphic tunable leucophores and iridocytes.

Loliginid squid use tunable multilayer reflectors to modulate the optical properties of their skin for camouflage and communication. Contained inside specialized cells called iridocytes, these photonic structures have been a model for investigations into bio-inspired adaptive optics. Here, we describe two distinct sexually dimorphic tunable biophotonic features in the commercially important species Doryteuthis opalescens: bright stripes of rainbow iridescence on the mantle just beneath each fin attachment and a bright white stripe centered on the dorsal surface of the mantle between the fins. Both of these cellular features are unique to the female; positioned in the same location as the conspicuously bright white testis in the male, they are completely switchable, transitioning between transparency and high reflectivity. The sexual dimorphism, location and tunability of these features suggest that they may function in mating or reproduction. These features provide advantageous new models for investigation of adaptive biophotonics. The intensely reflective cells of the iridescent stripes provide a greater signal-to-noise ratio than the adaptive iridocytes studied thus far, while the cells constituting the white stripe are adaptive leucophores--unique biological tunable broadband scatterers containing Mie-scattering organelles activated by acetylcholine, and a unique complement of reflectin proteins.

The Tentacular Strike Behavior in Squid: Functional Interdependency of Morphology and Predatory Behaviors During Ontogeny.

This study examines the relationship between morphology and predatory behaviors to evaluate the ontogeny of the specialized tentacular strike (TS) in Doryteuthis opalescens squid reared under laboratory conditions [hatching to 80 day-old; 2-16 mm mantle length (ML)]. Ontogenetic morphological changes in the arm-crown and the role played by the arms and tentacles during predatory behavior was correlated with prey types captured and revealed interconnected morphological and behavior traits that enabled paralarvae to perform the TS. Hatchlings have a poorly developed arm-crown and tentacles that resemble and function as arms, in which tentacular clubs (suckerfull non-contractile portion) and stalks (suckerless contractile portion) have not yet formed. Only a basic attack (BA) behavior was observed, involving arms and tentacles, which were not ejected during prey capture. A more elaborated behavior, the arm-net (AN) was first employed by 30 day-old (>4.7 mm ML) paralarvae, in which the tentacles were eject down, but not toward the prey. The TS was first observed in 40-50 day-old (6.7-7.8 mm ML) squid, which stay stationary by sustainable swimming prior to ejecting the tentacles toward the prey. Thus, the ability to perform sustainable swimming and acquisition of swimming coordination (schooling behavior) are prerequisites for the expression of the TS. The arms played the same roles after prey was captured: hold, subdue and manipulate the prey, while the actions performed by the tentacles truly defined each behavior. Prey size captured increased with increasing squid size. Morphometric data showed that hatchlings have little ability of elongating their tentacles, but this ability increases significantly with size. Squid older than 40 days could elongate their tentacles up to 61% of their ML, whereas early paralarvae 13% on average. Paralarvae were frequently observed elongating and contracting their tentacles, while not attempting to capture prey, which could perhaps serve to adjust muscle activity and development, while specializations for the strike - stalks, clubs, muscle fibers, arm-crown and swimming coordination - are still being developed. The expression of the TS is constrained by development in early paralarvae as it involves interdependency of morphology and behavior and as such, represents a major developmental milestone in the early life history of squid.

Development of Swimming Abilities in Squid Paralarvae: Behavioral and Ecological Implications for Dispersal.

This study investigates the development of swimming abilities and its relationship with morphology, growth, and nourishment of reared Doryteuthis opalescens paralarvae from hatching to 60 days of age. Paralarvae (2.5-11 mm mantle length - ML) were videotaped, and their behavior quantified throughout development using computerized motion analysis. Hatchlings swim dispersed maintaining large nearest neighbor distances (NND, 8.7 ML), with swimming speeds (SS) of 3-8 mm s-1 and paths with long horizontal displacements, resulting in high net to gross displacement ratios (NGDR). For 15-day-old paralarvae, swimming paths are more consistent between jets, growth of fins, length, and mass increases. The swimming pattern of 18-day-old paralarvae starved for 72 h exhibited a significant reduction in mean SS and inability to perform escape jets. A key morphological, behavioral, and ecological transition occurs at about 6 mm ML (>35-day old), when there is a clear change in body shape, swimming performance, and behavior, paths are more regularly repeated and directional swimming is evident, suggesting that morphological changes incur in swimming performance. These squid are able to perform sustained swimming and hover against a current at significantly closer NND (2.0 ML), as path displacement is reduced and maneuverability increases. As paralarvae reach 6-7 mm ML, they are able to attain speeds up to 562 mm s-1 and to form schools. Social feeding interactions (kleptoparasitism) are often observed prior to the formation of schools. Schools are always formed within areas of high flow gradient in the tanks and are dependent on squid size and current speed. Fin development is a requisite for synchronized and maneuverable swimming of schooling early juveniles. Although average speeds of paralarvae are within intermediate Reynolds numbers (Re < 100), they make the transition to the inertia-dominated realm during escape jets of high propulsion (Re > 3200), transitioning from plankton to nekton after their first month of life. The progressive development of swimming capabilities and social interactions enable juvenile squid to school, while also accelerates learning, orientation and cognition. These observations indicate that modeling of the lifecycle should include competency to exert influence over small currents and dispersal patterns after the first month of life.

Experimental determination of refractive index of condensed reflectin in squid iridocytes.

Loliginid squid dynamically tune the structural iridescence of cells in their skin for active camouflage and communication. Bragg reflectors in these cells consist of membrane-bound lamellae periodically alternating with low refractive index extracellular spaces; neuronal signalling induces condensation of the reflectin proteins that fill the lamellae, consequently triggering the expulsion of water. This causes an increase in refractive index within the lamellae, activating reflectance, with the change in lamellar thickness and spacing progressively shifting the wavelength of reflected light. We used micro-spectrophotometry to measure the functionally relevant refractive index of the high-index lamellae of the Bragg reflectors containing the condensed reflectins in chemically fixed dermal iridocytes of the squid, Doryteuthis opalescens. Our high-magnification imaging spectrometer allowed us to obtain normalized spectra of optically distinct sections of the individual, subcellular, multi-layer Bragg stacks. Replacement of the extracellular fluid with liquids of increasing refractive index allowed us to measure the reflectivity of the Bragg stacks as it decreased progressively to 0 when the refractive index of the extracellular medium exactly matched that of the reflectin-filled lamellae, thus allowing us to directly measure the refractive index of the reflectin-filled lamellae as ncondensed lamellae ≈ 1.44. The measured value of the physiologically relevant ncondensed lamellae from these bright iridocytes falls within the range of values that we recently determined by an independent optical method and is significantly lower than values previously reported for dehydrated and air-dried reflectin films. We propose that this directly measured value for the refractive index of the squid's Bragg lamellae containing the condensed reflectins is most appropriate for calculations of reflectivity in similar reflectin-based high-index layers in other molluscs.

Emergence of sensory structures in the developing epidermis in sepia officinalis and other coleoid cephalopods.

Embryonic cuttlefish can first respond to a variety of sensory stimuli during early development in the egg capsule. To examine the neural basis of this ability, we investigated the emergence of sensory structures within the developing epidermis. We show that the skin facing the outer environment (not the skin lining the mantle cavity, for example) is derived from embryonic domains expressing the Sepia officinalis ortholog of pax3/7, a gene involved in epidermis specification in vertebrates. On the head, they are confined to discrete brachial regions referred to as "arm pillars" that expand and cover Sof-pax3/7-negative head ectodermal tissues. As revealed by the expression of the S. officinalis ortholog of elav1, an early marker of neural differentiation, the olfactory organs first differentiate at about stage 16 within Sof-pax3/7-negative ectodermal regions before they are covered by the definitive Sof-pax3/7-positive outer epithelium. In contrast, the eight mechanosensory lateral lines running over the head surface and the numerous other putative sensory cells in the epidermis, differentiate in the Sof-pax3/7-positive tissues at stages ∼24-25, after they have extended over the entire outer surfaces of the head and arms. Locations and morphologies of the various sensory cells in the olfactory organs and skin were examined using antibodies against acetylated tubulin during the development of S. officinalis and were compared with those in hatchlings of two other cephalopod species. The early differentiation of olfactory structures and the peculiar development of the epidermis with its sensory cells provide new perspectives for comparisons of developmental processes among molluscs.

Optical parameters of the tunable Bragg reflectors in squid.

Cephalopods (e.g. octopus, squid and cuttlefish) dynamically tune the colour and brightness of their skin for camouflage and communication using specialized skin cells called iridocytes. We use high-resolution microspectrophotometry to investigate individual tunable Bragg structures (consisting of alternating reflectin protein-containing, high-refractive index lamellae and low-refractive index inter-lamellar spaces) in live and chemically fixed iridocytes of the California market squid, Doryteuthis opalescens. This subcellular, single-stack microspectrophotometry allows for spectral normalization, permitting use of a transfer-matrix model of Bragg reflectance to calculate all the parameters of the Bragg stack-the refractive indices, dimensions and numbers of the lamellae and inter-lamellar spaces. Results of the fitting analyses show that eight or nine pairs of low- and high-index layers typically contribute to the observed reflectivity in live cells, whereas six or seven pairs of low- and high-index layers typically contribute to the reflectivity in chemically fixed cells. The reflectin-containing, high-index lamellae of live cells have a refractive index proportional to the peak reflectivity, with an average of 1.405 ± 0.012 and a maximum around 1.44, while the reflectin-containing lamellae in fixed tissue have a refractive index of 1.413 ± 0.015 suggesting a slight increase of refractive index in the process of fixation. As expected, incremental changes in refractive index contribute to the greatest incremental changes in reflectivity for those Bragg stacks with the most layers. The excursions in dimensions required to tune the measured reflected wavelength from 675 (red) to 425 nm (blue) are a decrease from ca 150 to 80 nm for the high-index lamellae and from ca 120 to 50 nm for the low-index inter-lamellar spaces. Fixation-induced dimensional changes also are quantified, leading us to suggest that further microspectrophotometric analyses of this iridocyte system can be used as a model system to quantify the effects of various methods of tissue fixation. The microspectrophotometry technique described can be expected to provide deeper insights into the molecular and physical mechanisms governing other biophotonically active cells and structures.