Contact chemoreception in multi-modal sensing of prey by Octopus.

Octopuses have keen vision and are generally considered visual predators, yet octopuses predominantly forage blindly in nature, inserting their arms into crevices to search and detect hidden prey. The extent to which octopuses discriminate prey using chemo- versus mechano-tactile sensing is unknown. We developed a whole-animal behavioral assay that takes advantage of octopuses' natural searching behavior to test their ability to discriminate prey from non-prey tastes solely via contact chemoreception. This methodology eliminated vision, mechano-tactile sensing and distance chemoreception while testing the contact chemosensory discriminatory abilities of the octopus arm suckers. Extracts from two types of prey (crab, shrimp) and three types of non-prey (sea star, algae, seawater) were embedded in agarose (to control for mechano-tactile discrimination) and presented to octopuses inside an artificial rock dome; octopuses reached their arms inside to explore its contents - imitating natural prey-searching behavior. Results revealed that octopuses are capable of discriminating between potential prey items using only contact chemoreception, as measured by an increased amount of sucker contact time and arm curls when presented with prey extracts versus non-prey extracts. These results highlight the importance of contact chemoreception in the multi-modal sensing involved in a complex foraging behavior.

Expression of squid iridescence depends on environmental luminance and peripheral ganglion control.

Squid display impressive changes in body coloration that are afforded by two types of dynamic skin elements: structural iridophores (which produce iridescence) and pigmented chromatophores. Both color elements are neurally controlled, but nothing is known about the iridescence circuit, or the environmental cues, that elicit iridescence expression. To tackle this knowledge gap, we performed denervation, electrical stimulation and behavioral experiments using the long-fin squid, Doryteuthis pealeii. We show that while the pigmentary and iridescence circuits originate in the brain, they are wired differently in the periphery: (1) the iridescence signals are routed through a peripheral center called the stellate ganglion and (2) the iridescence motor neurons likely originate within this ganglion (as revealed by nerve fluorescence dye fills). Cutting the inputs to the stellate ganglion that descend from the brain shifts highly reflective iridophores into a transparent state. Taken together, these findings suggest that although brain commands are necessary for expression of iridescence, integration with peripheral information in the stellate ganglion could modulate the final output. We also demonstrate that squid change their iridescence brightness in response to environmental luminance; such changes are robust but slow (minutes to hours). The squid's ability to alter its iridescence levels may improve camouflage under different lighting intensities.

Dynamic camouflage by Nassau groupers Epinephelus striatus on a Caribbean coral reef.

This field study describes the camouflage pattern repertoire, associated behaviours and speed of pattern change of Nassau groupers Epinephelus striatus at Little Cayman Island, British West Indies. Three basic camouflaged body patterns were observed under natural conditions and characterized quantitatively. The mean speed of pattern change across the entire body was 4.44 s (range = 0.97-9.87 s); the fastest pattern change as well as contrast change within a fixed pattern occurred within 1 s. Aside from apparent defensive camouflage, E. striatus used camouflage offensively to approach crustacean or fish prey, and three successful predation events were recorded. Although animal camouflage is a widespread tactic, dynamic camouflage is relatively uncommon and has been studied rarely in marine teleosts under natural conditions. The rapid changes observed in E. striatus suggest direct neural control of some skin colouration elements, and comparative studies of functional morphology and behaviour of colour change in other coral-reef teleosts are likely to reveal new mechanisms and adaptations of dynamic colouration.

Neural control of tuneable skin iridescence in squid.

Fast dynamic control of skin coloration is rare in the animal kingdom, whether it be pigmentary or structural. Iridescent structural coloration results when nanoscale structures disrupt incident light and selectively reflect specific colours. Unlike animals with fixed iridescent coloration (e.g. butterflies), squid iridophores (i.e. aggregations of iridescent cells in the skin) produce dynamically tuneable structural coloration, as exogenous application of acetylcholine (ACh) changes the colour and brightness output. Previous efforts to stimulate iridophores neurally or to identify the source of endogenous ACh were unsuccessful, leaving researchers to question the activation mechanism. We developed a novel neurophysiological preparation in the squid Doryteuthis pealeii and demonstrated that electrical stimulation of neurons in the skin shifts the spectral peak of the reflected light to shorter wavelengths (greater than 145 nm) and increases the peak reflectance (greater than 245%) of innervated iridophores. We show ACh is released within the iridophore layer and that extensive nerve branching is seen within the iridophore. The dynamic colour shift is significantly faster (17 s) than the peak reflectance increase (32 s), revealing two distinct mechanisms. Responses from a structurally altered preparation indicate that the reflectin protein condensation mechanism explains peak reflectance change, while an undiscovered mechanism causes the fast colour shift.

To be seen or to hide: visual characteristics of body patterns for camouflage and communication in the Australian giant cuttlefish Sepia apama.

It might seem obvious that a camouflaged animal must generally match its background whereas to be conspicuous an organism must differ from the background. However, the image parameters (or statistics) that evaluate the conspicuousness of patterns and textures are seldom well defined, and animal coloration patterns are rarely compared quantitatively with their respective backgrounds. Here we examine this issue in the Australian giant cuttlefish Sepia apama. We confine our analysis to the best-known and simplest image statistic, the correlation in intensity between neighboring pixels. Sepia apama can rapidly change their body patterns from assumed conspicuous signaling to assumed camouflage, thus providing an excellent and unique opportunity to investigate how such patterns differ in a single visual habitat. We describe the intensity variance and spatial frequency power spectra of these differing body patterns and compare these patterns with the backgrounds against which they are viewed. The measured image statistics of camouflaged animals closely resemble their backgrounds, while signaling animals differ significantly from their backgrounds. Our findings may provide the basis for a set of general rules for crypsis and signals. Furthermore, our methods may be widely applicable to the quantitative study of animal coloration.

Dark scene elements strongly influence cuttlefish camouflage responses in visually cluttered environments.

This study investigated how cuttlefish (Sepia officinalis) camouflage patterns are influenced by the proportions of different gray-scales present in visually cluttered environments. All experimental substrates comprised spatially random arrays of texture elements (texels) of five gray-scales: Black, Dark gray, Gray, Light gray, and White. The substrates in Experiment 1 were densely packed arrays of square texels that varied over 4 sizes in different conditions. Experiment 2 used substrates in which texels were disks separated on a homogeneous background that was Black, Gray or White in different conditions. In a given condition, the histogram of texel gray-scales was varied across different substrates. For each of 16 cuttlefish pattern response statistics c, the resulting data were used to determine the strength with which variations in the proportions of different gray-scales influenced c. The main finding is that darker-than-average texels (i.e., texels of negative contrast polarity) predominate in controlling cuttlefish pattern responses in the context of cluttered substrates. In Experiment 1, for example, substrates of all four texel-sizes, activation of the cuttlefish "white square" and "white head bar" (two highly salient skin components) is strongly influenced by variations in the proportions of Black and Dark gray (but not Gray, Light gray, or White) texels. It is hypothesized that in the context of high-variance visual input characteristic of cluttered substrates in the cuttlefish natural habitat, elements of negative contrast polarity reliably signal the presence of edges produced by overlapping objects, in the presence of which disruptive pattern responses are likely to achieve effective camouflage.

Stretchable surfaces with programmable 3D texture morphing for synthetic camouflaging skins.

Technologies that use stretchable materials are increasingly important, yet we are unable to control how they stretch with much more sophistication than inflating balloons. Nature, however, demonstrates remarkable control of stretchable surfaces; for example, cephalopods can project hierarchical structures from their skin in milliseconds for a wide range of textural camouflage. Inspired by cephalopod muscular morphology, we developed synthetic tissue groupings that allowed programmable transformation of two-dimensional (2D) stretchable surfaces into target 3D shapes. The synthetic tissue groupings consisted of elastomeric membranes embedded with inextensible textile mesh that inflated to within 10% of their target shapes by using a simple fabrication method and modeling approach. These stretchable surfaces transform from flat sheets to 3D textures that imitate natural stone and plant shapes and camouflage into their background environments.

Brain pathways of the chromatophore system in the squid Lolliguncula brevis.

Brain pathways controlling the chromatophores of the squid Lolliguncula brevis are described using cobalt iontophoresis. The results show several input and output pathways of the anterior and posterior chromatophore and lateral basal lobes. These connections allow coordination and modification of the chromatophore motor program throughout the motor pathway. Unlike other cephalopod species, there seems to be no direct input from the optic lobes to the lateral basal lobes in L. brevis. This species displays only a few simple patterns; therefore the underlying neural pathways for chromatophore control may be different from those of other cephalopods with more extensive patterning repertoires.

Chromatophore motoneurons in the brain of the squid, Lolliguncula brevis: an HRP study.

The location of the motoneuron somata controlling activity of the chromatophore muscles was studied in the squid Lolliguncula brevis. Retrograde transport of horseradish peroxidase from injection sites in the skin or in the mantle muscle established that the chromatophore motoneurons are situated in the subesophageal mass of the brain while at least some of the mantle muscle motoneurons are in the stellate ganglia. Motoneurons to chromatophores in the mantle have their somata in the posterior subesophageal mass, mainly in the chromatophore or fin lobes. Motoneurons to chromatophores in the head are located in the anterior pedal lobes and those to the chromatophores in the arms project mainly from the anterior chromatophore lobes. However, some neurons in the posterior chromatophore lobes project to the head or arm regions. A few cells in both the anterior and posterior chromatophore lobes project contralaterally. Somata in other lobes of the subesophageal mass are also labelled by injections in the skin or in the mantle muscle. Evidence presented here suggests that some of the neurons labelled outside the chromatophore lobes are chromatophore motoneurons.

Cuttlefish use polarization sensitivity in predation on silvery fish.

Cephalopods are sensitive to the linear polarization characteristics of light. To examine if this polarization sensitivity plays a role in the predatory behavior of cuttlefish, we examined the preference of Sepia officinalis when presented with fish whose polarization reflection was greatly reduced versus fish whose polarization reflection was not affected. Cuttlefish preyed preferably on fish with normal polarization reflection over fish that did not reflect linearly polarized light (n = 24, chi 2 = 17.3, P < 0.0001), implying that polarization sensitivity is used during predation. We suggest that polarization vision is used to break the countershading camouflage of light-reflecting silvery fish.

Experimental evidence for spatial learning on octopuses (octopus bimaculoides).

Octopuses forage far from temporary home dens to which they return for shelter. Spatial tasks may assess learning. Octopuses (Octopus bimaculoides) were placed in a novel arena, and their movements were tracked for 72 hr. Movements around the arena decreased across time, consistent with exploratory learning. Next, octopuses were given 23 hr to move around an arena; after a 24-hr delay, their memory of a burrow location was tested. Most remembered the location of the open burrow, demonstrating learning in 1 day. Finally, octopuses were trained to locate a single open escape burrow among 6 possible locations. Retention was tested after a week and was immediately followed by reversal training (location rotated 180 degrees ). Octopuses learned the original location of the burrow, remembering it for a week. Path lengths increased significantly after reversal, gradually improving and showing relearning. Octopuses show exploratory behavior, learning, and retention of spatial information.

Pattern of inheritance of microsatellite loci in the squid Loligo pealeii (Mollusca: Cephalopoda).

Six microsatellite loci are described for the squid Loligo pealeii. All loci exhibit some degree of allelic diversity. The pattern of inheritance was tested for 3 loci through an analysis of the filial genotypes from a female-male mating. At all 3 loci, the ratios of the filial genotypes conformed to the ratios expected by Mendelian inheritance. The hypervariable loci will be useful in studies on sexual selection in this species, whereas the relatively less variable loci will be useful to address questions of population structure.

Toxic exposure to ethylene dibromide and mercuric chloride: effects on laboratory-reared octopuses.

The effects of acute and chronic exposure to either ethylene dibromide (EDB) or mercuric chloride (MC) were studied in laboratory-reared Octopus joubini, O. maya and O. bimaculoides. The advantages of using octopuses were that the responses were immediate, highly visible and sensitive. All species demonstrated signs of toxicity to acute and chronic exposure to EDB and to MC. A dosage-sensitive relationship for the loss and subsequent recovery of locomotor response and of chromatophore expansion was found for each species after acute exposure. For each species the LC50 for chronic exposure occurred within 12 hr at 100 mg/l for EDB and within 3 hr at 1,000 mg/l for MC. This study demonstrated the potential usefulness of laboratory-reared octopuses in evaluating the toxicity of marine environmental pollutants.

Biological characteristics and biomedical applications of the squid Sepioteuthis lessoniana cultured through multiple generations.

Providing squids--especially their giant axons--for biomedical research has now been achieved in 10 mariculture trials extending through multiple generations. The noteworthy biological characteristics of Sepioteuthis lessoniana are (1) this species is behaviorally and morphologically well suited to the laboratory environment; (2) the life cycle is completed in 4-6 months; (3) growth is rapid (12% and 5% wet body weight d-1 for 100 d and for the life span, respectively), with adult size ranging from 0.4-2.2 kg; (4) feeding rates are high (30% wet body weight d-1), and a variety of live crustaceans and fishes are eaten; (5) crowding is tolerated (about 4 squids m-3); (6) the incidence of disease and cannibalism is low; and (7) reproduction in captivity allows culture through three successive generations. Engineering factors contributed to culture success: (1) physical design (i.e., size, shape, and painted pattern) of the culture tanks; (2) patterns of water flow in the culture tanks; (3) water filtration systems; and (4) spawning substrates. Initial production (a few hundred squids per year) suggests that large-scale culture will be able to supply the needs of the biomedical research community. The size (> 400 microns in diameter) and characteristics of the giant axons of Sepioteuthis are appropriate for experimentation, and other studies indicate that the eye, oculomotor/equilibrium system, olfactory system, blood, and ink are equally suitable for research.

An ethogram of body patterning behavior in the biomedically and commercially valuable squid Loligo pealei off Cape Cod, Massachusetts.

Squids have a wide repertoire of body patterns; these patterns contain visual signals assembled from a highly diverse inventory of chromatic, postural, and locomotor components. The chromatic components reflect the activity of dermal chromatophore organs that, like the postural and locomotor muscles, are controlled directly from the central nervous system. Because a thorough knowledge of body patterns is fundamental to an understanding of squid behavior, we have compiled and described an ethogram (a catalog of body patterns and associated behaviors) for Loligo pealei. Observations of this species were made over a period of three years (> or = 440 h) and under a variety of behavioral circumstances. The natural behavior of the squid was filmed on spawning grounds off Cape Cod (northwestern Atlantic), and behavioral trials in the laboratory were run in large tanks. The body pattern components--34 chromatic (including 4 polarization components), 5 postural, and 12 locomotor--are each described in detail. Eleven of the most common body patterns are also described. Four of them are chronic, or long-lasting, patterns for crypsis; an example is Banded Bottom Sitting, which produces disruptive coloration against the substrate. The remaining seven patterns are acute; they are mostly used in intraspecific communication among spawning squids. Two of these acute patterns--Lateral Display and Mate Guarding Pattern--are used during agonistic bouts and mate guarding; they are visually bright and conspicuous, which may subject the squids to predation; but we hypothesize that schooling and diurnal activity may offset the disadvantage presented by increased visibility to predators. The rapid changeability and the diversity of body patterns used for crypsis and communication are discussed in the context of the behavioral ecology of this species.

Choreography of the squid's "nuptial dance".

A mass spawning of squid resembles, at first glance, a chaotic "nuptial dance" (1). But for the first time, we have applied 3-D, radio-linked acoustic positioning (RAP) to this confusing process, and our early results now reveal a choreography that is, in fact, well organized in time and space. Remote tracking with RAP of individual Loligo vulgaris reynaudii off South Africa has provided insights into the daily sequence of behaviours that lead these animals to aggregate for sexual selection. Each dawn, the squid navigate for several kilometers, towards the shore, to small, well-defined zones near egg beds on the substrate. After several hours of circling above the egg beds, a pelagic, 3-D lek-like aggregation of large males forms: females are drawn in, and the aggregation condenses as the females and males pair, mate, and lay eggs. Smaller "sneaker males" remain on the periphery of the mating arena and, from this station, attempt extra-pair copulations (EPCs). The mating system of squids is thus unexpectedly complex, rivaling those of mammals and birds (2, 3). Commercial squid-jigging fishermen in South Africa have recently been attracted to the spawning grounds, and they have been successful. Moreover, their activities may be selective for large males. Thus, attention should be devoted to ensuring that such targeted fishing does not alter the characteristics of squid population genetics. Remote tracking and video observations, in combination with genetic analyses, may offer a new opportunity to monitor mating effort and reproductive success, and thus to manage the fishery.

A closed marine culture system for rearing Octopus joubini and other large-egged benthic octopods.

The system consists of 2 adjoining 150 litre aquaria, one functioning as the water-conditioning tank and the other as the principal rearing tank. Water quality remained high with biological and mechanical filtration, physical adsorption and ultraviolet-light disinfection taking place exclusively in the conditioning tank. The pH ranged from 7.58 to 8.00 and ammonia and nitrite levels never exceeded 0.004 mg/l and 0.198 mg/l, respectively. Nitrate levels were maintained at 40 mg/l or less with no adverse affects. Adult octopuses readily mated and females produced 50-150 eggs, with 95% hatching success. When fed small liver crabs, the octopus hatchlings were reared to sexual maturity either in groups or individually in about 120-150 days. Growth rates (4% bodyweight/day) and food conversion efficiences (30-40%) were as high as those obtained in open systems by previous workers.

Vertical visual features have a strong influence on cuttlefish camouflage.

Cuttlefish and other cephalopods use visual cues from their surroundings to adaptively change their body pattern for camouflage. Numerous previous experiments have demonstrated the influence of two-dimensional (2D) substrates (e.g., sand and gravel habitats) on camouflage, yet many marine habitats have varied three-dimensional (3D) structures among which cuttlefish camouflage from predators, including benthic predators that view cuttlefish horizontally against such 3D backgrounds. We conducted laboratory experiments, using Sepia officinalis, to test the relative influence of horizontal versus vertical visual cues on cuttlefish camouflage: 2D patterns on benthic substrates were tested versus 2D wall patterns and 3D objects with patterns. Specifically, we investigated the influence of (i) quantity and (ii) placement of high-contrast elements on a 3D object or a 2D wall, as well as (iii) the diameter and (iv) number of 3D objects with high-contrast elements on cuttlefish body pattern expression. Additionally, we tested the influence of high-contrast visual stimuli covering the entire 2D benthic substrate versus the entire 2D wall. In all experiments, visual cues presented in the vertical plane evoked the strongest body pattern response in cuttlefish. These experiments support field observations that, in some marine habitats, cuttlefish will respond to vertically oriented background features even when the preponderance of visual information in their field of view seems to be from the 2D surrounding substrate. Such choices highlight the selective decision-making that occurs in cephalopods with their adaptive camouflage capability.