SUMMARY

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.

INTRODUCTION

Cephalopods are famous for their remarkable ability to camouflage. They use adaptive skin texture, pigmentation and physical colors not only to fool predators and prey but also for intraspecific communication (Hanlon and Messenger, 1996; Mäthger and Hanlon, 2007; Mäthger et al., 2009; Messenger, 2001). Of particular optical interest are their systems of structural reflectance and coloration resulting from the interaction of incident light with the structures organized at length scales near optical wavelengths to produce constructive interference (Land, 1972). Iridocytes and leucophores are two specialized cell types that produce such structural scattering in cephalopods (Cloney and Brocco, 1983; Mäthger et al., 2009). Squid iridocytes have been the subject of several investigations into the molecular mechanisms of adaptive bio-photonics (DeMartini et al., 2013; Izumi et al., 2010; Tao et al., 2010) and are an attractive model for the development of bio-inspired adaptive optical materials (Fudouzi, 2011; Kreit et al., 2013).

The iridocytes of squid, octopuses and cuttlefish contain stacks of plates of high refractive index separated by low refractive index space to form multilayer reflectors, the periodicity of which reflects specific wavelengths via constructive interference of reflected light (Cloney and Brocco, 1983; Denton and Land, 1971; Land, 1972; Mirow, 1972). Like many other iridescent biological materials, the iridocytes in most cephalopods are static; however, in certain regions in the dermis of loliginid squid these optical structures are adaptive (Hanlon et al., 1990; Mäthger et al., 2004; Tao et al., 2010). Through neuronal control (Wardill et al., 2012), via the neurotransmitter acetylcholine (ACh) (Cooper and Hanlon, 1986), the squid are able to tune the parameters of the multilayer reflector to control the wavelength and intensity of the reflected light (Cooper et al., 1990). In response to a signal transduction cascade activated by ACh, differential phosphorylation of specialized reflectin proteins drives the reversible assembly and condensation of the reflectins, increasing the refractive index of the protein-containing lamellae (Cooper and Hanlon, 1986; Izumi et al., 2010; Tao et al., 2010); this in turn modulates the water content of the lamellae (DeMartini et al., 2013), further increasing their refractive index and changing their periodicity, thereby changing the intensity and wavelength of the reflected light (Cooper et al., 1990; Tao et al., 2010).

Leucophores also use structural organization of contrasting refractive index materials to interact with light. Instead of the periodic lamellae seen in iridocytes, the leucophores contain polydisperse, micrometer-scale, non-absorbing, non-periodic intracellular bodies that scatter all wavelengths to produce broadband (white) scattering (Cloney and Brocco, 1983). Static leucophore structures in cuttlefish have recently been described, and their broadband reflectance analyzed by the Lorenz–Mie theory (Mäthger et al., 2013). To date, there have been no reported adaptive (i.e. tunable) leucophores (Mäthger et al., 2009; Mäthger et al., 2013). Among the many biological materials functioning specifically for broadband scattering, there are only a few documented examples of reversible broadband scattering structures in nature. One of these is the reversible opacity of the siphonophore Hippopodius hippopus in which the reversible precipitation of a protein changes the scattering properties of mesogleal cells (Mackie and Mackie, 1967).

Here, we report two previously undescribed sexually dimorphic photonic features in the dermis of female Doryteuthis (formerly Loligo) opalescens (Berry 1911): bright stripes of iridescent iridocytes on the mantle below the fins and a white stripe of leucophores centered between the fins on the dorsal mantle. We have examined these regions spectrophotometrically and by light and electron microscopy, and compared them with the corresponding regions in the male squid. We demonstrate that both features are adaptive, responsive to acetylcholine and likely to be under neuronal control. Moreover, these stripes potentially mimic the male testis and may serve to mitigate male mating harassment, although alternative functions in the mating behavior of this species also are possible. We anticipate that these cells will provide useful models for ongoing research into the mechanisms of adaptive biophotonics.

MATERIALS AND METHODS

Definitions

Iridocyte: a single cell with intracellular periodic lamellae of alternating refractive index material that produces iridescent colors via constructive interference. Earlier workers have referred to these cells variously as iridocytes, iridophores, iridescent cells or reflective cells. We prefer to use the unambiguous convention of contemporary cell biology, referring to them as iridocytes (literally ‘iridescent cells’), with no specific photonic mechanism implied.

Leucophore: a single cell with intracellular bodies (leucosomes) of higher refractive index than the surrounding medium that scatters all wavelengths of visible light to produce the observed ‘white color’.

In reference to the iridocytes and the leucophores, we use the distinctions ‘adaptive’ (physiological response drives changes to the intracellular structural elements controlling the cell's optical properties) and ‘static’ (not responsive and the internal structural color elements do not change).

Specimen collection and observations

Live D. opalescens specimens were purchased from Outerbanks Commercial Fisheries (Oxnard, CA, USA), and transported under constant oxygen aeration to the University of California, Santa Barbara, where they were kept in 2 m circular tanks connected to an open seawater system. Male and female squid were separated and housed separately. Each squid was inspected visually, by lifting the fin to view the dermis covering the mantle just below the fin attachment, to assess the absence or presence of the iridocyte stripe (absence: transparent dermis and a clear view of the slightly opaque mantle muscle and internal organs; presence: a conspicuous bright iridescent stripe in the dermis that occludes viewing into the mantle). The iridescent stripes were surveyed (1) upon arrival at the facility, (2) immediately before squid decapitation, and (3) 10–15 min following decapitation. Similarly, the presence (brilliant white) or absence (transparent) of the leucophore stripe was also surveyed in dead specimens.

Microscopy sample preparation

Squid were killed by decapitation immediately prior to dissection. The mantle was cut along the ventral surface and the internal organs and gladius were removed; the mantle was then washed with artificial seawater (ASW, mmol l−1: 470 NaCl, 10 KCl, 27 MgCl2, 29 MgSO4, 11 CaCl2 and 10 Hepes, pH 7.8) and pinned out flat in a dissection tray. Areas of the dermis were carefully dissected away from the mantle muscle and pinned out to the original dimensions in Sylgard 184 (Dow Corning, Midland, MI, USA)-coated dishes. These samples were fixed for electron microscopy (2% formaldehyde, 2% glutaraldehyde in ASW, 2 h at room temperature, RT). Following fixation, the tissue was washed with deionized water (freshly degassed) at least three times, then post-fixed (2% OsO4, 1 h at RT). The tissue was dehydrated in a series of graded ethanol solutions (% ethanol): 25, 50, 75, 90, 100, 100 and 100, followed by an exchange into propylene oxide:ethanol solutions (% propylene oxide): 33, 66, 100, 100 and 100. The tissue was transferred to Spurr's resin (Ted Pella Inc., Redding, CA, USA) through a graded series of Spurr's resin:propylene oxide solutions (% Spurr's resin): 33, 66, 100 and 100. The tissue samples were transferred to silicon molds, overlaid with fresh resin and cured (overnight, 55°C). The hardened blocks were trimmed and sectioned on a Leica EM UC6 ultramicrotome (Leica Microsystems, Wetzlar, Germany). Semi-thin sections (500–1000 nm) were stained (0.5% Toluidine Blue, 0.25% Methylene Blue, 0.25% Azure B, 10% sodium borate) and imaged under a Zeiss Axioplan light microscope. Ultrathin sections (80–100 nm) were collected onto formvar-coated copper mesh grids and post-stained using 4% uranyl acetate (10 min) followed by four water washes, and 2% lead citrate (2 min) followed by four more washes. Samples were imaged on a JEOL 123 Transmission Electron Microscope operating at 80 kV.

Optical measurements

The optical spectra of the squid tissues were measured by a USB2000 spectrophotometer (Ocean Optics, Dunedin, FL, USA) through a 0.6 mm multi-mode collection fiber surrounded by a coaxial illumination fiber positioned normal to the skin surface at a distance of 2 mm. The reflectance measurements were taken under illumination (also normal to the surface) with a broadband halogen white light source (DH-2000, Ocean Optics). All spectra were standardized to a diffuse reflection standard WS-1 (Ocean Optics), had the electric dark subtracted, and used an integration time of 0.75 s. Where stated, the leucophore and iridocyte stripes were stimulated by dermal injections (50 μl) of ASW containing 100 μmol l−1 acetylcholine. ASW alone was used for control injections.

Protein analysis

Tissue samples taken for protein analysis were carefully dissected away from adjacent tissue under a dissecting microscope and placed in sterile phosphate-buffered saline (PBS, pH 7.4) containing 5 mmol l−1 EDTA and protease inhibitors (Halt Protease Inhibitor, Pierce Biotechnology, Rockford, IL, USA). Tissues were homogenized in ground-glass tissue grinders. Insoluble proteins were collected by centrifugation (20,000 g, 15 min); the pellets contained the reflectin proteins. Protein samples were heated (95°C, 5 min) in sodium dodecyl sulfate (SDS) gel loading buffer [100 mmol l−1 Tris-HCl pH 6.8, 4% (w/v) SDS, 0.2% (w/v) Bromophenol Blue, 20% (v/v) glycerol, 200 mmol l−1 β-mercaptoethanol] and separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) on a 12% polyacrylamide gel (125 V, 1.5 h). The BenchMark protein ladder (Invitrogen, Carlsbad, CA, USA) provided internal standards for determination of the relative molecular mass of the protein bands. Protein bands from freshly resolved SDS-PAGE gels were transferred to PVDF membranes in transfer buffer [25 mmol l−1 Tris, 192 mmol l−1 glycine, and 20% (v/v) methanol] using a semi-dry blotter (25 V, 15 min). Western blots for the detection of Doryteuthis pealeii reflectin-like protein-A1 (RA1, GenBank accession no. FJ824804) and D. pealeii reflectin-like protein A2 (RA2, GenBank accession no. FJ824805) were performed using a rabbit polyclonal IgG antibody against recombinant RA1 (Tao et al., 2010). Membranes were blocked overnight at 4°C in blocking buffer [PBS, 0.1% Tween-20, 3% (w/v) BSA, pH 7.4]. The membranes were incubated in a 1:100 dilution of the primary antibodies in blocking buffer for 1 h at room temperature, then washed in blocking buffer three times for 10 min. The membranes were then incubated in a 1:5000 dilution of secondary antibodies (goat anti-rabbit conjugated HRP; cat. no. 31460, Thermo Scientific, Waltham, MA, USA) in blocking buffer for 1 h at room temperature. The membranes were again washed in blocking buffer three times. Immunoreactivity was visualized by chemiluminescence (SuperSignal Dura West Chemiluminescence Substrate, cat. no. 37071, Thermo Scientific). For western blot detection of D. pealeii reflectin-like protein-B1 (RB1, GenBank accession no. FJ824806), we used a custom-made polyclonal chicken IgY antibody generated against the peptide: DDHYMENDRFLYPHD (Aves Labs, Tigard, OR, USA). The protocol was the same as outlined above, except the blocking buffer had 3% (w/v) non-fat milk instead of BSA, the primary antibodies were diluted 1:1000, and the secondary antibody (goat anti-chicken IgY HRP no. H-1004, Aves Labs) was diluted 1:5000. Blots and protein gels were imaged and analyzed on a ChemiDoc XRS+ imaging system with Image Lab software (Bio-Rad, Hercules, CA, USA).

Fig. 1.

Doryteuthis opalescens contains sexually dimorphic structural color features. (A) Dorsal aspect of juxtaposed male and female squid. These show the pronounced testis within the mantle of the male and the correspondingly positioned white stripe in the dermis of the female; the iridescent stripes in the dermis on the dorsal–lateral surface of the mantle just beneath the fin attachment in the female can also be faintly seen. (B) Squid mantles laid out flat after being cut longitudinally on the ventral side and after removal of all the internal organs. The two on the left are males and the two on the right are females. The two dominant structural color features, the iridescent stripe (iridocytes) and the white stripes (leucophores), are present only in the female squid. The relatively fainter iridescence of the typical dorsal iridocytes can be seen in the dermis of both males and females. (C) Diagram of the male and female squid and the location of the associated white and iridescent structures; the horizontal dashed line indicates the location of the cross-section (through the posterior region of the mantle, between the fins). The diagram also shows the observed reversible changes in the presentation of these optical structures. (D) Histogram showing the presence of the iridescent stripe by sex upon arrival in the laboratory (Nmale=296, Nfemale=115), pre-mortem (Nmale=131, Nfemale=64) and post-mortem (Nmale=179, Nfemale=89) (error bars represent ±1 s.d.) (see supplementary material Table S1 for more details).

Fig. 1.

Doryteuthis opalescens contains sexually dimorphic structural color features. (A) Dorsal aspect of juxtaposed male and female squid. These show the pronounced testis within the mantle of the male and the correspondingly positioned white stripe in the dermis of the female; the iridescent stripes in the dermis on the dorsal–lateral surface of the mantle just beneath the fin attachment in the female can also be faintly seen. (B) Squid mantles laid out flat after being cut longitudinally on the ventral side and after removal of all the internal organs. The two on the left are males and the two on the right are females. The two dominant structural color features, the iridescent stripe (iridocytes) and the white stripes (leucophores), are present only in the female squid. The relatively fainter iridescence of the typical dorsal iridocytes can be seen in the dermis of both males and females. (C) Diagram of the male and female squid and the location of the associated white and iridescent structures; the horizontal dashed line indicates the location of the cross-section (through the posterior region of the mantle, between the fins). The diagram also shows the observed reversible changes in the presentation of these optical structures. (D) Histogram showing the presence of the iridescent stripe by sex upon arrival in the laboratory (Nmale=296, Nfemale=115), pre-mortem (Nmale=131, Nfemale=64) and post-mortem (Nmale=179, Nfemale=89) (error bars represent ±1 s.d.) (see supplementary material Table S1 for more details).

RESULTS

The female-specific iridescent stripes

All our observations were carried out on sexually mature specimens of D. opalescens caught in and around the Santa Barbara Channel. When the squid mantles are dissected and pinned out, some distinct optical features become apparent: two colorful stripes of iridescence flanking a single, central, bright white stripe (Fig. 1A,B; supplementary material Figs S1, S2). The pronounced iridescent stripes are on the mantle just beneath the fin attachment (Fig. 1C). Interestingly, these iridescent features are only present in the female squid, exhibiting complete sexual dimorphism in the dead specimens we surveyed (N=268). The iridescent stripes are transient in live female squid, with the animal able to controllably switch the iridescence between transparent (‘off’) and reflecting (‘on’) states. Fig. 1D shows the percentage of individuals displaying these stripes upon arrival at our facility (observed while segregating them by sex), before dissection and >10 min after dissection. There was a high variability in the number of live females presenting the iridescent stripes (49.0±34.5%). Fewer females presented the stripes following segregation (22.5±11.1%) and all females developed the bright iridescent stripes following decapitation regardless of stripe presentation prior to death (100±0%). There were no instances of males showing iridescence in the same region either pre- or post-mortem.

Fig. 2.

Optical measurements of the female iridescent stripes. (A) Representative spectra of the female iridescent stripes in their reflective state, from four different females, compared with the same region in male squid and the background non-iridescent region immediately adjacent to the female stripe. Measurements were taken without interference from the fin. (B) Progression of a single female iridescent stripe following decapitation of the squid. This stripe was initially non-iridescent. The spectra are shown for successive 4 min intervals over a 56 min period following decapitation. Measurements were taken through the fin.

Fig. 2.

Optical measurements of the female iridescent stripes. (A) Representative spectra of the female iridescent stripes in their reflective state, from four different females, compared with the same region in male squid and the background non-iridescent region immediately adjacent to the female stripe. Measurements were taken without interference from the fin. (B) Progression of a single female iridescent stripe following decapitation of the squid. This stripe was initially non-iridescent. The spectra are shown for successive 4 min intervals over a 56 min period following decapitation. Measurements were taken through the fin.

Fig. 2A shows representative reflection spectra from the iridescent stripes compared with the same region in the male squid and the non-reflective tissue immediately adjacent to the stripe. Spectra were collected on the skin surface beneath the fins with all the dermal layers intact. These measurements show the large variability in peak wavelength and intensity seen between female iridescent stripes. In the males, the corresponding regions show no optical features that are significantly different from the background. Spectrophotometric measurements of the iridescent stripes in the females indicate a maximum reflectance of 36.6±9.5% relative to a diffuse white reflectance standard with both viewing and illumination angles normal to the skin surface (Fig. 2A, Table 1). The reflectance intensity is almost an order of magnitude brighter than the iridocyte patches on the dorsal surface of the mantle, which measured 6.6±3.2% reflective following stimulation by ACh. The female iridescent stripe begins to develop almost immediately after death; Fig. 2B shows the natural progression of the stripe reflectance of an individual squid from ‘off’ to ‘on’ over a 56 min period (without the addition of ACh). The time-lapse spectra were collected through the fins (prepared as in Fig. 1B), decreasing the observed intensity (in comparison to Fig. 2A) because of the diffusion and absorption of light by the overlying tissue, but this was necessary in order to shorten sample preparation time and perturbation. The stripe is initially transparent; it turns red and then progressively shifts to the blue end of the spectrum. In many cases the stripes remained brilliant for more than 24 h. Decapitated males did not develop any color in the stripe region over the same period of time. Dermal injections of the neurotransmitter ACh induced stripe iridescence in all live female specimens tested (N=6) within 120 s, while ASW injections had no effect (N=6). Conversely, injections of ACh (or ASW) had no effect in the corresponding region of live male specimens.

Comparison of the ultrastructure of the iridescent stripe region in males (Fig. 3A–C) and females (Fig. 3D–F) shows a clear morphological distinction between the sexes. Table 1 contains measurements of the iridocyte layer from the various regions of the dermis. The female iridescent stripes contain a particularly thick layer of iridocytes (41.3±14.9 μm) with the multilayer stacks being oriented roughly parallel to the plane of the skin surface and a large number of periodic lamellae per stack. The analogous space in the male dermis, in contrast, has only a thin and sparsely populated iridocyte layer (6.6±5.5 μm thick), and does not contain the dense iridocytes with a high number of periodic lamellae. These sparse iridocytes in the male exhibit only irregular lamellae oriented roughly perpendicular to the skin surface, in these respects being similar to the iridocytes of the ventral dermis (Mäthger and Denton, 2001). The poor periodicity, non-uniform thickness, small number of lamellae and orientation of these lamellae all explain why these sparsely distributed iridocytes found in the relevant area of the male are incapable of producing constructive interference in the visible range, consistent with the observed spectral data (Fig. 2A). Therefore, the males are incapable of producing iridescent stripes because they lack the necessary ultrastructure. Their failure to respond in this region to the injection of ACh confirms that this is not a question of stimulation.

Table 1.

Properties of the iridocyte/leucophore dermal layer from various locations

Properties of the iridocyte/leucophore dermal layer from various locations
Properties of the iridocyte/leucophore dermal layer from various locations
Fig. 3.

The female iridescent stripe is histologically distinct from the corresponding region in the male squid. Micrographs at progressively higher magnification of the same anatomical space from the male (A–C) and female (D–F) dermis corresponding to the bright iridescent structure in the females. (A,D) Relatively low magnification light micrographs of semi-thin (0.5 μm) sections showing the various dermal strata: epidermis (e), chromatophore layer (c), iridocyte layer (i) and the underlying muscle (m). (B,E) Intermediate magnification transmission electron micrographs (TEMs) of the iridocytes, showing the iridocyte platelets (ip) and nucleus (n). (C,F) Higher magnification TEMs of the iridocyte platelets. All the images are arranged such that the vertical axis is normal to the tissue surface.

Fig. 3.

The female iridescent stripe is histologically distinct from the corresponding region in the male squid. Micrographs at progressively higher magnification of the same anatomical space from the male (A–C) and female (D–F) dermis corresponding to the bright iridescent structure in the females. (A,D) Relatively low magnification light micrographs of semi-thin (0.5 μm) sections showing the various dermal strata: epidermis (e), chromatophore layer (c), iridocyte layer (i) and the underlying muscle (m). (B,E) Intermediate magnification transmission electron micrographs (TEMs) of the iridocytes, showing the iridocyte platelets (ip) and nucleus (n). (C,F) Higher magnification TEMs of the iridocyte platelets. All the images are arranged such that the vertical axis is normal to the tissue surface.

The female stripe iridocytes contain a particularly high number of lamellae in each multilayer stack relative to other iridocytes (including the dorsal iridocytes typically studied; supplementary material Fig. S3) (Table 1), making each individual cell brighter (Ghoshal et al., 2013; Land, 1972). The iridocyte stripe also has more cells in cross-section than the iridocyte patches in the dermis. Therefore, the pronounced brightness of the female-specific iridescent stripes is attributed to both more iridocytes and brighter individual iridocytes.

Table 2.

Reflectance intensity of the testis and white stripe at various wavelengths

Reflectance intensity of the testis and white stripe at various wavelengths
Reflectance intensity of the testis and white stripe at various wavelengths

The female-specific white stripe and adaptive leucophores

The second sexually dimorphic feature is the large, central white stripe on the dorsal mantle between the fins (Fig. 1A–C). This stripe covers the analogous space above the testis in the male (Fig. 1C; supplementary material Figs S4 and S5). Similar to the iridescent stripes, this white stripe – the result of a dense population of white-reflecting leucophores – is a characteristic specific to the female. The white leucophore stripes are also responsive to ACh, which induces a transition from transparent to broadband (white) reflectance with a progressive increase in intensity. Fig. 4A shows a representative spectral response of a female squid to a dermal injection of ACh. The white stripe region begins with reflectance properties similar to the surrounding tissue and over a 200 s period the stripe progressively becomes brighter, reaching a maximum of ~20% reflectance. We also observed endogenous regulation of the white stripe presentation by live female squid in captivity. This feature is thus tunable or adaptive.

Fig. 5 shows the reflectance spectra of this white stripe, the male testis and other regions of the mantle for comparison. As expected, the white stripe in the females exhibits a broadband reflectance spectrum. The leucophore stripes are optically similar to the male testis measured both through the mantle and in isolation. The mean percentage reflectance and standard deviations for specific wavelengths are shown in Table 2. For reference, the absorption λmax for the squid visual pigment is 493 nm (Hubbard and St George, 1958); at this wavelength the reflectance intensity of the isolated testis is 18.08±2.1%; the testis in the mantle is 15.25±2.1%; and the white stripe is 18.72±2.8%. Our background reflectance measurements (through the mantle immediately adjacent to the testis or white stripe, and through the gill tissue) showed background reflectance of ~1–3%.

Fig. 4.

Female white stripes are adaptive broadband scatterers. (A) Spectra of the female white stripe progressively increasing in reflectance as a function of time following stimulation by acetylcholine (ACh). The reflectance stopped changing after 200 s. (B) Sequential images showing the development of the bright white stripe following addition of ACh (from left to right). The red cross on the image indicates the site of ACh injection. The black cross indicates the location of background spectra in A. (The iridescent stripes are seen laterally flanking both sides of the white stripe.)

Fig. 4.

Female white stripes are adaptive broadband scatterers. (A) Spectra of the female white stripe progressively increasing in reflectance as a function of time following stimulation by acetylcholine (ACh). The reflectance stopped changing after 200 s. (B) Sequential images showing the development of the bright white stripe following addition of ACh (from left to right). The red cross on the image indicates the site of ACh injection. The black cross indicates the location of background spectra in A. (The iridescent stripes are seen laterally flanking both sides of the white stripe.)

We examined the ultrastructure of this white scattering tissue and found that it comprised a thick layer of leucophores (239±70 μm thick) (Fig. 6A, Table 1). These leucophores reside within the dermal layer typically populated by iridocytes. Instead of containing densely staining lamellae, these cells are filled with densely staining (protein-rich), high-contrast subcellular bodies, called leucosomes, in the size range 0.5–4 μm (Fig. 6B–D) that scatter all wavelengths of incident visible light, hence producing the observed broadband reflection. These bodies range from round to bean-shaped in cross-section, although we recognize estimates of size and 3D shape from fixed and embedded thin sections is highly susceptible to error. Double membranes can be seen laminating these bodies (Fig. 6D), allowing us to recognize them as organelles. In comparison, we find that the male dermis from the same region is appropriately devoid of these scattering structures, containing a low density of the typical iridocytes characteristic of the rest of the dorsal dermis in both male and female squid.

Reflectin proteins in the adaptive leucophores

We observed that the adaptive leucophores possess a unique complement of reflectin proteins – members of the same abundant reflectin family associated with adaptive iridocytes. The protein banding profiles of the insoluble fraction [known to be enriched and dominated by the reflectin proteins, which represent the majority of the protein in the tissue (Crookes et al., 2004; Izumi et al., 2010; Tao et al., 2010)] are strikingly similar between the iridocytes and the leucophores. Subsequent immunoblots confirmed that the dominant bands represent the reflectins Reflectin-A1 (RA1), Reflectin-B1 (RB1) and Reflectin-A2 (RA2) (Fig. 7) in both the iridocyte and leucophore tissue. Three additional unknown relatively abundant protein bands appear to be enriched in the leucophore tissue, with apparent molecular masses of 136.4, 113.7 and 94.7 kDa (the 136.4 kDa protein being the most abundant). Similar to RA1, RB1 and RA2, the small ~14 kDa protein band is characteristic of the dermal iridocytes and will be described in a future publication.

Fig. 5.

The female white stripe is optically comparable to the white testis. The spectra represent the average of multiple spectral measurements of the testis taken through the mantle (N=6), the isolated testis (N=6) and the ACh-stimulated white stripe (N=11); for all three sets, s.d.<3%. For comparison, spectra from the male region corresponding to the location of the female white stripe (testis removed), the female tissue immediately adjacent to the white stripe, the digestive gland through the mantle and gills through the mantle are shown.

Fig. 5.

The female white stripe is optically comparable to the white testis. The spectra represent the average of multiple spectral measurements of the testis taken through the mantle (N=6), the isolated testis (N=6) and the ACh-stimulated white stripe (N=11); for all three sets, s.d.<3%. For comparison, spectra from the male region corresponding to the location of the female white stripe (testis removed), the female tissue immediately adjacent to the white stripe, the digestive gland through the mantle and gills through the mantle are shown.

DISCUSSION

Here, we have described the female-specific, adaptive optical features in D. opalescens, the bright iridescent stripes under the fins and the central white stripe of leucophores. It is surprising that these conspicuous iridescent features have not been described in detail previously. There is a brief mention of the female ‘leucophore’ stripe in Field's work on D. opalescens (Fields, 1965) but no sources have mentioned the iridescent stripes in this species, or their sexually dimorphic nature. There are several possible reasons that these features have gone unnoticed in D. opalescens. As the stripes are adaptive, their transience in live specimens may have contributed to their elusiveness. Most taxonomical studies are performed on chemically preserved specimens in which expansion of the overlying chromatophores, the induced opacity of the muscle and other tissues, and discoloration resulting from age and fixation would all make these features less pronounced. Likewise, after fixation, the rigidity of the overlying fin (now opaque) inhibits physical and visual access to the location of the iridescent stripe. Furthermore, females are generally more fragile during and following spawning. In our observations it was not uncommon for all of the females to be dead at the bottom of the ship's holding tank the day after being caught, while the males remained alive and healthy – possibly a result of the female's energy budget with respect to reproduction and/or male aggressiveness. Thus, rapid degradation of the live captive females may have favored more live observations of males. Whatever the reason, we are happy to report these previously undescribed sexually dimorphic features. Not only do these optical features have potentially important implications for the sexual interactions and mating behavior of this species but also they offer key insights into the molecular mechanisms of adaptive biological iridescence and lessons for dynamic optics in general. Additionally, they are useful key features for rapid sex determination in live and freshly dead D. opalescens specimens, and could even function as a reporter of the health and conditions of spawning aggregations.

Fig. 6.

The female white stripe contains a unique layer of leucophores. These are shown at progressively higher magnification. (A) Light micrograph of a semi-thin (0.5 μm) section of the female white stripe showing tissue strata: epidermis (e), chromatophore layer (c), thick female-specific leucophore layer (l) and underlying muscle (m). (B) Higher magnification light micrograph of the leucophore stripes in A, showing the nuclei (n) and the abundant, electron-dense, intracellular organelles called leucosomes (ls). (C) TEM of the leucophores. (D) Higher magnification TEM image of the leucosomes within the leucophores, showing their lamination by a double membrane (dm).

Fig. 6.

The female white stripe contains a unique layer of leucophores. These are shown at progressively higher magnification. (A) Light micrograph of a semi-thin (0.5 μm) section of the female white stripe showing tissue strata: epidermis (e), chromatophore layer (c), thick female-specific leucophore layer (l) and underlying muscle (m). (B) Higher magnification light micrograph of the leucophore stripes in A, showing the nuclei (n) and the abundant, electron-dense, intracellular organelles called leucosomes (ls). (C) TEM of the leucophores. (D) Higher magnification TEM image of the leucosomes within the leucophores, showing their lamination by a double membrane (dm).

While their precise function is yet to be determined, we can suggest at least three alternative mechanisms by which the sexually dimorphic and tunable stripes may operate in the mating and reproductive behaviors of this species. It is possible that the iridescent and white stripes offer a potential false testis, giving the females a system for reversible male mimicry. The switchable stripes of the female surround the corresponding space occupied by the male's bright white testis that develops at maturity. The same position inside the female mantle is occupied by the developing ovaries and does not strongly scatter light, so this region (when the stripes are ‘off’) is relatively transparent. Tunability of these stripes could provide the females with switchable protection from male harassment in mating assemblages, coupled with a means for female choice in mate selection. As seen in the well-studied examples of male mimicry in some insects and lizards (Svensson et al., 2009), this could provide a selective advantage during periods of male aggression, but a disadvantage when mates are scarce; in insects and lizards, this mimicry varies as a result of genetic polymorphism, while in the squid, it could vary dynamically under neuronal control. Cuttlefish, Sepia apama (Hanlon et al., 2005) and Sepia plangon (Brown et al., 2012), have been shown to use rapid reversible sexual mimicry in mating interactions, but in these cases it is the males that mimic female coloration to stealthily gain access to female mates.

Alternatively, the tunable stripes may communicate the female's reproductive state, attract mates or signal receptivity for mating. Another possibility is that the stripes play no role in communication during mating, but serve instead to protect gametes from harmful UV radiation, particularly when the female moves to shallower waters to spawn. However, these alternative hypotheses fail to explain why the ‘on’ state of the stripes approximates the male testis, or why optical features other than chromatophores are needed.

The female squid's dynamically tunable iridescent and white stripes provide specific advantages for further investigations into the mechanisms of adaptive bio-photonics. The especially bright iridocytes from the iridescent stripes yield a high reflectance signal, enabling our use of a specialized microspectrophotometer to obtain optical measurements of a single stack of high-index, reflectin-containing lamellae within a single cell. This has allowed us to directly calculate the parameters of the sub-cellular multilayer reflectors, including the number of lamellae and their thickness, spacing and refractive index (Ghoshal et al., 2013). The adaptive leucophores offer an interesting comparison to the adaptive iridocytes at an ultrastructural and protein level, providing further insights into the recently elucidated mechanism of reversible, ACh-dependent assembly of reflectins driving the changes in optical properties of the cells (DeMartini et al., 2013). We find that the adaptive leucophores are enriched in a unique complement of reflectin proteins (Fig. 7). The size of the high-index organelles in these cells is responsible for their broadband (white) Mie-scattering of light, as recently shown for the static leucocytes and their intracellular scattering bodies (Mäthger et al., 2013). The tunable, photonically active organelles in the adaptive leucophores provide the distinct advantage of being resolvable by optical microscopy, whereas the thin lamellae of the adaptive iridocytes studied to date did not afford that luxury. Further characterization of the adaptive leucophore structure, reflectin composition and dynamic changes in comparison to those of the adaptive iridocytes should reveal salient features needed to further understand the underlying mechanisms of tunable structural coloration and broadband reflectance.

Fig. 7.

SDS-PAGE and western blots of the leucophore proteins. The left panel is an SDS-PAGE of the insoluble leucophore and iridocyte proteins on a 12% acrylamide gel, against the Benchmark protein mass ladder. The middle and right panels are western blots of the same protein samples stained with primary antibodies against Reflectin-A1 (recognizes both Reflectin-A1 and Reflectin-A2) and Reflectin-B1. The several large enriched protein bands marked with asterisks have a molecular mass of 136.4, 113.7 and 94.7 kDa, respectively (top to bottom) (see supplementary material Fig. S6 for the standard curve).

Fig. 7.

SDS-PAGE and western blots of the leucophore proteins. The left panel is an SDS-PAGE of the insoluble leucophore and iridocyte proteins on a 12% acrylamide gel, against the Benchmark protein mass ladder. The middle and right panels are western blots of the same protein samples stained with primary antibodies against Reflectin-A1 (recognizes both Reflectin-A1 and Reflectin-A2) and Reflectin-B1. The several large enriched protein bands marked with asterisks have a molecular mass of 136.4, 113.7 and 94.7 kDa, respectively (top to bottom) (see supplementary material Fig. S6 for the standard curve).

Static leucophores play a key role in the coloration of many cephalopods (Cloney and Brocco, 1983; Mäthger et al., 2009; Mäthger et al., 2008), but it was thought that leucophores were not present in the Loliginids, with the possible exception of some Sepiateuthis species (Mäthger et al., 2009). Here, we report the first example of leucophores in a Loliginid squid and more importantly the first example of adaptive (i.e. dynamically switchable) leucophores. Our finding that these leucophores contain reflectin RB1, a member of the reflectin family that we previously found only in the tunable lamellae of the adaptive iridocytes (Izumi et al., 2010), led to our discovery that their broadband scattering is also tunable, or ‘adaptive’. In addition to containing high concentrations of unique members of the same sub-families of reflectins (RA1, RA2 and RB1), the adaptive leucophores and adaptive iridocytes exhibit several other similarities: both are stimulated by ACh and they respond to this neurotransmitter over a similar time scale; they occupy the same stratum of tissue; and the scattering components (i.e. sub-cellular lamellae or bodies) are laminated by a double membrane. The major difference, of course, is the morphology of their photonic structures. However, we note that some of the cells in the periphery of the leucophore stripe contain semi-periodic plate-like structures vaguely reminiscent of the iridocyte stacks, further suggesting a close relationship between the iridophores and the leucophores. We speculate that differences in gene expression and the resulting protein composition (potentially including the unidentified major protein bands and the unique relative ratios of the known reflectins we observe in electropherograms of the leucophore tissue) help direct the morphology of the high-index material controlling the optical properties of these otherwise similar cells. We suggest that it is this mesoscale assembly of the variety of reflectin protein subtypes (and their associated membrane structures) found throughout cephalopods that differentially creates distinct sub-cellular structures with specific optical properties ranging from specular to diffuse reflectance (Holt et al., 2011; Sutherland et al., 2008), from static to adaptive reflectance (Izumi et al., 2010; Kramer et al., 2007; Tao et al., 2010) and from narrowband to broadband reflectance (Mäthger et al., 2009; Mäthger et al., 2013), with colors tunable from red to blue (Tao et al., 2010) and from transparent to highly reflective (Ghoshal et al., 2013; Kramer et al., 2007). This multitude of reflectin-based structures and their photonic properties is increasingly recognized as an interesting model of mesoscale assembly and its control for the design of functional materials.

In conclusion, female squid of the species D. opalescens exhibit stripes of adaptive iridescent cells (iridocytes) and adaptive broadband scattering cells (leucophores) in or proximate to the position occupied by the bright white testis of the male. We have characterized the ultrastructural features, content of reflectins and other proteins, responsiveness to the neurotransmitter ACh and optical performance of these cells. Possible roles of these sexually dimorphic, tunable biophotonic features in the mating and reproductive behaviors of the species are considered, potentially representing the first example of adaptive iridocytes and adaptive leucophores used for intraspecific communication. There are several examples of static iridocytes and static leucophores contributing to changeable coloration and communication as a result of tunable masking by overlying chromatophores, as in the mating displays of cuttlefish (Hanlon et al., 2005) and the flashing rings of the blue-ringed octopus (Hapalochlaena lunulata) (Mäthger et al., 2012). It is interesting to note that the Loliginids are the only family of cephalopods known thus far to produce adaptive iridocytes; we show here that they have novel adaptive leucophores as well. We anticipate that these unique cells will provide advantageous models to advance our understanding of the molecular and cellular mechanisms governing biologically tunable structural coloration and broadband reflectance.

Acknowledgements

We thank Professor William Gilly and Dr Eric Hochberg for helpful discussions regarding this work, Tim Athens of Outerbanks Commercial Fisheries for help in the collection of live, healthy squid, and an anonymous reviewer for his suggestions of possible alternative roles of the tunable photonic structures in reproduction.

FOOTNOTES

FUNDING

This research was supported by the Army Research Office [through contract W911NF-09-D-0001 to the Institute for Collaborative Biotechnologies and grant W911NF-10-1-0139 to D.E.M.] and by a subcontract from the Office of Naval Research via a MURI award to Duke University [no. N00014-09-1-1053]. Electron microscopy made use of instrumentation and facilities provided by UCSB's Materials Research Laboratory, a Materials Research Science and Engineering Center supported by the National Science Foundation [through grant DMR-0080034].

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COMPETING INTERESTS

No competing interests declared.

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