Water temperature effects on embryonic development and GnRH-like expression in kisslip cuttlefish (Sepia lycidas) hinting the potential role of GnRH-like peptide.

Gonadotropin-releasing hormone (GnRH)-like peptides are multifunctional neuropeptides involved in cardiac control, early ontogenesis, and reproduction in cephalopods. However, the precise role of GnRH-like peptides in embryonic development and juvenile growth in cephalopods remains unknown. In this study, we showed that GnRH-like peptides are involved in the embryonic development of kisslip cuttlefish (Sepia lycidas). We confirmed that higher water temperatures induced early hatching. Simultaneously, we found that brain GnRH-like peptide gene expression gradually increased with increasing hatching speed. However, the rise in water temperature within a suitable range had no effect on the juvenile sex ratio or early gonadal development. Our results indicate that GnRH-like peptides may play an accelerating role in embryonic development; however, they are not involved in sex determination or early gonadal development in kisslip cuttlefish.

Histological and scanning electron microscope observations on the developing retina of the cuttlefish (Sepia officinalis Linnaeus, 1758).

In this work we present a detailed study of the major events during retinal histogenesis of the cuttlefish Sepia officinalis from early embryos to newly hatched animals and juveniles. For this purpose, we carried out morphometric and histological analyses using light and scanning electron microscopy. From St19, the first embryonic stage analysed, to St23/24 the embryonic retina is composed of a pseudostratified epithelium showing abundant mitotic figures in the more internal surface. At St24 the first photoreceptor nuclei appear in the presumptive inner segment layer, while an incipient layer of apical processes of the future rhabdomeric layer become visible at St25. From this stage onwards, both the rhabdomeric layer and the inner segment layer increase in size until postnatal ages. In contrast, the width of the supporting cell layer progressively decreases from St25/26 until postnatal ages. S. officinalis embryos hatched in a morphologically advanced state, showing a differentiated retina even in the last stages of the embryonic period. However, features of immaturity are still observable in the retinal tissue during the first postnatal weeks of life, such as the existence of mitotic figures in the apical region of the supporting cell layer and migrating nuclei of differentiating photoreceptors crossing the basal membrane to reach their final location in the inner segment layer. Therefore, postnatal retinal neurogenesis is present in juvenile specimens of S. officinalis.

Embryonic development of the camouflaging dwarf cuttlefish, Sepia bandensis.

BACKGROUND: The dwarf cuttlefish Sepia bandensis, a camouflaging cephalopod from the Indo-Pacific, is a promising new model organism for neuroscience, developmental biology, and evolutionary studies. Cuttlefish dynamically camouflage to their surroundings by altering the color, pattern, and texture of their skin. The skin's "pixels" (chromatophores) are controlled by motor neurons projecting from the brain. Thus, camouflage is a visible representation of neural activity. In addition to camouflage, the dwarf cuttlefish uses dynamic skin patterns for social communication. Despite more than 500 million years of evolutionary separation, cuttlefish and vertebrates converged to form limbs, camera-type eyes and a closed circulatory system. Moreover, cuttlefish have a striking ability to regenerate their limbs. Interrogation of these unique biological features will benefit from the development of a new set of tools. Dwarf cuttlefish reach sexual maturity in 4 months, they lay dozens of eggs over their 9-month lifespan, and the embryos develop to hatching in 1 month. RESULTS: Here, we describe methods to culture dwarf cuttlefish embryos in vitro and define 25 stages of cuttlefish development. CONCLUSION: This staging series serves as a foundation for future technologies that can be used to address a myriad of developmental, neurobiological, and evolutionary questions.

Three-dimensional structural evolution of the cuttlefish Sepia officinalis shell from embryo to adult stages.

The cuttlefish shell is an internal structure with a composition and general organization unique among molluscs. Its formation and the structure-function relation are explored during Sepia officinalis development, using computerized axial tomography scanning (CAT-scan) three-dimensional analyses coupled to physical measurements and modelling. In addition to the evolution of the overall form, modifications of the internal structure were identified from the last third embryonic stages to adult. Most of these changes can be correlated to life cycle stages and environmental constraints. Protected by the capsule during embryonic life, the first internal chambers are sustained by isolated pillars formed from the dorsal to the ventral septum. After hatching, the formation of pillars appears to be a progressive process from isolated points to interconnected pillars forming a wall-delineated labyrinthine structure. We analysed the interpillar space, the connectivity and the tortuosity of the labyrinth. The labyrinthine pillar network is complete just prior to the wintering migration, probably to sustain the need to adapt to high pressure and to allow buoyancy regulation. At that time, the connectivity in the pillar network is compensated by an increase in tortuosity, most probably to reduce liquid diffusion in the shell. Altogether these results suggest adjustment of internal calcified structure development to both external forces and physiological needs.

Analyses of Sox-B and Sox-E Family Genes in the Cephalopod Sepia officinalis: Revealing the Conserved and the Unusual.

Cephalopods provide an unprecedented opportunity for comparative studies of the developmental genetics of organ systems that are convergent with analogous vertebrate structures. The Sox-family of transcription factors is an important class of DNA-binding proteins that are known to be involved in many aspects of differentiation, but have been largely unstudied in lophotrochozoan systems. Using a degenerate primer strategy we have isolated coding sequence for three members of the Sox family of transcription factors from a cephalopod mollusk, the European cuttlefish Sepia officinalis: Sof-SoxE, Sof-SoxB1, and Sof-SoxB2. Analyses of their expression patterns during organogenesis reveals distinct spatial and temporal expression domains. Sof-SoxB1 shows early ectodermal expression throughout the developing epithelium, which is gradually restricted to presumptive sensory epithelia. Expression within the nervous system appears by mid-embryogenesis. Sof-SoxB2 expression is similar to Sof-SoxB1 within the developing epithelia in early embryogenesis, however appears in largely non-overlapping expression domains within the central nervous system and is not expressed in the maturing sensory epithelium. In contrast, Sof-SoxE is expressed throughout the presumptive mesodermal territories at the onset of organogenesis. As development proceeds, Sof-SoxE expression is elevated throughout the developing peripheral circulatory system. This expression disappears as the circulatory system matures, but expression is maintained within undifferentiated connective tissues throughout the animal, and appears within the nervous system near the end of embryogenesis. SoxB proteins are widely known for their role in neural specification in numerous phylogenetic lineages. Our data suggests that Sof-SoxB genes play similar roles in cephalopods. In contrast, Sof-SoxE appears to be involved in the early stages of vasculogenesis of the cephalopod closed circulatory system, a novel role for a member of this gene family.

Nervous system development in cephalopods: How egg yolk-richness modifies the topology of the mediolateral patterning system.

Cephalopods possess the most complex centralized nervous system among molluscs and the molecular determinants of its development have only begun to be explored. To better understand how evolved their brain and body axes, we studied Sepia officinalis embryos and investigated the expression patterns of neural regionalization genes involved in the mediolateral patterning of the neuroectoderm in model species. SoxB1 expression reveals that the embryonic neuroectoderm is made of several distinct territories that constitute a large part of the animal pole disc. Concentric nkx2.1, pax6/gsx, and pax3/7/msx/pax2/5/8 positive domains subdivide this neuroectoderm. Looking from dorsal to ventral sides, the sequence of these expressions is reminiscent of the mediolateral subdivision in model species, which provides good evidence for "mediolateral patterning" conservation in cephalopods. A specific feature of cephalopod development, however, includes an unconventional orientation to this mediolateral sequence: median markers (like nkx2.1) are unexpectedly expressed at the periphery of the cuttlefish embryo and lateral markers (like Pax3/7) are expressed centrally. As the egg is rich with yolk, the lips of the blastopore (that classically organizes the neural midline) remain unclosed at the lateral side of the animal pole until late stages of organogenesis, therefore reversing the whole embryo topology. These findings confirm - by means of molecular tools - the location of both ventral and dorsal poles in cephalopod embryos.

Characterization of homeobox genes reveals sophisticated regionalization of the central nervous system in the European cuttlefish Sepia officinalis.

Cephalopod mollusks possess a number of anatomical traits that often parallel vertebrates in morphological complexity, including a centralized nervous system with sophisticated cognitive functionality. Very little is known about the genetic mechanisms underlying patterning of the cephalopod embryo to arrive at this anatomical structure. Homeodomain (HD) genes are transcription factors that regulate transcription of downstream genes through DNA binding, and as such are integral parts of gene regulatory networks controlling the specification and patterning of body parts across lineages. We have used a degenerate primer strategy to isolate homeobox genes active during late-organogenesis from the European cuttlefish Sepia officinalis. With this approach we have isolated fourteen HD gene fragments and examine the expression profiles of five of these genes during late stage (E24-28) embryonic development (Sof-Gbx, Sof-Hox3, Sof-Arx, Sof-Lhx3/4, Sof-Vsx). All five genes are expressed within the developing central nervous system in spatially restricted and largely non-overlapping domains. Our data provide a first glimpse into the diversity of HD genes in one of the largest, yet least studied, metazoan clades and illustrate how HD gene expression patterns reflect the functional partitioning of the cephalopod brain.

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.

[Effects of temperature on the embryonic development and larval growth of Sepia lycidas].

A single-factor experiment was conducted to study the effects of different temperature (15, 18, 21, 24, 27, 30, and 33 degrees C) on the embryonic development and larval growth of Sepia lycidas, aimed to search for the optimum temperature for the development and growth of S. lycidas. The results showed that temperature had significant effects on the embryonic development and larval growth of S. lycidas (P < 0.05). The suitable temperature for hatching ranged from 21 degrees C to 30 degrees C, and the optimum temperature was 24 degrees C. At the optimum temperature, the hatching rate was (93.3 +/- 2.9)%, incubation period was (24.33 +/- 0.58) d, hatching period was (6.00 +/- 1.00) d, completely absorked rate of yolk sac was (96.4 +/- 3.1)%, and newly hatched larvae mass was (0.258 +/- 0.007) g. The effective accumulated temperature model was N = 284.42/(T-12.57). The suitable temperature for the larval survival and growth ranged from 21 degrees C to 30 degrees C, and the optimum temperature was from 24 degrees C to 27 degrees C. At the optimum temperature, the survival rate ranged from 70.0% to 73.3%, and the specific growth rate was from 2.4% to 3.8%.

Delayed and asynchronous ganglionic maturation during cephalopod neurogenesis as evidenced by Sof-elav1 expression in embryos of Sepia officinalis (Mollusca, Cephalopoda).

Among the Lophotrochozoa, centralization of the nervous system reaches an exceptional level of complexity in cephalopods, where the typical molluscan ganglia become highly developed and fuse into hierarchized lobes. It is known that ganglionic primordia initially emerge early and simultaneously during cephalopod embryogenesis but no data exist on the process of neuron differentiation in this group. We searched for members of the elav/hu family in the cuttlefish Sepia officinalis, since they are one of the first genetic markers of postmitotic neural cells. Two paralogs were identified and the expression of the most neural-specific gene, Sof-elav1, was characterized during embryogenesis. Sof-elav1 is expressed in all ganglia at one time of development, which provides the first genetic map of neurogenesis in a cephalopod. Our results unexpectedly revealed that Sof-elav1 expression is not similar and not coordinated in all the prospective ganglia. Both palliovisceral ganglia show extensive Sof-elav1 expression soon after emergence, showing that most of their cells differentiate into neurons at an early stage. On the contrary, other ganglia, and especially both cerebral ganglia that contribute to the main parts of the brain learning centers, show a late extensive Sof-elav1 expression. These delayed expressions in ganglia suggest that most ganglionic cells retain their proliferative capacities and postpone differentiation. In other molluscs, where a larval nervous system predates the development of the definitive adult nervous system, cerebral ganglia are among the first to mature. Thus, such a difference may constitute a cue in understanding the peculiar brain evolution in cephalopods.

Feel, smell and see in an egg: emergence of perception and learning in an immature invertebrate, the cuttlefish embryo.

It is now well established that prenatal sensory experience affects development itself and has long-term consequences in terms of postnatal behavior. This study focused on the functionality of the sensory system in cuttlefish in ovo. Embryos of stage 23, 25 and 30 received a tactile, chemical or visual stimulus. An increase of mantle contraction rhythm was taken to indicate a behavioral response to the stimulus. We clearly demonstrated that tactile and chemical systems are functional from stage 23, whereas the visual system is functional only from stage 25. At stage 25 and 30, embryos were also exposed to a repeated light stimulus. Stage 30 embryos were capable of habituation, showing a progressive decrease in contractions across stimulations. This process was not due to fatigue as we observed response recovery after a dishabituation tactile stimulus. This study is the first to show that cuttlefish embryos behaviorally respond to stimuli of different modalities and that the visual system is the last to become functional during embryonic development, as in vertebrate embryos. It also provides new evidence that the memory system develops in ovo in cuttlefish.

ESTs library from embryonic stages reveals tubulin and reflectin diversity in Sepia officinalis (Mollusca ­ Cephalopoda).

New molecular resources regarding the so-called "non-standard models" in biology extend the present knowledge and are essential for molecular evolution and diversity studies (especially during the development) and evolutionary inferences about these zoological groups, or more practically for their fruitful management. Sepia officinalis, an economically important cephalopod species, is emerging as a new lophotrochozoan developmental model. We developed a large set of expressed sequence tags (ESTs) from embryonic stages of S. officinalis, yielding 19,780 non-redundant sequences (NRS). Around 75% of these sequences have no homologs in existing available databases. This set is the first developmental ESTs library in cephalopods. By exploring these NRS for tubulin, a generic protein family, and reflectin, a cephalopod specific protein family,we point out for both families a striking molecular diversity in S. officinalis.

orthodenticle/otx ortholog expression in the anterior brain and eyes of Sepia officinalis (Mollusca, Cephalopoda).

The origin of cerebral structures is a major issue in both developmental and evolutionary biology. Among Lophotrochozoans, cephalopods present both a derived nervous system and an original body plan, therefore they constitute a key model to study the evolution of nervous system and molecular processes that control the neural organization. We characterized a partial sequence of an ortholog of otx2 in Sepia officinalis embryos, a gene specific to the anterior nervous system and eye development. By in situ hybridization, we assessed the expression pattern of otx2 during S. officinalis organogenesis and we showed that otx is expressed (1) in the eyes, from early to late developmental stages as observed in other species (2) in the nervous system during late developmental stages. The otx ortholog does not appear to be required for the precocious emergence of the nervous ganglia in cephalopods and is later expressed only in the most anterior ganglia of the future brain. Finally, otx expression becomes restricted to localized part of the brain, where it could be involved in the functional specification of the central nervous system of S. officinalis. These results suggest a conserved involvement of otx in eye maturation and development of the anterior neural structures in S. officinalis.

The dynamic nitric oxide pattern in developing cuttlefish Sepia officinalis.

BACKGROUND: Nitric oxide (NO) is implied in many important biological processes in all metazoans from porifera to chordates. In the cuttlefish Sepia officinalis NO plays a key role in the defense system and neurotransmission. RESULTS: Here, we detected for the first time NO, NO synthase (NOS) and transcript levels during the development of S. officinalis. The spatial pattern of NO and NOS is very dynamic, it begins during organogenesis in ganglia and epithelial tissues, as well as in sensory cells. At later stages, NO and NOS appear in organs and/or structures, including Hoyle organ, gills and suckers. Temporal expression of NOS, followed by real-time PCR, changes during development reaching the maximum level of expression at stage 26. CONCLUSIONS: Overall these data suggest the involvement of NO during cuttlefish development in different fundamental processes, such as differentiation of neural and nonneural structures, ciliary beating, sensory cell maintaining, and organ functioning.

FaRP cell distribution in the developing CNS suggests the involvement of FaRPs in all parts of the chromatophore control pathway in Sepia officinalis (Cephalopoda).

The FMRFamide-related peptide (FaRP) family includes a wide range of neuropeptides that have a role in many biological functions. In cephalopods, these peptides intervene in the peculiar body patterning system used for communication and camouflage. This system is particularly well developed in the cuttlefish and is functional immediately after hatching (stage 30). In this study, we investigate when and how the neural structures involved in the control of body patterning emerge and combine during Sepia embryogenesis, by studying the expression or the production of FaRPs. We detected FaRP expression and production in the nervous system of embryos from the beginning of organogenesis (stage 16). The wider FaRP expression was observed concomitantly with brain differentiation (around stage 22). Until hatching, FaRP-positive cells were located in specific areas of the central and peripheral nervous system (CNS and PNS). Most of these areas were implicated in the control of body patterns, suggesting that FaRPs are involved in all parts of the neural body pattern control system, from the 'receptive areas' via the CNS to the chromatophore effectors.

Evidence of early nervous differentiation and early catecholaminergic sensory system during Sepia officinalis embryogenesis.

Within Mollusca, cephalopods exhibit a particularly complex nervous system. The adult brain is formed from the fusion of several "typical" molluscan ganglia but it remains poorly understood how these ganglia emerge, migrate, and differentiate during embryogenesis. We studied the development of both central and peripheral nervous system by antibodies raised against alpha-tubulin and tyrosine hydroxylase (TH) in Sepia officinalis embryos to visualize neurites and catecholamine-containing neurons, respectively. In early embryos, when organs start delineating, some ganglia already exhibited a significant fiber network. TH-like immunoreactivity was detected in these fibers and in some primary sensory neurons in the embryo periphery. These data attest to the occurrence of an early embryonic sensory nervous system, likely effective, transient in part, and in relation to the perception of external cues. Concerning the peripheral nervous network, the stellate ganglia emerged as a plexus of numerous converging axons from TH-like immunoreactive sensory cells, first at the mantle edge, and then in the whole mantle surface. Later, TH-immunopositive motor fibers, originating from the stellate ganglia, penetrated the circular muscles of the mantle. These patterns reveal the setup of a mantle midline with likely attractive and repulsive properties. Our findings seem to challenge the widespread, still accepted, view of a late differentiation of cephalopod ganglia, and provides significant data for further investigations about axonal guidance during cephalopod development.

Biokinetics of Hg and Pb accumulation in the encapsulated egg of the common cuttlefish Sepia officinalis: radiotracer experiments.

Uptake and depuration kinetics of dissolved (203)Hg and (210)Pb were determined during the entire embryonic development of the eggs of the cuttlefish, Sepia officinalis (50d at 17 degrees C). (203)Hg and (210)Pb were accumulated continuously by the eggs all along the development time reaching load/concentration ratio (LCR) of 467+/-43 and 1301+/-126g, respectively. During the first month, most of the (203)Hg and (210)Pb remained associated with the eggshell indicating that the latter acted as an efficient shield against metal penetration. From this time onwards, (203)Hg accumulated in the embryo, indicating that it passed through the eggshell, whereas (210)Pb did not cross the chorion during the whole exposure time. It also demonstrated that translocation of Hg associated with the inner layers of the eggshell is a significant source of exposure for the embryo. This study highlighted that the maturing embryo could be subjected to the toxic effects of Hg in the coastal waters where the embryonic development is taking place.

Shh and Pax6 have unconventional expression patterns in embryonic morphogenesis in Sepia officinalis (Cephalopoda).

Cephalopods show a very complex nervous system, particularly derived when compared to other molluscs. In vertebrates, the setting up of the nervous system depends on genes such as Shh and Pax6. In this paper we assess Shh and Pax6 expression patterns during Sepia officinalis development by whole-mount in situ hybridization. In vertebrates, Shh has been shown to indirectly inhibit Pax6. This seems to be the case in cephalopods as the expression patterns of these genes do not overlap during S. officinalis development. Pax6 is expressed in the optic region and brain and Shh in gut structures, as already seen in vertebrates and Drosophila. Thus, both genes show expression in analogous structures in vertebrates. Surprisingly, they also exhibit unconventional expressions such as in gills for Pax6 and ganglia borders for Shh. They are also expressed in many cephalopods' derived characters among molluscs as in arm suckers for Pax6 and beak producing tissues, nuchal organ and neural cord of the arms for Shh. This new data supports the fact that molecular control patterns have evolved with the appearance of morphological novelties in cephalopods as shown in this new model, S. officinalis.

Phenoloxidase activation in the embryo of the common cuttlefish Sepia officinalis and responses to the Ag and Cu exposure.

The prophenoloxidase (proPO) system catalyzing the melanin production is considered as implicated in the innate immune system in invertebrates. The phenoloxidase (PO)-like activity was detected in the cuttlefish embryo sampled at the end of the organogenesis and few hours before hatching. Various modulators of the PO activity were used to assess the triggering of the proPO activating system. The results demonstrated the evidence of a true PO activity in the cuttlefish embryo. However, SDS and LPS granted contrasting effects on the PO-like activity between the developmental stages suggesting a progressive maturation of the proPO system from the embryonic to the juvenile stages. In eggs exposed to dissolved trace metals all along the embryonic development, Ag (1.2 microg L(-1)) inhibited the PO-like activity in the cuttlefish embryo except at hatching time, suggesting the synthesis of a new "juvenile" form of the PO enzyme. In similar conditions as for Ag, Cu (230 microg L(-1)) stimulated and then inhibited the PO-like activity according to a progressive metal accumulation within the egg and suggesting the occurrence of a threshold, above which the toxicity of the essential metal reduced the PO activity.

Impacts of seawater desalination on the giant Australian cuttlefish Sepia apama in the upper Spencer Gulf, South Australia.

With seawater desalination expanding rapidly, it is important that ecological studies are undertaken to determine the effects of brine discharge on the marine species in the area. The abundance of giant Australian cuttlefish (Sepia apama, Gray 1849) eggs and environmental data were recorded at nine sites near Point Lowly, Spencer Gulf, South Australia, an area where the largest desalination plant in the Southern hemisphere is proposed. In addition, the effects of different concentrations of desalination brine on the growth, survival and condition of cuttlefish embryos were investigated. The primary egg-laying sites for the cuttlefish were in the vicinity of Stony Point (sites 4 and 3) and the area with the least egg abundance was on the eastern and western areas around Point Lowly (sites 9 and 7) where no eggs were found. The survival of embryos decreased with an increase in salinity, with no embryos surviving to full term in salinities greater than 50 per thousand. Mean weight and mantle length also decreased with increasing salinity. Besides elevated salinity, the brine also had increased concentrations of Ba, Ca, K, Sr and Mg relative to water near Point Lowly. Brine discharge from seawater desalination poses a potential threat to the unique spawning aggregation of the giant Australian cuttlefish, in the upper Spencer Gulf, South Australia.