A practical staging atlas to study embryonic development of Octopus vulgaris under controlled laboratory conditions.

BACKGROUND: Octopus vulgaris has been an iconic cephalopod species for neurobiology research as well as for cephalopod aquaculture. It is one of the most intelligent and well-studied invertebrates, possessing both long- and short-term memory and the striking ability to perform complex cognitive tasks. Nevertheless, how the common octopus developed these uncommon features remains enigmatic. O. vulgaris females spawn thousands of small eggs and remain with their clutch during their entire development, cleaning, venting and protecting the eggs. In fact, eggs incubated without females usually do not develop normally, mainly due to biological contamination (fungi, bacteria, etc.). This high level of parental care might have hampered laboratory research on the embryonic development of this intriguing cephalopod. RESULTS: Here, we present a completely parameter-controlled artificial seawater standalone egg incubation system that replaces maternal care and allows successful embryonic development of a small-egged octopus species until hatching in a laboratory environment. We also provide a practical and detailed staging atlas based on bright-field and light sheet fluorescence microscopy imaging for precise monitoring of embryonic development. The atlas has a comparative section to benchmark stages to the different scales published by Naef (1928), Arnold (1965) and Boletzky (2016). Finally, we provide methods to monitor health and wellbeing of embryos during organogenesis. CONCLUSION: Besides introducing the study of O. vulgaris embryonic development to a wider community, this work can be a high-quality reference for comparative evolutionary developmental biology.

Amino and fatty acid dynamics of octopus (Octopus vulgaris) early life stages under ocean warming.

The oceans are becoming warmer, and the higher temperatures are expected to have a major impact on marine life at different levels of biological organization, especially at the most vulnerable early life stages. Thus, we hypothesize that the future warmer scenarios (here +3 °C) will affect the biochemical composition (amino acid - AA, and fatty acid-FA) of octopod (Octopus vulgaris) embryos and recently-hatched pelagic paralarvae. The main essential amino acids found in octopus embryos were arginine, leucine and lysine; while aspartic and glutamic acids, and taurine were the main non-essential amino acids. Palmitic, eicosapentaenoic and docosahexaenoic acids were the main FAs found in octopus tissues. Relevant ontogenetic changes were observed, namely a steep decrease in the content of many AAs, and a selective retention of FAs, thus evidencing the protein-based metabolism of these cephalopods. Temperature per si did not elicit significant changes in the overall FA composition, but was responsible for a significant decrease in the content of several AAs, indicating increased embryonic consumption.

Identification and Expression of Acetylcholinesterase in Octopus vulgaris Arm Development and Regeneration: a Conserved Role for ACHE?

Acetylcholinesterase (ACHE) is a glycoprotein with a key role in terminating synaptic transmission in cholinergic neurons of both vertebrates and invertebrates. ACHE is also involved in the regulation of cell growth and morphogenesis during embryogenesis and regeneration acting through its non-cholinergic sites. The mollusk Octopus vulgaris provides a powerful model for investigating the mechanisms underlying tissue morphogenesis due to its high regenerative power. Here, we performed a comparative investigation of arm morphogenesis during adult arm regeneration and embryonic arm development which may provide insights on the conserved ACHE pathways. In this study, we cloned and characterized O. vulgaris ACHE, finding a single highly conserved ACHE hydrophobic variant, characterized by prototypical catalytic sites and a putative consensus region for a glycosylphosphatidylinositol (GPI)-anchor attachment at the COOH-terminus. We then show that its expression level is correlated to the stage of morphogenesis in both adult and embryonic arm. In particular, ACHE is localized in typical neuronal sites when adult-like arm morphology is established and in differentiating cell locations during the early stages of arm morphogenesis. This possibility is also supported by the presence in the ACHE sequence and model structure of both cholinergic and non-cholinergic sites. This study provides insights into ACHE conserved roles during processes of arm morphogenesis. In addition, our modeling study offers a solid basis for predicting the interaction of the ACHE domains with pharmacological blockers for in vivo investigations. We therefore suggest ACHE as a target for the regulation of tissue morphogenesis.

Analysis of neurotransmitter distribution in brain development of benthic and pelagic octopod cephalopods.

The database on neurotransmitter distribution during central nervous system development of cephalopod mollusks is still scarce. We describe the ontogeny of serotonergic (5-HT-ir) and FMRFamide-like immunoreactive (Fa-lir) neurons in the central nervous system of the benthic Octopus vulgaris and Fa-lir distribution in the pelagic Argonauta hians. Comparing our data to previous studies, we aim at revealing shared immunochemical domains among coleoid cephalopods, i.e., all cephalopods except nautiluses. During development of O. vulgaris, 5-HT-ir and Fa-lir elements occur relatively late, namely during stage XII, when the brain neuropils are already highly differentiated. In stage XII-XX individuals, Fa-lir cell somata are located in the middle and posterior subesophageal mass and in the optic, posterior basal, and superior buccal lobes. 5-HT is predominately expressed in cell somata of the superior buccal, anterior basal, and optic lobes, as well as in the subesophageal mass. The overall population of Fa-lir neurons is larger than the one expressing 5-HT. Fa-lir elements are distributed throughout homologous brain areas of A. hians and O. vulgaris. We identified neuronal subsets with similar cell number and immunochemical phenotype in coleoids. These are located in corresponding brain regions of developmental stages and adults of O. vulgaris, A. hians, and the decapod squid Idiosepius notoides. O. vulgaris and I. notoides exhibit numerous 5-HT-ir cell somata in the superior buccal lobes but none or very few in the inferior buccal lobes. The latter have previously been homologized to the gastropod buccal ganglia, which also lack 5-HT-ir cell somata in euthyneuran gastropods. Among coleoids, 5-HT-ir neuronal subsets, which are located ventrally to the lateral anterior basal lobes and in the anterior middle subesophageal mass, are candidates for homologous subsets. Contrary to I. notoides, octopods exhibit Fa-lir cell somata ventrally to the brachial lobes and 5-HT-ir cell somata close to the stellate ganglia.

Pygmy squids and giant brains: mapping the complex cephalopod CNS by phalloidin staining of vibratome sections and whole-mount preparations.

Among bilaterian invertebrates, cephalopod molluscs (e.g., squids, cuttlefish and octopuses) have a central nervous system (CNS) that rivals in complexity that of the phylogenetically distant vertebrates (e.g., mouse and human). However, this prime example of convergent evolution has rarely been the subject of recent developmental and evolutionary studies, which may partly be due to the lack of suitable neural markers and the large size of cephalopod brains. Here, we demonstrate the usefulness of fluorescence-coupled phalloidin to characterize the CNS of cephalopods using histochemistry combined with confocal laser scanning microscopy. Whole-mount preparations of developmental stages as well as vibratome sections of embryonic and adult brains were analyzed and the benefits of this technique are illustrated. Compared to classical neuroanatomical and antibody-based studies, phalloidin labeling experiments are less time-consuming and allow a high throughput of samples. Besides other advantages summarized here, phalloidin reliably labels the entire neuropil of the CNS of all squids, cuttlefish and octopuses investigated. This facilitates high-resolution in toto reconstructions of the CNS and contributes to a better understanding of the organization of neural networks. Amenable for multi-labeling experiments employing antibodies against neurotransmitters, proteins and enzymes, phalloidin constitutes an excellent neuropil marker for the complex cephalopod CNS.

Biology of early life stages in cephalopod molluscs.

Recent literature on embryonic and post-embryonic development, biology and behavioural ecology of juvenile cephalopods is reviewed. Emphasis is placed on biological processes. Life-history patterns and phylogenetic systematics, which are important for a proper understanding of the evolutionary history of the cephalopods, are only briefly touched upon. Egg sizes in cephalopods range from less than 1 mm to about 30 mm in diameter, so the hatchlings emerging from the largest eggs are bigger than the adults of pygmy squid, the smallest known cephalopods. Developmental durations from spawning to hatching range from a few days (for very small eggs developing at high temperatures) to one or possibly several years (for very large eggs developing at low temperatures). Such important differences notwithstanding, the morphogenetic processes are very similar in all cephalopod embryos, the major variant being the size of the so-called outer yolk sac, which may be rudimentary in extremely small embryos. Several questions concerning the timing of hatching in relation to the developmental stage attained, especially in terms of yok absorption, need clarification. These questions concern the elimination of the transient closure of the mouth, the final differentiation of digestive gland cells, and the removal of the tranquilliser effect of the perivitelline fluid necessary for the onset of the hatching behaviour. Cephalopod hatchlings are active predators. They refine their behavioural repertoires by learning from individual experience in dealing with prey and would-be predators. There is no truly larval phase, and the ecologically defined term paralarva should be used with caution. Given the considerable resource potential of cephalopods, investigations into dispersal and recruitment are of particular interest to fishery biology, but they are also important for ecological biogeography. The related studies of feeding and growth involve field sampling and tentative age determination of caught specimens, in combination with laboratory studies to test food quality, measure feeding rates, and validation of periodicities in accretional growth structures (e.g. "daily rings" in statoliths).

Embryonic and paralarval development of the central nervous system of the loliginid squid Sepioteuthis lessoniana.

The embryonic development of the central nervous system (CNS) in the oval squid Sepioteuthis lessoniana is described. It has three distinct phases: (1) The ganglionic accumulation phase: Ganglionic cell clusters develop by ingression, migration, and accumulation of neuroblasts. (2) The lobe differentiation phase: Ganglia differentiate into lobes. The phase is identified by the beginning of an axogenesis. During this phase, neuropils are first formed in the suboesophageal mass, then in the basal lobe system, and finally in the inferior frontal lobes and the superior frontal-vertical lobe systems. (3) The neuropil increment phase: After the shape of the lobes reached its typical form, neuropil growth occurs, specifically in the vertical lobe. The paralarval central nervous system (CNS) is characterized by neuronal gigantism of the giant fibers and some suboesophageal commissures and connectives. The neuropil formation in the CNS of S. lessoniana occurs somewhat earlier than in Octopus vulgaris, although the principal developmental plan is quite conservative among the other coleoids investigated. Some phylogenetic aspects are discussed based on the similarities in the morphologic organization of their brains.

Distribution of three retinal proteins in developing octopus photoreceptors.

The expression of proteins unique to plasma membrane domains of developing photoreceptors is used as a marker for retinal differentiation in vertebrates. Invertebrate photoreceptors are also compartmentalized, but little information is available on the development of these compartments or the expression of retinal proteins specific to these cellular regions. Using routine electron microscopy techniques, we have made observations on the formation of photoreceptor organelles, including myeloid bodies and rhabdomeres, in embryonic octopus eyes from an early stage in development through hatching. Immunocytochemical experiments on the embryos demonstrate a timed expression of three retinal proteins during development, and the early separation of the octopus photoreceptor plasma membrane into distinct domains. Using polyclonal antibodies for opsin, retinochrome and retinal binding protein we have shown that opsin appears first and is confined to the distal end of the photoreceptor that will eventually differentiate into rhabdomeres. This membrane domain is separated from the proximal/inner segment plasma membrane by a septate junction. Retinochrome is expressed later when the myeloid bodies appear in the inner segments, and retinal binding protein is apparently not synthesized until sometime after hatching. These results suggest that, in the cephalopod retina, protein components of the retinoid cycling apparatus appear in a specific developmental sequence during the differentiation of this tissue.

Strontium is required for statolith development and thus normal swimming behaviour of hatchling cephalopods.

When cephalopod eggs were incubated in artificial sea water it was found that they sometimes resulted in hatchlings with defects of the statocyst suprastructure, leading to the severe behavioural defect of uncontrolled swimming. Experiments in defined media (seven basic salts mixed in deionized water) with seven species of cephalopods demonstrated clearly that there is 100% normal development of the aragonite statoliths when strontium levels were 8 mg l-1. Conversely, statoliths did not develop when strontium was absent. In cuttlefish, the growth of the cuttlebone was also affected adversely when strontium was absent. In mariculture production tanks, supplementing commercial artificial sea water with strontium to normal levels of 8 mg l-1 almost eliminated the occurrence of abnormal hatchlings. Circumstantial evidence indicates that there is a critical window in development during which strontium is required for normal development. The role of strontium in biomineralization during embryogenesis is unknown, but it appears to be important in the Mollusca.

Organogenesis in Cephalopoda: further evidence of blastodisc-bound developmental information.

The basic mechanisms of organ differentiation in the Cephalopod embryo (telolecithal egg, discoidal cleavage) are studied. The results of ligation experiments, performed in early cleavage stages, confirm earlier conclusions of the author, drawn from transplantation/explantation and heat-shock experiments. The developmental information for cellular differentiation is shown to reside in the blastodisc; the yolk syncytium, in which a large part of the original egg cortex is incorporated, acts as as nutritive substrate for the cellular material involved in organogenesis. On the basis of these results, Arnold's induction model supposing an undisplaceable determining informational pattern laid down in the uncleaved egg cortex must be rejected.