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

Abstract

BackgroundOctopus 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. ResultsHere, 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. ConclusionBesides 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.

Embryonic development of the camouflaging dwarf cuttlefish, Sepia bandensis

“…The blastomeres, which have undergone complete cleavage, lie in the center of the blastodisc and will give rise to the embryo proper (Figure 3). This separation of blastocones and blastomeres is found during cuttlefish, squid and octopus embryogenesis 8‐11,13,14,54 . The blastocones and blastomeres continue to divide every 2 hours (Stages 6‐8), and at 24 hours post‐fertilization a blastodisc of hundreds of cells sits on top of the yolk (Stage 9, Figure 3), bearing notable resemblance to the zebrafish blastula 3 hours post‐fertilization 56 …”

“…The first cell division occurs 8 hours post‐fertilization and produces a bilaterally symmetric embryo (Stage 2, Figure 3). When the second cell division occurs 2 hours later, an oblique cleavage plane creates the first asymmetry (Stage 3, Figure 3), a feature observed in cuttlefish and squid species, 8‐12,54 but not octopods 14 . At the 8‐cell stage (Stage 4) the cellular asymmetries have become more pronounced, and by the 16‐cell stage (Stage 5), the cells form two discrete populations.…”

“…This led us to a 25‐stage system of dwarf cuttlefish development, which contrasts with the 30‐stage systems of Sepia officinalis 8,9 and Sepia pharaonis 10 (see Table 2). Dwarf cuttlefish development can be divided into three major periods: cleavage, gastrulation and organogenesis, similar to other cephalopod staging systems 8‐11,13,14 …”

“…The disc begins to expand, bearing some spatial and behavioral resemblance to the mesendodermal cells of the zebrafish embryo 57 (Stage 11, Figure 4). Over the next 5 days, the marginal zone leads a migration of cells over the entire yolk in a process called epiboly (Stages 12‐15, Figure 4), a feature shared by coleoid cephalopods 8‐14,54 . By 7 days post‐fertilization, the majority of the yolk is enveloped by cells, and the cells that make up the embryo proper have begun the process of differentiation and reorganization 9 (Stage 15, Figure 4).…”

“…Although cephalopods are members of this group, they differ from many lophotrochozoans, undergoing meroblastic cleavage and direct development (no larval stage). The stages of embryogenesis have been described for a number of cephalopod species, including Sepia officinalis , 8,9 Sepia pharaonis , 10 Euprymna scolopes , 11 Doryteuthis pealeii , 12 Todarodes pacificus , 13 and Octopus vulgaris 14 . However, relatively little is known about the cellular and molecular basis of cephalopod embryogenesis.…”