# Collection, handling and care of cephalopod eggs

Roger Villanueva, Anne-Sophie Darmaillacq, Michael J Kuba, Katina

Roumbedakis, Jaruwat Nabhitabhata, Eduardo Almansa, Claudia

Caamal-Monsreal, Melanie Court, Ludovic Dickel, Erica D Durante, et al.

► To cite this version:

Roger Villanueva, Anne-Sophie Darmaillacq, Michael J Kuba, Katina Roumbedakis, Jaruwat Nabhitabhata, et al.. Collection, handling and care of cephalopod eggs. Reviews in Fish Biology and Fisheries, 2025, 36 (1), pp.17. 10.1007/s11160-025-10017-0 . hal-05413049

HAL Id: hal-05413049

https://hal.science/hal-05413049v1

Submitted on 12 Dec 2025

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# Collection, handling and care of cephalopod eggs

Roger Villanueva ID · Anne-Sophie Darmaillacq ID · Michael J. Kuba ID · Katina Roumedakis ID · Jaruwat Nabhitabhata ID · Eduardo Almansa ID · Claudia Caamal-Monsreal ID · Melanie Court ID · Ludovic Dickel ID · Erica D. Durante ID · Ali M. Elagoz ID · Oscar Escolar ID · Fernando Á. Fernández-Álvarez ID · Clara E. Galindo-Sánchez ID · Kostas Ganias ID · Connor J. Gibbons ID · Alice Goerger ID · Anaïd Gouveneaux · Bret Grasse ID · Ángel Guerra ID · Christelle Jozet-Alves ID · Thomas Lacoue-Labarthe ID · Lucía Martínez-Yebra · Giovanni D. Masucci · Sylvia L. S. Medeiros ID · Antoine Minet ID · Ryuta Nakajima ID · Owen C. Nichols ID · Nicolás Ortiz ID · James F. Peyla ID · Janina L. Röckner ID · Gabriela Rodríguez-Fuentes ID · Rui Rosa ID · Carlos Rosas ID · Satoshi Tomano · Lloyd A. Trueblood ID · Erica A. G. Vidal ID · Diego G. Vilarnau ID · Gjendine Voss ID

Received: 12 June 2025 / Accepted: 7 October 2025
© The Author(s) 2025

**Abstract** Research on cephalopod early ontogeny has significantly advanced in recent decades, including embryo organogenesis and neurogenesis and early behavioural adaptations, particularly in commercially important and coastal species. Within this context, here we compiled current knowledge on the collection, handling and care under experimental conditions and monitoring in the field of cephalopod eggs and

egg masses. It covers field observations and egg collection methods, as well as laboratory incubation protocols for eggs maintained with and without maternal care. It is emphasized how abiotic and biotic factors, including temperature, salinity, oxygen and maternal condition, shape embryonic development and hatchling survival and viability. Monitoring methods for cephalopod egg masses in the wild and the effects of natural threats such as storms and predators are reviewed. Anthropogenic impacts such as pollution,

**Supplementary Information** The online version contains supplementary material available at https://doi.org/10.1007/s11160-025-10017-0.

R. Villanueva (✉) · O. Escolar · D. G. Vilarnau
Institut de Ciencies del Mar (ICM-CSIC), Barcelona, Spain
e-mail: roger@icm.csic.es

A.-S. Darmaillacq · L. Dickel · A. Goerger ·
A. Gouveneaux
Université de Caen Normandie (UNICAEN), Université de Rennes, CNRS, EthoS (Ethologie Animale et Humaine), UMR 6552, Luc-sur-Mer, France

M. J. Kuba
University of Naples Federico II, Naples, Italy

K. Roumedakis
Instituto de Investigaciones Marinas, IIM-CSIC, Vigo, Spain

K. Roumedakis
Centro de Investigación y de Estudios Avanzados (Cinvestav-Unidad Mérida), Mérida, Mexico

J. Nabhitabhata
Centre for Marine and Coastal Studies (CEMACS), Universiti Sains Malaysia, Penang, Malaysia

J. Nabhitabhata
Excellence Center for Biodiversity of Peninsular Thailand, Prince of Songkla University, Hatyai, Songkhla, Thailand

E. Almansa
Centro Oceanográfico de Canarias, (COC-IEO), CSIC, Tenerife, Spain

C. Caamal-Monsreal · C. Rosas
Unidad Multidisciplinaria de Docencia e Investigación-Sisal, Facultad de Ciencias, Universidad Nacional Autónoma de México, Sisal, Yucatán, Mexico

M. Court · R. Rosa
MARE - Marine and Environmental Sciences Centre / ARNET - Aquatic Research Network, Laboratório Marítimo da Guia, Faculdade de Ciências, Universidade de Lisboa, Cascais, Portugal
fisheries and climate change are also discussed. Technological advances have enabled finer analysis of neural development as the embryo grows, while ethical considerations regarding embryonic sentience capacity and welfare conditions are becoming central to current experimental designs. Based on current knowledge, it is recommended to apply ethical considerations and the 3Rs principle (Replacement, Reduction, Refinement) for cephalopod embryos following the onset of organogenesis. Thus, a list of potential indicators of health and welfare that could be used for assessing and monitoring cephalopod embryos and hatchlings is proposed. Overall, this synthesis aims to serve as a guide for advancing egg collection and laboratory incubation methodologies, as well as adopting ethical handling protocols to improve cephalopod embryo care and quality.

E. D. Durante
University of South Australia, Mawson Lakes, Australia

A. M. Elagoz
Department of Biology, KU Leuven, Leuven, Belgium

F. Á. Fernández-Álvarez
Centro Oceanográfico de Gijón (COG-IEO), CSIC, Gijón, Spain

C. E. Galindo-Sánchez
Centro de Investigación Científica y de Educación Superior de Ensenada, Baja California, Mexico

K. Ganias
School of Biology, Aristotle University of Thessaloniki, Thessaloniki, Greece

C. J. Gibbons
Department of Neuroscience, Columbia University, NY, USA

B. Grasse
Marine Biological Laboratory, Woods Hole, USA

Á. Guerra
Instituto de Investigaciones Marinas (IIM-CSIC), Vigo, Spain

C. Jozet-Alves
Normandie Univ, Unicaen, CNRS, EthoS, Caen, France

T. Lacoue-Labarthe · A. Minet
UMR 7266 LIENSs, CNRS - La Rochelle Université, La Rochelle, France

L. Martínez-Yebra
Sociedad Española de Malacología, Madrid, Spain

G. D. Masucci · R. Nakajima
Okinawa Institute of Science and Technology Graduate University (OIST), Tancha, Onna-son, Okinawa, Japan

S. L. S. Medeiros
Brain Institute, Federal University of Rio Grande do Norte, Natal, Rio Grande do Norte, Brazil

R. Nakajima
Department of Art and Design, University of Minnesota Duluth, Duluth, USA

O. C. Nichols
Center for Coastal Studies, 5 Holway Avenue, Provincetown, USA

N. Ortiz
Laboratorio de Cefalópodos, Instituto de Biología de Organismos Marinos - CONICET, Puerto Madryn, Argentina

N. Ortiz
Facultad Regional Chubut - Universidad Tecnológica Nacional, Puerto Madryn, Argentina

J. F. Peyla
Science Department, Mount Saint Joseph High School, Baltimore, USA

J. L. Röckner
University of Genoa, Genoa, Italy

J. L. Röckner
Istituto Italiano di Tecnologia, NSYN, Genova, Italy

G. Rodríguez-Fuentes
Unidad Química Sisal, Universidad Nacional Autónoma de México, Yucatán, Mexico

S. Tomano
Atmosphere and Ocean Research Institute, University of Tokyo, Tokyo, Japan

L. A. Trueblood
Biology Department, La Sierra University, Riverside, USA

E. A. G. Vidal
Center for Marine Studies, University of Parana, Pontal do Paraná, Brazil

G. Voss
Marine Biological Laboratory, Woods Hole, MA, USA
**Keywords** Cephalopod embryology · Egg handling techniques · Embryonic development · Ethics in cephalopod research

## Introduction

Cephalopods are a group of exclusively marine molluscs with more than 850 extant described species living in benthic and pelagic habitats of all oceans and distributed from littoral to hadal environments (Boyle and Rodhouse 2008). While nautiloids (Ward 1987) and vampire squid (Hoving et al. 2015) are iteroparous, remaining cephalopod species are semelparous, with a single sexual maturity period that can be usually brief in coastal species and longer in the deep-sea (Rocha et al. 2001; Schwarz et al. 2018). Egg size and number vary notably among species, however, in comparison with other marine invertebrates, they are relatively larger and few in number (Boletzky 1998). Females spawn egg masses that usually develop without maternal care, except for incirrate octopods and a few species of oegopsid squids (Hoving et al. 2024). Eggs can be laid and incubated in different ways: (1) attached to hard structures or macroalgae on the sea floor (in nautiloids, sepiids, loliginid squids and most benthic octopods); (2) released in oceanic waters forming gelatinous pelagic masses (most squid families); (3) carried within the female arms and web (pelagic octopods and few benthic octopods and oegopsid squids); (4) attached to a calcareous shell that function as brood chamber (argonauts) and (5) carried inside long female distal oviducts (ovoviviparity) in the exceptional pelagic octopod *Ocythoe*.

Cephalopod eggs have been studied using different approaches, from biological monitoring in the field to experimental procedures in the laboratory. The current existing knowledge shows the relevance of the embryonic phase in the cephalopod life cycle and the adaptability of cephalopod embryos to the changing environment, opening new methodological questions for study. During the Cephalopod International Advisory Council (CIAC) Conference in Sesimbra, Portugal, during April 2022, a two-day workshop was held entitled "Collection, handling and care of cephalopod eggs and egg masses", aiming to provide an overview on recent methods and

advances in the study of cephalopod eggs. The present review is the outcome from the ideas and discussions resulting from that workshop. Comprehensive reviews of Sigurd von Boletzky (1987, 1989, 1998, 2003) brought together existing knowledge about cephalopod eggs and egg masses, and his translation of the immense work of Naef (1928), published in 2000, significantly contributed to our understanding of the embryonic life of cephalopods. The present review aims to synthesize the advances made during the past 25 years and to propose new approaches to the study of cephalopod eggs.

## General characteristics of cephalopod eggs and egg masses

Oocyte fertilisation sites and embryonic developmental scales

In cephalopods there is little evidence describing the exact place where the female gamete, the oocyte, is fertilised by spermatozoids and embryonic life starts. Sperm is transferred by the male in sperm packets named spermatophores, which eject other structures called spermatangia that can be the final place of sperm storage, or release the sperm which will migrate to the seminal receptacles placed in the buccal area of the female (Fernández-Álvarez et al. 2018). Nautiloids differ from coleoid cephalopods in having simpler spermatophores which are placed in the Valenciennes organ (Marian 2015), and virtually nothing is known about their fertilization. In the pygmy squid *Idiosepius paradoxus*, the female lays oocytes one by one between her arms, coating them with oviducal envelope first and then by nidamental envelope to fix them on a hard surface. Then, the female puts her seminal receptacle containing stored sperm near the oocyte, injecting a few spermatozoa (an average of 134±131 spermatozoa) into the envelope layers for each fertilisation (Iwata et al. 2019). In the flying squid *Todarodes pacificus*, females first create a large gelatinous mass, and then the sperm is ejected from their multiple seminal receptacles, followed by the oocytes (Yamato et al. 2019). Some squids, such as enoploteuthoids and sepiolines, have developed specialized areas for spermatangia implantation in the nuchal area or the end of the oviduct
(Marian 2015). In the case of loliginid squids, con-
sort males insert their spermatangia near the oviduct
opening and sneaker males near the seminal recepta-
cle (Apostolico et al. 2020), resulting in the paternity
being a mixture of both (Naud et al. 2016). In octo-
pods, fertilization is internal, either in the ovary or in
the spermatheca (Marian 2015).

Recognizing the different embryonic structures is
a key tool to determine the degree of development,
growth and health status of the embryo. The embry-
ology scales categorize development up to 34 mor-
phologically distinct stages, depending on the taxon
and classification system used. Naef (1928) provided
detailed descriptions for several cephalopod species.
During recent years, useful information and tables of
embryonic development has been published for nauti-
loids (Arnold 1987), cuttlefish (Boletzky et al. 2016),
loliginids (Arnold 1965; Segawa et al. 1988; Kuan
et al. 2022), ommastrephid squids (Watanabe et al.
1996), sepiolid squid (Lee et al. 2009), and incirrate
octopods (Deryckere et al. 2020).

Cephalopod egg diversity and incubation modes

Cephalopods lay eggs with different characteris-
tics, depending on phylogeny, habitat and maternal
behaviour (Table 1, Fig. 1). They can vary between
benthic individual eggs to large pelagic egg masses
and protective envelopes or capsules secreted by
oviducal and nidamental glands show great varia-
tion in shape (Boletzky 2003). Individual cephalopod
ggs attached to benthic substrates vary from small to
large, and can reach 45 mm in length, including its
protective egg capsule, in the exceptional nautiloids
(Arnold 1987, AZA 2025). Deep-sea cirrate octopods
lay also large, individual and non-brooded eggs cov-
ered by a hard egg capsule, reaching 26 mm in length,
attached to bathyal and abyssal substrates as cold
water octocorals (Shea et al. 2018; Ziegler et al. 2021;
Chen and Sigwart 2025). Sharing the same deep-sea
habitats, females of holobenthic incirrate octopods
brood very large eggs lacking egg capsule, with the
chorion directly exposed to the external environment;
that is also the case of meropelagic incirrate octopods
that produce small eggs.

The duration of embryogenesis is species-
specific and strongly influenced by temperature (see
Sect. "Main abiotic factors".) and egg size. In large-
egged octopods (i.e., *Graneledone*), females can

brood their eggs for more than 4 years attached to
rocky bottoms and eggs can reach 40 mm in length
(Voight and Drazen 2004; Robison et al. 2014). In
the deep-sea *Muusoctopus*, high temperatures from
hydrothermal vents might accelerate embryonic
development (Barry et al. 2023). Whereas in
oceanic, small-egged species (i.e., *Ommastrephes
bartrami*) with 1.5—2 mm egg length, the duration of
embryonic development could be as brief as 4–5 days
at warm temperatures (Vijai et al. 2015a). Age
estimation for *O. bartramii* is nearly one year (Yatsu
et al. 1997), suggesting that embryonic life represents
only around 1.4% of the life cycle. In coastal
species of reared cephalopods as *Acanthosepion
pharaonis*, *Sepiella inermis*, *Euprymna hyllebergi*
and *Amphioctopus aegina*, embryonic life represents
8 to 12% of the life cycle (Promboon et al. 2011).

In littoral, coastal and shelf benthic areas, the
cuttlefishes, sepiolids, loliginid squids and benthic
octopods lay their eggs associated with hard sea bot-
tom substrates with a variety of egg sizes and pro-
tective structures. Cuttlefish eggs have a medium to
large size, laid one by one and protected by an egg
capsule, where a single or several females can form
clusters from few to hundreds of eggs on the seabed
(Nabhitabhata and Nishiguchi 2014; Guerra et al.
2016). Loliginid squids also spawn small to large size
ggs covered by mucilaginous envelopes forming
finger-like capsules with two to more than three hun-
dred eggs inside, depending on the species. The egg
masses are formed by clusters of few to thousands of
capsules originating from one or many females and
are attached together, forming egg beds on the sea-
floor that develop until hatching without maternal
care (Steer et al. 2003; Laptikhovsky et al. 2021). In
the same coastal habitats, benthic incirrate octopods
brood their eggs, in egg masses with small to large
ggs, depending on species (Villanueva et al. 2016).
Benthic octopod females usually attach their eggs
to hard surfaces in protected shelters such as caves,
crevices or mollusc shells but, females of some spe-
cies, can brood their eggs within their arms (Tranter
and Augustine 1973; Miske and Kirchhauser 2006;
Nabhitabhata 2014a; Prasopsook et al. 2022).

In the oceanic epipelagic realm, female pelagic
octopods brood their small eggs in a variety of forms:
inside their oviducts, as the excepcional ovovivipa-
rous *Ocythoe*, where embryos develop inside the
female until hatching (Naef 1923, 1928; Laptikhovsky
<table><caption>**Table 1** Overview of egg characteristics from diverse cephalopod groups in relation to hatchling and adult life style. Egg size means the relative size of egg to adult size. Incirrate octopods are separated into three groups as small egg (a), large egg (b) and pelagic (c) species based on life styles of hatchlings and adults. In some groups there might be some exceptional taxa e.g., ¹Sepiella are benthic sepiid with pelagic hatchlings (Nabhitabbata 2014a, b, c); ²within bathyteuthid squids, *Bathyteuthis* displays maternal care (Bush et al. 2012) while the reproductive strategy of *Chitenopteryx* is unknown at present; ³gonatid squid are oegopsids with maternal care and few large eggs (Hoving et al. 2024); ⁴the ovovivorous *Ocythoe* is an incirrate pelagic octopod where embryos develop inside the female oviduct until hatching (Laptikhovsky et al. 2003)</caption><thead><tr><th>Group</th><th>Habitat</th><th>Adult life style</th><th>Egg size</th><th>Num. of embryos in each capsule</th><th>Egg structure and envelopes</th><th>Num. eggs</th><th>Maternal care</th><th>Hatchling life style</th><th>r/K strategy</th><th>r/K strategy</th></tr></thead><tbody><tr><td>Nautilid</td><td>Neritic/ oceanic</td><td>Pelagic</td><td>Large</td><td>single</td><td>Inner capsule formed in the oviduct, attached to the inside of the leathery outer capsule</td><td>Few</td><td>-</td><td>Pelagic</td><td>K</td><td>K</td></tr><tr><td>Spirulid</td><td>Oceanic</td><td>Pelagic</td><td>Large</td><td>?</td><td>?</td><td>Few?</td><td>-</td><td>Pelagic</td><td>K?</td><td>K</td></tr><tr><td>Sepiid¹</td><td>neritic</td><td>benthic</td><td>Large</td><td>single</td><td>Soft oviducal and nidamental envelopes possibly stained with ink</td><td>few</td><td>-</td><td>benthic</td><td>K</td><td>K</td></tr><tr><td>Sepiolid</td><td>Neritic/ oceanic</td><td>Benthic</td><td>Large</td><td>single</td><td>Oviducal and hard outer nidamental envelopes possibly stained with ink</td><td>Few</td><td>-</td><td>Pelagic/ benthic</td><td>r</td><td>r</td></tr><tr><td>Idiosepid</td><td>Neritic</td><td>Benthic</td><td>Large</td><td>single</td><td>Soft oviducal and nidamental envelopes</td><td>Few</td><td>-</td><td>Pelagic</td><td>r</td><td>r</td></tr><tr><td>Myopsid squid</td><td>Neritic</td><td>Pelagic</td><td>Small</td><td>several</td><td>Soft oviducal and nidamental envelopes, possibly spirally coiled</td><td>Many</td><td>-</td><td>Pelagic</td><td>r</td><td>r</td></tr><tr><td>Bathyteuthid squid²</td><td>Oceanic</td><td>Pelagic</td><td>Large</td><td>several</td><td>Soft oviducal and nidamental envelopes carried by the female</td><td>Few</td><td>+/+</td><td>Pelagic</td><td>K</td><td>K</td></tr><tr><td>Oegopsid squid³</td><td>Oceanic</td><td>Pelagic</td><td>Small</td><td>several/ many</td><td>Gelatinous oviducal and nidamental envelopes, possibly spirally coiled</td><td>Many</td><td>-/+</td><td>Pelagic</td><td>r</td><td>r</td></tr><tr><td>Incirrate octopod a</td><td>Neritic</td><td>Benthic</td><td>Small</td><td>Single</td><td>Without capsule, chorion directly in contact with seawater, fixed to a common oviducal cement</td><td>Many</td><td>+</td><td>Pelagic</td><td>r</td><td>r</td></tr><tr><td>Incirrate octopod b</td><td>Neritic/ oceanic</td><td>Benthic</td><td>Large</td><td>Single</td><td>Without capsule, chorion directly in contact with seawater, fixed to a common oviducal cement</td><td>Few</td><td>+</td><td>Benthic</td><td>K</td><td>K</td></tr><tr><td>Incirrate octopod c⁴</td><td>Oceanic</td><td>Pelagic</td><td>Small</td><td>Single</td><td>Without capsule, chorion directly in contact with seawater, fixed to a common oviducal cement</td><td>Many</td><td>+</td><td>Pelagic</td><td>r</td><td>r</td></tr><tr><td>Cirrate octopod</td><td>Oceanic</td><td>Benthic/ Pelagic</td><td>Large</td><td>Single</td><td>Single encapsulated eggs with hard capsule</td><td>Few</td><td>-</td><td>Pelagic/ benthic</td><td>K</td><td>K</td></tr><tr><td>Vampyromorph</td><td>Oceanic</td><td>pelagic</td><td>Large</td><td>Single</td><td>?</td><td>Few</td><td>-</td><td>Pelagic?</td><td>K</td><td>K</td></tr></tbody></table>

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►Fig. 1 Main types of cephalopod eggs. **a Nautilus macromphalus** eggs under aquarium conditions of low light intensity and temperature of 22 °C, Aquarium des Lagons, New Caledonia, Image: Sylvain Govan. **b Acanthosepion pharaonis** eggs laid under cement plates, Toucheng Township, Taiwan. Image: Anne-Sophie Darmaillacq. **c Ascarosepion pfefferi** eggs, Marine Biological Laboratory, Woods Hole, image: Bret Grasse. **d Sepia officinalis** egg mass on a marine rope, La Rochelle, Bay of Biscay, NE Atlantic. Image: Antoine Minet. **e Euprymna berryi** eggs incubated in laboratory, San Francisco State University, San Francisco, USA. Image: Anne-Sophie Darmaillacq. **f Rossia macrosoma** eggs laid in the Mediterranean Sea and incubated in the laboratory, Monaco. Image: Thomas Lacoue-Labarthe. **g Embryos of Loligo vulgaris** inside their capsule. Image: Thomas Lacoue-Labarthe. **h Loligo vulgaris** eggs from the Mediterranean incubated in a laboratory, Monaco, Mediterranean Sea. Image: Jean-Louis Teysisi. **i Sepioteuthis lessoniana** egg mass attached to *Sargassum* spp. found within a seagrass meadow, Kaichudoro, Okinawa, NW Pacific. Image: Ryuta Nakajima. **j A batch of eggs of the sharp-tail pygmy squid, Idiosepius pygmaeus**, attached to the aquarium glass wall. Scale bar: 5 mm. Image: Fernando Á. Fernández-Álvarez. **k Oegopsid squid egg mass 120–150 cm in diameter, probably Illex coindetii**, Tingelsaetted Island, Norway, 5 m depth, June 2017. Image: Erling Svensen. **m Cirrate octopod egg, probably Grimpoteuthis**, Aleutian Trench, North Pacific, 4627–4638 m depth, Agassiz trawl, August 2022, preserved in 95% ethanol (Senckenberg Research Institute and Museum of Natural History, Frankfurt, catalog number SMF373318). Scale bar: 5 mm. Image: Chen and Sigwart (2025). **n Callistoctopus macropus** captive laid eggs from a female collected in the Bay of Naples, Mediterranean Sea. Image: Michael J. Kuba. **o Octopus insularis** egg mass laid in an aquarium, Natal, Rio Grande do Norte, Brasil. Image: Sylvia Medeiros. **p Octopus maya** egg mass artificially incubated at the Laboratory of Applied Ecophysiology, National Autonomous University of Mexico, Sisal, Mexico. Image: Katina Roumbedakis. **q Octopus tehuelchus** egg mass within an empty shell of *Odontocymbiola magellanica*, Golfo Nuevo, Argentina. Image: Nicolás Ortiz. **r Robsonella huttoni** egg string, Portobello, Otago, New Zealand. Image: Erica Donlon Durante. **s Robsonella fontaniana** egg mass laid underneath a concrete plate, San José Gulf, Argentina. Image: Nicolás Ortiz

et al. 2003); within their brachial web and arms, as *Tremoctopus* (Mangold et al. 2018); or using a calcareous shell, as *Argonauta* (Finn 2013). In the same oceanic habitat, female squids can lay large gelatinous egg masses with thousands of small, 1–2 mm eggs, as in ommastrephid (Staaf et al. 2008; Birk et al. 2017; Vijai 2016; Ringvold et al. 2021), thysanoteuthid (Guerra et al. 2002; Miyahara et al. 2006; Brown et al. 2022) and enoploteuthid squids (Roberts et al.

2011) or carry gelatinous masses of medium sized (2–3 mm) to large (11 mm) eggs within their arms, as in gonatid (Seibel et al. 2000, 2005; Bower et al. 2012; Hoving et al. 2024) and bathyteuthid (Bush et al. 2012) squids in meso- to bathypelagic zones.

There is a great diversity in cephalopod egg capsule coloration (Fig. 1). In nautiloids, the large egg capsule is white to yellowish in colour. In some cuttlefish species, e.g., *Sepia officinalis* and *Sepiella inermis*, the capsule is coloured by ink at spawning (Boletzky 1986; Nabhitabhata 2014c). Interestingly, such cuttlefish eggs are laid in visible places whereas cuttlefish species that lay translucent eggs are hidden under rocks, among corals or in sponges. It has been hypothesized that ink acts as a protection against predators (Hanlon and Messenger 2018). Recently, it has been shown that *S. officinalis* embryos, exposed to UV-B light after removal of the black envelopes, present a higher mortality and developmental abnormalities (Molina-Carrillo et al. 2023). However, it is still not known whether this is due to the absence of ink or that of the coiled envelopes. Sand can also be used for egg protection, as in *Acanthosepium esculentum* (Hanlon and Messenger 2018) or in *Euprymna* sp. (Drerup et al. 2020; Fig. 1). In *Euprymna hyllebergi*, newly laid eggs have white gelatinous capsules that, after 2 h, harden and turn yellowish-brown in colour due to adhering diatoms (Nabhitabhata and Nishiguchi 2014, see their Fig. 15.3). This process could serve as protection and/or camouflage, however, the capsule will become increasingly transparent after 5 days, at which point the internal yolk sacs become visible (Nabhitabhata pers. comm.). In a similar way, recently spawned loliginid squid eggs have white colour that sometimes turns to creamish as algae and microorganisms grow on the capsule surface. Gelatinous pelagic egg masses of oegopsid squid are nearly transparent (Ringvold et al. 2021, see Fig. 1k, 1). In *Thysanoteuthis*, the gelatinous capsule is nearly translucid, and their egg masses change to whitish and pink in colour due to changes in embryo pigmentation as development advances (Brown et al. 2022). Cirrate octopods have a yellow to muddy brown and greenish coloured hard capsule (Ziegler et al. 2021). On the other hand, incirrate octopod eggs lack a capsule and the transparent chorion allows visualization that the embryos change from white to reddish brown as development and pigmentation advances (see Fig. 1 n-s).
## Monitoring egg masses in the wild

Continuous records of temperature, salinity, pH, water turbidity and oxygen on the spawning site are important to follow embryonic development conditions in situ, and attached tags on or close to the egg masses and photographic/video monitoring are useful to identify and recognize them later by SCUBA divers, cameras and/or ROV's. Likewise, it is important to characterize the biotic environment associated with the egg masses. Since cephalopod egg masses occur in varying spawning areas in the wild, depending on the group (see Sect. "Collection of cephalopod eggs from the wild"), their monitoring demands specific approaches.

### Monitoring eggs in shallow waters.

Egg masses of loliginids are traditionally the most monitored in coastal waters, as many species spawn in shallow depths making the eggs easily accessible. Also, these species are amongst the most relevant to the fishery. Main methods used to monitor and locate spawning grounds and egg distribution in loliginids are SCUBA diving (see among others, Steer et al. 2002; Oosthuizen and Roberts 2009; Shashar and Hanlon 2013; Garci et al. 2016), echosounder (Sauer et al. 1993; Roberts et al. 2002) and remotely operated vehicles (ROVs) (Hanlon et al. 2004; Zeidberg et al. 2012; Navarro et al. 2018). In addition, fishery-dependent data can provide seasonality of loliginid spawning and opportunities for in situ observations of spawning behaviour, egg deposition, embryonic development, and collection of samples (e.g., large passive gears such as fish weirs; Shashar et al. 2013, Nichols et al. 2019; see also Sect. "Collection of cephalopod eggs from the wild"). Long term data collection is especially valuable. For example, a continuous in situ monitoring of egg masses of *Loligo gahi* in the SW Atlantic showed that embryos developed at temperatures as low as 3.86 °C, which is the lowest temperature reported for any loliginid (Arkhipkin and Middleton 2003). When possible, non-invasive methods to follow embryo development are useful to reduce sampling impact in natural populations. For example, the recognition of embryonic developmental stages of *Loligo vulgaris* based only on the external features of egg capsules has been used as a non-invasive method for in situ

embryological data collection (Feyjoo et al. 2016). Determining the general structure of the egg mass and egg arrangement is also important. SCUBA-based monitoring of *Sepioteuthis australis* egg masses demonstrated that the specific position of an embryo within the egg mass and the size of the egg mass significantly affects its survival probability, growth rate, and likelihood of developing morphological abnormalities (Steer et al. 2002, 2003; Gowland et al. 2002; Steer and Moltschaniwskyj 2007).

Data collection by underwater visual transects of seagrass beds during SCUBA diving have been successfully used to estimate small-scale temporal and spatial variability in *Sepioteuthis australis* egg production in Tasmanian waters (Moltschaniwskyj and Pecl 2003) and to determine spawning habitat selection in *S. officinalis* (Guerra et al. 2016). In littoral waters, *Sepioteuthis lessoniana* egg abundance was monitored on seagrass meadows for several years, illustrating the positive role of the seasonal seagrass growth and development to support spawning (Kimoto et al. 2023). Further studies conducted in other littoral habitats by walking during low tide showed the importance of octocoral colonies as hard surfaces for attachment of *Sekiella inermis* eggs in the Arabian Sea (Gaikwad et al. 2021).

Monitoring of shallow octopus egg masses has been done in different ways. It ranged from monitoring of a single brooding female following embryonic development in situ (Garci et al. 2016) to underwater visual transects to estimate spawning dens density of *Octopus vulgaris* in the NE Atlantic (Guerra et al. 2015). In addition, shell shelters arranged along sampling lines were provided as potential refuges to assess their suitability as brood shelters for *Octopus tehuelchus* females in SW Atlantic to estimate their population density (Narvarte et al. 2013). Also, artificial dens anchored on rocky substrates in the western Mediterranean have been used to attract *O. vulgaris* females for spawning (Mereu et al. 2018). In the last few years, Citizen Science projects proved to be another source to gather cephalopod egg presence in the field. Laptikhovsky et al. (2021; 2025) used citizen science to map species-specific spawning grounds of *Loligo vulgaris* and *L. forbesii* in NE Atlantic and Mediterranean Sea and also helped to determine spatial and temporal variability on the distribution of *S. officinalis* eggs (Laptikhovsky et al. 2023).
Monitoring eggs in pelagic and deep-sea zones.

The presence of cephalopod eggs in pelagic and deep-sea zones have been poorly studied and require further scientific effort. In the pelagic realm, oegopid squid egg masses have not been routinely monitored at sea despite the high fishery value of some species. Their egg mass size and delicate gelatinous consistency makes them difficult to sample using pelagic nets. To date, information has been obtained primarily through opportunistic scientific sampling (Staaf et al. 2008; Birk et al. 2017) and observations and documentation from recreational diving activities in coastal waters (see between others, Roberts et al. 2011; Ringvold et al. 2021; Brown et al. 2022). These findings show the growing importance of citizen science in advancing cephalopod research.

In the benthic area, fishing trawl data obtained during demersal fisheries and research surveys has been used to estimate loliginid spawning aggregations (Roberts et al. 2012) and determine spawning extensions, in example, as for *Loligo forbesii* to 720 m in the Aegean Sea (Salman and Laptikhovsky 2002) or *Loligo vulgaris* to 550 m in NW Mediterranean (Laptikhovsky et al. 2025). Monitoring brooding females and egg masses of abyssal octopuses using ROVs and the human occupied vehicle (HOV) *Alvin* in hydrothermal springs generated one of the most surprising observations in cephalopod biology in recent years. Observations showed dense groups of brooding *Muusoctopus* females in NE Pacific at 3100—3300 m depth (Hartwell et al. 2018; Barry et al. 2023). Also of significance was the long duration of embryonic development in abyssal *Graneledone* octopuses that can last up to 53 months, information recorded by visiting the same brooding female at her egg-laying site 18 times at 1,397 m depth in the Monterey Submarine Canyon (Robison et al. 2014).

## Natural threats and anthropogenic impact on cephalopod egg masses

### Natural threats: storms and predators

The turbulence effects of storms have been reported in monitored loliginid egg masses by uprooting them from the sampling site (Arkhipkin and Middleton 2003; Moltschaniwskyj and Pecl 2003). Once

detached from the substrate, the abrasive effect of the seabed on the detached egg capsules with the movement of the sea currents greatly reduced or even abrogated the life expectancy of these embryos. After severe storms, Moltschaniwskyj and Pecl (2003) recorded losses to 54% of the monitored egg masses of *Sepioteuthis australis* in shallow areas (<10 m) of seagrass *Amphibolis antarctica*, showing that these events can have strong impact on embryonic squid populations. Stranding of *Thysanoteuthis* egg masses on intertidal seagrass bed (Brown et al. 2022) and *Argonauta* (Boletzky and Centelles 1979; Norman 2000; Finn 2013) and *Ocythoe* (Roper and Sweeney 1975) females carrying fertilized eggs and stranded on beaches are other examples of egg mass losses.

Fish predation on cuttlefish eggs has been reported for *Sepia officinalis* by the blenny *Parablennius gatorugine* (Guerra and González 2011) and the triggerfish *Balistes capriscus* (Blanc and Daguzan 1999). Fishes from the families Chaetodontidae, Balistidae, Monacanthidae and Zanclidae prey upon eggs of *Acanthosepion pharaonis* (Gutsal 1989) and the butterflyfish *Chaetodon ulietensis* prey on *Ascarosepion latimanus* eggs (Corner and Moore 1980). Within invertebrate predators, the gastropod *Hexaplex trunculus* prey on *S. officinalis* eggs (Melli et al. 2014) and laboratory experiments (Martins et al. 2018) showed also invertebrates feeding on cuttlefish eggs, such as sea snails (*Bolinus brandaris*), decapod crabs (*Cancer pagurus*, *Callinectes sapidus*, *Dardanus arrosor*), lobsters (*Homarus gammarus*), mantis shrimps (*Squilla mantis*), sea urchins (*Echinus melo*, *Cidaris* sp., *Paracentrotus lividus*) and starfishes (*Astropecten aranCIACUS*). Sepiolid *Rossia macrosoma* eggs are also preyed on by the sea urchin *Echinus esculentus* (Drerup et al. 2021).

In octopods, egg masses of *Eledone caparti* has been found in stomach contents of the blue shark *Prionace glauca* (Nesis and Nigmatullin 1981); egg masses of *Enteroctopus dofleini* were eaten by decapod crustaceans *Chorilia longipes*, *Scyra acutifrons* and *Oregonia gracilis* (Cosgrove 1993) and sea star *Evasterias troschelii* (Villanueva and Norman 2008); chitons *Chaetopleura isabellei* could remove eggs of *Octopus tehuelchus* from their dens (Navarte et al. 2013); and eggs of *Macroctopus maorum* were preyed on by the gastropod *Scutus breviculus* under aquarium conditions (Batham 1957). As far as we know, no predators have been reported
for loliginid eggs, perhaps indicating the existence of some unknown chemical or flavour protection. However, predators on spawning loliginid squids have been reported (Smale et al. 2001) and also the effect of predators in the disintegration of spawning squids concentrations (Lipinski and Soule 2007).

## Anthropogenic impact on cephalopod egg masses

### Ocean pollution

The increasing presence of marine debris has led many cephalopod species to use solid waste (e.g., cans, bottles, and snorkel mouthpieces) as alternative refuges. Freitas et al. (2022) identified seven species (*Amphioctopus marginatus*, *A. siamensis*, *Paroctopus cthulu*, *Octopus maya*, and *O. insularis*) using marine litter as brooding sites and most *Paroctopus cthulu* individuals were found sheltered in debris (Leite et al. 2021). These findings are particularly important for egg monitoring and collection in natural habitats and for minimizing the risk of inadvertently removing eggs during underwater cleanups. Furthermore, the use of debris as egg-laying sites by cephalopods raises concerns about the long-term effects of marine pollution on egg development, offspring viability and reproductive success. For instance, inadequate shelters have been shown to correlate with reduced clutch sizes (Narvarte et al. 2013).

In a recent study, García-Flores et al. (2025) reviewed the effects of 28 anthropogenic contaminants on cephalopod embryos, paralarvae, and juveniles and concluded that, cadmium, copper, zinc, fluoxetine, PAHs, organophosphorus compounds, and tributyltin were the only contaminants associated with adverse effects during early life. However, the literature is unbalanced as 67% of articles focused only on trace metals, and more than 70% had the European cuttlefish *S. oficinalis* embryo as a biological model.

Among legacy contaminants, trace element levels in cephalopods raised specific attention from more than 75 years. Consensus states that specific organs of cephalopods, such as digestive gland, branchial hearts and branchial heart appendages generally show the highest levels in line with their key role in contaminant detoxification (Penicaud et al. 2017; Lacoue-Labarthe et al. 2016). As model species, a considerable number of ecotoxicological studies have been carried out in *S. officinalis* embryos (see among

others, Lacoue-Labarthe et al. 2009, 2012, 2016; Bloor 2016; Rosa et al. 2015). In this species, the protective role of the egg capsule makes it difficult for most metals to penetrate into the egg. This phenomenon has been recorded both in embryos from the wild (Rosa et al. 2015) and from laboratory studies (Bustamante et al. 2002, 2004), and is selective for different metals. The shielding properties of the egg capsule are metal- and developmental stages dependent. Indeed, the development of cephalopod eggs is characterised by a swelling phenomenon (which is more important in singly encapsulated eggs compared to eggs contained in jelly masses or only encapsulated by the chorion; Boletzky 1998), during the last third of embryogenesis, which leads to an increase in the volume of perivitelline fluid and stretching of the egg capsule. Consequently, the egg capsule permeability to some trace elements (e.g., Ag, Cs, Zn, Hg) significantly increased during this period although still selective (e.g., Cd, Pb do not penetrate inside the egg). Importantly, the nature of the egg capsule layers (i.e., chorion, nidamental and oviducal secretions) appears to have metal-specific binding and retention properties (Lacoue-Labarthe et al. 2011). This means that the vulnerability of embryos to metal contamination is partially driven by the composition and structure of the egg capsule, which is highly diverse in cephalopods. Incirrate octopus eggs lack egg capsule, thus, their vulnerability to metal or other contaminants might be higher than for other cephalopods.

Microplastics (MPs) (size <5 µm) have been detected and characterised in embryos and yolk samples of *Sepia officinalis* (Chemello et al. 2023). Although there were MPs accumulated inside the embryos, they did not seem to interfere with organogenesis and no alterations in the morphology and functionality of the embryonic internal organs and tissues were observed. The pathway for MPs accumulation in both yolk and embryos is uncertain. Given that Oliveira et al. (2020) demonstrated preferential MP accumulation in the digestive gland of *S. officinalis* adults, Chemello et al. (2023) suggested maternal MP transfer via nutrients from the female's digestive gland to oocyte, ultimately reaching embryos and yolk. An alternative transfer pathway may occur through the nidamental glands, which were shown to accumulate microplastics in *Sekiella inermis* (Bar et al. 2024).
Persistent organic pollutants (POPs) are a set of toxic chemicals able to last for several years in the environment before breaking down. Several POPs have been reported to have negative effects on cephalopods embryos. For example, the pesticide Parathion produced alterations in *Octopus mimus* embryonic disc formation at the blastula stage, abnormal gastrulation and subsequent developmental arrest (Gutiérrez-Pajares et al. 2003); Phenanthrene inhibited the B-esterase activities in *O.maya* embryos (Aguilar et al. 2023); and Tributyltin led to yolk sac lysis, embryonic malformations and uneven pigment distributions in *Acanthosepion pharaonis* embryos (Jiang et al. 2023). A study on exposure to 16 polycyclic aromatic hydrocarbons (PAHs), a group of POPs, suggests that PAHs may disrupt developmental processes by dysregulation of homeobox gene networks involved in normal nervous system development and body growth in *S. officinalis* (Cocci et al. 2023). Due to their persistence in the environment, the study of the effects of polycyclic aromatic hydrocarbons (PAHs) on aquatic organisms has become a global priority (Chen et al. 2018). Among these, phenanthrene (Phe) has been reported in concentrations of up to 1540 µgL⁻¹ in the southern Gulf of Mexico, habitat of *O. maya*. In a recent study carried out on embryos of this species, Aguilar et al. (2023) reported that Phe inhibits the activity of β-esterases, particularly AChE and CbE. Due to its high solubility in water, Phe is rapidly absorbed into lipid matrices, such as those that make up the yolk of octopus embryos. Although additional studies are needed to fully characterize the effects of these contaminants, their impact on yolk composition strongly suggests profound consequences for *O. maya* embryonic development.

Emerging contaminants such as human pharmaceuticals and personal-care products have received attention for their potentially harmful effects on embryonic health. Fluoxetine, a pharmaceutical compound is transferred to the egg capsule of *S. officinalis* and impacts embryonic brain maturation causing damage at neurochemical, neurodevelopmental and immunological levels (Bidel et al. 2016). On the other hand, benzophenone-3 (BP3), an active ingredient in sunscreen lotions and personal-care products that protects against the damaging effects of ultraviolet rays, does not affect *O. maya* embryonic development (Moreno-Ortiz et al. 2023). Evaluations of the embryos' antioxidant

system and the potential oxidative damage associated with the presence of the contaminant were not statistically different from those of control embryos (Moreno Ortíz et al. 2023). That study also observed that BP3 did not affect the activities of acetylcholinesterase (AChE) or carboxylesterase (CbE). AChE is expressed in several tissues, including the nervous system and muscle, and its activity is essential during early development (Behra et al. 2002). The absence of alterations in the antioxidant system and esterase activity by BP3 suggests that either embryos have powerful detoxification mechanisms or that the chorion is an extraordinary barrier that prevents these substances from entering the egg's interior and affecting the embryos. More studies are necessary to evaluate the effects of BP3 and to better understand the mechanisms involved in UV filters' toxicokinetic and toxicodynamic.

Brine discharge from seawater desalination may critically impact coastal biodiversity, as demonstrated by the complete mortality of *Ascarosepion apama* embryos at salinities greater than 50, suggesting particular vulnerability of cephalopod developmental stages (Dupavillon and Gillanders 2009).

## Fisheries and underwater noise

In fishing areas, cephalopods that use benthic substrates to attach their egg masses may be affected by different fishing gears. In littoral and coastal waters, cuttlefish and loliginids use fishery lines, trammel nets, traps and other gears as hard substrate to lay their eggs (see among others, Pham et al. 2009; Nichols et al. 2019; Ganias et al. 2021; Barrett et al. 2022). In some trap fisheries, fishermen attach cephalopod eggs inside fish traps to attract squid and cuttlefish (Fig. 2a). Octopuses use traps or ceramic pots as refuge to lay eggs (Braga et al. 2021; Fig. 2b, c) and most of these egg masses are lost if they are not promptly and properly returned to the sea along with the gear. In the case of octopus species, the brooding female must also be returned to ensure the maternal care required during incubation. The impact of bottom trawling on benthic egg masses has been rarely quantified (Roberts et al. 2012) and the effect of pelagic nets on oegopsid pelagic egg masses is unknown.
<figure><img src="image_23.png" /><figcaption>**Fig. 2** Fishery impact on cephalopod egg masses. **a** A fisherman attaches a *Sepioteuthis lessoniana* egg mass inside a fishing trap to attract squid. Ta-Pong beach, Changwat Rayong, Gulf of Thailand. Image: Jaruwat Nabhitabata. **b** Egg mass</figcaption></figure>

Underwater noise from vessels, fishing operations and other anthropogenic activities has been increasing during recent decades. Although studies in the wild are difficult to conduct, underwater noise can induce stress responses and negatively impact cephalopod embryos and hatchlings. For instance, the exposure of *S. officinalis* embryos to pile-driving and drilling sounds in the laboratory resulted in hatchlings showing normal size and behaviour but reduced hatching success and increased juvenile mortality (Solé et al. 2022). These authors suggested that cuttlefish eggs exposed to a combination of both pile driving and drilling sound within 500 m of the operations will experience a reduced hatching percentage.

### Ocean acidification and warming

The marine environment is absorbing the increasing atmospheric carbon dioxide ($CO_2$) emission from anthropogenic activities, leading to ocean acidification, which could have uncertain or negative implications for cephalopod eggs. Warming is expected to shorten embryonic developmental time and diverse effects of increasing $pCO_2$ have been observed under experimental conditions. In *Idiosepius pygmeus*, elevated $pCO_2$ (940 µatm) produced embryos with yolk reserves and hatchlings that were 14% and 5% smaller respectively, with an 8% egg swelling increase (as a mechanism to maximize gas exchange with seawater) compared with embryos at control conditions (Spady et al. 2020). Similar effects are described for *S. officinalis*, resulting in increased egg swelling, hatching delay, and decrease in hatching success observed

and brooding *Octopus vulgaris* female on board, after being captured by an octopus trap; and (c) inside an octopus pot, Vilanova i la Geltrú, NW Mediterranean. Images **b**, **c**: Oscar Escolar

under ocean acidification conditions (Dorey et al. 2012; Rosa et al. 2013; Minet et al. 2025), affecting metabolites profiles in embryos until a metabolic suppression is observed at a seawater pH of 7.43 (Minet et al. 2025). As a result of the gradient of gas partial pressures between biological compartments, encapsulated cuttlefish embryos grow in a hypoxic and hypercarbic perivitelline fluid when eggs are in ambient seawater conditions. Under these conditions, they are able to form an internal $CaCO_3$ shell under the acidic environment and high $pCO_2$ prevailing in their egg environment (Gutowska and Melzner 2009). Elevated seawater $pCO_2$ (i.e. 980 µatm) does not seem to significantly affect the cuttlebone weight/area ratio of hatchlings (Otjacques et al. 2020) but, Dorey et al. (2012) found that seawater $pCO_2$ at 770 and 1400 µatm led to an enhanced Ca incorporation in cuttlebones in *S. officinalis*. However, no effects of acidification (1000 µatm; $\Delta pH=0.4$) were observed on development time, hatching success, survival rate and biometric data of *S. officinalis* hatchlings (Moura et al. 2019).

Intertidal habitats, where cuttlefish spawning can occur, are also ecologically challenging environments with periodic fluctuations in physical characteristics like temperature and aerial exposure (emersion) during tidal cycles. Coelho et al. (2023) suggest that *S. officinalis* early life stages exhibit considerable tolerance to tidal emersion and marine heatwave (MHW) conditions. Yet, Court et al. (2022) showed that while combined warming (+3 °C) and acidification (980 µatm$CO_2$) severely impacted cuttlefish hatching success, key behaviours of the hatchlings (like exploration and activity) were unaffected. MHW conditions also disrupt
cephalopod-luminous bacteria symbiosis. More specifically, besides decreased hatching success and survival, the bioluminescent hatchlings of the Hawaiian bobtail squid (*Euprymna scolopes*) required more luminous bacteria (*Vibrio fischeri*) from the surrounding seawater for successful colonisation under MHW conditions. Furthermore, the capacity for *Crypt*-specific core microbiota crypt 3 formation in the embryo's light organ, which is crucial for enhancing resilience under stress, declined with MHW conditions (Otjacques et al. 2025). In comparison with cuttlefish, pre-hatchlings of the squid *Loligo vulgaris* appear to be more impacted by warming via metabolic suppression than cuttlefish embryos (Pimentel et al. 2012). In addition, under acidified conditions (pH=7.5) and summer warming (19 °C) *L. vulgaris* showed a decrease in the survival rates of summer embryos to 47% in comparison with controls, with survivals ranging from 92 to 96% under a present-day environment (Rosa et al. 2014). Similarly, *Doryteuthis pealeii* embryos exposed to highly acidified conditions exhibited reduced size and morphological abnormalities, including degraded statoliths (Zakroff et al. 2019; Zakroff and Mooney 2020). Moreover, long-term environmental warming stress resulted in increased mortality and embryonic malformations in *Sepioteuthis lessoniana* (Kuan et al. 2022).

Fewer studies focused on the expected effects of ocean warming on octopus embryos. An incubation temperature increase (+3 °C) resulted in decreased survival and hatchling size together with increased metabolic costs in *O. vulgaris* embryos from NE Atlantic that seems to surpass the optimal thermal window of the species (Repolho et al. 2014). Also, *Octopus berrima* embryos exposed to future projected summer temperature (25 °C) until hatching, significantly reduced levels of key eye proteins such as S-crystallin and retinol dehydrogenase, suggesting the embryonic octopuses had impaired vision at elevated temperature (Hua et al. 2024). In the same way, in future thermal scenarios with quickly increasing temperatures at spawning areas after a short winter, *Octopus maya* embryos may suffer irreversible changes in the oxidative system due to the lack of thermal stability and long-term costs of thermal stress, so, this factor is likely to negatively affect recruitment and population dynamics of the species (Caamal-Monsreal et al. 2016; Sánchez-García et al. 2017; Vargas-Abúndez et al. 2025). More recently, it

was reported that in *O. maya* females, quantities of reactive oxygen species (ROS) together with genetic material (yolk), and the antioxidant glutathione, form part of the "package" that the embryos will use for development (Ramos-Rodríguez et al. 2024). At high temperatures, the transfer of ROS from the mother increases, overloading the embryos and provoking deformities (Olivares et al. 2019; Dominguez-Castanedo et al. 2023). In addition, recent results suggest that the transgenerational deleterious effects observed in *O. maya* could have their origin in epigenetic bases (Galindo-Torres et al. 2024), suggesting that high temperature produced greater DNA methylation in *O. maya* females and embryos, reinforcing the hypothesis that exposure to high temperatures influences the phenotype of the offspring through an effect on the *O. maya* epigene.

## Ocean deoxygenation and hypoxia

Ocean deoxygenation and hypoxia have received far less attention than the other environmental variables of marine climate change, namely ocean warming and acidification (see previous section) (Sampaio et al. 2021; Borges et al. 2022). Oxygen loss is shrinking fundamental niches, particularly in coastal areas, yet documented repercussions on cephalopod early development and behaviour are limited. Yet, recently Court et al. (2024) showed that exposure to hypoxia (2 and 5 mg O₂ l⁻¹) during development yielded lower survival rates, delayed hatching, smaller body sizes and inhibited predatory (increased latency to attack prey) and anti-predator (camouflage) behaviours in the cuttlefish *Sepia officinalis*. Moreover, these authors also showed that acute and chronic exposure to low oxygen produced similar effects on hatchling cognition, namely in the inability to learn socially, increased open-field activity levels, but had no changes in thigmotaxis.

## Collection of cephalopod eggs

Eggs can be collected from the wild, spawned in the laboratory and obtained from in vitro fertilization.
## Collection of cephalopod eggs from the wild

In the water column, cephalopod eggs can be found free floating (e.g., oegopsid egg masses) or attached to the brooding female (pelagic octopods and few oegopsid species). At the sea bottom, eggs can be found attached to natural or artificial hard structures as in cuttlefish, sepiolids, loliginid squids and benthic octopods and also transported by the female in some species of benthic octopods.

## Collection of pelagic egg masses

Collection of oegopsid squid egg masses from open ocean waters is difficult due to their relative large size and gelatinous consistency (e.g., O’Shea et al. 2004; Roberts et al. 2011; Birk et al. 2017; Puneeta et al. 2017; Ringvold et al. 2021; Brown et al. 2022) usually resulting in the collection of a sample of the whole mass (Staaf et al. 2008; Roberts et al. 2011;

Ringvold et al. 2021). Opportunistic incubation of portions of an egg mass aboard a vessel (Young et al. 1985a, b) and in the laboratory (Miyahara et al. 2006) has been done successfully during a few days until hatching, using the same incubation temperature as the collection sites and filtered seawater. Pelagic octopod eggs are usually collected together with the brooding female. *Argonauta* females secrete a shell that protects the eggs laid inside and *A. hians* brooding females were collected by purse seine, maintained onboard in PVC tanks with aeration, transported to the laboratory, and survived for 30 days until hatching of paralarvae (Sukhsangchan and Nabhitabhata 2007). In addition, 3–4 *A. hians* brooding females were maintained in 500 L aerated tanks covered with a black plastic sheet in order to reduce light intensity and visual stimuli and maintained under 27–29 °C and 32–35 salinity, with 1/3 of the water volume changed daily (Sukhsangchan et al. 2008).

<figure><img src="image_24.png" /><figcaption>**Fig. 3** Collection of benthic egg masses from the wild. **a** *Sepia officinalis* egg mass on vegetal remains; and **b** on a marine rope. La Rochelle, Bay of Biscay, NE Atlantic. Images: Antoine Minet. **c** *Sepia officinalis* egg mass showing white eggs over an octopus trap. Vilanova i la Geltrú, NW Mediterranean. Image: Oscar Escolar. **d** *Loligo vulgaris* egg mass</figcaption></figure>

attached to a trammel fishing net; and **e** attached to an egg laying device including the metal frame, hemp ropes and floats. Thermaikos Gulf, Eastern Mediterranean. Images: Kostas Ganias. **f** *Loligo vulgaris* egg mass attached to an octopus fishing trap. Vilanova i la Geltrú, NW Mediterranean. Image: Oscar Escolar
## Collection of benthic egg masses

Collection of cephalopod eggs attached to natural bottom structures such as rocks, coral, sponges, shells, tube worms, algae, and to anthropogenic structures such as fishery gears and lines is relatively common in all the seas (Fig. 3). Natural substrates could include invasive microalgae (Storero and Pereyra 2022) and anthropogenic substrates including litter (Leite et al. 2021; Freitas et al. 2022; Rizzo et al. 2022; Capillo et al. 2024). They are collected by diverse methods, including: by hand, snorkelling, SCUBA, trawling and ROVs. When possible, abrasive methods such as trawling should be avoided due to the mechanical stress produced to the embryos and the environmental impact of this gear. In the intertidal zone, egg masses of sepiids (Rosa et al. 2015) and some species of octopus (Narvarte et al. 2013) can be collected by hand. In shallow waters, snorkelling and SCUBA diving are the common methods used to collect or observe egg masses of cuttlefishes (see, among others, Guerra et al. 2016), loliginids (e.g., Villa et al. 1997; Hanlon et al. 2002; Steer et al. 2002), and octopods (e.g., Ortiz et al. 2006; Ortiz and Re 2011; Garci et al. 2016; Freitas et al. 2022). Based on a historical series of papers that used scientific diving for cephalopod collection purposes, Pak and Leite (2025) indicated that 18% of them were used for egg collection. Also, in shallow waters egg masses can be collected from fishery devices and lines (see, among others, Barret et al. 2022 for sepiids; Pham et al. 2009; Guerra et al. 2001; Nichols et al. 2019 for loliginids; and Braga et al. 2021 for octopuses).

Artificial structures dedicated to the collection of cephalopod eggs have shown positive results in shallow depths for cuttlefishes (Blanc and Daguzan 1998; Melli et al. 2014; Grati et al. 2018), loliginids (Villa et al. 1997; Baron 2001; Cinti et al. 2004; Cabanellas-Reboredo et al. 2014; Feyjoo et al. 2016; Ganias et al. 2021) and octopuses (Zheng et al. 2014). Ganias et al. (2021) described an experimental egg laying device for *Loligo vulgaris* made up from the metal frame from a cuttlefish trap with multiple potential egg laying surfaces. These surfaces included a small netting frame, a three-dimensional web-like structure of elastic ropes (5 mm in diameter, 1 m in length) equipped with floats to maintain their vertical orientation in the water (Fig. 3e) and the majority of squid egg masses

were detected on hemp ropes and web-like elastic ropes. The device was used with less intensity by cuttlefish *Sepia officinalis* to lay eggs.

In coastal areas far from SCUBA access, bottom trawling has been used to collect sepiolid (Deickert and Bello 2005) and loliginid egg masses (Augustyn 1990; Laptikhovsky et al. 2021). Deep-sea (> 200 m depth) bottom trawl has been used to collect eggs of sepiolids (Cuccu et al. 2007, 2010; Okutani and Sasaki 2007), loliginids (Laptikhovsky et al. 2021) and cirrate octopods (Ziegler et al. 2021; Chen and Sigwart 2025) and deep-sea fishing traps also collected female octopods that later laid egg masses in aquaria (Khen et al. 2022). ROVs used in bathyal depths collect live, advanced incirrate (Voight and Drazen 2004) and cirrate (Shea et al. 2018) octopod embryos.

## Collection of cephalopod eggs in the laboratory

At the present, cephalopods egg masses in the laboratory are obtained by spontaneous spawning of a broodstock or by in vitro fertilization. In Sepiidae, it is recommended to provide captive females with mesh or lattice-like structures for egg laying (Fig. 4a). In some species of Sepiidae such as *Ascarosepion bandense*, egg laying can be manipulated temporally through the addition and removal of preferred egg laying substrates on a routine basis (Connor J. Gibbons, pers. comm). Unlike in fish species, where reproductive control is relatively well-established for some species (Mylonas et al. 2010; Rodrigues-Filho et al. 2023), cephalopod reproduction remains unmanaged in culture systems. This limitation has been recognized as a critical knowledge gap in cephalopod culture (Villanueva et al. 2014).

## Techniques to accelerate/decelerate spawning in the laboratory

Currently little is known on techniques to control spawning in the laboratory. In cephalopods, maturation of the gonad is determined by the optic glands and presumably, upon light (Wells and Wells 1959) and temperature (Juárez et al. 2015). Darkness has been used to accelerate spawning in octopus (Zúñiga et al. 1995; Rosas et al. 2014) and light to decelerate spawning in cuttlefish (Richard 1968, 1971). Further studies are necessary to determine suitable protocols
<figure><img src="image_25.png" /><figcaption>**Fig. 4** Collection of cephalopod egg masses from the laboratory. **a** *Acanthosepion pharaonis* eggs attached to artificial substrate (fishing net) collected from the broodstock tank before being transferred to the incubation tanks. **b** Egg capsules of *Euprymna hyllebergi* attached to the artificial substrate (PVC pipe) for collection in the broodstock tank. Scale, 1 cm. Images</figcaption></figure>

as well as deepen our understanding of the hormones and neuropeptides associated with reproduction (Juárez et al. 2022, 2025; Di Cristo 2013; Zatylny-Gaudin et al. 2016, among others). There are visual cues for certain species, for instance, *S. lessoniana* will lay eggs repeatedly on human-made fake egg strings in the laboratory (Bret Grasse, pers. comm.). Additionally, the presence of sex pheromones in the cuttlefish egg capsule suggests that eggs act as pheromone diffusers (Gaudin-Zatylny et al. 2022).

Parents' stock density is another poorly understood factor. In *Acanthosepion esculentum*, Guo et al. (2022) indicated that: "with an increasing proportion of males, the average fecundity of females and the participation rate of males in paternity decreased. With increasing stocking density, the females had lower average daily egg production, and the males had a high variance in reproductive success and decreased participation rate in spawning." Protocols to maintain the suitable sex ratio of the parents stock are needed. To obtain *Octopus maya* spawns, adults are maintained in external ponds in proportions of 2:1 males: females, at densities of 1 octopus m⁻². During 10 days, individuals copulate, ensuring that, independently of wild copulations, all females will have fresh sperm to fertilize their oocytes. However, in a study of paternity under laboratory conditions, it was observed that an average of 10 males contributed to a single spawn, and that one of those males came from the group of males maintained in the external

**a** and **b**, Rayong Coastal Aquaculture Research and Development Center, Changwat Rayong, Gulf of Thailand. Images: Jaruwat Nabhitabhata. **c** *Octopus maya* egg masses artificially incubated at the Laboratory of Applied Ecophysiology, National Autonomous University of Mexico, Sisal, Mexico. Image: Carlos Rosas

pond during the conditioning period (López-Galindo et al. 2019). Those results suggest that to ensure spawns in the laboratory, independently of wild mating, it is better to maintain groups of adult females and males to promote matings.

### Pre-spawning signs

Detecting signs of pre-spawning is useful for having appropriate materials and protocols ready for egg collection in the laboratory, however, they are difficult to identify due to individual variability. As most coastal cephalopods spawn at the end of their life cycle, cephalopod workers can tentatively expect to obtain egg masses in the laboratory according to the existing knowledge of a given species reproductive seasonality. Notable reduction or cessation in food intake and/or increased reclusive behaviour in females of benthic coastal octopuses, using available material to protect the entrance of the den is sometimes a sign of pre-spawning behaviour, suggesting that egg laying is approaching (Montero-Ruíz et al. 2023). Internal gonad development could be examined through the mantle cavity in live, anesthetized individuals, providing useful information. The use of endoscopes in *S. officinalis* provides useful information for sex determination and maturity degree (Sykes et al. 2017). Also, gonadal maturation in anesthetized female octopus has been recorded using ultrasound devices, showing the potential of this method to
estimate the proximity of the spawning (Berrueta et al. 2025; Márquez et al. 2025).

Egg collection from in vitro fertilization and gamete preservation techniques

While in vitro fertilization techniques have been successfully developed for squids, no published information are available for sepioids or octopods. Recent updates for in vitro fertilization techniques are available for oegopsid (Villanueva et al. 2011, 2012; Vijai et al. 2015a, b a, b) and myopsid (Crawford et al. 2020) squids. Future research on the chemical properties of the oviducal and nidamental glands will likely help to obtain adequate egg chorion expansion, increasing survival by reducing premature hatching and ensuring proper development and hatchling size. For example, an active and unidentified salt soluble component from the oviducal gland induces chorion expansion and hatching during in vitro fertilization in Oegopsida, and that component may be similar in Myopsida (Adachi et al. 2016).

Cephalopod oocytes and eggs are relatively large with voluminous yolk reserves and their size prevents cryopreservation due to ice crystal formation during the freezing process, as observed in fishes and other marine invertebrates (Diwan et al. 2020). However, cephalopod sperm cryopreservation is possible using cryoprotectants to prevent cell damage during freezing and thawing, although these substances are usually toxic. Spermatozoa of the broadtail shortfin squid *Illex coindetii* remain viable with best cryopreservation protocol as: sodium citrate-glycine solution as the diluent with 15% DMSO as the cryoprotectant (Robles et al. 2013). Cryopreservation of whole spermatophores in *Amphioctopus fangsiao* using the same diluent and cryoprotectant, obtained 5–10% of motile spermatozoa after thawing (Hu et al. 2025).

To improve cephalopod sperm cryopreservation, freezing free cephalopod spermatozoa and testing diverse cryoprotectants may be attempted in the future. On the other hand, spermatophores of the cuttlefish *Ascarosepion apama* maintained in filtered seawater at 4 °C showed spermatozoa motility after two months (Naud and Havenhand 2006), thus, refrigeration is an alternative method to preserve spermatotozoa in the medium term. Long and medium-term cephalopod sperm storage would be a valuable tool for in vitro fertilization, helping to select particular

traits under culture and decoupling laboratory experiments from seasonal and geographical constraints.

## Transport of cephalopod eggs

Eggs should be collected carefully, without harming them, avoiding mechanical impact and periods out of well oxygenated seawater as much as possible. Benthic eggs could be carefully collected by cutting the base of the structure they are attached to (e.g., seagrass, tube worms, fishing devices). Octopus egg manipulation should preferably be carried out from the cementing agent (i.e., the material secreted by the oviducal gland to attach the eggs to the substrate) or from the chorion stalk, in order to reduce contact with the chorion. Record of the main collection data as date, hour, location, temperature and depth will be useful to register the source and acclimation for transport and maintenance. Records of egg images and/or preserving a sample in a fixative will help for posterior identification of the embryonic developmental stage at the collection site. For advanced embryos, the use of anaesthetics should be considered prior to fixation (see Sect. "Ethical recommendations for cephalopod embryo experimentation in scientific research" for ethical recommendations).

When possible, temperature, light intensity and photoperiod during maintenance in onboard aquariums or during transport should be maintained similar to the collection site until reaching laboratories with facilities to progressively adapt the egg masses to different incubation regimes or experimental protocols. However, for long-term transportation, lowering temperature and light intensity are recommended in order to reduce metabolic rate. Appropriate levels of oxygen (over 6 ppm), ammonia (<0.1 ppm), nitrite (<0.1 ppm), nitrate (<20 ppm) and pH (between 7.8–8.3) must be assured along the transport period (Feyjoo et al. 2011; Vidal et al. 2014; Fiorito et al. 2015, among others).

Few published studies described or analysed in detail cephalopod egg transport methods. In cuttlefish eggs, Jones et al. (2009) tested four egg packing methods using plastic bags: dry, wrapped in paper towel dampened in seawater, immersed in seawater, and immersed in seawater plus aeration. The eggs were transported 15 h after collection and the study concluded that hatching success did not differ among the treatments. Less volume of transport water reduces
<figure><img src="image_26.png" /><figcaption>**Fig. 5** Transport of cephalopod eggs. **a** Eggs masses of *Loligo vulgaris* inside a plastic bag filled with seawater and oxygen, prepared to be sent for 24 h transport between laboratories. Note that egg masses are tied to a float to ensure they do not rest on the bottom when the bag is upright. Image: Roger Vil-</figcaption></figure>

lanueva. **b** Aluminium containers used to transport *Octopus maya* eggs from Yucatan, Mexico to the Vienna aquarium. **c** Embryos (stages X -XI) of *O. maya* after transport. Images: Carlos Rosas and Gaetano Lorenzo

transport costs so, damp and dry methods will lower
freight costs and also reduce the risk of introducing
invasive species from the transport seawater (Jones
et al. 2009). *Loligo* egg masses could be transported
successfully within 24 h using double plastic bags con-
taining 30—50% of seawater with the remaining space
filled with pure oxygen (Vidal and Boletzky 2014)
(Fig. 5a). Egg masses of *Octopus maya* were success-
fully transported for more than 30 h from Mexico to
the Vienna aquarium, with early embryos (in stage X)
placed in a 1.2 L aluminium containers filled with ster-
ilized seawater previously oxygen-saturated at 22 °C.
The containers were filled to the maximum with sea-
water in order to avoid aerial space that could provoke
vigorous agitation during transport (Fig. 5b, c).

Whenever possible, shipping eggs in advanced
developmental stages should be avoided, and those in

early or intermediate stages of development is prefer-
able. In pre-hatching embryos, the sudden movements
generated during transport can cause premature
hatching and the higher energy consumption of these
stages due to their greater metabolic activity must
also be taken into account.

Biotic and abiotic factors influencing embryonic
development

Biotic factors

Maternal condition

In octopuses, maternal diet can influence the egg
protein and fatty acid composition (Quintana et al.
2015; Tercero et al. 2015) and females’ mixed diets produced better hatchling quality compared with single diets in *Octopus maya* (Caamal-Monsreal et al. 2015). Maternal ration correlated positively with egg size in *Euprymna tasmanica* (Steer et al. 2004) and *O. vulgaris* (Márquez et al. 2013), however, no effect of feed variety (fish only vs. fish and crustaceans) was found in *Enteroctopus megalocyathus*, where lower feed quantities reduced fecundity without affecting egg biochemical profiles (Farias et al. 2011), suggesting that more studies are necessary to determine female diet on the offsprings. For maternal behaviour and senescence see also Sect. "Incubation of eggs with maternal care". Maternal stress also influenced egg number and quality. *S. officinalis* females in confined space and repeatedly removed from the water spawned fewer eggs, fewer of them hatched and were more likely to spawn unfertilized eggs (O’Brien et al. 2018).

## Organisms associated with eggs

Organisms associated with the eggs can influence egg survival. For instance, the protozoan *Liburna oophaga* and the parasitic copepod *Ikanecator primus* were detected to co-infect *Acanthosepion pharaonis* eggs, resulting in mortality of embryos by inducing deformity in cuttlefish eggs. Immersion in peracetic acid and H₂O₂ reduced parasitic load and improved survival and hatchling size (Zoral et al. 2024a, b).

Infestation by siliceous sponges that penetrated the chorion of *S. officinalis* eggs were reported by Sykes et al. (2019) under culture conditions, probably due to malfunction of the seawater filter system. Gastropod eggs have been observed attached to cuttlefish egg masses in the field (Fig. 6a), although their possible effect is unknown. On the other hand, egg capsules of *Doryteuthis opalescens* containing the polychaete *Capitella ovincola* showed slightly higher survival rate than those without, and stable isotope analysis indicated that *C. ovincola* eat the mucilaginous capsule, not the paralarvae (Zeidberg et al. 2011). These authors concluded that provision of nutrients and shelter for the annelid and increased survival of the squid embryos suggests a symbiotic relationship between them.

## Main abiotic factors

### Temperature

The incubation temperature and its effects on embryonic development and hatchling characteristics is probably one of the most tested variables during laboratory experiments using cephalopod embryos (see, among others, Uriarte et al. 2012, 2015; Caamal et al. 2016; Nande et al. 2018; Donlon et al. 2020; Jiang et al. 2020; Zeng et al. 2024). Within a species’ natural temperature range, warmer temperatures generally accelerate embryonic development

<figure><img src="image_27.png" /><figcaption>**Fig. 6** Biotic factors influencing cephalopod embryonic development. **a** Gastropod eggs (cf. *Tritia mutabilis*) attached to a *Sepia officinalis* egg mass. **b** The gastropod *Bolinus brandaris* eating a *Sepia officinalis* egg after being captured inside a cuttlefish trap, both images from Vilanova i la Geltrú, NW Mediterranean Sea. Images: Oscar Escolar</figcaption></figure>

<figure><img src="image_28.png" /><figcaption></figcaption></figure>
(Boletzky 1987). Embryos abnormalities and/or mortality are detected beyond minimum and maximum temperatures. For this reason, previous knowledge of the thermal regimes tolerated by the species in the wild and, in particular, of its spawning sites is essential (e.g., Oosthuizen and Roberts 2009). Early blastula, blastopore closure, and optic vesicle formation stages were the most sensitive stages during high incubation temperatures in *Sepiella japonica* embryos (Liu et al. 2023). Increasing temperature stimulates gonadotropin-releasing hormone (GnRH)-like peptides that may play an accelerating role in embryonic development, however, they are not involved in sex determination or early gonadal development in *Acanthosepion lycidas* (Murata et al. 2024). Also, thermal stress produces disruption of embryonic chromatophore patterns in *Loligo forbesii* (Gowland et al. 2003). *Octopus maya* embryos exposed to thermal stress disable genes involved mainly in the development of the nervous system, morphogenesis and regulation of genetic expression, showing body malformations and immature nervous system development (Galindo-Torres et al. 2024). In addition, temperature can probably modify the expression of genes of the antioxidant system, essential for proper embryonic development (Ramos-Rodríguez et al. 2024). Maternal stress also influenced egg production as thermally stressed *O. maya* females at warm regimes produced smaller eggs and embryos with lower yolk content, resulting in low survival, depressed routine resting and high metabolic rates of hatchlings (Domínguez-Castanedo et al. 2023; Vargas-Abúndez et al. 2023). Zheng et al. (2024) evaluated the effects of multiple temperature fluctuations on embryos of *Amphioctopus fangsiao*, providing new insights into the physiological changes and molecular responses of cephalopod embryos following dynamic temperature stress. Recent studies showed the thermal time model of embryonic development duration (Márquez et al. 2023) and incubation of eggs at relatively low temperatures and biochemical content in octopods (Uriarte et al. 2024).

## Salinity and light

Salinities in the ocean are relatively stable, however, fluctuations are frequent in shallow water cephalopod

breeding grounds (e.g., coastal areas, sheltered bays). Most cephalopods are considered stenohaline and their habitats have salinity ranges from 27 to 37 (Vidal et al. 2014). High salinities (40 and higher) resulted in death of the embryo and low salinities (20 and lower) caused abnormalities in neritic hatchlings (Nabhitabhata et al. 2001; Peng et al. 2017). In addition, warmer temperatures appear to improve salinity tolerance, particularly in low-salinity conditions (Vidal et al. 2014).

Effects of light in cephalopod embryonic development are less studied. No relationship between light intensity and duration of embryonic development was found in *Heterololigo bleekeri* (Ikeda et al. 2004), however, abnormal development and premature hatching were observed in *Acanthosepion lycidas* incubated under high light intensities exceeding 10.2 µmol/(m²·s) (Peng et al. 2019a). Green light improves both hatching and survival rates in *Acanthosepion pharaonis*, in contrast, red and blue light lead to abnormal embryonic development (Huang et al. 2024) and constant light or darkness photoperiods also negatively affect its embryonic development (Jiang et al. 2022). The egg capsule could offer extra protection in some cephalopods, as for instance, for the cuttlefish *Sepia officinalis* could ensure successful embryonic development even at high doses of UV radiation (Molina-Carrillo et al. 2023). In addition, high light intensities can cause growth of algae and fungi on the surface of egg capsules in the laboratory, drastically reducing or even blocking oxygen supply to the embryos (Nabhitabhata and Segawa 2014).

## Oxygen, pH and ammonia

Egg masses should be maintained in well-oxygenated conditions, with levels near saturation. Oxygen levels decrease and pH drops significantly within developing loliginid egg capsules, often reaching metabolically stressful levels even for adults (Long et al. 2016). Embryos located in the interior of large egg masses experience low oxygen conditions, resulting in slower development and reduced survival compared to peripheral embryos. This explains why embryos from smaller egg masses develop faster than those from larger egg masses (Steer et al. 2003; Steer and Moltschaniwskyj 2007). The oxygen limitation appears particularly critical during late embryogenesis, when embryonic oxygen demand and ammonia
production peak, potentially triggering synchronous hatching (Vidal et al. 2014). Embryo rotation and movements inside the chorion probably enhances oxygen uptake and the maintenance of a balanced microenvironment in the perivitelline fluid, particularly in large gelatinous egg masses as in ommastrephids, with some embryos located far from the egg mass surface (Puneeta et al. 2018). Studies demonstrate that the oxygen consumption of *Octopus vulgaris* pre-hatching egg mass averages double that of an adult of average weight size (Parra et al. 2000). Therefore, for laboratory maintenance of a brooding female with late-stage eggs, it is important to consider that an oxygen supply three times greater than that required for a medium-sized octopus is required. Few data exist on the effects of ammonia in cephalopod embryos. Exposure to high concentrations of ammonia (>1 mg L⁻¹) causes multiple malformations and death in *Acanthosepion pharaonis* embryos (Peng et al. 2019b).

## Biochemical characterization of eggs

There is scarce information on the biochemical profiles of cephalopod eggs. During embryonic development, protein is the major component in the yolk of *Acanthosepion esculentum* (Lei et al. 2014). Essential and non-essential amino acid content decreased from mature ovary to eggs, except for phenylalanine, and then all of them decrease from eggs to hatchlings of *O. vulgaris* (Villanueva et al. 2004), a decreasing trend was also observed in cuttlefish *A. esculentum* (Lei et al. 2014). The chorion and the egg thread appear to be formed during a single continuous process and are biochemically uniform. Egg threads of *O. bimaculoides* appear to be made of a single protein, *octovafibrin*, that has 29 repeat Epidermal Growth Factor EGF-like domains flanked on either side by lectin binding domains (Wonderly et al. 2023).

Lipid and fatty acid content in cuttlefish *S. officinalis* eggs increased during embryonic development and lipid profiles showed predominant levels of phosphatidylcholine, phosphatidylethanolamine, cholesterol and triacylglycerol at the end of embryonic development (Sykes et al. 2009). Antioxidants such as vitamin E are regarded to be important for the prevention of lipid oxidation and are related to the high abundance of fatty acids in cephalopod eggs (Villanueva et al. 2009; Farias et al. 2011; Uriarte

et al. 2014; Estefanell et al. 2017). Vitamin C levels decrease throughout the embryonic development, whereas vitamin E content remains stable in *Enteroc-topus megalocyathus* (Hernández et al. 2019). Under hypo-osmotic stress, the same authors reported that vitamin C content was reduced and vitamin E levels did not vary. In *Octopus maya* embryos obtained from females fed with different mixed diets, it was observed that fatty acids as palmitic acid, linoleic acid, linolenic acid, and DHA were particularly high in the yolk of eggs, key fatty acids to embryo development (Caamal-Monsreal et al. 2015). Also, the most abundant essential amino acids in the yolk of those embryos were Arg, Lys and Leu, being, Lys the basis of carnitine synthesis and used in fatty acid transportation, while Leu (ketogenic amino acid) and Ile (glucogenic and energetic) play fundamental roles in embryonic development of *O. maya* (Caamal-Monsreal et al. 2015).

## Housing and care of eggs in the laboratory

### Incubation methods for eggs without maternal care

Nautiluses, cuttlefishes, bobtail squids, cirrate octopods and most squid eggs are naturally incubated without maternal care (see Fig. 7 for examples), in addition, artificial incubation methods also exist for octopuses. As a general rule, the use of the highest quality of filtered and oxygenated seawater available will ensure the best embryo quality and high hatching success. Egg masses should have the best possible ventilation to avoid anoxic zones in the egg mass that can cause fungal growth infections and mortality. This ventilation should be achieved with gentle water flows or artificial aeration between the eggs, that will make them move gently while avoiding excessive turbulence. Excessive turbulence can weaken the capsule and/or chorion membrane, causing direct damage and/or slow degradation. Preserving the integrity of the egg membranes is paramount in mitigating infections and premature hatching events.

Separating eggs from the dense egg masses in early stages of development can also improve individual oxygenation and growth. For incubation techniques, see among others, examples for nautiluses (AZA 2025), cuttlefishes (Sykes et al. 2006; Nabhitabhata 2014b, c; Montague et al. 2021), sepiolids
<figure><img src="image_29.png" /><figcaption>**Fig. 7** Incubation methods for cephalopod eggs without maternal care. **a** Incubation technique for eggs of *Ascarosepion pfefferi*, using plastic bottles (see text for details). Marine Biological Laboratory, Woods Hole. Image: Bret Grasse. **b** *Sepia officinalis* eggs incubated in polyethylene bowl-shaped containers at Institut de Ciències del Mar, Barcelona, Spain. Note some white eggs on the left bowls. Image: Roger Villanueva. **c** *Loligo vulgaris* incubated eggs hanging from a polyethylene float; to hold the egg mass in place, a nylon fishing line is threaded through its mucilaginous mass. Institut de Ciències del Mar, Barcelona, Spain. Image: Roger Villanueva. **d** *Sepio-teuthis lessoniana* egg masses maintained in baskets, floating</figcaption></figure>

in aerated seawater inside the incubation tank (150 cm diameter). Rayong Coastal Aquaculture Research and Development Center, Changwat Rayong, Gulf of Thailand. Image: Jaruwat Nabhitabhata. **e** *Octopus tehuelchus* egg strings maintained in cylindrical polyethylene containers suspended in a seawater recirculation system. Instituto de Biología de Organismos Marinos, Consejo Nacional de Investigaciones Científicas y Técnicas, Puerto Madryn, Argentina. Image: Nicolás Ortiz. **f** *Octopus vulgaris* egg strings in an open flow Kreisel tank. See also video S1. Centro Oceanográfico de Canarias (IEO, CSIC), Tenerife, Spain. Image: Eduardo Almansa

(Nabhitabhata and Nishiguchi 2014), ommastrephids (Puneeta et al. 2015) and loliginid squids (Nabhitabhata and Ikeda 2014; Vidal and Boletzky 2014). An incubation technique for eggs of *Ascarosepion pfefferi*, appropriate for cephalopod eggs that require higher flow, such as *A. pfefferi* is used in the Marine Biological Laboratory (Bret Grasse, pers. comm.) (see Fig. 7a). This model depicts two plastic bottles adhered together with silicon. The lower bottle cap is modified by drilling a large hole in the center and 3 mm polyurethane mesh is adhered with silicon over the bottle cap hole of the lower cap and between the two bottles. Flow is generated through an air stone placed in the upper bottle. Air exiting the upper bottle pulls seawater with it, which is replaced with

incoming seawater through the lower cap, thus driving incubation flow. This design provides gentle, consistent flow with limited egg rubbing and interaction with the interior walls.

In octopuses, egg strings separated from the brooding female were incubated with success using water flows for small-egged octopus species such as *Octopus vulgaris* (Deryckere et al. 2020) and *O. tetricus* (Joll 1976; Spreitzenbarth and Jeffs 2020) (Video S1). For large-egged octopuses such as *O. maya*, a useful system for egg collection and subsequent incubation was described by Rosas et al. (2014) (see Fig. 1p) and other simple artificial incubation methods has been used for *O. tehuelchus* (Braga et al. 2021), *Robsonella fontaniana* (Ortiz
and Ré 2011), *Octopus hubbsorum* (Alejo-Plata and Herrera-Alejo 2014) and *Amphioctopus fangsiao* (Jiang et al. 2020).

Incubation of eggs with maternal care

Only octopuses, aside from a few oegopsid squids, provide egg maternal care. After completing egg laying in a portable refuge, it is recommended to remove the female octopus and her egg mass from the broodstock tank. Isolation of the female in a tank will improve supervision and handling, prevent potential negative interactions with other individuals, and facilitate future harvesting of hatchlings. In addition, the fact that octopus females stop eating after laying eggs greatly improves the tank water quality when they are

isolated from the parental stock. Maternal health is a critical factor along the development of the octopus embryos. In *O. maya*, despite physiological changes, significant weight loss and depletion of energy reserves as a consequence of starvation, females maintained their immune functions and adequate health status to care for their offspring (Roumedakis et al. 2018). However, the female senescence process makes female octopuses more vulnerable as the incubation period progresses (Wang and Ragsdale 2018; Holst et al. 2022). When brooding (see Fig. 8), female octopuses continuously clean and ventilate the eggs with their suckers and water flushes, protecting eggs from potential predators and/or human presence in the laboratory. They also spread their own skin mucus over the eggs. The protective effects of

<figure><img src="image_30.png" /><figcaption>**Fig. 8** Incubation methods for eggs with maternal care. **a** *Octopus vulgaris* advanced egg mass and brooding female inside a ceramic pot, at the Institut de Ciències del Mar, Barcelona, Spain. Image: Roger Villanueva. **b** *Octopus maya* egg mass and brooding female inside a PVC tube. Laboratory of Applied Ecophysiology, National Autonomous University of Mexico, Sisal, Mexico. Image: Katina Roumedakis. **c** *Robsonella huttoni* egg strings and brooding female in a glass jar. University of Otago, New Zealand. Image: Erica Donlon Durante. **d** *Octopus berrima* eggs and brooding female inside</figcaption></figure>

a PVC tube. University of South Australia, Australia. Image: Erica Donlon Durante. **e** *Octopus tehuelchus* eggs and brooding female inside a PVC tube. Instituto de Biología de Organismos Marinos—Consejo Nacional de Investigaciones Científicas y Técnicas, Puerto Madryn, Argentina. Image: Nicolás Ortiz. **f** *Enteroctopus megalocyathus* brooding female with egg strings laid along the inner transparent wall of the aquarium. Instituto de Biología de Organismos Marinos—Consejo Nacional de Investigaciones Científicas y Técnicas, Puerto Madryn, Argentina. Image: Nicolás Ortiz.
this mucus remain poorly understood, but antifungal and antibacterial effects on the eggs can be suspected due to the presence of bioactive peptides (Maselli et al. 2020; Pérez-Polo et al. 2023, 2024; Vizcaino et al. 2023) and antimicrobial activity reported from the octopus skin mucus (Savin-Amador et al. 2024) that shows the importance of maternal care and contact with the eggs. On the other hand, in some cases, octopus females themselves could be highly responsible for the loss of eggs, detaching egg strings (Uriarte et al. 2014). Brooding female cannibalistic behaviour toward eggs has been observed under captive conditions in *Octopus cherchiae* (Rodaniche 1984), *Paroctopus digueti* (García-Flores et al. 2022), *O. tehuelchus* (Storero and Narvarte 2024), *O. parvus* (Gleadall et al. 2024), *Enteroctopus megalocyathus* (Ortiz et al. 2024) and *Robsonella huttoni* (Erica Donlon Durante, pers. comm.). The mechanical stimulation on the eggs provided by the brooding female may differ between nocturnal and diurnal octopus species, making maternal activity an unquantified factor that perhaps mechanically stimulates the hatching process.

## Egg pathologies

Eggs have their own, rich microbial community, more diverse in healthy eggs. For instance, the diversity of bacterial community of recently spawned *O. mimus* eggs is higher (with *Roseobacter* and *Bacteroidetes* as predominant clades), while the predominant species in the infected eggs was g-proteobacteria (*Pseudoalteromonas*, *Shewanella*, *Vibrio*) which may suggest being pathogenic candidates (Iehata et al. 2016; Hengst et al. 2025). Reported bacteria found in infected eggs of *E. megalocyathus* after 15 days of spawning were: *Neptunomonas naphthovorans*, *Pseudomonas fulva*, *Pseudoalteromonas atlantica*, *Sulfitobacter donghicola*, and *Sulfitobacter mediterraneus* (Uriarte et al. 2011). In *Euprymna scolopes* it has been pointed out that a bacterial consortium exists in the accessory nidamental glands of the female that then are transferred to the eggs where they have been hypothesized to help protecting them from fouling microorganisms and pathogens (Collins et al. 2012), with differential antimicrobial activity against a number of marine vibrios (Gromek et al. 2016). Regular removal of dead embryos from egg masses can prevent build-up of microbes and aquarium pests that

feed on or reproduce in dead eggs and help increase survival rates of remaining developing embryos.

There is little information concerning treatments for fungal and bacterial pathogen infections. However, proteins with antimicrobial activity on Gram-bacteria have been identified in the outer egg case of *Sepia officinalis* (Cornet et al. 2015). It has been proposed to ensure the survival of the species by providing physical and chemical protection to the embryos in the absence of maternal care. Sykes et al. (2019) reported mycelia covering the chorion of *Octopus insularis* and *O. vulgaris*. However, it is not unusual to detect mycelia over the eggs of *O. vulgaris* even under maternal care (Eduardo Almansa, pers. comm.) (Fig. 9), then seems not to have the same deleterious effects observed in cuttlefish eggs (Sykes et al. 2019). A diluted sodium hypochlorite (NaOCl) solution has been used as egg disinfectant for *O. insularis* (Maldonado et al. 2019) and NaOCl at 0.004% used also as egg disinfectant has a species-specific effect on early hatchlings as small-egg *O. sinensis* seems more sensitive that large-egg *A. fangsiao* (Spreitzenbarth et al. 2024). At the Marine Biological Laboratory, Woods Hole, two products has been used successfully to reduce microorganism proliferation on cephalopod eggs, povidone-iodine and ReVive Coral Cleaner® in form of immersion baths (Bret Grasse, pers. comm.). For ReVive Coral Cleaner®, successful results have been obtained for *Acanthosepion pharaonis*, *Ascarosepion bandense*, *Ascarosepion pfefferi*, and *Octopus bimaculoides*. For use, mixed 40 ml of ReVive Coral Cleaner® per 3.8 L of seawater and immerse cephalopod eggs, for no more than 10 min. Benefits are increased by gently moving the solution over the eggs using a baster, air stone, or by slowly moving the eggs up and down with a net. The treatment could be repeated weekly. *Euprymna* spp. embryos do not react well to this product and its use can lead to embryo mortality.

## Ethical recommendations for cephalopod embryo experimentation in scientific research

The rich and diverse sensory world of cephalopods makes them sentient animals capable of experiencing pain, suffering or distress, and therefore there are ethical recommendations available for their proper handling under scientific research (see, among
<figure><img src="image_31.png" /><figcaption>**Fig. 9** Cephalopod egg pathologies. **a** *Octopus vulgaris* fungus-infected eggs with the mycelia reaching the embryo; **b** or on the chorion only, obtained by stereo microscope (4x); **c** and others, Fiorito et al. 2014, 2015; Ponte et al. 2023).</figcaption></figure>

Regulations on animal welfare for research purposes vary across countries. For example, in the European Union, the welfare of cephalopods used for scientific purposes is regulated by the Directive 2010/63/ EU (and its Annex III, as amended by Commission Delegated Directive EU 2024/1262). Other countries such as Australia, Canada, New Zealand, Norway, Switzerland and the United Kingdom adopted similar frameworks for cephalopods within their ethical oversight. All of these regulations apply to cephalopods from hatching, excluding embryos. However, as scientific research increases regarding cephalopod embryonic development, there is growing discussions about whether and how ethical considerations should be extended to embryos.

**d** Fungal mycelia on the chorion of an *O. vulgaris* infected egg obtained by scanning electron microscopy. Centro Oceanográfico de Canarias (IEO-CSIC), Spain. Images: Eduardo Almansa

Sensory and cognitive development of cephalopod embryos.

Refined embryonic neural development in cephalopods (see, among others, Shigeno et al. 2001; Yamamoto et al. 2003; Deryckere et al. 2021; Elagoz et al. 2022, 2024; Styfhals et al. 2022; Zolotarov et al. 2022) suggests a complex sensitive world in embryos. Current knowledge of the sensory and cognitive sophistication of embryos is largely based on studies conducted in cuttlefish. *Sepia officinalis* embryos develop tactile, chemosensory, and visual systems sequentially. Tactile and chemical sensitivities emerge earlier, with embryos responding to touch and chemical cues such as predator odours around stage 23 of the embryonic development. Visual system functionality appears later, around stage 25 of embryonic development,
approximately three weeks before hatching (Romagny et al. 2012). In *Acanthosepion pharaonis*, vision becomes functional around stage 22, before the chemosensory system at stage 23 (Mezrai et al. 2019). Amazingly, *S. officinalis* develops in dark eggs, whereas *A. pharaonis* in whitish translucent ones. Whether this is the reason for such a difference between the two species is not known; further studies in other white egg species are needed. Regardless of such developmental differences, in the late stages, embryos can perceive chemical and visual stimuli, which is crucial for post-hatching behaviours like prey recognition and predator avoidance. This complex sensory—nervous system allows cuttlefish embryos to learn a week before hatching. Research has demonstrated that cuttlefish embryos are capable of learning through exposure to environmental stimuli. For instance, embryos exposed to specific prey items, such as crabs, for at least a week before hatching showed a subsequent visual preference for those prey types after hatching (Darmaillacq et al. 2008). Additionally, studies have shown that cuttlefish embryos can undergo habituation to a repeated light stimulus. It led to decreased behavioural responses in embryos at stage 30, suggesting the presence of learning mechanisms even before hatching. Learning is also involved in predator recognition. Mezrai et al. (2020) showed that cuttlefish embryos exhibit both innate and learned responses to predatory threats. When exposed to chemical and visual cues from predators, embryos showed a significant increase in ventilation rate. This response was not observed when embryos were exposed to non-predatory species, indicating an innate ability to differentiate between harmful and harmless stimuli.

In addition, embryonic exposure to visual and olfactory predator cues altered cuttlefish's postnatal visual lateralisation and primary defensive behaviours (Jozet-Alves and Hébert 2013; Lee et al. 2020). The ability of cuttlefish embryos to learn and adapt based on sensory experiences has significant implications for their survival. This capacity has so far not been documented in octopuses (Portela et al. 2014; Hua et al. 2023). Embryonic learning allows for early adaptation to environmental conditions, enabling hatchlings to recognize and respond appropriately to prey and predators. This early learning capacity underscores the cognitive complexity of cephalopods

and highlights the importance of prenatal experiences in shaping behaviour. It also has implications in the frame of conservation and this alerts us to the potential risks of disrupting the development of the nervous and sensory systems on the survival of hatchlings. Given the sensory capabilities exhibited by embryos prior to hatching, should be considered to investigate whether housing conditions with sensory enrichment (visual, olfactory, auditory stimuli, or the presence of potential moving prey) could be beneficial—potentially enhancing their well-being and making them more capable of adapting to their environment after hatching. As mentioned above, most of these studies have been conducted with cuttlefish, specifically with *Sepia officinalis*. Further research is urgently needed to explore these learning and sensory embryonic abilities in other groups of cephalopods.

Developmental stage at which it is expected that embryos may first experience pain, distress, suffering and lasting harm

As the embryo grows, it can be assumed that its sensory and neural capacity also develop. The developmental stage at which cephalopod embryos are capable of perceiving pain, distress, suffering and lasting harm is unknown, because the presence of a functioning nociceptive system and conscious awareness in embryos has not been empirically demonstrated. We hypothesize that during organogenesis, embryos may possess the neuroanatomical substrate necessary for nociceptive processing once the central nervous system becomes functionally active, sensory structures are morphologically differentiated, and coordinated motor responses are observable. From this point onwards, it could be assumed that cephalopod embryos have a sensory nervous system able to perceive pain. Due to the uncertainty and lack of extensive scientific knowledge, we recommended applying the precautionary principle and ethical considerations for cephalopod embryos after the start of the organogenesis period. This involves minimizing invasive or stressful procedures, use of anaesthetics and humane endpoints where appropriate, and justifying the use of advanced embryos with rigorous harm-benefit analyses. This period of embryo development will further be referred to as advanced embryo.
Potential indicators of discomfort and suffering in advanced embryos and hatchlings

An adequate control of chemical/physical param-
eters of the water quality, cleanliness of the rearing
tank, thermal, salinity, light and oxygen conditions
within the species ranges and suitable embryo den-
sity are key aspects to maintain during the incubation
period to ensure the best embryo development (see
Sect. "Biotic and abiotic factors influencing embry-
onic development"). Embryos should be monitored
for parameters that could indicate their health and
welfare and allow the early detection of any devia-
tions from optimal status and indicate discomfort and/
or suffering. Here, we propose a list of potential indi-
cators to monitor the health and welfare of advanced
cephalopod embryos and hatchlings, based on their
appearance, behaviour and clinical signs, listed in
Tables 2 and 3. The feasibility of monitoring these
welfare indicators in embryos, given their small size,
opaque egg capsules in some species, and limited
behavioural visibility, may present a challenge, par-
ticularly in high-throughput or field-based research.
Likewise, laboratory observations of embryos under
a binocular microscope must be carried out avoid-
ing abrupt changes in light and temperature that can
affect the embryos.

The principles of the 3Rs (Replacement, Reduction and Refinement) and its application to cephalopod advanced embryos and hatchlings

When studying live animals for research, minimizing animal use is a fundamental ethical consideration. This can be achieved by adhering to the 3Rs principle, initially proposed by Russell and Burch (1959), which ensures that research quality is maintained while prioritizing animal welfare. We recommend the use of these three key principles adapted to cephalopod advanced embryos and hatchlings, summarized as follows:

(a) *Replacement* Whenever possible, procedures that may cause potential pain, suffering, distress or lasting harm should be avoided and researchers should replace cephalopod embryos and hatchlings with alternative materials and/or methods, such as in vitro techniques, to achieve the same scientific results. *Example:* development of cell-

based assays as have been described in Nesher
et al. (2019) or Maselli et al. (2018).

(b) *Reduction* The number of cephalopod embryos and hatchlings to be used in research should be kept to a minimum, without compromising scientific outcomes and robust statistical analysis. Experimental protocols, including pilot studies, must be properly designed in advance to avoid unnecessary sampling repetitions. In addition, researchers should consider possibilities of sharing embryos, tissue samples or organs with other research laboratories that can further reduce the need for additional animals, and the use of non-invasive monitoring methods. *Examples:* when working from egg masses spawned in laboratory, incubating only a selected number of embryos expected to be used during planned experiments allows for early discarding of the remaining eggs at the beginning of their development, thus, avoiding future euthanasia of many advanced embryos and hatchlings that will not be used for research. These expected discarded embryos could be selected before they reach the organogenesis stage. In addition, artificial incubation techniques make it possible to collect only a limited number of cuttlefish, squid or octopus eggs from the wild, rather than removing the entire egg mass, thus reducing the impact on natural populations.

(c) *Refinement* Experimental procedures and study designs must be optimized to minimize pain, suffering, distress, or lasting harm to the cephalopod embryos and hatchlings involved in research procedures. It is essential to maintain optimal conditions according to the species needs to support healthy development of the embryos and adequate hatching. Continuous monitoring of health and welfare parameters (see Sect. "Potential indicators of discomfort and suffering in advanced embryos and hatchlings") can help to identify unfavourable conditions and inform early interventions, if needed. The use of humane endpoints, such as terminating the experiment if abnormal development or high mortality occurs, should be considered. *Examples:* the use of anaesthetics, whenever needed, and the use of gentle handling and minimal physical disturbance.
<table><caption>**Table 2** Potential indicators of health and welfare that could be used for assessing and monitoring cephalopod embryos. These indicators attempt to be as general as possible and applicable to a wide spectrum of embryos of different taxa, thus, specific indicators should be defined in the future for each specific genus/family. Adapted from Fiorito et al. 2015</caption><thead><tr><th>Categories</th><th>Types of sign</th><th>Positive welfare status<br>No concerns; routine<br>monitoring</th><th>Minor deviations in welfare<br>status<br>Monitoring of eggs with<br>increased frequency;<br>additional checks of water<br>quality</th><th>Signs of stress or abnor-<br>malities<br>Monitor for signs of resolu-<br>tion or increased severity.<br>Intervention/treatment may<br>be needed</th><th>Significant welfare compro-<br>mise<br>Immediate action required<br>(intervention or humane<br>endpoint)</th></tr></thead><tbody><tr><td rowspan="4">Appearance (Physical state)</td><td>Growth rate, development,<br>survival rates</td><td>Normal growth rate and<br>development aligned<br>with the thermal regime;<br>low or no mortality</td><td>Slight delays in growth rate<br>and development; isolated<br>mortality events</td><td>Moderate delays in growth<br>rates, frequent develop-<br>ment anomalies, increased<br>mortality; some prema-<br>ture hatching</td><td>Abrupt growth rate and<br>development interruption;<br>Mortality significantly<br>increases; mass premature<br>hatching</td></tr><tr><td>Chorion colour and shape</td><td>Intact, transparent chorion;<br>regular egg shape</td><td>Minor chorion deformities<br>in some eggs</td><td>Moderate chorion deformi-<br>ties; translucent chorion</td><td>White chorion or the chorion<br>appears opaque; chorion<br>rupture; severely deformed<br>eggs</td></tr><tr><td>Embryo morphology and<br>pigmentation</td><td>Symmetrical body;<br>expected chromatophore<br>pattern according to the<br>developmental stage</td><td>Mild body asymmetry;<br>chromatophore irregulari-<br>ties; small embryo rela-<br>tive to the expected size<br>at the current embryonic<br>developmental stage</td><td>Moderate body asymmetry;<br>chromatophore irregulari-<br>ties; clearly underdevel-<br>oped embryo</td><td>Underdeveloped embryo and<br>major deformities; abnor-<br>mal chromatophore pat-<br>terns; areas of permanently<br>contracted or expanded<br>chromatophores, mantle<br>detached, eye dimorphism,<br>inhibited arm development</td></tr><tr><td>External yolk morphology</td><td>Uniform yolk morphology</td><td>Mild yolk asymmetry</td><td>Fragmented, fissured,<br>pinched or convoluted<br>yolk</td><td>Severe yolk irregularities</td></tr><tr><td rowspan="2">Behaviour (Psychological<br>state)</td><td>Movement patterns; rota-<br>tion and inversion</td><td>Regular body contractions,<br>rotations and inversion<br>(secondary inversion in<br>some species)</td><td>Slightly irregular contrac-<br>tions and rotations</td><td>Reduced or intermittent<br>body movements and<br>rotations (no embryo sec-<br>ondary inversion reached<br>in some species)</td><td>No body movements;<br>spasms; inking inside the<br>egg</td></tr><tr><td>Clinical signs (Physiologi-<br>cal state)</td><td>Ventilation rate<br>Pathogens</td><td>Normal ventilation rates<br>according to the species<br>and temperature<br>Absence of pathogens,<br>clean eggs</td><td>Small ventilation changes<br>(increase or decrease),<br>but remain coordinated<br>Low pathogen presence,<br>such as a few mycelia in<br>the eggs' surface</td><td>Moderate ventilation<br>changes (increase or<br>decrease)<br>Increase in the pathogens<br>presence such as mycelia<br>or bacteria in the eggs'<br>surface; small organisms<br>(like worms) around the<br>eggs; dirty environment</td><td>Major changes in ventilation<br>changes; uncoordinated<br>ventilation<br>Pathogens reach the embryos<br>causing death in a sig-<br>nificant percentage; opaque<br>eggs in species with trans-<br>parent chorion</td></tr></tbody></table>

<figure><img src="image_32.png" /></figure>
<table><caption>**Table 3** Potential indicators of health and welfare that could be used for assessing and monitoring cephalopod hatchlings. These indicators attempt to be as general as possible and applicable to a wide spectrum of hatchlings of different taxa, thus, specific indicators should be defined in the future for each specific genus/family. Adapted from Fiorito et al. 2015</caption><thead><tr><th>Categories</th><th>Types of sign</th><th>Positive welfare status<br>No concerns; routine monitoring</th><th>Minor deviations in welfare status<br>Monitoring of hatchlings with increased frequency; additional checks of water quality</th><th>Signs of stress or abnormalities<br>Monitor for signs of resolution or increased severity.<br>Intervention/treatment may be needed</th><th>Significant welfare compromise<br>Immediate action required (intervention or humane endpoint)</th></tr></thead><tbody><tr><td rowspan="4">Appearance (Physical state)</td><td>Skin colour and integrity</td><td>Normal skin colour pattern; skin and chromatophores intact, with no visible damage, over the entire body</td><td>Occasional abnormal skin colour changes; small skin damage on body, arms and/or fins</td><td>Frequent abnormal skin colour displays; uncoordinated colour patterns; evident skin damages</td><td>Pale; areas of permanently contracted or expanded chromatophores, severe skin damage</td></tr><tr><td>Body morphology and pigmentation</td><td>Normal body morphology and chromatophore pattern</td><td>Small body size and chromatophore irregularities</td><td>Moderate body asymmetry, uneven arm lengths, missing suckers</td><td>Severe deformities in the mantle, fins and/or arms; mantle detached</td></tr><tr><td>Yolk absorption</td><td>Absence or minimal outer yolk sac at hatching</td><td>Slight yolk retention; presence of small outer yolk sac</td><td>Moderate yolk retention; presence of large outer yolk sac</td><td>Large outer yolk sac, limiting mobility/swimming and/or ventilation</td></tr><tr><td>Swimming ability</td><td>Stable, coordinated swimming; good buoyancy; normal orientation and stability in the water column</td><td>Occasional disorientation in the water column; occasional uncoordinated swimming</td><td>Unstable swimming; repeated contact with tank's objects or walls</td><td>Inability to swim; erratic movements or complete immobility on the tank bottom</td></tr><tr><td rowspan="4">Behaviour (Psychological state)</td><td>Feeding at hatching or few days later (reactivity towards prey)</td><td>Low latency in detecting prey and capturing food at hatching or few days after hatching</td><td>No detection of prey few days after hatching; high latency to capture food</td><td>High latency to capture food; possible release food partially or completely without eating it few days after hatching</td><td>No signs of prey detection or prey capture within few days after hatching</td></tr><tr><td>Ventilation rate</td><td>Normal ventilation rate according to the species and temperature</td><td>Small ventilation changes (increased or decreased), but remain coordinated</td><td>Moderate ventilation changes; periods of tachypnea/apnea/dyspnea/hyperpnea</td><td>Major changes in ventilation rate; frequent periods of tachypnea/apnea/dyspnea/hyperpnea</td></tr><tr><td>Stress response and general condition</td><td>Responsive to stimuli; normal environment interaction</td><td>Slight withdrawal or delayed response to stimuli</td><td>Lethargic; reduced interaction, or apparent hyperactive behaviour; inking</td><td>Persistent inking in the absence of an overt stimulus</td></tr></tbody></table>

<figure><img src="image_33.png" /></figure>
## Anaesthetics for advanced cephalopod embryos and hatchlings

There are very few studies on the use of anaesthetics in embryos and hatchlings. This area urgently needs research to provide standardized protocols across different species and developmental stages. Sprecher et al. (2022) used ethanol (EtOH) and magnesium chloride ($MgCl_2$) at different concentrations to anesthetize *Loligo vulgaris* late stage embryos, and concluded that at 14 °C, 2–3% EtOH was the most efficient substance as EtOH-treated embryos regain their heart and respiratory rates quickly and recovered physiological responsiveness more rapidly in comparison with $MgCl_2$ treated embryos. Escánez et al. (2018) after comparing $MgCl_2$, clove oil and EtOH in *Octopus vulgaris* hatchlings also concluded that at 20 °C, 1% EtOH produced best results as an anaesthetic agent, discarding clove oil due to the adverse effects on the paralarvae. Furthermore, Roumedakis et al. (2020) recommended anaesthetising *O. maya* juveniles maintained at 24 °C with 3% EtOH or exposing them to cold sea water (11 °C) prior to short-term handling; however, the authors emphasise that the use of cold water in potentially invasive procedures should not be considered. Thus, EtOH seems to be a suitable anaesthetic of early life stages of cephalopods and concentration used will depend on the species and rearing temperature. On the other hand, the use of $MgCl_2$ may cause adverse effects in early life stages. Escolar et al. (2024) recorded that 5% $MgCl_2$ during 30 min used to euthanize juvenile *O. vulgaris* weighing 0.2–4.4 g produces dehydration and loss of weight of 18±5% (range 8–34%). A similar or higher effect could be suspected in hatchlings and/or embryos and this possible weight loss and associated physiological consequences should be considered before using $MgCl_2$ for early life stages due to its welfare implications. Extreme adverse effects, such as inking, mucous release and critical damages in the tegument, have been observed in ommastrephid paralarvae obtained through in vitro fertilization when using $MgCl_2$ (Fernández-Álvarez, pers. comm.), but not with EtOH (Fernández-Álvarez et al. 2017a). However, the potential limitations of EtOH, such as species-specific responses or long-term effects, should be determined.

## Methods to euthanize advanced cephalopod embryos and hatchlings

Embryos can be humanely killed by an overdose of anaesthetic agents, by using either an increased concentration (EtOH to 4%) and/or longer exposure time than required for anaesthesia. After anaesthesia, death can be confirmed by mechanical brain/head destruction, however, this might not be feasible for embryos and hatchlings of all cephalopod species or experimental needs. For morphological studies, immersion in a fixative after anaesthesia overdose can be used as confirmation of death (Fernández-Álvarez et al. 2017b). For instance, in some cases and depending on the species, several thousands of embryos and hatchlings not used for research could be accumulated in the laboratory, as occurs with small egg octopus species. When a large number of small individuals accumulate, the use of quick freezing (at -80 °C or liquid nitrogen), after anesthesia, can be considered for humane killing.

The accumulation of a large number of hatchlings in the laboratory that are not intended to be cultured can cause a problem. Releasing live individuals to the environment could represent a sanitary risk for natural cephalopod populations, and might require an authorization from local authorities. In several countries regulations prohibit the release of laboratory subjects into the environment. This for example applies to marine organisms hatched or previously maintained in experimental aquatic facilities in the European Union (Directive 2016/429/EU). However, it must be taken into account that if the broodstocks have not been under an experimental procedure (for example, only for embryos or hatchlings production) and have been reared according to standardised protocols ensuring their welfare and health, these animals (including the embryos or hatchlings not to be used in experimental procedures) are not affected by the aforementioned Directive. In consequence, the release may be permitted upon the provision of the necessary authorization from the local authorities.

## Future research on cephalopod eggs

Most studies on cephalopod eggs have been done with coastal species. It is clear that many new adaptations await discovery particularly for oceanic and
deep-sea species (see for example, Robison et al. 2014). Recent laboratory techniques could help to visualize development of some embryonic structures, e.g., by using CRISPR-Cas9 methods, producing albino phenotypes (Crawford et al. 2020; Ahuja et al. 2023). The patterns of formation of different organs and tissues during embryonic development across species should be identified (e.g., Kimbara et al. 2022). Image and microscopy techniques as magnetic resonance imaging, micro-computed tomography (Shea et al. 2018; Ziegler et al. 2021) and light sheet fluorescence microscopy (Deryckere et al. 2021; Villanueva et al. 2021; Vilarnau et al. 2024) could help to better visualise the anatomy of embryos and hatchlings. Recently, a chromosome-level octopus genome assembly showed that the female octopus has just one sex chromosome (ZO karyotype) and males have two (ZZ karyotype) (Coffing et al. 2025) and a qPCR-based method also determined genetic sex (Lee et al. 2025). These techniques open the way for recognizing sex in cephalopod embryos. Factors that trigger the hatching process are unclear and oxygen conditions on either side of the chorionic membrane may shed some light on this matter and need to be clarified. Learning and sensorial embryonic abilities in different groups of cephalopods needs further research. Karasch and Ward (2025) suggested fish embryos are sensitive to the presence of adjacent conspecifics, suggesting the potential for social mechanisms of embryonic learning in addition to non-social environmental cues. The same authors also suggest that it will be interesting to understand how embryonic interactions change as a function of nest composition, an interesting field to explore in cephalopods. In the future, it will be necessary to integrate routine laboratory practices and techniques that contemplate the welfare of cephalopod embryos and hatchlings, along with further research to improve and standardize embryo handling, anesthesia, and euthanasia protocols.

## Conclusions

The diversity of egg morphologies and incubation strategies reflects an evolutionary plasticity that has enabled cephalopods to exploit a wide range of marine environments. However, this diversity also presents practical challenges in sampling, species

identification, transport, and laboratory care and welfare. Environmental threats, from pollution to ocean warming and acidification, present further pressures that may alter development, hatchling fitness, and ultimately population dynamics and biodiversity. It is essential to improve laboratory and field methodologies to detect these possible effects. Advances in embryo incubation systems and imaging techniques now allow detailed studies of embryonic development across taxa. Yet, critical knowledge gaps remain in some areas, particularly the lack of control of parent reproduction in cephalopods to ensure timing for egg collection in laboratory, long-term gamete preservation, and embryo viability under climate stressors. As cephalopods gain prominence in fisheries, aquaculture, ecotoxicology, and neurobiology fields, research on their early life stages becomes increasingly relevant. Moreover, the study of cephalopod eggs has entered a new era of methodological refinement and ethical awareness. Ethical considerations, such as the use of anaesthesia and euthanasia methods in advanced embryos, urge the application of the 3Rs principle (Replacement, Reduction, Refinement) and the development of protocols to ensure embryo and hatchling welfare and the responsible use of these organisms. To ensure ethical standards in cephalopod research, future policies should mandate the integration of welfare-conscious practices and protocols into routine laboratory procedures involving embryos and hatchlings.

**Acknowledgements** We would like to thank Chong Chen (JAMSTEC), Sylvain Govan (Aquarium des Lagons), Julia D. Sigwart (Senckenberg Research Institute and Natural History Museum), Erling Svensen (underwater photographer) and Jean-Louis Teyssié (IAEA Environment Laboratories) for kindly providing images included in Fig. 1. Authors wish to express their gratitude to the organizers and support staff of the Cephalopod International Advisory Council (CIAC) conference held in Sesimbra, Portugal, during April 2022 for the facilities provided to develop of a two-day workshop on the collection, handling, and care of cephalopod eggs.

**Author contributions** Roger Villanueva: Conceptualization, Writing—original draft. Katina Roumedakis: Image editing. All authors: Writing—revision & editing.

**Funding** Open Access funding provided thanks to the CRUE-CSIC agreement with Springer Nature. Project funding to RV, OE, FAFA, EA and DGV was provided by the Spanish Ministry of Science, Innovation and Universities (OCTOSET project RTI2018-097908-B-I00; ECOPHYN project PID2021-126824NB-C32 and C31 funded by MICIU/AEI/https://doi.
org/10.13039/501100011033 and "ERDF/EU"; PIE Project ECOCEPH 202230E094; CEREBAL (CSIC—AECID), project INTER23001 and the Spanish government through the "Severo Ochoa Center of Excellence" Grant CEX2024-001494-S funded by AEI https://doi.org/10.13039/501100011033. FAFA was supported by a Ramón y Cajal fellowship (Ref. RYC2023-043494-I) funded by MCIN/AEI /https://doi.org/10.13039/501100011033 and FSE+. KR gratefully acknowledges funding from the European Union's Horizon Europe research and innovation program under the Marie Skłodowska-Curie Postdoctoral Fellowship (Grant Agreement No. 101107839). RR acknowledges Fundação para a Ciência e Tecnologia, I.P. (FCT) for the strategic project UIDB/04292/2020 granted to MARE, and the project LA/P/0069/2020 granted to the Associate Laboratory ARNET. GV is supported by a postdoctoral fellowship from the Swedish Wenner-Gren Foundations. ASD and LD acknowledge the French National Agency of Research (PReSTO'Cog ANR-13-BSV7-0002). CR and CC are supported by Project PRONAI2-2024-70 From SECITHI, Mexico. AME was supported by the Research Foundation—Flanders (FWO) (FR/11D4120N). NO was supported by the National Scientific and Technical Research Council—Argentina. EAGV was supported the Brazilian National Research Council- CNPq (grant #316391/2021-2).

**Data availability** No datasets were generated or analysed during the current study.

**Declarations**

**Conflict of interest** The authors declare no competing interests.

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