The genetic program for cartilage development has deep homology within Bilateria.

The evolution of novel cell types led to the emergence of new tissues and organs during the diversification of animals. The origin of the chondrocyte, the cell type that synthesizes cartilage matrix, was central to the evolution of the vertebrate endoskeleton. Cartilage-like tissues also exist outside the vertebrates, although their relationship to vertebrate cartilage is enigmatic. Here we show that protostome and deuterostome cartilage share structural and chemical properties, and that the mechanisms of cartilage development are extensively conserved--from induction of chondroprogenitor cells by Hedgehog and ß-catenin signalling, to chondrocyte differentiation and matrix synthesis by SoxE and SoxD regulation of clade A fibrillar collagen (ColA) genes--suggesting that the chondrogenic gene regulatory network evolved in the common ancestor of Bilateria. These results reveal deep homology of the genetic program for cartilage development in Bilateria and suggest that activation of this ancient core chondrogenic network underlies the parallel evolution of cartilage tissues in Ecdysozoa, Lophotrochozoa and Deuterostomia.

Embryonic development of the camouflaging dwarf cuttlefish, Sepia bandensis.

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

De novo transcriptome sequencing and analysis of the cuttlefish (Sepiella japonica) with different embryonic developmental stages.

In this study, embryos of Sepiella japonica from eye primordium formation to the larval growing stage were collected and used for RNA-Seq analysis. A total of 183,542,186 clean reads were assembled de novo into 58,054 unigenes consisting of 54,118,228 bp, with the average length at 932 bp and the N50 at 1667 bp. 21,469 (36.98%) unigenes were annotated at least in one of four databases including non-redundant protein (NR), Swiss-Prot, clusters of orthologous groups of proteins (KOG) and Kyoto Encyclopedia of Genes and Genomes (KEGG). 4460 (7.68%) unigenes were annotated in all databases. Analysis of differentially expressed genes (DEGs) was carried out on embryos at Eye primordium formation stage (SJ1), organ differentiation stage (SJ2), and hatching stage (SJ3). Overall, the current study provided the de novo assembly of S. japonica transcriptome and identified the DEGs and pathways during embryonic development, which will provide a fundamental genetic resource for further functional research.

Orientation Patterns of Japanese Flying Squid Todarodes pacificus Embryos within Egg Masses and Responses of Paralarvae to Light.

Squid embryos develop in the perivitelline fluid inside the chorion, which is an envelope secreted by the ovarian follicle. The onset of hatching initiates local dissolution of the chorion when the hatching gland enzyme facilitates the release of the developed paralarvae. In the present study, we investigated the pre-hatching behavioral patterns of Todarodes pacificus embryos and their responses to light after hatching. Observations of orientation were conducted using embryos developing inside chorions embedded within intact egg masses, while phototactic experiments were conducted on paralarvae that hatched from these egg masses. Within the restricted chorion and along the animal-vegetal axis, the embryos demonstrated a variety of orientation patterns that were categorized as swirls, glides, and somersaults. The contributions of these orientations to enhance oxygen diffusion and stimulate paralarval swimming immediately after hatching are discussed. Paralarvae exhibited normal diel vertical migration and responded positively to light sources. Vertical migration and phototaxis in T. pacificus paralarvae could have great adaptive significance because they hatch in neritic environments and are transported by ocean currents during their planktonic life.

Observations on the spawning behavior, egg masses and paralarval development of the ommastrephid squid Todarodes pacificus in a laboratory mesocosm.

The spawning behavior of ommastrephid squids has never been observed under natural conditions. Previous laboratory observations of Japanese flying squid (Todarodes pacificus) suggest that pre-spawning females might rest on the continental shelf or slope before they ascend above the pycnocline to spawn, and that the egg masses might settle in the pycnocline. Here, two mesocosm experiments were conducted in a 300 m(3) tank that was 6 m deep to investigate this hypothesis. In the first experiment, a thermocline (2.5-3.5 m) was established in the tank by creating a thermally stratified (17-22°C) water column. In the second experiment, the temperature was uniform (22°C) at all depths. Prior to spawning, females did not rest on the tank floor. In the stratified water column, egg masses remained suspended in the thermocline, but in an unstratified water column, they settled on the tank bottom, collapsed and were infested by microbes, resulting in abnormal or nonviable embryos. Eleven females spawned a total of 18 egg masses (17-80 cm in diameter), indicating that females can spawn more than once when under stress. Paralarvae hatched at stage 30/31 and survived for up to 10 days, allowing us to observe the most advanced stage of paralarvae in captivity. Paralarvae survived after consumption of the inner yolk, suggesting they might have fed in the tank.

Differential impacts of ocean acidification and warming on winter and summer progeny of a coastal squid (Loligo vulgaris).

Little is known about the capacity of early life stages to undergo hypercapnic and thermal acclimation under the future scenarios of ocean acidification and warming. Here, we investigated a comprehensive set of biological responses to these climate change-related variables (2°C above winter and summer average spawning temperatures and ΔpH=0.5 units) during the early ontogeny of the squid Loligo vulgaris. Embryo survival rates ranged from 92% to 96% under present-day temperature (13-17°C) and pH (8.0) scenarios. Yet, ocean acidification (pH 7.5) and summer warming (19°C) led to a significant drop in the survival rates of summer embryos (47%, P<0.05). The embryonic period was shortened by increasing temperature in both pH treatments (P<0.05). Embryo growth rates increased significantly with temperature under present-day scenarios, but there was a significant trend reversal under future summer warming conditions (P<0.05). Besides pronounced premature hatching, a higher percentage of abnormalities was found in summer embryos exposed to future warming and lower pH (P<0.05). Under the hypercapnic scenario, oxygen consumption rates decreased significantly in late embryos and newly hatched paralarvae, especially in the summer period (P<0.05). Concomitantly, there was a significant enhancement of the heat shock response (HSP70/HSC70) with warming in both pH treatments and developmental stages. Upper thermal tolerance limits were positively influenced by acclimation temperature, and such thresholds were significantly higher in late embryos than in hatchlings under present-day conditions (P<0.05). In contrast, the upper thermal tolerance limits under hypercapnia were higher in hatchlings than in embryos. Thus, we show that the stressful abiotic conditions inside the embryo's capsules will be exacerbated under near-future ocean acidification and summer warming scenarios. The occurrence of prolonged embryogenesis along with lowered thermal tolerance limits under such conditions is expected to negatively affect the survival success of squid early life stages during the summer spawning period, but not winter spawning.

Reflectin genes and development of iridophore patterns in Sepia officinalis embryos (Mollusca, Cephalopoda).

BACKGROUND: In the cuttlefish Sepia officinalis, iridescence is known to play a role in patterning and communication. In iridophores, iridosomes are composed of reflectins, a protein family, which show great diversity in all cephalopod species. Iridosomes are established before hatching, but very little is known about how these cells are established, their distribution in embryos, or the contribution of each reflectin gene to iridosome structures. RESULTS: Six reflectin genes are expressed during the development of iridosomes in Sepia officinalis. We show that they are expressed in numerous parts of the body before hatching. Evidence of the colocalization of two different genes of reflectin was found. Curiously, reflectin mRNA expression was no longer detectable at the time of hatchling, while reflectin proteins were present and gave rise to visible iridescence. CONCLUSION: These data suggest that several different forms of reflectins are simultaneously used to produce iridescence in S. officinalis and that mRNA production and translation are decoupled in time during iridosome development.

Lower hypoxia thresholds of cuttlefish early life stages living in a warm acidified ocean.

The combined effects of future ocean acidification and global warming on the hypoxia thresholds of marine biota are, to date, poorly known. Here, we show that the future warming and acidification scenario led to shorter embryonic periods, lower survival rates and the enhancement of premature hatching in the cuttlefish Sepia officinalis. Routine metabolic rates increased during the embryonic period, but environmental hypercapnia significantly depressed pre-hatchling's energy expenditures rates (independently of temperature). During embryogenesis, there was also a significant rise in the carbon dioxide partial pressure in the perivitelline fluid (PVF), bicarbonate levels, as well as a drop in pH and oxygen partial pressure (pO2). The critical partial pressure (i.e. hypoxic threshold) of the pre-hatchlings was significantly higher than the PVF oxygen partial pressure at the warmer and hypercapnic condition. Thus, the record of oxygen tensions below critical pO2 in such climate scenario indicates that the already harsh conditions inside the egg capsules are expected to be magnified in the years to come, especially in populations at the border of their thermal envelope. Such a scenario promotes untimely hatching and smaller post-hatching body sizes, thus challenging the survival and fitness of early life stages.

Embryonic exposure to predator odour modulates visual lateralization in cuttlefish.

Predation pressure acts on the behaviour and morphology of prey species. In fish, the degree of lateralization varies between high- and low-predation populations. While lateralization appears to be widespread in invertebrates, we do not know whether heredity and early experience interact during development as in vertebrates. Here we show, for the first time, that an exposure to predator odour prior to hatching modulates visual lateralization in newly hatched cuttlefish. Only cuttlefish that have been exposed to predator odour display a left-turning bias when tested with blank seawater in a T-shaped apparatus. Exposure to predator odour all the incubation long could appear as an acute predictor of a high-predation surrounding environment. In addition, cuttlefish of all groups display a left-turning preference when tested with predator odour in the apparatus. This suggests the ability of cuttlefish to innately recognize predator odour. To our knowledge, this is the first clear demonstration that lateralization is vulnerable to ecological challenges encountered during embryonic life, and that environmental stimulation of the embryo through the olfactory system could influence the development of subsequent visual lateralization.

New insights into ion regulation of cephalopod molluscs: a role of epidermal ionocytes in acid-base regulation during embryogenesis.

The constraints of an active life in a pelagic habitat led to numerous convergent morphological and physiological adaptations that enable cephalopod molluscs and teleost fishes to compete for similar resources. Here, we show for the first time that such convergent developments are also found in the ontogenetic progression of ion regulatory tissues; as in teleost fish, epidermal ionocytes scattered on skin and yolk sac of cephalopod embryos appear to be responsible for ionic and acid-base regulation before gill epithelia become functional. Ion and acid-base regulation is crucial in cephalopod embryos, as they are surrounded by a hypercapnic egg fluid with a Pco(2) between 0.2 and 0.4 kPa. Epidermal ionocytes were characterized via immunohistochemistry, in situ hybridization, and vital dye-staining techniques. We found one group of cells that is recognized by concavalin A and MitoTracker, which also expresses Na(+)/H(+) exchangers (NHE3) and Na(+)-K(+)-ATPase. Similar to findings obtained in teleosts, these NHE3-rich cells take up sodium in exchange for protons, illustrating the energetic superiority of NHE-based proton excretion in marine systems. In vivo electrophysiological techniques demonstrated that acid equivalents are secreted by the yolk and skin integument. Intriguingly, epidermal ionocytes of cephalopod embryos are ciliated as demonstrated by scanning electron microscopy, suggesting a dual function of epithelial cells in water convection and ion regulation. These findings add significant knowledge to our mechanistic understanding of hypercapnia tolerance in marine organisms, as it demonstrates that marine taxa, which were identified as powerful acid-base regulators during hypercapnic challenges, already exhibit strong acid-base regulatory abilities during embryogenesis.

In situ recordings of large gelatinous spheres from NE Atlantic, and the first genetic confirmation of egg mass of Illex coindetii (Vérany, 1839) (Cephalopoda, Mollusca).

In total, 90 gelatinous spheres, averaging one meter in diameter, have been recorded from ~ 1985 to 2019 from the NE Atlantic Ocean, including the Mediterranean Sea, using citizen science. More than 50% had a dark streak through center. They were recorded from the surface to ~ 60-70 m depth, mainly neutrally buoyant, in temperatures between 8 and 24°C. Lack of tissue samples has until now, prohibited confirmation of species. However, in 2019 scuba divers secured four tissue samples from the Norwegian coast. In the present study, DNA analysis using COI confirms species identity as the ommastrephid broadtail shortfin squid Illex coindetii (Vérany, 1839); these are the first confirmed records from the wild. Squid embryos at different stages were found in different egg masses: (1) recently fertilized eggs (stage ~ 3), (2) organogenesis (stages ~ 17-19 and ~ 23), and (3) developed embryo (stage ~ 30). Without tissue samples from each and every record for DNA corroboration we cannot be certain that all spherical egg masses are conspecific, or that the remaining 86 observed spheres belong to Illex coindetii. However, due to similar morphology and size of these spheres, relative to the four spheres with DNA analysis, we suspect that many of them were made by I. coindetii.

Animal development in the microbial world: The power of experimental model systems.

The development of powerful model systems has been a critical strategy for understanding the mechanisms underlying the progression of an animal through its ontogeny. Here we provide two examples that allow deep and mechanistic insight into the development of specific animal systems. Species of the cnidarian genus Hydra have provided excellent models for studying host-microbe interactions and how metaorganisms function in vivo. Studies of the Hawaiian bobtail squid Euprymna scolopes and its luminous bacterial partner Vibrio fischeri have been used for over 30 years to understand the impact of a broad array of levels, from ecology to genomics, on the development and persistence of symbiosis. These examples provide an integrated perspective of how developmental processes work and evolve within the context of a microbial world, a new view that opens vast horizons for developmental biology research. The Hydra and the squid systems also lend an example of how profound insights can be discovered by taking advantage of the "experiments" that evolution had done in shaping conserved developmental processes.

Squid vascular endothelial growth factor receptor: a shared molecular signature in the convergent evolution of closed circulatory systems.

The highly specialized cephalopod cardiovascular system has long been considered a valuable model for understanding the evolution of circulatory systems. Despite the number of studies devoted to this topic, the developmental regulatory mechanisms remain largely unexplored. Here, we focus on the vascular endothelial growth factor receptor (VEGFR). This factor is known to mediate levels of endothelial growth factor that is involved in hematopoiesis and vasculogenesis including multichambered heart development in vertebrates. We found a squid VEGFR ortholog that is expressed in the developing blood vessels, notably in the sheet-like endothelial cells of the systemic and branchial hearts. The highly restricted localization of VEGFR in the vascular endothelial cells and its shared expression pattern in the developing hearts of cephalopods and vertebrates suggest a shared molecular signature of closed circulatory systems that has been independently elaborated during evolution.

Expression of serotonin (5-HT) during CNS development of the cephalopod mollusk, Idiosepius notoides.

Cephalopods are unique among mollusks in exhibiting an elaborate central nervous system (CNS) and remarkable cognitive abilities. Despite a profound knowledge of the neuroanatomy and neurotransmitter distribution in their adult CNS, little is known about the expression of neurotransmitters during cephalopod development. Here, we identify the first serotonin-immunoreactive (5-HT-ir) neurons during ontogeny and describe the establishment of the 5-HT system in the pygmy squid, Idiosepius notoides. Neurons that are located dorsally to each optic lobe are the first to express 5-HT, albeit only when the lobular neuropils are already quite elaborated. Later, 5-HT is expressed in almost all lobes, with most 5-HT-ir cell somata appearing in the subesophageal mass. Further lobes with numerous 5-HT-ir cell somata are the subvertical and posterior basal lobes and the optic and superior buccal lobes. Hatching squids possess more 5-HT-ir neurons, although the proportions between the individual brain lobes remain the same. The majority of 5-HT-ir cell somata appears to be retained in the adult CNS. The overall distribution of 5-HT-ir elements within the CNS of adult I. notoides resembles that of adult Octopus vulgaris and Sepia officinalis. The superior frontal lobe of all three species possesses few or no 5-HT-ir cell somata, whereas the superior buccal lobe comprises many cell somata. The absence of 5-HT-ir cell somata in the inferior buccal lobes of cephalopods and the buccal ganglia of gastropods may constitute immunochemical evidence of their homology. This integrative work forms the basis for future studies comparing molluscan, lophotrochozoan, ecdysozoan, and vertebrate brains.

Highly Efficient Knockout of a Squid Pigmentation Gene.

Seminal studies using squid as a model led to breakthroughs in neurobiology. The squid giant axon and synapse, for example, laid the foundation for our current understanding of the action potential [1], ionic gradients across cells [2], voltage-dependent ion channels [3], molecular motors [4-7], and synaptic transmission [8-11]. Despite their anatomical advantages, the use of squid as a model receded over the past several decades as investigators turned to genetically tractable systems. Recently, however, two key advances have made it possible to develop techniques for the genetic manipulation of squid. The first is the CRISPR-Cas9 system for targeted gene disruption, a largely species-agnostic method [12, 13]. The second is the sequencing of genomes for several cephalopod species [14-16]. If made genetically tractable, squid and other cephalopods offer a wealth of biological novelties that could spur discovery. Within invertebrates, not only do they possess by far the largest brains, they also express the most sophisticated behaviors [17]. In this paper, we demonstrate efficient gene knockout in the squid Doryteuthis pealeii using CRISPR-Cas9. Ommochromes, the pigments found in squid retinas and chromatophores, are derivatives of tryptophan, and the first committed step in their synthesis is normally catalyzed by Tryptophan 2,3 Dioxygenase (TDO [18-20]). Knocking out TDO in squid embryos efficiently eliminated pigmentation. By precisely timing CRISPR-Cas9 delivery during early development, the degree of pigmentation could be finely controlled. Genotyping revealed knockout efficiencies routinely greater than 90%. This study represents a critical advancement toward making squid genetically tractable.

Developmental expression of apterous/Lhx2/9 in the sepiolid squid Euprymna scolopes supports an ancestral role in neural development.

The transcription factors Apterous/Lhx2/9 play many pivotal roles in the development of protostomes and deuterostomes, most notably limb patterning, eye morphogenesis, and brain development. Full-length apterous/lhx2/9 homologs have been isolated from several invertebrate species, but hitherto not from a lophotrochozoan. Here, we report the isolation, characterization, and spatio-temporal expression of apterous in the sepiolid squid Euprymna scolopes. The isolated composite cDNA encodes a hypothetical protein of 448 amino acid residues with a typical LIM-homeodomain (LIM-HD) structure and the greatest overall sequence similarity to vertebrate Lhx2/9 proteins. The Euprymna scolopes apterous (Es-ap) expression patterns provided no indication of a role in the early dorso/ventral patterning or growth of the arm crown that showed expression only in two ventral cords running in parallel inside the arms and tentacles and at the base of the suckers, a region rich in nerve endings and chemosensory neurons. The Es-ap hybridization signal was also conspicuous in the eyes, olfactory organs, optic lobes, and in several lobes of the supraesophageal mass, among these the olfactory and vertical lobes, and paravertical bodies. The observed expression patterns suggest gene involvement in eye morphogenesis and neural wiring of sensory structures, including those for olfaction and vision.

Cartilage differentiation in cephalopod molluscs.

Amongst the various metazoan lineages that possess cartilage, tissues most closely resembling vertebrate hyaline cartilage in histological section are those of cephalopod molluscs. Although elements of the adult skeleton have been described, the development of these cartilages has not. Using serial histology of sequential developmental stages of the European cuttlefish, Sepia officinalis, we investigate these skeletal elements and offer the first description of the formation of any cellular invertebrate cartilage. Our data reveal that cuttlefish cartilage most often differentiates from uncondensed mesenchymal cells near the end of embryonic development, but that the earliest-forming cartilages differentiate from a cellular condensation which goes through a protocartilage stage in a manner typical of vertebrate primary cartilage formation. We further investigate the distribution and degree of differentiation of cartilages at the time of hatching in an additional four cephalopod species. We find that the timing of cartilage development varies between elements within a single species, as well as between species. We identify a tendency towards cartilage differentiation from uncondensed connective tissue in elements that form at the end of embryogenesis or after hatching. These data suggest a form of metaplasia from connective tissue is the ancestral mode of cartilage formation in this lineage.

Morphological changes of the optic lobe from late embryonic to adult stages in oval squids Sepioteuthis lessoniana.

The optic lobe is the largest brain area within the central nervous system of cephalopods and it plays important roles in the processing of visual information, the regulation of body patterning, and locomotive behavior. The oval squid Sepioteuthis lessoniana has relatively large optic lobes that are responsible for visual communication via dynamic body patterning. It has been observed that the visual behaviors of oval squids change as the animals mature, yet little is known about how the structure of the optic lobes changes during development. The aim of the present study was to characterize the ontogenetic changes in neural organization of the optic lobes of S. lessoniana from late embryonic stage to adulthood. Magnetic resonance imaging and micro-CT scans were acquired to reconstruct the 3D-structure of the optic lobes and examine the external morphology at different developmental stages. In addition, optic lobe slices with nuclear staining were used to reveal changes in the internal morphology throughout development. As oval squids mature, the proportion of the brain making up the optic lobes increases continuously, and the optic lobes appear to have a prominent dent on the ventrolateral side. Inside the optic lobe, the cortex and the medulla expand steadily from the late embryonic stage to adulthood, but the cell islands in the tangential zone of the optic lobe decrease continuously in parallel. Interestingly, the size of the nuclei of cells within the medulla of the optic lobe increases throughout development. These findings suggest that the optic lobe undergoes continuous external morphological change and internal neural reorganization throughout the oval squid's development. These morphological changes in the optic lobe are likely to be responsible for changes in the visuomotor behavior of oval squids from hatching to adulthood.

Transcriptome analysis of gene expression patterns during embryonic development in golden cuttlefish (Sepia esculenta).

Golden cuttlefish (Sepia esculenta) is an important economic species in China. Because of the rapid decline of its natural resource, researchers are exploring breeding technique for this species. The major obstacle that hinders artificial breeding of S. esculenta is the low larvae survival rate. Mortality is especially high during the mouth-opening stage. Investigating the embryogenesis before the first feed could provide theoretical guidance for reproduction control and breeding of S. esculenta and other Sepia species. In this study, we analyzed the dynamics of the S. esculenta transcriptome along different stages of embryonic development by mRNA-sEq. Our bioinformatics protocol identified 1492 differentially expressed genes (DEGs) across the early developmental stages. Gene ontology enrichment analysis showed that the DEGs were significantly involved in developmental processes and molecular functions, including chitin metabolic process, peptidase activity, catalytic activity, and calcium ion binding. Our results indicated that genes related to cuttlebone development and gene regulation functions were active during the early life phase of S. esculenta. Hierarchical clustering of the DEGs reflected the successiveness of the developmental stages, revealing that gene expression patterns of neighboring stages were similar. The DEG analysis allowed us to identify specific genes and relevant biological pathways to better understand the molecular mechanisms during each developmental stage. This study provides novel insights into the processes underlying the early developmental stages of S. esculenta. The transcriptomic data and identified genes will serve as valuable references for the developmental biology of this species and will help promote its aquaculture research.

Development of Embryonic Market Squid, Doryteuthis opalescens, under Chronic Exposure to Low Environmental pH and [O2].

The market squid, Doryteuthis opalescens, is an important forage species for the inshore ecosystems of the California Current System. Due to increased upwelling and expansion of the oxygen minimum zone in the California Current Ecosystem, the inshore environment is expected to experience lower pH and [O2] conditions in the future, potentially impacting the development of seafloor-attached encapsulated embryos. To understand the consequences of this co-occurring environmental pH and [O2] stress for D. opalescens encapsulated embryos, we performed two laboratory experiments. In Experiment 1, embryo capsules were chronically exposed to a treatment of higher (normal) pH (7.93) and [O2] (242 µM) or a treatment of low pH (7.57) and [O2] (80 µM), characteristic of upwelling events and/or La Niña conditions. The low pH and low [O2] treatment extended embryo development duration by 5-7 days; embryos remained at less developed stages more often and had 54.7% smaller statolith area at a given embryo size. Importantly, the embryos that did develop to mature embryonic stages grew to sizes that were similar (non-distinct) to those exposed to the high pH and high [O2] treatment. In Experiment 2, we exposed encapsulated embryos to a single stressor, low pH (7.56) or low [O2] (85 µM), to understand the importance of environmental pH and [O2] rising and falling together for squid embryogenesis. Embryos in the low pH only treatment had smaller yolk reserves and bigger statoliths compared to those in low [O2] only treatment. These results suggest that D. opalescens developmental duration and statolith size are impacted by exposure to environmental [O2] and pH (pCO2) and provide insight into embryo resilience to these effects.