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A: Na accumulation in cells on the skin and yolk of S. lessoniana embryo showing the morphology of the animal divided in mantle, head, and yolk. On the skin in the mantle and head region Sodium Green, a Nadependent fluorescent reagent, predominantly stains round-to oval-shaped cells scattered on the skin surface of stage 28 embryos (B and C). C: higher cell densities are found on the lateral side of the head region. D: high magnification of the yolk sac surface demonstrating the presence of Sodium Green signals equally distributed in epithelial cells. Note the grain-like pattern of the fluorescence signal in these cells.
Source publication
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;...
Contexts in source publication
Context 1
... with ConA- labeled cells. Instead ConA-labeled cells on the entire skin of squid embryos were characterized by high Na /H exchanger (NHE3) immunoreactivity and sodium accumulation (Fig. 3). We termed these cells sodium-rich cells (SRCs). Highest densities of SRCs were found on the lateral side of the head, arms, and close to the olfactory organ (Fig. 4, A-C). Furthermore, no SRCs were found on the yolk sac epithelium. Instead, small vesicles located inside large flattened epidermal cells that resemble pavement cells/accessory cells (51) of the entire yolk epithelium show positive sodium reactivity (Fig. 4D). 3. Localization of NKA, NHE3, and the glycoprotein concavilin A (ConA) in epider- ...
Context 2
... of SRCs were found on the lateral side of the head, arms, and close to the olfactory organ (Fig. 4, A-C). Furthermore, no SRCs were found on the yolk sac epithelium. Instead, small vesicles located inside large flattened epidermal cells that resemble pavement cells/accessory cells (51) of the entire yolk epithelium show positive sodium reactivity (Fig. 4D). 3. Localization of NKA, NHE3, and the glycoprotein concavilin A (ConA) in epider- mal cells on the skin of S. lessoniana embryos (stages 26 and 27). Epidermal cells that are positively labeled by ConA are not colocalized with NKA antibody (NaR) cells (left). These NaR cells are not colocalized with cells that exhibit positive NHE3 ...
Citations
... Encouraged by these results, we decided to do a comprehensive study of conditions triggering protopeptide self-assembly into macroscopic structures as hydrogels, which could resemble the conditions found in nature. Cephalopod tissues present a tight acid-base control to maintain pH close to 7.4 to avoid acidosis, which is also ideal for their survival in marine water (Hu et al., 2011;Kaplan et al., 2013), while, in their stomach, the digestion occurs in an acidic medium at pH 3-4 (Bastos et al., 2020). The protopeptide has a pI of 4 (determined experimentally, Supplementary Figure S3) and a specific composition of amino acids. ...
Reflectins are a family of intrinsically disordered proteins involved in cephalopod camouflage, making them an interesting source for bioinspired optical materials. Understanding reflectin assembly into higher-order structures by standard biophysical methods enables the rational design of new materials, but it is difficult due to their low solubility. To address this challenge, we aim to understand the molecular self-assembly mechanism of reflectin’s basic unit—the protopeptide sequence YMDMSGYQ—as a means to understand reflectin’s assembly phenomena. Protopeptide self-assembly was triggered by different environmental cues, yielding supramolecular hydrogels, and characterized by experimental and theoretical methods. Protopeptide films were also prepared to assess optical properties. Our results support the hypothesis for the protopeptide aggregation model at an atomistic level, led by hydrophilic and hydrophobic interactions mediated by tyrosine residues. Protopeptide-derived films were optically active, presenting diffuse reflectance in the visible region of the light spectrum. Hence, these results contribute to a better understanding of the protopeptide structural assembly, crucial for the design of peptide- and reflectin-based functional materials.
... For instance, routine oxygen consumption rates have been shown to be higher in pelagic squid when compared to benthic species, such as octopuses (Seibel 2007;Rosa et al. 2009). Increased energetic costs have been shown during acclimation to environmental changes such as increasing temperature, and are tightly linked to adjustments to neuronal circuits, epithelial ion transports, or behavioural responses (Hu et al. 2011(Hu et al. , 2013Chung and Marshall 2017). Indeed, temperature-induced changes to life-history characteristics in cephalopods (e.g., growth, behaviour, reproduction, etc.) are known to be mediated by modifications to metabolism Pörtner and Lannig 2009). ...
... Recently, Kuan et al. (2022) Cephalopods have been hypothesized to be less vulnerable to OA-induced stress compared to other organisms, due to their naturally high metabolic rates and to their high ion-and acid-base regulation capabilities Gutowska et al. 2010). However, several species have been shown to undergo metabolic depression in either high IPCC-relevant pCO 2 levels , and under extreme hypercapnic conditions (Hu et al. 2011;Kaplan et al. 2013). The depression of metabolic rates has been shown to occur virtually across all major animal phyla, when exposed to environmental stress (Guppy and Withers 1999;Storey and Storey 2007) such as hypercapnic conditions (Barnhart 1989;Rees and Hand 1990). ...
... officinalis (Sigwart et al. 2016), while acutely high pCO 2 induced shorter mantle lengths and body mass in cuttlefish embryos and hatchlings (Hu et al. 2011). Notwithstanding, juvenile sepiids have been shown to maintain normal growth in extremely high pCO 2 levels for 6 weeks (Gutowska et al. 2008). ...
Aside from being one of the most fascinating groups of marine organisms, cephalopods play a major role in marine food webs, both as predators and as prey, while representing key living economic assets, namely for artisanal and subsistence fisheries worldwide. Recent research suggests that cephalopods are benefitting from ongoing environmental changes and the overfishing of certain fish stocks (i.e., of their predators and/or competitors), putting forward the hypothesis that this group may be one of the few 'winners' of climate change. While many meta-analyses have demonstrated negative and overwhelming consequences of ocean warming (OW), acidification (OA), and their combination (OWA) for a variety of marine taxa, such a comprehensive analysis is lacking for cephalopod molluscs. In this context, the existing literature was surveyed for peer-reviewed articles featuring the sustained (≥24h) and controlled exposure of cephalopod species (Cephalopoda Class) to these factors, applying a comparative framework of mixed-model meta-analyses (784 control-treatment comparisons, from 47 suitable articles). Impacts on a wide set of biological categories at the individual level (e.g., survival, metabolism, behaviour, cell stress, growth) were evaluated and contrasted across different ecological attributes (i.e., taxonomic lineages, climates, and ontogenetic stages). Contrary to what is commonly assumed, OW arises as a clear threat to cephalopods, while OA exhibited more restricted impacts. In fact, OW impacts were ubiquitous across different stages of ontogeny, taxonomical lineages (i.e., octopuses, squids, and cuttlefishes). These results challenge the assumption that cephalopods benefit from novel ocean conditions, revealing an overarching negative impact of OW in this group. Importantly, we also identify lingering literature gaps, showing that most studies to date focus on OW and early life stages of mainly temperate species. Our results raise the need to consolidate experimental efforts in a wider variety of taxa, climate regions, life stages, and other key environmental stressors such as deoxygenation and hypoxia, to better understand how cephalopods will cope with future climate change.
... For the purpose of ensuring that the regulatory proteins are inhibited by the specific inhibitors (ethylisopropyl amiloride (EIPA) for NHE; bafilomycin A1 (BafA1) for VHA), the sample collection should begin after the saline has been in the gill for at least 30 minutes. We prepared perfusion saline by adding different dosages of EIPA that could compete for Na + binding sites to determine whether NHEs are responsible for regulating cephalopod gill function (Schaffhauser et al., 2016) and could be acted on squid as well (Hu et al., 2011). The role of VHA in the regulation of homeostasis in the gills of squid and octopus was examined by preparing perfusion saline containing different dosages of BafA1, which is noncovalently binding with VHA to prohibit H + conduction. ...
Cephalopods are ancient mollusks that can be found in many different ecological niches in the ocean ranging from the intertidal zone to the deep-sea abyss. In order to adapt to a lifestyle in various habitats, cephalopods have evolved a variety of locomotory modes to accommodate their respective habitats. Most cephalopods have relatively high metabolic rates due to their less efficient swimming mode by jet propulsion. This lifestyle is characterized by a high level of energy expenditure, fueled exclusively by protein diets that are rapidly digested and may produce metabolic nitrogenous waste NH3/ NH 4 + accumulation and acid-base disturbances. This study observed that the NH 4 + transport rate in pelagic bigfin reef squid (Sepioteuthis lessoniana) is two times faster than benthic common octopus (Octopus vulgaris). Inhibition of Na⁺/H⁺ exchangers (NHEs) showed significant disruption of NH 4 + and H⁺ excretory processes in gills of octopus but not in squid. However, inhibition of vacuolar-type H⁺-ATPase (VHA) significantly disrupts NH 4 + and H⁺ transport rates in gills of both animals. Accordingly, for NH 4 + and H⁺ homeostasis, benthic octopus with lower aerobic respiration rates utilize both active and Na⁺-driven secondary transport machinery. In order to avoid NH 4 + accumulated in the blood, pelagic squids with higher aerobic respiration rates prefer active NH 4 + and H⁺ transport mechanisms that consume ATP intensively.
... It is proposed that ammonia transporters from the Rh family in combination with NHE3, expressed in HR cells are key players in mediating the active secretion of ammonia and protons in seawater teleost [22,48]. A previous study demonstrated that NHE3 expressing epidermal ionocytes of cephalopod embryos are also involved in active secretion of acid equivalents [49]. Together, different expression patterns of C. nasus NHEs during multiple embryonic development stages seem to involve in ammonia excretion, Na + uptake, and maintaining cellular pH homeostasis, or other physiological processes. ...
Background
In aquatic environments, pH, salinity, and ammonia concentration are extremely important for aquatic animals. NHE is a two-way ion exchange carrier protein, which can transport Na ⁺ into cells and exchange out H ⁺ , and also plays key roles in regulating intracellular pH, osmotic pressure, and ammonia concentration.
Results
In the present study, ten NHEs , the entire NHE gene family, were identified from Coilia nasus genome and systemically analyzed via phylogenetic, structural, and synteny analysis. Different expression patterns of C. nasus NHEs in multiple tissues indicated that expression profiles of NHE genes displayed tissue-specific. Expression patterns of C. nasus NHEs were related to ammonia excretion during multiple embryonic development stages. To explore the potential functions on salinity challenge and ammonia stress, expression levels of ten NHEs were detected in C. nasus gills under hypotonic stress, hypertonic stress, and ammonia stress. Expression levels of all NHEs were upregulated during hypotonic stress, while they were downregulated during hypertonic stress. NHE2 and NHE3 displayed higher expression levels in C. nasus larvae and juvenile gills under ammonia stress.
Conclusions
Our study revealed that NHE genes played distinct roles in embryonic development, salinity stress, and ammonia exposure. Syntenic analysis showed significant difference between stenohaline fish and euryhaline fishes. Our findings will provide insight into effects of C. nasus NHE gene family on ion transport and ammonia tolerance and be beneficial for healthy aquaculture of C. nasus .
... In cephalopods, specialized ionocytes have been found in their embryonic skin and yolk, as well as in their adult gill epithelium, which are responsible for maintaining ionic balance, a feature also found in fish. The potential for H + secretion from epithelium has also been observed in larvae of cephalopod and fish (Hu et al., 2015;Hu et al., 2011a). ...
Climate changes, such as extreme temperature shifts, can have a direct and significant impact on animals living in the ocean system. Ectothermic animals may undergo concerted metabolic shifts in response to ambient temperature changes. The physiological and molecular adaptations in cephalopods during their early life stages are largely unknown due to the challenge of rearing them outside of a natural marine environment. To overcome this obstacle, we established a pelagic bigfin reef squid (Sepioteuthis lessoniana) culture facility, which allowed us to monitor the effects of ambient thermal elevation and fluctuation on cephalopod embryos/larvae. By carefully observing embryonic development in the breeding facility, we defined 23 stages of bigfin reef squid embryonic development, beginning at stage 12 (blastocyst; 72 h post-egg laying) and continuing through hatching (~1 month post-egg laying). Since temperature recordings from the bigfin reef squid natural habitats have shown a steady rise over the past decade, we examined energy substrate utilization and cellular/metabolic responses in developing animals under different temperature conditions. As the ambient temperature increased by 7 °C, hatching larvae favored aerobic metabolism by about 2.3-fold. Short-term environmental warming stress inhibited oxygen consumption but did not affect ammonium excretion in stage (St.) 25 larvae. Meanwhile, an aerobic metabolism-related marker (CoxI) and a cellular stress-responsive marker (HSP70) were rapidly up-regulated upon acute warming treatments. In addition, our simulations of temperature oscillations mimicking natural daily rhythms did not result in significant changes in metabolic processes in St. 25 animals. As the ambient temperature increased by 7 °C, referred to as heatwave conditions, CoxI, HSP70, and antioxidant molecule (SOD) were stimulated, indicating the importance of cellular and metabolic adjustments. As with other aquatic species with high metabolic rates, squid larvae in the tropical/sub-tropical climate zone undergo adaptive metabolic shifts to maintain physiological functions and prevent excessive oxidative stress under environmental warming.
... In mollusks, these molecules were suggested to play a major role in acid-base balance (Wang et al., 2017), mediating the accumulation of calcium in mantle and gill tissue, and enabling the biomineralization process (Cudennec et al., 2006;Miyamoto et al., 1996). It has also been proposed that carbonic anhydrases are involved in ion regulation processes (osmoregulation) by generating HCO 3 − that can serve as counter-ions in sodium (Na + ) and potassium (K + ) uptake (Henry and Saintsing, 1983;Hu et al., 2011). While carbonic anhydrases have been found abundantly in the gills of bivalves (Duvail et al., 1998;Henry and Saintsing, 1983), it remains unclear why this protein is more abundant in the dorsal tract. ...
In the oyster Crassostrea virginica , the organization of the gill allows bidirectional particle transport where a dorsal gill tract directs particles meant to be ingested while a ventral tract collects particles intended to be rejected as pseudofeces. Previous studies showed that the transport of particles in both tracts is mediated by mucus. Consequently, we hypothesized that the nature and/or the quantity of mucosal proteins present in each tract is likely different. Using endoscopy-aided micro-sampling of mucus from each tract followed by multidimensional protein identification technologies, and in situ hybridization, a high spatial resolution mapping of the oyster gill proteome was generated. Results showed the presence in gill mucus of a wide range of molecules involved in non-self recognition and interactions with microbes. Mucus composition was different between the two tracts, with mucus from the ventral tract shown to be rich in mucin-like proteins, providing an explanation of its high viscosity, while mucus from the dorsal tract was found to be enriched in mannose-binding proteins, known to be involved in food particle binding and selection. Overall, this study generated high-resolution proteomes for C. virginica gill mucus and demonstrated that the contrasting functions of the two pathways present on oyster gills are associated with significant differences in their protein makeup.
... Zakroff et al. (2019) discussed potential mechanisms of acidification impact to DML and YV, in the scope of a limited energy store and energy budget, suggesting that reductions in growth and YV under hatching are a potential product of upregulation and increased activity of energetically costly proton secreting transporters in ion-transport epithelia (Hu et al., 2010. The increased proportion of paralarvae showing inflation of the membrane around the eyes under increased OA may further suggest a breakdown in osmoregulatory controls, particularly given the prevalence of ionocytes in the epidermis of cephalopod embryos (Hu et al., 2011). Though it is also plausible that this inflation is related to poorly known osmotic mechanisms that cause the swelling of the egg capsule during development (Hu and Tseng, 2017). ...
Ocean acidification (OA) and warming seas are significant concerns for coastal systems and species. The Atlantic longfin squid, Doryteuthis pealeii, a core component of the Northwest Atlantic trophic web, has demonstrated impacts, such as reduced growth and delayed development, under high chronic exposure to acidification (2200 ppm), but the combined effects of OA and warming have not been explored in this species. In this study, D. pealeii egg capsules were reared under a combination of several acidification levels (400, 2200, and 3500 ppm) and temperatures (20 and 27°C). Hatchlings were measured for a range of metrics [dorsal mantle length (DML), yolk sac volume (YV), malformation, and hatching success] in three trials over the 2016 breeding season (May – October). Although notable resistance to stressors was seen, highlighting variability within and between clutches, reduced DML and malformation of the embryos occurred at the highest OA exposure. Surprisingly, increased temperatures did not appear to exacerbate OA impacts, although responses were variable. Time to hatching, which increased with acidification, decreased much more drastically under warming and, further, decreased or removed delays caused by acidification. Hatching success, while variable by clutch, showed consistent patterns of greater late stage loss of embryos under acidification and greater early stage loss under warming, highlighting the potential difference in timing between these stressors for this system, i.e., that acidification stress builds up and causes impacts over time within the egg capsule as the embryos grow and respire. High OA-exposed hatchlings from the warmer conditions often showed reduced impacts compared to those reared in ambient temperatures. This may be due to the increased developmental rate and subsequently reduced OA exposure time of embryos in the higher temperature treatment. These results indicate a substantive potential plasticity to multiple stressors during the embryonic development of this species of squid, but do not predict how this species would fare under these future ocean scenarios.
... Based on our results and from studies previously conducted on other cephalopod species, it appears that elevated CO 2 elicits a range of aerobic responses in cephalopods, which may be life stage dependent. The reduced oxygen uptake observed during the embryonic period in some cephalopods at elevated CO 2 (Rosa et al., 2014;Sigwart et al., 2016) might be expected, as acid-base regulatory mechanisms in cephalopods often remain rudimentary until respiration switches from cutaneous (via skin) to branchial (via gills) (Hu et al., 2011b). Although little is known about the acidbase regulatory capabilities of pygmy squid, those of bigfin reef squid have been thoroughly investigated. ...
... Although little is known about the acidbase regulatory capabilities of pygmy squid, those of bigfin reef squid have been thoroughly investigated. Bigfin reef squid, among other cephalopods, have evolved ion regulatory epithelia in both the gills (Hu et al., 2011a(Hu et al., , 2014 and skin cells (Hu et al., 2011b(Hu et al., , 2013, which are effective in coping with acid-base disturbances. ...
Squid and many other cephalopods live continuously on the threshold of their environmental oxygen limitations. If the abilities of squid to effectively take up oxygen are negatively affected by projected future carbon dioxide (CO2) levels in ways similar to those demonstrated in some fish and invertebrates, it could affect the success of squid in future oceans. While there is evidence that acute exposure to elevated CO2 has adverse effects on cephalopod respiratory performance, no studies have investigated this in an adult cephalopod after relatively prolonged exposure to elevated CO2 or determined any effects on aerobic scope. Here, we tested the effects of prolonged exposure (≥20% of lifespan) to elevated CO2 levels (∼1000 μatm) on the routine and maximal oxygen uptake rates, aerobic scope and recovery time of two tropical cephalopod species, the two-toned pygmy squid, Idiosepius pygmaeus and the bigfin reef squid, Sepioteuthis lessoniana. Neither species exhibited evidence of altered aerobic performance after exposure to elevated CO2 when compared to individuals held at control conditions. The recovery time of I. pygmaeus under both control and elevated CO2 conditions was less than 1 hour, whereas S. lessoniana required approximately 8 hours to recover fully following maximal aerobic performance. This difference in recovery time may be due to the more sedentary behaviours of I. pygmaeus. The ability of these two cephalopod species to cope with prolonged exposure to elevated CO2 without detriment to their aerobic performance suggests some resilience to an increasingly high CO2 world.
... Depending on the mechanism through which pH balance is achieved, an organism may reach its limit through either increasing energetic costs or through the accumulation of bicarbonate (Fabry et al. 2008). Cephalopods are highly effective at pH balancing through ion transport, but this process is considered energetically costly (Hu et al. 2011b. The results presented here indicate an OA threshold for the case of embryonic D. pealeii, which have a finite energy reserve, but this "threshold" may not apply to post-hatch paralarvae and later stages of development which are potentially capable of moving out of stressful pH environments and may supplement energy through feeding (Vidal and Haimovici 1998;Bartol et al. 2008). ...
Coastal squids lay their eggs on the benthos, leaving them to develop in a dynamic system that is undergoing rapid acidification due to human influence. Prior studies have broadly investigated the impacts of ocean acidification on embryonic squid, but have not addressed the thresholds at which these responses occur or their potential variability. We raised squid, Doryteuthis pealeii (captured in Vineyard Sound, Massachusetts, USA: 41°23.370'N 70°46.418′W), eggs in three trials across the breeding season (May–September, 2013) in a total of six chronic pCO2 exposures (400, 550, 850, 1300, 1900, and 2200 ppm). Hatchlings were counted and subsampled for mantle length, yolk volume, hatching time, hatching success, and statolith morphology. New methods for analysis of statolith shape, rugosity, and surface degradation were developed and are presented (with code). Responses to acidification (e.g., reduced mantle lengths, delayed hatching, and smaller, more degraded statoliths) were evident at ~ 1300 ppm CO2. However, patterns of physiological response and energy management, based on comparisons of yolk consumption and growth, varied among trials. Interactions between pCO2 and hatching day indicated a potential influence of exposure time on responses, while interactions with culture vessel highlighted the substantive natural variability within a clutch of eggs. While this study is consistent with, and expands upon, previous findings of sensitivity of the early life stages to acidification, it also highlights the plasticity and potential for resilience in this population of squid.
... Depending on the mechanism through which pH balance is achieved, an organism may reach its limit through either increasing energetic costs or through the accumulation of bicarbonate (Fabry et al. 2008). Cephalopods are highly effective at pH balancing through ion transport, but this process is considered energetically costly (Hu et al. 2011b. The results presented here indicate an OA threshold for the case of embryonic D. pealeii, which have a finite energy reserve, but this "threshold" may not apply to post-hatch paralarvae and later stages of development which are potentially capable of moving out of stressful pH environments and may supplement energy through feeding (Vidal and Haimovici 1998;Bartol et al. 2008). ...
... Zakroff et al. (2019) discussed potential mechanisms of acidification impact to DML and YV, in the context of a limited energy store and energy budget, suggesting that reductions in growth and yolk volume under hatching are a potential product of upregulation and increased activity of energeticallycostly proton secreting transporters in ion-transport epithelia (Hu et al. 2010. The increased proportion of paralarvae showing inflation of the membrane around the eyes under increased OA further suggests a breakdown in osmoregulatory controls, particularly given the prevalence of ionocytes in the epidermis of cephalopod embryos (Hu et al. 2011b). Though it is also plausible that this inflation is related to poorly known osmotic mechanisms that cause the swelling of the egg capsule during development (Hu and Tseng 2017). ...
