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° 23.370N 70° 46.418´W), eggs in three trials across the breeding season (May-September, 2013) in a total of six chronic pCO 2 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 CO 2. However, patterns of physiological response and energy management, based on comparisons of yolk consumption and growth, varied among trials. Interactions between pCO 2 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.

Chronic embryonic exposure to ocean acidification (OA) has been shown to degrade the aragonitic statolith of paralarval squid, Doryteuthis pealeii, a key structure for their swimming behavior. This study examined if day-of-hatching paralarval D. pealeii from eggs reared under chronic OA demonstrated measurable impairments to swimming activity and control. This required the development of a novel, cost-effective, and robust method for 3D motion tracking and analysis. Squid eggs were reared in pCO 2 levels in a dose-dependent manner ranging from 400-2200 ppm. Initial 2D experiments showed paralarvae in higher acidification environments spent more time at depth. In 3D experiments, velocity, particularly positive and negative vertical velocities, significantly decreased from 400 to 1000 ppm pCO 2 , but showed non-significant decreases at higher concentrations. Activity and horizontal velocity decreased linearly with increasing pCO 2 , indicating a subtle impact to paralarval energetics. Patterns may have been obscured by notable individual variability in the paralarvae. Responses were also seen to vary between trials on cohort or potentially annual scales. Overall, paralarval swimming appeared resilient to OA, with effects being slight. The newly developed 3D tracking system provides a powerful and accessible method for future studies to explore similar questions in the larvae of aquatic taxa.

27Young animals found future cohorts and populations but are often particularly susceptible 28 to environmental changes. This raises concerns that future conditions, influenced by 29 anthropogenic changes such as ocean acidification and increasing oxygen minimum zones, will 30 greatly affect ecosystems by impacting developing larvae. Understanding the potential impacts 31 requires addressing present tolerances and the current conditions in which animals develop. 32Here, we examined the changes in oxygen and pH adjacent to and within normally-developing 33 squid egg capsules, providing the first observations that the egg capsules, housing hundreds of 34 embryos, had extremely low internal pH (7.34) and oxygen concentrations (1.9 µmol L -1

The whale shark Rhincodon typus is found throughout the world's tropical and warm‐temperate ocean basins. Despite their broad physical distribution, research on the species has been concentrated at a few aggregation sites. Comparing DNA sequences from sharks at different sites can provide a demographically neutral understanding of the whale shark's global ecology. Here, we created genetic profiles for 84 whale sharks from the Saudi Arabian Red Sea and 72 individuals from the coast of Tanzania using a combination of microsatellite and mitochondrial sequences. These two sites, separated by approximately 4500 km (shortest over‐water distance), exhibit markedly different population demographics and behavioral ecologies. Eleven microsatellite DNA markers revealed that the two aggregation sites have similar levels of allelic richness and appear to be derived from the same source population. We sequenced the mitochondrial control region to produce multiple global haplotype networks (based on different alignment methodologies) that were broadly similar to each other in terms of population structure but suggested different demographic histories. Data from both microsatellite and mitochondrial markers demonstrated the stability of genetic diversity within the Saudi Arabian aggregation site throughout the sampling period. These results contrast previously measured declines in diversity at Ningaloo Reef, Western Australia. Mapping the geographic distribution of whale shark lineages provides insight into the species’ connectivity and can be used to direct management efforts at both local and global scales. Similarly, understanding historical fluctuations in whale shark abundance provides a baseline by which to assess current trends. Continued development of new sequencing methods and the incorporation of genomic data could lead to considerable advances in the scientific understanding of whale shark population ecology and corresponding improvements to conservation policy.

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.

Background A recent global study of whale shark population genetics has allowed for better understanding of genetic connections between aggregations in both the Indo-Pacific and Atlantic. This overview included an aggregation found within the Red Sea near Al Lith, Saudi Arabia, however the Mafia Island, Tanzania, aggregation was not part of the study. The ecological behavior of these aggregations differs with the Saudi Arabian individuals showing strong seasonality, while acoustic telemetry data revealed cryptic residency at Mafia Island. Approach Genetic analysis using 11 microsatellite markers was performed on whale sharks from both locations. A combination of primers sourced from previous studies and newly designed primers were used to compare both aggregations and the individuals within. The Red Sea population was compared between 5 seasons spanning 6 years from 2010–2015. The Tanzanian population was compared for 2 field seasons from 2012–2014. Temporal genetic diversity was examined using allelic richness on only the Saudi Arabian individuals due to a short sampling period in Tanzania. Kinship for both aggregations was tested using COLONY and KINALYZER. Results Over a 6 year period, genetic diversity in the Red Sea showed no significant change. Contrasting to other whale shark aggregations, allelic richness in the Red Sea shows no sign of reduction. Kinship analysis using COLONY found two potential sibling pairs in Tanzania. One pair had a high probability (.993) of being a full sibling dyad while the other had a lower probability (.357). There were no sibling pairs identified in the Red Sea. Conclusions The lack of significant change of genetic diversity in Al Lith, Saudi Arabia, differs from a trend at Ningaloo Reef, Australia that showed a decrease in genetic diversity. Although these differences could be driven by location, this should encourage further long term genetic sampling at aggregations to better understand whale shark population trends. The potential of sibling pairs being found within one aggregation warrants further investigation into kinship within and between aggregations throughout the Indo-Pacific.

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