Crustacean larvae have served as distinguished models in the field of Ecological Developmental Biology (“EcoDevo”) for many decades, a discipline that examines how developmental mechanisms and their resulting phenotype depend on the environmental context. A contemporary line of research in EcoDevo aims at gaining insights into the immediate tolerance of organisms and their evolutionary potential to adapt to the changing abiotic and biotic environmental conditions created by anthropogenic climate change. Thus, an EcoDevo perspective may be critical to understand and predict the future of organisms in a changing world. Many decapod crustaceans display a complex life cycle that includes pelagic larvae and, in many subgroups, benthic juvenile–adult stages so that a niche shift occurs during the transition from the larval to the juvenile phase. Already at hatching, the larvae possess a wealth of organ systems, many of which also characterise the adult animals, necessary for autonomously surviving and developing in the plankton and suited to respond adaptively to fluctuations of environmental drivers. They also display a rich behavioural repertoire that allows for responses to environmental key factors such as light, hydrostatic pressure, tidal currents, and temperature. Cells, tissues, and organs are at the basis of larval survival, and as the larvae develop, their organs continue to grow in size and complexity. To study organ development, researchers need a suite of state-of-the-art methods adapted to the usually very small size of the larvae. This review and the companion paper set out to provide an overview of methods to study organogenesis in decapod larvae. This first section focuses on larval rearing, preparation, and fixation, whereas the second describes methods to study cells, tissues, and organs.

Cells and tissues form the bewildering diversity of crustacean larval organ systems which are necessary for these organisms to autonomously survive in the plankton. For the developmental biologist, decapod crustaceans provide the fascinating opportunity to analyse how the adult organism unfolds from organ Anlagen compressed into a miniature larva in the sub-millimetre range. This publication is the second part of our survey of methods to study organogenesis in decapod crustacean larvae. In a companion paper, we have already described the techniques for culturing larvae in the laboratory and dissecting and chemically fixing their tissues for histological analyses. Here, we review various classical and more modern imaging techniques suitable for analyses of eidonomy, anatomy, and morphogenetic changes within decapod larval development, and protocols including many tips and tricks for successful research are provided. The methods cover reflected-light-based methods, autofluorescence-based imaging, scanning electron microscopy, usage of specific fluorescence markers, classical histology (paraffin, semithin and ultrathin sectioning combined with light and electron microscopy), X-ray microscopy (µCT), immunohistochemistry and usage of in vivo markers. For each method, we report our personal experience and give estimations of the method’s research possibilities, the effort needed, costs and provide an outlook for future directions of research.

Current understanding of species capacities to respond to climate change is limited by the amount of information available about intraspecific variation in the responses. Therefore, we quantified between- and within- population variation in larval performance (survival, development, and growth to metamorphosis) of the shore crab Carcinus maenas in response to key environmental drivers (temperature, salinity) in 2 populations from regions with contrasting salinities (32-33 PSU: Helgoland, North Sea; 16-20 PSU: Kerteminde, Baltic Sea). We also accounted for the effect(s) of salinity experienced during embryogenesis, which differs between populations. We found contrasting patterns between populations and embryonic salinity conditions. In the Helgoland population, we observed a strong thermal mitigation of low salinity stress (TMLS) for all performance indicators, when embryos were kept in seawater. The negative effects of low salinity on survival were mitigated at increased temperatures; only at high temperatures were larvae exposed to low salinity able to sustain high growth rates and reduced developmental time, thereby metamorphosing with comparable levels of carbon and nitrogen to those reared in seawater. By contrast, larvae from the Kerteminde population showed a detrimental effect of low salinity, consistent with a maladaptive response and a weak TMLS. Low salinity experienced during embryogenesis pre-empted the development of TMLS in both populations, and reduced survival for the Kerteminde population, which is exposed to low salinity. Our study emphasises the importance of evaluating species responses to variation in temperature and salinity across populations; the existence of maladaptive responses and the importance of the maternal habitat should not be underestimated.

Species’ responses to climate change may vary considerably among populations. Various response patterns define the portfolio available for a species to cope with and mitigate effects of climate change. Here, we quantified variation in larval survival and physiological rates of Carcinus maenas among populations occurring in distant or contrasting habitats (Cádiz: Spain, Helgoland: North Sea, Kerteminde: Baltic Sea). During the reproductive season, we reared larvae of these populations, in the laboratory, under a combination of several temperatures (15–24 °C) and salinities (25 and 32.5 PSU). In survival, all three populations showed a mitigating effect of high temperatures at lower salinity, with the strongest pattern for Helgoland. However, Cádiz and Kerteminde differed from Helgoland in that a strong thermal mitigation did not occur for growth and developmental rates. For all populations, oxygen consumption rates were driven only by temperature; hence, these could not explain the growth rate depression found at lower salinity. Larvae from Cádiz, reared in seawater, showed increased survival at the highest temperature, which differs from Helgoland (no clear survival pattern), and especially Kerteminde (decreased survival at high temperature). These responses from the Cádiz population correspond with the larval and parental habitat (i.e., high salinity and temperature) and may reflect local adaptation. Overall, along the European coast, C. maenas larvae showed a diversity of responses, which may enable specific populations to tolerate warming and subsidise more vulnerable populations. In such case, C. maenas would be able to cope with climate change through a spatial portfolio effect.