A “deep trench of knowledge” separates developmental and ecological biologists focusing on marine crustaceans (Torres et al., 2019). The former mostly focus on specific processes that occur within a given developmental stage (e.g., neurogenesis) under optimal conditions. In contrast, ecological biologists usually attempt to link plastic responses (e.g., growth rates or trophic ecology consequences) to some key (yet specific) factors. Nevertheless, these two research lines share the need for information on real-world environmental contexts. Such information is key to address questions that link environmental conditions to inter-individual variability and its fitness consequences (Bosch et al., 2014; Gilbert, 2017; Sultan, 2017). In particular, inter-individual variability is typically associated with the levels of adaptability and resilience for a given species (Sunday et al., 2011; Foo et al., 2012; Schlegel et al., 2012; Forsman and Wennersten, 2016), which highlights the importance of understanding the key drivers of variability for both developmental and ecological disciplines.

Studying inter-individual variability in marine invertebrates with complex life cycles (i.e., those who experience metamorphosis from pelagic larvae to benthonic juveniles) is, however, beset by in situ logistic limitations to secure the monitoring of complete larval periods, and by the inherent difficulty of sampling in oceanic environments (Rey et al., 2019). As such, the extent of inter-individual variability in this type of organisms and the possible effects of varying environmental conditions on this trait have been largely overlooked (Calado and Leal, 2015). Indeed, marine invertebrates with complex life cycles often inhabit contrasting environments in their larval and adult forms, and shift dramatically from a pelagic to a benthic environment (e.g., Torres et al., 2021 and references therein). This change results in multiple potential drivers of inter-individual variability throughout their life cycle, thus highlighting the role of marine invertebrates with this type of development as model species to assess phenotypic variability throughout ontogeny (Duarte et al., 2014; Torres et al., 2019).

Aside from environmental drivers, inter-individual variability can also be affected by developmental processes. When considering embryogenesis, for example, it is important to know if the natural variability present in newly extruded embryos is propagated to later embryonic stages (Fernández et al., 2000; Leal et al., 2013) or if, on the other hand, initial embryonic inter-individual variability decreases over embryogenesis (Rosa et al., 2003; Rey et al., 2016a). Embryogenesis in marine invertebrates with complex life cycles, such as decapod crustaceans, is heavily dependent on lipidic reserves (Petersen and Anger, 1997 and references therein), which are known to vary throughout embryogenesis (Rey et al., 2015a) and with environmental conditions (e.g., Brillon et al., 2005) and/or with the nutritional status of females (e.g., Tuck et al., 1997; Racotta et al., 2003; Calado et al., 2010). A contrasting allocation of lipids from females to their embryos due to environmental or dietary drivers can determine the level of initial lipidic reserves available to fuel embryogenesis. Thence, the fitness of subsequent life stages can either be compromised or enhanced, and this effect likely repercusses on inter-individual variability throughout embryonic development (e.g., Rosa et al., 2007; Rey et al., 2016a). Ultimately, the performance and survival of decapod crustaceans with complex life cycles can be already compromised at hatching (e.g., Giménez, 2010; Rey et al., 2015b; Rey et al., 2016b) or even at oviposition if maternal effects are taken into consideration (see review by Calado and Leal, 2015). Indeed, early life stages can provide key information on how contrasting environmental conditions affect inter-individual variability (and performance) of these invertebrates with bi-phasic life cycles (Pechenik, 2006; Giménez, 2010; Rey et al., 2019; Rey et al., 2022).

The green crab Carcinus maenas develops through four planktonic zoeal stages, a single megalopae stage (semi-benthonic), and then undergoes metamorphosis to settle as a benthic juvenile that grows onto adulthood (Rice and Ingle, 1975). This species is a highly successful global invader (Carlton and Cohen, 2003; Young and Elliott, 2019), which makes it an optimal candidate to study the influence of environmental parameters (such as contrasting oceanographic conditions, namely downwelling and upwelling) as drivers of inter-individual variability. For instance, downwelling and upwelling repercuss on planktonic community composition and abundance (e.g., Ospina-Alvarez et al., 2010; Wilson et al., 2021). In turn, this affects C. maenas throughout its different life stages, as they ingest plankton during larval life (Pihl, 1985) and predate on important planktotrophs as adults (Palacios and Ferraro, 2003). Consequently, the variability experienced by C. maenas in such natural settings provides an excellent opportunity to investigate how these oceanographic events shape the inter-individual variability of this model species (Domingues et al., 2011).

The present study determined the effects of different developmental and environmental factors on inter-individual variability throughout the early life stages of the model species C. maenas. The following hypotheses were tested: i) the dominant oceanographic condition (up- or downwelling) is an important determinant of heterogeneity among embryos, and ii) variability at hatching (irrespective of the source) affects the degree of inter-individual variability of later developmental stages. The hypotheses were addressed by assessing the stable isotopic niche variability of both individual embryos and megalopae, and the elemental composition of the latter, sampled during contrasting oceanographic events (downwelling and upwelling). We also determined the biochemical profile (phospholipid, elemental composition and isotopic signature) of newly extruded and late-stage embryos to assess maternal effects and embryogenesis as potential sources of inter-individual variability. Altogether, the collected data provide an overview of different sources of inter-individual variability and their potential fitness consequences (e.g., Tang and Dam, 1999).

Two main questions addressed in this study using the brachyuran crab C. maenas as a model species were: i) whether downwelling and upwelling events differently affect inter-individual variability of organisms with complex life cycles, and ii) how different developmental stages can drive variability among individuals. To answer these questions we focused on the early life stages of this species, namely their embryos and megalopae, as their influence on early benthic life stages is already documented (e.g., Giménez, 2010; Rey et al., 2015b; Rey et al., 2016b) through carry-over effects (also known as legacy effects) with potential phenotypic and fitness consequences (e.g., Pechenik, 2006; Giménez, 2010; Rey et al., 2019). In summary, downwelling and upwelling events differently affected inter-individual variability, particularly through the trophic ecology of females and of megalopae during planktonic life.

One possible source of inter-individual variability in early life stages concerns maternal effects, which can be defined as “the effects of adult conditioning on offspring quality” as in Pechenik (2006) and references therein. Here, maternal effects were assessed through the consequences of female biomass on the volume and elemental composition of their embryos ( Figure 1 ), despite acknowledging that these effects can also be accounted using other parameters (e.g., cortisol levels in McCormick, 1998). The reason why female size was considered as a plausible source of maternal effects concerns the fact that larger females can secure more and/or better food, which improves the allocation of reserves to their embryos (Calado and Leal, 2015) and ultimately allow larvae to hatch in better conditions than those from smaller females. Indeed, energetic reserves are significantly correlated with embryonic size in various decapod species (Rosa et al., 2007). We thus hypothesized that stage 1 embryos from larger C. maenas females could be fitter than those from smaller females, and consequently display a higher volume and/or C:N ratio. However, neither the volume of stage 1 embryos ( Figure 1A ) nor the C:N ratio ( Figure 1B ) were affected by the size of the ovigerous female (p > 0.05 on all cases), suggesting that female size does not significantly contribute as a driver of inter-individual variability. While this observation is in agreement with previous findings for Paralithodes camtschaticus, where female weight did not affect embryo size (Swiney et al., 2013), it contrasts with the findings for the European lobster Homarus gammarus (Moland et al., 2010).

A common feature of embryogenesis in crustaceans is the catabolism of lipidic reserves (Petersen and Anger, 1997; Rosa et al., 2003; Rosa et al., 2005), which is supported by the differences observed between the lipidic reserves of stage 1 and stage 3 embryos ( Table 1 ). This consumption of the available lipid pool throughout embryo development could also explain the lower C and N contents, and C:N ratios observed in late-stage embryos, when compared with newly-extruded ones (e.g., Dawirs, 1981; Rosa et al., 2007). Nevertheless, it is worth mentioning that the C:N ratio remained unaltered within a given embryonic stage (p > 0.05 – Figure 1 ), despite these originating from females sampled at different oceanographic events, which is a clear sign of homeostasis in C. maenas’ embryos. Irrespective of developmental stage, different oceanographic events affected embryonic lipid content (PL content), with these being significantly lower during downwelling (p< 0.05 on all cases – Table 1 ). These differences in energetic reserves between supply events are natural (e.g., Brillon et al., 2005; Calado et al., 2010) and are expected to repercuss on the success of larval life, as well as post-metamorphosis (e.g., Giménez, 2010; Rey et al., 2015b; Rey et al., 2016b). Indeed, upwelling causes an ascent of nutrients from cooler and deeper waters to the surface, which reflects heavily on the food chain composition (e.g., Wilson et al., 2021) that becomes diatom-dominated (e.g., Fenchel, 1988; Sommer et al., 2002; Vidal et al., 2017). This shift in the dominant planktonic group alters the entire food chain, including the trophic ecology and nutritional status of C. maenas females that feed on planktivorous species (Pardal et al., 2006), as well as the extent of their energetic reserves (Tuck et al., 1997; Racotta et al., 2003).

The most prominent effect of different oceanographic events was recorded on the isotopic signature of embryos ( Figure 2 ). As these tissues display fast turnover rates (e.g., Dimitrijević et al., 2018), their isotopic signature is strongly shaped by maternal diet prior oviposition. Thus, the isotopic signature of embryos can be used as a reliable proxy to estimate the isotopic niche of ovigerous females (e.g., Kaufman et al., 2014). A species’ isotopic niche is tightly correlated with its trophic niche, despite not being the same concept (Post, 2002; Jackson et al., 2011). The trophic niche of a predator is determined by its diet (Moore and Semmens, 2008; Parnell et al., 2010), whereas the isotopic niche is often beset by the consumer physiology (although in predictable amounts, thus the value of such comparison – e.g., (Post, 2002), Bearhop et al. (2002); Fox-Dobbs et al. (2007). In this regard, the δ¹³C values can be used to determine the variability of dietary carbon sources (e.g., Jackson et al., 2011; Yeakel et al., 2016) due to different incorporation rates across prey species (Layman et al., 2007). On the other hand, δ¹⁵N values are better suited to determine the trophic level of individuals (Layman et al., 2007; Jackson et al., 2011). Therefore, one can observe a direct effect of the supply event on the trophic ecology of ovigerous females: during downwelling females eat more diverse food (higher δ¹³C variability and range) and show a notably higher trophic level than females collected during upwelling conditions (higher δ¹⁵N value). The average δ¹⁵N signature during downwelling was ca. 1.97‰ higher than that during upwelling, which denotes an enrichment within the expected values of a one-level trophic variation; between 1.3 and 5.3‰, with an average of ca. 3.4‰ (Minagawa and Wada, 1984; Wada et al., 1987; Fry, 1988). In other words, C. maenas females predate on a shorter food chain during upwelling than they do during downwelling, with the diversity of prey items being lower. Adults C. maenas predate on important consumers of planktonic communities (e.g., Pihl, 1985; Palacios and Ferraro, 2003), whose abundance (e.g., Ospina-Alvarez et al., 2010) and composition (e.g., Wilson et al., 2021) is heavily influenced by oceanographic events and seasons (Pardal et al., 2006). Altogether, results suggest that downwelling increases the diversity of food sources being used and the length of the food chain, thus shaping the trophic ecology of adult C. maenas ( Figure 2 ). These consequences of downwelling are in agreement with previous studies (e.g., Tarran et al., 1999). Indeed, the lack of a dominant planktonic group during downwelling events, such as diatoms that dominate the phytoplankton community during upwelling conditions (Fenchel, 1988; Sommer et al., 2002), might force a diversification of prey items (and increase competition for resources – e.g., Duarte et al., 2017); under this scenario the range of δ¹³C values increases for a given species (Jackson et al., 2011; Yeakel et al., 2016). Therefore, the change in the trophic ecology of C. maenas in response to contrasting oceanographic events can be deemed responsible for the changes in the lipidic reserves displayed by its embryos ( Table 1 ). Additionally, results suggest that the higher inter-individual variability observed in the isotopic profile of ovigerous females during downwelling conditions is retained throughout embryogenesis.

Cascade-like effects, where early life histories determine the success or demise of later developmental stages, have been previously investigated in marine invertebrates, particularly in decapod crustaceans (e.g., Giménez, 2010; Rey et al., 2015b). Yet, few studies have addressed such impacts at the individual level due to the inherently complex process of sampling and analysing individual larvae (Rey et al., 2019). Here we assessed whether the inter-individual variability during embryonic development was associated with maternal effects, as well as contrasting oceanographic conditions. One other aspect was whether megalopae would still exhibit measurable inter-individual variability that could be traced back to the influence of distinct larval supply events. It is worth noticing that despite having data from distinct developmental stages in C. maenas, our study does not provide information on the effect of development per se as a possible source of inter-individual variability, as this would require following the same individuals of a given population across multiple developmental stages.

The larger megalopae of C. maenas observed during upwelling ( Figure 3 ) suggest that the higher lipidic reserves observed in upwelling embryos ( Table 1 ) likely benefited the organisms in subsequent developmental stage (also confirmed by the higher C:N ratio recorded during upwelling – Figure 4 ). Yet, it could have been beneficial to account for other biochemical parameters (other than lipidic reserves) that also affect a larvae’s development into megalopae, such as the protein or carbohydrate content, for example (McAlister and Moran, 2012). In any case, our observations are in accordance with Rey et al. (2017), who suggested that upwelling produced higher quality lipids in C. maenas embryos and improved larval quality and chances to successfully metamorphose. Furthermore, our results match the model predictions of Rey et al. (2016b), where being a zoea during upwelling conditions promoted the growth of juvenile crabs. The larger an organism is, the faster it undergoes metamorphosis (Giménez, 2010; Calado and Leal, 2015), which can be useful both from a prey’s but also from a predator’s point of view. From the prey’s perspective, metamorphosis can make an organism large enough to attain a so called “safe size”, hence escape predation (Werner, 1991; Abrams et al., 1996) or, in the specific case of C. maenas, most likely cannibalism (Moksnes, 2004; Almeida et al., 2011). As a predator, metamorphosis in C. maenas involves the formation of claws and improves the overall strength of the organism, which grants it a competitive advantage over its competitors for food (Duarte et al., 2014). As such, the biomass data record (larger megalopae during upwelling conditions) suggest that individuals are better suited to deal with various biotic constraints during upwelling than during downwelling, as confirmed by the C:N ratio ( Figure 4 ) and the significant interaction of supply event ( Table 2 ). The reason behind having higher biomass and C:N ratios during upwelling may be explained by the dietary shifts that occur in the plankton, a direct consequence of shifting oceanographic conditions (see discussion below). Indeed, the fact that megalopae feed on plankton paired with the observations of healthy megalopae during downwelling, imply that embryonic reserves cannot completely explain the success or demise of later life stages. In other words, a lipid-poor embryo does not necessarily imply failure as megalopae since success also depends on the conditions and resources (such as food) encountered as a megalopae, thus compensating the lack of initial energetic reserves (see Swearer et al., 2019 and references therein). It is also important to highlight that the higher and less variable C:N ratio (in megalopae) during upwelling suggests no absolute homeostasis and implies that these individuals are in better conditions when upwelling is the dominant oceanographic condition. Our results thus agree with previous observations that organisms deal with a changing environment by adjusting homeostatic mechanisms over time i.e., exhibit rheostasis (e.g., Villar-Argaiz et al., 2002; Leal et al., 2017).

Another important aspect to highlight is the lower inter-individual variability of megalopae isotopic signature during upwelling than downwelling. As previously discussed, the available range of preys greatly affects the trophic ecology of C. maenas. The trophic level of the megalopae ( Figure 5 ) suggests that, during upwelling, the organisms are a part of a shorter and more efficient food chain (e.g., Fenchel, 1988; Sommer et al., 2002). As observed by Duarte et al. (2017) for a different decapod species, a habitat with a surplus of food (equivalent to upwelling conditions in our study) enables feeding on preferred food items, which narrows down the isotopic niche of a species. In the opposite scenario (downwelling), megalopae cannot be selective and must feed on whatever preys are available. This generalist feeding behaviour often implies a higher competition for resources, with intra/inter-specific competition being known to affect the magnitude of inter-individual variability within a population (Araújo et al., 2011). Altogether, this information supports our observation of a wider (i.e., more variable) isotopic niche in megalopae developing during downwelling events when contrasted to conspecifics developing during upwelling ( Figure 5 ).

Our combined observations in C. maenas adults, newly extruded and late-stage embryos, and megalopae suggest that contrasting oceanographic conditions of downwelling and upwelling greatly affect inter-individual variability. However, it must be highlighted that the conclusions attained at adult level (i.e., with ovigerous females) should be taken with caution, as sampling 20 females per oceanographic event may underestimate diversity at population level. Overall, variability was higher during downwelling irrespective of the life stage, which can indicate that organisms can be more adaptable and resilient during these oceanographic events (Sunday et al., 2011; Foo et al., 2012; Schlegel et al., 2012). However, upwelling resulted in higher lipidic reserves and C:N ratios in embryos. Altogether, it appears that hatching and growing during upwelling conditions results not only in larger individuals but also in better fitness conditions and, ultimately, more resilient populations. Nevertheless, during downwelling the higher inter-individual variability recorded highlights the remarkable ability of C. maenas to cope and thrive in highly variable environments, likely being key for this species success as a global invader.

Data is available at Zenodo and can be cited using this reference: Leal, Miguel Costa, Rey, Felisa, Ferreira, Guilherme Duarte, Domingues, M. Rosário, & Calado, Ricardo. (2022). Inter-individual variability of early life stages of a model marine invertebrate with a bi-phasic life cycle is shaped by contrasting oceanographic conditions [Data set]. https://doi.org/10.3389/fmars.2022.984569.

ML, FR, MD, and RC conceptualized the experiments. RC, MD and ML acquired funding and provided materials and facilities. FR collected the samples and conducted the laboratory experiments. ML performed the laboratory analysis. ML and GF analysed the data and prepared the figures. GF and ML prepared the original draft manuscript. All authors commented and contributed to the final version of the manuscript thus justifying all authorships.

This work was supported by national funds, through Fundação para a Ciência e Tecnologia (FCT), in the scope of the Individual Call to Scientific Employment Stimulus 2017 with a Junior Researcher contract [CEECIND/00580/2017 to FR] and an Assistant Researcher contract [CEECIND/01618/2020 to ML]. We also acknowledge financial support to CESAM by FCT/MCTES (UIDP/50017/2020+UIDB/50017/2020+ LA/P/0094/2020), through national funds, and to national funding through FCT, within the research project, NO RESET PTDC/BIA-BIC/116871/2010. ML was also supported by EAWAG funding. The authors also acknowledge the contribution of Serge Robert and Inês Carvalho during the laboratory analysis.

The authors acknowledge the contribution of Serge Robert and Inês Carvalho during the laboratory analysis.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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