Shell mineralogy and organic composition
This is the first study to characterize the calcium carbonate polymorphs and organic composition of T. atra shells. Here, we found that T. atra has a shell comprising of two calcium carbonate forms (i.e., calcite and aragonite), that consists of a calcite layer at the outer shell portions and a thick aragonite layer at the inner shell area. The results obtained an organic composition of the shell showing that carbonate signals were inversely proportional to relative organic composition content (e.g., proteins). Similar results on organic compounds and carbonate signal relationships have been reported for other mollusc species such as limpets (e.g., García- Huidobro et al. 2020), and scallops (e.g., Lagos et al. 2021). The aragonite content of the mollusc shells has been proposed to be more vulnerable than those with calcite mineral in response to environmental stressors (e.g., low pH/high pCO2 levels; Barclay et al. 2019). Thus, more studies to evaluate the implications of environmental stressors on shell formation and/or the compensatory mechanisms of herbivore calcifying organisms are needed, and our results suggest that these investigations are warranted (see Lagos et al. 2021). Considering that consuming food of higher nutritional quality can provide more energy for calcifying organisms, the energy intake through food consumption can influence their shell quality/integrity (e.g., organic compounds, resistance to breakage, etc). Therefore, the quality of food becomes even more important to better understand potential vulnerability and/or adaption of organisms under climate change scenarios (e.g., Leung et al. 2019) at different levels of the trophic web (e.g., algae-herbivore interactions). Trade-offs between energetic physiology and skeletal production (i.e., biomineralization) under environmental stressors have been reported in other snail species (e.g., Littorina littorea; Melatunan et al. 2013), (e.g., Eatoniella mortoni; Leung et al. 2019).
Nutritional quality of kelp species and feeding behavior
Our results show that feeding preference of T. atra appears to be based on nutritional quality of kelps. The macroalgae L. spicata presented the highest nutritional value (i.e., organic matter and protein content) compared with M. pyrifera and was preferentially consumed by T. atra. Furthermore, the snails reared with L. spicata (i.e., the most nutritious kelp) achieved the highest oxygen uptake and absorption efficiency but not growth rate.
Nutritional quality of macroalgae affects the feeding behavior of marine herbivorous organisms (Pennings et al. 1998; Pennings and Paul 1992; Pansch et al. 2008), and their biochemical components (e.g., protein content), which has been described as one of the most important constituents to influence food preferences (Barile et al. 2004). Thus, the algae that contain the greatest amount of protein are mostly consumed by grazers (e.g., Barile et al. 2004). Preferential feeding on algae with high content of organic matter and/or proteins have also been reported for other herbivorous organisms such as echinoderms (e.g., Loxechinus albus; González et al. 2008), amphipods (e.g., Orchestoidea tuberculata; Duarte et al. 2010, 2011; Gammarus mucronatus and Elasmopus levis; Cruz-Rivera and Hay 2000), gastropods (e.g., Aplysia californica; Barile et al. 2004; Diloma nigerrima; Quintanilla-Ahumada et al. 2018), and another species of Tegula genus (e.g., Tegula funebralis; Steinberg 1985 and Tegula brunnea; Thornber et al. 2008). The results obtained in this study agree with these previous studies, as the nutritional quality of macroalgae affected the feeding behavior of T. atra, and also, the alga with the highest protein content (i.e., L. spicata) was preferred by this snail. In other species of Tegula the nutritional quality of algae species, and their influence on feeding preferences, have shown contrasting results. Previous studies have demonstrated a significant influence (e.g., T. funebralis; Steinberg 1985), influence in part (e.g., T. brunnea; Thornber et al. 2008), as well as no influence (e.g., T. brunnea, T. montereyi and T. pulligo; Watanabe 1985) on feeding preferences of grazers. However, herbivore food choice, and algal traits (e.g., nutritional quality, morphology differences etc), that this snail species uses to choose its food has scarcely been documented. Nevertheless, it is important to emphasize here that in addition to nutritional quality, palatability (i.e. morphology, shape and toughness), presence of chemical defences (e.g., secondary metabolites), availability and vertical gradient of macroalgae, and/or the combination of these factors (Thornber et al. 2008), have also been shown to influence the food choices of herbivores (Pennings et al. 1998; Pavia and Toth 2000; Granado and Caballero 2001; Jormalainen et al. 2005; Vergés et al. 2007; Rotini et al. 2018), which could modify the feeding behavior of grazers. Therefore, we do not exclude consideration of these factors which can influence the feeding behavior and performance of T. atra.
Physiological traits and growth rate
The increase in the quality and/or quantity of food items may increase the metabolic demands of gastropods (e.g., McSkimming et al. 2015). Shumway et al. (1993). These studies showed that the respiration rate of periwinkles Littorina littorea and Littorina obtusata increased by 40-60% when fed their preferred algae. More recently, Remy et al. (2017) demonstrated that amphipods Gammarus aequicauda reared with food with high nutritional quality showed higher respiration and absorption rates, and enhanced fitness, than amphipods reared with food items with low nutritional value. In this study, oxygen uptake of individuals reared on a diet of a single algal species was significantly different. More specifically, snails reared with L. spicata (the preferred kelp species) had significantly higher oxygen uptake than those fed with M. pyrifera. The higher oxygen uptake of T. atra reared with L. spicata was supported by high absorption efficiency that corresponded to high values of energy intake. Thus, snails fed with L. spicata incorporated more energy to take advantage of the high nutritional quality of this kelp species. Furthermore, individuals reared with M. pyrifera reduced their respiration rate to cope with an unsuitable food source (i.e., low nutritional value of these food items), which could be a response aimed at improving energy balance. Therefore, the reduction of metabolism could be interpreted as a compensatory mechanism of T. atra to maintain its growth rate under diets of low nutritional value.
A higher growth rate of individuals fed with the most preferred algae with the highest nutritional value would have been expected. However, in this study, the feeding preference of T. atra on L. spicata did not allow for an increase in growth rate compared to those snails fed with M. pyrifera. We hypothesized that the additional energetic cost of consuming L. spicata, and/or their processing capacity, could be greater than consuming M. pyrifera. For example, post-ingestive mechanisms of processing food, change of digestive enzymes, and increases in absorption efficiency of nutrients, have been demonstrated as some physiological adjustments involved to optimize nutrient extraction and utilization by herbivorous organisms (Foster et al. 1999; Cox and Murray 2006; Secor 2009). Hence, our results show that those individuals reared with the most nutritious alga, indicated that the energetic cost of consuming this alga was high, which would be reflected in its metabolic rate (i.e., highest oxygen consumption). In addition, the mechanical cost and digestive process associated with consuming dietary food that has a macronutrient composition can increase energy expenditure. For example, carbohydrates have more complex molecules and may require a higher energetic cost to process and be digested by T. atra. In addition, snails did not increase consumption of food items of comparatively lower nutritional quality (i.e., M. pyrifera), to achieve optimal growth in the absence of better-quality food. Our results also suggest that a decrease in metabolic activity was the behavioral/physiological mechanism used for snails to compensate for the low nutritional value of M. pyrifera, to maintain a positive growth rate. Consequently, our results showed no compensatory feeding strategy for T. atra. We do not know if the compensatory physiological performance (i.e., decreased metabolic rate) observed in T. atra fed with food of low nutritional quality is sustainable for longer periods of time, and/or if it would affect the fitness of the snail. It is important to emphasize that T. atra had high absorption efficiency, and acclimatation capacity, in the experimental conditions used. Our results indicate that individuals reared with both kelp species present positive and similar values for growth. Thus, we demonstrated that T. atra had high behavioral/physiological plasticity in response to nutritional quality by consuming the two kelp species studied here. This susceptibility for plasticity makes T. atra an ideal model for studying such plastic responses and associated trade-offs. The results of this study could help to better understand physiological traits of T. atra upon consuming kelp species (that differ in their nutritional quality). These kelp species and their grazers, commonly inhabit intertidal-subtidal areas of the Chilean coast and are important ecologically and economically. Considering that the impacts of global stressors (e.g., increasing seawater temperature and pCO2 levels) in marine environments will most likely increase, the net physiological demands on calcifying organisms. In addition, as herbivores are dependent on the nutritional quality of seaweeds to meet their energetic demands, any alterations in their palatability and/or nutritional content, due to the influence of these environmental stressors (or their interactions), may affect in a complex way their physiological traits (Duarte et al. 2016; Leung et al. 2019; Kinnby et al. 2021a, b), and also their fitness. Calcifying organisms have been shown to be particularly vulnerable to changing environments and because of this, understanding how molluscs construct and maintain their shells can help to better comprehend potential vulnerability and/or adaptative mechanisms of marine organisms to confront climate change (Barclay et al. 2020; Lagos et al. 2021). In coastal environments, grazer-seaweed interactions may be facing one of their biggest challenges, as environmental stress imposed on these relationships could influence community structure in the future (e.g., Jellison and Gaylord 2019; Grilo et al. 2019; Fieber and Bourdeau 2021; Kinnby et al. 2021a; Burnam et al. 2022). This highlights the need to understanding and to predict how rocky-shore systems may change in the near-future, and how these effects can modify marine herbivore-algae interactions.