Our study highlights that altered thermal regimes change the interaction among ICS and their impacts on leaf-litter breakdown in streams. As hypothesized, temperature increase enhanced leaf-litter breakdown and FPOM production by ICS, but this effect was species specific because it was only observed for P. clarkii. Interactions among ICS were antagonistic, consequently decreasing the impacts on this important ecosystem process. However, in a temperature increase scenario, the effects of both ICS in interspecific treatments became additive.
Contrary to our hypothesis, the indirect presence of ICS did not alter the activity of native invertebrates and microbes on leaf-litter breakdown. Because leaf decomposition is an ecosystem process where many species across multiple trophic levels are involved, some species, including omnivores, may display strong influence in the process dynamics by affecting biodiversity (through predation) or by reducing basal resources (Gessner et al. 2010; Carvalho et al. 2016). Invasive crayfish can, for example, change nutrient dynamics through their activity including feeding or burrowing behaviour (Shin-ichiro et al. 2009; Harvey et al. 2014) and consequently change nutrient concentrations affecting leaf-litter breakdown rates by freshwater microbes, particularly at higher temperatures (Fernandes et al. 2014). However, in our study, we were not able to detect differences in microbial leaf-litter breakdown among crayfish treatments. We used a short-term experiment and included a water renewal in the middle of the experiment, which probably led to low nutrient concentrations associated with crayfish activity and low accumulation of excreted products, consequently masking a potential response of microbial communities to crayfish. Microbial leaf-litter breakdown was enhanced by increasing temperature in our study. Other studies demonstrated that warming temperature might intensify leaf-litter breakdown by microbial communities in streams (Fernandes et al. 2014). Impacts of climate change have been widely studied particularly at individual or species population levels but few have used multi-trophic approaches (but see Woodward et al. 2010).
Also, native invertebrates (Sericostoma sp.) were only affected by temperature but not by the indirect risk of predation imposed by the crayfish presence. Naive responses of native invertebrates to invasive crayfish were previously reported in other studies (e.g. Carvalho et al. 2016). Exposure to predation risk may change the behaviour of native species leading to a decrease in their foraging activity or changing their strategies with potential indirect consequences for ecosystem functions (Klose and Cooper 2012). In our experiment, invertebrates were indeed protected from direct predation by crayfish and oak leaves inside their cages were able to provide shelter and food resources that could possibly explain no change in their behaviour. Beyond that, Sericostoma sp. individuals were captured at a site where they have low contact with the invasive crayfish and there are no native crayfish in those rivers. This situation is consistent with their naive behaviour. We should note that this lack of response might change in the future due to evolutionary pressures imposed by crayfish dispersion and population growth and co-occurrence between ICS with native invertebrates (e.g. Klose and Copper 2012; Carvalho et al. in press). Moreover, climate change is expected to increase co-occurrence between native and invasive species increasing the probability of competitive interactions for resource availability (Zeng and Yeo 2018). Because native invertebrates might have to compete for resources and avoid predation by invasive crayfish, this will probably constitute a challenge in the future. In this sense, extreme events such as droughts, which are predicted to increase in number and intensity in the studied region (Santos et al. 2015; Sousa et al. 2018; Nogueira et al. 2021), may indirectly favour crayfish impacts due to habitat dysconnectivity (intermittence) where in limited space and lower volume of water crayfish can easily affect direct resource consumption or predate on native species (Martinez 2012).
Temperature affected leaf-litter breakdown by crayfish, although those effects were species specific. Only P. clarkii increased leaf breakdown when compared with interspecific treatments at lower temperature. Because crayfish species are ectotherms, increasing temperatures are expected to enhance crayfish activity, including foraging and feeding behaviour (Rahel 2002). Invasive crayfish can tolerate temperatures around 30ºC (Souty-Grosset et al. 2006) and may be highly tolerant to desiccation (Larson et al. 2009) but crayfish responses to temperature changes may be species specific. For example, annual mean temperature is the main driver in the projected distributions of P. clarkii and P. leniusculus for the future, but it will affect both species in different ways benefiting mostly P.clarkii in Europe (Zhang et al. 2020). Impacts of invasive crayfish on leaf-litter breakdown and the associated biota may also be species specific (Dunoyer et al. 2014). In our experiment, the effect of P. leniusculus on leaf-litter breakdown did not vary with increasing temperature. Invasive P. leniusculus captured in the UK showed increasing feeding rates at higher temperatures (Rodríguez Valido et al. 2021) but its maximum performance was reached at a higher temperature (24ºC) than the maximum used in our experiment (18ºC). Males of P. leniusculus showed its maximum feeding rate at 20ºC (Simčič et al. 2014). No significant differences were found between the individual effects of both ICS, suggesting that both species have a strong impact on leaf-litter breakdown. Other studies suggested that ICS may functionally substitute vulnerable native invertebrate species (Stenroth and Nyström 2003). Our results show that P. clarkii. significantly increased their leaf-litter breakdown at higher temperatures. Other studies demonstrated that heat waves may shift the diet of P. clarkii towards an increasing consumption of plant material (Carreira et al. 2017).
Interestingly, interactive effects between P. leniusculus and P. clarkii significantly decreased their expected individual impacts on leaf-litter consumption possible due to competition. Procambarus clarkii is expected to have a more aggressive and dominant behaviour when foraging for food sources when compared with P. leniusculus (Meira et al. 2019). Other studies showed that native P. leniusculus occupies a higher trophic position than P. clarkii in lake ecosystems (Larson et al. 2017). However, in our experiment, crayfish were only able to feed on leaf litter. Although the invasion meltdown hypothesis postulated that invasive species may benefit from other invasive by facilitation or mutualism leading to a synergistic or additive effect on ecosystems (Simberloff and von Holle 1999), interactions among multiple invaders might also be negative (antagonistic) particularly in freshwater ecosystems and are more severe in omnivore species (Jackson 2015). Our results show that these antagonistic interactions on leaf-litter breakdown change with increasing temperature where effects between both species become additive. Based on our data, we can hypothesize that this effect resulted from enhanced leaf-litter breakdown by P. clarkii. Nonetheless we should carefully interpret our results because in our experiment leaf litter was the only food source for ICS although food availability was not limited. However, in natural ecosystems niche partitioning between invasive species may also facilitate the spread of multiple invasions and amplify their impacts on native biodiversity and ecosystems (Jackson et al. 2014). This indicates that although we used a simplified food web model, our results may help to clarify how these species interact and affect important ecosystem processes in freshwaters.
FPOM production by P. clarkii increased at higher temperature and was higher than that of P. leniusculus at higher temperature. ICS can functionally act as detritivores and play a significant role in detritus-based food webs contributing to produce FPOM, an important source of energy and carbon to stream biota (Carvalho et al. 2016). Again, we should carefully interpret our results because we limited food sources to leaf litter inside aquariums, oversimplifying natural conditions. For example, ICS may also have negative indirect impacts on detritus-based food webs through trophic cascades (Greig and McIntosh 2006). Our results showed expected positive and significant correlations between leaf-litter breakdown and FPOM production in intraspecific treatments, but interspecific crayfish interaction led to a non-significant correlation. Here competition may play an important role if crayfish increase the uptake of food resources to invest in body mass. Other studies reported increase in energy uptake and body mass in crustaceans when subjected to predation pressures (Glazier et al. 2020) and competition interaction may possibly lead to the same trends.