Discussion
This study provides, for the first time, information on burrowing behavior of the freshwater mussel C. ambigua using two distinct morphotypes, which are shaped by hydrodynamics and/or sexual dimorphism (Simeone et al., 2022). In our experiment, C. ambigua presented different patterns of shell exposure linked with shell shape and density. Although statistically significant differences were not found, both morphotypes tended to carry out greater horizontal movements at higher densities. Mussels may burrow to avoid predation or due to desiccation and/or flooding (Lymbery et al., 2021). Transporting mussels from their natural habitat to a manipulated one may have increased the stress of C. ambigua specimens. However, the experiment was carried out after a period of adaptation to reduce the effects of stress. Castalia ambigua has a wide distribution in South America (Pereira et al., 2014), therefore, our results may be used as a baseline to understand its burrowing behavior in other regions, taking into account potentially different shell morphologies.
The C. ambigua Morphotype I with a more elongated shell, remained less exposed in the lower density treatment (4 mussels). In this combination, all mussels remained with only the posterior margin above the sediment. Excavation is an important behavioral response of many mussel species to persist during periods of high flow (Lymbery et al., 2021; Sansom et al., 2022). Morphotype I is associated with high hydrodynamics (Simeone et al., 2022), suggesting that their burrowing behavior may be linked with their functional morphology (Hernández et al., 2021). For example, more elongated shells may allow mussels to remain buried more deeply and yet maintain their filtration apertures extended close to the sediment surface, facilitating filtration in more hydrodynamic areas. Interestingly, Morphotype I presented more exposed shells in treatments with higher densities (8 and 16 mussels). In these combinations, we observed more horizontal movements and a tendency for aggregation. These patterns are similar to those observed in all density treatments for C. ambigua Morphotype II with rounded shells that are mainly found in low hydrodynamic energy habitats. Extrapolating to the natural habitat in the Caeté River, the densities of 8 and 16 mussels used in our trials are equivalent to 32 and 64 mussels per m2 (Simeone et al., 2021b). We suggest that patches with high mussel densities may stabilize the substrate (Daniel et al., 2018; Christian et al., 2020), reducing the effect of hydrodynamics around their shells (Randklev et al., 2019). May & Pryor (2016) found similar results, with dense mussel beds stabilizing the riverbed in high hydrodynamic energy habitats. Remaining buried for long periods may prevent feeding from the water column (Curley et al., 2021, 2022). Therefore, a stable riverbed environment, likely created by the aggregation of C. ambigua, may allow the species to be more exposed, facilitating the filtration of food particles. Similar results are observed for sculptured mussels in temperate rivers, which remain more exposed in habitats with lower hydrodynamic energy (Hornbach et al., 2010; Newton, Zigler & Brian, 2015).
We suggest that C. ambigua aggregation may be associated with intrinsic factors of the species. However, there is a lack of information about the factors that initiate mussel aggregation (Archambault, Cope & Kwak, 2014). Studies suggest that this pattern may be associated with reproduction (Amyot & Downing, 1998; Schwalb, Morris & Cottenie, 2015; Kemble et al., 2020). For example, mussels may move horizontally and aggregate during the spawning period (Schwalb & Pusch, 2007). This behavior facilitates the capture of sperm by females that fertilize the eggs in the suprabranchial chamber (Schwalb et al., 2015; Kemble et al., 2020). Castalia ambigua can remain reproductively active throughout the year (Vale, Beasley & Tagliaro, 2004). However, we may not directly infer that the aggregation pattern is linked to reproduction, since our experiment was carried out in short, experimental trials in the laboratory. Another hypothesis is that aggregation may be associated with mussel feeding, in order to better access food in areas with higher concentrations of seston (Schwalb & Pusch, 2007).
The biomechanics of mussel burrowing may influence ecosystem processes in rivers (Allen & Vaughn, 2009), since their burrowing may mix sediments increasing oxygen inputs (Boeker et al., 2016) and the release of nutrients to the water column (Schwalb & Pusch, 2007). In our study, C. ambigua carried out more horizontal movements and are more exposed at the sediment surface at high densities, which may increase bioturbation (Vaughn & Hakenkamp, 2001). This behavior may directly affect associated macroinvertebrates, such as Chironomidae that generally occupy the infaunal layer of the riverbed (Simeone et al., 2021a).
In summary, the shell exposure and horizontal behavior observed in our study may be species dependent, since burrowing patterns may differ between mussel species (Allen & Vaughn, 2009). Castalia ambigua may vary in shell shape due to sexual dimorphism and effects of habitat hydrological variability (Simeone et al., 2022). Thus, additional studies using other mussel species belonging to the families Hyriidae and Mycetopodidae and the inclusion of different morphotypes are important, since the behavior of these mussels in the sediment may provide useful information on their functional roles in the river (Schwalb & Pusch, 2007).
 
Acknowledgments
This work was carried out as a part of GCR’s Master degree in Environmental Biology, Universidade Federal do Pará (UFPA). GCR thanks the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) for a postgraduate scholarship and the Programa de Pós-Graduação em Biologia Ambiental at the UFPA for logistic support. We are also grateful to the people of the Mocajuba settlement on the Caeté river for their kind assistance. Fieldwork was carried out under licenses 21864 and 21865 from the Instituto Chico Mendes de Conservação da Biodiversidade – ICMBio.
Data Availability Statement
Data associated with this manuscript will be available on Dryad after acceptance.
Conflict of Interest Statement
The authors declare that they have no conflicts or financial interest.
Author Contributions
Gisele do Carmo Reis: Conceptualization (equal); Experiment procedures (equal); Writing-original draft (lead). Diego Simeone: Conceptualization (equal); Experiment procedures (equal); Writing-review & editing (equal). Colin Robert Beasley: Conceptualization (equal); Formal analysis (lead); Writing-review & editing (equal).
 
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