Procedures in the laboratory and experiment
Prior to the experiments, we cultivated microalgae in the laboratory as food for the mussels, using a liquid NPK (nitrogen, phosphorus and potassium) gardening supplement, which resulted in excellent microalgal growth (Simeone, Tagliaro & Beasley, 2021a).
For the experiments, we prepared microcosms equipped with aerators that simulated a water current velocity of 0.2 m/s, which is the average velocity found in the habitats of C. ambigua in the Caeté River (Simeone, Tagliaro & Beasley, 2021c). Each microcosm contained water from the Caeté River and a sediment layer composed of sand and silt, approximating the sediment composition of the Caeté River (Simeone, Tagliaro & Beasley, 2021c).
For the experiments, we used three different Morphotype treatments:
· MI – Morphotype I of C. ambigua
· MII – Morphotype II of C. ambigua
· MI+II – Morphotype I + Morphotype II in equal proportions
We combined each morphotype treatment with three different mussel densities (4, 8 and 16 mussels; hereafter D4, D8 and D16). Three microcosms (replicates) were maintained simultaneously for each combination of morphotype and density: for example, three microcosms for MI with D4, and so on. All microcosms were maintained for 6 days, with three observations made on days two, four and six. A total of 81 observations (9 combinations × 3 replicates × 3 days of observations) were obtained for the experiment, similar to that obtained by Allen & Vaughn (2009). Between each set of combinations, we replaced the water in the microcosms.
We marked each mussel on the central and posterior parts of the shell using shellfish glue-on tags. Thus, horizontal positions and shell exposure could be recorded for each set of observations. Horizontal movements (cm) were calculated by summing changes in the position of each mussel, using 5×5 cm grids that were placed over the microcosms without touching the water. The average values of shell exposure at the sediment-water interface were estimated by measuring (mm) the exposed part of each mussel shell using a caliper (precision of 0.05 mm).
Statistical analysis
All statistical analyzes were carried out in GNU R (R Core Team, 2022). We used a two-way analysis of variance (ANOVA) with repeated measures to test whether the burrowing behavior of C. ambigua varied between the three mussel densities and among the two morphotypes. The treatments Morphotype (MI, MII and MI+II) and Density (D4, D8 and D16) were used as fixed effects. The random effect was included with the factor Replicate (n = 3 replicates) nested in the factor Day (days of observations: two, four and six) as a repeated measure. Mixed-effects models provide a flexible and powerful tool for modeling the within-group correlation often present in repeated measures data (Pinheiro & Bates, 2000; Zuur et al., 2009).
Results
Castalia ambigua presented a significantly high shell exposure for MI in treatments D8 and D16, but not for D4 (Table 1; Figure 2). On the other hand, MII and MI+II presented high shell exposure for all mussel densities (Figure 2). For horizontal movements, Morphotype effect and the Morphotype by Density interaction were not significant (Table 1; Figure 2). However, both morphotypes showed a marginally significant increase in horizontal movements in the treatment D16 for MI and in the treatments D8 and D16 for MII (Table 1; Figure 2).