## 4 Discussion  
It is well known that changes in environmental conditions, such as temperature, salinity, dissolved oxygen, pH, food availability, and contamination, produce alterations in the immune and physiological functions of bivalves [20; 46; 47]. One of the most important environmental factors involved in immunomodulation is water temperature and salinity [48]. Several studies have shown that changes in temperature or salinity compromise different defense mechanisms of bivalves [20; 49]. Earlier works with _C. gallina_ and other bivalve species (e.g., _Crassostrea gigas_, _Ostra edulis_, and _Mytilus_ sp.) have found that LMS is sensitive to temperature fluctuations in both laboratory and field studies [50; 51; 52]. In contrast, few studies have evaluated the combined effects of both factors on LMS, in species such as _Ostra edulis_, _Ruditapes philippinarum_ and _Anadara trapezia_[53; 54; 55]. While it has been observed that phagocytic activity in _C. gallina_ is altered differently depending on whether environmental parameters change individually or in combination [56], this study is the first to assess the combined effect of the environmental factors temperature and salinity on the stability of the lysosomal membrane in _C. gallina_ and _D. trunculus_. Figure 6: **(a)** Neutral red retention time (NRRT) and **(b)** lysosomal membrane stability percentage (%LMS) in _D. trunculus_ after 21 days of exposure to combined conditions of temperature (low, medium, and high) and salinity ranges (27–28, 32–33, and 37–38). Background assessment criteria (BAC). Environmental assessment criteria (EAC). Data are reported as mean \(\pm\) SE, \(n=16\). Lowercase letters above the bars indicate inclusion within homogeneous subsets (2-way ANOVA; [PERSON]’s post hoc test; \(p\)-value \(<0.001\)). In this study, we found that certain combined conditions of seawater temperature and salinity maintained for at least 21 days are capable, by themselves, of causing significant cellular stress by affecting lysosomal membrane permeability in the clam species _D. trunculus_ and _C. gallina_. We found a differential response to temperature, dependent on acclimation salinity, which has also been observed in other bivalves [53]. In _C. gallina_, at salinities of 32-33, the neutral red was retained within the lysosomal compartment for the shortest duration at 20 \({}^{\circ}\)C, increasing as the acclimation salinity was raised or lowered (Figure 2). However, an opposite pattern was observed in the treatments conducted at 12 \({}^{\circ}\)C, with longest retention times at 32-33 salinities. At the highest temperature (27.5 \({}^{\circ}\)C), no significant changes in lysosomal membrane stability were found at different salinities. Furthermore, our findings show that this differential response varied between the clam species under study. The lysosomal permeability to intermediate salinities (32-33) at 12 \({}^{\circ}\)C and 20 \({}^{\circ}\)C in _C. gallina_ has opposite patterns in comparison to _D. trunculus_. Different patterns of variation in total hemocytes counts has been described between _C. gallina_ and _Mytilus galloprovincialis_ submitted to the same environmental conditions [56], which partially supports the differential interspecies responses found on LMS in our study. In both species, the LMS at 27.5 \({}^{\circ}\)C showed little response to changes in salinity. Consequently, our findings show that the adaptive capacity of the cellular processes in these species was reduced at the highest temperature tested. For _D. trunculus_, at the highest tested temperature (27.5 \({}^{\circ}\)C), the NR remained within the lysosomes for only a short time (Figure 6), signaling that the lipid membranes within the cell were less stable. It is known that the interaction between two or more environmental variables may cause changes in other variables [57], such as a decrease in dissolved oxygen (DO) in the water, which may cause stress in organisms [58; 59].