I'm planning an experimental design to investigate the gene expression of mussels under thermal stress conditions, to compare their responses.

The experiment includes a control tank and a treatment tank, with enough mussels to sample 4 time points (60 mussels per time point per treatment).

In our facilities, I have two separate recirculation systems (with heaters and coolers) with two tanks each.

Since the two tanks of one recirculating system are connected they cannot be considered as isolated tanks and therefore they are not replicates. I'm planning to distribute the control samples between the two tanks of system 1 and the treatment samples between the two tanks of system 2, and at each time point sample from both tanks of one system and pooling them together (e.g. 30 samples from tank 1 and 30 samples from tank 2 of the control system)

My question is: do I need to have tank replicates for this experiment? Or can I sample three groups of 60 mussels per timepoint and per treatment between the two tanks of one system? i.e. have sample replicates within the control temperature and the treatment temperature tanks at each timepoint?

Thanks!