In one set of experimental infections, we supplemented D. magna with l-arginine, the NOS substrate, or with l-NAME, a NOS antagonist, and found this to result in lower and higher infection levels, respectively, which is at least compatible with the notion that NO may aid defence against Pasteuria. A second experiment indicated that NOS transcription does not increase following exposure to Pasteuria. Thus, the function of NOS in Daphnia immunity remains uncertain, but the pattern of gene duplication and subsequent divergence suggests evolution via neo- or subfunctionalization. Here, NOS2 is linked to infection.