The C. elegans protein kinase G ortholog, EGL-4, has been demonstrated to be involved in C. elegans egg laying and the initiation of dwelling, a behavioral state characterized in part by low rates of locomotion (Trent et al. 1983; Hao et al. 2011). Serotonin regulates C. elegans egg laying through all five identified serotonin receptors, and serotonin promotes the dwelling state through the mod-1 serotonin receptor (Trent et al. 1983; Hapiak et al. 2009; Flavell et al. 2013; Brewer et al. 2019). Given that serotonin plays a role in both the egg laying and dwelling behaviors that egl-4 regulates, we sought to determine if protein kinase G is required for C. elegans to respond to serotonin. Exogenous serotonin paralyzes wild-type C. elegans (Grel et al. 2012). The assays shown in Figure 1A indicate that one loss-of-function mutant of egl-4 and two independent loss-of-function mutants of the egl-4 paralog, pkg-2 are resistant to paralysis by serotonin, similar to the previously-known serotonin resistant mutant elpc-3(ok2452) (Grel et al.2012).The pkg-2(tm3878)and pkg-2(tm5814) alleles each carry deletions of sequences coding large portions of the conserved catalytic cGMP-dependent protein kinase domain of the PKG-2 protein, including the active site and ATP binding site (Hofmann et al. 1992), and thus we predict them to be null alleles. The egl-4(n479)mutation is an early stop codon prior to the kinase domain and n479 is thus also predicted to be a null allele (Fujiwara et al. 2002). The Mak lab isolated an egl-4 gain-of-function allele mg410 that gives rise to a K162N single amino acid substitution (Hao et al. 2011). This K162N mutation lies in the conserved pseudo-substrate motif of EGL-4, and is predicted to result in auto-phosphorylation even in the absence of cGMP, leading to constitutively active EGL-4 (Hao et al. 2011). Figure 1B demonstrates that this egl-4 gain-of-function mutant is hypersensitive to paralysis by serotonin, like the previously-known serotonin hypersensitive mutant mod-5(n3314) (Grel et al. 2012), and in contrast to the serotonin-resistant egl-4 and pkg-2 loss-of-function mutants. Taken together these data indicate that the protein kinase G, egl-4, and its paralog pkg-2 mediate serotonin-induced paralysis of C. elegans. How do the protein kinase G orthologs, EGL-4 and PKG-2, mediate serotonin signaling in C. elegans? Previously our lab performed a forward genetic screen to identify proteins involved in serotonin signaling (Grel et al. 2012). The proteins identified includedtwo serotonin receptors, SER-4 and MOD-1, and several of the other proteins were predicted to act in the SER-4 or MOD-1 pathways. SER-4 is a G protein coupled receptor (Olde and Mccombie 1997) and MOD-1 is a serotonin-gated chloride channel (Ranganathan et al. 2000). It is possible that protein kinase G is acting in the SER-4 or MOD-1 pathways to control the effects of serotonin on C. elegans locomotion. Alternatively, protein kinase G could act with the MOD-5 serotonin transporter (SERT). Prior work indicates that phosphorylation of mammalian SERT increases its activity and that protein kinase G acts in a pathway to stimulate SERT, although protein kinase G may not directly phosphorylate SERT (Miller and Hoffman 1994; Kilic et al. 2003; Ramamoorthy et al. 2007; Wong et al. 2012; Zhang et al. 2016). However, additional studies indicate that stimulation of cGMP pathways reduces SERT activity in certain cell types (Pogun et al. 1994; Asano et al. 1997). It is possible that C. elegans protein kinase G negatively regulates MOD-5 function.