5-Hydroxytryptamine (5-HT) plays a vital role in the reproductive process of vertebrates and is also present in many invertebrates. The cDNA of the Sepiella japonica 5-HT₆ receptor (Sj5-HT₆r) was first cloned by RACE (Rapid Amplification of cDNA Ends). The length was 1450 bp, and the predicted open reading frame (ORF) was 1116 bp, which encoded 371 amino acids. Sequence characteristics analysis showed that Sj5-HT₆r shares a high degree of identity with 5-HT₆r from other cephalopods and forms a sister branch to bivalves. Subcellular localization showed that Sj5-HT₆r protein was localized on the HEK293T cell membrane surface. Quantitative Real-time PCR (qPCR) analysis demonstrated that Sj5-HT₆r was highly expressed in reproductive organs of both sexes. In particular, transcripts with significant expression were observed at stage III of female gonadal development in tissues of the ovary and nidamental gland, and at stage IV in tissues of the accessory nidamental gland. In situ hybridization (ISH) experiment results indicated that Sj5-HT₆r mRNA was primarily distributed in all regions of the optic lobes except the plexiform zone. These results may provide a basis for the future exploration of the reproductive regulation of 5-HT and 5-HT₆ receptors in S. japonica.

Neuropeptide F (NPF), an invertebrate homolog of vertebrate neuropeptide Y (NPY), exerts pleiotropic functions through its interaction with the G protein-coupled receptor (GPCR) neuropeptide F receptor (NPFR). However, the role of the NPF/NPFR system in the Chinese common cuttlefish Sepiella japonica Sasaki, 1929—a commercially and scientifically important cephalopod species in East China Sea aquaculture—remains unclear. In the present study, SjNPF/SjNPFR genes were cloned from S. japonica. Multiple alignments demonstrated that SjNPF/SjNPFR exhibited a high identity with that of other cephalopods. Spatio-temporal expression patterns revealed that SjNPF and SjNPFR transcripts were relatively highly expressed in the central nervous and digestive systems across all developmental stages. In situ hybridization (ISH) monitored clear and stable positive signals of SjNPF and SjNPFR mRNA at the junction of the subvertical lobe and the vertical lobe, as well as in the brachial lobe, pedal lobe and the palleovisceral lobe. Subcellular localization studies showed that SjNPF was primarily localized in the cytoplasm, whereas SjNPFR was membrane-localized. Moreover, under feeding-regulatory conditions (5-day starvation followed by 3-day refeeding), mRNA expression levels of SjNPF and SjNPFR in the treated group were positively correlated with starvation and negatively correlated with refeeding. These findings provide valuable insights for future investigations into the pleiotropic functional roles of the NPF/NPFR system in S. japonica and the peptidergic regulation of this system in cephalopods.