Neuropeptidome of the Cephalopod Sepia officinalis: Identification, Tissue Mapping, and Expression Pattern of Neuropeptides and Neurohormones during Egg Laying

Abstract

Cephalopods exhibit a wide variety of behaviors such as prey capture, communication, camouflage, and reproduction thanks to a complex central nervous system (CNS) divided into several functional lobes that express a wide range of neuropeptides involved in the modulation of behaviors and physiological mechanisms associated with the main stages of their life cycle. This work focuses on the neuropeptidome expressed during egg-laying through de novo construction of the CNS transcriptome using an RNAseq approach (Illumina sequencing). Then, we completed the in silico analysis of the transcriptome by characterizing and tissue-mapping neuropeptides by mass spectrometry. To identify neuropeptides involved in the egg-laying process, we determined (1) the neuropeptide contents of the neurohemal area, hemolymph (blood), and nerve endings in mature females and (2) the expression levels of these peptides. Among the 38 neuropeptide families identified from 55 transcripts, 30 were described for the first time in Sepia officinalis, 5 were described for the first time in the animal kingdom, and 14 were strongly overexpressed in egg-laying females as compared with mature males. Mass spectrometry screening of hemolymph and nerve ending contents allowed us to clarify the status of many neuropeptides, that is, to determine whether they were neuromodulators or neurohormones.

Egg-Laying in the Cuttlefish Sepia officinalis

“…Thanks to the sequencing of the neurotranscriptome, several neuropeptide families involved in the regulation of egg-laying were identified on the basis of expression pattern and tissue localization out of the 38 families composing the cuttlefish neuropeptidome [10]. Finally, the RNA sequencing of ovary tissue revealed that most of the ovarian regulatory peptides involved in oocyte release were cleaved from a single yolk protein (unpublished results).…”

“…Most of the eight neuropeptides predicted from the two protein precursors were detected by nLC-MS/MS in cuttlefish CNS, oviduct gland, main nidamental glands, and ovarian stroma [10]. It is the only neuropeptide family detected in the ovarian stroma, suggesting a putative involvement in the release of smooth oocytes into the genital coelom.…”

“…The identification of the cuttlefish neuropeptidome by de novo RNAseq and mass spectrometry screening was the next step that provided an overview of the neuropeptidome via a deep structural and functional investigation [10].…”

“…The three neuropeptides predicted from the protein precursor of CCAPs were detected by nLC-MS/MS in the CNS, the oviduct gland and the main nidamental glands [10]. CCAPs are also overexpressed in the sub-esophageal mass of egg-laying females (as opposed to mature males), which is the only part of the CNS that innervates the genital apparatus.…”

“…Myomodulins were detected by nLC-MS/MS in the CNS, the oviduct gland and the main nidamental glands [10], and so were mRNAs, as described for APGWamide and CCAPs. They appear to be closely associated to accessory sex glands involved in egg capsule secretion.…”