Characterization and tissue distribution of neuropeptide F in the eyestalk and brain of the male giant freshwater prawn, Macrobrachium rosenbergii

Abstract

We previously analyzed the central nervous system (CNS) transcriptome and found three isotypes of long neuropeptide F (MrNPF-I, -II, -III) and four isoforms of short NPF (sMrNPF) in the giant freshwater prawn, Macrobrachium rosenbergii. We now validate the complete sequences of the MrNPF-I and -II precursor proteins, which show high similarity (91-95 %) to NPFs of the penaeus shrimp (PsNPF). MrNPF-I and -II precursors share 71 % amino acid identity, whereas the mature 32-amino-acid MrNPF-I and 69-amino-acid MrNPF-II are identical, except for a 37-amino-acid insert within the middle part of the latter. Both mature MrNPFs are almost identical to PsNPF-I and -II except for four amino acids at the mid-region of the peptides. Reverse transcription plus the polymerase chain reaction revealed that transripts of MrNPF-I and -II were expressed in various parts of CNS including the eyestalk, brain and thoracic and abdominal ganglia, with the highest expression occurring in the brain and thoracic ganglia and with MrNPF-I showing five- to seven-fold higher expression than MrNPF-II. These peptides were also expressed in the midgut hindgut, and hepatopancreas, with MrNPF-I expression in the former two organs being at the same level as that in the brain and thoracic ganglia and about 4-fold higher than NPF-II. The expression of NPFs was also detected in the testes and spermatic duct but appeared much weaker in the latter. Other tissues that also expressed a considerable amount of NPF-I included the hematopoeitic tissue, heart and muscle. By immunohistochemistry, we detected MrNPFs in neurons of clusters 2, 3 and 4 and neuropils ME, MT and SG of the optic ganglia, neurons in cluster 6 and neuropils AMPN, PMPN, PT, PB and CB of the medial protocerebrum, neurons in clusters 9 and 11 and neurophils ON and OGTN of the deutocerebrum and neurons in clusters 14, 15 and 16 and neuropils TN and AnN of the tritocerebrum. Because of their high degree of conservation and strong and wide-spread expression in tissues other than CNS, we believe that, in addition to being a neuromodulator in controlling feeding, MrNPFs also play critical roles in tissue homeostasis. This should be further explored.

Comparative neuroanatomical distribution and expression levels of neuropeptide F in the central nervous system of the female freshwater prawn, Macrobrachium rosenbergii, during the ovarian cycle

“…In M. rosenbergii, our research group has identified and characterized M. rosenbergii NPF (MrNPF) sequences from the CNS transcriptomic analysis and found that there are three isoforms of long MrNPF, namely, MrNPF-I, MrNPF-II, and MrNPF-III, as well as four isoforms of shortMrNPF (Suwansa-Ard et al, 2015). We further validated the complete sequences of the MrNPF-I and MrNPF-II precursor proteins, which showed high similarity (about 91-95%) to NPFs of the penaeid shrimp (Thongrod et al, 2017). In male prawn, M. rosenbergii, MrNPF was detected in several regions of the CNS.…”

“…We named neuronal clusters of the ES based on the description of Sandeman et al (1992) and Thongrod et al (2017), while the naming of various parts of the BR, that is, medial protocerebrum (PC), deuto-cerebrum (DC), and tritocerebrum (TC) was based on that described by Sandeman et al (1992) and Thongrod et al (2017). In addition, groups of neurons and neuropils SEG, TG, and AG were named following the description of Tinikul et al (2011) and Thongrod et al (2019).…”

“…A polyclonal antiserum against MrNPF was produced previously, and its specificity was also rigorously tested, as previously described (Thongrod et al, 2017(Thongrod et al, , 2019. This primary antiserum was used for subsequent immunolabeling in this study.…”

“…The whole-mount immunohistochemistry protocol for immunolabeling of MrNPF in the BR and VNC tissues was based on Tinikul et al (2011) and Thongrod et al (2017Thongrod et al ( , 2019…”

“…NPF is a homolog of neuropeptide Y (NPY) in vertebrates, and these neuropeptides are included within the subgroup of FMRFamide-like peptides, but with a major difference as the former possesses the Cterminal motif-GRPRFamide and tyrosine residues at positions 10 and 17 from their C-termini (Christie et al, 2011;Nässel & Wegener, 2011;Thongrod et al, 2017). The neuronal distribution of NPF has been extensively mapped, and its roles have been described in several insect species.…”