In this work, we report the expression of critical neuropeptides in Ny. albimanus, with increased expression during infection with a malaria parasite. Of the 29 putative neuropeptide transcripts, that predicted at least 60 possible biopeptides, 27 had coding sequence (CDS).
In addition, we describe transcripts mainly associated with translation, oxidation-reduction process, protein binding, ATP binding, integral components of membrane and ribosomes. Previously, we reported 19 brain proteins of this mosquito with differential expression after P. berghei infection, and we identified 17 transcripts of proteins previously reported [92] (Additional file 7. Figure S3). All transcripts reported herein have orthologues in other insect disease vectors [4, 16, 50, 93–95] and in relevant agricultural insect pest [5, 96, 97]. Although most of the encoded peptides are identical or similar to orthologs in other species, some of them exhibit amino acid sequence variations. Alike to other mosquito species (An. darlingi, An. gambiae, Ae. aegypti and Culex quinquefasciatus) [98]. Our results in Ny. albimanus indicate that the allatostatin-A transcript has the potential to generate seven biopeptides; similar to the allatostatin-A transcripts of D. melanogaster and M. domestica that can generate six and five biopeptides, respectively. Whereas the tachykinin-related peptide transcript of Ny. albimanus is identical to that of Ny. darlingi and has the potential for generating four biopeptides, in Ae. aegypti this is limited to only one biopeptide. Other organisms, such as Procambarus clarkii (Decapoda: Cambaridae) and Phenacogrammus interruptus (Characiformes; Alestiidae) can generate three biopeptides. Insulin-like peptides presented the most remarkable amino acid sequence variation. This results could be related to the long size of these neuropeptides and the diversity of its functions [99–101].
The Ny. albimanus myosuppressin sequence is identical to that of other insects, including several anophelines orthologs [85] (Fig. 3F). A close analysis of the An. albimanus AALB009255-RA, transcript revealed that the first two exons, coding for a signal peptide, are highly similar to that of An. gambiae myosuppressin precursor (Fig. 2B), but the downstream sequence lacked myosuppressin homology (Fig. 3A). Full reconstruction of the myosuppressin precursor, including the first two exons of AALB009255-RA and the novel exon predicted a full ORF with the canonical structure of the myosuppressin precursor gene. This observation indicates that AALB009255-RA annotation was mischaracterized, since it codes for two or more different transcripts, one of which includes a myosuppressin (Fig. 3B-E). The predicted myosuppressin sequence was identified in the second intron of the genomic sequence (Fig. 3B and E) and the Locus_31294_Length_691 of the annotated An. albimanus transcriptome version 2 [56] (Fig. 3C). This locus has an ORF of 97 amino acid residues; the first 75 amino acid residues are identical to those of the peptide AALB009255-PA. This transcript encodes a signal peptide of 20 amino acid residues (Fig. 3C and D, highlighted in red) and a predicted neuropeptide-coding sequence of 11 amino acid residues corresponding to myosuppressin (TDVDHVFLRFG) (Fig. 3D, highlighted in green).
Despite the importance of neuropeptides in adult insects, information about their function is limited, especially for anophelines. Neuropeptides are associated with most of the physiological processes in insects, including the immune response activation [102–105] and suppression [106] after infection by different microorganisms. Interestingly, we detected a significant increase in the transcription of ACP, pk/PBAN, and corazonin in Ny. albimanus infected with P. berghei.
The study of adipokinetic hormone/corazonin-related peptide (ACP) [88], initially characterized as adipokinetic hormone II (AKH II) [107–109], structurally intermediate between corazonin and adipokinetic hormone, has provided new evidence and questions on the role of neuropeptides in insects. The structural and sequential analysis described this neuropeptide in several insects, including anophelines. ACP is primarily expressed in the nervous system and, to a lesser extent, in other insect organs and tissues [110, 111]. ACP transcripts were detected in the head and thorax of larvae, pupae, and adult of Ae. aegypti and An. gambiae [109]. Furthermore, ACP transcripts expression increases prominently in the brain and thoracic ganglia of Ae. aegypti after adult eclosion, suggest this neuropeptide may function in the regulation of post-ecdysis activities [112].
ACP transcripts in the brain of Ny. albimanus is consistent with previous results in An. gambiae where ACP (called AKHII) is expressed 72 h after feeding (blood or sugar) [109]. The differential expression of ACP between uninfected blood-fed and P. berghei-infected Ny. albimanus suggests that the midgut invasion by this parasite activates or modifies physiological pathways dependent on ACP. [14, 15, 17, 24]. Thus, it is possible that the increased level of this neuropeptide in Plasmodium infected mosquitoes is part of the immune response activation against this parasite. Further studies are necessary to unravel the function of the ACP system; currently, no definitive function for ACP has been determined and, functional studies in other insects revealed that ACP does not perform AKH and corazonin functions [112–114].
Pyrokinins have been identified in various insects [95, 110, 115–118] and are generated from the capa and pk/pban genes. Capa produces at least two peptides with sequence XXFPRV and XXWFGPRL, while pk/pban produces at least one peptide with sequence XXWFGPRL. Two genes pk/pban-like (capa and hugin) encode pyrokinins PK1 (XXXXWFGPRL) and PK2 (XXXXXXRPPFAPRL) respectively in D. melanogaster. Several functions for these peptides were described, including stimulation of pheromone biosynthesis [119], induction of melanization [120], induction of embryonic diapause [121], stimulation of visceral muscle contraction [122] and termination of pupal diapause development [123]. But no evidence exists for the participation of pyrokinins in immune response mechanisms. Here, we show that a pk/pban and capa transcripts coding for PK, PVK-1, and PVK-2 peptides (Table 2) increase in Plasmodium-infected Ny. albimanus; suggesting that the pyrokinins PK-1, PK-2, and PK-3 could be involved in signaling or activation mechanisms of the immune response of mosquitoes to these parasites.
Corazonin is widely conserved across insect genera [124] with various functions including cuticular melanization [32]. Its increased expression in brains of P. berghei-infected Ny. albimanus could be related to these two functions, but other not yet identified roles of this neuropeptide in mosquito defenses await identification.
Insulin-like peptides are involved in several processes that are conserved among vertebrates and invertebrates, including immunity [125–127]. ILPs have been identified in Ae. aegypti [128], An. gambiae [129], and An. stephensi (42). In An. stephensi, the ingestion of P. falciparum-infected blood, increased ILPs expression (42), and the inhibition of this induction reduced parasite development, indicating the possibility that ILP induction is a parasite mechanism to avoid elimination by the mosquito.t [106]. However, while inhibition of ILP4 induced the expression of immune genes prior to parasite invasion of the mosquito midgut, the inhibition of ILP3 increased immune gene expression at 24 hours after infection, when parasites had already invaded the midgut, indicating that the relationship between ILP mediated mechanism and immunity could be multiple. Our results documented an increase in of ILP-5 transcription in Ny. albimanus at times when P. berghei parasites had initiated development on the mosquito midgut. Although this increase was consistent among mosquito samples, it was not statically significant. On the other hand, no difference in ILP 3 transcription was observed between infected and uninfected mosquitoes. These results require further studies with a bigger sample size, but they may indicate that ILPs also participate in P. berghei infection, and that the induction of ILP is a generalized mechanism of Plasmodium parasites survival in mosquitoes. Our results on Tachykinin-related peptide transcription were also inconclusive.
The neuropeptide receptors identified in this work, have characteristic domains, almost all are G-protein coupled receptors rhodopshin like (GPCRs) (Table 1). However, the functional characterization of these receptors to understand the neuropeptide-receptor interaction and the role they play in the mosquito-parasite interaction awaits investigation.
In summary, we provide a brain neuropeptidome repertoire composed by 29 transcripts coding for at least 60 potential biopeptides in Ny. albimanus. Particularly we report the increased expression of the adipokinetic hormone/corazonin-related peptide (ACP), pk/pban pirokinin, and corazonin transcripts after P. berghei infection. At present, the functions of neuropeptides in insects during infection with parasites or viruses are partially understood. Further investigation is required as to whether significant changes in ACP and pk/PBAN transcription in the brain reflect an increase in the production or release of the respective biopeptides.
Most of the neuropeptides identified here showed high similarity with previously reported in other insects and other mosquito vectors. However, it is still necessary to explore the mechanisms triggered by neuropeptides up-regulated in the Ny. albimanus brain by a Plasmodium infection.