Prediction of neuropeptide precursors and differential expression of adipokinetic hormone/corazonin-related peptide, hugin and corazonin in the brain of malaria vector Nyssorhynchus albimanus during a Plasmodium berghei infection

Abstract

Insect neuropeptides, play a central role in the control of many physiological processes. Based on an analysis of Nyssorhynchus albimanus brain transcriptome a neuropeptide precursor database of the mosquito was described. Also, we observed that adipokinetic hormone/corazonin-related peptide (ACP), hugin and corazonin encoding genes were differentially expressed during Plasmodium infection. Transcriptomic data from Ny. albimanus brain identified 29 pre-propeptides deduced from the sequences that allowed the prediction of at least 60 neuropeptides. The predicted peptides include isoforms of allatostatin C, orcokinin, corazonin, adipokinetic hormone (AKH), SIFamide, capa, hugin, pigment-dispersing factor, adipokinetic hormone/corazonin-related peptide (ACP), tachykinin-related peptide, trissin, neuropeptide F, diuretic hormone 31, bursicon, crustacean cardioactive peptide (CCAP), allatotropin, allatostatin A, ecdysis triggering hormone (ETH), diuretic hormone 44 (Dh44), insulin-like peptides (ILPs) and eclosion hormone (EH). The analysis of the genome of An. albimanus and the generated transcriptome, provided evidence for the identification of myosuppressin neuropeptide precursor. A quantitative analysis documented increased expression of precursors encoding ACP peptide, hugin and corazonin in the mosquito brain after Plasmodium berghei infection. This work represents an initial effort to characterize the neuropeptide precursors repertoire of Ny. albimanus and provides information for understanding neuroregulation of the mosquito response during Plasmodium infection.

Keywords: AKH, adipokinetic hormone; Adipokinetic hormone/corazonin-related peptide; BLAST, Basic Local Alignment Search Tool; CCAP, crustacean cardioactive peptide; CDS, Coding sequence; CNS, central nervous system; EH, eclosion hormone; ETH, ecdysis triggering hormone, Dh44, diuretic hormone 44, ILP, insulin-like peptide; GFP, green fluorescent protein; GO, Gene ontology; GPA2, glycoprotein 2; Hugin; K, lysine; Myosuppressin; NPLP1, neuropeptide-like precursor 1; Neuropeptide precursors; Nyssorhynchus albimanus; PBURS, partner of bursicon; PDH, pigment dispersing hormone; R, arginine; qPCR, quantitative polymerase chain reaction; sNPF, short neuropeptide F.

Graphical abstract

Fig. 1

Venn diagram depicting transcripts detected in uninfected (947) and Plasmodium berghei-infected brains (1220) of Ny. albimanus, 1644 transcripts were identified in both groups.

Fig. 2

Neuropeptides transcripts identified in Ny. albimanus brain transcriptome. SIFamide (AALB005004-RA), pigment dispersing hormone (AALB006094-RA) and insulin like peptide 3 (AALB010411-RA) transcripts with more represented.

Fig. 3

Myosuppressin identification. A) Partial deduced amino acid sequence of AALB009255-PA (red, signal peptide and yellow, polypeptide). B) Structure of AALB009255 gene, exons (14 red boxes) and introns (13 red lines) are shown, besides reads from the transcriptome brain of Ny albimanus that aligned with the second intron of AALB009255 gene are indicate by red arrows. C) Magnification from the zone of introns 1 and 2. D) Partial genomic sequence of AALB009255, the first two exons (purple italic letters) and introns (black letters) and a sequence that should correspond to intron 2 (black boxes) are shown, in the second intron we identified the sequence for the neuropeptide myossuppresin (green), KR cleavage sites (red), and a stop codon (gray). E) Deduced nucleotide sequence of Locus_31294_Length_691 of the transcriptome of An. albimanus in red and yellow shows the sequence of the ORF identical to AALB009255-PA and the sequence encoding myossupressin neuropeptide (green), amidation site (blue), KR cleavage sites (red) and a stop codon (gray). F) Deduced amino acid sequence of myosuppressin of An. albimanus. G) Multiple sequence alignment of the An. albimanus myosuppressin prepropeptide with homologs from other insects. Note the conserved peptide DVDHVFLRFamide. D. melanogaster, C. capitata, M. domestica, Ae. aegypti, An. gambiae, A. mellifera, P. clarkii and R. prolixus.

Fig. 3

Myosuppressin identification. A) Partial deduced amino acid sequence of AALB009255-PA (red, signal peptide and yellow, polypeptide). B) Structure of AALB009255 gene, exons (14 red boxes) and introns (13 red lines) are shown, besides reads from the transcriptome brain of Ny albimanus that aligned with the second intron of AALB009255 gene are indicate by red arrows. C) Magnification from the zone of introns 1 and 2. D) Partial genomic sequence of AALB009255, the first two exons (purple italic letters) and introns (black letters) and a sequence that should correspond to intron 2 (black boxes) are shown, in the second intron we identified the sequence for the neuropeptide myossuppresin (green), KR cleavage sites (red), and a stop codon (gray). E) Deduced nucleotide sequence of Locus_31294_Length_691 of the transcriptome of An. albimanus in red and yellow shows the sequence of the ORF identical to AALB009255-PA and the sequence encoding myossupressin neuropeptide (green), amidation site (blue), KR cleavage sites (red) and a stop codon (gray). F) Deduced amino acid sequence of myosuppressin of An. albimanus. G) Multiple sequence alignment of the An. albimanus myosuppressin prepropeptide with homologs from other insects. Note the conserved peptide DVDHVFLRFamide. D. melanogaster, C. capitata, M. domestica, Ae. aegypti, An. gambiae, A. mellifera, P. clarkii and R. prolixus.

Fig. 4

Adipokinetic hormone/corazonin-related peptide (ACP). (A) Nucleotide sequence and deduced amino acid sequence. Signal peptide (green), ACP neuropeptide (blue), glycine residue for amidation (G), K-R cleavage site (red) and stop codon (*) are shown. (B) Multiple alignment of the An. albimanus ACP prepropeptide with homologs from other insects. Note the conserved peptide motif QVTFSRDWNA amide An.darlingi, An. gambiae, An. stephensi, An. funestus, Ae. albopictus P. papatasi, C. quinquefasciatus C. lectularius and Ae. aegypti.

Fig. 5

Hugin. (A) Nucleotide sequence and deduced amino acid sequence. Signal peptide (green), neuropeptides XXXXXPRLG (blue), glycine residue for amidation (G), K-R, R-K, R-R and R cleavage site (red) and stop codon (*) are shown. (B) Multiple alignment of the An. albimanus Pk/PBAN prepropeptide with homologs from other insects. Note the conserved peptide motifs XXXXXPRLamide and XXXWFGPRLamide of An. darlingi, An. gambiae, Ae. aegypti, T. castaneum, N.lugens and B. mori.

Fig. 6

Corazonin. (A) Nucleotide sequence and deduced amino acid sequence. Signal peptide (green), corazonin neuropeptide (blue), glycine residue for amidation (G), K-R cleavage site (red) and stop codon (*) are shown. (B) Multiple alignment of the An. albimanus corazonin prepropeptide with homologs from other insects. Note the conserved peptide motif QTFQYSRGWTN amide. An. darlingi, An. dirus, Ae. aegypti, An. albopictus, M. domestica, I. scapularis, N. vitripennis O. furnacalis, G. morsitans, G. pallidipes, and C. lectularius.

Fig. 7

Relative expression of neuropeptide transcripts in the brain of Ny. albimanus mosquitoes infected with P. berghei ookinetes. Expression of neuropeptide transcripts in the uninfected group was normalized to a fold change of 1, to which the other condition (infected) was compared. ACP (4.94*), tachykinin-related peptide (1.34), SIFamide (1.64), Myosuppressin (1.54), hugin (4.41*), corazonin (1.75*), ILP-2 (1.04), ILP-3 (1.00) and ILP-5 (1.46). For statistical analysis, data were analyzed by ANOVA followed by Kruskal-Wallis post-test. () = Fold change vs uninfected group, * = significative fold change Kruskal-Wallis post-test, neuropeptide infected group vs uninfected group (α= 0.05).