Table 1.
Neuropeptide/peptide hormones predicted based on transcriptome mining of Balanus amphitrite.
Figure 1.
Sequence alignment of barnacle pigment dispersing hormone, SIFamide and neuropeptide F with arthropods.
(A) Sequence alignment of mature pigment dispersing hormone. Decapods peptide sequences are from: Uca pugilator P08871, Procambarus clarkii Q9TWW7 and Cancer productus ABV68725; isopod peptide is from Eurydice pulchra ACX49752; insect peptides are from: Drosophila melanogaster AAL49303, Schistocerca gregaria ACY02888 and Musca domestica Q76JT9. (B) Alignment of barnacle SIFamide mature peptides. Sequences are from: decapods Homarus americanus, Cancer borealis; branchiopod Daphnia pulex; insects Drosophila melanogaster, Tribolium castaneum, Bombyx mori, Rhodnius prolixus and Ixodes scapularis [38]. (C) Alignment of barnacle NPF mature peptide. Insect NPF are from: Drosophila melanogaster AF117896, Bombyx mori NP_001124361, Locusta migratoria CO854418 and Pediculus humanus EEB15547. Decapods NPF are from shrimps Marsupenaeus japonicus CI998017 and Litopenaeus vannamei AEC12204; copepod NPF is from Lepeophtheirus salmonis FK933794; branchiopods NPF are from Daphnia magna EG565358 and Daphnia pulex EFX90018. Asterisks “*” represent typical motif residues of each peptide. “x” indicates unique amino acid residues of B. amphitrite compared to other arthropods. Line marks the unique five amino acids insertion of NPF in B. amphitrite.
Figure 2.
Comparison of barnacle sulfakinin peptide with other arthropods.
(A) Sequence alignment of mature sulf. (B) Precursor structure of barnacle sulf compared with other crustaceans and insects. Insects Sulf are from: Drosophila melanogaster AAF52173, Tribolium castaneum EFA04708, Anopheles gambiae AAR03495, Bombyx mori NP_001124354 and Culex quinquefasciatus XP_001846221; branchiopod is from Daphnia pulex EFX80896; decapods Sulf are from Homarus americanus and Penaeus monodon [38]. Asterisks “*” represent typical motif residues of each peptide. “x” indicates unique amino acid residues of B. amphitrite compared to other arthropods.
Figure 3.
Comparison of barnacle tachykinin-related peptide with that of insects and crustaceans.
(A) TRP sequence alignment. Decapods TRP are from: Cancer borealis [72] and Homarus americanus ACB41786; isopod TRP is from Eurydice pulchra CO869025; branchiopod TRP is from Daphnia pulex [38]. Insects TRP are from: Tribolium castaneum EFA09176, Drosophila melanogaster AAF89172, Bombyx mori NP_001124364 and Rhyparobia maderae AAX11211. (B) Barnacle TRP precursor structure compared with other crustaceans and insects. Decapods TRP are from: Homarus americanus ACB41786 and Procambarus clarkia BAC82426. Insects TRP are from: Drosophila melanogaster AAF89172, Camponotus floridanus EFN66667 and Periplaneta americana AAX11212. Branchiopod TRP is from Daphnia pulex [38]. Asterisks “*” represent typical motif residues of each peptide.
Figure 4.
Comparison of barnacle orcokynin with other arthropods.
(A) Sequence alignment mature orco. (B) Precursor structure of barnacle orco compared with other crustaceans and insects. Sequences of insects Orco are from: Nasonia vitripennis XP_003426062, Bombyx mori NP_001124366, Danaus plexippus EHJ77769, Harpegnathos saltator EFN80782 and Apis mellifera XP_001120650; copepods Orco are from: Calius rogercresseyi and Lernaeocera branchialis [22]; decapods Orco are from: Litopenaeus vannamei [1], Marsupenaeus japonicas [73], Homarus americanus ACD13197, Procambarus clarkii [73], Carcinus maenas [74] and Orconectes limosus [75]. Branchiopod Orco is from Daphnia pulex EFX70781. Asterisks “*” represent typical motif residues of each peptide.
Figure 5.
Comparison of barnacle B-type allatostatin with other arthropods.
(A) Mature ASTB sequences alignment. (B) Precursor structure of barnacle ASTB compared with other crustaceans and insects. Insects ASTB are from: Bombyx mori P82003, Tribolium castaneum B8XQ58 and Gryllus bimaculatus Q5QRY7; branchiopod ASTB is from Daphnia pulex E9GSK4. Asterisks “*” represent conserved motif residues.
Figure 6.
Peptide alignment of insulin-related peptide of Balanus amphitrite with arthropods.
Isopods IRP are from: Armadillidium vulgare AB029615 and Porcellio dilatatus AB089811; decapods IRP are from: Marsupenaeus japonicus AB029615, Penaeus monodon GU208677.1 and Cherax quadricarinatus DQ851163; branchiopod IRP is from Daphnia pulex [38]; insects IRP are from: Bombyx mori NP_001233285, Locusta migratoria P15131, Drosophila melanogaster CG14173, Anopheles gambiae AAQ89693 and Camponotus floridanus EFN61735. Asterisks “*” mark the conserved Cysteine residues, black frame indicates specific amino acids insertion and deletion in IRP of isopod and decapod species, compared to insects, branchiopod and barnacle.
Figure 7.
Sequence comparison of barnacle A-type allatostatin with homologs in arthropods.
Decapod ASTAs are from Macrobrachium rosenbergii Q1AHE3, Panulirus interruptus A6BL33 and Procambarus clarkii Q3LI53; insect ASTAs are from Bombyx mori NP_001037036, Drosophila melanogaster AAF97792. Asterisk “*” represents typical motif residues of ASTA peptide.
Figure 8.
Sequence alignment of barnacle bursicon peptides.
(A) Alignment of bursicon α subunit. Sequences are from: euphausiacean Euphausia superba [22]; decapods Litopenaeus vannamei [22], Homarus gammarus ADI86242.1 and Carcinus maenas EU139428; branchiopod Daphnia pulex [38]; insects Tribolium castaneum DQ138190, Drosophila melanogaster NM_142726, Apis mellifera NM_001098234, Musca domestica EF424614 and Bombyx mori NM_001098375; chelicerate Ixodes scapularis XM_002407468. (B) Alignment of bursicon β subunit. Sequences are from: decapods Homarus americanus [22], Homarus gammarus ADI86243.1, Litopenaeus vannamei [22] and Carcinus maenas EU139429; branchiopod Daphnia pulex [38]; insects Tribolium castaneum DQ156997, Drosophila melanogaster NM_135868, Apis mellifera NM_001040262, Musca domestica EF424613 and Bombyx mori NM_001043824; chelicerate Ixodes scapularis XM_002407469. Asterisk “*” mark the conserved cysteine that will form dimers.
Figure 9.
Sequence alignment of calcitonin and eclosion hormone of barnacle.
(A) Sequence alignment of barnacle calcitonin isoform A and B. Sequences are from: decapods Homarus americanus ACX46386.1; copepods Caligus clemensi [22], Lepeophtheirus salmonis ADD38663; branchiopod Daphnia pulex EFX90445; insects Tribolium castaneum EEZ99367, Drosophila melanogaster NP_523514, Anopheles gambiae XP_321755, Bombyx mori NP_001124379, Camponotus floridanus EFN61187 and Nasonia vitripennis XP_001599948. (B) Alignment of barnacle eclosion hormone. Sequences are from: decapods Callinectes sapidus CV224237, Penaeus monodon [22], Marsupenaeus japonicus [22]; branchiopods Daphnia pulex [37], Triops cancriformis [22]; insects Acromyrmex echinatior EGI68318, Anopheles gambiae XP_001230805, Drosophila melanogaster NP_524386, Bombyx mori NM_001043842 and Tribolium castaneum XP_969164. Asterisks “*” mark the six conserved Cysteine residues in EH.
Figure 10.
Real-time PCR results of predicted neuropeptide genes in four developmental stages.
Stages include the late nauplius VI (NauVI), cyprid (Cyp), young juvenile (Juv) and the adult (Adu). Gene expression was measured for (A) A-type allatostatin (asta), (B) B-type allatostatin (astb), (C) C-type allatostatin (astc), (D) bursicon subunit β (burb), (E) calcitonin-like diuretic hormone isoform A (calcia), (F) calcitonin-like diuretic hormone isoform B (calcib), (G) eclosion hormone (eh), (H) insulin-related peptide (irp), (I) neuropeptide F (npf), (J) orcokinin (orco), (K) pigment dispersing hormone (pdh), (L) SIFamide (sif), (M) sulfakinin (sulf), (N) tachykinin-related peptide (trp). Values are showed as mean ± SD from three biological replicates. Asterisks indicate significant difference detected by one-way ANOVA comparing to gene expression level in juvenile stage (Tukey test, * p<0.05, **p<0.01).
Figure 11.
Proprotein convertase inhibitor bioassay result.
Autoclaved filtered seawater (AFSW) and 0.1% DMSO served as positive control. Error bar represents mean ± SD for three biological replicates. Asterisks indicate significant difference detected by one-way ANOVA comparing the treatments to the positive control (Tukey test, **p<0.01 and ***p<0.001).