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. 2015 Oct 6:5:14784.
doi: 10.1038/srep14784.

Multicopper oxidase-1 is required for iron homeostasis in Malpighian tubules of Helicoverpa armigera

Xiaoming Liu  1 , Chengxian Sun  1 , Xiaoguang Liu  1 , Xinming Yin  1   2 , Baohai Wang  3 , Mengfang Du  1 , Shiheng An  1
Affiliations

Multicopper oxidase-1 is required for iron homeostasis in Malpighian tubules of Helicoverpa armigera

Xiaoming Liu et al. Sci Rep. .

Abstract

Multicopper oxidases (MCOs) are enzymes that contain 10 conserved histidine residues and 1 cysteine residue. MCO1 has been extensively investigated in the midgut because this MCO is implicated in ascorbate oxidation, iron homeostasis and immune responses. However, information regarding the action of MCO1 in Malpighian tubules is limited. In this study, Helicoverpa armigera was used as a model to investigate the function of MCO1 in Malpighian tubules. Sequence analysis results revealed that HaMCO1 exhibits typical MCO characteristics, with 10 histidine and 1 cysteine residues for copper ion binding. HaMCO1 was also found to be highly abundant in Malpighian tubules. Temporal expression patterns indicated that HaMCO1 is mainly expressed during larval molting stages. Hormone treatments [the molting hormone 20-hydroxyecdysone (20E) and juvenile hormone (JH)] revealed that 20E inhibits HaMCO1 transcript expression via its heterodimer receptor, which consists of ecdysone receptor (EcR) and ultraspiracle (USP), and that JH counteracts the action of 20E to activate HaMCO1 transcript expression via its intracellular receptor methoprene-tolerant (Met). HaMCO1 knockdown caused a significant decrease in iron accumulation and also significantly reduced transferrin and ferritin transcript expression. Therefore, HaMCO1 is coordinately regulated by 20E and JH and is required for iron homeostasis in Malpighian tubules.

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Figures

Figure 1
Figure 1. Nucleotide sequence and putative amino acid sequence of HaMCO1.
The underlining indicates the initiation codon or stop codon. The predicted signal peptide is indicated in italicized text. The putative carboxyl-terminal transmembrane region is delineated with a blue line.
Figure 2
Figure 2. Multiple sequence alignments of the deduced amino acid sequences of HaMCO1, MsMCO1, BmMCO1 DpMCO1, AmMCO1, TcMCO1, ApMCO1, CqMCO, AaMCO1, DmMCO1 and SaFet3p.
The numbering on the right represents the position of the last amino acid in that line. A black box indicates 100% identity, and a grey box presents 100% similarity. The numbers 1, 2 and 3 located above the sequence represent the amino acids involved in coordinating the T1, T2 and T3 copper centers. The sequences (with GenBank accession numbers) for the alignment analysis were as follows: Helicoverpa armigera: HaMCO1 (KP318028); Manduca sexta: MsMCO1(AAN17506.1); Bombyx mori: BmMCO1 (XP_004933574.1); Danaus plexippus: DpMCO1 (EHJ67706.1); Apis mellifera: AmMCO1 (XP_001120790.2); Tribolium castaneum: TcMCO1 (NP_001034514.1); Acyrthosiphon pisum: ApMCO1(XP_001948070.1); Culex quinquefasciatus: CqMCO (XP_001862911.1); Aedes aegypti: AaMCO (AAY29698.1); Drosophila melanogaster: DmMCO1 (NP_609287.3); Saccharomyces arboricola: SaFet3p (XP_011104804.1).
Figure 3
Figure 3. Phylogenetic tree of insect MCO proteins.
The phylogenetic tree was constructed using the MEGA4 program (Tamura et al., 2007) according to the neighbor-joining method with a Poisson correction model. The sequences (with GenBank accession numbers) for the phylogenic analysis were as follows: Helicoverpa armigera: HaMCO1 (KP318028); Manduca sexta: MsMCO1(AAN17506.1); Bombyx mori: BmMCO1 (XP_004933574.1); Danaus plexippus: DpMCO1 (EHJ67706.1); Plutella xylostella: PxMCO1 (XP_011552373.1); Bombus terrestris: BtMCO1 (XP_003394771.1); Bombus impatiens: BiMCO1 (XP_003485633.1); Apis mellifera: AmMCO1 (XP_001120790.2); Apis dorsata: AdMCO1 (XP_006611755.1); Harpegnathos saltator: HsMCO1 (XP_011135308.1); Tribolium castaneum: TcMCO1 (NP_001034514.1); Camponotus floridanus: CfMCO1 (XP_011255366.1); Acyrthosiphon pisum: ApMCO1(XP_001948070.1); Pogonomyrmex barbatus: PbMCO1 (XP_011645337.1); Bemisia tabaci: BtMCO1 (AGC83693.1); Culex quinquefasciatus: CqMCO (XP_001862911.1); Aedes aegypti: AaMCO (AAY29698.1); Culex quinquefasciatus: CqMCO2 (XP_001867157.1); Culex pipiens pallens: CpMCO2 (ACG63789.1); Drosophila melanogaster: DmMCO1 (NP_609287.3); Ceratosolen solmsi marchali: CsMCO1 (XP_011500453.1); Fopius arisanus: FaMCO1 (XP_011300938.1); Nephotettix cincticeps: NcMCO1(BAJ06131.1); Nasonia vitripennis: NvMCO1 (XP_008214237.1); Helicoverpa armigera: HaMCO2 (AHA15412.1); Bombyx mori: BmMCO2A (BAG70891.1); Antheraea pernyi: ApMCO2 (AII19522.1), Manduca sexta: MsMCO2 (AAN17507.1); Danaus plexippus: DpMCO2A (EHJ72220.1); Papilio machaon: PmMCO2(BAJ07600.1); Papilio xuthus: PxMCO2 (BAI87829.1); Papilio polytes: PpMCO2(BAJ07602.1); Bombyx mori: BmMCO2B (DAA06287.1); Biston betularia: BbMCO2 (AEP43806.1); Anopheles sinensis: AsMCO2A (KFB50921.1); Anopheles gambiae: AgMCO2A (AAX49501.); Drosophila melanogaster: DmMCO2A (NP_724412.1); Drosophila melanogaster: DmMCO2F (NP_724413.2); Anopheles gambiae: AgMCO2B (AAX49502.1); Drosophila melanogaster: DmMCO2D (NP_001137606.1); Drosophila melanogaster: DmMCO2E (NP_610170.2); Drosophila melanogaster: DmMCO2G (NP_001260709.1); Tribolium castaneum: TcMCO2B (AAX84203.2); Monochamus alternatus: MaMCO2 (ABU68466.1); Riptortus pedestris: RpMCO2 (BAJ83487.1); Nephotettix cincticeps: NcMCO2 (BAJ06133.1); Megacopta punctatissima: MpMCO2 (BAJ83488.1); Gryllus bimaculatus: GbMCO2 (BAM09185.1); Apis mellifera: AmMCO2 (ACK57559.2); Nysius plebeius: NpMCO2 (BAJ83489.1); Danaus plexippus: DpMCO2B (EHJ72219.1); Saccharomyces arboricola: SaFet3p (XP_011104804.1).
Figure 4
Figure 4. Temporal expression profile of HaMCO1.
E1, day 1 egg; E2, day 2 egg; E3, day 3 egg; L1-1, day 1 of 1st-instar larva; L1-2, day 2 of 1st-instar larva; L3M, molting stage of 3rd-instar larva; L3-1, day 1 of 3rd-instar larva; L3-2, day 2 of 3rd-instar larva; L4M, molting stage of 4th-instar larva; L4-1, day 1 of 4th-instar larva; L4-2, day 2 of 4th-instar larva; L5M, molting stage of 5th-instar larva; L5-0, 0 h of 5th-instar larva; L5-1, day 1 of 5th-instar larva; L5-2, day 2 of 5th-instar larva; L5-3, day 3 of 5th-instar larva; L5-4, day 4 of 5th-instar larva; L5-5, day 5 of 5th-instar larva; PP, prepupa; P0, 0 h pupa; P1, day 1 of pupa; P2, day 2 of pupa; P3, 3 day of pupa; P5, 5 day of pupa; P7, 7 day of pupa; P9, 9 day of pupa; A1, day 1 of adult; A2, day 2 of adult; A3, day 3 of adult; A5, day 5 of adult; A7, day 7 of adult.
Figure 5
Figure 5. Tissue distributions of HaMCO1.
EP: epidermis; MD: midgut; FB: fat body; TR: trachea; HE: hemocyte; SG: salivary gland; MT: Malpighian tubule; PG: pheromone gland; MS: muscle; HP: hairpencil.
Figure 6
Figure 6. Regulation by 20E of HaMCO1 transcript expression.
(A) The effect of 20E treatment on the expression of HaMCO1 transcript. (B) qPCR analysis of ECR and USP dsRNAi efficiency on ECR and USP transcript levels, respectively. (C) The effect of ECR and USP knockdown on HaMCO1 transcript level. The 18S rRNA was used as the housekeeping gene for normalization in all qPCR analyses. The data represent the mean ± SD of three biological replicates. The significance of comparisons were determined by Student’s t-test (*p < 0.05, **p < 0.01, ***p < 0.001).
Figure 7
Figure 7. Regulation by JH of HaMCO1 transcript expression.
(A) The effect of JH treatment on the expression of HaMCO1 transcript. (B) qPCR analysis of MET dsRNAi efficiency on the MET transcript level. (C) The effect of MET knockdown on HaMCO1 transcript levels. The 18S rRNA was used as the housekeeping gene for normalization in all qPCR analyses. The data represent the mean ± SD of three biological replicates. The significance of comparisons were determined by Student’s t-test (*p < 0.05, **p < 0.01, ***p < 0.001).
Figure 8
Figure 8. Effect of HaMCO1 knockdown on iron accumulation and the expression of transferrin and ferritin transcripts.
(A) qPCR analysis of HaMCO1 dsRNAi efficiency on the HaMCO1 transcript level. The 18S rRNA was used as the housekeeping gene for normalization in all qPCR analyses. The data represent the mean ± SD of three biological replicates. The significance of comparisons are marked with ***(p < 0.001), as determined by Student’s t-test. (B) The effect of HaMCO1 knockdown on iron accumulation. (B-1) Larval Malpighian tubules from the control (EGFP dsRNA) and treatment (HaMCO1 dsRNA) were analyzed. (B-2) The relative blue color density of the control (EGFP dsRNA) and treatment (HaMCO1 dsRNA). The relative blue color density was measured using Quantity One software. The data represent the mean ± SD of three biological replicates. Statistically significant differences were assessed by Student’s t-test (***p < 0.001). (C) The effect of HaMCO1 knockdown on the expression of transferrin and ferritin transcripts. qPCR analysis of the effects of HaMCO1 dsRNA injection on transferrin and ferritin transcript levels. The 18S rRNA was used as the housekeeping gene for normalization in all qPCR analyses. The data represent the mean ± SD of three biological replicates. The significance of comparisons are marked with ***(p < 0.001), as determined by Student’s t-test.
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