Figures
Abstract
Despite the established role of Culex quinquefasciatus as a vector of various neurotropic viruses, such as the Rift Valley and West Nile viruses, as well as lymphatic filariasis, little is known regarding the organism’s reproductive physiology. As in other oviparous animals, vitellogenin, the most important source of nutrients for the embryo development, is digested by intracellular proteases. Using mass spectrometry, we have identified two cathepsin B homologues partially purified by self-proteolysis of Cx. quinquefasciatus total egg extract. The transcriptional profile of these two cathepsin B homologues was determined by quantitative RT-PCR, and the enzymatic activity associated with the peptidase was determined in ovaries after female engorgement. According to the VectorBase (vectorbase.org) annotation, both cathepsin B homologues shared approximately 66% identity in their amino acid sequences. The two cathepsin B genes are expressed simultaneously in the fat body of the vitellogenic females, and enzymatic activity was detected within the ovaries, suggesting an extra-ovarian origin. Similar to the transcriptional profile of vitellogenin, cathepsin B transcripts were shown to accumulate post-blood meal and reached their highest expression at 36 h PBM. However, while vitellogenin expression decreased drastically at 48 h PBM, the expression of the cathepsins increased until 84 h PBM, at which time the females of our colony were ready for oviposition. The similarity between their transcriptional profiles strongly suggests a role for the cathepsin B homologues in vitellin degradation.
Citation: Moura AS, Cardoso AF, Costa-da-Silva AL, Winter CE, Bijovsky AT (2015) Two Cathepsins B Are Responsible for the Yolk Protein Hydrolysis in Culex quinquefasciatus. PLoS ONE 10(2): e0118736. https://doi.org/10.1371/journal.pone.0118736
Academic Editor: Pedro Lagerblad Oliveira, Universidade Federal do Rio de Janeiro, BRAZIL
Received: July 31, 2014; Accepted: January 20, 2015; Published: February 24, 2015
Copyright: © 2015 Moura et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Data Availability: All relevant data are within the paper and its Supporting Information files.
Funding: This work was funded by FAPESP - Fundação de Amparo à Pesquisa do Estado de São Paulo (http://www.fapesp.br/) (Grant numbers: 2010/51241-0; 2013/09211-4). The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors confirm that co-author Carlos Eduardo Winter is a PLOS ONE Editorial Board member. This does not alter the authors' adherence to PLOS ONE Editorial policies and criteria.
Introduction
Culex quinquefasciatus (Diptera: Culicidae) is a cosmopolitan mosquito that is highly anthropophilic and completely adapted to urban conditions. This mosquito is a competent vector of neurotrophic viruses such as the St. Louis and Japanese encephalitis viruses, the eastern and western equine encephalomyelitis viruses and the Rift Valley and West Nile viruses [1–4]. Moreover, Cx. quinquefasciatus is the most important Brazilian vector of Wuchereria bancrofti, the lymphatic filariasis agent [5].
Similar to other oviparous animals, Cx. quinquefasciatus generates and stores within the oocytes the nutrients needed for the embryonic development. Nutrient reserves are synthesised in the maternal fat body, a tissue analogous in function to the vertebrate liver. The primary source of amino acids and lipids for embryonic development is vitellogenin (Vg), a glycosylated phospholipoprotein that is secreted into the haemolymph and then incorporated via receptor mediated endocytosis by the developing ovarian follicles [6] and stored into the yolk platelets as vitellin [7].
The use of yolk protein as a nutrient reserve involves enzyme-mediated hydrolysis, a process that has been described to depend on various enzymes in different insect orders. Among these, the most frequently reported enzymes are cysteine proteinases, which have been described in Diptera: Drosophila melanogaster [8], Musca domestica [9,10] and Aedes aegypti [11,12]; Lepidoptera: Bombyx mori [13–18] and Helicoverpa armigera [19–21]; Dictyoptera: Blatella germanica [22] and Periplaneta americana [23]. While previous works in Culex pipiens pallens [24,25] have implicated cathepsins B and L in the atretic process of ovarian follicles degrading not only yolk proteins but also the follicular structure itself, it remains unclear whether these enzymes are required for yolk protein degradation during embryogenesis.
Haematophagous mosquitoes of the Aedes, Anopheles and Culex genera share multiple biochemical, morphological, developmental and behavioural characteristics. However, Cx. quinquefasciatus diverges from mosquitoes of other genera in the fine structure of their salivary glands, saliva composition [26,27], cellular and biochemical mechanisms governing blood digestion and haem detoxification [28,29] and their response to odorants and biting behaviour [30].
In the following study, we build on our initial description of the morphofunctional aspects of Cx. quinquefasciatus oogenesis and identify two cathepsin B proteinases, which are present in the Cx. quinquefasciatus eggs, expressed in the female fat bodies following a blood meal and are involved in promoting yolk protein degradation.
Materials and Methods
Ethics Statement
The protocols used in this work were approved by the Animal Experimentation Ethics Committee of the Institute of Biomedical Sciences (University of São Paulo, São Paulo, Brazil—process number CEAU 103/2012).
Animals
Cx. quinquefasciatus (PIN strain) [31] mosquitoes were raised at 27°C, with 70–80% relative humidity and a photoperiod of 12 h dark-12 h light. Larvae were fed with ground fish food (Seravipan, Germany), and adults were fed ad libitum on 10% sucrose solution. As needed, 4–5 day-old adult females were fed on Balb/c mice anaesthetised with 0.3 mg/kg of xylazine hydrochloride (Calmiun, Agner União, Brazil) plus 30 μg/kg of acepromazine (Acepran, Univet S.A., Brazil).
Egg extract
Approximately 1,500 eggs (dark eggs, harvested 24 h after oviposition) were ground with a Pellet Pestle Motor (Kontes, USA) in ice bath in a microcentrifuge tube in 200 μl of 10 mM sodium acetate buffer pH 5.0. Following centrifugation at 10,000 xg for 5 s, the supernatant was transferred to a new tube and the pellet was resuspended in 200 μl of sodium acetate buffer, mixed, and centrifuged, after which the supernatants were combined to obtain a final volume of 400 μl. The total protein concentration was estimated according to Bradford [32] using BSA protein as the standard.
Alternatively, white (harvested 2 h after oviposition) or dark eggs were ground as described above in a microcentrifuge tube in 200 μl of PBS pH 7.0 containing 50 μM E-64 and 1 μl/ml of a cocktail of protease inhibitors (50 μg/ml leupeptin, 5 μg/ml pepstatin, 5 μg/ml chymostatin, 5 μg/m; antipain, 5 μg/ml PMSF).
All extracts were immediately used or stored at -20°C until needed.
Ovary extract
Ovaries of adult females 96 and 120 hours post blood meal (PBM) were processed as described above for white eggs.
Determination of cathepsin B activity
Total protein from egg extract (40 ng) was incubated for 30 min at 27°C with 20 μM of Z-Arg-Arg-NHMec (benzyloxycarbonylarginyl-arginine 4-methylcoumarin-7-amide) in 10 mM sodium acetate buffer pH 5.0 at a final volume of 200 μl. The fluorescent product was detected using a Fluoroskan Ascent Microplate Fluorometer (Thermo Scientific, USA) with an excitation wavelength of 360 nm and emission of 450 nm. Control samples were established by adding 50 μM E-64 (N-[N-(L-3-trans-carboxyirane-2-carbonyl)-L-leucyl]-agmatine), a specific inhibitor of cysteine proteases [33]. A standard curve was obtained by measuring the fluorescence of 0.8 nM 7-amino-4-methyl Coumarin solution (AMC). The cathepsin proteolytic activities were expressed in units (U) defined as the amount of enzyme that hydrolyses 1 μmol of substrate per minute.
Enzyme partial purification
Total egg extract was incubated at 27°C for 18 h in 10 mM sodium acetate buffer pH 5.0 supplemented with 0.5 mM phenylthiocarbamide. The degradation profile was analysed by SDS-PAGE and the enzymatic activity of the proteolytic product was determined as described above.
Inhibition of proteolytic activity
The following protease inhibitors were added to the total egg extract to evaluate the inhibition of the reaction: 50 μM E-64 (cysteine proteases inhibitor), 1 mM PMSF (phenylmethanesulfonyl fluoride; serine proteases inhibitor), 2 mM EDTA (ethylenediamine tetraacetic acid; metalloproteases inhibitor), 10 mM pepstatin (aspartyl proteases inhibitor), and 1 mM CA-074 (N.(L-3.trans-propylcarbamoyloxirane-2-carbonyl)-L-isoleucyl-L-proline; a specific inhibitor of cathepsin B) [34]. Control reactions were incubated without protease inhibitors. The proteolytic products were analysed by SDS-PAGE, and their enzymatic activity was measured as described above.
Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE)
Total egg extract and proteolytic products were mixed with an equal volume of 2x sample buffer (62 mM Tris, 50 mM DTT, 0.2% SDS, 10% glycerol, and 0.01% bromophenol blue). Proteins were resolved by SDS-PAGE using a 12% gel [35] and visualised by staining with 0.2% Coomassie Brilliant Blue R250 (w/v) dissolved in ethanol, acetic acid, and water (45:10:45, v/v/v) (Bio-Rad Laboratories, Brazil), and the molecular masses were estimated using the following protein standards: phosphorylase B (101.4 kDa), bovine serum albumin (87.5 kDa), ovalbumin (52.7 kDa), carbonic anhydrase (35.8 kDa), soybean trypsin inhibitor (22.8 kDa), and lysozyme (18.8 kDa) (Bio-Rad Laboratories, USA).
Total ovary extracts were resolved using 8% SDS-PAGE [35] by the same process described above, and molecular masses were estimated using the following protein standards: myosin (202.8 kDa), β-galactosidase (115.5 kDa), bovine serum albumin (98.2 kDa), ovalbumin (51.4 kDa) (Bio-Rad Laboratories, USA).
Mass spectrometry analyses
Protein bands of interest were excised from the gel and in-gel digestion was performed as previously described [36]. Following desalting with Zip-Tip (Millipore, USA), tryptic fragments were analysed using a nano-HPLC-MS/MS (LCQDuo ion trap mass spectrometer; Finnigan, Thermo Fisher Scientific Inc. Waltham, Massachusetts, USA) through a 120 min gradient from 5% to 56% acetonitrile in 0.2% formic acid. The digestion spectra were analysed with the Sequest program (Thermo Fisher Scientific Inc.) using a NCBI (National Center for Biotechnology Information, http://blast.ncbi.nlm.nih.gov/blast) non-redundant database of Culex and confirmed with the specific vector database (https://www.vectorbase.org/). Peptides were validated using protein probability ≤ 1 x 10–7, dCN ≥ 0.05, and Xcorr values of 1.5, 2.2, and 2.7 for singly, doubly or triply charged peptides, respectively. Proteins with a minimum of two validated peptides were considered.
Oligonucleotide design
Primers for RT-PCR amplification, detection and quantification of rp49 ribosomal protein and vitellogenin transcripts were designed using the Primer3 program (http://frodo.wi.mit.edu/) [37], and primers for enzyme-coding transcripts were manually designed over the 3’ noncoding region (3’UTR) of each transcript. All primers were designed using the transcript sequences available at VectorBase as template (Table 1) and synthesised by Exxtend (Brazil). The amplification efficiency for each primer was: RP49: 90,8%; CatB1: 96,82%; CatB2: 100,84%; and Vg: 101,03%.
RNA extraction
Total RNA was extracted using Trizol (Life Technologies, USA) from dissected fat bodies of adult females fed on sucrose (SUC) and at 12, 24, 36, 48, 60, 72, and 84 hours post blood meal (PBM). Alternatively, whole adult females 24 hours PBM were dissected at different times and total RNA was extracted as described above. In all samples, residual genomic DNA was removed by incubation with DNase I, Amp Grade (Invitrogen, USA) and RNA integrity was evaluated by agarose gel electrophoresis. RNA was quantified using a NanoDrop ND-1000 spectrophotometer (Thermo Scientific, USA).
RT-PCR
Reverse transcription (RT) was carried out with 2.2 μg of total RNA primed with 500 ng of Oligo dT (Life Technologies, USA) using the SuperScript II first-strand synthesis system (Life Technologies, USA) according to the manufacturer’s instructions.
PCR was performed with 1 μl of complementary DNA (cDNA) as template and 0.4 μM of each primer (Table 1). Reactions were performed in a T3 Biometra (Biometra, Germany) thermocycler as follows: 2 min at 94°C, followed by 40 cycles of 94°C for 20 s, 57°C for 20 s, and 72°C for 40 s. The amplified products were resolved on 1.0% agarose gels, stained with Gel Red (Uniscience, Brazil), and visualised using an Image Quant 300 (GE Life Sciences, USA).
Quantitative RT-PCR
Quantitative RT-PCR (qRT-PCR) was performed using a StepOne Real-Time PCR System (Applied Biosystems, USA) in 96-well optical reaction plates (Applied Biosystems, USA). One microliter of cDNA template, 0.6 μM of each specific primer (Table 1), and 1 μl of Maxima SYBR Green (Thermo Scientific, USA) were used for each reaction. The thermal cycling program was: 10 min at 95°C, followed by 40 cycles of 95°C for 15 s, 57°C for 1 min, and 60°C for 1 min. The threshold cycle was normalised according to the rp49 ribosomal protein expression, and relative gene expression was calculated by the 2-ΔΔCT method [38], using expression data generated from adult females fed on sucrose as a reference condition. All depicted data correspond to three independent biological samples analysed in triplicate.
Sequencing and analysis
Partial fragments amplified from genomic DNA and cDNA for all the analysed genes were sequenced on both strands with BigDye Terminator v3.1 (Applied Biosystems, USA) on an automatic sequencer model ABI 3100 (Applied Biosystems, USA). The resulting sequences were subjected to BLASTn searches using the major databases, NCBI and VectorBase. The sequence analyses after sequencing were performed using Bioedit (http://www.mbio.ncsu.edu/bioedit/bioedit.html [39] and aligned by ClustalW (http://www.ebi.ac.uk/Tools/msa/clustalw2 [40]) software. Signal peptides were predicted by the SignalP 4.0 algorithm (http://www.cbs.dtu.dk/services/SignalP/ [41]). The phylogenetic tree was constructed using maximum likelihood bootstrap with 1000 replicates, built in the MEGA 6 program [42].
Determination of cathepsin B activity during vitellogenesis
Three pairs of ovaries dissected from females fed on sucrose (SUC) and at 12, 24, 36, 48, 60, 72, and 84 hours post blood meal (PBM) were homogenised in 60 μl of PBS in the absence of protease inhibitors. Enzymatic activity was measured using 10 μl of homogenate as described above.
Statistical analysis
All experiments were repeated three times and the data were analysed using one-way or two-way analysis of variance (ANOVA) and the Tukey's HSD (Honestly Significant Difference) test with confidence intervals of 95% with the GraphPad InStat 3.02 software package (GraphPad Software Inc., USA). Experimental values were obtained from three independent assays and expressed as the means ± standard error.
Results and Discussion
Enzymatic activity profile of cathepsin B
The activity of cathepsin B has been previously characterised by hydrolysis of the specific synthetic substrate, Z-Arg-Arg-NHMec [43], at an acidic pH in ovaries and eggs of Diptera such as Drosophila melanogaster [8], Musca domestica [9,10] and Aedes aegypti [11]. For all of these insects, cathepsin B has been shown to play a role in the degradation of yolk during embryogenesis.
Based on these data, we confirmed cathepsin B activity in total extract from dark eggs (approximately 24 h after oviposition) of Cx. quinquefasciatus with Z-Arg-Arg-NHMec at an optimum pH level between 5.0 and 5.5 (Fig. 1), similar to the conditions described previously [8–11]. Based on these data, all future experiments were conducted at pH 5.0 at 27°C.
Enzymatic activity of total extract of dark eggs (approximately 24 h after oviposition) of Cx. quinquefasciatus on Z-Arg-Arg-NHMec was assessed at different pHs at 27°C. The proteolytic activities are expressed in units (U) defined as the amount of enzyme that hydrolyses 1 μmol of substrate per minute. Each point represents the mean expression ± standard error of three independent biological experiments.
Self-proteolysis
The SDS-PAGE profile of the dark eggs’ total extract (Fig. 2A) revealed a drastic decrease in band number compared to extracts from vitellogenic ovaries or white eggs (S1 Fig.). These results confirm recent data [44], which describes advanced embryonic development at 24 h after oviposition.
A: 12% SDS-PAGE of the total extract (TE) before or after incubation at 27°C for 18 h without protease inhibitors (WI) or in the presence of specific inhibitors of various proteases: 50 mM E-64 (cysteine proteases); 1 mM CA-074 (cathepsin B); 1 mM PMSF (serine proteases); 2 mM EDTA (metalloproteases); 10 mM pepstatin (aspartyl proteases); WI: without inhibitor. A total of 2 μg of protein was loaded into each lane, and the molecular weight (MW) is shown in kiloDaltons. The arrow points to the approximately 30 kDa band, which was analysed by mass spectrometry. B: The proteolytic activity, calculated at pH 5.0, of the total extract from Cx. quinquefasciatus dark eggs prior to and after incubation in the presence or not of the same specific protease inhibitors. Proteolytic activity is expressed in units (U) defined as the amount of enzyme that hydrolyses 1 μmol of substrate per minute. Each column represents the mean expression ± standard error of three independent biological experiments; asterisks denote statistically significant divergences compared to the standard condition (total extract) as determined by one-way ANOVA (*: p < 0.001; **: p < 0.0001) and the Tukey's HSD (Honestly Significant Difference) test.
Only one protein band was observed at approximately 30 kDa (the approximate mass of the dipteran cathepsin B homologues previously described [9–11]; Fig. 2A, arrow) within egg extracts that were incubated without protease inhibitors for 18 hours at pH 5.0 and 27°C [10] compared to the total egg extract prior to incubation (Fig. 2A).
Together, these results suggested that at least one active protease remains within the egg extract (Fig. 2B). The residual enzymatic activity was shown to be inhibited by E-64 and CA-074, a specific inhibitor of cysteine proteases [33] and cathepsin B [34], respectively, but not by any other enzyme inhibitors tested, including PMSF (serine proteases), EDTA (metalloproteases) and pepstatin (aspartyl proteases; Fig. 2B).
Mass spectrometry identifies cathepsin B peptides
The 30 kDa band (Fig. 2A) was excised from the gel and submitted for in gel reduction, alkylation and trypsinisation. The resulting tryptic peptides were individually analysed by mass spectrometry. Sequest analysis [45] of MS/MS data revealed that the peptides corresponding to the protein band shared identity with two cathepsins B of Cx. quinquefasciatus deposited in the VectorBase database with codes CPIJ015761 and CPIJ015762 (S1 Table). In this work, we will refer to CPIJ015761 as CatB1 and CPIJ015762 as CatB2.
The protein sequences of CatB1 and CatB2 identified by peptide analysis were found to display high alignment similarity with a lysosomal cathepsin B of Homo sapiens (AAH10240.1) [43] and with a cathepsin B of Ae. aegypti (AAEL007585) [11,12] (S2 Fig.; S2 Table). This analysis also identified the catalytic dyad, which consists of a cysteine and histidine residue and is specific to cysteine proteases [43].
The alignment of the deduced amino acid sequences of CatB1 (342 aa) and CatB2 (353 aa) revealed approximately 66% shared similarity (S2 Fig.; S2 Table).
The nucleotide sequence analysis of the two Cx. quinquefasciatus cathepsins B genes obtained from the VectorBase database showed 73% shared identity, with the exception of non-coding regions within the 3' UTR region (S3 Fig.), which allowed for the design and synthesis of specific reverse primers for each gene (Table 1). Oligonucleotide specificity was confirmed by sequencing PCR products and by the dissociation curve analysis generated by real-time PCR, which revealed a single peak for each cathepsin B transcript (data not shown).
Expression profiling of cathepsins B1 and B2
Previous work, performed in several insect species, has highlighted the role of a single acting vitellolytic cathepsin B in yolk protein degradation. However, it remains unclear whether multiple peptidases may contribute to targeted protein degradation within the oocyte. In support of potential enzyme cooperativity, Price and co-workers [12] reported the e transcription of four genes of cathepsin B within the fat body of Ae. aegypti female following a blood meal; however, the enzymatic activities were not analysed. In contrast, Snigirevskaya and colleagues [46] reported only a single cathepsin B at the yolk granule periphery in the oocytes of the same mosquito. As vitellogenin, this enzyme is synthesised by the vitellogenic fat body as a 44 kDa precursor protein, which is then secreted into the haemolymph and internalised by the developing oocytes. Following fertilisation, it is activated by the acidification of the granule to produce an active enzyme of 33 kDa [11,46].
A single cathepsin B was also described in other dipterans as an enzyme of 39 kDa in Drosophila melanogaster [8], 41 kDa in Musca domestica [9,10], and 30 kDa in Blatella germanica (Dictyoptera) [22] and Bombyx mori (Lepidoptera) [16].
These findings led us to investigate whether CatB1 and CatB2 of Cx. quinquefasciatus are co-expressed. For this purpose, Cx. quinquefasciatus females 24 hours PBM were individually tested by quantitative relative real-time PCR and revealed that CatB1 and CatB2 were simultaneously expressed in each mosquito analysed (Fig. 3).
Quantitative relative real-time PCR was used to examine the transcript levels of CatB1 (dotted bars) and CatB2 (grey bars) from 9 individual Cx. quinquefasciatus females 24 h PBM. The mean expression ratio between CatB1 and CatB2 is 1.2 in all of the subjects. Each column represents the mean expression ± standard error of the nine individuals.
Based on the finding that the cathepsins B1 and B2 of Cx. quinquefasciatus were expressed simultaneously and reside within the same phylogenetic tree constructed from the amino acid sequences of cathepsins B of Cx. quinquefasciatus and Ae. aegypti [11,12,47], we hypothesise that they are likely the product of a successful gene duplication [48,49] (S4 Fig.).
In addition to simultaneous expression, both enzymes were shown to be upregulated post blood meal (Fig. 4), with peak expression occurring at 36 h PBM, similar to vitellogenin (Fig. 4). Interestingly, while the expression of vitellogenin showed a sharp decrease at 60 h PBM, CatB1 and CatB2 expression remained high until 84 h PBM, which coincides with female entry into oviposition (Patricia S. Yogi, unpublished data). The amount of cDNA was confirmed using the RP49 gene, which was also used as housekeeping control gene for the qRT-PCR (S5 Fig.).
Quantitative real-time PCR was used to examine the transcript levels of CatB1 (A), CatB2 (B), and vitellogenin (Vg; C) of females fed on sucrose (SUC) and every 12 h PBM (12, 24, 36, 60, 72, 84). Each column represents the mean expression ± standard error of three independent biological replicates; asterisks denote data with a statistically significant differences compared to the standard condition (SUC), as determined by one-way ANOVA (*: p < 0.05; **: p <0.001; ***: p < 0.001) and the Tukey's HSD (Honestly Significant Difference) test.
Enzyme activity profiling of cathepsins B during vitellogenesis
While the transcription of both cathepsins B was shown to be upregulated at 12 h PBM (Fig. 4A and 4B), the enzymatic activity was only detectable at 24 h PBM and continuously increased until reaching a plateau at 60 h PBM (Fig. 5). We hypothesise that this observation may be explained by cessation of incorporation, as this time is synonymous with endochorion secretion by the follicular cells [50].
Enzymatic activity of cathepsin B was measured in a single ovary of adult females fed on sucrose (SUC) and every 12 h PBM (12, 24, 36, 60, 72, 84). U/ovary: Units per ovary, where unit is defined as the amount of enzyme that hydrolyses 1 μmol of substrate per minute. Each column represents the mean expression ± standard error of three independent biological replicates; asterisks denote data with a statistically significant differences compared to the standard condition (SUC), as determined by one-way ANOVA (*: p < 0.05; **: p < 0.001) and Tukey's HSD (Honestly Significant Difference) test.
Conclusions
In this paper, we describe the transcriptional profile of two cathepsin B homologues, which are expressed simultaneously in the fat body of Cx. quinquefasciatus vitellogenic females from 12 h PBM.
Both cathepsins were identified by mass spectrometry from the total extract of Cx. quinquefasciatus eggs after self- proteolysis. While in silico analysis suggested that both sequences harbour catalytic residues that may contribute to the observed enzymatic activity, it was not possible to determine whether both enzymes are simultaneously activated.
Finally, the transcriptional profile of the two cathepsin B genes is similar to that of vitellogenin, which supports the notion that the two enzymes likely cooperate in vitellin degradation.
Supporting Information
S1 Fig. Analysis of the total extract from Cx. quinquefasciatus ovaries and eggs.
8% SDS-PAGE was used to visualise the total extract of ovaries (OVA) at 96 and 120 h PBM and eggs 2 h (white eggs) and 24 h (dark eggs) after oviposition. In each lane, 2 g of total protein was loaded, and the molecular weight is shown in kiloDaltons.
https://doi.org/10.1371/journal.pone.0118736.s001
(TIF)
S2 Fig. Alignment of the deduced amino acid sequences of different cathepsins B.
Sequences of Homo sapiens (AAH10240.1; NCBI) [43], Ae. aegypti (AAEL007585; VectorBase) [11,12] and Cx. quinquefasciatus (CatB1: CPIJ015761 and CatB2: CPIJ015762; VectorBase) were compared. Box region: signal peptides; black background indicate identical amino acids; asterisks indicate catalytic site residues: cysteine (C) and histidine (H).
https://doi.org/10.1371/journal.pone.0118736.s002
(TIF)
S3 Fig. Alignment of the nucleotide sequences of Cx. quinquefasciatus cathepsins B.
A: Alignment of the 3'UTR regions of CatB1 (CPIJ015761) and CatB2 (CPIJ015762) of Cx. quinquefasciatus. Boxes indicate the sequences used to design reverse primers. B: Alignment of the nucleotide sequences of the Cx quinquefasciatus cathepsin B genes: CatB1 (CPIJ015761) and CatB2 (CPIJ015762). Sequences highlighted in black represent conserved nucleotides.
https://doi.org/10.1371/journal.pone.0118736.s003
(TIF)
S4 Fig. Phylogenetic tree of the cathepsins B.
A phylogenetic tree was constructed using the cathepsins B amino acid sequences of Cx. quinquefasciatus (CPIJ015761, CPIJ015762; VectorBase) and Ae. aegypti (AAEL007585, AAEL007590, AAEL007599; VectorBase).
https://doi.org/10.1371/journal.pone.0118736.s004
(TIF)
S5 Fig. Cathepsins B and vitellogenin expression during the vitellogenic process of Cx. quinquefasciatus.
The expression of CatB1, CatB2, vitellogenin (Vg), and ribosomal protein 49 (RP49) was calculated using RT-PCR in females fed on sucrose (SUC) and every 12 h PBM (12, 24, 36, 60, 72, 84). C+: positive control (genomic DNA); C-: negative control (without DNA).
https://doi.org/10.1371/journal.pone.0118736.s005
(TIF)
S1 Table. The physicochemical properties of the peptide sequences of Cx. quinquefasciatus cathepsins B.
Listed are each of the peptides detected by mass spectrometry of the 30 kDa band and the percentage of similarity observed between each peptide and the protein sequences of each cathepsin B (CPIJ015761 and CPIJ015762) using BLASTp. The bolded amino acid (H) represents the catalytic histidine. The cross correlation (Xcorr) function was used to assess the quality of peptide spectra matches. The Delta Correlation (DeltaCN) represents the difference between the normalised Xcorrs of the primary and secondary matches.
https://doi.org/10.1371/journal.pone.0118736.s006
(DOCX)
S2 Table. Comparison of the amino acid sequences of different cathepsins B.
The percent of similarity between the amino acid sequences of cathepsins B of Homo sapiens (AAH10240.1; NCBI; Barrett and Kirschke, 1980), Ae. aegypti (AAEL007585; VectorBase; Cho et al., 1999; Price et al., 2010) and Cx. quinquefasciatus (CatB1: CPIJ015761 and CatB2: CPIJ015762; VectorBase) is depicted.
https://doi.org/10.1371/journal.pone.0118736.s007
(DOCX)
Acknowledgments
We are grateful to Claudia Blanes Angeli for her technical support with mass spectrometry and to Aparecida Sadae Tanaka and Pedro Ismael da Silva Junior for suggestions during the development of this work.
Author Contributions
Conceived and designed the experiments: ASM CEW ATB. Performed the experiments: ASM AFC. Analyzed the data: ASM AFC ALCS CEW ATB. Contributed reagents/materials/analysis tools: CEW ATB. Wrote the paper: ASM AFC ALCS CEW ATB. Ethics protocols were obtained by: ASM ATB.
References
- 1. Gaunt MW, Sall AA, de Lamballerie X, Falconar AKI, Dzhivanian TI, Gould EA. Phylogenetic relationships of flaviviruses correlate with their epidemiology, disease association and biogeography. J Gen Virol. 2001;82: 1867–1876. pmid:11457992
- 2. Vaidyanathan R, Scott TW. Geographic variation in vector competence for West Nile virus in the Culex pipiens (Diptera: Culicidae) complex in California. Vector Borne Zoonot. 2007;7: 193–198. pmid:17627438
- 3. Hamer GL, Kitron UD, Brawn JD, Loss SR, Ruiz MO, Goldberg TL, et al. Culex pipiens (Diptera: Culicidae): A bridge vector of West Nile virus to humans. J Med Entomol. 2008;45: 125–128. pmid:18283952
- 4. Richards SL, Anderson SL, Lord CC. Vector competence of Culex pipiens quinquefasciatus (Diptera: Culicidae) for West Nile virus isolates from Florida. Trop Med Int Health. 2014;19: 610–617. pmid:24898274
- 5.
Rey L. Parasitologia. Rio de Janeiro: Guanabara Koogan; 2008.
- 6. Roth TF, Porter KR. Yolk protein uptake in the oocyte of the mosquito Aedes aegypti. L. J Cell Biol. 1964;20: 313–332. pmid:14126875
- 7. Raikhel AS, Dhadialla TS. Accumulation of yolk proteins in insect oocytes. Annu Rev Entomol. 1992;37: 217–251. pmid:1311540
- 8. Medina M, León P, Vallejo CG. Drosophila cathepsin B-like proteinase: A suggested role in yolk degradation. Arch Biochem Biophys. 1988;263: 355–363. pmid:3132106
- 9. Ribolla PEM, Daffre S, de Bianchi AG. Cathepsin B and acid phosphatase activities during Musca domestica embryogenesis. Insect Biochem Mol Biol. 1993;23: 217–223.
- 10. Ribolla PEM, de Bianchi AG. Processing of procathepsin from Musca domestica eggs. Insect Biochem Mol Biol. 1995;25: 1011–1017. pmid:8541883
- 11. Cho WL, Tsao SM, Hays AR, Walter R, Chen JS, Snigirevskaya ES, et al. Mosquito cathepsin B-like protease involved in embryonic degradation of vitellin is produced as a latent extraovarian precursor. J Biol Chem. 1999;274: 13311–13321. pmid:10224092
- 12. Price DP, Nagarajan V, Churbanov A, Houde P, Milligan B, Drake LL, et al. The fat body transcriptomes of the yellow fever mosquito Aedes aegypti, pre- and post- blood meal. PLoS One 2011;6: e22573. pmid:21818341
- 13. Takahashi SY, Yamamoto Y, Shionoya Y, Kageyama T. Cysteine proteinase from the eggs of the silkmoth, Bombyx mori: identification of a latent enzyme and characterization of activation and proteolytic processing in vivo and in vitro. J Biochem. 1993;114: 267–272. pmid:8262908
- 14. Takahashi SY, Yamamoto Y, Watabe S, Kageyama T. Autolytic activation mechanism of Bombyx acid cysteine protease (BCP). Biochem Mol Biol Int. 1997;42: 591–600. pmid:9247717
- 15. Takahashi SY, Yamamoto Y, Zhao XF, Watabe S. Bombyx acid cysteine proteinase. Invertebrate Reprod Dev. 1996;30: 265–281.
- 16. Yamamoto Y, Takahashi SY. Cysteine proteinase from Bombyx eggs: role in programmed degradation of yolk proteins during embryogenesis. Comp Biochem Phys B. 1993;106: 35–45. pmid:8403852
- 17. Yamamoto Y, Yamahama Y, Katou K, Watabe S, Takahashi SY. Bombyx acid cysteine protease (BCP): hormonal regulation of biosynthesis and accumulation in the ovary. J Insect Physiol. 2000;46: 783–791. pmid:10742527
- 18. Yamamoto Y, Takimoto K, Izumi S, Toriyama-Sakurai M, Kageyama T, Takahashi SY. Molecular cloning and sequencing of cDNA that encodes cysteine proteinase in the eggs of the silkmoth, Bombyx mori. J Biochem. 1994;116: 1330–1335. pmid:7706225
- 19. Zhao XF, Wang JX, Wang YC. Purification and characterization of a cysteine proteinase from eggs of the cotton boll worm, Helicoverpa armigera. Insect Biochem Mol Biol. 1998;28: 259–264.
- 20. Zhao XF, Wang JX, Xu XL, Schmid R, Wieczorek H. Molecular cloning and characterization of the cathepsin B-like proteinase from the cotton boll worm, Helicoverpa armigera. Insect Mol Biol. 2002;11: 567–575. pmid:12421414
- 21. Zhao XF, Wang JX, Li FX, Sueda S, Kondo H. Analysis of substrate specificity and endopeptidyl activities of the cathepsin B-like proteinase from Helicoverpa armigera. Protein J. 2005;24: 219–225. pmid:16283544
- 22. Liu XD, McCarron RC, Nordin JH. A cysteine protease that processes insect vitellin. Purification and partial characterization of the enzyme and the proenzyme. J Biol Chem. 1996;271: 33344–33351. pmid:8969194
- 23. Oliveira DMP, Ramos IB, Reis FCG, Lima APCA, Machado EA. Interplay between acid phosphatase and cysteine proteases in mediating vitellin degradation during early embryogenesis of Periplaneta americana. J Insect Physiol. 2008;54: 883–891. pmid:18499122
- 24. Uchida K, Nishizuka M, Ohmori D, Ueno T, Eshita Y, Fukunaga A. Follicular epithelial cell apoptosis of atretic follicles within developing ovaries of the mosquito Culex pipiens pallens. J Insect Physiol. 2004;50: 903–912. pmid:15518658
- 25. Uchida K, Ohmori D, Ueno T, Nishizuka M, Eshita Y, Fukunaga A, et al. Preoviposition activation of cathepsin-like proteinases in degenerating ovarian follicles of the mosquito Culex pipiens pallens. Dev Biol. 2001;237: 68–78. pmid:11518506
- 26. da Cunha Sais T, de Moraes RM, Ribolla PE, de Bianchi AG, Marinotti O, Bijovsky AT. Morphological aspects of Culex quinquefasciatus salivary glands. Arthropod Struct Dev. 2003;32: 219–226. pmid:18089007
- 27. Ribeiro JM, Charlab R, Pham VM, Garfield M, Valenzuela JG. An insight into the salivary transcriptome and proteome of the adult female mosquito Culex pipiens quinquefasciatus. Insect Biochem Mol Biol. 2004;34: 543–563. pmid:15147756
- 28. Okuda K, de Almeida F, Mortara RA, Krieger H, Marinotti O, Bijovsky AT. Cell death and regeneration in the midgut of the mosquito, Culex quinquefasciatus. J Insect Physiol. 2007;53: 1307–1315. pmid:17716685
- 29. Okuda K, de Souza Caroci A, Ribolla PE, de Bianchi AG, Bijovsky AT. Functional morphology of adult female Culex quinquefasciatus midgut during blood digestion. Tissue Cell 2002;34: 210–219. pmid:12182814
- 30. Van Essen PH, Kemme JA, Ritchie SA, Kay BH. Differential responses of Aedes and Culex mosquitoes to octenol or light in combination with carbon dioxide in Queensland, Australia. Med Vet Entomol. 1994;8: 63–67. pmid:8161847
- 31. Bracco JE, Barata JM, Marinotti O. Evaluation of insecticide resistance and biochemical mechanisms in a population of Culex quinquefasciatus (Diptera: Culicidae) from Sao Paulo, Brazil. Mem Inst Oswaldo Cruz 1999;94: 115–120. pmid:10029921
- 32. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976;72: 248–254. pmid:942051
- 33. Barrett AJ, Kembhavi AA, Brown MA, Kirschke H, Knight CG, Tamai M, et al. L-trans-Epoxysuccinyl-leucylamido(4-guanidino)butane (E-64) and its analogues as inhibitors of cysteine proteinases including cathepsins B, H and L. Biochem J. 1982;201: 189–198. pmid:7044372
- 34. Murata M, Miyashita S, Yokoo C, Tamai M, Hanada K, Hatayama K, et al. Novel epoxysuccinyl peptides. Selective onhibitors of cathepsin-B, in vitro. FEBS Letters 1991;280: 307–310. pmid:2013328
- 35. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685. pmid:5432063
- 36. Shevchenko A, Tomas H, Havlis J, Olsen JV, Mann M. In-gel digestion for mass spectrometric characterization of proteins and proteomes. Nat Protoc. 2006;1: 2856–2860. pmid:17406544
- 37. Untergasser A, Cutcutache I, Koressaar T, Ye J, Faircloth BC, Remm M, et al. Primer3-new capabilities and interfaces. Nucleic Acids Res. 2012;40.
- 38. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 2001;25: 402–408. pmid:11846609
- 39. Dunkov BC, Georgieva T, Yoshiga T, Hall M, Law JH. Aedes aegypti ferritin heavy chain homologue: feeding of iron or blood influences message levels, lengths and subunit abundance. J Insect Sci. 2002;2: 7. pmid:15455041
- 40. Higgins DG, Sharp PM. CLUSTAL: a package for performing multiple sequence alignment on a microcomputer. Gene 1988;73: 237–244. pmid:3243435
- 41. Nielsen H, Engelbrecht J, Brunak S, von Heijne G. A neural network method for identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Int J Neural Syst. 1997;8: 581–599. pmid:10065837
- 42. Tamura K, Stecher G, Peterson D, Filipski A, Kumar S. MEGA6: Molecular Evolutionary Genetics Analysis version 6.0. Mole Biol Evol. 2013;30: 2725–2729. pmid:24132122
- 43. Barrett AJ, Kirschke H. Cathepsin B, Cathepsin H, and cathepsin L. Methods Enzymol. 1981;80 Pt C: 535–561. pmid:7043200
- 44. Vargas HC, Farnesi LC, Martins AJ, Valle D, Rezende GL. Serosal cuticle formation and distinct degrees of desiccation resistance in embryos of the mosquito vectors Aedes aegypti, Anopheles aquasalis and Culex quinquefasciatus. J Insect Physiol. 2014;62: 54–60. pmid:24534672
- 45. Eng JK, Mccormack AL, Yates JR. An approach to correlate tandem mass spectral data of peptides with amino acid sequences in a protein database. J Am Soc Mass Spectrom. 1994;5: 976–989. pmid:24226387
- 46. Snigirevskaya ES, Hays AR, Raikhel AS. Secretory and internalization pathways of mosquito yolk protein precursors. Cell Tissue Res. 1997;290: 129–142. pmid:9377633
- 47. Dissanayake SN, Ribeiro JM, Wang MH, Dunn WA, Yan G, James AA, et al. aeGEPUCI: a database of gene expression in the dengue vector mosquito, Aedes aegypti. BMC Res Notes 2010;3: 248. pmid:20920356
- 48. Neurath H, Walsh KA, Winter WP. Evolution of Structure and Function of Proteases—Amino Acid Sequences of Proteolytic Enzymes Reflect Phylogenetic Relationships. Science 1967;158: 1638–1644. pmid:4862530
- 49. Shimeld SM. Gene function, gene networks and the fate of duplicated genes. Semin Cell Dev Biol. 1999;10: 549–553. pmid:10597639
- 50. Cardoso AF, Cres RL, Moura AS, de Almeida F, Bijovsky AT. Culex quinquefasciatus vitellogenesis: morphological and biochemical aspects. Mem Inst Oswaldo Cruz 2010;105: 254–262. pmid:20512237