Molecular characterization of Rhodnius prolixus' embryonic cuticle.

Insect Biochemistry and Molecular Biology xxx (2014) 1e12

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Molecular characterization of Rhodnius prolixus’ embryonic cuticle Paula S. Souza-Ferreira a, Mônica F. Moreira b, c, Geórgia C. Atella a, c, Ana Lúcia Oliveira-Carvalho a, Roberto Eizemberg d, David Majerowicz a, Ana C.A. Melo b, c, Russolina B. Zingali a, Hatisaburo Masuda a, c, * a Universidade Federal do Rio de Janeiro, Instituto de Bioquímica Médica, Programa de Biologia Molecular e Biotecnologia, 21941-902 Rio de Janeiro, RJ, Brazil b Universidade Federal do Rio de Janeiro, Instituto de Química, 21941-909 Rio de Janeiro, RJ, Brazil c Instituto Nacional de Ciência e Tecnologia em Entomologia Molecular, 21941-902 Rio de Janeiro, Brazil d Universidade Federal do Rio de Janeiro, Escola de Educação Física e Desportos, 21941-599 Rio de Janeiro, RJ, Brazil

a r t i c l e i n f o

a b s t r a c t

Article history: Received 12 August 2013 Received in revised form 5 November 2013 Accepted 9 December 2013

The embryonic cuticle (EC) of Rhodnius prolixus envelopes the entire body of the embryo during hatching and provides physical protection, allowing the embryo to pass through a narrow chorionic border. Most of the knowledge about the EC of insects is derived from studies on ultrastructure and secretion processes during embryonic development, and little is known about the molecular composition of this structure. We performed a comprehensive molecular characterization of the major components extracted from the EC of R. prolixus, and we discuss the role of the different molecules that were identified during the eclosion process. The results showed that, similar to the post-embryonic cuticles of insects, the EC of R. prolixus is primarily composed of carbohydrates (57%), lipids (19%), and proteins (8%). Considering only the carbohydrates, chitin is by far the major component (approximately 70%), and it is found primarily along the body of the EC. It is scarce or absent in its prolongations, which are composed of glycosaminoglycans. In addition to chitin, we also identified amino (15%), neutral (12%) and acidic (3%) carbohydrates in the EC of R. prolixus. In addition carbohydrates, we also identified neutral lipids (64.12%) and phospholipids (35.88%). Proteomic analysis detected 68 proteins (55 were identified and 13 are hypothetical proteins) using the sequences in the R. prolixus genome (http://www.vectorbase.org). Among these proteins, 8 out of 15 are associated with cuticle metabolism. These proteins are unequivocally cuticle proteins, and they have been described in other insects. Approximately 35% of the total proteins identified were classified as having a structural function. Chitin-binding protein, amino peptidase, amino acid oxidase, oxidoreductase, catalase and peroxidase are all proteins associated with cuticle metabolism. Proteins known to be cuticle constituents may be related to the function of the EC in assisting the insect during eclosion. To our knowledge, this is the first study to describe the global molecular composition of an EC in insects. Ó 2014 Elsevier Ltd. All rights reserved.

In memoriam of our colleague Dr. Alexandre Peixoto. Keywords: Embryonic cuticle Rhodnius prolixus Carbohydrates Lipids Proteins Eclosion

1. Introduction In recent years, possibly due to the availability of the Rhodnius prolixus genome (http://www.vectorbase.org), an increasing number of studies at the molecular level have performed to understand the biology of this species. R. prolixus is one of the main vectors of Chagas disease in Latin America (Schofield, 2000). This species produces approximately 40 eggs per female

* Corresponding author. Instituto Nacional de Ciência e Tecnologia em Entomologia Molecular, 21941-902 Rio de Janeiro, Brazil. E-mail address: [email protected] (H. Masuda).

after a single blood meal (Atella et al., 2005). Oviposition occurs in the environment, and embryonic development lasts approximately 15 days and varies with ambient temperature (Kelly and Huebner, 1989; Melllanby, 1935; Wigglesworth, 1972). The main hurdle faced by R. prolixus nymphs during eclosion is exiting the chorion through the narrow chorionic border orifice. The EC envelopes the entire body of the hatching insect, providing physical protection and serving as a conducting channel as the insect passes through the chorionic border (Wigglesworth, 1972). The presence of an EC has been reported in several insect orders (Konopova and Zrzavy, 2005). Moussian et al. (2006) showed that cuticle differentiation occurs in three phases during Drosophila melanogaster embryogenesis that is genetic controlled (Payre,

0965-1748/$ e see front matter Ó 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.ibmb.2013.12.005

Please cite this article in press as: Souza-Ferreira, P.S., et al., Molecular characterization of Rhodnius prolixus’ embryonic cuticle, Insect Biochemistry and Molecular Biology (2014), http://dx.doi.org/10.1016/j.ibmb.2013.12.005

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P.S. Souza-Ferreira et al. / Insect Biochemistry and Molecular Biology xxx (2014) 1e12

2004). The layers that are established partially simultaneously in the first stage thicken in the second stage. After the secretion of materials ceases in the third phase, the chitin laminae acquires its final orientation and the epicuticle darkens. The EC is described as a highly elastic, transparent layer that separates the embryo from the yolk membrane during embryogenesis (Louvet, 1974), but its biology and formation have not been thoroughly investigated (Fewkes, 1968; Konopova and Zrzavy, 2005; Provine, 1976; Wigglesworth, 1972). The knowledge of embryonic cuticles (EC) is based on data on the ultrastructure and the secretion processes during the embryonic development of insects. Conversely, studies on post-embryonic cuticles are more comprehensive and include more detailed data on their structure, chemical composition, morphogenesis and the protein products of the cuticular genes (Dombrovsky et al., 2003; Juarez and Fernandez, 2007; Wigglesworth, 1972). The post-embryonic cuticle of insects gives them shape, flexibility, strength, freedom of movement, defense, and protection; assists in homeostasis; protects against excessive moisture and desiccation; and prevents penetration by microorganisms. The success of these functions is directly related to the biochemical composition of the cuticle, and there is a direct correlation between its lipid composition and its permeability, resistance, and malleability. In triatomines, post-embryonic cuticles are primarily composed of neutral lipids, and these cuticles also have high concentrations of triacylglycerol, diacylglycerol, fatty acids, and hydrocarbons. Phospholipids are primarily composed of phosphatidylethanolamine and phosphatidylcholine (Gibbs, 2002; Juarez and Fernandez, 2007). The rigidity of the exoskeleton is conferred by sugar polymers such as chitin (Ostrowski et al., 2002; Richards, 1978). Chitin is a b-glycoside biopolymer composed of units of b-(1-4)-N-acetyl-D-glucosamine, which is the major carbohydrate that is found in the postembryonic cuticle of insects. In general, chitin corresponds to approximately 20e50% of the animal’s dry weight, and variations in the chitin composition of insect cuticles are found among different species, stages, and body parts in the same individual and between sexes in the same species (Moussian et al., 2006; Okot-Kotber et al., 1994; Ostrowski et al., 2002; Richards, 1978; Wigglesworth, 1972). Proteins are the group of macromolecules with the highest number and diversity in insect cuticles, and they are largely responsible for the most significant differences that are found in cuticles. Proteins associated with chitin confer strength and rigidity to cuticles, in addition to flexibility and freedom of movement, and they are essential in adaptation, response, and regulatory processes (Kramer and Muthukrishnan, 2005; Merzendorfer and Zimoch, 2003). Some of the information about insect cuticular proteins (CuPs) comes from sequencing of the N-terminal region of a few proteins extracted from insect cuticles (Hojrup et al., 1986), proteomic analysis (He et al., 2007) and protein sequences derived from the transcription of cDNAs (Asano et al., 2013; Dombrovsky et al., 2003), which were identified based on their similarities to proteins extracted from post-embryonic cuticles (Dombrovsky et al., 2007; Togawa et al., 2008; Willis, 2010). Although more information is available on post-embryonic than ECs, much remains to be discovered and understood in both cases. Possibly due to the difficulty in obtaining enough material from ECs, little is known about their molecular composition. Taking advantage of our access to a large colony of R. prolixus, we were able to directly extract lipids, carbohydrates, and proteins from a vast quantity of EC for analysis. Lipids and carbohydrates were analyzed by standard methods such as TLC, FTIR spectroscopy, HPLC, and fluorescence microscopy, and proteins were analyzed by proteomic technology.

Insect cuticles are comprised of various materials including a mixture of proteins, lipids, carbohydrates, and other minor components. The mechanical properties of the cuticle depend largely on its composition, as well as on the number of interactions that occur among its constituents. Thus, a variety of mechanical characteristics can be obtained, allowing the multimolecular structure to fit the needs of a particular physiological function. In Rhodnius, the composition of an extensible cuticle has been studied by Hillerton (1978). Here, we performed a comprehensive molecular characterization of the major components extracted from the EC of R. prolixus, and we compare our results with published data on postembryonic cuticles. We then discuss the importance of these macromolecules on the eclosion process of first-instars nymphs. 2. Materials and methods 2.1. Reagents All reagents were analytical grade and were acquired from Sigma Chemical Co. (St Louis, MO, USA), Merck (Rio de Janeiro, Brazil), Reagen (Rio de Janeiro, Brazil) or Vetec (Rio de Janeiro, Brazil) and Invitrogen (São Paulo, Brazil). All solutions were prepared using double-distilled water from a Milli-Q system with ionexchange resins (Millipore Corp, Bedford, MA, USA). 2.2. Preparation of Rhodnius prolixus embryonic cuticles (EC) R. prolixus females were reared and maintained at 28 C and 70e 80% relative humidity, and they were fed rabbit blood in the Laboratory of Insect Biochemistry, Federal University of Rio de Janeiro, Brazil. Fertilized eggs were collected hourly, handled in a sterile environment, and stored in sterile vials under the same climatic conditions as the colony. Embryonic cuticles (EC) were collected after eclosion of first-instars nymphs using sterile sieves, spatulas, and tweezers. All animal care and experimental protocols were conducted following the guidelines of the institutional care and use committee (Committee for Evaluation of Animal Use for Research from the Federal University of Rio de Janeiro, CAUAP-UFRJ, Brazil). The protocols were approved by CAUAP-UFRJ under registry #IBqM001. 2.3. Dry weight composition of embryonic cuticle of R. prolixus To estimate the dry weight of different components of R. prolixus’ EC, 3 mg of EC was dehydrated and weighed (total). Next, the material was placed in conical tubes, and total lipid extraction was initiated by adding 4 mL of methanol:chloroform:double-distilled water solution (2:1:1 v/v), followed by intermittent agitation every 5 min for 2 h. The samples were then left standing for 12 h at 4 C. The mixture was centrifuged for 15 min at 1500 g, and the hydrophobic phase was saved. Then, 1 mL of chloroform was added to the remaining material, and the procedure was repeated twice. The hydrophobic phases were pooled, dried in polypropylene tubes, and weighed to measure the amount of lipids extracted (lipid fraction). The remaining fraction was dried and weighed again before treatment with papain (1 mg/ mL in 5 mM sodium acetate, pH 5.0, containing 5 mM EDTA and 5 mM cysteine) for 24 h at 65 C to remove proteins. The remaining material was washed by centrifugation for at 1550 g for 15 min, and the supernatant (A) and precipitate (B) were saved. Ethanol (1:1 v/v) was added to the supernatant (A) to precipitate soluble carbohydrates for 24 h at 4 C, and then they were separated by centrifugation at 1500 g for 15 min. Hexane and H2O (1:1 v/v) were added to the precipitate (B) to remove other hydrophobic



components under agitation for 2 h, and then the insoluble carbohydrates were separated by centrifugation at 1500 g for 15 min (chitin-enriched fraction). The difference in weight before and after the treatment with papain corresponds to the fraction of proteins. 2.4. Acquisition of proteins for proteomic analysis A batch of EC was used exclusively for protein extraction. Three milligrams of EC was homogenized in a Potter-Elvehjem (PE) homogenizer in the presence of 8 M urea and 0.03 M DTT (Bouts et al., 2007) to remove proteins. The material was centrifuged at 1500 g for 30 min to remove the debris, and the supernatant was dialyzed at 4 C for 8 h in 2 L of 0.02 M TriseHCl buffer at pH 8.4. The buffer solution was exchanged four times. The sample was vacuum concentrated in a Savant SVC100 centrifuge and saved in a polypropylene tube at 4 C until use. After urea removal by dialysis and vacuum concentration of the sample, EC proteins were quantified according to the protocol described by Lowry et al. (1951) using bovine serum albumin as a standard. 2.5. Lipid extraction for analysis The lipids for TLC analysis were obtained as described above (Section 2.3). The obtained lipids were stored at 4 C until use. 2.6. Lipid analysis by TLC Neutral lipids were separated by thin layer chromatography (TLC) on one-dimensional CCF-C/25 silica plates (5 20 cm, Merck) using the solvent system described in Kawooya and Law (1988). Neutral lipid standards (Sigma) were added to the TLC plate simultaneously with the sample. After separation, the TLC plate was stained according to the method described by Bitman et al. (1982) and developed in a developing chamber according to Ruiz and Ochoa (1997). Then, TLC plates were digitized, and densitometric analysis of lipid bands was conducted using “ImageMaster TotalLab v1.11” software. The one-dimensional separation of phospholipids was performed using thin layer chromatography (TLC) on CCF-C/25 silica plates (5 20 cm, Merck) using the solvent system of Horwitz and Perlman (1987). The two-dimensional separation of phospholipids was conducted by TLC on CCF-C/25 silica plates (20 20 cm, Merck) using the solvent/buffer system of Yavin and Zutra (1977). Phospholipid standards (Sigma Chemical Co. St Louis. MO, USA) were applied to the TLC plate simultaneously with the sample. Twodimensional TLC plates were first stained with iodine vapors and digitized and then stained with ninhydrin according to the method of Wagner and Bladt (1995). Then, the TLC plates were digitized, and densitometric analysis of lipid bands was conducted using “ImageMaster TotalLab v1.11” software. 2.7. Extraction of carbohydrates The extraction of EC carbohydrates consisted of two steps. As described in Section 2.3, lipids were first extracted from a batch of 12,000 units of EC, and the proteins in the remaining material were digested with papain at 65 C for 24 h according to the protocol of Vieira et al. (1991). Carbohydrates were isolated from the remaining material as described in Section 2.3. Carbohydrates present in both extraction steps (soluble and chitin-enriched fraction) were stored in polypropylene tubes and maintained at 4 C until further analysis.

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2.8. Carbohydrate determination The total carbohydrate content was determined by the phenolsulfuric acid method described by Dubois et al. (1951). The uronic acid content was determined by the carbazole method of Bitter and Muir (1962). 2.9. Carbohydrate analysis by TLC A sample of EC carbohydrates obtained in the second extraction step was hydrolyzed in TFA (1:1 v/v) at 100 C for 6 h. Neutral carbohydrates were separated by thin layer chromatography (TLC) on one-dimensional CCF-C/25 silica plates (5 10 cm, Merck) using the solvent system of Ovodov et al. (1967). Additionally, 7.5 mg of neutral carbohydrate standard (Sigma) was simultaneously applied to the TLC plate. After separation, TLC plates were first stained with ninhydrin according to the method of Wagner and Bladt (1995) and then with orcinol according to the method described by Ovodov et al. (1967). TLC plates were digitized, and densitometric analysis of carbohydrate bands was conducted using “ImageMaster TotalLab v1.11” software. 2.10. Carbohydrate analysis by FTIR spectrometry A sample of EC carbohydrates obtained in the second extraction step was dried, weighed, mixed with potassium bromide (1:1 m/ m), and applied at 0.63 cm/s to a DQI infrared spectrophotometer with a DTGS detector according to the protocols of Potts (1963) and Moreira et al. (2007). The obtained spectrum was compared to the standard spectra of several polysaccharides. 2.11. Carbohydrate analysis by HPLC on HPX-87C column A sample of EC carbohydrates obtained in the second extraction step was placed in a closed vial and subjected to acid hydrolysis in 5.7 N hydrochloric acid solutions for 48 h at 105 C. The hydrolyzed sample was neutralized in 10 N sodium hydroxide solutions, and its concentration was adjusted to 0.5% Brix. Chromatography was performed using HPLC with an HPX-87C column (BIORAD, Hercules, California, USA) at 85 C and a flow rate of 0.35 mL/min according to the protocols of Moreira et al. (2007) and Mansur et al. (2010). The same procedure was repeated with standard commercial chitin to hydrolyze it. To examine the presence of chitin associated with EC, the retention time of the standard glucosamine peak was compared with the retention time of the glucosamine peaks derived from the hydrolysis of commercial chitin and the ECextracted chitin. 2.12. Fluorescence microscopy of the embryonic cuticle of Rhodnius prolixus To localize the b-glycosides in the EC of R. prolixus, ECs were incubated in Calcofluor White M2R, 25 mM sodium phosphate, pH 6.35, for 15 min (Moussian et al., 2006). The excess was removed by washing three times with 25 mM sodium phosphate buffer, pH 6.5. To determine chitin’s location in the EC of R. prolixus, we incubated ECs with a chitin-binding domain associated with fluorescein isothiocyanate (FITC-CBD; New England BioLabs, CO, USA) (Arakane et al., 2005; Mansur et al., 2010). The excess was removed by washing three times with 25 mM sodium phosphate buffer, pH 6.5. The samples were placed on slides and observed under an Axioskop 40 HBO 100 fluorescence microscope equipped with an EBQ 100 fluorescence illuminator, and digital images were acquired on a Zeiss Axio Cam MRc 5. As a control for autofluorescence, we washed


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the EC with 25 mM sodium phosphate buffer, pH 6.5, and visualized these samples under the same conditions. 2.13. Polyacrylamide gel electrophoresis Proteins (90 mg) were subjected to gel electrophoresis (10% SDSPAGE, 75 mm wide) (Laemmli, 1970) in a BioRad vertical gel system at a constant current of 20 mA for 45 min. The proteins were stained with colloidal Coomassie blue G-250 (CBB-G250) (Neuhoff et al., 1988) or silver (Shevchenko et al., 1996; Switzer et al., 1979). Gel images were digitized with Canon Scan using Photoshop CS3 software. 2.14. Protein digestion The gel was cut into regular 0.5 cm sections along the entire column using a ruler and a scalpel, resulting in a total of 12 gel strips per column, regardless of the presence of protein bands. Gel sections were placed in a polypropylene tube, and tryptic gel digestion was conducted according to the protocol described by Shevchenko et al. (1996) with some modifications. Gel fragments were de-stained by immersion in 25 mM ammonium bicarbonate, pH 7.8, with 50% acetonitrile (ACN) for 12 h at 25 C. The material was homogenized in Vivax VM3000 vortex agitator for 1 min. Then, the initial solution was discarded, and the samples were dehydrated by immersion in 100% ACN for 10 min. Subsequently, ACN was discarded, and disulfide bonds in the proteins were reduced by immersion in 25 mM ammonium bicarbonate, pH 7.8, containing 10 mM DTT at 56 C for 1 h. The proteins were then alkylated in a dark room by immersion in 25 mM ammonium bicarbonate containing 50 mM iodoacetamide (IAA) for 45 min at room temperature. Then, proteins were dehydrated by immersion in 100% ACN for 10 min and rehydrated by immersion in 25 mM ammonium bicarbonate, pH 7.8, containing 2 ng/mL trypsin (Promega, Madison, WI, USA) and incubated for 24 h at 37 C. Finally, 0.1% trifluoroacetic acid (TFA) solution with 50% (v/v) ACN was added to the gel fragments, and the samples were subjected to sonication for 20 min. This procedure was repeated five times. The solutions containing the extracted peptides were pooled in a polypropylene tube. The samples were vacuum concentrated in a Savant SVC100 centrifuge and reconstituted in 0.1% v/v trifluoroacetic acid (TFA) solution. 2.15. Analysis of tryptic peptides on a C18 column The columns were mounted on 200 mL pipettes with a small amount of fiber glass (Sigma #F7144) to control the flow of the resin. We used 20 mL of C18 POROS R2 resin solution (Applied Biosystems) in 40 mg/mL 100% ACN. The column was rinsed with 200 mL of 100% isopropanol, activated with 100 mL of 100% methanol, and equilibrated with 200 mL of 0.05% TFA solution. After sample application, the column was washed with 200 mL of 0.05% TFA solution. The peptides were eluted in 80% ACN with 0.05% TFA. The samples were vacuum concentrated in a Savant SVC100 centrifuge and reconstituted in a 1% v/v formic acid solution containing 3% ACN. 2.16. Mass spectrometry protein analysis Samples derived from 1D SDS-PAGE gels were analyzed using a Micromass ESI-Q-ToF mass spectrometer (Waters Corporation e USA) coupled to a NanoUPLC (NanoAcquity e Waters) located in the Proteomics and Mass Spectrometry Unit, Health Sciences Center (Federal University of Rio de Janeiro, Rio de Janeiro, Brazil). The settings for the mass spectrometry platform and the configuration of chromatographic runs were established according to guidelines

recommended by MIAPE (Minimum Information About Proteomics Experiments; http://psidev.info/miape) (Taylor et al., 2007). The raw data obtained in the spectrometric identification were processed using Data Explorer 2.4 software to facilitate the search and identification of proteins using MASCOT software. All results regarding the identification of peptides were visually compared to the original spectra obtained, and only peptides with five residues sequenced consecutively in complementary B- or Y-ion series were considered valid identifications.

2.17. Protein identification and annotation Peptide fragments were identified by comparing the sequences obtained from R. prolixus-CDC_PEPTIDES_RproC1.1.fa.gz (available at http://www.vectorbase.org) using Mascot v2.1 software (Matrix Science Ltd; London, UK) licensed to Brazilian Synchrotron Light Laboratory-Campinas, SP, Brazil. The search parameters were as follows: the loss of one cleavage site (one missed cleavage) by trypsin hydrolysis; 0.2 Da mass tolerance for precursor and ion fragments; ESI-QUAD-ToF instrumental profile; cysteine carbamidomethylation as a fixed modification and methionine oxidation as a variable modification; peptide charge set as þ2/þ3; and MS/MS monoisotopic mass spectrum. The criteria for positive identification included the minimum reliability score provided by Mascot v2.1 software and the percentage of sequence coverage. The ion score was 10 log (P), and the significance level was set at p < 0.5. To determine the rate of false-positives, we included reverse analysis of the data and discarded results with p > 0.5. The analysis in Mascot v2.1 software yielded protein sequences in FASTA format and their related peptides. Validation of the identified proteins was performed by determining whether peptides had 100% identity with the protein (Zhang et al., 2013). Sequences were correlated to the NCBI database via protein BLAST analysis for annotation and verification. The protein selection parameters used were as follows: E-value tending to 0; belongs to Insecta class; and has the largest possible sequence coverage. To assess the sequences and eliminate contaminants, sequences were correlated with proteins from the bacterium Rhodococcus rhodnii. Data from the identification and annotation steps were then processed in UniProt (Universal Protein Resource). We restricted our search to the Insecta class. UniProt mapped and classified sequences according to the Gene Ontology (GO) database classification. We manually adjusted the classification suggested by the database whenever necessary. The presence of a secreted signal peptide was determined using TargetP 1.1 Server (Emanuelsson et al., 2000; Nielsen et al., 1997), SignalP 4.0 Server (Petersen et al., 2011) software. The presence of domains characteristic of structural cuticular proteins defining twelve CP families as described in Willis (2010) were determined using BlastP 2.2.21þ (Altschul et al., 1997 and Schaffer et al., 2001).

3. Results The movements of R. prolixus nymphs during hatching were filmed under a microscope, and the video is available at http:// archive.org/details/EC-Rhodnius. Fig. 1A shows a microphotographic sequence of the process, including the rupture of the EC. Supplementary data related to this article can be found online at http://dx.doi.org/10.1016/j.ibmb.2013.12.005. The major components of the EC of R. prolixus were ranked according to dry weight (Fig. 1B), and their percentages were estimated. The EC is primarily composed of carbohydrates (57%), followed by lipids (19%), proteins (8%), water (13%), and other substances (3%).



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Fig. 1. A) Photomicrography of Rhodnius prolixus emergence. A.1) The insect exits the chorion and pauses to rest. A.2) Simultaneous elevation of the proboscis and rupture of the embryonic cuticle on the dorsal region of the nymph. A.3e5) With repetitive movements, the insect frees itself from the protection of the embryonic cuticle. A.6) Isolated embryonic cuticle. The arrows indicate the embryonic cuticle limits. B) Diagram showing the estimated dry weight of Rhodnius prolixus embryonic cuticle components.

Fig. 2. Characterization of amino monosaccharide associated with the embryonic cuticle of Rhodnius prolixus. 2.1) Comparative analysis by FTIR-DQI coupled to a DTGS detector of monosaccharide samples extracted from the embryonic cuticle with standard chitin. 2.2) Analysis by histolabelling with Calcofluor White M2R (A and B), and FITC-conjugated chitin-binding domain protein (C and D). Left plates (A and C): phase-contrast; Right plates (B and D): fluorescent mode. 2.3) Comparative analysis by HPLC (HPX-87C column) of embryonic cuticle hydrolysate, commercial chitin hydrolysate, and commercial glucosamine.


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3.1. Carbohydrates The FTIR spectrum of the carbohydrates extracted from EC showed a pattern that was typical of chitin and similar to the one exhibited by the commercial chitin used as a control (Fig. 2.1). Typical diagnostic peaks of chitin were observed at approximately 3500, 1675, and 1085 cm-1. The b-glycoside marker Calcofluor White M2R revealed that fluorescence was evenly distributed on the EC (Fig. 2.2A and B). The FITC-conjugated chitin-binding protein (CBD) preferentially marked the body of the EC but not its projections (Fig. 2.2C and D). The peak of amino glycoside extracted from the EC exhibited the same retention time as the product of chitin hydrolysis and the glucosamine standard (Fig. 2.3). The percentage of amino carbohydrates (47.11%) was higher than those of neutral carbohydrates (41.87%) and acid carbohydrates (11.02%) (Table 1). Chromatographic separation showed that neutral monosaccharides were primarily composed of fructose and fucose. 3.2. Lipids The percentage of neutral lipids (64.12%) was higher than that of phospholipids (35.88%) (Table 2). In addition, fatty acids and hydrocarbons were the major components among neutral lipids, whereas phosphatidic acid was the major component among phospholipids, exceeding the abundance of phosphatidylethanolamine and phosphatidylcholine.

Table 2 Characterization of lipid groups associated with the embryonic cuticle of Rhodnius prolixus. Densitometric analysis of lipid groups by TLC CCF-C/25 e N ¼ 5. Lipids groups

Lipids identified by retention frame

Percentage found

Neutral lipids

Fatty acids Hydrocarbons Cholesterol Sterified cholesterol Monoacylglycerol Triacylglycerol Other TOTAL Phosphatidic acid Phosphatidyl ethanolamine Phosphatidyl choline Lysophosphatidyl choline Phosphatidyl inositol Other TOTAL

21.82% 17.10% 7.80% 5.61% 3.89% 2.95% 4.95% 64.12% 15.39% 9.20% 6.09% 1.95% 0.91% 2.34% 35.88%

Phospholipids

by Willis (2010), and 33 proteins were identified with at least one motif that is representative of a cuticular family. All families were represented among these 33 proteins (Supplementary data File S4). When a peptide summary was correlated with NCBI databases, we identified 15 proteins that were somehow related with the insect cuticle, such as cuticulin, lectin, cuticular proteins, N-acetylglucosaminidase, collagen, and chitinase (Tables 3 and Table 4). Among these 15 proteins associated with cuticles, 8 are characteristic cuticle proteins, as shown in Table 4.

3.3. Proteins 4. Discussion The proteins extracted from the EC of R. prolixus were separated by SDS PAGE, but they were poorly stained with Coomassie blue (Fig. 3A); thus, we conducted simple one-dimensional electrophoresis separation to avoid protein loss. The gel was sequentially fragmented (Fig. 3A), and the proteomic analysis was conducted after tryptic digestion as described in the Materials and Methods. To ensure that our results did not include proteins from possible contaminants of the symbiont Rhodococcus rhodnii, we blasted our protein sequences against the sequences available in the databank of Rhodococcus available at NCBI. No Rhodococcus proteins were identified. We identified a total of 68 proteins (Supplementary data File S1) and classified them based on Gene Ontology (Supplementary data File S2). The proteins were grouped into twelve protein classes according to molecular function (Fig. 3B.1), eight classes according to cytolocation (Fig. 3B.2) and fourteen classes according to biological processes (Fig. 3B.3). When needed, we adapted the classification suggested by the Gene Ontology database to include proteins that have been identified but not classified in the database. Among the 68 identified proteins, 24 proteins were determined to have a high probability of containing secretion signal peptides (Supplementary data File S3) and (Fig. 3B.4). Additionally, we searched for the presence of cuticular protein motifs as suggested Table 1 Characterization of monosaccharide groups associated with the embryonic cuticle of Rhodnius prolixus. Densitometry analysis of monosaccharide groups by TLC CCF-C/ 25. Saccharide groups

Percentage found

Monosaccharide identified by retention frame

Percentage found

Neutral saccharide

41.87%

Fructose Fucose

33.13% 08.74%

Amino saccharide Acid saccharide

47.11% 11.02%

We performed a comprehensive characterization of the major components extracted from the embryonic cuticle (EC) of R. prolixus, and we found that it is primarily composed of carbohydrates, lipids, and proteins. We focused on the molecular composition of the EC and its role in the eclosion process. This study was only possible because we took advantage of our access to a large colony of R. prolixus; thousands of tiny ECs were available, yielding enough material for biochemical and molecular analyses. Carbohydrates and lipids are the major components extracted from the EC of R. prolixus, which is a result that is similar to that of other studies where the composition, differentiation and genetic control of post-embryonic cuticles is already known (Moussian et al., 2006; Ostrowski et al., 2002; Richards, 1978; Wigglesworth, 1972). 4.1. Carbohydrates Although the extraction method used in the TLC analysis does not favor the extraction of N-acetylglucosamine, a large amount of this saccharide was detected in the sample. This finding was also supported by the results of HPLC and DQI infrared spectrophotometry with a DTGS detector (Mansur et al., 2010; Moreira et al., 2007). The infrared spectrum was similar to the spectrum of standard commercial chitin (N-acetylglucosamine polymer) and different from that of chitosan (data not shown). In addition, the retention time observed by HPLC was consistent with the retention time of glucosamine, which is a product of N-acetylglucosamine hydrolysis. Labeling of the EC with Calcofluor White M2R and FITCconjugated chitin-binding domain (CBD) showed the distribution of chitin in the cuticle. While calcofluor labeled the entire area of the EC, CBD primarily marked the main body of the EC, but not its projections. This fact strongly suggests the presence of glycosaminoglycans in EC projections because calcofluor is a b-glycoside



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Fig. 3. (A) 10% SDS-PAGE profile of proteins extracted from the embryonic cuticle of Rhodnius prolixus. Lane 1: Molecular weight standard (Sigma). Lane 2: 30 mg of total protein extract from the embryonic cuticle of R. prolixus stained with Comassie blue G. Lane 3: 30 mg of total protein extract from the embryonic cuticle of R. prolixus stained with silver. Lane 4: Schematic diagram of the gel slices. (B) Diagram of embryonic cuticle proteins of R. prolixus. Embryonic cuticle proteins (ECuPs) of R. prolixus were annotated using UniProt (The Universal Protein Resource) and manually classified according to Gene Onthology classification guidelines of signal peptide prediction softwares (TargetP 1.1 Server, SignalP 4.0 Server, and SecretomeP 2.0 Server). (B1) Classification of ECuPs according to molecular functions. (B2) Classification of ECuPs according to component cytolocation. (B3) Classification of ECuPs according to biological processes. (B4) Classification of ECuPs according to their likelihood of being secreted.

marker that is not specific for chitin (Moussian et al., 2006), and it interacts with any N-acetylglucosamine present in the EC, either in the form of glycosaminoglycans or chitin, whereas CBD binds exclusively to the biopolymer chitin (Arakane et al., 2005; Mansur et al., 2010). The presence of acid monosaccharides and N-acetylglucosamine in the sample is further indicative of the occurrence of chitin in the EC. In fact, histolabeling of the EC of R. prolixus with alcian blue stain at pH 1.0 supports the occurrence of glycosaminoglycans in the EC (data not shown). Moreover, the presence of glycosaminoglycans has previously been reported in the ovaries and eggs of R. prolixus (Costa-Filho et al., 2004, 2001; Souza et al., 2004). The percentage of carbohydrates in the EC of Rhodnius is higher than that commonly observed in post-embryonic cuticles. Because

chitin is the major carbohydrate, the EC contains proportionally more chitin than the post-embryonic cuticle. The physiological meaning of this difference is not clear, but it could be associated with the protection of the embryo during hatching. Nymphs, larvae, and pupae usually have a higher percentage of chitin in their cuticles than adults (Moussian et al., 2006; Ostrowski et al., 2002; Richards, 1978; Wigglesworth, 1972). The presence of carbohydrates other than chitin, such as the neutral monosaccharides fucose and fructose in addition to acid and amino monosaccharides, requires further investigation, but we believe that it is related to the presence of proteoglycans and glycoproteins. Monosaccharides such as fucose and uronic acid, on the other hand, have been linked to formation, maturation, regulatory, and signaling processes during embryonic development (Haltiwanger

Table 3 Other proteins related to cuticle embryonic proteins identified at NCBI. Protein ID (VectorBase)

Protein Occurrence Protein NCBI NCBI NCBI e protein ID mass (Da) score E-value coverage (%) (correlated) organism

1. RPRC003338-PA

48539

3

40

3e-135

100

2. RPRC005064-PA

85580

4

63

0.0

94

3. RPRC002707-PA

63357

3

83

2e-66

75

4. RPRC001863-PA 220942

3

42

6e-07

45

5. RPRC010177-PA

77630

3

154

0.0

99

6. RPRC000478-PA

25221

3

134

5e-78

99

7. RPRC008125-PA

68915

3

70

0.0

99

EGI57614.1 Acromyrmex echinatior XP_001661365.1 Aedes aegypti EHJ75523.1 Danaus plexippus EHJ75523.1 Danaus plexippus EFN80467.1 Harpegnathos saltator ACR78452.1 Heliothis virescens AAQ97603.1 Manduca sexta

Domain/function

Domain/ Reference Willis, 2010 CuPs

Chitinase

-x-

Unpublished

Chorion peoxidase -x-

Nene et al., 2007

Colagen

CPR-RR3 CPAP3 CPR-RR2 CPAP3 -x-

Unpublished

CPR-RR3 CPR-144 BCNCPI

Shelby and Popham, 2009

Collagen Cuticulin Lectin C N-acetylglucosamidase

Unpublished Bonasio et al., 2010

Zen et al., 1996

Sample score >30; Sample Decoy ¼ 0.


8


Table 4 Summary of cuticle embryonic proteins identified at NCBI. Protein ID (VectorBase)

Protein mass (Da)

Occurrence

Protein score

NCBI E-value

NCBI coverage (%)

NCBI e protein ID (correlated) organism

Domain/ Function

Domain/ Willis, 2010 CuPs

Reference

CPR-RR1 CPR-RR2 CPR-RR3 CPR144 CPR-RR1 CPR-RR2 CPR-RR1 CPR-RR2 CPF

Werren et al., 2010

1.

RPRC001981-PA

28191

3

89

2e-20

52

NP_001161364.1 Nasonia vitripennis

Cuticle protein CPR e RR1

2.

RPRC000582-PA

134469

3

67

2e-66

56

Cuticular protein

3.

RPRC000069-PA

18966

3

42

1e-50

100

4.

RPRC006462-PA

15304

4

51

5e-22

89

5.

RPRC011606-PA

38949

3

47

6e-47

76

6.

RPRC000117-PA

670502

2

34

5e-25

34

NP_001165114.1 Bombyx mori BAG30751.1 Papilio xuthus AAR15418.1 Antheraea pernyi ACY06906.1 Bombyx mori EFA10573.1 Tribolium castaneum

7.

RPRC013845-PA

18471

2

41

3e-25

91

8.

RPRC000618-PA

26589

3

98

5e-132

82

NP_001156724.1 Acynthosiphon pisum BAM18925.1 Papilio polytes

Cuticular protein Cuticular protein Cuticular protein

Futahashi et al., 2008 Futahashi and Fujiwara, 2008 unpublished unpublished

Cuticular protein

CPR-RR1 CPR-RR2 CPR-RR1 CPR-RR2 18 aminoacids repeated CPAP3

Cuticular protein

-x-

Futahashi et al., 2012

Cuticular protein

Richards et al., 2008

Jasrapuria et al., 2010

Sample score >30; Sample Decoy ¼ 0.

and Lowe, 2004), and they could be related to EC formation in R. prolixus. Other saccharides that are found in insect cuticles include glucose, N-acetylglucosamine, N-acetylgalactosamine, and galactose, which are associated with glycoproteins and mucopolysaccharides (Okot-Kotber et al., 1994). It is known that chitin is responsible for cuticle structure (Merzendorfer and Zimoch, 2003) and that its differentiation occurs in three phases Moussian et al. (2006). The association of chitin fibers with cuticular proteins confers different physical properties that allow the cuticle to support a variety of functions as discussed previously in Dombrovsky et al. (2003); Samuels and Paterson (1995). Thus, the higher amount of chitin in the EC could play a role in this critical stage of embryo life (Souza-Ferreira et al, 2014). 4.2. Lipids The lipids that are associated with the EC are normally used in post-embryonic cuticles to cover the cuticular surface to prevent desiccation, microorganism penetration and communication (Hadley, 1981; Juarez and Fernandez, 2007; Wigglesworth, 1947). Although the proportions and compositions of these macromolecules varies according to stage and species (Dombrovsky et al., 2003; Juarez and Fernandez, 2007; Samuels and Paterson, 1995; Wigglesworth, 1972), the proportion of lipids in post-embryonic cuticles is usually higher than that of proteins, as observed in the EC of R. prolixus. Additionally, the percentage of neutral lipids in the EC of R. prolixus was higher than that of phospholipids. Nevertheless, the percentage of phospholipids is well above the 15% usually reported in the integument of insects (Gibbs, 2002; Juarez and Fernandez, 2007). The characterization of neutral lipids was similar to that reported in the cuticle of fifth-instar nymphs of R. prolixus (Juarez et al., 2001), except for the presence of cholesterol (7.8%) and the absence of diacylglycerol. However, there were some differences in the characterization of phospholipids. Although Gibbs (2002) and Juarez and Fernandez (2007) reported that phosphatidylethanolamine (PE) and phosphatidylcholine (PC) are the main phospholipids in the integument of insects, phosphatidic acid was the most abundant phospholipid (15.39%) in our study. Phosphatidic acid is a potential precursor of other

phospholipids, but it is also associated with pathophysiological processes, cellular signaling events, inflammation, and apoptosis (Zhao et al., 2012). These results are consistent with the physiology of the EC, which needs to extricate itself from the cuticle of the hatching nymph to conclude the eclosion process, which potentially requires a well-organized flux of information. The high percentage of phospholipids associated with the EC and high cholesterol concentration suggest that these lipid components could originate from cells that are associated with the EC that are shed with the cuticle after eclosion (e.g., residue from extraembryonic cells originating from the amnion and serosa that could help the insect during eclosion), although we were unable to identify cellular components (data not shown). In fact, during part of embryogenesis, the embryo is immersed in yolk (Kelly and Huebner, 1989; Wigglesworth, 1972), and although the study of Kelly and Huebner (1989) suggested that after katatrepsis, the embryo of R. prolixus initiates a stage of blastokinesis with the fusion and extinction of the amnion and serosa, it is not certain that all the material from these structures are completely degraded; thus, some of it could be incorporated into the EC. Alternatively, the shedding of the EC associated with the eggshell during hatching could be similar to the ecdysis of nymphs, in which epithelial cells produce a basal membrane and external cuticle, with separation of the epidermis and cuticle, and secretion of epicuticulin to form a new epicuticle. Then, a fluid with proteinases and chitinases is secreted, and the old endocuticle is digested (Samuels et al., 1993; Samuels and Paterson, 1995). However, this explanation does not allow us to reconcile all our data, as significant differences were observed between the EC of R. prolixus and post-embryonic cuticles, which could be because both possess different structures and functions. The presence of a high phosphatidic acid content in the EC compared to the cuticle of a fifth-instar nymph (Juarez and Fernandez, 2007) suggests that ECs have different characteristics from cuticles that are shed during post-embryonic ecdysis. It worth noting that the EC is protected by the eggshell most of the time, makes contact with the external environment very briefly and is discarded during hatching. The ECs are more flexible than postembryonic ones, and their main role is likely to protect the embryo during eclosion, serving as a safe conducting channel while the embryo exits the chorion. Thus, ECs require chitin and proteins



for rigidity and functionality. The inhibition of chitin deposition by chitin synthase dsRNA hinders the function of the EC by making it physiologically incompetent (Souza-Ferreira et al, 2014). 4.3. Proteins Proteomic analysis detected the presence of 456 peptides from 68 proteins extracted from the EC of R. prolixus and listed in the R. prolixus non-annotated genome database at VectorBase. The number of proteins identified can be considered satisfactory, but it should be noted that some proteins in the EC may not have been identified due to problems during extraction or detection by MS/ MS, or because individual peptides are bound to glycosides or compounds that are involved in sclerotization mechanisms (Kerwin et al., 1999). The correlation of proteins with the NCBI database via protein BLAST analysis resulted in the identification of 15 proteins that are somehow associated with insect cuticles. Among these, 8 proteins were unequivocally cuticle proteins and have also been identified in Papilio polytes (Futahashi et al., 2012), Nasomia vitripennis (Werren et al., 2010), Bombyx mori (Futahashi et al., 2008), Papilio xuthus (Futahashi and Fujiwara, 2008), Antheraea pernyi, Tribolium castaneum (Richards et al., 2008) and Acynthosiphon pisum (Jasrapuria et al., 2010). This number of authentic identified cuticular proteins is likely due to the fact that we isolated them directly from a large quantity of EC. Another group of seven proteins that are somehow associated with insect cuticles are possibly involved in the metabolism of the cuticle. These proteins were also identified in Manduca sexta (Zen et al., 1996), Acromyrmex echinatior, Danaus plexippus, Heliothis virescens (Shelby and Popham, 2009), Camponotus floridanus (Bonasio et al., 2010), Aedes aegypti (Nene et al., 2007) and Herpegnathos saltator (Bonasio et al., 2010). The classification suggested by the database had to be manually reassessed because some of the proteins included in the group of unclassified proteins, such as Rp45 and RP30 (Bouts et al., 2007), had been characterized and studied by our research group, but they were not available in the VectorBase. Many of the cuticular proteins described here originated from the transcription of cDNAs or genomic sequences and were designated as such for sharing similarities with proteins extracted from the post-embryonic cuticles of insects (Dombrovsky et al., 2007; He et al., 2007; Togawa et al., 2008). Many proteins of insect cuticles were extracted and sequenced as reviewed by Andersen et al. (2000), and Willis (2010). In Anopheles gambiae, He et al. (2007), also identified cuticular proteins based on the isolation and sequencing of proteins from post-embryonic cuticles, and they contributed to the identification of a significant number of cuticular proteins. Here, we present the first study to focus on proteins that were unequivocally extracted from the EC of an insect. This is only a preliminary assessment of authentic cuticular proteins from an EC because we did not attempt to remove and analyze the tightly bound proteins of R. prolixus. In addition, it is possible that many cuticular proteins were removed by molting fluid that was potentially present in the embryo prior to hatching. Among the 68 identified proteins, we identified 33 proteins that contained domains that were representative of all cuticular protein families as described by Willis (2010). Among the 8 proteins that were considered cuticle proteins, 7 contained domains that were typical of the cuticle protein family. The following families were determined to be associated with Rhodnius EC proteins: CPR-RR1, CPR-RR2, CPF, CPAP3 and 18 amino acid motifs. Six out of eight proteins contained CPR-RR1 and CPR-RR2. Among the other seven proteins that were recognized to be associated with cuticles, four contained domains of the cuticle family, including the two collagens, N-acetylglucosaminidase and lectin C. It is debatable whether the fact that 33 proteins exhibit at

9

least one motif that is characteristic of cuticular proteins is sufficient to consider them as belonging to the cuticle. It is possible that other criteria, such as the presence of a secretion signal peptide, should be added. It should be noted that among the 68 proteins identified by proteomic analysis, only 23 had evidence for the presence of a secretion signal peptide in their sequence. If we consider all the proteins containing motifs that are characteristic of cuticular families along with a secretion signal peptide, 11 proteins remain. Secretion signal peptides are commonly used as markers for post-embryonic cuticle proteins (Willis, 2010). Thus, the remaining proteins without a signal peptide are not typical candidates for cuticular proteins. However, because we worked directly with the EC, we decided not to exclude them from our analysis, but we included the presence or absence of the signal peptide associated with the protein. Among the eight proteins that are considered cuticle proteins, six contained the signal peptides and the other two had medium and low probabilities of possessing signal peptides. Among the other 7 proteins associated with cuticles, 4 contained signal peptides. These proteins were chitinase, lectin, glucosaminidase and chorion peroxidase. It is necessary to keep in mind that it is always possible to identify proteins that are secreted by an unconventional secretory pathway. The other 21 proteins, including collagens, alkaline phosphatase, and myosin, that also contained cuticular proteins domains but were not recognized to be associated with the cuticle remain to be studied. Multifunctional proteins could explain these results. Among the proteins without a secretion signal peptide, we identified lipophorin and apolipophorin proteins that are known to be secreted (Jakubowiak et al., 1988; Jenkins and Ellison, 1989; Nunez and Tapia, 1999; Smolenaars et al., 2005). Moreover, proteins such as vitellogenin that have secretion signal peptides are known to be non-cuticular. The small number of cuticle proteins that were identified in our study, compared with the number of cuticular proteins discussed in Willis (2010), could be due to the large functional differences between embryonic and post-embryonic cuticles. In A. gambiae, the genes for only 240 cuticular proteins have been identified (He et al., 2007). Another possible explanation for this is that we did not investigate the tightly bound proteins of the EC. All the proteins investigated here were extracted in 8 M urea. Proteomic characterization of ECs of other insect species is essential to better understand this structure. Thus, the acquisition of new data will help to determine whether the differences between ECs and postembryonic cuticles are due to differences in their function. 4.4. Analysis of proteins via Gene Ontology The 68 proteins identified were processed in UniProt and correlated and classify their sequences according to Gene Ontology. The identification of the main molecular functions (12 classes), cytolocation (8 classes), and biological processes (14 classes) associated with proteins extracted from the EC of R. prolixus enabled us to establish the importance of these proteins in the EC and during insect development. 4.4.1. Molecular function The presence of structural and polysaccharide-binding proteins with oxidase or peroxidase activity and oxide reductase activity is usually expected to occur in the cuticle. The presence of proteins with protease or peptidase activity or phosphatase activity and other regulatory proteins seems to be essential for the physiology of the EC. The presence of proteins associated with molecular binding and transport should also be appreciated in terms of the intrinsic requirements of the embryo, which needs to extricate its EC during


10


emergence, which is a complex process that may require signaling mechanisms. The presence of proteins for energy reserve (3%) was not expected and is likely to be caused by residue from yolk, amnion, or serosa. 4.4.2. Protein location Regarding protein location, a large percentage of proteins were classified as extracellular and integral to the membrane, and a smaller percentage were classified as intracellular and included proteins associated with the mitochondrial matrix, vesicles and the nucleus. In addition, the observed percentage of intracellular proteins (19%) is quite high for a cuticular structure. This can be attributed to the fact that ECs are different from post-embryonic ones, with different structures and functions that are limited to a highly specific period of insect development. As discussed above, although Kelly and Huebner (1989) have suggested that amnion and serosa are absent during embryogenesis, it is possible that residues of these tissues are associated with the EC. 4.4.3. Biological process The presence of proteins associated with the biological processes of the cuticle, such as proteins involved in contraction, proteolysis, regulation or signaling; metabolic processes; and chitin metabolism may be related to the physiological state of the insect in its critical stage, which is the eclosion of a new life. The EC is of fundamental importance in this process because it protects the nymph while it passes through a narrow chorionic border. To our knowledge, this is the first successful attempt to directly extract molecules from an EC of an insect. The analysis of this material aided the elucidation of the role of these molecules in the process of hatching of the first instar nymph of R. prolixus. 4.5. Final considerations Because cuticles are composite materials and their mechanical properties depend on the interactions of their constituents such as proteins, lipids, carbohydrates and other minor components, a large number of mechanical properties can be obtained by varying the type and number of components, as well as the number of interactions among them, to assemble a final multimolecular structure that will regulate a particular physiological function. It worth noting that Rhodnius’s EC is synthesized inside the eggshell and envelopes the entire embryo before hatching. During hatching, with the embryo mostly outside the eggshell, the expansion of the embryo inside the cuticle creates enough pressure to rupture the EC. As soon as the EC is ruptured, the EC retracts, which helps the nymph to shed the EC. Thus, we can hypothesize that the mechanical properties of this EC are very different from those of postembryonic cuticles with respect to the function of their different chemical compositions. Future studies addressing this question must be conducted. 5. Essential title page information Molecular Characterization of Embryonic Cuticle. Acknowledgments This work was supported by grants from Conselho Nacional de Desenvolvimento Científico e Tecnológico e CNPq, Coordenação de Aperfeiçoamento de Pessoal de Nível Superior e CAPES, Fundação de AmparoàPesquisa do Estado do Rio de Janeiro e FAPERJ, Instituto Nacional de Ciência e Tecnologia em Entomologia Molecular e INCTEM and Fundação José Bonifácio e FUJB. We thank Heloísa S. L. Coelho, Lilian S.C. Gomes, José S. Lima Júnior, Litiane M. Rodrigues

and Gustavo T. Ali for their excellent technical assistance, and Dr. Gustavo L. Rezende, Dr. Pedro L. Oliveira, Dr. Kátia C. Gondim, Dr. Claudio A. Masuda, Dr. Gabriela O. Paiva-Silva for helpful discussions.

Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.ibmb.2013.12.005.

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Molecular characterization and possible biological roles of allatotropin in Rhodnius prolixus.

Effects of azadirachtin on Rhodnius prolixus: immunity and Trypanosoma interaction.

Role of phospholipids in the lipophorin particles of Rhodnius prolixus.

The biochemical basis of tolerance to malathion in Rhodnius prolixus.

Rhodnius prolixus: modulation of antioxidant defenses by Trypanosoma rangeli.

Rhodnius prolixus: From classical physiology to modern developmental biology.

Insulin-like immunoreactivity and molting in Rhodnius prolixus.

Identification and functional characterization of FGLamide-related allatostatin receptor in Rhodnius prolixus.

Allatotropin modulates myostimulatory and cardioacceleratory activities in Rhodnius prolixus (Stal).

Identification and Characterization of the Corazonin Receptor and Possible Physiological Roles of the Corazonin-Signaling Pathway in Rhodnius prolixus.

Relative susceptibility of different stages of Rhodnius prolixus to the entomopathogenic hyphomycete Beauveria bassiana.

Mariner transposons are sailing in the genome of the blood-sucking bug Rhodnius prolixus.

Development of data for the identification and characterization of proteins found in Rhodnius prolixus, Triatoma lecticularia and Panstrongylus herreri.

Functional characterization and expression analysis of the myoinhibiting peptide receptor in the Chagas disease vector, Rhodnius prolixus.

Chitin deposition on the embryonic cuticle of Rhodnius prolixus: the reduction of CHS transcripts by CHS-dsRNA injection in females affects chitin deposition and eclosion of the first instar nymph.

Adipokinetic hormone signalling system in the Chagas disease vector, Rhodnius prolixus.

Identification of yolk platelet-associated hydrolases in the oocytes of Rhodnius prolixus.

Behavioral and toxicological responses of Rhodnius prolixus and Triatoma infestans (Hemiptera: Reduviidae) to 10 monoterpene alcohols.

Serotonin triggers cAMP and PKA-mediated intracellular calcium waves in Malpighian tubules of Rhodnius prolixus.

Embryonic cuticle establishment: the great (apoplastic) divide.

Morphology of the dorsal vessel in the abdomen of the blood-feeding insect Rhodnius prolixus.

An insight into the transcriptome of the digestive tract of the bloodsucking bug, Rhodnius prolixus.

In vitro photosensitivity of ecdysteroid synthesis by prothoracic glands of Rhodnius prolixus (hemiptera).

Autogeny in three species of Triatominae: Rhodnius prolixus, Triatoma rubrovaria, and Triatoma infestans (Hemiptera: Reduviidae).