A second gene encodes the anti-diuretic hormone in the insect, Rhodnius prolixus

Molecular and Cellular Endocrinology 317 (2010) 53–63

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A second gene encodes the anti-diuretic hormone in the insect, Rhodnius prolixus Jean-Paul Paluzzi ∗ , Ian Orchard Department of Biology, University of Toronto Mississauga, Mississauga, Ontario, Canada L5L 1C6

a r t i c l e

i n f o

Article history: Received 4 August 2009 Received in revised form 30 September 2009 Accepted 10 November 2009 Keywords: CAPA CAP2b Anti-diuresis Peptide Hormone Insect

a b s t r a c t In the haematophagous insect, Rhodnius prolixus, a rapid diuresis following engorgement of vertebrate blood is under the control of two main diuretic hormones: a corticotropin-releasing factor (CRF)-related peptide andserotonin (5HT). A CAP2b (CAPA)-related peptide is involved in the termination of this diuresis, and we have recently identified a gene, now referred to as RhoprCAPA-␣, encoding CAPA peptides in R. prolixus. Here we identify a second gene, RhoprCAPA-␤, which also encodes CAPA peptides and characterize its expression in fifth-instar and adults. The RhoprCAPA-␤ gene is more highly expressed in the CNS than the RhoprCAPA-␣ gene, but neither gene is expressed in other tested adult tissues. Both genes are expressed in a subset of immunoreactive neurons identified using an antisera which recognizes CAP2b-related peptides. The expression of each paralog is modified by feeding and we propose this to be a result of requirements of anti-diuretic regulation during salt and water homeostasis. © 2009 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Insect diuresis and anti-diuresis typically involves hormonal control of the excretory system, which includes the Malpighian tubules (MTs) and hindgut (Coast et al., 2002). Specifically, diuresis is induced by stimulating fluid secretion by the MTs, using peptidergic hormones, and/or biogenic amines, such as serotonin (5-hydroxytryptamine or 5HT). In contrast, it has been demonstrated that anti-diuresis involves fluid reabsorption by the hindgut, whereby essential salts and water are retrieved, and in some cases a concurrent decrease in action of diuretic hormones on the MTs. In the blood-feeding reduviid bug, Rhodnius prolixus, there are extended periods of starvation between successful foraging events in search of an appropriate vertebrate blood meal. It is well documented that the rapid diuresis in R. prolixus following blood gorging involves stimulation of MTs by a peptidergic hormone, likely related to corticotropin-releasing factor (CRF) peptides identified in other insects, and the amine, 5HT, acting via cAMP (Maddrell et al., 1993; Lange et al., 1989; Te Brugge et al., 1999; Te Brugge and Orchard, 2002). In R. prolixus, the anterior midgut (or crop) also plays an essential role in the rapid diuresis following gorging on a blood meal. The excess salt and water, derived from the plasma portion of the blood meal, is absorbed into the haemolymph, for subsequent elimination by the MTs, while the

∗ Corresponding author at: Department of Biology, University of Toronto Mississauga, Room 3016A/B, South Building, 3359 Mississauga Road North, Mississauga, Ontario, Canada L5L 1C6. Tel.: +1 905 828 5333; fax: +1 905 828 3792. E-mail address: [email protected] (J.-P. Paluzzi). 0303-7207/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.mce.2009.11.004

nutritive red blood cell-containing component is stored for eventual digestion. Serotonin and a CRF-related peptide have also been shown to increase the cAMP levels and increase absorption by the anterior midgut (Te Brugge et al., 2009). It was earlier believed that the cessation of diuresis in R. prolixus was the result of decreased titres of diuretic hormones. Studies by Quinlan et al. (1997) and Quinlan and O’Donnell (1998), however, suggested that a unique anti-diuretic mechanism might exist in this haematophagous insect, revealed by the physiological response to exogenously applied Manduca sexta cardioaccelatory peptide 2b (ManseCAP2b). This peptide inhibits MTs fluid secretion stimulated by 5HT (Quinlan et al., 1997), with the intracellular second messenger, cGMP, possibly playing a role (Quinlan and O’Donnell, 1998; Orchard, 2006). More recently, we identified the endogenous CAP2b-related peptide in R. prolixus central nervous system (CNS) that inhibits MT secretion stimulated by 5HT (Paluzzi and Orchard, 2006; Paluzzi et al., 2008). In addition, this peptide, RhoprCAPA-2, also abolishes the decrease in cGMP levels in MTs stimulated with 5HT, suggesting that cGMP may be involved in the anti-diuretic effect observed in MTs. Interestingly, RhoprCAPA-2 also abolishes the 5HT-stimulated increase in absorption by the anterior midgut (Orchard and Paluzzi, 2009). CAP2b-related peptides have also been referred to as periviscerokinins (PVKs) since they are the most abundant peptide class in the abdominal perivisceral neurohaemal system in many insects and have myotropic effects on visceral muscle such as the heart and gut (for a review, see Predel and Wegener, 2006). The first genes encoding CAP2b and CAP2b-related peptides, called capability (CAPA) genes, were identified in Drosophila melanogaster (Kean et al., 2002) and later in M. sexta (Loi and Tublitz, 2004). These

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genes each encode two CAP2b-related peptides and a pyrokinin peptide. With the increase in insect genome projects completed or in progress, homologous genes have been annotated in other genera such as several Dipteran, Lepidopteran, and Coleopteran species. In addition, we have observed homologous CAPA genes encoding CAP2b-related peptides in other organisms such as the human body louse, Pediculus humanus corporis (EEB10638) and aphids, Acyrthosiphon pisum (XP 001946149) and Myzus persicae (EE570800.1). Recently it was demonstrated that the CAP2b-related peptides in Bombyx mori were produced by a single gene, which can undergo alternative splicing to produce an extended pyrokinin peptide (Roller et al., 2008), although the distribution of these variant peptides within the CNS has yet to be established. The RhoprCAPA gene in fifth-instar R. prolixus is expressed primarily in the CNS, consistent with previous immunological identification of cells containing CAP2b-related peptides (Paluzzi and Orchard, 2006; Paluzzi et al., 2008), although expression was also observed in immature male reproductive tissue (Paluzzi et al., 2008). Here, we present data demonstrating that a second, closely related gene exists in this haematophagous insect and we characterize the expression levels of these paralogs in fifth-instar and adult insects. In the spatial analysis, we compare expression levels of each paralog in several different tissues and also examine the expression level distribution within the CNS. In the temporal analysis we investigate expression levels over a period of several hours and also over a few weeks following feeding, which we term as a micro- and macro-temporal time-scale, respectively. Using a combined immunohistochemical and in situ hybridization approach, we have localized neurons containing CAP2b-related peptides, and have verified that a considerable subset of these immunopositive neurons express one or both RhoprCAPA gene paralogs. We rename the first identified paralog gene as RhoprCAPA-␣, since this will avoid confusion in future comparison with the second RhoprCAPA paralog gene, RhoprCAPA-␤, identified herein. 2. Methods 2.1. Animals Fifth-instar R. prolixus Stål were reared at high relative humidity in incubators at 25 ◦ C and routinely fed on rabbits’ blood. Tissues were dissected from insects under physiological saline prepared as described previously (Paluzzi et al., 2008) in nuclease-free water. 2.2. Screening of fifth-instar CNS cDNA library A cDNA library from the CNS of fifth-instar R. prolixus (Paluzzi et al., 2008) was screened using a modified rapid amplification of cDNA ends (RACE) PCR approach. A series of gene-specific primers (gsp) were designed, based on several initial clones identified during the isolation of the RhoprCAPA-␣ gene, and used in combination with the pDNR-LIB plasmid primers described previously (Paluzzi et al., 2008). These partial cDNA clones although similar, demonstrated consistent differences from the RhoprCAPA-␣ gene and thus were pursued in this study. Amplified fragments were isolated and cloned using the pGEM-T Easy Vector System (Promega, Madison, WI). Sequencing was carried out at the Centre for Applied Genomics at the Hospital for Sick Children (MaRS Centre, Toronto, Ontario, Canada), and sequences were confirmed from several independent clones to ensure base accuracy and differentiate between the previously identified RhoprCAPA-␣ gene. 2.3. Sequence analysis of RhoprCAPA-ˇ prepropeptide The deduced RhoprCAPA-␤ prepropeptide sequence was analyzed for potential processing in the secretory pathway using online software for signal peptide prediction (SignalP 3.0; Bendtsen et al., 2004). To confirm the processing and presence of the RhoprCAPA-␤ peptides, CNS material was fractionated by RP-HPLC (as described previously, Paluzzi et al., 2008) and collected fractions were analyzed by mass spectrometry at the Toronto Integrative Proteomics Lab (Ontario Cancer Biomarker Network, Toronto, ON) and The Advanced Protein Technology Centre (Hospital for Sick Children, Toronto, ON). The two RhoprCAPA prepropeptide sequences (RhoprCAPA-␣, ABS17680; RhoprCAPA-␤, ACH70295) and homologs from several insects (D. melanogaster, AAF56969; Anopheles gambiae, EAL41227; Aedes aegypti, EAT43089; B. mori, NP 001124357 and AB362228.1; M. sexta, AAT69684; A. pisum, XP 001946149; M. persicae, EE570800.1; P. humanus cor-

poris, EEB10638; Tribolium castaneum, GLEAN 08429) were compared using ClustalX (Larkin et al., 2007) and the final figure prepared using the BOXSHADE 3.21 server (http://www.ch.embnet.org/software/BOX form.html). The prepropeptide sequence alignment was used to produce a phylogenetic tree prepared in MEGA 4.02 using either the neighbor-joining or parsimony method and bootstrapping with 1000 iterations (Tamura et al., 2007). A putative CAPA/PVK prepropeptide consensus sequence from the Arachnid, Ixodes scapularis, was generated from EST data (EW933575.1) and included in the analysis and imposed as the outgroup. 2.4. Northern blot hybridization Tissues of adult R. prolixus were dissected in nuclease-free PBS and stored at −20 ◦ C in RNAlater reagent (Ambion, Austin, TX). Total RNA was isolated using the SV Total RNA Isolation System (Promega, Madison, WI). Once purified, 1 ␮g of total RNA from each tissue (anterior midgut; posterior midgut; salivary gland; hindgut; dorsal vessel (DV), fat body and diaphragm; MTs; testis; and CNS) was prepared with 2× RNA loading dye (Fermentas, Burlington, ON) and denatured at 75 ◦ C for 5 min and subsequently cooled on ice. The samples were then run at 5 V/cm on a 1–2% formaldehyde-agarose gel (20 mM MOPS, 5 mM NaOAc, 10 mM EDTA, pH 7.0, 2.2 M formaldehyde) that was precast and allowed to set for at least 1 h. Once RNA samples were appropriately separated, as determined by brief visualization under UV transilluminator, gels were rinsed for 30–40 min in nuclease-free water to remove excess formaldehyde. Gels were then prepared for downward capillary transfer to positively charged nylon membranes (Roche Applied Science, Laval, QC). Transfer was carried out with 20× SSC for 12–16 h and blots were then briefly rinsed (<10 s) in nuclease-free water to remove excess SSC. Blots were then transferred onto dampened blotting paper for UV crosslinking at a setting of 30 mJ/cm2 (UVP CL-1000, Upland, CA). The blots were then allowed to air-dry for 3–4 h and stored at 4 ◦ C in a sealed plastic bag until used in hybridization. Digoxigenin (DIG)-labelled RNA anti-sense probes were prepared for each paralog following methods described previously (Paluzzi et al., 2008). Briefly, using CNS cDNA as template, PCR was used to generate a 713 bp RhoprCAPA-␣ fragment using the sense primer, CAPAalpha GSPfor (5 -GCATGCGACATTGTTTTTTC-3 ) and a 703 bp RhoprCAPA-␤ fragment using the sense primer, CAPAbeta GSPfor (5 GCATGCGACATTTTTGACC-3 ). Both fragments utilized the same anti-sense primer, CAPArev1 (5 -ATGAAAAGGCACATTTATTGTATGC-3 ), designed over a region with no difference in sequence between the two paralogs. For analysis of RNA quality and quantity, a fragment specific for actin 5c (beta-actin, FJ851423.1) was generated using the sense primer, actin GSPfor (5 -ACTAACTGGGACGACATGG-3 ) and anti-sense primer, actin GSPrev (5 -GTGGCCATTTCCTGTTC-3 ). These products were ligated into the pGEM-T Easy vector and the directionality of the insert was confirmed via sequencing (as described above) in order to use the appropriate RNA polymerase (SP6/T7) for in vitro transcription. Plasmid miniprep DNA for each clone was then linearized using a restriction enzyme cutting the vector on the 3 end of the anti-sense strand in order to generate anti-sense-labeled RNA probes. 2.5. Fluorescent in situ hybridization and immunohistochemistry Cell-specific transcript expression was localized in adult CNS as described previously for fifth-instars (Paluzzi et al., 2008) with the following changes and adaptations. To reduce non-specific background staining associated with endogenous peroxidase activity, tissues were incubated in a 1% H2 O2 phosphate-buffered saline for 10 min at room temperature following initial tissue fixation in 4% paraformaldehyde. Following this endogenous peroxidase quenching step, tissues were washed five times with PBS and 0.1% Tween 20 (PBT) and subsequently incubated in 4% Triton X-100 (Sigma–Aldrich, Oakville, Ontario, Canada) in PBT for 1 h at room temperature. The detergent containing buffer was then removed and tissues washed for approximately 30 min in PBT changing the wash solution every 5 min. The remainder of the in situ hybridization was carried out as previously described. DIG-labeled RNA probes were prepared as described in the northern hybridization methods section (see above). For control treatments, restriction enzymes cutting on the 3 end of the sense strand were used to generate sense-labeled RNA probe. Following the in situ preparations, tissues were briefly observed under a Nikon epifluorescence microscope in PBS and were then processed for immunohistochemical localization of CAPA-related peptides using an antisera described previously (Paluzzi and Orchard, 2006), identifying peptides containing the C-terminal motif, PRX-amide (where X = I, L, M or V; Zitnan et al., 2003). Subsequent incubations were carried out with preparations protected from light exposure in order to minimize photo-bleaching of in situ hybridization red–orange-fluorescent Alexa Fluor® 568 (Molecular Probes, Eugene, OR). Since tissues had already been digested for the in situ preparation, tissues were treated directly with antisera that had been preincubated overnight at 4 ◦ C in PBS containing 0.4% Triton X-100, 2% bovine serum albumin and 2% normal sheep serum. This incubation with the primary antisera (1:500) was carried out for 36–48 h at 4 ◦ C with gentle shaking on a flat bed shaker and tissues were then washed in PBS for a minimum of 6 h at room temperature on a rocking platform. To detect immunolocalization of PRX-amide-related peptides, FITC-conjugated goat anti-rabbit IgG (BioCan Scientific, Mississauga, ON) was used at a dilution of 1:200 in PBS containing 10% normal goat serum and preparations were incubated

J.-P. Paluzzi, I. Orchard / Molecular and Cellular Endocrinology 317 (2010) 53–63 overnight at 4 ◦ C. The following day, tissues were washed for approximately 2 h at room temperature changing PBS every 15 min. Tissues were then mounted onto slides in glycerol and viewed under a laser scanning confocal microscope equipped with a helium-neon (543 nm line) and Argon laser (488 nm line) for excitation of Alexa Fluor 568 and FITC fluorescent molecules, respectively. Images were acquired using LSM Image browser software (LSM 510; Zeiss, Jena, Germany). 2.6. Reverse transcriptase quantitative PCR (RT-qPCR) analysis of RhoprCAPA paralog expression Tissues were dissected in nuclease-free PBS and stored in RNAlater reagent (Ambion, Austin, TX). Total RNA was extracted as described above for northern analysis and 100 ng of total RNA was used as template for first-strand cDNA synthesis using the RevertAidTM H Minus M-MuLV Reverse Transcriptase and supplied oligo(dT)18 primer (Fermentas, Burlington, ON). Tissues for temporal expression analysis were staged from the end of a 15 min feeding regime on defibrinated rabbits blood (time = zero). For the micro-temporal analysis, CNS from six insects of either sex were dissected within 5 min of each time-point, which consisted of insects immediately prior to feeding (unfed), immediately following feeding (time = 0) and 1, 2, 3, 4, 5, 6, 7 and 8 h post-feeding. For the macro-temporal analysis, CNS tissues from six insects were dissected at each time-point (at the same time of day, approximately 09:00 ± 0:30), which consisted of insects immediately prior to feed-

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ing (unfed), immediately following feeding (time = 0) and 4, 7, 11, 14, 18, 21, 25, 28 and 32 days post-feeding. Insects were fed as fifth-instars in both micro- and macrotemporal analyses; however in the latter, insects dissected at 21 days post-feeding and beyond were adult stage insects that had ecdysed at 19–20 days post-feeding. For spatial expression quantitative analyses, tissues were dissected from fifth-instar or adult stage insects of the same age since the last blood meal (approximately 5–6 weeks). Quantitative PCR (qPCR) analyses were carried out on a Mx4000® Multiplex Quantitative PCR System (Stratagene, La Jolla, CA) using the MaximaTM SYBR Green qPCR Master Mix (Fermentas, Burlington, ON). Primers were optimized to amplify target fragments of similar size across all experimental (RhoprCAPA paralogs) and housekeeping control genes (rp49 and actin 5c). In addition, each primer set consisted of at least one primer designed over an exon–exon splice boundary to ensure target amplification was solely cDNA synthesized from the RNA isolation. Primers utilized were as follows: RhoprCAPA-␣, sense CAPAalpha GSPfor (5 -GCATGCGACATTGTTTTTTC-3 ) and anti-sense CAPAalpha qPCRrev (5 -CGCTTGTTTTTGTCATCACC-3 ); RhoprCAPA-␤, sense CAPAbeta GSPfor (5 -GCATGCGACATTTTTGACC-3 ) and anti-sense CAPAbeta qPCRrev (5 -TCGTTTGTATTTGTCATCACCG-3 ); RhoprRP49, sense rp49 qPCRfor (5 -GTGAAACTCAGGAGAAATTGGC-3 ) and anti(5 -AGGACACACCATGCGCTATC-3 ); RhoprACTIN-5C, sense rp49 qPCRrev sense actin qPCRfor (5 -AGAGAAAAGATGACGCAGATAATGT-3 ) and anti-sense actin qPCRrev (5 -GTTCGGCTGTGGTGATGA-3 ). Cycling conditions for qPCR utilized a typical three-step protocol as follows: initial denaturation at 95 ◦ C for 10 min,

Fig. 1. Deduced amino acid prepropeptide of the R. prolixus CAPA-␤ (RhoprCAPA-␤) gene. (A) Sequences are numbered on the right starting with the first nucleotide in the 5 untranslated region (UTR) and initial methionine start codon (capitalized), respectively. The three encoded peptides are shown in boxes, with N-terminal dibasic and C-terminal monobasic post-translational cleavage sites in bold. The highly predicted signal peptide required for processing in the secretory pathway is underlined with predicted cleavage occurring between alanine24 and aspartate25. The polyadenylation signal sequences occur within the 3 UTR (bases 686–692 and 724–730). (B) Preliminary organization of the CAPA genes in the R. prolixus genome. Exons are represented within boxes with sizes denoted and introns are represented by lines connecting the exons with sizes also indicated (or predicted) beneath them.

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followed by 40 cycles of 95 ◦ C for 15 s, annealing for 30 s and extension for 30 s with data acquisition during the extension step. To validate the specificity of the SYBR green detected products, a melting curve analysis along with gel electrophoresis was performed to validate specificity of the products generated (data not shown). The expression levels were quantified using the standard curve method and quantities were normalized to either of the housekeeping genes (mentioned above), with no difference in results obtained based on housekeeping gene chosen. Results are representative of at least two biological replicates with each individual assay measurement quantified in duplicate or triplicate. In every assay, a no template control was included to assess reagent contamination or primer dimer generation, and in addition, a no reverse transcriptase control (RNA not synthesized into cDNA) was included to ensure absence of contaminating genomic DNA in the samples.

3. Results 3.1. Two genes encode the CAPA-related peptides in R. prolixus We have identified a cDNA, which we refer to as RhoprCAPA␤ (GenBank accession number: EU937527), with high sequence similarity to a gene identified previously, RhoprCAPA-␣ (GenBank accession number: EF989016; Paluzzi et al., 2008). In fact, at the nucleotide level, the R. prolixus paralogs share 88.7% identity in nucleotide sequence. At the amino acid level, both paralogs encode a prepropeptide of 157 residues sharing 85.4% similarity. The first encoded peptide, RhoprCAPA-␤1 (SPITSIGLLPFLRAA), deviates considerably from its paralog in the RhoprCAPA-␣ gene, RhoprCAPA-␣1 (73.3% identity, see Table 1). Interestingly, it is modified on its C-terminus, with an additional alanine residue and lacking the glycine residue required for amidation of the mature peptide (see Fig. 1A). The second peptide is fully conserved with its related paralog on the RhoprCAPA-␣ gene; however, to distinguish its location on the RhoprCAPA-␤ gene, we give it a unique nomenclature, RhoprCAPA-␤2 (EGGFISFPRV-NH2 , see Table 1). The third encoded peptide, RhoprCAPA-␤PK-1 (IGGGGNGGGLWFGPRLNH2 ), shares near perfect identity with its paralog from the RhoprCAPA-␣ gene, nonetheless the N-terminal most asparagine residue has been substituted by isoleucine (see Fig. 1A and Table 1). All peptide masses predicted by processing of the RhoprCAPA␤ prepropeptide were identified by mass spectrometry (data not shown), as were those for the RhoprCAPA-␣ prepropeptide (see also Paluzzi et al., 2008). To our knowledge, this is the first report demonstrating a duplication event, in any insect species, for the CAPA/CAP2b peptide-encoding genes. 3.2. Genomic organization of the R. prolixus CAPA genes Preliminary genome assembly data from the ongoing R. prolixus genome sequencing project was screened using a local BLASTN analysis revealing an initial molecular organization of each paralog (see Fig. 1B). The open reading frame of each paralog is encoded by four exons separated by three introns. The RhoprCAPA-␣ exons are 153, 144, 174 and 253 bp in length and the RhoprCAPA-␤ exons are identical in size with the exception of the first exon which is 142 bp. Although the introns are located at identical positions, they diverge greatly in size and the RhoprCAPA-␣ gene spans a greater region of the genome than does the RhoprCAPA-␤ paralog. However, given Table 1 Sequences deduced from the RhoprCAPA genes which arise following posttranslational processing and detected by mass spectrometry (data not shown). Gene

Peptide name

Sequence

[M+H]+

RhoprCAPA-␣

RhoprCAPA-␣1 RhoprCAPA-␣2 RhoprCAPA-␣PK1

SPISSVGLFPLRA-NH2 EGGFISFPRV-NH2 NGGGGNGGGLWFGPRL-NH2

1489.85 1107.59 1514.76

RhoprCAPA-␤

RhoprCAPA-␤1 RhoprCAPA-␤2 RhoprCAPA-␤PK1

SPITSIGLLPFLRAA-OH EGGFISFPRV-NH2 IGGGGNGGGLWFGPRL-NH2

1555.92 1107.59 1513.80

the current stage of the R. prolixus genome sequencing project, it was not possible to confirm precisely the size of all of the introns (see Fig. 1B for predicted intron sizes). 3.3. Sequence and phylogenetic analysis of CAPA-precursors The CAPA prepropeptide sequences in insects generally share sequence identity on the C-terminus of each encoded peptide (see Fig. 2A). Implementing a 75% conservation threshold and considering all species where a prepropeptide sequence is known, the region containing the first peptide has the consensus motif LX1 X2 FX3 RVGR (where X1 = L, I, F, T or Y; X2 = A or P; X3 = L or P), with some minor exceptions (such as the substitutions observed in both R. prolixus precursors and that in T. castaneum). The conserved motif in the region producing the second peptide is FPRVGR, again with some minor exceptions (A. pisum and T. castaneum have isoleucine substituted for valine). The region producing the pyrokinin-related peptide contains the conserved motif WFGPRLG, normally followed by a single or dibasic residue (with the exception of the recently identified ‘CAPA-B’ splice variant in B. mori, which may not be processed at this site as it lacks basic residues required for propeptide cleavage). In comparison to other insect species, our phylogenetic analysis indicates the RhoprCAPA prepropeptide sequences are most similar with the precursor identified in T. castaneum (see Fig. 2B), which together generate a monophyletic group having strong support (90% bootstrap percentage). The sister group to these taxas’ sequences was one which included all other insect sequences except the aphid taxa sequences. Interestingly, the Dipteran representatives (the Drosophilidae and Culicidae) did not form a monophyletic group. Instead, the Culicidae formed a sister taxa to a monophyletic group including the Drosophilidae, Lepidopteran and Hymenopteran sequences. A second group of Hemipteran representatives (the Aphidoidea), are placed at the basal position of all insect representatives having the greatest difference to all the insect CAPA-precursors known. 3.4. CAPA paralog spatial expression profile in fifth-instars Expression of the RhoprCAPA paralogs in fifth-instar tissues was assessed by Northern blot hybridization. A band was detected in lanes containing CNS RNA at an approximate size of 0.7–0.8 kb after hybridization with a DIG-labelled anti-sense RNA probe generated using either the RhoprCAPA-␣ (data not shown) or RhoprCAPA-␤ cDNA (Fig. 3A). With the exception of a faint, similarly sized, band in the testis RNA sample, no other tissues displayed presence of RhoprCAPA paralog transcript. A probe for beta-actin (actin 5c) was used as a control to verify the quantity and integrity of RNA samples loaded in each lane. To corroborate these findings and more accurately measure the expression levels of each paralog, a two-step RT-qPCR approach was utilized. Both paralogs are expressed in the CNS, while only the RhoprCAPA-␣ transcript is detected in testis samples (Fig. 3B). The expression of the RhoprCAPA-␣ transcript in the testis represents only a marginal level compared to the total neuronal expression levels of both paralogs, but it is interesting that only the one transcript (RhoprCAPA-␣) was detected in this non-neuronal tissue. When comparing expression levels within the CNS however, the RhoprCAPA-␤ transcript shows approximately 30% higher levels. Within the different regions of the CNS, divided as the brain and suboesophageal ganglion (SOG), prothoracic ganglion (PRO), and mesothoracic ganglionic mass (MTGM), RhoprCAPA-␤ expression is consistently higher than that of RhoprCAPA-␣ transcript (Fig. 3C), albeit highest levels are observed in the MTGM and lowest levels in the PRO.

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Fig. 2. ClustalW2 alignment and phylogenectic analysis of known and predicted insect CAPA encoding prepropeptide sequences. (B) The phylogenetic relationship of the prepropeptide sequences of CAPA-related peptides of R. prolixus with representatives of the insect Coleopteran, Dipteran, Lepidopteran, Hemipteran, Phthirapteran and Hymenopteran orders as well as a species of the arachnid order, Acari. The tree was constructed using neighbor-joining method. Numbers at the nodes are for percent support in 1000 bootstrapping. A parsimonious tree provided a similar tree topology.

3.5. CAPA paralog temporal expression profile relative to blood meal Upon engorging a blood meal, R. prolixus undergoes a rapid diuresis whereby the excess water and salt loads are excreted. In order to better understand the role of CAPA peptides in serving to regulate the termination of this rapid diuresis, we investigated

expression levels of the RhoprCAPA genes following a blood meal. The analysis was carried out in both a micro (hours) and macro (days/weeks) time-scale. The outcome of the micro-temporal analysis reveals that both genes undergo an immediate decrease in expression levels following blood gorging. Transcript levels in CNS dissected immediately following feeding show an approximate 25% reduction in

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expression for both paralogs and the levels remain lower than those prior to feeding over the next couple of hours (Fig. 4A). The RhoprCAPA-␣ transcript has its lowest levels at 2 h post-feeding, reaching a level 40% lower than that prior to feeding. Subsequent to this, levels of RhoprCAPA-␣ begin to increase, reaching a titer similar to the unfed state at 4 h post-feeding. However, at 5 and 6 h after feeding, the RhoprCAPA-␣ levels are again 20% lower, and are then restored to near-unfed levels at 7 h. At 8 h, the transcript level of RhoprCAPA-␣ is again lower by about 20% and remains low up to 24 h post-feeding. For the most part, the RhoprCAPA-␤ transcript level changes mirror those of RhoprCAPA-␣, but levels are consistently higher by a range of 20–40% at each time-point (a ratio similar to that observed in the spatial expression profile, see Fig. 3). Similar to its paralog, the RhoprCAPA-␤ transcript had lowest levels at 2 and 3 h after feeding, and levels begin increasing thereafter reaching levels comparable to the unfed state at 5–7 h after the blood meal (Fig. 4A). As shown for RhoprCAPA-␣, a decrease in RhoprCAPA-␤ transcript level is also observed at 8 h post-feeding with a small increase occurring 24 h post-feeding. The macro-temporal analysis reveals that both RhoprCAPA paralogs undergo fluctuations during the days/weeks following a blood meal (Fig. 4B). As observed in the micro-temporal analysis, expression immediately following feeding is lower relative to the levels in the unfed insect. Transcript levels of both paralogs reach lowest levels at 12 days post-feeding and subsequently increase, whereby they reach their highest levels at time-points subsequent to the nymph-adult ecdysis (21, 25, 28 and 32 days post-feeding), which occurs on days 19–20 post-feeding. As observed in other quantitative analyses of these two paralogs in CNS, the changes in expression are generally mirrored between the two paralogs – again, the RhoprCAPA-␤ transcript has levels ranging approximately 20–40% higher than the RhoprCAPA-␣ transcript.

3.6. CAPA paralog expression in adult R. prolixus

Fig. 4. Quantitative reverse transcriptase PCR in central nervous system following a blood meal for RhoprCAPA-␣ and RhoprCAPA-␤. Pools of tissues dissected from 5 to 6 insects were used for total RNA isolation and subsequent cDNA synthesis. The averages and standard deviations of three biological replicates are shown. (A) Micro-temporal analysis of RhoprCAPA-␣ and RhoprCAPA-␤ expression during the hours following a blood meal (t = 0). (B) Macrotemporal analysis of RhoprCAPA-␣ and RhoprCAPA-␤ expression following a blood meal (t = 0) during development from fifth-instar to adults. Triangles and squares denote RhoprCAPA-␣ and RhoprCAPA-␤ expression, respectively.

Investigation of the expression distribution of the two transcripts in various adult tissues (CNS, thoracic muscle, hindgut, reproductive tissue, anterior midgut, salivary gland, dorsal vessel,

Fig. 3. Expression analysis of RhoprCAPA genes in fifth-instar R. prolixus. (A) Northern blot analysis demonstrates RhoprCAPA genes are expressed primarily in the central nervous system. Detection of transcript size and abundance was similar in experiments using digoxigenin-labelled anti-sense RNA probes for RhoprCAPA-␣ or RhoprCAPA-␤. (B) Quantitative RT-PCR analysis of transcript levels in fifth-instar tissues. (C) Transcript levels in regions of the central nervous system were assessed to determine spatial expression profile of the two CAPA gene paralogs.

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CNS tissues, expression levels are similar to those found in fifthinstars (regardless of gender). Conversely, in adult female CNS, transcript levels are approximately half those levels observed in their male counterparts. Neither paralog is detected in adult reproductive tissues. 3.7. Immunohistochemical localization of CAPA-related peptides and CAPA gene expression in R. prolixus adult central nervous system

Fig. 5. Tissue expression analysis of RhoprCAPA genes in adult male (A) and female (B) R. prolixus and comparison of expression between fifth-instar and adult neuronal and reproductive tissues (C). Regardless the sex and stage of the insect, expression was primarily identified in the central nervous system. Pools of tissues dissected from 5 to 6 insects were used for total RNA isolation and subsequent cDNA synthesis. In (A) and (B) numbers denote as follows: 1, CNS; 2, thoracic muscle; 3, hindgut; 4, reproductive tissue; 5, anterior midgut; 6, salivary gland; 7, dorsal vessel; 8, Malpighian tubules; 9, posterior midgut; and 10, fat body, diaphragm and trachea. In (C) numbers denote the following: 1, fifth male CNS; 2, fifth female CNS; 3, fifth male reproductive; 4, fifth female reproductive; 5, adult male CNS; 6, adult female CNS; 7, adult male reproductive; and 8, adult female reproductive.

MTs, posterior midgut and a combined sample composed of fat body, diaphragm and trachea) in male (Fig. 5A) and female (Fig. 5B) R. prolixus demonstrates that transcript expression is confined to the CNS in both sexes. Since we found that the RhoprCAPA␣ transcript was also expressed in fifth-instar male reproductive tissues, and that expression levels of the transcripts fluctuated during development following a blood meal, we sought to quantitate transcript levels in tissue types that had demonstrated expression of one or both transcripts. This analysis (Fig. 5C) reveals that in fifth-instars, transcript levels are similar in male and female CNS; however outside of the CNS, male reproductive tissue (testis) also contains low levels of the RhoprCAPA-␣ transcript. In adult male

We have previously determined the distribution of PRX-amidelike immunoreactivity in fifth-instar R. prolixus and located cells expressing the RhoprCAPA-␣ transcript in CNS (and presumably, given the high sequence identity between these two paralogs, the RhoprCAPA-␤ transcript expression). Since we observed some differences in expression within the CNS of male and female adults (see Fig. 5C), we performed combined fluorescent in situ hybridization and immunohistochemical assays to determine which cells might be expressing higher or lower levels of these transcripts. A comparison of adult male and female CNS reveals no difference in cell number or location; however, cell staining-intensity was noticeably greater in adult male CNS. We have not observed any differences in cell distribution with regard to the paralog (RhoprCAPA-␣ or RhoprCAPA-␤) that was utilized as a probe. The cells expressing RhoprCAPA transcript and those positive for PRX-amide-like immunoreactivity are shown in Figs. 6 and 7. The double-labelling reveals that all cells positive for RhoprCAPA transcript expression are also positive for PRX-amide-like immunoreactivity. However, as might be expected for an antiserum that recognizes families of peptides with similar C-termini, there are PRX-amide-like immunoreactive cells that are negative for RhoprCAPA transcript expression. These cells probably express the PBAN family or pyrokinin family of neuropeptides which share some sequence similarity to the CAPA-related neuropeptide family (see Rafaeli, 2009). A bilateral pair of cells is present in the dorsal-lateral region of the brain bordering the optic lobe (Fig. 6A), which are positive for RhoprCAPA transcript and PRX-amide-like immunoreactivity. The PRX-amide-like immunoreactivity reveals processes arising from the cell bodies that enter the central neuropile of the brain (Fig. 6B). Additional PRX-amide-like positive cells, but lacking RhoprCAPA transcript expression, are found within the central region of the brain, and these are consistant with being the medial neurosecretory cells identified in fifth-instars. The colocalization of transcript expression and the peptide products within the adult brain using merged images is shown in Fig. 6C. In the SOG, two pairs of cells (Fig. 6D and F) demonstrate strong transcript abundance with the more posterior pair being approximately twice the diameter of the anterior pair. Another group of cells displaying RhopCAPA transcript expression lie adjacent to the oesophageal foramen, but are less strongly stained. Both groups of cells are PRX-amide-like immunoreactive; however a number of other immunoreactivepositive cells do not show RhoprCAPA transcript expression (Fig. 6E and F). The central region of the PRO contains two pairs of cells on the ventral surface which exhibit RhoprCAPA transcript expression (Fig. 6G and I) and are also positive for PRX-amide-like immunoreactivity (Fig. 6H and I). A schematic of cells which colocalize for RhoprCAPA transcript expression and PRX-amide-like immunoreactivity in the adult brain, SOG and PRO is illustrated in Fig. 6J. Within the MTGM, three pairs of strongly staining cells are present within the abdominal neuromeres on the posterior ventral surface that express RhoprCAPA transcript (Fig. 7A and C) and these same cells demonstrate PRX-amide-like immunoreactive staining (Fig. 7B and C). More anteriorly, two additional pairs of cells and one ventral unpaired median neuron lie within the meso- and

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Fig. 6. RhoprCAPA-␣ and RhoprCAPA-␤ transcript expression (Alexa568/red) and PRX-amide-like immunoreactivity (FITC/green) in the brain, suboesophageal ganglion (SOG) and prothoracic ganglion (PRO) of adult R. prolixus. Over the dorsal surface of the brain, a pair of lateral neurosecretory cells show PRX-amide-like immunoreactivity and RhoprCAPA transcript expression (A–C). In addition, a number of medial neurosecretory cells show PRX-amide-like immunoreactivity; however, they do not show any RhoprCAPA transcript expression. Over the ventral surface of the SOG (D–F), two pairs of cells with prominent expression are observed located medially and an additional three pairs of cells, which stained more weakly, are observed just posterior of the oeshophogeal foramen. Over the ventral prothoracic ganglion (G–I), two small pairs of cells are observed lying medially. A schematic diagram (J) summarizes the cells within the brain, SOG and PRO which show both PRX-amide-like immunoreactivity and RhoprCAPA transcript expression. Outlined cells are located on the ventral surface while filled cells are located on the dorsal surface. Scale bar in A–I is 50 ␮m and in J is 200 ␮m. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

Fig. 7. RhoprCAPA-␣ and RhoprCAPA-␤ transcript expression (Alexa568/red) and PRX-amide immunoreactivity (FITC/green) in the mesothoracic ganglionic mass (MTGM) of adult R. prolixus. The ventral posterior segment of the MTGM (A–C) contains three bilaterally paired RhoprCAPA gene-expressing cells within the abdominal neuromeres. In the more anterior segment of the ventral MTGM (the meso- and metathoracic neuromeres), two additional pairs of cells are positive for PRX-amide-like immunoreactivity but RhoprCAPA transcript expression was not detected. In the dorsal region of the posterior MTGM, two pairs of lateral neurosecretory cells were immunopositive for PRX-amide like peptides (D–F), however, these cells did not show any RhoprCAPA transcript expression. A schematic diagram (G) summarizes the cells within the MTGM on the ventral surface which show both PRX-amide-like immunoreactivity and RhoprCAPA transcript expression. Scale bar in A–F is 50 ␮m and in G is 200 ␮m. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

metathoracic neuromeres and contain PRX-amide-like immunoreactivity, but these cells show no RhoprCAPA transcript expression (Fig. 7B and C). No cells on the dorsal surface of the MTGM have RhoprCAPA transcript expression (Fig. 7D). Immunoreactive processes that arise from the three pairs of strongly stained and doublelabelled neurosecretory cells on the ventral surface are however observed (Fig. 7E and F). These processes continue posteriorly and produce extensive neurohaemal sites on abdominal nerves two, three and four (as previously shown for fifth-instars). Two lateral pairs of cells which are PRX-amide-like immunoreactive, but negative for RhoprCAPA transcript expression, lie on the dorsal surface of the MTGM (Fig. 7E and F). A schematic of cells which colocalize for

RhoprCAPA transcript expression and PRX-amide-like immunoreactivity in the adult MTGM is illustrated in Fig. 7G. 4. Discussion CAPA gene peptides include the CAP2b-related/perviscerokinins and pyrokinins and are an important family of peptides which appear to regulate central and peripheral physiological processes in insects. In Dipteran species, the CAP2b-related peptides stimulate fluid secretion by the MTs through increasing levels of nitric oxide and cyclic GMP, which in turn elevates calcium levels by opening cyclic-nucleotide gated calcium channels (Pollock et al.,

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2004). In contrast, while members of this family may have no effect on MTs in insects such as the Orthopteran, Schistocerca gregaria, these peptides are known for their anti-diuretic role in R. prolixus (Quinlan et al., 1997; Paluzzi and Orchard, 2006; Paluzzi et al., 2008), where they inhibit tubule secretion stimulated by 5HT. In addition, unlike the increase in cGMP over unstimulated levels that is induced in Dipteran MTs by CAPA-related peptides (Pollock et al., 2004), resting R. prolixus tubules do not exhibit an increase in cGMP in response to CAPA-related peptides (Quinlan et al., 1997; Paluzzi and Orchard, 2006; Paluzzi et al., 2008). Importantly, however, the decrease in cGMP levels that is observed when tubules are stimulated with 5HT, is abolished in the presence of CAPA-related peptides (Quinlan et al., 1997; Paluzzi and Orchard, 2006; Paluzzi et al., 2008). CAPA-related peptides have been identified in a number of insect species including Dipterans (Kean et al., 2002; Pollock et al., 2004; Riehle et al., 2002; Predel et al., 2003a,b; Nachman et al., 2006), at least two Lepidopterans (Loi and Tublitz, 2004; Huesmann et al., 1995; Predel et al., 2003a,b), Blattarian species (Predel et al., 1998; Predel et al., 2000; Predel and Gade, 2005; Roth et al., 2009), the Coleopteran, T. castaneum (Li et al., 2008), four polyphagous Hemipteran species (Predel et al., 2006; Predel et al., 2008) and the haematophagous Hemipteran, R. prolixus (Paluzzi et al., 2008 and this study). Genes encoding CAPA-related peptides have not been abundantly resolved in insects, however they have been identified in Dipterans (Kean et al., 2002), Lepidopterans (Loi and Tublitz, 2004), Hemipterans (Paluzzi et al., 2008) and Coleopterans (Li et al., 2008). Genome sequencing of the human body louse, P. humanus corporis (EEB10638), and the pea aphid, A. pisum (XP 001946149), as well as EST data of the green peach aphid, M. persicae (EE570800.1), reveals the presence of CAPA genes in these additional insects of medical and agricultural importance. Interestingly, a putative CAPA gene exists in the genome of the Arachnid, I. scapularis (EW933575.1), although the resulting prepropeptide lacks many of the conserved motifs seen in the Insecta sequences. In support of the above gene prediction, the first neuropeptide in ticks was identified using a combined immunocytochemical and mass spectrometric approach where a CAPA-related peptide was sequenced from analysis of single cells from two species, Ixodes ricinus and Boophilus microplus (Neupert et al., 2005). In the current study, we show that R. prolixus contains a second gene, RhoprCAPA-␤, encoding CAPA-related peptides. This gene shares high sequence identity (88.7%) with the previously identified gene, RhoprCAPA-␣, and also the prepropeptides demonstrate strong similarity (85.4%). The RhoprCAPA-␤ gene produces peptides that share exact or near-identical sequence to the products of the RhoprCAPA-␣ paralog; however, the first encoded peptide contains the greatest variation and lacks a glycine residue at its carboxyl terminus required for amidation. Each predicted peptide in the RhoprCAPA-␤ prepropeptide is flanked on their amino termini by a dibasic cleavage site and a monobasic site on their carboxyl termini, thus providing the necessary sites for post-translation processing into biologically active forms (Veenstra, 2000). In support of this predicted processing, molecular masses matching these novel peptides have been confirmed by mass spectrometry (data not shown). Phylogenetic analysis of CAPA prepropeptide sequences and those predicted from EST databases reveals the R. prolixus sequences share the closest similarity with the T. castaneum precursor. Interestingly, precursors from the more evolutionaryrelated Aphidoidean species, are not grouped closely with their haematophagous relatives, suggesting the CAPA genes may undergo divergent species-specific selection. A similar phenomenon is observed between representatives of the Dipteran order, where the Culicidea group more closely with non-Dipteran

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insects, as opposed to the more closely related Drosophilidae. Thus, this suggests the CAPA prepropeptides may not be a good representative for determining phylogenetic relationships above the species level in all insects. Nonetheless, a recent phylogenetic analysis using CAPA peptides from numerous cockroaches and the termite, Mastotermes darwiniensis, demonstrated mature peptides from species within the Dictyopteran group may be suitable for reconstruction of phylogenetic relationships (Roth et al., 2009). Previously we demonstrated the expression of the RhoprCAPA␣ paralog in various tissues in fifth-instar R. prolixus as well as all post-embryonic stages using a non-quantitative RT-PCR approach (Paluzzi et al., 2008). Using northern blot hybridization, we demonstrate strong CAPA gene expression within the CNS of R. prolixus, regardless of which paralog was used to synthesize the anti-sense RNA probe. In order to differentiate between the expression levels of the two paralogs and quantify the levels identified in the CNS and testis tissues, we designed gene-specific primers taking advantage of a 10 bp sequence (‘TGTTTTTTCT’, residue 63–72) that was present in the RhoprCAPA-␣ transcript, but absent in RhoprCAPA-␤. We confirmed that both paralogs are expressed within the CNS, with the RhoprCAPA-␤ gene having moderately higher levels (∼30%). Expression outside of the CNS was restricted to the fifth-instar male reproductive tissue, where only the RhoprCAPA-␣ transcript was detected. This is consistent with that found previously (Paluzzi et al., 2008), albeit a level greatly lower than expression levels found in the CNS. Within each region of the CNS, relative expression levels between the two paralogs was consistent with that found for the CNS as a whole, where the RhoprCAPA-␤ transcript was approximately 30–40% more highly expressed. Expression of both paralogs was highest in the MTGM, followed by the brain & SOG and lowest levels were localized to the PRO. The neuronal expression patterns corroborate well with that described for other insects where the CAPA genes have been studied. In D. melanogaster, the CAPA gene peptides were immunolocalized in the larval and adult CNS to three pairs of ventral neuroendocrine cells in the abdominal neuromeres, as well as a number of cells in the SOG and brain (Kean et al., 2002). In M. sexta, in situ hybridization identified cells expressing the CAPA gene in similar midline cells within the abdominal ganglia; however, many additional cells were identified in the larval brain (Loi and Tublitz, 2004) that were not found in D. melangaster (Kean et al., 2002) nor in R. prolixus (Paluzzi et al., 2008). CAPA-related peptides have also been identified via immunolocalization or mass spectrometric analyses of neuroendocrine cells in the abdominal ganglia, or analogous regions, in other insects (Predel et al., 2003a,b, 1998, 2008, 2004; Clynen et al., 2003; Verleyen et al., 2004) and two tick species (Neupert et al., 2005). The appearance of PRX-amidelike immunoreactive neurons in R. prolixus CNS that do not show RhoprCAPA gene expression illustrates the cross-reactivity of the anti-PRX-amide antiserum with some other insect peptide families such as PBAN family of peptides, which contain similar C-terminal sequences (Zitnan et al., 2003), but are not encoded by CAPA genes. The temporal expression profile of CAPA genes has not previously been comprehensively studied. Given the importance of a subset of the R. prolixus CAPA peptides in regulating anti-diuresis (Paluzzi et al., 2008), we questioned if gene expression was altered following feeding. Interestingly, both R. prolixus CAPA paralogs show a decrease immediately following feeding, but levels are soon recovered in the hours that follow the rapid diuresis. Thus, this suggests that the CAPA genes may undergo transcriptional regulation associated with feeding, although admittedly, little is known of the transcriptional control of regulators of diuresis in R. prolixus. Previously we showed that immunoreactivity within neuroendocrine cells in abdominal neuromeres of the MTGM, and their associated neurohaemal release sites, is greatly decreased at 3–4 h post-feeding (Paluzzi and Orchard, 2006). Thus, one possible

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explanation for the recovery of gene expression observed between 4 and 7 h post-feeding may be to facilitate the restocking of these peptides that have been depleted by release into the haemolymph in order to cease diuresis. The expression profile observed over the days following feeding and leading up to and beyond the nymphadult ecdysis suggest that the R. prolixus CAPA genes may also undergo regulation beyond the rapid diuresis following engorgement. The decrease observed in the early days that follow a blood meal (a fed state) could be indicative of the insect not requiring starved-state amounts of anti-diuretic peptide, where the insect must ensure maintenance of water and essential salts since the previous blood meal has been fully digested. Instead, as the insect prepares for the nymph-adult ecdysis, our results suggest that increases in CAPA gene transcripts may reflect the need to synthesize higher amounts of anti-diuretic peptides to ensure osmotic balance during the nymph-adult ecdysis, where the insect is susceptible to desiccation. Naturally, we must also recognize that the quantification of gene expression may not correlate directly with levels of biologically mature peptides. We also assessed the expression of the two paralogs in various adult tissues and determined that expression is exclusively localized to the CNS in both female and male insects. Expression was not localized to reproductive tissues in either male or female adults. The role of the fifth-instar male-specific expression of the RhoprCAPA-␣ paralog remains unclear. Two genes encode the CAPA-related peptides in the haematophagous Chagas’ disease vector, R. prolixus. From our combined immunohistochemical and in situ hybridization analysis, it is evident that the neurons in the CNS of R. prolixus synthesize at least a two-fold greater number of CAPA-related peptides than that known in other insects. In addition, given the sequence variation that has occurred over the first encoded peptide in each prepropeptide, it is unlikely that the first peptide will elicit the same anti-diuretic effect as shown by RhoprCAPA-␣2 (-␤2). Physiological studies utilizing these peptides clearly demonstrate that the first encoded peptides do not inhibit serotonin-stimulated Malpighian tubule secretion nor do they abolish absorption by the anterior midgut (data not shown). Thus, one or both of the first encoded peptides, namely RhoprCAPA-␣1 (-␤1), in these paralogous genes may have a distinct role, and utilize a unique receptor, in accordance with the theory of ligand-receptor coevolution (Park et al., 2002). Our results suggest that expression levels of the CAPA genes in R. prolixus are modified following gorging on a blood meal, which we suspect allows the restocking of peptide stores that are depleted from the neuroendocrine cells (and associated neurohaemal sites) during the termination of the rapid diuresis. Finally, the RhoprCAPA genes undergo changes in expression as the fifth-instar digests the blood meal for a period of days and weeks following feeding and prepares for the nymph-adult ecdysis. At times when water must be conserved in R. prolixus (such as the end of the rapid diuresis and nymph-adult ecdysis), our results indicate the CAPA genes may be activated to ensure sufficient anti-diuretic peptide is present to meet that physiological demand. Acknowledgements The authors wish to thank Nikki Sarkar for assistance in the feeding experiments. This research was made possible through an NSERC Discovery Grant to I.O. and an NSERC Canada Graduate Scholarship to J.P.P. References Bendtsen, J.D., Nielsen, H., von Heijne, G., Brunak, S., 2004. Improved prediction of signal peptides: SignalP 3.0. J. Mol. Biol. 340 (4), 783–795.

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