Orcokinin neuropeptides regulate ecdysis in the hemimetabolous insect Rhodnius prolixus

Accepted Manuscript Orcokinin neuropeptides regulate ecdysis in the hemimetabolous insect Rhodnius prolixus Juan Pedro Wulff, Ivana Sierra, Marcos Sterkel, Michiel Holtof, Pieter Van Wielendaele, Flavio Francini, Jozef Vanden Broeck, Sheila Ons PII:

S0965-1748(17)30003-6

DOI:

10.1016/j.ibmb.2017.01.003

Reference:

IB 2915

To appear in:

Insect Biochemistry and Molecular Biology

Received Date: 10 August 2016 Revised Date:

14 December 2016

Accepted Date: 4 January 2017

Please cite this article as: Wulff, J.P., Sierra, I., Sterkel, M., Holtof, M., Van Wielendaele, P., Francini, F., Broeck, J.V., Ons, S., Orcokinin neuropeptides regulate ecdysis in the hemimetabolous insect Rhodnius prolixus, Insect Biochemistry and Molecular Biology (2017), doi: 10.1016/j.ibmb.2017.01.003. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT TITLE: “ORCOKININ NEUROPEPTIDES REGULATE ECDYSIS IN THE

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HEMIMETABOLOUS INSECT RHODNIUS PROLIXUS”

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AUTHOR NAMES: Juan Pedro Wulff (1), Ivana Sierra(1), Marcos Sterkel(2), Michiel

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Holtof(3), Pieter Van Wielendaele(3), Flavio Francini(4), Jozef Vanden Broeck(3) and Sheila

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Ons(1) *.

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*Author to whom correspondence should be addressed.

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AUTHOR ADDRESSES:

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(1) Laboratory of Genetics and Functional Genomics. Regional Center for Genomic

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Studies. Faculty of Exact Sciences. National University of La Plata Bvd 120 y 62 (1900).

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La Plata. Buenos Aires. Argentina.

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(2) Institute of Medical Biochemistry. Federal University of Rio de Janeiro. Av. Carlos

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Chagas Filho, 373, bloco D. Prédio do CCS. Ilha do Fundão. Rio de Janeiro - 21941-902

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– Brazil.

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(3) Molecular Developmental Physiology and Signal Transduction, Division of Animal

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Physiology and Neurobiology, Zoological Institute, K.U. Leuven, Leuven, Belgium

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(4) Centre of Experimental and Applied Endocrinology, National University of La Plata,

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School of Medicine. 60 street y 120 (1900). La Plata. Buenos Aires. Argentina.

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AUTHOR E-MAIL ADDRESSES

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[email protected]

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[email protected]

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[email protected]

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[email protected]

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[email protected]

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[email protected]

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[email protected]

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[email protected] ABSTRACT To grow and develop insects must undergo ecdysis. During this process, the individual sheds the old cuticle to emerge as the following developmental stage. During

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ecdysis, different programed behaviors are regulated by neuropeptidergic pathways. In

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general, components of these pathways are better characterized in crustacean and

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holometabolous insects than in hemimetabola. In insects, the orkoninin gene produces

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two different neuropeptide precursors by alternative splicing: orcokinin A and orcokinin

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B. Although orcokinins are well conserved in insect species, their physiological role

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remains elusive. Here we describe a new splicing variant of the orcokinin gene in the

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hemimetabolous triatomine Rhodnius prolixus. We further analyze the expression pattern

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and the function of the alternatively spliced RhoprOK transcripts by means of

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immunohistochemistry and RNAi-mediated gene silencing. Our results indicate that

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orkoninis play an essential role in the peptidergic signaling pathway regulating ecdysis in

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the hemimetabolous insect Rhodnius prolixus.

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Keywords: Chagas' disease, post-embryonic development, molting, brain-gut

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neuropeptides, RNA interference (RNAi)

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1. Introduction

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Animals are capable of performing programed behaviors that are stereotypic and

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critical for survival in the absence of previous experience, including escape strategies,

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nurturing and courtship (Zitnan, Ross et al. ,1999; Kim, Zitnan et al., 2006). In insects,

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development occurs through successive stages of feeding and molting that culminate in an

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endocrine-regulated process called ecdysis. During ecdysis, insects undergo a sequence of

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programed behaviors to shed the cuticle surrounding the external surface and the lining of

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the foregut, hindgut and tracheae.

ACCEPTED MANUSCRIPT Ecdysis is coordinated by a complex network of hormones leading to a cascade of

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(neuro)peptides that are secreted after the decline of ecdysteroid levels (Zitnan, Ross et al.

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1999; Zitnan and Adams 2012), in a process that was studied mainly in two

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holometabolous species: the moth Manduca sexta and the fruit fly Drosophila melanogaster

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(Zitnan and Adams, 2012). In hemimetabola, the regulation of ecdysis by neuropeptides

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has not been thoroughly characterized, and only scattered information is available.

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In insects, the orcokinin (OK) gene produces two peptide precursors by alternative

splicing: orcokinin A (OKA) encoding mainly OKA peptides, and orcokinin B (OKB)

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encoding OKB mature neuropeptides (Sterkel, Oliveira et al. 2012). Even though OKs are

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well conserved in insect species, their role in the regulation of insect physiology has been

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poorly studied, compared to other neuroendocrine systems. Two reports dealt with the

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physiological effects of synthetic OKA. These studies showed an in vitro

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prothoracicotropic action in the lepidopteran Bombyx mori (Yamanaka, Roller et al. 2011)

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and the regulation of circadian locomotor activity in the cockroach Leucophaea maderae

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(Hofer and Homberg 2006). Recently, studies using gene silencing by RNAi suggested a

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role of the OK gene (OKA and OKB isoforms) in oogenesis in the cockroach Blattella

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germanica (Ons, Belles et al. 2015) and in the regulation of “awakening” escape behavior in

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the beetle Tribolium castaneum (Jiang, Kim et al. 2015).

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Rhodnius prolixus is a medically relevant insect species as a vector of Chagas' disease, a

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neglected disease affecting approximately 7 million people around the world (Rassi, Rassi

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et al. 2010). Larvae remain in an arrested developmental state after molting; blood feeding

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triggers the neuroendocrinological response that conducts to molting and ecdysis to the

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following life stage (Maddrell 1964, Maddrell 1966). This characteristic allows

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experimental timing during physiological assays, making R. prolixus a convenient

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experimental model for the study of post-embryonic development and ecdysis.

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ACCEPTED MANUSCRIPT In the present work, we study the RhoprOK encoding transcripts both structurally and

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functionally. The expression pattern as well as the phenotype obtained with the depletion

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of the transcript by RNAi point to RhoprOKA, and to a lesser extent at RhoprOKB, as

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regulators of ecdysis. This is the first study demonstrating the crucial role of OKs in

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peptidergic pathways regulating ecdysis, highlighting the relevance of this hormonal

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system in the regulation of insect physiology.

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2. Material and Methods 2.1 Insect rearing

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A colony of R. prolixus was maintained in the laboratory under a 12 h light/dark

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schedule at 28oC. Insects were weekly fed on chickens, which were housed, cared, fed

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and handled in accordance with resolution 1047/2005 (National Council of Scientific

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and Technical Research, CONICET) regarding the National Reference Ethical

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Framework for Biomedical Research with Laboratory, Farm, and Nature Collected

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Animals. This framework is in accordance with the standard procedures of the Office

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for Laboratory Animal Welfare, Department of Health and Human Services, NIH, and

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the recommendations established by the 2010/63/EU Directive of the European

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Parliament, related to the protection of animals used for scientific purposes. Biosecurity

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considerations are in agreement with CONICET resolution 1619/2008, which is in

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accordance with the WHO Biosecurity Handbook (ISBN 92 4 354 6503).

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2.2 RhoprOKC transcript identification and tissue profiling A public EST database from R. prolixus whole body and tissues

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(http://rhodnius.iq.ufrj.br/index.php?option=com_content&view=article&id=22&Item

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id=34) was used for the homology-based search, performed by local TBLASTN by using

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BLOSUM62 and RhoprOKB sequence as query. In addition to the known RhoprOKB

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sequence, we found an EST encoding a potentially new isoform of RhoprOK. The latter

ACCEPTED MANUSCRIPT was used to design specific primers to sequence the complete open reading frame of the

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RhoprOKC transcript by using RACE-PCR. For the 3’ region, we used the primers

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OKA/B fwd (Table 1) and the 3’ GeneRacer primer from the GeneRacer® Kit

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(Invitrogen, Argentina). One µl of this reaction was used as template for amplification

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with the primers OKC fwd (Table 1) and 3’GeneRacer nested primer (Invitrogen,

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Argentina). Template cDNA was prepared from fifth instar R. prolixus starved for 4-5

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weeks, using Trizol Reagent (Invitrogen, Carlsbad, CA, USA). PCR fragments were

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purified by ethanolic precipitation and directly sequenced in Macrogen (Seoul, Korea).

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The 5’ region of the transcript was complete in the EST database. The sequences

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obtained were compared to R. prolixus genome (http://rprolixus.vectorbase.org/). For

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prediction of convertase cleavage sites, the rules described for insect neuropeptide

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precursors (Veenstra 2000) were followed. Signal peptide sequence was predicted with

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the

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(http://www.cbs.dtu.dk/services/SignalP/).

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Tissues were microdissected from adult R. prolixus at different times postfeeding. Total RNA was isolated from tissues using Trizol (Ambion, São Paulo, Brazil). cDNA was

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synthesized using MMLV Reverse Transcriptase (Promega, USA), following the

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manufacturer’s instructions. An aliquot of the cDNA from each tissue was used as

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template to perform the subsequent PCR reactions. The following temperature-cycling

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parameters were used to amplify the RhoprOKC specific fragment in parallel reactions:

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95°C, 5 min (1 cycle); 94°C, 35 sec; 54°C, 35 sec; 72°C, 1.5 min (35 cycles); 72 °C, 10 min

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(1 cycle). For a positive control to test the quality of the template cDNA, a 300 bp

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fragment from RhoprActin was used (see primer sequences in Table 1). The procedure was

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repeated three times using independent biological samples.

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2.3 Immunohistochemistry

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The dorsal cuticle was removed from 5th instar larvae, or male and female adult R. prolixus starved since last eclosion (2 weeks before dissections). Cuticle removal was

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performed under phosphate buffered saline pH 7 (PBS), exposing nervous structures and

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organs. Tissues were fixed in situ in 2% paraformaldehyde (pH 7) for 12 h at 4°C.

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Nervous system and organs were removed, washed with PBS, transferred to 4% Triton X-

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100 with 2% BSA and 10% normal goat serum (NGS) in PBS for 1 h at room

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temperature, and then washed several times with PBS at room temperature. Primary

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antisera were preincubated at a dilution of 1:5000 in a 0.4% solution of Triton X-100 in

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PBS with 2% BSA and 2% NGS for 12 h at 4°C prior to use. Organs and tissues were

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incubated in primary antiserum for 48 h at 4°C, and then washed for 18-24 h at 4°C in

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PBS. Primary rabbit antiserum anti-Asn13-orcokinin (OKA) was generously provided by

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Dr. H. Dircksen, (Department of Zoology, Stockholm University). The primary rabbit

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antiserum against OKB9 peptide (present in RhoprOK-B and RhprOK-C isoforms) was

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produced by Genscript (Piscataway, NJ, USA). The antiserum was generated in rabbits

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injected with a peptide with sequence EFLDPLGGGHLIC and T-Max™ as adjuvant

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(Genscript, Piscataway, NJ, USA). Once produced, the antiserum was affinity purified to

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the antigen. Finally, the sensitivity of the antiserum was characterized by ELISA (reported

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titer= 1:512000).

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Tissues were incubated in Cy3-labeled goat anti-rabbit immunoglobulin IgG

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solution (Jackson, USA) at a dilution of 1:500 with 10% NGS in PBS for 12 h at 4 °C,

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and finally rinsed several times in PBS at room temperature. Control experiments

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omitting the primary antiserum or in which the primary antiserum was preincubated for

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24 h at room temperature with 1 mM of the specific synthetic peptide (Genscript,

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Piscataway, NJ, USA) were also performed. Tissues were mounted on microscope slides

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in Mowiol. Images were obtained using a confocal microscope and LSM510 viewing

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software (LSM510-Zeiss, Germany).

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2.3 RNA Interference To generate dsRNAs targeting RhoprOKs, specific fragments were PCR-amplified using Pegasus Taq polymerase (Productos Bio-Lógicos, Argentina) and primers

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containing T7 sequence at 5’ end, designed for each target (see sequences in Table 1). One

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µl of the PCR product was used for a secondary PCR using T7-promoter primer (Table

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1). RNAs were generated by in vitro transcription using T7-RNA polymerase (Promega,

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USA). To obtain dsRNAs, the products of transcription were heated for 10 min at 95°C,

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cooled down slowly to room temperature, and treated with DNAse and RNAseA

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(Fermentas, USA). The formation of dsRNAs was confirmed by running a 1.5% agarose

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gel and quantified using the software ImageJ from an image of the gel. The dsRNAs were

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stored at -20°C until use. Three fragments were used to generate three different dsRNAs:

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a 315 fragment spanning positions 389 to 703 for depleting RhoprOKA mRNA (dsOKA), a

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348 fragment spanning positions 341 to 689 for depleting RhproOKB, and a 343 fragment

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spanning positions 1 to 343, a part of the molecule that is common to all isoforms of the

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gene (dsRhoprOKs). A heterologous 808-fragment from the ampicillin resistance genes,

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which was PCR-amplified from the pBluescript plasmid, was used as a control (dsAmpi)

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(see primers in Table 1). Once we confirmed that there were no differences between

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dsAmpi and water injected controls (Table 2), injection of water was used as control in

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further experiments. Depending on the experiment, 4th or 5th instar R. prolixus larvae 1-2

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weeks after molt and starved since ecdysis were ventrally injected into the abdomen with 2

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µg dsRNA (2 µl) diluted in saline solution. Forty-eight hours after injection, the insects

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were fed on chickens. In all the experiments performed, the insects that died during the

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firsts 5 days after dsRNA injection were discarded from the analysis; early mortality

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equally occurred in all the experimental and control groups.

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2.6 qRT-PCR

ACCEPTED MANUSCRIPT Total RNA was isolated using Trizol (Ambion, São Paulo, Brazil) and treated with

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DNase (Promega, USA). cDNA was synthesized using MMLV Reverse Transcription Kit

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(Applied Biosystems, São Paulo, Brazil), following the manufacturer’s instructions. cDNA

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amplifications were performed in triplicate, in a 20 µl final volume (primers are shown in

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Supplementary Table 1). cDNA levels were quantified using FastStart SYBR Green

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Master (Roche) in iQ single color in a Mini Opticon/Miniopticon Real-Time PCR

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Detection System (Bio-Rad). The schedule used for the amplification reaction was as

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follows: (i) 95°C for 5 min; (ii) 95°C for 30 sec; (iii) 58°C for 30 sec; (iv) steps (i) and (ii)

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were repeated for 40 cycles. Real-time data were collected through the iQ5 optical system

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software v.2.0 (Bio-Rad). A control without a template was included in all batches. We

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used Elongation Factor 1 (RpEF1) as reference transcript to determine the efficiency of

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gene silencing. For the study of the expression of RhoprOKa and RhoprOKB at different

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times postfeeding and after 20E injection, we normalized the expression levels using two

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reference genes: RpEF1 and tubulin (RpTub). The reference primers used were previously

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validated as stable targets in R. prolixus (Majerowicz, Alves-Bezerra et al. 2011, Omondi,

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Latorre-Estivalis et al. 2015). All primer pairs used for qRT-PCRs (Table 1) were tested

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for dimerization, efficiency, and amplification of a single product.

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2.7 Identification of RhoprE75 transcript sequence A TBLASTN search was performed using online BLAST tool (Altschul, Gish et al.

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1990) in RhoprC3.1 geneset database available on Vectorbase (www.vectorbase.org). The

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amino-acidic sequence of DromeE75B (CG8127) was used as query. With this strategy, we

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identified the transcript RPRC000853-RA as RhoprE75 (E value: 2e-87).

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2.8 Ecdysteroid extraction of hemolymph samples and quantification

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Hemolymph samples from control and dsOK treated 4th instar larvae were taken

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at different time points after blood feeding. Each sample was collected in 95 µl of cold

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methanol, the pellet was washed twice more with 100 µl of cold methanol and stored at -

ACCEPTED MANUSCRIPT 20°C until further processing. The samples were extracted three times with 100%

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methanol by cyclic centrifugation and collection of the supernatants. The different

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supernatants originating from one sample were combined and dried under vacuum.

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When completely dry, the samples were diluted in a sample buffer for enzyme

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immunoassay (EIA) measurement (0.1 M phosphate buffer, pH 7.4) and stored at -20°C.

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Ecdysteroid levels were evaluated using EIA, as described in Van Wielendaele et

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al. (Van Wielendaele, Dillen et al. 2012). A peroxidase conjugated form of 20-hydroxy-

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ecdysone (20E) was used as tracer. The antibody used was a rabbit polyclonal antibody

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against ecdysteroids. The antibody and tracer were a kind gift from Dr. De Reggi

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(Marseille, France) and Dr. Delbecque (Bordeaux, France). In order to quantify the

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ecdysteroid levels, a serial dilution (ranging from 1028 M to 10212 M) of 20E was placed

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on each 96-well plate. Results were determined by comparison with dose-response curves

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obtained using these diluted 20E standards. Hence, the values were calculated and

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expressed as 20E equivalents.

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2.9 Treatments in vivo with 20E

Fourth instar larvae from control or dsOK treated groups were injected at day 4

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and day 6 PBM with 20E (Sigma-Aldrich, Missouri, USA) at doses of 0 (water injected), 5

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or 10 µg per specimen in 2 µl of distilled water.

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In another experiment, unfed 4th instar larvae 2 weeks post-eclosion were injected

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with either water or 10 µg/2 µl 20E. Six days after injections the insects were processed

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as described above, and transcript levels were measured by qRT-PCR.

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2.10 Statistical analysis

The statistical significance of qRT-PCR results (transcript abundance) for the

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different transcripts under study, as well as the molting/mortality day results, was analyzed

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using one-way ANOVA. For the EIA assays, the statistical significance of 20E

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concentration in hemolymph was analyzed by two-way ANOVA with “experimental

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treatment” and “days PBM” as the main factors. Log-transformation of data was

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performed to achieve homogeneity of variances when appropriate.

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3. Results 3.1 Identification and characterization of a new transcript isoform encoding RhoprOK

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A TBLASTN search in an EST database from R. prolixus whole body and tissues

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revealed the presence of a partial open reading frame (ORF) containing the first two

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exons of the gene (encoding the signal peptide) followed by a third exon, which was

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absent in the previously known isoforms. To obtain the complete sequence of this

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transcript, we performed RACE-PCR on cDNA from R. prolixus whole body. The

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amplified cDNA (RhoprOKC, GenbankTM accession number KF179047) spans 1803

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nucleotides, with an ORF encoding a prepropeptide precursor of 422 amino acid

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residues (Figure 1A). RhoprOKC shares two exons with RhoprOKA and three exons with

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RhoprOKB, but also includes a specific exon (Figure 1B). Dibasic and monobasic cleavage

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sites were predicted from RhoprOKC sequence to generate 18 putative mature peptides.

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From these, 15 show the characteristic OKB core domains previously described (Sterkel,

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Oliveira et al. 2012), being actually OKB-like mature peptides. The other three peptides

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deduced from the RhoprOKC sequence are specific OKC-derived neuropeptides (Figure

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1A). These three neuropeptides lack the characteristic OKA or OKB core domains, or

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any other conserved sequence, being considered “spacer peptides” (Wegener and

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Gorbashov (2008). Therefore, all the peptides with conserved core sequence encoded in

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the RhoprOK gene are OKA or OKB peptides, similar to other species studied (Chen,

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Choi et al. 2015; Jiang, Kim et al. 2015; Sterkel et al. 2012, Ons et al. 2015, Veenstra and

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Ida 2014).

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To determine the expression pattern of the RhoprOKC transcript, RT-PCR tissue

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profiling was performed (three independent trials). Results show that RhoprOKC shows a

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peripheral expression restricted to anterior midgut, but no expression in the central

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nervous system (CNS), where most neuropeptides are expressed (including RhoprOKA

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and RhoprOKB) (Figure 1C).

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3.2 Expression pattern of OKA and OKB peptides in central nervous system and peripheral organs determined by immunohistochemistry The immunoreactivity of OKA and OKB peptides was determined in CNS,

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salivary glands, anterior midgut, posterior midgut, hindgut, and reproductive organs. 3.2.1 OKA-like immunoreactivity

OKA-like immunoreactivity (OALI) was observed in neurons that were

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distributed extensively throughout the CNS. Figure 2A shows a schematic representation

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of the more intensely stained cell groups and processes in the CNS. Approximately 80-

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100 big neurosecretory cells (NSCs) presented intense OALI in the protocerebrum. In the

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optic lobes, small neurons were profusely stained (Figure 2B). Groups of OALI cells were

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located at the boundary between the protocerebrum and the optic lobes, in a cluster of

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NSCs characterized as the master circadian clock in the insect's brain (Steel and

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Vafopoulou 2006, Reischig and Stengl 2003, Helfrich-Forster, Shafer et al. 2007). NSCs in

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posterior and lateral margins of the brain presented OALI, in neurons showing

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posteromedial processes. A cluster of paired medial NSCs stained strongly in each lobe of

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the brain. Posterior neurons send processes that could be traced to the subesophageal

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ganglion (SOG). A cluster of immunoreactive (IR) cells was found in the posterior margin

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of the protocerebral lobes. Processes from this group of cells join in a small nerve that

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exits the brain through the corpora cardiaca (CC), a neurohemal release site where OALI

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was also found (Figure 2B).

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Extensive neuropile and 20-30 neuroendocrine cells were IR in the SOG (Figure

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2C-D). There were strongly stained bilaterally paired cells in the lateral margins of the

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SOG, all of them sending anterior projections. The strongest stained were a pair of NSCs

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was situated at the posterior end, next to the connection with the prothoracic ganglion

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(PRO). Ventral view revealed OALI neuropile and neuronal bodies in the medial part

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(Figure 2C). Dorsally, OALI neuropile traversed the SOG in an anteroposterior direction;

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the stained projections formed a cross in the midline. Neuronal bodies sending anterior

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projections were observed in the dorsal-lateral margins (Figure 2D). Two pairs of axonal processes could be traced from the anterior end of the SOG to

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the mesothoracic ganglionic mass (MTGM), traversing the PRO. Strongly stained bilateral

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cells were present in the anterior and in the posterior margins of the PRO (Figure 2E). A

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group of cells in the anterior margin sent posterior projections, whereas cells in the

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posterior margin sent processes to the anterior side. Extensive IR neuropile and neurons

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were observed throughout all the structure, both in ventral and in dorsal view. The

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MTGM contained approximately 80 OALI cells (Figure 2F). Extensive IR neuropile was

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present throughout all the structure. Axonal tracts could be traced running transversally in

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the middle line, from PRO to the posterior margin of the MTGM. The posterior lateral

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NSC groups contained 5 bilaterally paired strongly stained cells, sending anterior

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projections to a group of unpaired medial moderately stained neurons and posterior

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projections to abdominal nerves (ABN) 1 and 2. These NSCs contain neuropeptides that

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are known to regulate diuresis (Coast 2009). OALI positive projections were also

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observed in mesothoracic nerves 2 and 3 (Figure 2G).

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IHC in reproductive organs revealed OALI in cells over the common oviduct and

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testes, and processes associated with the two former structures and the seminal vessel

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(Figure 2H-L). The other organs and tissues analyzed (anterior midgut, posterior midgut,

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rectum, salivary glands, fat body, and Malpighian tubules) were positive neither for cells

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nor for neuropili .

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3.2.2 OKB/C-like immunoreactivity (OBCLI) OBCLI in brain and ganglia was more restricted than OALI (Figure 3), as has been observed in D. melanogaster (Chen, Choi et al. 2015). The main places of OBCLI were

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optic lobes (Figure 3B). Posterior and lateral margins of the brain presented scattered

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OBCLI neurons (Figure 3B). OBCLI was also found in the CC (Figure 3C). Only one

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unpaired neuroendocrine cell was intensely stained in the SOG (Figure 3C). This cell was

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located in the posterior lateral margin, sending an anterior projection (Figure 3C). Even

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though this expression pattern is not frequent for neuropeptides, an unpaired OKB

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expressing cell was also found for DromeOKB in the abdominal ganglion (Chen, Choi et

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al. 2015). In the PRO, two paired neurons were stained at the posterior lateral margins,

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sending anterior projections (Figure 3D). In the MTGM 16 paired cells presented intense

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OBLI. From these, one pair was in the anterior margin, one pair was medial-lateral, and

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one pair was in the posterior end (Figure 3E). The five neurons of the posterior lateral

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NSC groups stained strongly, and sent projections to the ABN 1 and 2 (Figure 3E-F).

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OBCLI was observed in endocrine-like cells distributed throughout the anterior midgut (Figure 3G), and in a few cells scattered in the posterior midgut (Figure 3H).

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These cells appear to be of the open type with processes extending towards the midgut

324

lumen. This pattern of expression is consistent with previous reports in D. melanogaster

325

(Veenstra and Ida 2014, Chen, Choi et al. 2015) and differs from T. castaneum, where

326

OKB (and OKA) are detected in the posterior but not in the anterior midgut (Jiang, Kim

327

et al. 2015).

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Cells positively stained for OBCLI were observed in ovaries (Figure 3I).

329

Processes were associated with seminal vessel (Figure 3J), and a few IR cells were

330

detected in testes (Figure 3K). Rectum, salivary glands, fat body, and Malpighian tubules

331

were negative for OBCLI.

332

3.3 Disruption of RhoprOKs by in vivo RNAi affects ecdysis

333

Insects were treated with a dsRNA spanning a region common to RhoprOKA and

334

RhoprOKB/C precursors (dsRhoprOKs), in order to silence all these transcript isoforms.

ACCEPTED MANUSCRIPT We included an experimental group injected with both dsRNAOKA and dsRNAOKB/C

336

(2 µg each, diluted in 2 µl saline solution), to discard off-target effects. Controls were

337

equivalently treated with either water (control water) or dsRNAAmpi (unrelated gene;

338

control Ampi). Forty-eight hours after injection, the insects were allowed to feed ad

339

libitum.

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As a consequence of the treatments, the mRNA levels of RhoprOKA and

RhoprOKB/C were dramatically reduced for dsRhoprOKs (85% and 92%, respectively) as

342

well as for dsRhoprOKA+dsRhoprOKB injected insects (99.5% and 84%, respectively)

343

(Figure 4).

The insects belonging to the different experimental groups did not show

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phenotypic differences until the expected ecdysis time (12-13 days post blood meal;

346

PBM). At this time, the insects belonging to dsAmpi (N=20) and water (N=39) control

347

groups molted to normal 5th instar larvae. Conversely, those insects treated with dsOKs

348

(n=34) or with a conjunct injection of dsOKA+dsOKB (n=18) did not molt at the

349

expected time; 100% of them stopped any movement and died 1-2 days later (Table 2;

350

Figure 5). Treated insects did not reach the stage when they fold their heads, the first step

351

of the ecdysis sequence in R. prolixus (DoLee et al. 2013), which would suggest that they

352

are arrested before ecdysis initiation.

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The larvae treated with dsOKs or dsOKA+dsOKB showed duplicated and

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superimposed ectodermally derived structures, such as the proboscis (Figure 5A).

355

Furthermore, the manual removal of the cuticle revealed that the new cuticle was already

356

sclerotized and tanned under the old one (Figure 5B), indicating that the 5th larva was

357

already formed but failed to shed its old cuticle.

358

To test whether the effects of RhoprOKs on ecdysis were restricted to the larval

359

molt or were also necessary in the imaginal molt, we repeated the experiment using 5th

360

(last) instar insects. As was observed for 4th instar larvae, all the insects normally survived

ACCEPTED MANUSCRIPT 361

until the expected ecdysis time. The individuals belonging to the control group normally

362

molted to adults (Table 2), whereas 24 to 48 h later 100% of dsOKs treated larvae stopped

363

moving and finally died (Table 2). All the treated insects showed the opened ecdysis line,

364

but ecdysis was aborted at this stage (Figure 5C-D). Once we analyzed the effects of the simultaneous depletion of both RhoprOK types

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on ecdysis, we aimed to determine the involvement of isoforms RhoprOKA and

367

RhoprOKB/C separately. We therefore used dsRNA fragments specifically targeting the

368

RhoprOKA or RhoprOKB/C precursors transcripts. Injections provoked a marked

369

isoform-specific depletion of both precursors (>99%) (Figure 4 E-H). Interestingly, all the

370

dsOKA treated insects died 1-2 days after the expected ecdysis (Table 2). On the other

371

hand, all the dsOKB injected animals reached a successful molt, albeit with a significant

372

delay compared to the control group (14.5 days PBM) (p<0.001; n=13) (Table 2).

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3.5. Disruption of RhoprOKs by in vivo RNAi does not affect ecdysteroidogenesis We injected 20E to 4th instar larvae from dsOKs treated and control groups, to

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study whether this hormone would rescue the ecdysis for treated insects. The different

376

experimental groups were injected with either water or 5 µg/10 µg of 20E. Every

377

individual insect received two identical injections, one on day 4 and a second on day 6

378

PBM. While all the insects from the control group molted to 5th instar at the expected

379

time PBM, none of the insects from the dsOKs group underwent ecdysis. All of them died

380

between days 12 and 14 PBM (Table 2). The molt occurred significantly earlier in control

381

insects injected with 10 µg of 20E (around day 12 PBM) compared to the control water

382

injected group (around day 13.5 PBM) (p<0.05, n=7-10). In parallel, dsOKs+10 µg 20E

383

injected animals died around day 12 PBM, significantly earlier than the dsOKs+water

384

injected group, which died around day 13 PBM (p<0.05, n=13). The former could be

385

explained given that RNAi treatments did not prevent molting to be initiated, but

386

prevented shedding of the old cuticle as expected to occur during ecdysis. Ecdysteroid

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ACCEPTED MANUSCRIPT 387

injection caused the earlier initiation of the molting process. Therefore, the ecdysis

388

deficiencies caused by the RNAi treatment manifested earlier.

389

In an indirect way to measure ecdysteroid signaling, we evaluated the expression of RhoprE75 (RPRC000853), a primary response gene encoding a transcription factor,

391

whose expression was demonstrated to be depending on and directly correlating with the

392

concentration of 20E in hemolymph (Mané-Padrós et al. 2008). The qRT-PCRs indicated

393

that RhoprE75 was not differentially expressed in water and dsOKs injected groups (n=9-

394

10) (Figure 6A).

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As a direct way to measure possible failures in ecdysteroidogenesis, we analyzed the 20E levels in the hemolymph of both water and dsOKs treated 4th instar larvae at 2, 4,

397

6, 8, 10 and 12 days PBM. The 20E equivalents in the hemolymph reached a peak around

398

day 6 PBM in both experimental groups, recovering to basal levels at day 12, just before

399

the expected ecdysis time. No significant differences were observed in ecdysteroid

400

concentration throughout all the time points analyzed (Two way ANOVA; n=4-10/

401

group/ time point (Figure 6B).

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3.5. RhoprOKA and RhoprOKB/C are upregulated just before ecdysis The expression of RhoprOKA and RhoprOKB was measured in 4th instar larvae, in

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the same individuals where the 20E levels were analyzed in the hemolymph. RhoprOKA

405

mRNA levels increased throughout the cycle, reaching a peak on day 12, just before

406

ecdysis (Figure 6C). RhoprOKB levels were 10-20-fold overexpressed on day 12, compared

407

to previous time points (Figure 6D). To test whether the overexpression of RhoprOKs on

408

day 12 PBM could be regulated by the 20E peak on day 6 PBM, we injected unfed 4th

409

instar larvae with either water or 10 µg/2µl 20E. Six days after injection the insects were

410

processed, and the transcript levels for RhoprOKA and RhoprOKB/C were evaluated. We

411

did not find significant differences between the experimental groups (N=10/group).

412

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4. Discussion

ACCEPTED MANUSCRIPT 413

Our results indicate for the first time that RhoprOKs are essential for successful ecdysis in R. prolixus. The experimental evidence obtained here suggests a dramatic

415

involvement of RhoprOKA in controlling ecdysis, whereas the involvement of RhoprOKB

416

appears to be less critical. Even though the exact participation of each RhoprOK isoform

417

in the peptidergic network regulating ecdysis remains to be elucidated, we propose that

418

these molecules are important regulators in this network.

419

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The expression in LCCs is coherent with the observed effects of RhoprOKs on

controlling ecdysis, which is a circadian-regulated behavior. In addition, we observed that

421

the pattern of neurosecretory cells presenting OKB/C-like IR in MTGM is identical to

422

the pattern obtained for RhoprCCAP, a neuropeptide involved in ecdysis in several insect

423

species including R. prolixus (Lee and Lange 2011; Lee, Orchard et al. 2013). The co-

424

expression of both neuropeptides in similar neurosecretory cells reinforces the

425

observations regarding an involvement of RhoprOKs in regulating ecdysis. Furthermore,

426

the expression of CCAP is also elevated during ecdysis in the crab Carcinus maenas, the

427

crayfish Orconectes limosus, and R. prolixus (Phlippen, Webster et al. 2000; Lee, Orchard et

428

al. 2013). Our results show that RhoprOKA and RhoprOKB/C expression levels are

429

elevated just before ecdysis, supporting the hypothesis of a role of these molecules in the

430

regulation of ecdysis behavior, as previously described for CCAP.

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The physiological characterization of OKs in insects is still incipient. This is the first

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432

report involving OK neuropeptides in post-embryonic development. However, the

433

expression of this gene in anterior midgut and adult gonads suggests that this probably is

434

a pleiotropic (neuro) peptide system. The identification of the OK receptor(s), which has

435

not been done identified in any species to date, will be fundamental in the future study of

436

this neuronal/endocrine signaling system, which is now emerging as a central regulator in

437

the physiology of insects.

438

5. Acknowledgments

ACCEPTED MANUSCRIPT 439

We thank Raúl Stariolo and Patricia Lobbia from Centro de Referencia de Vectores, for their generous assistance in the rearing of insects, and to Natalia Esponda for the

441

illustration on Rhodnius prolixus CNS. This work was supported by grants from the Consejo

442

Nacional de Investigaciones Científicas y Técnicas (CONICET Grant PIP 2012-2014 049

443

to S.O), Agencia Nacional de Ciencia y Tecnología (ANPCyT Grant PICT2011-194 and

444

PICT2014-1399 to S.O), the Interuniversity Attraction Poles program (Belgian Science

445

Policy Grant IAP P7/40 to J.VdB), and the International Research Collaboration between

446

Argentina and Flanders (MINCyT and FWO: grant number 1204/VS.045.16N and 1505 to

447

S.O. and J.VdB). S.O. and F.F are investigators of the CONICET. J.P.W is a recipient of a

448

research fellowship from CONICET. I.S is supported by a fellowship from CONICET.

449

M.H. was supported by a PhD fellowship of the IWT (Agency for Innovation by Science

450

and Technology, Flanders - Belgium).

451

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6. References

453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475

Altschul, S. F., W. Gish, et al. (1990). "Basic local alignment search tool." J Mol Biol 215(3): 403-410. Arakane, Y., B. Li, S. Muthukrishnan, R. W. Beeman, K. J. Kramer and Y. Park (2008). "Functional analysis of four neuropeptides, EH, ETH, CCAP and bursicon, and their receptors in adult ecdysis behavior of the red flour beetle, Tribolium castaneum." Mech Dev 125(11-12): 984-995. Brezden, B. L., M. S. Yeoman, D. R. Gardner and P. R. Benjamin (1999). "FMRFamideactivated Ca2+ channels in Lymnaea heart cells are modulated by "SEEPLY," a neuropeptide encoded on the same gene." J Neurophysiol 81(4): 1818-1826. Chen, J., M. S. Choi, A. Mizoguchi, J. A. Veenstra, K. Kang, Y. J. Kim and J. Y. Kwon (2015). "Isoform-specific expression of the neuropeptide orcokinin in Drosophila melanogaster." Peptides 68: 50-57. Coast, G. M. (2009). "Neuroendocrine control of ionic homeostasis in blood-sucking insects." J Exp Biol 212(Pt 3): 378-386. García, E. Feder, D., Gomes J.E., Azambuja, P. (1987). “Effects of precocene and azadirachtin in Rhodnius prolixus: some data on development and reproduction.” Mem. Inst. Oswaldo Cruz 82(3): 67-73. Helfrich-Forster, C., O. T. Shafer, C. Wulbeck, E. Grieshaber, D. Rieger and P. Taghert (2007). "Development and morphology of the clock-gene-expressing lateral neurons of Drosophila melanogaster." J Comp Neurol 500(1): 47-70. Hofer, S., H. Dircksen, P. Tollback and U. Homberg (2005). "Novel insect orcokinins: characterization and neuronal distribution in the brains of selected dicondylian insects." J Comp Neurol 490(1): 57-71.

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Hofer, S. and U. Homberg (2006). "Evidence for a role of orcokinin-related peptides in the circadian clock controlling locomotor activity of the cockroach Leucophaea maderae." J Exp Biol 209(Pt 14): 2794-2803. Jiang, H., H. G. Kim and Y. Park (2015). "Alternatively spliced orcokinin isoforms and their functions in Tribolium castaneum." Insect Biochem Mol Biol. Kim, Y. J., D. Zitnan, K. H. Cho, D. A. Schooley, A. Mizoguchi and M. E. Adams (2006). "Central peptidergic ensembles associated with organization of an innate behavior." Proc Natl Acad Sci U S A 103(38): 14211-14216. Lee, D., I. Orchard and A. B. Lange (2013). "Evidence for a conserved CCAP-signaling pathway controlling ecdysis in a hemimetabolous insect, Rhodnius prolixus." Front Neurosci 7: 207. Maddrell, S. H. (1964). "Excretion in the Blood-Sucking Bug, Rhodnius Prolixus Stal. Ii. The Normal Course of Diuresis and the Effect of Temperature." J Exp Biol 41: 163-176. Maddrell, S. H. (1966). "Nervous control of the mechanical properties of the abdominal wall at feeding in Rhodnius." J Exp Biol 44(1): 59-68. Majerowicz, D., M. Alves-Bezerra, R. Logullo, A. L. Fonseca-de-Souza, J. R. MeyerFernandes, G. R. Braz and K. C. Gondim (2011). "Looking for reference genes for realtime quantitative PCR experiments in Rhodnius prolixus (Hemiptera: Reduviidae)." Insect Mol Biol 20(6): 713-722. Omondi, B. A., J. M. Latorre-Estivalis, I. H. Rocha Oliveira, R. Ignell and M. G. Lorenzo (2015). "Evaluation of reference genes for insect olfaction studies." Parasit Vectors 8: 243. Ons, S., X. Belles and J. L. Maestro (2015). "Orcokinins contribute to the regulation of vitellogenin transcription in the cockroach Blattella germanica." J Insect Physiol. Ons, S., M. Sterkel, L. Diambra, H. Urlaub and R. Rivera-Pomar (2011). "Neuropeptide precursor gene discovery in the Chagas disease vector Rhodnius prolixus." Insect Mol Biol 20(1): 29-44. Petersen, T. N., S. Brunak, G. von Heijne and H. Nielsen (2011). "SignalP 4.0: discriminating signal peptides from transmembrane regions." Nat Methods 8(10): 785-786. Phlippen, M. K., S. G. Webster, J. S. Chung and H. Dircksen (2000). "Ecdysis of decapod crustaceans is associated with a dramatic release of crustacean cardioactive peptide into the haemolymph." J Exp Biol 203(Pt 3): 521-536. Rassi, A., Jr., A. Rassi and J. A. Marin-Neto (2010). "Chagas disease." Lancet 375(9723): 1388-1402. Reischig, T. and M. Stengl (2003). "Ectopic transplantation of the accessory medulla restores circadian locomotor rhythms in arrhythmic cockroaches (Leucophaea maderae)." J Exp Biol 206(Pt 11): 1877-1886. Steel, C. G. and X. Vafopoulou (2006). "Circadian orchestration of developmental hormones in the insect, Rhodnius prolixus." Comp Biochem Physiol A Mol Integr Physiol 144(3): 351-364. Sterkel, M., P. L. Oliveira, H. Urlaub, S. Hernandez-Martinez, R. Rivera-Pomar and S. Ons (2012). "OKB, a novel family of brain-gut neuropeptides from insects." Insect Biochem Mol Biol 42(7): 466-473. Sterkel, M., H. Urlaub, R. Rivera-Pomar and S. Ons (2011). "Functional proteomics of neuropeptidome dynamics during the feeding process of Rhodnius prolixus." J Proteome Res 10(8): 3363-3371. Van Wielendaele, P., S. Dillen, E. Marchal, L. Badisco and J. Vanden Broeck (2012). "CRFlike diuretic hormone negatively affects both feeding and reproduction in the desert locust, Schistocerca gregaria." PLoS One 7(2): e31425. Veenstra, J. A. (2000). "Mono- and dibasic proteolytic cleavage sites in insect neuroendocrine peptide precursors." Arch Insect Biochem Physiol 43(2): 49-63.

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539

FIGURE LEGENDS

540

Figure 1: Structure of RhoprOK gene. (A) Complete nucleotide and amino acid sequence

541

of RhoprOKC transcript. The predicted signal peptide sequence is underlined. The mono

542

and dibasic post-translational cleavage sites are highlighted in black. The RhoprOKC

543

exclusive exon and predicted mature peptides are in boldface. Arrows indicate intron sites.

544

M+H+ for each peptide is indicated. (B) Structure of the orcokinin precursor gene, and the

545

three splicing variants in Rhodnius prolixus. The numbers into each exon (black boxes)

546

indicate its size in nucleotides. Lines represent introns. Exon number is indicated. (C) RT-

547

PCR of RhoprOKC in adult R. prolixus. A fragment of RhoprOKC is PCR amplified in

548

cDNA from anterior midgut. CNS: central nervous system; AM: anterior midgut; PM:

549

posterior midgut; HG: hindgut; OV: ovaries; T: testes; MW: molecular weight marker;

550

RhoprAmpi was PCR amplified from tissues as a positive control to test the quality of the

551

template cDNAs (n=3 biological replicates). Numbers on the right indicate molecular

552

weight in base pairs.

553

Figure 2: Orkokinin A-like immunoreactivity (OALI) in R. prolixus. (A) Illustration

554

representing the most intensely stained neurosecretory cells and processes in brain,

555

subesophageal ganglion (SOG), prothoracic ganglion (PRO), mesothoracic ganglionic

556

mass (MTGM), corpora cardiaca (CC), corpora allata (CA), and abdominal nerves (ABN).

557

(B) Dorsal view of the 5th instar brain. Note the presence of OALI in the CC and lateral

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526 527 528 529 530 531 532 533 534 535 536 537 538

ACCEPTED MANUSCRIPT clock cells (LCCs). (C) Ventral view of the 5th instar brain and SOG. (D) Dorsal view of

559

the 5th instar SOG. (E) 5th instar PTG. (F) 5th instar MTGM. (G) Detail of one of the

560

posterior lateral neurosecretory cell groups in the 5th instar MTGM and ABN. (H) OALI

561

processes are associated with the common oviduct in adults. (I) Positively stained cells

562

from the common oviduct in adults (arrows). (J) Positively stained cells from the adult

563

testes (arrows). (K) OALI processes over the adult testes. (L) OALI processes are

564

associated with the seminal vessel in adults. Bar=100 μm.

565

Figure 3: Orkokinin B-like immunoreactivity (OBLI) R. prolixus. (A) Illustration

566

representing the most intensely stained neurosecretory cells and processes in brain,

567

subesophageal ganglion (SOG), prothoracic ganglion (PRO), mesothoracic ganglionic

568

mass (MTGM), corpora cardiaca (CC), corpora allata (CA), and abdominal nerves (ABN).

569

(B) Dorsal view of the 5th instar brain. Note the presence of OBLI in the lateral clock cells

570

(LCCs). (C) Dorsal view of the 5th instar subesophageal ganglion and corpora cardiaca

571

(arrow). (D) Prothoracic ganglion. (E) Mesothoracic ganglionic mass (MTGM). (E) (F)

572

Detail of one of the posterior-lateral neurosecretory cell groups in the MTGM and

573

abdominal nerves. (G) OBLI in endocrine-like cells from 5th instar anterior midgut

574

(arrows). (H) OBLI in a cell from posterior midgut (arrow). (I) The arrow indicates a

575

group of OBLI positive cells in adult lateral oviduct. (J) OBLI is observable in processes

576

associated with the seminal vessel. (K) An immunoreactive cell present in testes (arrow).

577

Bar=100 μm.

578

Figure 4: Effect of dsRNA injections in/on the expression levels of RhoprOKA and

579

RhoprOKB/C. C: control. n=3-4. (A) dsOK injection effect on the expression of RhoprOKA.

580

(B) dsOK injection effect on the expression of RhoprOKB. (C) dsOKA+dsOKB injection

581

effect on the expression of RhoprOKA. (D) dsOKA+dsOKB injection effect on the

582

expression of RhoprOKB. (E) dsOKA injection effect on the expression of RhoprOKA. (F)

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ACCEPTED MANUSCRIPT dsOKA injection effect on the expression of RhoprOKB. (G) dsOKB injection effect on the

584

expression of RhoprOKA. (H) dsOKB injection effect on the expression of RhoprOKB.

585

Figure 5: RhoprOKs in vivo RNAi in nymphs of R. prolixus. (A) Arrested specimens show

586

the larvae 4th proboscis (black arrow) above the parathe larva 5th proboscis (white arrow).

587

(B) The old cuticle (black arrow) was manually removed in a dsOK treated 4th larva that

588

died at the time of ecdysis. The new cuticle (white arrow, surrounded by a dotted line) is

589

hardened and tanned under the old one. (C) Fifth instar larvae belonging to the control

590

group molt at the expected time, showing the normal adult appearance. (D) Fifth instar

591

larvae belonging to the dsOK group did not undergo ecdysis and died as a parathe adult. A

592

gap is observed in the pronotum region.

593

Figure 6: (A) RhoprE75 expression levels were evaluated in control and dsOK treated 4th

594

instar larvae. (B) Ecdysteroid levels were evaluated by EIA in hemolymph samples from

595

4th larvae R. prolixus belonging to control and dsOKs groups. Hemolymph samples were

596

taken from insects at different times post blood meal (PBM) (n=4-10/group/time point).

597

(C) RhoprOKA mRNA levels were evaluated by qRT-PCR in 4th instar larvae at different

598

times PBM. (D) RhoprOKB/C mRNA levels were evaluated by qRT-PCR in 4th instar

599

larvae at different times PBM.

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GenRacerTM 3’ Nested Primer RhoprACTIN Fwd RhoprACTIN Rev qPCR_OKA Fwd qPCR_OKA Rev qPCR_OKB Fwd qPCR_OKB Rev qPCR_E75a Fwd qPCR_E75a Rev qPCR_RpEF-1 Fwd qPCR_RpEF-1 Rev qPCR_TUB Fwd qPCR_TUB Rev dsOKA Fwd dsOKA Rev dsOKB Fwd dsOKB/C Rev dsOKs Fwd dsOKs Rev dsAmpi Fwd dsAmpi Rev T7-Promotor Primer Oligo dT

5'-3' Sequence TGCGGCTAGAGTTAAGAGGA TGACATATTTAAAAGCGCAGCA GATGATCAACATGCTGTCGT GCTGTCAACGATACGCTACGTAACG CGCTACGTAACGGCATGACAGTG ACACCCAGTTTTGCTTACGG GTTCGGCTGTGGTGATGA GATGATCAACATGCTGTCGT AGGTTTGGATGAGCCCTGAGT GAGCGAAATTATGGACCAGCA ATGACCTCCACCCAAACCAT AGAGGGAGTCAATACATAGTGGA AATCTCCGGATCTGTCAGCC GATTCCACTGAACCGCCTTA GCCGGGTTATATCCGATTTT TGTGCCCAAGGATGTGAACG CACAGTGGGTGGTTGGTAGTTGAT TAATACGACTCACTATAGGGGAAGCGGTTTTGATGGTTTTGT TAATACGACTCACTATAGGGGGGATTCTTTGCATAAATGGTCA TAATACGACTCACTATAGGGTAGACGGTGTTATCGTAGAG TAATACGACTCACTATAGGGAATGACCTCCACCCAAACCAT TAATACGACTCACTATAGGGTCACTATCGCTGCGTCTCTGT TAATACGACTCACTATAGGGCTAAAGTATCCAAATTTCGGCCCTC TAATACGACTCACTATAGGGGAACTGGATCTCAACAG TAATACGACTCACTATAGGGGGATCTTCACCTAGATC ATAGAATTCTCTCTAGAAGCTTAATACGACTCACTATAGGG TTTTTTTTTTTTTTTTTTTTTTTT

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Sequence of the primers used for qRT-PCR Primer name PCR_OKC Fwd PCR_OKC Rev PCR_OKA/B fwd GenRacerTM 3’ Primer

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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT Table 2: Effect of experimental treatments in Rhodnius prolixus nymphs ecdysis and mortality Larval stage

Experimental treatment water

Phenotype obtained Molting (days PBM) Mortality (days PBM)

n

4th

100% (12,3 +/- 0.63)

39

th

-

dsAmpi

4

100% (12.9 +/- 0.42)

-

20

dsOKs

4th

-

100% (13.6 +/- 0.14)

34

dsOKA+dsOKB/C

4th

-

100% (14.67 +/- 0.47)

18

dsOKA

4th

-

100% (14.18 +/- 0.37)

dsOKB

4th

100% (14.46+/-0.63)****

water

5th

100% (19.4 +/- 0.35)

-

dsOKs

5th

-

100% (21.5 +/- 1.77)

6

water + water (2x2µl)

4th

100% (13.5 +/- 0.62)

-

10

water+20E (2x5 µg)

4th

100% (13.0 +/- 0.37)

-

5

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th

17 13

water+20E (2x10 µg)

4

100% (12.3 +/- 0.35)*

-

dsOKs + water (2x2µl)

4th

-

100%(13.2 +/- 0.7)

5

7

13

AC C

EP

TE D

M AN U

SC

4th dsOKs+20E (2x5 µg) 100% (12.5 +/- 0.49) 11 4th 100% (12.2 +/- 0.45)& dsOKs+20E (2x10 µg) 13 ****= p<0.001 respect to water injected; *=p<0.05 respect to water + water injected; &=p<0.05 respect to dsOKs + water injected

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

ct to dsOKs + water injected

tcactatcgctgcgtctctgttagcgtctcggacatttcattggtccccatatttctacc atcatcgaggagcgaatcaaacatcccccctcttttctccaacgtatatatactcaatcc aaatttcgttttcaagcaacaatttgagagagagagagacagacaaccgcaaacaaccat ACCEPTED MANUSCRIPT ccgatgatcaacatgctgtcgttgactattcttgcaatggctgttgcagttacttcggca M I N M L S L T I L A M A V A V T S A

A

tttccaaggggcgagcttggtgtggaggaaggtaatttatatcctggtctttaccgagat F P R G E L G V E E G N L Y P G L Y R D cagacaatggaagataaagagggccgaaatttggatactttagattcattacagaataac Q T M E D K E G R N L D T L D S L Q N N

RI PT

agattaaatgcggctagagttaagaggaattcaccagaaattcaaaaaagcaattggtca R L N A A R V K R N S P E I Q K S N W S aaacgagacggtgttatcgtagagccaatctatggtctcagacccgataagaaggaaggc K R D G V I V E P I Y G L R P D K K E G ggcacattggacagtcttggaggcggacatcttattagaaatctaaaagaaaacgaagac G T L D S L G G G H L I R N L K E N E D

SC

aattacgaacatgaaattaaaaaagaagctttaaaaaccaaatttcatacagatacaagt N Y E H E I K K E A L K T K F H T D T S

M AN U

aaagcgtttttaccaaacaaattgtcaaatacgaatcattttatttctaaaagtttagaa K A F L P N K L S N T N H F I S K S L E agtgttaatggtaggagcgaaattatggaccagcatttgttaagaaacttggtgtatggt S V N G R S E I M D Q H L L R N L V Y G aaaggaagacgcttcaaacgtgatttcctggatggtttgggtggaggtcatttgctaaga K G R R F K R D F L D G L G G G H L L R gataatattgatccatttgtggatgtccatcctgtgaaaggaacgagtaaaattccatat D N I D P F V D V H P V K G T S K I P Y

TE D

acaggcccaatatcttcagacggtgatcaacaggtgtctgtcaatttttcaaatggtaga T G P I S S D G D Q Q V S V N F S N G R cattttgtaagagaatttttagacccattaggaggtggacatttaatcagaggaatagat H F V R E F L D P L G G G H L I R G I D

EP

tccatagggggtggtcatttattaagaggattagatgctaaagagaatggaaatttagga S I G G G H L L R G L D A K E N G N L G

AC C

aaagaacttacggacggatttagaagtggttattccattagtggaggcccattagtaaga K E L T D G F R S G Y S I S G G P L V R gaatttttggatccattaggtggaggtcatttaatcagaggattagattcaaaaagtggt E F L D P L G G G H L I R G L D S K S G gatcattttaagagagaatttttagatccacttggaggtggtcatttaataagaggttta D H F K R E F L D P L G G G H L I R G L gattccattggcggtggtcatttagtgagggaatttttggatccattaggtggaggtcat D S I G G G H L V R E F L D P L G G G H ttaatcagaggattagattccaaaggaggagatcattttaagagagaatttttagatcca L I R G L D S K G G D H F K R E F L D P cttggaggcggtcatttaataagaggtttagattccattggtggtggtcatttagtgagg L G G G H L I R G L D S I G G G H L V R gagtttttggatccattaggaggtggtcatttaatcagaggattagattctgaaggtgac E F L D P L G G G H L I R G L D S E G D agtcatccttaggagatcattagaaaattaatcaggacaatgtaatttaagactccatta S H P - aaaggctttttgggttcttaagagggagatatcttcttagaaataattaaatatagattg aacctaactaaaaaccaactataatagcttttaatgcattattttccataacaatatcat

tatgacatatttaaaagcgcagcatttctggctacctcaaaaaaactgaaattaaatctc agaatttaagatttccatgtgtttttttatttacttaaaaattttatgcattcatacaaa ttatatatatttttttccatatattgtagctcattagtaataatataataataaaaccgc ACCEPTED MANUSCRIPT ttt

Predicted mature peptides encoded absents in RhoprOKA and RhoprOKB isoforms: RhoprOKC1: NLDTLDSLQNNRLNAARV

(2027.06)

RhoprOKC2: NSPEIQKSNWS (1289.61)

RI PT

RhoprOKC3: DGVIVEPIYGLRPD (1542.82)

AC C

EP

TE D

M AN U

SC

B

C

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT

Highlights RhoprOKC is a new isoform of RhoprOK gene > the expression pattern of the three isoforms of the gene was studied in central nervous system and periphery > RNAi showed

RI PT

that RhoprOKA peptides are key regulator of ecdysis in Rhodnius prolixus > RhoprOKB type peptide are also involved in regulating ecdysis but less critically than RhoprOKA>

AC C

EP

TE D

M AN U

SC

RhoprOKs does not regulate ecdysteroidogenesis