Identification of RNAi-related genes and transgenerational efficiency of RNAi in Artemia franciscana

Accepted Manuscript RNA interference in Artemia franciscana

Dung Nguyen Viet, Olivier Christiaens, Duy V.B. Le, Stephanie De Vos, Thomas H. MacRae, Guy Smagghe, Peter Bossier PII: DOI: Reference:

S0044-8486(17)32601-7 doi:10.1016/j.aquaculture.2018.09.042 AQUA 633566

To appear in:

aquaculture

Received date: Revised date: Accepted date:

31 December 2017 21 August 2018 18 September 2018

Please cite this article as: Dung Nguyen Viet, Olivier Christiaens, Duy V.B. Le, Stephanie De Vos, Thomas H. MacRae, Guy Smagghe, Peter Bossier , RNA interference in Artemia franciscana. Aqua (2018), doi:10.1016/j.aquaculture.2018.09.042

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ACCEPTED MANUSCRIPT RNA interference in Artemia franciscana

Dung Nguyen Vieta,b, Olivier Christiaensc, Duy V.B. Lea , Stephanie De Vosa , Thomas H. MacRaed, Guy Smagghec, Peter Bossiera,* [email protected] a

Laboratory of Aquaculture & Artemia Reference Center, Ghent University, Ghent 9000,

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Belgium b

Research Institute for Aquaculture No.2, Ho Chi Minh City 7000, Vietnam

Department of Crop Protection, Faculty of Bioscience Engineering, Ghent University,

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c

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Coupure Links 653, 9000 Ghent, Belgium d

*

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Department of Biology, Dalhousie University, Halifax, Nova Scotia, B3H 4R2, Canada Corresponding author at: Laboratory of Aquaculture & Artemia Reference Center, Ghent

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University, Belgium.

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Abstract

Using a transcriptome database, nine RNA interference (RNAi) core genes were identified

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and characterized in the crustacean Artemia franciscana, including two Dicer, three Argonaute, two dsRNA binding protein, a Drosha and a Sid-1 transcript, together with

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evidence of a Pasha and Exportin-5 transcript. Cloning, sequencing and phylogenetic analysis confirmed the identity of these genes and revealed that genes homologous to those in both the siRNA and miRNA pathway in arthropods are present in A. franciscana. Interestingly, we found that a known cofactor of Dicer in arthropods, namely R2D2, is not present in A. franciscana. However, we found a new dsRNA-binding protein which was not yet identified . This study provides the first look at the core genes of the RNAi machinery of A. franciscana. Additionally, egg-sac microinjection in the female was used to elicit a successful RNAi response in offspring nauplii. Injecting dsRNA targeting the caudal gene

ACCEPTED MANUSCRIPT into the female egg-sac led to a successful RNAi phenotype in 93-96% of nauplii in the first two broods, characterized by a shortened abdomen, and quantitative PCR analysis showed that transcript silencing in the second brood was ~93%. Efficient RNAi silencing could be found in nauplii broods released up to 17 days after injection of the female. This demonstrates the reliability of the egg-sac microinjection method, providing a useful tool for

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gene function studies during early development of this model crustacean species.

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Keywords: RNA interference, RNAi, Artemia franciscana, dsRNA.

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1. Introduction

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RNA interference (RNAi) is used extensively in research, applied as a functional genomic tool. It is a biological process in which the expression of a specific gene is silenced post-

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transcriptionally by mRNA degradation after the introduction of gene-specific doublestranded RNA (dsRNA) into a cell or organism (Fire et al., 1998; Marc et al., 2010; Meister,

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Tuschl, 2004). To activate RNAi, dsRNAs are introduced into experimental animals by strategies such as injection, immersion in dsRNA solution, or uptake through the digestive

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tract. One of the natural functions of RNAi is to protect cells against foreign genetic material coming into the cell, for example from viruses (Labreuche et al., 2010). Two parallel and

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closely related RNA pathways exist in arthropods: the siRNA (small interfering RNA) and miRNA (micro-RNA) pathways. In Drosophila melanogaster, Dicer-1, Loquacious and Ago1 are involved in the miRNA pathway, whereas Dicer-2, R2D2 and Ago-2 function in the siRNAi pathway. A number of other genes are involved in RNAi, such as Drosha, Pasha and Exportin-5, which process and transport miRNAs from the nucleus to the cytoplasm (Bohnsack, 2004; Denli et al., 2004). The miRNAs are small noncoding RNAs that function as regulators of gene expression. Furthermore, in Caenorhabditis elegans, systemic RNA interference deficiency-2 (SID-2 is responsible for the cellular uptake of dsRNA, and

ACCEPTED MANUSCRIPT systemic RNA interference deficiency-1 (SID-1), mediates dsRNA transport from cell to cell which spreads the signals needed to activate RNAi in target tissues. Next to its commercial value in fish and shrimp aquaculture, Artemia sp. is used as a model organism to study biological processes in crustaceans. In this context, RNAi has been used to

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examine stress resistance and diapause (Liu et al., 2009), cell division (Chen et al., 2016), development and differentiation (Copf et al., 2004), and reproduction (Dai et al., 2010). Up to

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now, only microinjection has been used to deliver dsRNA into Artemia. The microinjection

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technique allows the study of genes during embryonic development (Dai et al., 2008), to obtain eggs with a specific phenotype (Dai et al., 2011; King, MacRae, 2012; Li et al., 2012;

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Liu et al., 2009; Zhao et al., 2012), and to study larvae development (Copf et al., 2004). In this study, RNAi core genes were identified in an A. franciscana transcriptome database

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developed at Ghent University. Subsequently, we performed an RNAi bioassay for the caudal gene which is a homeo box transcription factor, by injecting females in the egg-sac. Our

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objective was to investigate if there was a transgenerational RNAi, meaning that successful

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gene knockdown in the offspring could be obtained by delivery of the dsRNA to the mother. Successful RNAi was evaluated by scoring the number of nauplii with a shortened abdomen (Copf et al., 2004). Our data are useful in basic research of Crustaceans and in therapeutic

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applications, such as increasing the resistance of crustaceans to viral diseases in the offspring and over different generations. 2. Material and methods

2.1 Culture of A. franciscana A. franciscana cysts used in this study were obtained from the Artemia Reference Center of Ghent University. Cysts were incubated at 28 o C with strong aeration in autoclaved natural sea water (salinity 28 – 30 ppt) until hatching. The harvested larvae were cultured and maintained

ACCEPTED MANUSCRIPT at 28o C with aeration in autoclaved sea water under fluorescent light (300 lux, 24/24 hours) and fed with green alga, Tetraselmis suecica. 2.2. Identification of RNAi core genes in Artemia franciscana 2.2.1 Candidate RNAi core genes

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Amino acid sequences of homologues for the RNAi core genes were used as reference sequences to search for contigs in the A. franciscana transcriptome database at Ghent

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University. The selected contig sequences were subsequently used in BLASTX searches to

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verify their identity with known proteins in GenBank. The contigs showing a match to RNAi

cDNAs.

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2.2.2 RNA extraction and cDNA synthesis

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core genes were used to design PCR primers for the preparation of their corresponding

A pool of A. franciscana consisting of life history stages from 5 day larvae to adults was used

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to extract total RNA using the RNeasy mini kit (Qiagen, Hilden, Germany). The total RNA (3 g) was used to synthesize cDNA in a 20 µL reaction using an oligo-dT primer and the

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RevertAid H Minus First Strand cDNA Synthesis Kit (Thermo Science, Waltham, Massachusetts, USA). The cDNA was then used as PCR template in subsequent steps.

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2.2.3 Amplification of RNAi core genes The polymerase chain reaction (PCR) primers were designed to amplify the longest possible cDNA corresponding to the RNAi core gene of interest. The conditions used in the first PCR were applied for all genes in this study with the PCR reaction containing 2.0 L cDNA, 5 L reaction buffer, 1.0 L dNTPs (10 mM), 0.5 mM forward and reverse primers (first primer, table 1) and 0.25 L DreamTaq DNA polymerase (Thermo Fisher Scientific, Waltham, Massachusetts, USA). The PCR reaction was carried out in a thermal cycler (2720 Thermal Cycler, Applied Biosystems, Foster, California USA) programmed for 94 C for 4 minutes,

ACCEPTED MANUSCRIPT 40 cycles at 94 C for 30 seconds, 55 C for 30 seconds, and 72 C for 5 minutes; followed by 72 C for 10 minutes. The amplicons were then separated in agarose gels, cleaned using Wizard® SV Gel and PCR Clean-Up System (Promega, Madison, Wisconsin, USA) and sequenced by the Sanger method using the same PCR primers. For long amplicons, the complete sequence was obtained by repeated sequencing with walking primers. The cDNA

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sequence obtained for each gene was compiled with Vector NTI software (Invitrogen,

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Waltham, Massachusetts, USA), while the open reading frame was predicted by ORF Finder

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from NCBI. Missing 5’UTR and 3’UTR sequences were obtained by Rapid amplification of cDNA ends (RACE).

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2.2.4 RACE of cDNA ends

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Based on the sequences that were obtained from the first PCR, gene specific primers (Table 1) were designed to amplify the UTR of each gene using the SMARTer RACE Kit (Clontech, Mountain View, California, USA). The PCR thermal cycle to amplify the UTR was

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denaturation for 3 minutes at 95 C, followed by 35 cycles of amplification (30 seconds at 95

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C, 30 seconds at 55 C. 3 minutes at 72 C). The reaction was extended for 10 minutes at 72 C and then cooled to 4 C. The UTR PCR products were purified from agarose gels using

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Wizard® SV gel and PCR Clean-up System (Promega, Madison, Wisconsin, USA) and then ligated into the pGEM®-T easy vector (Promega, Madison, Wisconsin, USA) using T4 ligase (LigaFast™ Rapid DNA Ligation System, Promega, Madison, Wisconsin, USA). The recombinant vectors were transformed into TOP10 competent E. coli (Thermo Fisher Scientific, Waltham, Massachusetts, USA). Recombinant vectors bearing the UTR sequence from transformants were purified with the Wizard® Plus SV Minipreps DNA Purification System kit (Promega, Madison, Wisconsin, USA) and both strands were sequenced using T7 and SP6 primers (Promega, Madison, Wisconsin, USA). The full length UTR sequence was assembled by Vector NTI software.

ACCEPTED MANUSCRIPT 2.2.5 Signal peptide, domain prediction and phylogenetic analysis The domain architecture of the proteins identified in this study was analyzed by Scan Prosite (http://prosite.expasy.org/prosite.html) (De Castro et al., 2006). The signal peptide sequence was predicted by the SignalP 4.1 Server (http://www.cbs.dtu.dk/services/SignalP). The phylogeny tree was generated from http://www.phylogeny.fr/ by using PhyML, a phylogeny

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software based on the maximum-likelihood principle (Guindon et al., 2010), where the usual

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bootstrapping procedure is replaced by a fast approximate likelihood ratio test (aLRT)

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(Anisimova et al., 2006). The polypeptide sequences derived from the isolated transcripts and

2.3 RNA interference in A. franciscana

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2.3.1 Microinjection of dsRNA

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known homologs from NCBI were introduced under FASTA format.

The dsRNA caudal template was produced by PCR using a cDNA template and caudal-

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specific primers (ArCad-for ATCTGTATATACAACCCGATACTTG and ArCad-rev CAA CAGACCTATAACAAGAG) with a T7 promoter sequence (TAATACGACTCACTA

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TAGGGAGA) added to the 5’ end of each primer. After cleaning with a PCR cleanup kit (Promega, Madison, Wisconsin, USA), the PCR product was employed for in vitro dsRNA

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synthesis using the MEGAscript RNAi kit (Ambion, Waltham, Massachusetts, USA). The quantity and quality of the dsRNA were respectively determined by NanoDrop and agarose gel electrophoresis. The dsRNA was stored at -20 °C prior to further use. For the negative control, dsRNA which is specific to green fluorescent protein gene (gfp) was used. The protocol for dsRNA-gfp production was the same as the caudal dsRNA. Specific primers for gfp

were

GFP355F

AGGAGCGCACCATCTTCTTC.

CTGATCGCGCTTCTCG

and

GFP355R

ACCEPTED MANUSCRIPT dsRNA for caudal and gfp were prepared for injection by dilution to 320 ng/µL in 0.5% phenol red in Dulbecco’s phosphate buffered saline (Sigma-Aldrich, Missouri, USA). Just before the injection, adult females were placed on a 2% agarose gel and excess seawater was removed using tissue paper. Approximately, 250 nL of the dsRNA solution was injected into the egg-sac. Injection was executed with a Nanoject II microinjector (Drummond Scientific

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Co., Broomall, PA, USA) using a micropipette prepared with a preset program 45 on a P-97

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Flaming/Brown Micropipette Puller (Sutter Instrument Co., Novato, CA, USA). Forty

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females were used per treatment and after injection they were kept in 6-well plates. Only animals which still had a red color two hours after injection (indicating absence of leaking of

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the dsRNA solution) were used for mating in two days, due to the fact that injected females needed time to heal the wound at the injection site. Each couple was reared separately in 10

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ml sea water using a 6-well plate, and fresh sea water with algae added every day. The RNAi phenotypes of released nauplii were examined periodically with a stereoscope for evaluating

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of RNAi effectiveness.

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2.3.2 Real time PCR

To confirm the success of RNAi silencing, caudal mRNA was quantified by RT-qPCR using

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gene specific primers for caudal and tubulin, the latter as reference gene (King et al., 2013). To confirm the qPCR efficiency, standard curves for caudal and tubulin amplification were prepared by serial dilution of known copy numbers of PCR product. The qPCR was used as a tool to double check that caudal mRNA was downregulated by silencing in nauplii from the second brood. The pooled nauplii of three different females, considered as three biological samples, were separately used for RNA extraction and cDNA preparation. Two replications were used in the qPCR experiment for each cDNA sample. Briefly, 30-50 nauplii from the same brood were pooled for total RNA extraction using Purelink RNA minikit (Thermo Fisher Scientific, Waltham, Massachusetts, USA). At the last step of RNA purification, 40

ACCEPTED MANUSCRIPT µL nuclease free water was added for elution. cDNA was then prepared with the SuperScript IV First-Strand Synthesis System (Thermo Fisher Scientific, Waltham, Massachusetts, USA) in a reaction of 20 µL using 300 ng total RNA as template. The qPCR was carried out using QuantiFast SYBR Green PCR kit (Qiagen, Hilden, Germany) on Rotor-Gene Q Platform

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(Qiagen, Hilden, Germany). 3. Results

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3.3.1 Dicer

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Two A. franciscana cDNAs amplified by RACE were sequenced. The predicted ORF of these cDNAs encoded polypeptides with several domains corresponding to Dicer. The

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cDNAs were named ArDicer-1 and ArDicer-2 (Fig. 1). The ArDicer-1 cDNA (GenBank

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accession no. KY609157) is 5973 bp in length with an ORF that encodes a polypeptide of 1932 amino acid (aa) residues, while the ArDicer-2 cDNA (GenBank accession no.

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KY609158) is 4875 bp in length, bearing an ORF that encodes a polypeptide of 1518 aa

3.3.2 Argonaute

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residues.

Based on the transcriptome of A. franciscana, specific primers were designed for PCR and

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RACE, resulting in amplification and cloning of three cDNAs named ArAgo-1, ArAgo-2 and ArAgo-3. RACE was used to isolate ArAgo-1 (GenBank accession no. KY609160, 3875 bp) and ArAgo-2 (GenBank accession no. KY609161, 3515 bp), while ArAgo-3 (GenBank accession no. KY661908, 2772 bp) was obtained after the first-round PCR. The sequence analysis showed that a full coding DNA sequence (CDS) was cloned for ArAgo1 and ArAgo2, but for ArAgo-3 only a partial CDS was retrieved. The three ArAgo’s encoded different proteins bearing typical Argonaute domains including one PAZ and one Piwi domain. The phylogenetic tree confirmed that the Argonaute candidates in this study belong to the cluster

ACCEPTED MANUSCRIPT of AGO proteins and did not cluster together with the related and similar Aubergine/Piwi protein clade (data not shown). 3.3.3 dsRNA-binding proteins Searching the A. franciscana transcriptome database for dsRNA binding proteins using

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homologs of the RNA-binding protein (TRBP), Loquacious (Loqs) and R2D2 yielded three cDNA sequences that match TRBP. Two cDNA sequences encoding for proteins containing

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dsRNA binding domains were then isolated. The first protein, ArTRBP-1 (GenBank

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accession no. KY661910), has three double stranded RNA binding domains (dsRBDs) and

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the second protein ArStaufen (GenBank accession no. KY680793) was comprised of four dsRBDs and one Staufen C-terminal domain. In addition, another contig designed as

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ArTRBP-like (GenBank accession no. KY996554) encodes a polypeptide bearing three dsRBDs. The phylogenetic tree was used to identify the ArTRBP-1 and ArTRBP-like

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proteins by comparing them to RNAi-related proteins identified in other species. The ArTRBP-1 is located in the same cluster with TRBP of another shrimp, while the ArTRBP-

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like is located close to the cluster of Staufen (Fig. 2). 3.3.4 Drosha, Pasha and Exportin-5

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An A. franciscana cDNA of 4342 nucleotides, encoding for a protein of 1358 aa, (GenBank accession no. KY609159) which contained two RNase III domains and a dsRNA-binding domain at the C-terminal was isolated and named ArDrosha. Using Pasha from L. vannamei as a homologous sequence in the Blast search, a transcript was found in the A. franciscana transcriptome database that encoded a polypeptide of 589 aa and contained the amino acid sequence

“SYICILHEYTQHIIRKLPKYEFSELENSSSPYGCTISIDNVKYGAAQGASKA

KLEAAKKALEILIP”, a dsRNA-binding motif found in the Pasha homolog. Another CDS

ACCEPTED MANUSCRIPT that encodes a polypeptide which shares 27% identity with Crassostrea gigas Exportin-5 gene was also found. 3.3.5 Uptake and transport of dsRNA The polypeptide sid-1 of C. elegans was used in a BLAST search of the A. franciscana database

yielding a cDNA termed

ArSid-1

(GenBank

accession no.

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transcriptome

KY661909) encoding for a C-terminal polypeptide of 560 aa bearing a complete systemic

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RNA interference deficiency-1 (SID-1) domain.

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3.4 RNAi in A. franciscana

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Forty females were injected with ArCad dsRNA of which twenty-four survived and released nauplii. The surviving females were divided into three groups based on the number of broods

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of nauplii that they released. The four females in group A released only one brood of nauplii exhibiting an RNAi phenotype and then produced cysts or died. Group B contained thirteen

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females which died or produced cysts after the second brood of nauplii. Of those thirteen females, eleven produced two broods of nauplii exhibiting an RNAi phenotype and two

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females produced only normal nauplii. Group C, containing seven females, produced three broods of nauplii exhibiting RNAi phenotype, after which they died or produced cysts. The

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average sizes of the 1st, 2nd, 3rd broods were respectively about 36, 28 and 25 nauplii. Female A. franciscana in groups A, B and C injected with ArCad dsRNA released 95.81.5% (762/795) nauplii exhibiting RNAi phenotype in the first brood. Similarly, A. franciscana females in groups B and C injected with ArCad dsRNA released 93.41.6% (487/511) nauplii exhibiting RNAi phenotype in the second brood. However, only 2% (5/254) of nauplii in the third brood from group C exhibited RNAi phenotype (Fig. 4). The nauplii exhibiting RNAi phenotype were reared in new sea water and most died after seven days. The two surviving nauplii showed a shortened abdomen and missing appendages. Quantification of

ACCEPTED MANUSCRIPT ArCad mRNA for the second brood nauplii, revealed 93.4% silencing, indicating that RNA knockdown was very effective even in the second brood (Fig. 4). 4. Discussion 4.1 RNAi

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DICER, the first enzyme in the RNAi pathway, binds to dsRNA and cuts it into small

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interfering RNA (siRNAs) before transferring them to Argonaute. DICERs have similar structures and are highly conserved between species, normally consisting of seven main

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domains including the N-terminal ATP-binding Helicase type-1 domain, a second helicase

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domain, double-stranded Dicer binding fold, PAZ, two Ribonuclease III domains and a Cterminal dsRBD (Gao et al., 2014). Invertebrate Dicers can be divided into Dicer-1 or Dicer-

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2 subfamilies (Gao et al., 2014). In Drosophila, DmDicer-1 and DmDicer-2 generate different classes of small RNAs, with DmDicer-1 processing pre-miRNAs originating from

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the nucleolus, while DmDicer-2 processes dsRNAs into siRNAs in the cytoplasm (Lee et al., 2004; Liu et al., 2007). The number of DICERs varies among different organisms. For

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example, four DICERs are present in Arabidopsis thaliana, two in D. melanogaster, and one in C. elegans and Schizosaccharomyces pombe. DICERs usually exhibit conserved domain

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structure across species but some domains were lost during evolution, such as the DEAD domain in DICER-1 of mollusks, annelids, platyhelminths and most arthropods (Gao et al., 2014). In Gardia intestinalis, DICER lacks the N-terminal helicase domain and the dsRBD at its C-terminal (Svobodova et al., 2016). A. franciscana possess one dicer-1 and one dicer-2 gene with domain prediction showing that the C-terminal dsRBD is present in ArDICER-1 but not in ArDICER-2. In human DICER, the dsRBD binds siRNAs and longer dsRNAs with overhangs (Zhang et al., 2004). Crustacean DICER-2 sequences available in the NCBI

ACCEPTED MANUSCRIPT GenBank, including those from L. vannamei (AEB54796.1), M. japonicus (BAM37458.1) and P. monodon (AGL08684.1), lack the dsRBD (data not shown). Argonaute proteins are characterized by PAZ and Piwi domains and they are classified into three

subfamilies: AGO,

PIWI

and

worm-specific

Argonautes

(WAGO) (Carthew,

Sontheimer, 2009). Argonaute family members DmAgo-1 and DmAgo-2, belonging to the

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Ago subfamily, were identified in D. melanogaster. DmAgo-1 is required for mature miRNA

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production in the siRNA pathway, while DmAgo-2 is part of the RISC complex of the siRNA

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pathway (Okamura et al., 2004). Three genes that encode for different Argonaute proteins were identified in A. franciscana. Unlike in insects, some crustaceans are characterized by a

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variable number of AGO subfamily proteins. For instance, three AGO proteins were found in Daphnia pulex (McTaggart et al., 2009) and four in Parhyale hawaiensi (Kao et al., 2016)

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and P. monodon (Leebonoi et al., 2015; Phetrungnapha et al., 2013; Unajak et al., 2006; Yang et al., 2014). Three Ago cDNAs were isolated in L. vannamei using the RACE method. These

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species share a single-copy homolog of Ago-1, and a variable set of multiple copies of Ago-2 homologs. Among the three isolated AGOs of A. franciscana, only ArAGO-1 was found in

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the same cluster with other AGO-1 homologs. Hence, ArAGO-1 of A. franciscana is homologous to AGO-1 homologs in other arthropods, suggesting a role in the miRNA

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pathway. In P. monodon, PmAgo1, PmAgo2 and PmAgo3 are activated by bacterial and viral infection, and involved in dsRNA-mediated gene silencing. PmAgo4 may not play a role in dsRNA-mediated gene silencing or antiviral defense, but it appeared to participate in the control of transposons (Leebonoi et al., 2015). Therefore, the Ago-2 subfamily in crustacean not only silences exogenous RNA but may have other activities during development. ArAgo2 and ArAgo-3 were found in a separate cluster in the phylogenetic tree, which is located inside a bigger crustacean and insect Ago-2 homolog clade, indicating that these Artemia Ago’s belong to the Ago-2 subfamily.

ACCEPTED MANUSCRIPT To recruit Argonaute proteins and form the core of the RNA-induced silencing complex (RISC), DICERs act in cooperation with several proteins which have a dsRBD, such as Loquacious (LOQS) and R2D2 in D. melanogaster (Forstemann et al., 2005; Okamura et al., 2011), or a HIV-1 transactivating response (TAR) RNA-binding protein (TRBP) in humans (Chendrimada et al., 2005; Haase et al., 2005). In D. melanogaster, the LOQS contains three

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dsRBDs with dsRBD1 and dsRBD2 being classical RNA-binding domains which interact

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with dsRNA during DICER processing (Jakob et al., 2016). In contrast, R2D2 has only two

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dsRBDs and it chooses the guide strand of the siRNA duplex to be loaded into the RISC. The DICER-2/R2D2 complex binds to siRNA and enhances sequence-specific messenger RNA

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degradation mediated by the RNA-initiated silencing complex (RISC) (Liu et al., 2006). In humans, TRBP plays a role in the recruitment of the DICER complex to AGO-2

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(Chendrimada et al., 2005). TRBP consists of three dsRBDs (Daviet et al., 2000; Haase et al., 2005). ArTRBP-1 and ArTRBP-like from A. franciscana encode for a polypeptide containing

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three double strand binding protein domains (dsRBDs). The phylogeny analysis showed that ArTRBP-1 is more closely related to the TRBP of crustaceans than the LOQS and R2D2 of

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insects. This result suggests that ArTRBP-1 interacts with ArDICER-2 and plays a similar role as RBP of others shrimp such as L. vannamei and F. chinensis. TRBP-1, DICER-2 and

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AGO-2 of L. vannamei have been proven to interact by co-immunoprecipitation assays and pull-down assays (Chen et al., 2011). Evidence of interaction between TRBP and RISC complex has also been found indirectly in F. chinensis (Wang et al., 2009). So far there is no evidence for a R2D2 homolog in A. franciscana. The absence of R2D2 was reported for the brown shrimp (Crangon crangon) (Christiaens et al., 2015). In this study, the ArTRBP-like of A. franciscana was found to contain three dsRBDs but does not cluster with any TRBP known in other crustaceans (Fig 2.). No protein from other crustaceans was found by Protein BLAST in NCBI with a structure similar to ArTRBP-like. In addition, signal peptide

ACCEPTED MANUSCRIPT prediction confirmed the absence of a signal peptide in the primary polypeptides of ArTRBPlike which suggests that ArTRBP-like plays its role only in the cytoplasm. Given these facts, it is not unlikely that ArTRBP-like could have a similar function as R2D2 in D. melanogaster, interacting with ArDICER-1 to assist in loading of siRNA to the RISC complex. Further research, investigating the interaction between ArTRBP-like and ArDicer-1,

protein

has

diverged

considerably,

or

even

evolved

differently,

in

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dsRNA-binding

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is required to confirm this. If correct, these results suggest that the RISC complex with its

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crustaceans and insects.

The initiation of the siRNA pathway is triggered by extracellular dsRNAs in the cell. These

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dsRNAs are transported or taken up into the cell through systemic RNAi and environmental RNAi mechanisms. Environmental RNAi comprises the uptake of dsRNAs from the

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environment such as the digestive system of shrimp, while cell to cell or tissue to tissue transport of dsRNA is called systemic RNAi. The identification of these proteins allows

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exploration of suitable dsRNA delivery strategies to activate tRNAi. In C. elegans, RNAi signal spreading is coordinated by sid-1 (Feinberg, Hunter, 2003), the intercellular transporter

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of dsRNAs, while sid-2 plays a role in uptake of dsRNA from the environment (McEwan et al., 2012). The sid-1 homolog is considered as a key component in the uptake of dsRNA in

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certain arthropods (Gu, Knipple, 2013), however some insects such as D. melanogaster have no sid-1 homologs. Therefore, receptor-mediated endocytosis most probably plays a role in dsRNA uptake (Saleh et al., 2006). Sid-1 homologs have been identified in crustaceans, such as L. vannamei (Labreuche et al., 2010) and C. crangon (Christiaens et al., 2015), but not in Parhyale hawaiensis (Kao et al., 2016). One copy of a ArSid-1 homolog was found in the A. franciscana transcriptome, which might explain the success of RNAi in this species by using dsRNA injection (Copf et al., 2004). 4.2 Effectiveness and transgenerational RNAi

ACCEPTED MANUSCRIPT The introduction of dsRNA into the body cavity or egg-sac of females by microinjection induces RNAi in oocytes and leads to silencing in the progeny. It is not clear whether the dsRNA directly enters the embryo or the RNAi signal spreads from the female body to the embryo. The Sid-1 gene found in A. franciscana may be involved in the transport of dsRNA and maintenance of the signal for RNAi in the host. In C. elegans, maternal SID-1 transports

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extracellular dsRNA into the germline where it can silence maternally deposited mRNAs and

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segregate to embryos to silence embryonically expressed mRNAs. Extracellular dsRNA is

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also endocytosed into oocytes and it requires SID-1 and SID-5 in the embryo to silence embryonically expressed genes (Hinas et al., 2012; Wang, Hunter, 2017). In Artemia, the

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effectiveness of RNAi is usually evaluated through qPCR or the immunoprobing of western blots. In some cases, the effectiveness of RNAi can be evaluated visually through scoring

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phenotypes of cysts (Dai et al., 2011; Yang et al., 2013) or abnormal embryonic development (Zhao et al., 2012). However, the efficiency of RNAi on living nauplii released from the

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RNAi treated females have not been reported yet. Due to the typical RNAi phenotype at the nauplii stage, the ArCad gene is a good candidate gene to investigate transgenerational RNAi

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from treated A. franciscana adult females to their offspring. In our study, RNAi as evaluated through the RNAi phenotype of released nauplii, demonstrated that ArCad RNAi-treated

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maternal Artemia produced high numbers of nauplii with a strong RNAi phenotype for at least two broods within about 17 days post injection. In contrast, the nauplii exhibited RNAi phenotype numbers in the 3rd brood was significantly reduced. In C. elegans, dsRNA is directly transferred between generations via ingestion of dsRNA by larvae resulting in silencing 100% of the progeny (Marré et al., 2016). In insects such as the flour beetle Tribolium castaneum, the injection of dsRNA into the mother's hemocoel knocks down zygotic genes and nearly 100% of the offspring embryos exhibit RNAi phenotypes (Bucher et al., 2002). Although biochemical evidence for intergenerational transfer of dsRNA was not

ACCEPTED MANUSCRIPT documented in this study, the RNAi phenotype of released nauplii provide circumstantial evidence for intergenerational RNAi transfer, from the females to their progenies, facilitating RNAi research in Artemia. In shrimps, the duration of RNAi has been reported in WSSV challenges of L. vannamei where animals treated with dsRNA targeting viral genes vp26 and vp28 gradually lost the

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antiviral effect 10 days after the initiation of RNAi treatment (Mejía-Ruíz et al., 2011; Nilsen

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et al., 2017). In P. monodon, protection against WSSV lasts 13 days upon RNAi treatment via

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viral-specific dsRNA injection (Westenberg et al., 2005). The presence of the RNAi ArCad phenotype in A. franciscana nauplii suggests that RNAi is sustained for at least up two weeks

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in dsRNA treated females. Additionally, the qPCR of ArCad in released nauplii of the second brood (Fig. 4) and the presence of nauplii exhibited RNAi phenotype (Fig. 3) which could not

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survive beyond day 7 suggested that RNAi is probably happening in nauplii after being released from the females.

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Recently, nauplii released by dsRNA-treated maternal Artemia have been used to study the involvement of HSP70 in protection against pathogenic bacterial (Iryani et al., 2017) or to

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study stress-resistance related proteins such as Ste20-Like kinase (Zhou et al., 2014). The high numbers of nauplii RNAi phenotypes obtained from ArCad dsRNA-treated maternal

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Artemia demonstrated the reliability of the egg-sac injection method. Hence it can be used to produce nauplii for functional studies of developmental genes. The nauplii, which are released within the first 17 days post injection by this method, are suitable for studying different genes, including for example immune system genes and stress resistance genes. 5. Conclusions In this study, RNAi core genes belonging to the RNAi pathways generating miRNA and siRNA pathway were identified in A. franciscana. Previously only one copy of a dsRNAbinding protein homolog was reported in crustaceans, but in this study, two dsRNA-binding

ACCEPTED MANUSCRIPT protein genes were found in A. franciscana. The ArTRBP-like is considered to be a new member of this protein family. RNAi is a component of the immune system in crustaceans, so RNAi core genes can be used in immunological studies on A. franciscana. Egg-sac microinjection was used to prepare a nauplii population with a desired phenotype. High numbers of nauplii with the desired phenotype were released from RNAi-treated

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females in the first 2 broods, whereas in the 3rd brood the RNAi phenotype was mostly lost.

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The RNAi phenotypes in the nauplii suggested that dsRNA molecules are transgenerationally

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transmitted, meaning that successful gene knockdown in the offspring could be obtained by delivery of the dsRNA to the mother. This offers new possibilities in using RNAi to elucidate

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early development processes in this model crustacean species. 6. Acknowledgements

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This work was funded by the Vietnam International Education Development (VIED) and Research Foundation - Flanders (FWO, Belgium). Olivier Christiaens is a recipient of a

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postdoctoral fellowship from the Research Foundation – Flanders (FWO).

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Primer names

Sequences (5’-3’)

F5

CTGGTCCCTTGGGCCCTAGTAC CGACCATTATCAAGAGAATCAGTTCTAAGC

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Gene name

R3

Ago 3

Dicer 1

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AAATGAGGCGGAAATACCGCG TGCTGGTCTACAGCTTGTTGTTGTTG TTGGTGCCTCTTGAGGAGAAGATGC CGTGGGCATTTATCAGGTTGAATGGACACATC CGGTTAGCACTACTTATAAGGGTGAGGATG CACAAAAAAAGCATATCACTATGACGTAGAA GGCAAGAAGGTGAGCATAATAGCTCGGAGC CCACCGGGTACAGTCGTTGACTCCTTG ACGACCTCAAATTCCCTAGCTCTCTCTTCG CTCCCTGAGGTTGCGTAGGTCTAGGTCCCC AGTAATAGTGTTGAGATCGATCTCAGCAGC CCACCGGGTACAGTCGTTGACTCCTTG CAGCCAGGTGCATGGGGACAAGGACCTCAG GACTGGACCATGAGTTTTGACACACACGAC ATGGGAGCTCATACGCAACAATCC TGAGCTGAAATTGTTCTCATCAAAAAGGACAC CGATGCGAAACAGCTAGTGATATTG TCAACTCATTTTCACAAATTGTATGGGATG CAAGCTCTGACAATGTCAAATGC AAGGTTCTGAGACTTTTAGAGATC GATGATTACTGTCATGAAGATGATG CCGAGTCCATCAATCATAGTTAAATTG TCTTTTCGTACTGTGGAAGACAATCTG TCAGTTGGATCTGGCAAGCTATTC GAAAAGTCTCGACGCTGCATCTTGAG

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Ago 2

F2 F4 R4 R5 F8 F2 R3 F3 R5 R6 Ago2.5nRACE Ago2.3nRACE ArAgo2.3URTF ArAgo2.3URTR ArAgo3aF ArAgo3aR Dicer1-F4 Dicer1-3UTR1 Dicer1F1 Dicer1F5 Dicer1F8 Dicer1R3 Dicer1 R5 Dicer1.5nRACE

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Ago 1

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Table 1. The primers used in this study

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resistance to environmental stress. PLoS One. 9, e92234.

F R

ACAAAGAATAGCCAAAGTAAGTAATTTATTGCTCC

Information First PCR First PCR, confirming primer Sequencing Sequencing Sequencing 5’RACE primer Confirm PCR First PCR First PCR Sequencing Sequencing Sequencing 5’RACE primer 3’RACE primer confirm primer confirm primer

First PCR First PCR Sequencing Sequencing Sequencing Sequencing Sequencing 5’RACE primer confirming primer confirming primer

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Sid-1 dsRNA binding protein

StaufenCDS R

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Drosha

CTTATATCCACGAGTGTATTGGAAGAAGGG GAGGGCACTAACGTCATTGAAAGATTGTGG CTACAATAACCCGCGAAAATTTGGCCCTGG GGAATCTAGGTATACAGCACCGCATATAG GACTGCGTCACCAAGGAACTCTAGCCTCTG GGACAGTTAAATCAGGCCAAGCACTGAC CCAGCAACGCCACGTAAGTAGTTTCCATC CCAGCAACGCCACGTAAGTAGTTTCCATC GAACAAATGACGAAAACAGCAGCTTTCGGAC GTCCTTCATCACTAAGAATAGAAGC GTCCATCATTTGGGAGGGATGAGG CATGTACACTTTCCACTTTCGCTTCC GGGATCCATGTTTGAGGTGAAAAG AGCCATACAGGCTGAGTATTTCTC CATCTTTAAGACAATCTTCTTTAAGCTAACG GCCAATAAAGAAATGCAAGGAAGGC

First PCR First PCR Sequencing Sequencing Sequencing Sequencing Sequencing 5’RACE primer First PCR First PCR First PCR First PCR First PCR First PCR First PCR First PCR

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Dicer 2

ArDicer2F1 ArDicer2UTRR Dicer2F2 Dicer2R1 Dicer2R2 Dicer2R3 Dicer2R4 Dicer2.5nRace F1 R1 F R TRBPCDS-F TRBPCDS-R StaufenCDS-F

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Figure 1. The structure of Dicer proteins in Artemia franciscana, named ArDicer1 and ArDicer2. LvDicer-1 and Lvdicer-2 are Dicer proteins from L. vannamei. DMDicer-1 and DMdicer-2 are Dicer

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from D. melanogaster. The domains in order from left to right: ATP-binding helicase type-1 (HELICASE_ATP_BIND_1), C-terminal helicase (HELICASE_CTER), Double-stranded Dicer -binding fold

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(DICER_DSRBF), PAZ, two Ribonuclease III (RNASE_3_2) and Double stranded RNA-binding domain

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(DS_RBD).

Figure 2. The phylogenetic tree of three isolated dsRNA binding proteins from A. franciscana with

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homologs from other shrimp and insect species. The full polypeptide sequences derived from the isolated transcripts and known homologs from NCBI were used for generating the phylogenetic tree using PhyML, a phylogeny software based on the maximum-likelihood principle (Guindon et al.,

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2010). The numbers are the branch support values.

Figure 3. Egg sac microinjection in A. franciscana and the RNAi phenotype in nauplii. Arrows indicate individuals with the ArCad RNAi- phenotype. A: The needle penetrating the egg sac; the pink color indicates that the dsRNA solution was injected successfully. B and C, the ArCad RNAi phenotype. D: the larval phenotype 9 days post-release. There are two small deformed nauplii caused by ArCad RNAi in maternal Artemia and one bigger nauplius with the normal wild type phenotype caused by the gfp control.

Figure 4. mRNA knockdown and abnormal nauplii. A: The qPCR shows different silencing efficiencies

ACCEPTED MANUSCRIPT for ArCad RNAi treatment (orange) relative to control group (blue) (tubulin was used as a reference gene, AU: arbitrary units). Error bars represent the standard deviation. B: The percentage of RNAi phenotype of nauplii in first brood (released at day 8 to 10) and second brood (released from day 14 to 17) is different compared to the third brood (released at day 20 to 21).

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Statement of Relevance This study investigates the functionality of RNA interference in in Artemia franciscana and shows

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that microinjection of dsRNA in the egg-sac of female A. franciscana can lead to successful RNAi

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phenotypes in the offspring. This offers a useful tool to investigate embryonic and developmental processes in this model crustacean species. Furthermore, genes that may be involved in RNAi

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interference, including two ArDicer, three ArAgo, two ArTRBP, and one each of ArDrosha, ArPasha, ArSid-1 and ArExportin-5 were identified in A. franciscana. Interestingly, we found that a known

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cofactor of Dicer in arthropods, namely R2D2, is not present in A. franciscana. However, we found a

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new dsRNA-binding protein which was previously not identified yet.

Highlights

Homologs of the RNA interference core genes belonging to the miRNA and siRNA

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

pathway were identified in the Artemia franciscana transcriptome. 

One dsRNA binding protein, ArTRBP-like, is a new member of the dsRNA binding protein family.



R2D2, which is an important cofactor of Dicer-2 in most insects, was not found in A. franciscana.



The egg-sac microinjection method in female Artemia franciscana was used successfully to obtain a desired RNAi phenotype in offspring nauplii.

Figure 1

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Figure 6