2 and apoptosis in mosquito salivary glandssalivary glands (SGs), from where they are expectorated...

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JNK pathway restricts DENV, ZIKV and CHIKV infection by activating complement 1

and apoptosis in mosquito salivary glands 2

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Avisha Chowdhury,α, Cassandra M. Modahl1,α, Siok Thing Tan1, Benjamin Wong Wei 6

Xiang2, Dorothée Missé3, Thomas Vial2, R. Manjunatha Kini1,* and Julien Francis 7

Pompon2,3,* 8

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1Department of Biological Sciences, National University of Singapore, Singapore 12

2Emerging Infectious Diseases, Duke-NUS Medical School, Singapore 13

3MIVEGEC, IRD, CNRS, Univ. Montpellier, Montpellier, France 14

αAuthors contributed equally 15

*corresponding authors: [email protected]; [email protected] 16

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Key words: dengue, Zika, chikungunya, Aedes aegypti, immunity, salivary glands, JNK 19

pathway 20

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.CC-BY-NC-ND 4.0 International license(which was not certified by peer review) is the author/funder. It is made available under aThe copyright holder for this preprintthis version posted March 3, 2020. . https://doi.org/10.1101/2020.03.01.972026doi: bioRxiv preprint

https://doi.org/10.1101/2020.03.01.972026

http://creativecommons.org/licenses/by-nc-nd/4.0/

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Abstract (150 words) 23

Arbovirus infection of Aedes aegypti salivary glands (SGs) determines transmission. 24

However, there is a dearth of knowledge on SG immunity. Here, we characterized SG 25

immune response to dengue, Zika and chikungunya viruses using high-throughput 26

transcriptomics. The three viruses regulate components of Toll, IMD and JNK pathways. 27

However, silencing of Toll and IMD components showed variable effects on SG infection by 28

each virus. In contrast, regulation of JNK pathway produced consistent responses. Virus 29

infection increased with depletion of component Kayak and decreased with depletion of 30

negative regulator Puckered. Virus-induced JNK pathway regulates complement and 31

apoptosis in SGs via TEP20 and Dronc, respectively. Individual and co-silencing of these 32

genes demonstrate their antiviral effects and that both may function together. Co-silencing 33

either TEP20 or Dronc with Puckered annihilates JNK pathway antiviral effect. We 34

identified and characterized the broad antiviral function of JNK pathway in SGs, expanding 35

the immune arsenal that blocks arbovirus transmission. 36

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Introduction 39

In recent decades, dengue (DENV), Zika (ZIKV) and chikungunya (CHIKV) viruses have 40

emerged as global public health issues, with over 50% of the world population at risk for 41

infection (Kraemer et al., 2019). DENV and ZIKV belong to the Flavivirus genus 42

(Flaviviridae family), while CHIKV belongs to the Alphavirus genus (Togaviridae family). 43

DENV infects an estimated 390 million people yearly, causing a wide range of clinical 44

manifestations from mild fever to shock syndrome and fatal hemorrhage (Bhatt et al., 2013). 45

ZIKV recently emerged as an epidemic virus, infecting 1.5 million people over the past five 46

years (Hamel et al., 2016). Although ZIKV infection is mostly asymptomatic, it can result in 47

life-debilitating neurological disorders including Guillain-Barré syndrome in adults and 48

microcephaly in prenatally-infected newborns. CHIKV emerged as an epidemic virus in 49

2004, and has infected more than 6 million people (Silva & Dermody, 2017). It causes mild 50

fever but can result in musculoskeletal inflammation, leading to long-term polyarthralgia. 51

Amplified by urban growth, climate change and global travel, arboviral outbreaks are 52

unlikely to recede in the near future (Mayer et al., 2017). 53

DENV, ZIKV and CHIKV are primarily transmitted by Aedes aegypti mosquitoes. In 54

the absence of efficient vaccines (Wilder-Smith & Gubler, 2015) and curative drugs(Barrows 55

et al., 2018), targeting this common vector is the best available strategy to control the spread 56

of all three viruses. However, current vector control methods that rely on chemical 57

insecticides are not effective in preventing outbreaks (Wilder-Smith et al., 2017), partly due 58

to insecticide resistance (Liu, 2015). A novel strategy employing Wolbachia to reduce virus 59

transmission by mosquitoes has been deployed as a trial in several countries (Anders et al., 60

2018). However, its long-term efficacy may be compromised by converging bacteria and 61

virus evolution (Ritchie et al., 2018). Other promising approaches utilize genetic engineering 62

technology to develop refractory vector populations (Kistler et al., 2015). Mosquito innate 63


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immunity can drastically reduce virus transmission, and characterization of mosquito immune 64

pathways and mechanisms will aid in identifying gene candidates for transformation. 65

Immunity in midguts, the first organ to be infected following a blood meal, has been 66

extensively studied by using transcriptomics. DENV and ZIKV activate the Toll, Immune 67

Deficiency (IMD) and Janus Kinase (JAK)/Signal Transduction and Activators of 68

Transcription (STAT) immune pathways (Angleró-Rodríguez et al., 2017; Sim et al., 2013; 69

Souza-Neto et al., 2009; Xi et al., 2008). Selective gene silencing studies have demonstrated 70

the anti-DENV impact of Toll and JAK/STAT pathways. However, JAK/STAT transgenic 71

activation was not effective in reducing ZIKV and CHIKV infection (Jupatanakul et al., 72

2017). Immune effectors downstream of these pathways include antimicrobial peptides 73

(AMPs) and thioester containing proteins (TEPs) (Angleró-Rodríguez et al., 2017; 74

Jupatanakul et al., 2017; Ramirez & Dimopoulos, 2010; Sim et al., 2013; Souza-Neto et al., 75

2009). AMPs can have direct antiviral activity (Luplertlop et al., 2011), while TEPs that 76

belong to the complement system tag pathogens for lysis, phagocytosis or melanization 77

(Blandin, 2004; Xiao et al., 2014). Additionally, the RNA interference (RNAi) pathway 78

cleaves viral RNA genomes (Sanchez-Vargas et al., 2004). However, its impact against 79

arbovirus infection is uncertain (Olmo et al., 2018). In Drosophila melanogaster, the Jun-N-80

terminal Kinase (JNK) pathway regulates a range of biological functions including immunity 81

and apoptosis (Kockel et al., 2001). In Anopheles gambiae, the JNK pathway mediates an 82

anti-malaria response through complement activation (Garver et al., 2013). Currently, the 83

impact of JNK pathway on arbovirus infection remains unexplored. 84

Following midgut invasion, arboviruses propagate to remaining tissues, including 85

salivary glands (SGs), from where they are expectorated with saliva during subsequent bites. 86

Despite the critical role of SGs in transmission, only three studies have examined DENV-87

responsive differential gene expression in SGs. These studies revealed activation of the Toll 88


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and IMD pathways (Bonizzoni et al., 2012; Luplertlop et al., 2011; Sim et al., 2012) and 89

identified the anti-DENV functions of Cecropin (Luplertlop et al., 2011), putative Cystatin 90

and ankyrin-repeat proteins (Sim et al., 2012). Here, we characterized the SG immune 91

response to DENV, ZIKV and CHIKV. We performed the first high throughput RNA-92

sequencing (RNA-seq) in infected SGs and observed differentially expressed genes (DEGs) 93

related to immunity, apoptosis, blood-feeding and lipid metabolism. Using gene silencing, we 94

discovered that upregulated components of the Toll and IMD pathways had variable effects 95

against DENV, ZIKV and CHIKV. However, silencing of a JNK pathway upregulated 96

component increased, and silencing of a negative regulator decreased infection by the three 97

viruses in SGs. Further, we show that the JNK pathway is activated by all viruses and triggers 98

a cooperative complement and apoptosis response in SGs. This work identifies and 99

characterizes the JNK antiviral response that reduces DENV, ZIKV and CHIKV in A. aegypti 100

SGs. 101

102

Results 103

Transcriptome regulation by DENV, ZIKV and CHIKV in SGs 104

SGs were collected at 14 days post oral infection (dpi) with DENV and ZIKV, and at seven 105

dpi with CHIKV (Supplementary Figure 1) to account for variability between virus extrinsic 106

incubation periods (EIP) (Mbaika et al., 2016; Salazar et al., 2007). Differentially expressed 107

genes (DEGs) were calculated with DESeq2, edgeR and Cuffdiff 2, and showed little overlap 108

among the algorithms (Supplementary Figure 2). To validate DEGs and select which 109

software to use, we quantified the expression of 10 genes in a biological repeat with RT-110

qPCR and compared these values to the output from each algorithm. RNA-seq gene 111

expression obtained with DESeq2 correlated best with RT-qPCR values (DENV: r2 = 0.69; 112

ZIKV: r² = 0.81; CHIKV: r² = 0.79; Supplementary Figure 3), and only these DEGs are 113


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discussed. 114

The SG transcriptome was the most regulated by CHIKV infection (966 DEGs), 115

followed by ZIKV (396) and DENV (202) (Figure 1a; Supplementary Table 1). Only 19 116

DEGs were common amongst the three virus infections (Supplementary text), indicating a 117

virus-specific transcriptome response. Comparison between DEGs from the current DENV 118

infection and the three previous DENV studies with SGs collected at 14 dpi showed little 119

overlap among them (Supplementary Figure 4) (Bonizzoni et al., 2012; Luplertlop et al., 120

2011; Sim et al., 2012). However, despite technical variations (e.g. mosquito colony, virus 121

strain, transcriptomics technology), DEGs belonged to similar functional groups across the 122

studies. We observed that immunity, apoptosis, blood-feeding and lipid metabolism related 123

genes were highly regulated by DENV, ZIKV and CHIKV (Figure 1b; Supplementary text). 124

125


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126

Figure 1. DENV, ZIKV and CHIKV transcriptome regulation in A. aegypti salivary glands. 127

a, Venn diagram shows numbers of uniquely and commonly regulated differentially 128

expressed genes (DEGs) in DENV (green), ZIKV (red) and CHIKV (blue) infected salivary 129

glands. Colorless area shows total number of unchanged genes. Arrows indicate the direction 130

of regulation for the corresponding color code. b, Functional annotation of DEGs in DENV, 131

ZIKV or CHIKV infected salivary glands. APO, apoptosis; BF, blood feeding; CS, 132

cytoskeleton and structure; DIG, digestion; DIV, diverse functions; IMM, immunity; MET, 133


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metabolism; PROT, proteolysis; RSM, redox, stress and mitochondria; RTT, replication, 134

transcription and translation; TRP, transport; UNK, unknown functions. c, Transcriptomic 135

regulation of the IMD and JNK pathways in DENV, ZIKV and CHIKV infected salivary 136

glands. First column represents a scheme of the JNK and IMD pathways partially, modified 137

from (Sim et al., 2014). In the following columns, boxes indicate DEGs with AAEL number 138

below for DENV, ZIKV and CHIKV. Arrows indicate the direction of regulation. Pink-filled 139

boxes indicate genes regulated by more than one virus. Boxes with dotted line indicate DEGs 140

selected for functional studies. Dark and light shaded green areas differentiate IMD from 141

JNK pathways. 142

143

144

A high proportion of DEGs were related to immunity with DENV, ZIKV and CHIKV 145

modulating 20, 118 and 86 immune genes, respectively (Figure 1b; Supplementary Table 1). 146

Differential regulation of the RNAi, Toll, IMD, JAK/STAT and JNK pathways were 147

observed. The RNAi pathway is initiated when Dicer2 (Dcr2) recognizes and cleaves viral-148

derived dsRNA into small-interfering RNAs (siRNA) (Supplementary Figure 5). siRNAs are 149

loaded onto the RNA-induced silencing complex (RISC) that includes Argonaute2 (Ago2). 150

RISC unwinds siRNA to allow binding to complementary viral RNA fragments and cleavage 151

(Sanchez-Vargas et al., 2004). Strikingly, Dcr2 was upregulated by all viruses, while Ago2 152

was upregulated by DENV (Supplementary Figure 5), suggesting activation of RNAi. The 153

Toll pathway is triggered when extracellular pattern recognition receptors (PRR) (e.g. PGRP 154

and GNBP) bind to pathogen-derived ligands and activate a proteolytic cascade that leads to 155

activation of pro-Spätzle to Spätzle by Spätzle processing enzyme (SPE) (Supplementary 156

Figure 6) (DeLotto & DeLotto, 1998). Spätzle binding to transmembrane receptor Toll 157


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induces a cytoplasmic cascade that leads to nuclear translocation of NF-κB transcription 158

factor Rel1a to initiate effector gene transcriptions. Among PRRs, we observed that ZIKV 159

and CHIKV upregulated GNBPA1 and downregulated GNBPB6, while ZIKV also 160

upregulated PGRPS1 and CHIKV downregulated GNBP2 (Supplementary Figure 6). 161

Numerous serine proteases [e.g. CLIPs including Snake-likes (Snk-like) or Easter-likes (Est-162

like)] and serine protease inhibitors (Serpins) were upregulated by the viruses, except for Snk-163

like AAEL002273 and CLIPB41 downregulation by CHIKV, and SPE and CLIPB37 164

downregulation by DENV. SPE downregulation by DENV is reminiscent of downregulation 165

of Toll pathway components by subgenomic flaviviral RNA (sfRNA) in SGs (Pompon et al., 166

2017). The IMD pathway, similarly to Toll, is triggered by microbial ligand recognition by 167

PRRs, which activate PGRP-LC transmembrane protein (Figure 1c). In the ensuing 168

cytoplasmic cascade, NF-κB transcription factor Rel2 is phosphorylated by IKK2 and cleaved 169

by DREDD to induce its nuclear translocation. IMD pathway is repressed by Caspar (Sim et 170

al., 2014), an Ectoderm-expressed 4-containing module (Ect4) (Akhouayri et al., 2011) and 171

PGRP-LB (Gendrin et al., 2017). We observed an induction of IMD pathway by DENV and 172

ZIKV through PGRP-LB downregulation, and by CHIKV through Caspar downregulation 173

and IKK2 upregulation (Figure 1c). Ect4 upregulation by CHIKV may have controlled the 174

pathway activation. The JAK/STAT pathway is triggered by binding of Unpaired (Upd) to 175

transmembrane protein Domeless (Dome), which dimerizes and phosphorylates Hopscotch 176

(Hop) (Supplementary Figure 7) (Sim et al., 2014). The activated Dome/Hop complex 177

phosphorylates STATs, which upon dimerization translocate to nucleus and initiate 178

transcription. The phosphorylation of STATs is controlled by Suppressor of Cytokine 179

Signaling (SOCS) family of inhibitors (Callus & Mathey-Prevot, 2002). We observed an 180

upregulation of SOCS36E by CHIKV (Supplementary Figure 7), suggesting inhibition of the 181

pathway. The JNK pathway is triggered by Hemipterous (Hep) phosphorylation through 182


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tumor necrosis-associated factor 4 (Traf4) or Tak1, the latter being an IMD pathway 183

component (Figure 1c). Hep activates Basket (Bask) that phosphorylates c-Jun and Kayak 184

(Kay) transcription factors. Activated c-Jun and Kay dimerize to form the activator protein-1 185

(AP-1) complex, which translocates to nucleus and induces transcription. Puckered (Puc) 186

transcription is induced by the JNK pathway and represses Bask (Martín-Blanco et al., 1998), 187

acting as a feedback loop. Intriguingly, CHIKV upregulated most of the JNK pathway 188

components (i.e. Bask, c-Jun, Kay and Puc; Figure 1c), indicating activation of the pathway 189

in SGs. Furthermore, Traf4 upregulation by CHIKV suggested an IMD-independent 190

activation. 191

192

Upregulated components from JNK but not from Toll and IMD pathway reduce DENV, 193

ZIKV and CHIKV 194

To determine how immune response influences DENV, ZIKV and CHIKV multiplication in 195

SGs, we silenced nine previously uncharacterized immune genes that were upregulated by at 196

least one of the viruses (Table 1). They included four protease genes that initiate the Toll 197

pathway (CLIPB13A, CLIPB21, one of the Est-like, one of the Snk-like), two genes from the 198

cytoplasmic signaling of the IMD pathway (IKK2 and Ect4), one transcription factor gene of 199

the JNK pathway (Kay), and two putative immune genes - Galectin-5 (Gale5) and a juvenile 200

hormone induced gene (JHI). Gale5 shows antiviral function against O’nyong-nyong in A. 201

gambiae (Waldock et al., 2012), while juvenile hormone treatment regulates immune gene 202

expression in D. melanogaster (Flatt et al., 2008) and JHI was upregulated by all three 203

viruses in our study (Table 1). RNAi-mediated gene silencing efficacy ranged from 35-85 % 204

in SGs (Supplementary Figure 8a) and did not affect mosquito survival (Supplementary 205

Figure 9a). To bypass the midgut barrier, we intrathoracically inoculated mosquitoes with a 206

non-saturating inoculum of DENV, ZIKV or CHIKV, resulting in 70-80% infected SGs 207


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(Supplementary Figure 10). This permitted the evaluation of an increase or decrease in 208

infection upon gene silencing. At 10 days post inoculation, viral infection in SGs was 209

measured using two parameters, infection rate and infection intensity. Infection rate was 210

defined as the percentage of infected SGs out of 20 inoculated ones and represents 211

dissemination of intrathoracically injected viruses into SGs. Infection intensity was calculated 212

as viral genomic RNA (gRNA) copies per individual infected SGs and indicates virus 213

replication. Of note, the two infection parameters do not reflect isolated biological 214

phenomenon. For instance, a negative impact on infection intensity may lower infection rate 215

due to virus clearance. 216


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Table 1. Impact of immune response on DENV, ZIKV and CHIKV infection in salivary glands 217

1Induction in SGs as measured with RNA-seq. 2Determined by RNAi-mediated silencing studies in SGs. 218

219

220

221

222

223

Pathway Gene Virus Induced1 Function2

Proviral Antiviral

Dissemination Replication Dissemination Replication

Toll CLIPB13A

(AAEL003243)

DENV

ZIKV

CHIKV

CLIPB21

(AAEL001084)

DENV

ZIKV

CHIKV

Est-like

(AAEL012775)

DENV

ZIKV

CHIKV

Snk-like

(AAEL002273)

DENV

ZIKV

CHIKV

IMD IKK2

(AAEL012510)

DENV

ZIKV

CHIKV

Ect4

(AAEL014931)

DENV

ZIKV

CHIKV

JNK Kayak

(AAEL008953)

DENV

ZIKV

CHIKV

Putative

immune

JHI

(AAEL000515)

DENV

ZIKV

CHIKV

Gale5

(AAEL003844)

DENV

ZIKV

CHIKV


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Silencing of most of the immune genes had a virus-specific effect on infection 224

intensity and infection rate (Figure 2a-c and Table 1). Silencing of CLIPB13A increased 225

ZIKV infection rate, but decreased DENV and CHIKV infection rates. This suggests that 226

CLIPB13A hinders ZIKV dissemination and facilitates DENV and CHIKV dissemination into 227

SGs. However, it has a minor role in regulating virus replication in SGs. CLIPB21 silencing 228

increased ZIKV infection rate but decreased its infection intensity. Est-like silencing 229

decreased, whereas Snk-like silencing increased CHIKV infection rate. Overall, Toll pathway 230

upregulated components showed mostly a virus-specific impact on dissemination into SGs 231

(Figure 2a-c and Table 1). Silencing of IKK2 increased infection rate and decreased infection 232

intensity for ZIKV. Ect4 silencing increased DENV infection intensity and decreased CHIKV 233

infection rate. Overall, IMD pathway response appears both proviral and antiviral (Figure 2a-234

c and Table 1). Gale5 silencing decreased both ZIKV infection intensity and infection rate, 235

and enhanced CHIKV infection rate. Although JHI was upregulated by all viruses, it had no 236

effect on any infections (Figure 2a-c and Table 1). The silencing studies reflect a complex 237

interaction between transcriptomic response and antiviral functions (Table 1) with the notable 238

exception of Kay. Kay silencing increased infection rate to 100% for both ZIKV and CHIKV, 239

and increased infection intensities by 8.6-, 6.75- and 17.65-fold for DENV, ZIKV and 240

CHIKV, respectively (Figure 2a-c and Table 1). These results reveal a broad antiviral 241

function of the JNK pathway in SGs. To determine whether JNK pathway also restricts virus 242

in midguts, we orally infected Kay-silenced mosquitoes with ZIKV (Supplementary Figure 243

8b). At seven dpi, while infection rate was already saturated at 100% in the control, infection 244

intensity increased in midguts (Figure 2d). Overall, our functional studies discovered the 245

antiviral and proviral roles of several immune-related genes and established that JNK 246

pathway has a broad ubiquitous antiviral function. 247

248


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249

Figure 2. Kayak depletion but not Toll or IMD component depletion increases salivary 250

glands infection by DENV, ZIKV and CHIKV, and midgut infection by ZIKV. Four days 251

post dsRNA injection, mosquitoes were infected by intrathoracic inoculation with DENV, 252

ZIKV or CHIKV, or by oral feeding on ZIKV. Viral genomic RNA (gRNA) was quantified 253

at 10 days post inoculation in salivary glands and 7 days post oral infection in midguts. a-c, 254

Effect of immune-related gene silencing on gRNA copies and infection rate in salivary glands 255

infected with (a) DENV, (b) ZIKV, and (c) CHIKV. d, Impact of Kayak silencing on gRNA 256

number and infection rate in ZIKV-infected midgut. Bars show geometric mean ± 95% C.I. 257

from 20 individual pairs of salivary glands or 25 individual midguts. Each dot represents one 258

sample. dsCtrl, dsRNA against LacZ; dsCLIPB13A, dsRNA against CLIP domain serine 259


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protease B13A; dsCLIPB21, dsRNA against CLIP domain serine protease B21; dsEst-like, 260

dsRNA against Easter-like; dsSnk-like, dsRNA against Snake-like; dsIKK2, dsRNA against 261

Inhibitor of nuclear factor kappa-B kinase; dsKay, dsRNA against Kayak; dsEct4, dsRNA 262

against Ectoderm expressed-4; dsJHI, dsRNA against Juvenile hormone inducible; dsGale5, 263

dsRNA against Galectin 5. *, p-value < 0.05; **, p-value < 0.01; ***, p-value < 0.001, 264

determined by post hoc Dunnett’s with dsCtrl, unpaired t-test or Z-test for infection rate. 265

266

JNK pathway is induced by DENV, ZIKV and CHIKV and reduces infection in SGs 267

SG transcriptomics showed that Kay was induced by CHIKV at seven dpi, but not by DENV 268

or ZIKV at 14 dpi (Figure 1c). To test whether JNK pathway is activated by the three viruses, 269

we monitored the kinetics of Kay expression in SGs after oral infection with either of the 270

three viruses. Kay was similarly induced by DENV, ZIKV and CHIKV at three and seven 271

dpi, but not at 14 dpi (Figure 3a), corroborating the RNA-seq data. We quantified Puc 272

expression, which is induced as a negative regulator (Martín-Blanco et al., 1998). Although 273

not regulated at three and 14 dpi, Puc expression was increased at seven dpi for all three 274

viruses (Figure 3b). We then tested the impact of Puc silencing in ZIKV-inoculated 275

mosquitoes (Supplementary Figure 8c). Puc silencing did not alter mosquito survival 276

(Supplementary Figure 9b). Although SG infection rate was not affected, SG infection 277

intensity was decreased by 8.85-fold at 10 days post inoculation (Figure 3c). Altogether our 278

data demonstrate that the JNK pathway is induced by DENV, ZIKV and CHIKV at a time 279

that corresponds to the onset of infection in SGs (Salazar et al., 2007) and further support the 280

JNK antiviral function. 281

282


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283

Figure 3. Kayak and Puckered expressions are induced by DENV, ZIKV and CHIKV 284

infection, and Puckered depletion restricts ZIKV infection in salivary glands. a, Kayak (Kay) 285

and b, Puckered (Puc) expressions in salivary glands at 3, 7 and 14 days post oral infection 286

with DENV, ZIKV and CHIKV. Gene expression was quantified in pools of 10 salivary 287

glands. Actin expression was used for normalization. Bars show arithmetic means ± s.e.m. 288

from three biological replicates. c, Effect of Puc depletion on infection in salivary glands at 7 289

days post ZIKV inoculation. Four days prior infection, mosquitoes were injected with 290

dsRNA. Bars show geometric means ± 95% C.I. from 20 individual salivary glands. Each dot 291

represents one sample. *, p-value < 0.05, as determined by Dunnett’s test within time points 292

with mock infection as control (a, b) or unpaired t-test (c). 293

294

The JNK pathway mediates an antiviral complement and apoptosis response in SGs 295

JNK pathway can regulate complement system (Garver et al., 2013), apoptosis (McEwen, 296

2005) and autophagy (Wu et al., 2009). To determine whether JNK pathway induces these 297

functions in SGs, we monitored the impact of Kay silencing on expressions of four TEPs 298

(TEP20, TEP24, TEP15 and TEP2), two pro-apoptotic (Caspase8 and Dronc) and two 299

autophagy-related (ATG14 and ATG18A) genes at 10 dpi with ZIKV. All these genes were 300


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upregulated by infection in the transcriptomic data (Figure 1c; Supplementary Table 1). 301

Control mosquitoes were injected with control dsRNA (dsCtrl). Kay silencing significantly 302

reduced expressions of TEP20 and Dronc, and moderately reduced TEP15 and TEP24 303

(Figure 4a). TEP2 expression was increased and the two ATGs and Caspase 8 were 304

unaffected. These results suggest that upon infection the JNK pathway induces the 305

complement system through TEPs, and apoptosis through Dronc, but not Caspase8. We then 306

tested the antiviral function of TEP20 and Dronc in SGs by challenging either TEP20- or 307

Dronc-silenced mosquitoes (Supplementary Figure 8d) by ZIKV intrathoracic inoculation. 308

Similar to Kay-silencing, TEP20- and Dronc-silencing increased SG infection intensity by 309

21-fold to 2.6 x 106 gRNA and by 12-fold to 1.3 x 106 gRNA, respectively (Figure 4b). Since 310

both complement and apoptosis can interact for cell clearance (Fishelson, 2001), we 311

determined whether TEP20 and Dronc act in the same antiviral pathway by evaluating their 312

synergistic effect when both were co-silenced (Supplementary Figure 8e). We did not 313

observe a clear difference in SG infection between TEP20 and Dronc individual or co-314

silencing (Figure 4c,d). As the infection conditions did not saturate the infection intensity 315

(higher inoculum resulted in 108 gRNA per infected SG; Supplementary Figure 10), the lack 316

of synergism when TEP20 and Dronc are co-silenced suggests that TEP20 and Dronc 317

function in the same antiviral pathway. To show that TEP20 and Dronc mediate the JNK 318

antiviral response in SGs, we induced JNK pathway by silencing of Puc and co-silenced 319

TEP20 or Dronc before ZIKV inoculation. Control mosquitoes were injected with the same 320

quantity of dsCtrl or dsRNA against Puc. While infection intensity decreased upon Puc 321

silencing, co-silencing of Puc with TEP20 or Dronc restored viral gRNA copies to the level 322

in dsCtrl-injected SGs (Figure 4d). Altogether, these results demonstrate that the JNK 323

pathway is induced by DENV, ZIKV or CHIKV in SGs and eliminates viruses through the 324

complement system and apoptosis. 325


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326

Figure 4. Virus-induced JNK pathway activates an antiviral response through complement 327

and apoptosis. a, Impact of Kayak depletion on expression of genes related to complement 328

(TEP20, TEP24, TEP15 and TEP2), apoptosis (Caspase8 and Dronc) and autophagy (ATG14 329

and ATG18A) at 10 days post ZIKV oral infection. Gene expression was quantified in pools 330

of 10 SGs. Actin expression was used for normalization. Bars show arithmetic means ± s.e.m. 331

from three biological replicates. b-d, Effect of depletion of (b) Dronc or TEP20 alone, (c) 332

Dronc and TEP20 simultaneously, and (d) Puc alone, or Puc and Dronc simultaneously, or 333

Puc and TEP20 simultaneously on SG infection at 7 days post ZIKV inoculation. Four days 334

prior infection, mosquitoes were injected with dsRNA. Bars show geometric means ± 95% 335


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C.I. from 20 individual salivary glands. Each dot represents one sample. TEP, Thioester-336

containing protein; ATG, Autophagy related gene; Kay, Kayak; Puc, Puckered; Ctrl, control. 337

*, p-value < 0.05; **, p-value < 0.01; ***, p-value < 0.001, as determined by unpaired t-test 338

(a) or Dunnett’s test with dsCtrl as control (b-d). 339

340

Discussion 341

Dengue, Zika and chikungunya are widespread mosquito-borne diseases primarily 342

transmitted by A. aegypti. Disease mitigation through insecticide-based vector control has 343

mostly remained ineffective to prevent outbreaks (Wilder-Smith & Gubler, 2015). Currently, 344

insecticide-free vector-control strategies are being extensively investigated. The improved 345

understanding of the vector-virus interaction at the molecular level proposed here paves the 346

way to manipulate mosquito biology to make them refractory towards arboviruses. This study 347

discovers the antiviral function of the JNK pathway in SGs against three major arboviruses, 348

DENV, ZIKV and CHIKV belonging to two families. Further, we determine that the antiviral 349

response is mediated through a combined action of complement system and apoptosis. 350

Several studies have functionally characterized the immune response in A. aegypti 351

midgut, the first barrier in the mosquito body, determining the antiviral function of Toll, IMD 352

and JAKSTAT pathways (Angleró-Rodríguez et al., 2017; Bonizzoni et al., 2012; Dong et 353

al., 2017; Sim et al., 2013). However, only a couple of studies tested the impact of SG (exit 354

barrier) immune response (Luplertlop et al., 2011; Sim et al., 2012). Luplertlop et al., 2011 355

used Digital Gene Expression tag profiling to quantify the impact of DENV infection in SGs. 356

They reported overexpression of Toll pathway components, but did not test their functions. 357

Instead, they characterized the most abundant protein, an AMP from the Cecropin family, and 358

revealed its broad antiviral function in vitro. This Cecropin was not regulated in our 359


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transcriptomic analysis, although two other Cecropins (Cecropin A and G) were differentially 360

modulated by the different viruses. Sim et al., 2012 used microarrays to identify SG-specific 361

transcripts as compared to carcasses. An enrichment in immune-related genes suggested the 362

ability to mount an immune response. Further, the same authors determined the transcriptome 363

upon DENV infection. Similar to our data, they reveal a high regulation of serine proteases 364

that could initiate the different immune pathways or play a role in blood feeding when 365

secreted. While they did not functionally test the immune pathways, Sim et al. revealed the 366

antiviral and proviral functions of three DENV-upregulated genes. This supports the complex 367

interaction between gene regulation and function that we also observed. In our study, we 368

functionally tested for the first time the impact of upregulated components from Toll, IMD 369

and JNK pathways on DENV, ZIKV and CHIKV in SGs. JAK/STAT pathway was not 370

activated at the studied time-points. The lack of impact against DENV, ZIKV and CHIKV 371

when silencing the components of Toll and IMD pathways may not reflect the antiviral 372

capabilities of these pathways. Indeed, it is possible that a complete shutdown of the 373

signaling cascades increases virus infection. Importantly, we discovered the antiviral impact 374

of the JNK pathway against DENV, ZIKV and CHIKV in SGs. 375

JNK pathway activation by DENV, ZIKV and CHIKV in SGs occurred early during 376

infection at 3 dpi. This time corresponds to the onset of SG infection (Salazar et al., 2007). 377

Such an early induction may affect virus dissemination to SGs as reflected in ZIKV and 378

CHIKV infection rates. The JNK pathway can be induced either through IMD-mediated 379

pathogen recognition or oxidative stress. Tak1 activates the IMD and JNK pathways through 380

IKK2 and Hep, respectively (Takatsu et al., 2000). Since Tak1-mediated JNK activation is 381

transient, lasting less than one hour (Park, 2004), the observed JNK activation (lasting 3-7 382

dpi) is probably due to an IMD-independent activation. Upon oxidative stress, the JNK 383

pathway is induced through p53 upregulation of Traf4 that then phosphorylates Hep (Lu et 384


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al., 2017; Myers et al., 2018; Sax & El-Deiry, 2003). Activated JNK pathway then induces 385

other oxidative stress-associated genes, such as FoxO and κ-B Ras (AAEL003817) (Essers et 386

al., 2004). In our transcriptomic data, we reported the upregulation of several oxidative 387

stress-associated genes upon infection including p53, Traf4, FoxO, CYPs and κ-B Ras (Table 388

S1). These results suggest that JNK pathway is activated in SGs as a result of infection-389

induced oxidative stress. 390

Using in vivo functional studies, we showed that the JNK antiviral function depends 391

on complement system and apoptosis inductions. The complement system is activated when 392

TEPs bind to pathogens and trigger lysis, phagocytosis (Levashina et al., 2001) or even AMP 393

production (Xiao et al., 2014). TEPs such as TEP15 and AaMCR can restrict DENV in 394

mosquitoes (Cheng et al., 2011; Xiao et al., 2014), although TEP22 does not (Jupatanakul et 395

al., 2017). Apoptosis was previously reported in SGs infected with different flaviviruses and 396

alphaviruses (Day et al., 1966; Girard et al., 2005; Kelly et al., 2012). Apoptosis antiviral 397

function in mosquitoes is supported by two observations. Firstly, an arbovirus with an 398

apoptosis-inducing transgene was selected out during mosquito infection (O’Neill et al., 399

2015), and secondly, there is an association between the ability to induce apoptosis and 400

colony refractoriness in different virus-mosquito systems (Clem, 2016; Ocampo et al., 2013; 401

Vaidyanathan & Scott, 2006). These indicate that apoptosis can define vector competence, 402

emphasizing its importance as a target to control transmission. In our study, we reported the 403

antiviral functions of a complement system component, TEP20, and an apoptotic component, 404

Dronc, extending our understanding of the two mechanisms in SGs. Based on a lack of 405

synergistic effect when both TEP20 and Dronc were silenced, we propose that complement 406

system and apoptosis function in the same cascade to reduce virus infection. The complement 407

system could mediate apoptotic cell clearance as in mammals (Fishelson, 2001) and in testes 408

of A. gambiae (Pompon & Levashina, 2015). Alternatively, apoptosis-triggered nitration of 409


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virus surface could be required to direct TEP binding, as in Anopheles mosquitoes infected 410

with parasites (Kumar et al., 2010; Oliveira et al., 2012). 411

A recent study established a negative association between the presence of efficient 412

Plasmodium-killing immune response in mosquitoes and epidemics in Africa, confirming the 413

long-suspected impact of mosquito immunity on epidemiology for arthropod-borne diseases 414

(Gildenhard et al., 2019). Upon close inspection of field-derived A. aegypti colony 415

transcriptomes (Sim et al., 2013), we found that DENV-refractory colonies expressed a higher 416

level of Kay, c-Jun and TEP20. This suggests that variation in vector competence among these 417

colonies is partially related to JNK pathway. Consistent with our study, this points to a role of 418

JNK pathway in determining the arbovirus transmission dynamics in the field. Because JNK 419

pathway is sensitive to various microbes (Guntermann & Foley, 2011), differential activation 420

by distinct microbes present in natural habitats may also represent a trigger that influences 421

transmission. 422

Development of transgenic refractory mosquitoes have gained prominence in 423

preventing arboviral transmission (Wilke et al., 2018). Engineered overexpression of a 424

JAK/STAT activator in mosquitoes reduced DENV propagation and established its proof-of-425

concept (Jupatanakul et al., 2017). However, JAK/STAT is ineffective against ZIKV and 426

CHIKV. To our knowledge, no promising candidates have been identified to be antiviral 427

against DENV, ZIKV and CHIKV. In this context, our work reveals that the JNK pathway 428

components could be harnessed to develop effective transmission blocking tools against a 429

broad range of arboviruses. 430

431

Acknowledgments 432


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We thank the Professor Eng Eong Ooi from Duke-NUS Medical School, for providing the 433

DENV and ZIKV stock, and Professor Lisa Ng from Singapore Immunology Network (SIgN, 434

A*STAR, Singapore), for providing the CHIKV stock. This work was funded by the Tier-3 435

grant from the Ministry of Education, Singapore (MOE 2015-T3-1-003) and the Duke-NUS 436

Signature Research Programme funded by the Agency for Science, Technology and Research 437

(A*STAR), Singapore, and the Ministry of Health, Singapore. 438

439

Author Contributions 440

J.F.P. and R.M.K. designed the project. A.C., S.T.T. and B.W. performed the experiments. 441

C.M.M. performed bioinformatics analysis. A.C., C.M.M., D.M., T.V., R.M.K. and J.F.P. 442

analyzed the data. A.C. and C.M.M. made the figures. A.C., C.M.M., R.M.K. and J.F.P. 443

wrote the manuscript. All authors reviewed, critiqued and provided comments on the text. 444

445

Declarations of Interests 446

The authors declare no conflict of interest. 447


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Material and Methods 448

Mosquitoes 449

Aedes aegypti mosquitoes were collected in Singapore in 2010. Eggs were hatched in MilliQ 450

water and larvae were fed with a mixture of fish food (TetraMin fish flakes), yeast and liver 451

powder (MP Biomedicals). Adults were reared in cages (Bioquip) supplemented with 10% 452

sucrose and water. The insectary was held at 28°C and 50% relative humidity with a 12h:12h 453

light:dark cycle. 454

455

Virus isolates 456

The dengue virus serotype 2 PVP110 (DENV) was isolated from an EDEN cohort patient in 457

Singapore in 2008 (Christenbury et al., 2010). The Zika virus Paraiba_01/2015 (ZIKV) was 458

isolated from a febrile female in the state of Paraiba, Brazil in 2015 (Tsetsarkin et al., 2016). 459

The chikungunya virus SGP011 (CHIKV) was isolated from a patient at the National 460

University Hospital in Singapore (Her et al., 2010). DENV and ZIKV isolates were 461

propagated in C6/36 and CHIKV in Vero cell line. Virus stocks were titered with BHK-21 462

cell plaque assay as previously described (Manokaran et al., 2015), aliquoted and stored at -463

80°C. 464

465

Oral infection 466

Three-to-five day-old female mosquitoes were starved for 24 h and offered an infectious 467

blood meal containing 40% volume of washed erythrocytes from serum pathogen free (SPF) 468

pig’s blood (PWG Genetics), 5% 10 mM ATP (Thermo Scientific), 5% human serum 469

(Sigma) and 50% virus solution in RPMI media (Gibco). Mosquitoes were left to feed for 1.5 470


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h using Hemotek membrane feeder system (Discovery Workshops) covered with porcine 471

intestine membrane (sausage casing). The virus titers in blood meals were 2 x 107 pfu/ml for 472

DENV, 6 x 106 pfu/ml for ZIKV, and 1.5 x 108 pfu/ml for CHIKV, and validated in plaque 473

assay using BHK-21 cells. Control mosquitoes were fed with the same blood meal 474

composition except for virus. Fully engorged females were selected and kept in a cage with 475

ad libitum access to a 10% sucrose solution in an incubation chamber with conditions similar 476

to insect rearing. 477

478

Virus inoculation 479

Mosquitoes were cold anesthetized and intrathoracically inoculated with either DENV, ZIKV 480

or CHIKV using Nanoject-II (Drummond). The same volume of RPMI media (ThermoFisher 481

Scientific) was injected as control. Functional studies were conducted by inoculating 0.1 pfu 482

of DENV, 0.01 pfu of ZIKV and 0.2 pfu of CHIKV. 483

484

SG collection, library preparation and RNA-sequencing 485

SGs from DENV- and ZIKV-orally infected mosquitoes were dissected at 14 dpi. Those from 486

CHIKV-orally infected mosquitoes were dissected at 7 dpi. Controls for CHIKV were 487

dissected at 7 days post feeding on a non-infectious blood meal, and at 14 days post feeding 488

for DENV and ZIKV. Inoculum resulted in 100% infected SGs (Supplementary Figure 1). 489

Fifty pairs of SGs per condition were homogenized using a bead mill homogenizer (FastPrep-490

24, MP Biomedicals). Total RNA was extracted using E.Z.N.A Total RNA kit I (OMEGA 491

Bio-Tek). RNA-seq libraries were prepared using True-Seq Stranded Total RNA with Ribo-492

Zero Gold kit (Illumina), according to manufacturer’s instructions. Following quantification 493

by RT-qPCR using KAPA Library Quantification Kit (KAPA Biosystems), libraries were 494


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pooled in equimolar concentrations for cluster generation on cBOT system (Illumina) and 495

sequenced (150 bp pair-end) on a HiSeq 3000 instrument (Illumina) at the Duke-NUS 496

Genome Biology Facility, according to manufacturer’s protocols. Two repeats per condition 497

were processed. The need for more than two repeats was mitigated by using a large number 498

of tissues from different mosquitoes in each repeat. Raw sequencing reads from RNA-seq 499

libraries are available online under NCBI accessions: SRR8921123-8921132. 500

501

RNA-seq data processing and identification of differentially expressed genes (DEGs) 502

Reads were quality checked with FastQC (www.bioinformatics.babraham.ac.uk) to confirm 503

that adapter sequences and low-quality reads (Phred+33 score < 20) had been removed. The 504

reads were then aligned against the A. aegypti genome [VectorBase(Giraldo-Calderón et al., 505

2015)] AaegL3.3) using TopHat v2.1.0 (Kim et al., 2013) with parameters –N 6 –read-gap-506

length 6 –read-edit-dist. 6 set to account for regional genetic variation between the RNA-seq 507

and genome. SAM tools v0.1.19 (Li et al., 2009) and HTSeq v0.6.0 (Anders et al., 2015) 508

were used to format and produce count files for gene expression analysis. DEGs were 509

identified using DESeq2 (Love et al., 2014) with at least 1.4-fold change between control and 510

infected conditions, an adjusted False Discovery Rate (FDR) of 0.05. edgeR (Robinson et al., 511

2010) and Cuffdiff 2 (Trapnell et al., 2013) (following the Cufflinks pipeline (Trapnell et al., 512

2012)) were also used to identify DEGs following the same criteria. 513

514

Gene annotations 515

DEGs were annotated through several pipelines. Predicted gene protein sequences were 516

searched against the NCBI nr database (accessed July 2017) (BLASTp; e-value 1.0E-5 and 517

word size 3), VectorBase (Giraldo-Calderón et al., 2015) Aedes peptide database 518


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(downloaded July 2017) with BLAST+ (Camacho et al., 2009) (BLASTp; e-value 1.0E-5 519

threshold and word size 2), and FlyBase (Gramates et al., 2017) D. melanogaster peptide 520

database (downloaded August 2017) with BLAST+ (BLASTp; e-value 1.0E-5 threshold and 521

word size 2) for identification of orthologues. Gene ontology terms were assigned in 522

BLAST2GO (Conesa et al., 2005) (program default parameters). Signal peptides were 523

identified using SignalP v4.1 (Petersen et al., 2011). Functional annotations were also 524

assigned based on literature. 525

526

Double-stranded mediated RNAi 527

Mosquito cDNA was used to amplify dsRNA targets with gene specific primers tagged with 528

T7 promoter as detailed in Supplementary Table 2. The amplified products were in vitro 529

transcribed with T7 Scribe kit (Cellscript). dsRNAs were annealed by heating to 95°C and 530

slowly cooling down to 4°C using a thermocycler. Three to five-day-old adult female 531

mosquitoes were cold-anesthetized and intrathoracically injected with 2 or 4 µg of dsRNA 532

using Nanoject II. The same quantity of dsRNA against the bacterial gene LacZ was injected 533

as control (dsCtrl). Validation of gene silencing was conducted 4 days post injection by 534

pooling 10 SGs or 5 midguts. 535

536

Gene expression quantification using RT-qPCR 537

Total RNA was extracted from 10 SGs or 5 midguts using E.Z.N.A. Total RNA kit I, DNAse 538

treated using Turbo DNA-free kit (Thermo Fisher Scientific), and reverse transcribed using 539

iScript cDNA synthesis kit (Biorad). Gene expression was quantified using qPCR with 540

SensiFast Sybr no-rox kit (Bioline) and gene specific primers detailed in Supplementary 541

Table 2, 3 and 4. Actin expression was used for normalization. The reactions were performed 542


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using the following cycle conditions: an initial 95°C for 10 min, followed by 40 cycles of 543

95°C for 5 s, 60°C for 20 s and ending with a melting curve analysis. The delta delta method 544

was used to calculate relative fold changes. 545

546

Quantification of virus genome RNA (gRNA) copies using RT-qPCR 547

Individual pairs of either SGs or midguts were homogenized with a bead Mill Homogenizer 548

in 350 µl of TRK lysis buffer (E.Z. N. A Total RNA kit I). Total RNA was extracted using 549

the RNA extraction kit and reverse-transcribed using iScript cDNA synthesis kit. DENV 550

gRNA copies were quantified by RT-qPCR using i-Taq one step universal probes kit 551

(BioRad) and ZIKV and CHIKV gRNA copies with i-Taq one step universal sybr kit 552

(BioRad) with primers detailed in Supplementary Table 5. Amplification was run on CFX96 553

Touch Real-Time PCR Detection System (BioRad) with the following thermal profile: 50°C 554

for 10 min, 95 °C for 1 min, followed by 40 cycles at 95 °C for 10 s, 60 °C for 15 s. A melt-555

curve analysis was added for Sybr qPCR. 556

Absolute quantification of gRNA was obtained by generating a standard curve for 557

each virus target. Viral cDNA was used to amplify qPCR target using qPCR primers with T7-558

tagged forward primer. RNA fragments were generated with T7-Scribe kit and RNA 559

concentration calculated by Nanodrop was used to estimate concentration of RNA fragments. 560

Ten-time serial dilutions were quantified with RT-qPCR and used to generate absolute 561

standard equations. Three standard dilutions were used in each subsequent RT-qPCR plate to 562

adjust for inter-plate variation. 563

564

Statistical analyses and software used 565


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One-way ANOVA and post-hoc Dunnett’s test or unpaired T-test were used to test 566

differences in gene expression and log10-transformed gRNA copies. These analyses were 567

done using GraphPad Prism 5. Z-score was used to test differences in infection rate with 568

www.socscistatistics.com/tests/ztest/. Standard error for sample proportion was calculated 569

with www.easycalculation.com. 570

571

References 572

Akhouayri, I., Turc, C., Royet, J., & Charroux, B. 2011. Toll-8/Tollo negatively regulates 573

antimicrobial response in the Drosophila respiratory epithelium. PLOS Pathogens, 574

7(10), e1002319. https://doi.org/10.1371/journal.ppat.1002319 575

Anders, K. L., Indriani, C., Ahmad, R. A., Tantowijoyo, W., Arguni, E., Andari, B., Jewell, 576

N. P., Rances, E., O’Neill, S. L., Simmons, C. P., & Utarini, A. 2018. The AWED 577

trial (applying Wolbachia to eliminate dengue) to assess the efficacy of Wolbachia-578

infected mosquito deployments to reduce dengue incidence in Yogyakarta, Indonesia: 579

Study protocol for a cluster randomised controlled trial. Trials, 19(1). 580

https://doi.org/10.1186/s13063-018-2670-z 581

Anders, S., Pyl, P. T., & Huber, W. 2015. HTSeq—A Python framework to work with high-582

throughput sequencing data. Bioinformatics, 31(2), 166–169. 583

https://doi.org/10.1093/bioinformatics/btu638 584

Angleró-Rodríguez, Y. I., MacLeod, H. J., Kang, S., Carlson, J. S., Jupatanakul, N., & 585

Dimopoulos, G. 2017. Aedes aegypti molecular responses to Zika virus: modulation 586

of infection by the Toll and Jak/STAT immune pathways and virus host factors. 587

Frontiers in Microbiology, 8, 2050. https://doi.org/10.3389/fmicb.2017.02050 588


http://www.socscistatistics.com/tests/ztest/

https://doi.org/10.1101/2020.03.01.972026


30

Barrows, N. J., Campos, R. K., Liao, K.-C., Prasanth, K. R., Soto-Acosta, R., Yeh, S.-C., 589

Schott-Lerner, G., Pompon, J., Sessions, O. M., Bradrick, S. S., & Garcia-Blanco, M. 590

A. 2018. Biochemistry and molecular biology of flaviviruses. Chemical Reviews, 591

118(8), 4448–4482. https://doi.org/10.1021/acs.chemrev.7b00719 592

Bhatt, S., Gething, P. W., Brady, O. J., Messina, J. P., Farlow, A. W., Moyes, C. L., Drake, J. 593

M., Brownstein, J. S., Hoen, A. G., Sankoh, O., Myers, M. F., George, D. B., 594

Jaenisch, T., Wint, G. R. W., Simmons, C. P., Scott, T. W., Farrar, J. J., & Hay, S. I. 595

2013. The global distribution and burden of dengue. Nature, 496(7446), 504–507. 596

https://doi.org/10.1038/nature12060 597

Blandin, S. 2004. Thioester-containing proteins and insect immunity. Molecular Immunology, 598

40(12), 903–908. https://doi.org/10.1016/j.molimm.2003.10.010 599

Bonizzoni, M., Dunn, W. A., Campbell, C. L., Olson, K. E., Marinotti, O., & James, A. A. 600

2012. Complex modulation of the Aedes aegypti transcriptome in response to dengue 601

virus infection. PLOS One, 7(11), e50512. 602

https://doi.org/10.1371/journal.pone.0050512 603

Callus, B. A., & Mathey-Prevot, B. 2002. SOCS36E, a novel Drosophila SOCS protein, 604

suppresses JAK/STAT and EGF-R signalling in the imaginal wing disc. Oncogene, 605

21(31), 4812–4821. https://doi.org/10.1038/sj.onc.1205618 606

Camacho, C., Coulouris, G., Avagyan, V., Ma, N., Papadopoulos, J., Bealer, K., & Madden, 607

T. L. 2009. BLAST+: Architecture and applications. BMC Bioinformatics, 10(1), 421. 608

https://doi.org/10.1186/1471-2105-10-421 609

Cheng, G., Liu, L., Wang, P., Zhang, Y., Zhao, Y. O., Colpitts, T. M., Feitosa, F., Anderson, 610

J. F., & Fikrig, E. 2011. An in vivo transfection approach elucidates a role for Aedes 611

aegypti thioester-containing proteins in flaviviral infection. PLOS One, 6(7), e22786. 612

https://doi.org/10.1371/journal.pone.0022786 613


https://doi.org/10.1101/2020.03.01.972026


31

Christenbury, J. G., Aw, P. P. K., Ong, S. H., Schreiber, M. J., Chow, A., Gubler, D. J., 614

Vasudevan, S. G., Ooi, E. E., & Hibberd, M. L. 2010. A method for full genome 615

sequencing of all four serotypes of the dengue virus. Journal of Virological Methods, 616

169(1), 202–206. https://doi.org/10.1016/j.jviromet.2010.06.013 617

Clem, R. J. 2016. Arboviruses and apoptosis: The role of cell death in determining vector 618

competence. Journal of General Virology, 97(5), 1033–1036. 619

https://doi.org/10.1099/jgv.0.000429 620

Conesa, A., Gotz, S., Garcia-Gomez, J. M., Terol, J., Talon, M., & Robles, M. 2005. 621

Blast2GO: A universal tool for annotation, visualization and analysis in functional 622

genomics research. Bioinformatics, 21(18), 3674–3676. 623

https://doi.org/10.1093/bioinformatics/bti610 624

Day, M. F., Marshall, I. D., & Mims, C. A. 1966. Cytopathic Effect of Semliki Forest Virus 625

in the Mosquito Aedes Aegypti. The American Journal of Tropical Medicine and 626

Hygiene, 15(5), 775–784. https://doi.org/10.4269/ajtmh.1966.15.775 627

DeLotto, Y., & DeLotto, R. 1998. Proteolytic processing of the Drosophila Spätzle protein by 628

Easter generates a dimeric NGF-like molecule with ventralising activity. Mechanisms 629

of Development, 72(1–2), 141–148. https://doi.org/10.1016/S0925-4773(98)00024-0 630

Dong, S., Behura, S. K., & Franz, A. W. E. 2017. The midgut transcriptome of Aedes aegypti 631

fed with saline or protein meals containing chikungunya virus reveals genes 632

potentially involved in viral midgut escape. BMC Genomics, 18(1). 633

https://doi.org/10.1186/s12864-017-3775-6 634

Essers, M. A. G., Weijzen, S., de Vries-Smits, A. M. M., Saarloos, I., de Ruiter, N. D., Bos, J. 635

L., & Burgering, B. M. T. 2004. FOXO transcription factor activation by oxidative 636

stress mediated by the small GTPase Ral and JNK. The EMBO Journal, 23(24), 637

4802–4812. https://doi.org/10.1038/sj.emboj.7600476 638


https://doi.org/10.1101/2020.03.01.972026


32

Fishelson, Z. 2001. Complement and apoptosis. Molecular Immunology, 38(2–3), 207–219. 639

https://doi.org/10.1016/S0161-5890(01)00055-4 640

Flatt, T., Heyland, A., Rus, F., Porpiglia, E., Sherlock, C., Yamamoto, R., Garbuzov, A., 641

Palli, S. R., Tatar, M., & Silverman, N. 2008. Hormonal regulation of the humoral 642

innate immune response in Drosophila melanogaster. Journal of Experimental 643

Biology, 211(16), 2712–2724. https://doi.org/10.1242/jeb.014878 644

Garver, L. S., de Almeida Oliveira, G., & Barillas-Mury, C. 2013. The JNK pathway is a key 645

mediator of Anopheles gambiae antiplasmodial immunity. PLOS Pathogens, 9(9). 646

https://doi.org/10.1371/journal.ppat.1003622 647

Gendrin, M., Turlure, F., Rodgers, F. H., Cohuet, A., Morlais, I., & Christophides, G. K. 648

(2017). The peptidoglycan recognition proteins PGRPLA and PGRPLB regulate 649

Anopheles immunity to bacteria and affect infection by Plasmodium. Journal of 650

Innate Immunity, 9(4), 333–342. https://doi.org/10.1159/000452797 651

Gildenhard, M., Rono, E. K., Diarra, A., Boissière, A., Bascunan, P., Carrillo-Bustamante, P., 652

Camara, D., Krüger, H., Mariko, M., Mariko, R., Mireji, P., Nsango, S. E., Pompon, 653

J., Reis, Y., Rono, M. K., Seda, P. B., Thailayil, J., Traorè, A., Yapto, C. V., … 654

Levashina, E. A. 2019. Mosquito microevolution drives Plasmodium falciparum 655

dynamics. Nature Microbiology. 4(6), 941-947. https://doi.org/10.1038/s41564-019-656

0414-9 657

Giraldo-Calderón, G. I., Emrich, S. J., MacCallum, R. M., Maslen, G., Dialynas, E., Topalis, 658

P., Ho, N., Gesing, S., the VectorBase Consortium, Madey, G., Collins, F. H., & 659

Lawson, D. 2015. VectorBase: An updated bioinformatics resource for invertebrate 660

vectors and other organisms related with human diseases. Nucleic Acids Research, 661

43(D1), D707–D713. https://doi.org/10.1093/nar/gku1117 662


https://doi.org/10.1101/2020.03.01.972026


33

Girard, Y. A., Popov, V., Wen, J., Han, V., & Higgs, S. 2005. Ultrastructural study of West 663

Nile virus pathogenesis in Culex pipiens quinquefasciatus (diptera: culicidae). Journal 664

of medical entomology, 42(3), 429-44. https://doi.org/10.1093/jmedent/42.3.429 665

Gramates, L. S., Marygold, S. J., Santos, G. dos, Urbano, J.-M., Antonazzo, G., Matthews, B. 666

B., Rey, A. J., Tabone, C. J., Crosby, M. A., Emmert, D. B., Falls, K., Goodman, J. 667

L., Hu, Y., Ponting, L., Schroeder, A. J., Strelets, V. B., Thurmond, J., Zhou, P., & 668

the FlyBase Consortium. 2017. FlyBase at 25: Looking to the future. Nucleic Acids 669

Research, 45(D1), D663–D671. https://doi.org/10.1093/nar/gkw1016 670

Guntermann, S., & Foley, E. 2011. The protein Dredd is an essential component of the c-Jun 671

N-terminal Kinase pathway in the Drosophila immune response. The Journal of 672

Biological Chemistry, 286(35), 30284–30294. 673

https://doi.org/10.1074/jbc.M111.220285 674

Hamel, R., Liégeois, F., Wichit, S., Pompon, J., Diop, F., Talignani, L., Thomas, F., Desprès, 675

P., Yssel, H., & Missé, D. 2016. Zika virus: epidemiology, clinical features and host-676

virus interactions. Microbes and Infection, 18(7–8), 441–449. 677

https://doi.org/10.1016/j.micinf.2016.03.009 678

Her, Z., Malleret, B., Chan, M., Ong, E. K. S., Wong, S.-C., Kwek, D. J. C., Tolou, H., Lin, 679

R. T. P., Tambyah, P. A., Rénia, L., & Ng, L. F. P. 2010. Active infection of human 680

blood monocytes by chikungunya virus triggers an innate immune response. The 681

Journal of Immunology, 184(10), 5903–5913. 682

https://doi.org/10.4049/jimmunol.0904181 683

Jupatanakul, N., Sim, S., Angleró-Rodríguez, Y. I., Souza-Neto, J., Das, S., Poti, K. E., 684

Rossi, S. L., Bergren, N., Vasilakis, N., & Dimopoulos, G. 2017. Engineered Aedes 685

aegypti JAK/STAT pathway-mediated immunity to dengue virus. PLOS Neglected 686

Tropical Diseases, 11(1). https://doi.org/10.1371/journal.pntd.0005187 687


https://doi.org/10.1101/2020.03.01.972026


34

Kelly, E. M., Moon, D. C., & Bowers, D. F. 2012. Apoptosis in mosquito salivary glands: 688

Sindbis virus-associated and tissue homeostasis. The Journal of General Virology, 689

93(11), 2419–2424. https://doi.org/10.1099/vir.0.042846-0 690

Kim, D., Pertea, G., Trapnell, C., Pimentel, H., Kelley, R., & Salzberg, S. L. 2013. TopHat2: 691

Accurate alignment of transcriptomes in the presence of insertions, deletions and gene 692

fusions. Genome Biology, 14(4), R36. https://doi.org/10.1186/gb-2013-14-4-r36 693

Kistler, K. E., Vosshall, L. B., & Matthews, B. J. 2015. Genome-engineering with CRISPR-694

Cas9 in the mosquito Aedes aegypti. Cell Reports, 11(1), 51–60. 695

https://doi.org/10.1016/j.celrep.2015.03.009 696

Kockel, L., Homsy, J. G., & Bohmann, D. 2001. Drosophila AP-1: lessons from an 697

invertebrate. Oncogene, 20(19), 2347–2364. https://doi.org/10.1038/sj.onc.1204300 698

Kraemer, M. U. G., Reiner, R. C., Brady, O. J., Messina, J. P., Gilbert, M., Pigott, D. M., Yi, 699

D., Johnson, K., Earl, L., Marczak, L. B., Shirude, S., Davis Weaver, N., Bisanzio, D., 700

Perkins, T. A., Lai, S., Lu, X., Jones, P., Coelho, G. E., Carvalho, R. G., … Golding, 701

N. 2019. Past and future spread of the arbovirus vectors Aedes aegypti and Aedes 702

albopictus. Nature Microbiology, 4(5), 854–863. https://doi.org/10.1038/s41564-019-703

0376-y 704

Kumar, S., Molina-Cruz, A., Gupta, L., Rodrigues, J., & Barillas-Mury, C. 2010. A 705

Peroxidase/Dual oxidase system modulates midgut epithelial immunity in Anopheles 706

gambiae. Science, 327(5973), 1644–1648. https://doi.org/10.1126/science.1184008 707

Levashina, E. A., Moita, L. F., Blandin, S., Vriend, G., Lagueux, M., & Kafatos, F. C. 708

(2001). Conserved Role of a Complement-like Protein in Phagocytosis Revealed by 709

dsRNA Knockout in Cultured Cells of the Mosquito, Anopheles gambiae. 104(5), 710

709-718. https://doi.org/10.1016/s0092-8674(01)00267-7 711


https://doi.org/10.1101/2020.03.01.972026


35

Li, H., Handsaker, B., Wysoker, A., Fennell, T., Ruan, J., Homer, N., Marth, G., Abecasis, 712

G., Durbin, R., & 1000 Genome Project Data Processing Subgroup. 2009. The 713

sequence alignment/map format and SAMtools. Bioinformatics, 25(16), 2078–2079. 714

https://doi.org/10.1093/bioinformatics/btp352 715

Liu, N. 2015. Insecticide resistance in mosquitoes: impact, mechanisms, and research 716

directions. Annual Review of Entomology, 60(1), 537–559. 717

https://doi.org/10.1146/annurev-ento-010814-020828 718

Love, M. I., Huber, W., & Anders, S. 2014. Moderated estimation of fold change and 719

dispersion for RNA-seq data with DESeq2. Genome Biology, 15(12). 720

https://doi.org/10.1186/s13059-014-0550-8 721

Lu, T.-Y., MacDonald, J. M., Neukomm, L. J., Sheehan, A. E., Bradshaw, R., Logan, M. A., 722

& Freeman, M. R. 2017. Axon degeneration induces glial responses through Draper-723

TRAF4-JNK signalling. Nature Communications, 8, 14355. 724

https://doi.org/10.1038/ncomms14355 725

Luplertlop, N., Surasombatpattana, P., Patramool, S., Dumas, E., Wasinpiyamongkol, L., 726

Saune, L., Hamel, R., Bernard, E., Sereno, D., Thomas, F., Piquemal, D., Yssel, H., 727

Briant, L., & Missé, D. 2011. Induction of a peptide with activity against a broad 728

spectrum of pathogens in the Aedes aegypti salivary gland, following infection with 729

dengue virus. PLOS Pathogens, 7(1), e1001252. 730


Manokaran, G., Finol, E., Wang, C., Gunaratne, J., Bahl, J., Ong, E. Z., Tan, H. C., Sessions, 732

O. M., Ward, A. M., Gubler, D. J., Harris, E., Garcia-Blanco, M. A., & Ooi, E. E. 733

2015. Dengue subgenomic RNA binds TRIM25 to inhibit interferon expression for 734

epidemiological fitness. Science, 350(6257), 217–221. 735

https://doi.org/10.1126/science.aab3369 736


https://doi.org/10.1101/2020.03.01.972026


36

Martín-Blanco, E., Gampel, A., Ring, J., Virdee, K., Kirov, N., Tolkovsky, A. M., & 737

Martinez-Arias, A. 1998. Puckered encodes a phosphatase that mediates a feedback 738

loop regulating JNK activity during dorsal closure in Drosophila. Genes & 739

Development, 12(4), 557–570. https://doi.org/10.1101/gad.12.4.557 740

Mayer, S. V., Tesh, R. B., & Vasilakis, N. 2017. The emergence of arthropod-borne viral 741

diseases: A global prospective on dengue, chikungunya and Zika fevers. Acta 742

Tropica, 166, 155–163. https://doi.org/10.1016/j.actatropica.2016.11.020 743

Mbaika, S., Lutomiah, J., Chepkorir, E., Mulwa, F., Khayeka-Wandabwa, C., Tigoi, C., 744

Oyoo-Okoth, E., Mutisya, J., Ng’ang’a, Z., & Sang, R. 2016. Vector competence of 745

Aedes aegypti in transmitting chikungunya virus: effects and implications of extrinsic 746

incubation temperature on dissemination and infection rates. Virology Journal, 13. 747

https://doi.org/10.1186/s12985-016-0566-7 748

McEwen, D. G. 2005. Puckered, a Drosophila MAPK phosphatase, ensures cell viability by 749

antagonizing JNK-induced apoptosis. Development, 132(17), 3935–3946. 750

https://doi.org/10.1242/dev.01949 751

Myers, A. L., Harris, C. M., Choe, K.-M., & Brennan, C. A. 2018. Inflammatory production 752

of reactive oxygen species by Drosophila hemocytes activates cellular immune 753

defenses. Biochemical and Biophysical Research Communications, 505(3), 726–732. 754

https://doi.org/10.1016/j.bbrc.2018.09.126 755

Ocampo, C. B., Caicedo, P. A., Jaramillo, G., Ursic Bedoya, R., Baron, O., Serrato, I. M., 756

Cooper, D. M., & Lowenberger, C. 2013. Differential expression of apoptosis related 757

genes in selected strains of Aedes aegypti with different susceptibilities to dengue 758

virus. PLOS One, 8(4), e61187. https://doi.org/10.1371/journal.pone.0061187 759


https://doi.org/10.1101/2020.03.01.972026


37

Oliveira, G. d. A., Lieberman, J., & Barillas-Mury, C. 2012. Epithelial nitration by a 760

Peroxidase/NOX5 system mediates mosquito antiplasmodial immunity. Science, 761

335(6070), 856–859. https://doi.org/10.1126/science.1209678 762

Olmo, R. P., Ferreira, A. G. A., Izidoro-Toledo, T. C., Aguiar, E. R. G. R., de Faria, I. J. S., 763

de Souza, K. P. R., Osório, K. P., Kuhn, L., Hammann, P., de Andrade, E. G., Todjro, 764

Y. M., Rocha, M. N., Leite, T. H. J. F., Amadou, S. C. G., Armache, J. N., Paro, S., de 765

Oliveira, C. D., Carvalho, F. D., Moreira, L. A., … Marques, J. T. 2018. Control of 766

dengue virus in the midgut of Aedes aegypti by ectopic expression of the dsRNA-767

binding protein Loqs2. Nature Microbiology, 3(12), 1385–1393. 768

https://doi.org/10.1038/s41564-018-0268-6 769

O’Neill, K., Olson, B. J. S. C., Huang, N., Unis, D., & Clem, R. J. 2015. Rapid selection 770

against arbovirus-induced apoptosis during infection of a mosquito vector. PNAS, 771

112(10), E1152–E1161. https://doi.org/10.1073/pnas.1424469112 772

Park, J. M. 2004. Targeting of TAK1 by the NF- B protein Relish regulates the JNK-773

mediated immune response in Drosophila. Genes & Development, 18(5), 584–594. 774

https://doi.org/10.1101/gad.1168104 775

Petersen, T. N., Brunak, S., von Heijne, G., & Nielsen, H. 2011. SignalP 4.0: Discriminating 776

signal peptides from transmembrane regions. Nature Methods, 8(10), 785–786. 777

https://doi.org/10.1038/nmeth.1701 778

Pierson, T. C., & Graham, B. S. 2016. Zika Virus: immunity and vaccine development. Cell, 779

167(3), 625–631. https://doi.org/10.1016/j.cell.2016.09.020 780

Pompon, J., & Levashina, E. A. 2015. A new role of the mosquito complement-like cascade 781

in male fertility in Anopheles gambiae. PLOS Biology, 13(9), e1002255. 782

https://doi.org/10.1371/journal.pbio.1002255 783


https://doi.org/10.1101/2020.03.01.972026


38

Pompon, J., Manuel, M., Ng, G. K., Wong, B., Shan, C., Manokaran, G., Soto-Acosta, R., 784

Bradrick, S. S., Ooi, E. E., Missé, D., Shi, P.-Y., & Garcia-Blanco, M. A. 2017. 785

Dengue subgenomic flaviviral RNA disrupts immunity in mosquito salivary glands to 786

increase virus transmission. PLOS Pathogens, 13(7), e1006535. 787


Ramirez, J. L., & Dimopoulos, G. 2010. The Toll immune signaling pathway control 789

conserved anti-dengue defenses across diverse Aedes aegypti strains and against 790

multiple dengue virus serotypes. Developmental and Comparative Immunology, 791

34(6), 625–629. https://doi.org/10.1016/j.dci.2010.01.006 792

Ritchie, S. A., van den Hurk, A. F., Smout, M. J., Staunton, K. M., & Hoffmann, A. A. 2018. 793

Mission accomplished? we need a guide to the ‘post release’ world of Wolbachia for 794

Aedes -borne disease control. Trends in Parasitology, 34(3), 217–226. 795

https://doi.org/10.1016/j.pt.2017.11.011 796

Robinson, M. D., McCarthy, D. J., & Smyth, G. K. 2010. edgeR: A Bioconductor package 797

for differential expression analysis of digital gene expression data. Bioinformatics, 798

26(1), 139–140. https://doi.org/10.1093/bioinformatics/btp616 799

Salazar, M. I., Richardson, J. H., Sánchez-Vargas, I., Olson, K. E., & Beaty, B. J. 2007. 800

Dengue virus type 2: replication and tropisms in orally infected Aedes aegypti 801

mosquitoes. BMC Microbiology, 7,9. https://doi.org/10.1186/1471-2180-7-9 802

Sanchez-Vargas, I., Travanty, E. A., Keene, K. M., Franz, A. W. E., Beaty, B. J., Blair, C. D., 803

& Olson, K. E. 2004. RNA interference, arthropod-borne viruses, and mosquitoes. 804

Virus Research, 102(1), 65–74. https://doi.org/10.1016/j.virusres.2004.01.017 805

Sax, J. K., & El-Deiry, W. S. 2003. Identification and characterization of the cytoplasmic 806

protein TRAF4 as a p53-regulated proapoptotic gene. Journal of Biological 807

Chemistry, 278(38), 36435–36444. https://doi.org/10.1074/jbc.M303191200 808


https://doi.org/10.1101/2020.03.01.972026


39

Silva, L. A., & Dermody, T. S. 2017. Chikungunya virus: epidemiology, replication, disease 809

mechanisms, and prospective intervention strategies. Journal of Clinical 810

Investigation, 127(3), 737–749. https://doi.org/10.1172/JCI84417 811

Sim, S., Jupatanakul, N., & Dimopoulos, G. 2014. Mosquito immunity against arboviruses. 812

Viruses, 6(11), 4479–4504. https://doi.org/10.3390/v6114479 813

Sim, S., Jupatanakul, N., Ramirez, J. L., Kang, S., Romero-Vivas, C. M., Mohammed, H., & 814

Dimopoulos, G. 2013. Transcriptomic profiling of diverse Aedes aegypti strains 815

reveals increased basal-level immune activation in dengue virus-refractory 816

populations and identifies novel virus-vector molecular interactions. PLOS Neglected 817

Tropical Diseases, 7(7), e2295. https://doi.org/10.1371/journal.pntd.0002295 818

Sim, S., Ramirez, J. L., & Dimopoulos, G. 2012. Dengue virus infection of the Aedes aegypti 819

salivary gland and chemosensory apparatus induces genes that modulate infection and 820

blood-feeding behavior. PLOS Pathogens, 8(3), e1002631. 821


Souza-Neto, J. A., Sim, S., & Dimopoulos, G. 2009. An evolutionary conserved function of 823

the JAK-STAT pathway in anti-dengue defense. PNAS, 106(42), 17841–17846. 824

https://doi.org/10.1073/pnas.0905006106 825

Takatsu, Y., Nakamura, M., Stapleton, M., Danos, M. C., Matsumoto, K., O’Connor, M. B., 826

Shibuya, H., & Ueno, N. 2000. TAK1 participates in c-Jun N-Terminal Kinase 827

signaling during Drosophila development. Molecular and Cellular Biology, 20(9), 828

3015–3026. https://doi.org/10.1128/MCB.20.9.3015-3026.2000 829

Trapnell, C., Hendrickson, D. G., Sauvageau, M., Goff, L., Rinn, J. L., & Pachter, L. 2013. 830

Differential analysis of gene regulation at transcript resolution with RNA-seq. Nature 831

Biotechnology, 31(1), 46–53. https://doi.org/10.1038/nbt.2450 832


https://doi.org/10.1101/2020.03.01.972026


40

Trapnell, C., Roberts, A., Goff, L., Pertea, G., Kim, D., Kelley, D. R., Pimentel, H., Salzberg, 833

S. L., Rinn, J. L., & Pachter, L. 2012. Differential gene and transcript expression 834

analysis of RNA-seq experiments with TopHat and Cufflinks. Nature Protocols, 7(3), 835

562–578. https://doi.org/10.1038/nprot.2012.016 836

Tsetsarkin, K. A., Kenney, H., Chen, R., Liu, G., Manukyan, H., Whitehead, S. S., Laassri, 837

M., Chumakov, K., & Pletnev, A. G. 2016. A full-length infectious cDNA clone of 838

Zika virus from the 2015 epidemic in Brazil as a genetic platform for studies of virus-839

host interactions and vaccine development. MBio, 7(4), e01114-16. 840

https://doi.org/10.1128/mBio.01114-16 841

Vaidyanathan, R., & Scott, T. W. 2006. Apoptosis in mosquito midgut epithelia associated 842

with West Nile virus infection. Apoptosis, 11(9), 1643–1651. 843

https://doi.org/10.1007/s10495-006-8783-y 844

Waldock, J., Olson, K. E., & Christophides, G. K. 2012. Anopheles gambiae antiviral 845

immune response to systemic O’nyong-nyong infection. PLOS Neglected Tropical 846

Diseases, 6(3), e1565. https://doi.org/10.1371/journal.pntd.0001565 847

Wilder-Smith, A., & Gubler, D. J. 2015. Dengue vaccines at a crossroad. Science, 350(6261), 848

626–627. https://doi.org/10.1126/science.aab4047 849

Wilder-Smith, A., Gubler, D. J., Weaver, S. C., Monath, T. P., Heymann, D. L., & Scott, T. 850

W. 2017. Epidemic arboviral diseases: priorities for research and public health. The 851

Lancet Infectious Diseases, 17(3), e101–e106. https://doi.org/10.1016/S1473-852

3099(16)30518-7 853

Wilke, A. B. B., Beier, J. C., & Benelli, G. 2018. Transgenic mosquitoes – fact or fiction? 854

Trends in Parasitology, 34(6), 456–465. https://doi.org/10.1016/j.pt.2018.02.003 855


https://doi.org/10.1101/2020.03.01.972026


41

Wu, H., Wang, M. C., & Bohmann, D. 2009. JNK protects Drosophila from oxidative stress 856

by trancriptionally activating autophagy. Mechanisms of Development, 126(8–9), 857

624–637. https://doi.org/10.1016/j.mod.2009.06.1082 858

Xi, Z., Ramirez, J. L., & Dimopoulos, G. 2008. The Aedes aegypti Toll Pathway controls 859

dengue virus infection. PLOS Pathogens, 4(7), e1000098. 860


Xiao, X., Liu, Y., Zhang, X., Wang, J., Li, Z., Pang, X., Wang, P., & Cheng, G. 2014. 862

Complement-related proteins control the flavivirus infection of Aedes aegypti by 863

inducing antimicrobial peptides. PLOS Pathogens, 10(4), e1004027. 864


866


https://doi.org/10.1101/2020.03.01.972026


2 and apoptosis in mosquito salivary glandssalivary glands (SGs), from where they are expectorated...

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