Journal of Insect Physiology

Journal of Insect Physiology

http://dx.doi.org/10.1016/j.jinsphys.2014.08.003http://dx.doi.org/http://dx.doi.org/10.1016/j.jinsphys.2014.08.003

Review article

microRNAs as mediators of insect host-pathogen interactions and

immunity

Mazhar Hussain and Sassan Asgari*

Australian Infectious Disease Research Centre, School of Biological Sciences, The

University of Queensland, Brisbane, QLD 4072, Australia

* To whom correspondence should be addressed: Asgari, S, Australian Infectious Disease

Research Centre, School of Biological Sciences, The University of Queensland, St Lucia

QLD 4072, Tel: (617) 3365-2043; Fax: (617) 3365-1655; Email: s.asgari@uq.edu.au.

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Abstract 1

Insects are the most successful group of animals on earth, owing this partly to their very effective 2

immune responses to microbial invasion. These responses mainly include cellular and humoral 3

responses as well as RNA interference (RNAi). Small non-coding RNAs (snRNAs) produced 4

through RNAi are important molecules in the regulation of gene expression in almost all living 5

organisms; contributing to important processes such as development, differentiation, immunity as 6

well as host-microorganism interactions. The main snRNAs produced by the RNAi response 7

include short interfering RNAs, microRNAs and piwi-interacting RNAs. In addition to the host 8

snRNAs, some microorganisms encode snRNAs that affect the dynamics of host-pathogen 9

interactions. In this review, we will discuss the latest developments in regards to the role of 10

microRNA in insect host-pathogen interactions and provide some insights into this rapidly 11

developing area of research. 12

1. Introduction 13

1.1. Unfolding the multiple layers of insect responses to microbial infection 14

Multiple mechanisms of attack and counter-attack or host defences have evolved during the natural 15

process of the host-pathogen arms race. Animals defend themselves against microorganisms in two 16

major ways: innate and/or acquired immune systems. The main difference between the two systems 17

is the involvement of germline encoded factors for recognizing and destroying pathogens in innate 18

immunity, while in acquired immunity effector molecules are produced to recognize antigens 19

leading to the development of immunological memory. In the insect world, the immune system is 20

based on innate immune responses that can be further categorised into three main parts: (1) 21

pathogen immobilization, (2) core immune signalling pathways (Toll, Imd and JAK-STAT) and (3) 22

the RNAi pathway. Pathogen immobilization is a cellular response towards fungal and bacterial 23

infections in the form of phagocytosis, engulfment, melanisation, coagulation and autophagy 24

(Dushay, 2009; Jiravanichpaisal et al., 2006). These responses help in immobilizing pathogens, 25

which are then destroyed extracellularly by antimicrobial peptides (AMPs) or eliminated 26

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intracellularly. In response to systemic infection, AMPs are produced and secreted in the 27

haemolymph that directly attack microbes (Bulet et al., 1999). 28

Upon pathogen invasion, activation of immune signalling pathways usually ensues. In insects, these 29

are mainly the Toll, Imd and JAK/STAT pathways. The Toll pathway is activated through an 30

endogenous ligand namely the Nerve Growth Factor-related cytokine Spätzle (Spz) (Weber et al., 31

2003). Binding of Spz triggers a conformational change in the Toll receptor which activates the 32

downstream signalling pathway (Gangloff et al., 2008). The Toll-Spz interaction is the convergence 33

point for the activation of three pathogen recognition pathways; one triggered by fungal or bacterial 34

proteases, one induced by recognition of fungal cell wall and one activated by Lysine-type bacterial 35

peptidoglycan (El Chamy et al., 2008; Gottar et al., 2006; Ligoxygakis et al., 2002). Following 36

receptor activation, MyD88 proteins interact with Toll through their respective Toll/Interleukin-1 37

receptor domains and recruit the Drosophila melanogaster IRAK homologue, the kinase Pelle that 38

phosphorylates Cactus for degradation. Upon Cactus degradation, the NF-κB homologues, Dorsal 39

or Dif, are freed to move to the nucleus and regulate hundreds of target genes (Tauszig-Delamasure 40

et al., 2002; Towb et al., 2001). 41

The Immune deficiency (Imd) pathway is activated by meso-diaminopimelic acid (DAP)-type 42

bacterial peptidoglycans that form the cell wall of Gram-negative bacteria and some Gram-positive 43

Bacilli. Pathogen recognition in Imd occurs through the transmembrane receptor PGRP-LC and the 44

intracellular receptor PGRP-LE that are critical for recognizing extracellular bacteria. Flies deficient 45

in both of these proteins are unable to induce AMPs in response to Gram-negative bacteria and are 46

therefore highly susceptible to these infections (Kaneko et al., 2006). 47

The JAK/STAT signalling pathway has been shown to be involved in insect immunity, in particular 48

anti-viral responses and hemocyte development (Agaisse and Perrimon, 2004; Dostert et al., 2005); 49

although the mechanism of activation of the pathway by viruses is not known. The signal 50

transduction pathway consists of unpaired 1-3 ligands, the receptor domeless, the kinase JAK and 51

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the transcription factor STAT. Upon activation of the pathway, STAT is phosphorylated and 52

translocated into the nucleus, thus, activating several target genes. 53

1.2. RNA interference an effective anti-viral defence against viruses 54

RNA interference (RNAi) is another arm of the multilayered defence system in insects involved in 55

gene regulation, genome protection and anti-viral responses. Short interfering RNAs (siRNAs), 56

microRNAs (miRNAs) and piwi-interacting RNAs (piRNAs) are the three main types of small non-57

coding RNAs (snRNAs) central to the RNAi response pathway. Recently, a connection between the 58

JAK/STAT signalling transduction and the RNAi pathway has been established in mosquitoes as 59

part of the anti-viral immunity through the secretion of the signalling molecule, vago (Paradkar et 60

al., 2012). This suggests the effective communications between different pathways associated with 61

the innate immune responses in insects. Likewise, involvement of several miRNAs generated by 62

host cells or encoded by viral genomes in host-pathogen interactions have recently opened up new 63

research avenues in insect-microbe interactions. As the field of RNAi is rapidly expanding, in this 64

review article, we will mainly focus on the role of miRNAs in insect host-pathogen crosstalk and 65

immunity with the majority of examples from viruses as more relevant literature is available. For 66

other types of small RNAs and their role in anti-viral responses, readers are referred to recent 67

reviews (e.g. Sabin and Cherry, 2013; Bronkhorst and van Rij, 2014). 68

2. miRNA biogenesis at a glance 69

Since the discovery of the first miRNA from Caenorhabditis elegans just over two decades ago 70

(Lee et al., 1993) our understanding of miRNA biogenesis and its interaction with targets has 71

tremendously increased and is continuously evolving. In addition to the canonical pathway of 72

miRNA biogenesis, several non-canonical pathways have been described. Based on the canonical 73

pathway, a miRNA gene is transcribed in the nucleus by RNA polymerase II producing the primary 74

miRNA (pri-miRNA) transcript that could be variable in length but contains one or several stem-75

loop structures (Fig. 1). A nuclear residing ribonuclease, Drosha, in association with Pasha (in 76

insects) cleaves the stem-loop producing a ∼70 nt hairpin precursor miRNA (pre-miRNA) that is 77

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translocated into the cytoplasm by Exportin-5. In the cytoplasm, the hairpin head is cleaved by 78

another ribonuclease enzyme, Dicer, producing a short duplex RNA molecule. In insects, two dicer 79

proteins have been identified: Dicer-1 that is mainly involved in the miRNA biogenesis, and Dicer-80

2 that produces siRNAs. The miRNA duplex triggers the formation of the RNA induced silencing 81

complex (RISC) in which an Argonaut (Ago) protein is the main component. Most miRNAs are 82

loaded into RISC that includes Ago1 and siRNAs into Ago2 complexes; however, recent research 83

indicates that miRNAs can also be loaded into Ago2 complexes (Ghildiyal et al., 2010; Rubio and 84

Belles, 2013), especially as the insect ages (Abe et al., 2014). Once the miRNA duplex becomes 85

associated with the RISC, one of the strands is degraded (referred to as miRNA* strand) and the 86

other one forms the mature miR-RISC complex. In some instances, the miRNA* strand is retained 87

and loaded into Ago2 and can be functional (Ghildiyal et al., 2010). Finally, the mature miRNA 88

guides the complex to target mRNA(s) in a sequence specific manner, leading to post-89

transcriptional gene regulation, or the complex might be transported into the nucleus where the 90

miRNA normally binds to the promoter regions of target genes regulating gene expression at the 91

transcriptional level. Notably, each miRNA may have multiple mRNA targets and each mRNA may 92

be targeted by multiple miRNAs. 93

3. Role of microRNAs as moderators of host-pathogen interactions 94

While there is a lot known about the role of miRNAs in insect development, less is understood 95

about their function in host-pathogen interactions. Nevertheless, accumulating evidence in recent 96

years has suggested their effects on host-microorganisms interactions and cross-kingdom 97

communications. For example, several cellular as well as viral miRNAs have been found to be 98

involved in assisting or limiting virus replication (see below). What is commonly observed in 99

various host-pathogen systems is the differential abundance of host miRNAs following an infection, 100

which varies at different stages of infection and type of pathogen involved. Concurrently, a large 101

number of host genes are found down- or upregulated that could be regulated by miRNAs or other 102

types of snRNAs. These changes in miRNA/mRNA profiles could be part of the host anti-pathogen 103

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response or controlled by pathogen manipulation, including miRNAs expressed by viruses. Below, 104

we will examine several relevant examples of the role miRNAs play in mediating host-pathogen 105

interactions. 106

3.1. Host-virus interactions 107

3.1.1. Impact of viral infection on the host miRNA profile 108

In recent years, based on deep sequencing or microarray analysis, several publications have 109

appeared reporting changes in the expression levels of cellular miRNAs in insects in response to 110

pathogen infection, although in the majority of these cases functional analyses are still lacking. For 111

example, the abundance of a large number of miRNAs was changed in Spodoptera frugiperda (Sf9) 112

cells following infection with Autographa californica multiple nucleopolyhedrovirus (AcMNPV). 113

These included several upregulated miRNAs (e.g. miR-184, miR-998 and miR-10) at 24 h post-114

infection (hpi), with expression of some of those downregulated at 72 hpi (Mehrabadi et al., 2013). 115

In addition, three of the novel S. frugiperda miRNAs identified in the study showed upregulation 116

upon virus infection. Relevant to baculoviruses, Helicoverpa armigera single nucleopolyhedrovirus 117

(HaSNPV) was shown to cause changes in the abundance of host miRNAs (Jayachandran et al., 118

2013). Among those, miR-14 that positively regulates H. armigera ecdysone receptor (Ha-EcR) in 119

this insect was downregulated upon infection providing an example of virus-medicated 120

manipulation of host physiology. 121

In Bombyx mori larvae infected with B. mori cytoplasmic polyhedrosis virus (BmCPV), 58 122

miRNAs were found significantly up or downregulated in the midgut as compared with that of non-123

infected larvae at 72 and 96 hpi (Wu et al., 2013). Based on gene ontology (GO) analysis, the 124

putative target genes of six of the differentially expressed miRNAs (bmo-miR-275, miR-14, miR-125

1a, N-50, N-46 and N-45) were associated with response to stimulus and immune system process. 126

The authors speculated that significant increases in miR-275 levels following BmCPV infection 127

could explain the stunted growth phenotype in virus-infected B. mori, since the miRNA has been 128

shown to regulate development during metamorphosis (Liu et al., 2010). In another study, 129

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microarray analysis showed differential expression of at least 20 miRNAs in a Heliothis zea fat 130

body (HzFB) cell line upon infection with Heliothis virescens ascovirus (HvAV3e) with three of 131

them (Hz-miR-24, -22, -7) significantly downregulated (Hussain and Asgari, 2010). 132

Arthropod-borne viruses (arboviruses) have also been demonstrated to alter the host miRNA 133

profile. For example, in Culex quinquefasciatus infected with West Nile virus (WNV) 134

downregulation of miR-989 and upregulation of miR-92 was noted (Skalsky et al., 2010). Deep 135

sequencing analysis of Aedes aegypti mosquitoes infected with dengue virus (DENV) at 2, 4 and 9 136

days post-infection revealed differential abundance of 35 miRNAs. Among those miR-5119-5p, 137

miR-34-3p, miR-87-5p and miR-988-5p were upregulated (Campbell et al., 2013). As more of these 138

studies emerge, it will become possible to carry out comparative studies to find out which miRNAs 139

are commonly involved in host-pathogen responses in different host insects and whether particular 140

miRNAs could be specifically regulated in response to particular groups of pathogens (e.g. viruses, 141

bacteria, etc). 142

3.1.2. Cellular miRNAs target viral genes 143

Given alteration of the abundance of host miRNAs following infection, it is likely that some of 144

those could directly target viral genes. For instance, in H. zea HzFB cells, miR-24 downregulates 145

DNA dependent RNA polymerase II RPC2 (DdRP; ORF64) and DdRP β subunits (DdRP; ORF82) 146

of HvAV3e (Hussain and Asgari, 2010). Interestingly, this miRNA is selectively downregulated 147

early in infection allowing transcription of viral genes required for virus replication suggesting that 148

the virus may actively suppress host miRNAs that directly target its genes. Relevant to this, in B. 149

mori, several targets of the cellular bmo-miR-8 were predicted in Bombyx mori 150

nucleopolyhedrovirus (BmNPV) genes (see below). Inhibition of this miRNA led to 8-fold increase 151

in viral titre in the fat bodies of infected larvae (Singh et al., 2012). 152

A recent paper highlighted the possibility of utilizing cellular miRNAs as a tool to target virus 153

genes as a control strategy (Zhang et al., 2014a). This work was conducted in B. mori against 154

BmNPV using three cellular miRNAs; bmo-miR-279, bmo-miR-92b, and bmo-miR-2764. 155

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Accordingly, the cellular pre-miRNA sequences were used as backbone to replace the natural 156

miRNA duplex sequences with artificial siRNA duplex sequences targeting the viral lef-11 gene, 157

which is involved in virus replication (Zhang et al., 2014a). For this, a virus inducible artificial 158

miRNA (amiRNAs) expression cassette was constructed targeting the viral gene. Infection with the 159

virus stimulates the baculovirus-induced promoter 39K included in the expression cassette leading 160

to production of pre-amiRNA and eventually formation of siRNA duplexes. The mature amiRNAs 161

successfully inhibited BmNPV proliferation by downregulating the lef-11 gene in silkworm BmN-162

SWU1 cells. It will be interesting to see if the approach could be utilized in whole insects to inhibit 163

BmNPV replication without compromising the insect’s fitness. 164

Viruses might also subvert host miRNAs to their own benefit. For example, in American eastern 165

equine encephalitis virus, binding of the mammalian host miR-142-3p to the 3’UTR of the virus 166

genome in myeloid-lineage cells restricts replication of the virus in this particular cell type leading 167

to minimal interferon induction and hence suppression of the host innate immune system (Trobaugh 168

et al., 2013). Interestingly, this study showed that these same binding sites in the virus 3’UTR are 169

also required for efficient infection of the mosquito vector Aedes taeniorhynchus. However, it is not 170

known whether a mosquito miRNA might interact with the sequences facilitating its replication in 171

the insect host similar to the mammalian host. 172

3.1.3 Virus-encoded miRNAs can target host genes 173

Recent investigations have revealed that insect virus miRNAs, similar to those reported from human 174

viruses, might target and regulate host genes to establish infection and successfully replicate. 175

However, the information is very limited in respect to insect viruses. The impact of the miRNA 176

might be via regulation of a specific gene leading to the modification of a particular pathway or 177

might cause a global reduction in the host miRNA depending on the target genes. For example, 178

baculovirus encoded BmNPV-miR-1 downregulates the nuclear Exportin-5 cofactor Ran resulting 179

in the blockage of the export of cellular pre-miRNAs into the cytoplasm generally affecting the 180

abundance of cellular mature miRNAs in infected cells (Singh et al., 2012) (Table 1). This may 181

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affect regulation of cellular as well as viral genes. For instance, predicted targets of four selected 182

host miRNAs affected by downregulation of Ran via BmNPV-miR-1 were mostly related to 183

immunity. In addition, targets of these miRNAs were also predicted in the viral genome. For 184

example, bmo-miR-8, which is downregulated following infection, targets the immediate early-1 185

(ie1) gene (Singh et al., 2012). Downregulation of the miRNA is obviously to the benefit of the 186

virus. However, it remains to be elucidated how viral miRNAs are produced while the host miRNA 187

biogenesis pathway is interrupted. 188

In addition, a WNV (Kunjin strain, WNVKUN) encoded miRNA, KUN-miR-1, derived from the 189

3’SL stem-loop located at the end of the 3’UTR of the virus in mosquito cells positively regulates 190

the host GATA-binding protein 4 (GATA-4) (Hussain et al., 2012) (Table 1). RNAi-mediated 191

silencing of GATA-4 led to a significant reduction in virus RNA replication, suggesting that 192

GATA-4-induction via KUN-miR-1 plays an important role in virus replication. However, the 193

mechanism remains to be explored. Members of the GATA family are transcription factors that 194

regulate various biological processes in insects and vertebrates (Shapira et al., 2006). In particular, 195

GATA-4 has been shown to be involved in regulation of egg development, vitellogenesis, and lipid 196

metabolism in mosquitoes (Cheon et al., 2006; Park et al., 2006). 197

3.1.4. Virus-encoded miRNAs target viral own genes 198

Targeting virus genes by virus-encoded miRNAs appears to be common among different groups of 199

viruses to regulate their own replication, enter latency or suppress host anti-viral responses. The 200

first insect virus-encoded miRNA, HvAV-miR-1, was reported from the ascovirus HvAV-3e 201

originating from the major capsid protein (MCP) gene of the virus (Hussain et al., 2008). The virus-202

encoded miRNA was shown to autoregulate virus replication late in infection by downregulating 203

the virus’s own DNA polymerase (Table 1). In a recent report, it was revealed that BmNPV 204

expresses a miRNA, bmnpv-miR-3, early in infection, which targets and regulates expression of the 205

viral DNA binding protein (P6.9) as well as other late genes (Singh et al., 2014) (Table 1). This may 206

confer several benefits to the virus: (1) autoregulation of virus proliferation to avoid over 207

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replication, (2) regulation of the expression levels of the late genes to suppress their expression 208

early in infection, (3) modulation of the host immune response by regulating virus proliferation. In 209

regards to the latter, the investigators showed that virus load correlates with expression levels of the 210

AMP gloverin, which has been shown to be upregulated upon baculovirus infection and inactivates 211

budded viruses (Moreno-Habel et al., 2012). 212

A miRNA from the baculovirus AcMNPV, AcMNPV-miR-1, was found to be expressed from the 213

negative strand of the viral genome and target ODV-E25 transcripts on the opposite strand with 214

100% complementarity (Zhu et al., 2013) (Table 1). ODV-E25 is a late gene encoding a ∼25 kDa 215

structural protein of occlusion derived virus (ODV) and also found on the budded virus (BV) 216

envelope (Russell and Rohrmann, 1993; Wang et al., 2010). The protein is conserved in 217

baculoviruses that infect lepidopterans and important in ODV formation and BV infectivity (Chen 218

et al., 2012). AcMNPV-miR-1 was expressed late in infection starting from 12 hpi. Bioinformatics 219

analysis identified eight potential targets for AcMNPV-miR-1 in the virus genome with ODV-E25 220

showing 100% complementarity since it is expressed on the complementary strand of the miRNA. 221

Validation experiments confirmed that AcMNPV-miR-1 causes cleavage of ODV-E25 mRNA and 222

its higher expression levels coincided with reduced levels of the target mRNA levels. Notably, 223

using AcMNPV-miR-1 mimic, it was found that the small RNA had no effect on budded virus 224

production but affected their infectivity by reducing production of ODV-E25 (Chen et al., 2012). 225

Furthermore, the mimic increased ODV formation, and inhibition of AcMNPV-miR-1 dramatically 226

reduced the number of occluded ODVs. This implied that the miRNA promotes ODV formation. 227

In another example, a functional viral small RNA (DENV-vsRNA-5) produced from a stem-loop of 228

DENV-2 3’UTR was found in virus-infected Ae. aegypti mosquitoes and cell lines. The vsRNA was 229

shown to target the virus genome at NS1 gene region (Hussain and Asgari, 2014) (Table 1). 230

Synthetic RNA inhibitor or mimic of DENV-vsRNA-5 resulted in significant increases or decrease 231

in viral replication, respectively. The autoregulation of virus replication may facilitate low but 232

transmissible level of virus infection in the mosquito vector. 233

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In a number of vertebrate herpesviruses, it has been shown that virus-encoded miRNAs are 234

important in switching between lytic and latent phases (reviewed in Cullen, 2011). In insect viruses, 235

Heliothis zea nudivirus (HzNV-1) expresses only one non-protein coding gene, the persistency-236

associated gene 1 (pag1), during the latency phase (Wu et al., 2011) (Table 1). Two miRNAs 237

encoded by pag1 were shown to downregulate the viral early gene hhi1, which allows the virus to 238

enter the latent infection stage in cell culture. In the absence of the pag1 gene, the virus does not 239

enter latency phase. It will be interesting to find out whether virus-encoded miRNAs play any role 240

in latency or persistent infection observed in other insect viruses. 241

3.2. Bacterial infection can modulate host miRNAs in insects 242

A number of studies have revealed that similar to viruses bacterial infections can lead to changes in 243

cellular miRNAs in different insect species, with information again being limited. For example, 244

association of the endosymbiotic bacterium Wolbachia (wMelPop strain) with alterations in cellular 245

miRNAs and miRNA pathway proteins in Ae. aegypti has been documented (Hussain et al., 2011; 246

Hussain et al., 2013a; Mayoral et al., 2014; Osei-Amo et al., 2012; Zhang et al., 2013). Of note, this 247

strain causes life-shortening in the mosquito as well as inhibit replication of several vector-borne 248

pathogens especially RNA viruses such as DENV, WNV and Chikungunya virus (Moreira et al., 249

2009a; Moreira et al., 2009b). Initially, microarray analysis revealed that a number of mosquito 250

miRNAs were differentially expressed with two of them, aae-miR-2940 and -309a, showing high 251

abundance in Wolbachia-infected mosquitoes as compared with non-infected ones (Hussain et al., 252

2011). A target sequence of aae-miR-2940 was shown to reside in the 3’UTR of the mosquito’s 253

metalloprotease m41 ftsh transcripts; however, the miRNA was shown to positively regulate the 254

metalloprotease transcript levels. miRNA inhibition and silencing of the target gene revealed that 255

the metalloprotease gene and aae-miR-2940 are important in the maintenance of Wolbachia density 256

(Hussain et al., 2011). In a follow-up study, aae-miR-2940 was found to negatively regulate the 257

transcript levels of its other target, the DNA methyltransferase (AaDnmt2) (Zhang et al., 2013). 258

Overexpression of AaDnmt2 in Wolbachia-infected mosquito cells led to significant reductions in 259

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Wolbachia density, but significantly enhanced replication of DENV. This suggested that 260

downregulation of AaDnmt2 by aae-miR-2940 may contribute to inhibition of DENV replication in 261

Wolbachia-infected mosquitoes. Notably, significant hypomethylation of Ae. aegypti genome was 262

recognized in wMelPop infected mosquitoes (Ye et al., 2013). Given that AaDnmt2 is the only 263

DNA methyltransferase gene in the mosquito’s genome, Wolbachia may epigenetically regulate 264

expression of many host genes by suppression of AaDnmt2 via aae-miR-2940 induction. Further, 265

the instances above provide an example of a miRNA regulating more than one gene in opposite 266

ways, both benefiting the endosymbiotic bacterium. 267

Function of another Wolbachia induced cellular miRNA, aae-miR-12, was confirmed in regulation 268

of two target genes, DNA replication licensing factor MCM6 and monocarboxylate transporter 269

MCT1, and shown to be important for the maintenance of Wolbachia density in mosquito cells 270

(Osei-Amo et al., 2012). Recently, it was uncovered that Wolbachia blocks shuttling of Ago1 to the 271

nucleus through upregulation of aae-miR-981 (Hussain et al., 2013a) and that leads to changes in 272

the distribution of miRNAs in infected cells (Mayoral et al., 2014). Interestingly, this miRNA 273

downregulates expression of importin β-4 that facilitates Ago1 distribution to the nucleus (Hussain 274

et al., 2013a). 275

A study on honeybees (Apis mellifera) showed activation of AMPs and regulation of a large number 276

of immune-related genes in response to infection by the Gram-negative bacterium Serratia 277

marcescens (Lourenco et al., 2013). Analysis of miRNAs from the infected bees also revealed 278

differential expression of cellular miRNAs upon bacterial infection. Among the downregulated 279

miRNAs were ame-bantam, miR-12, -34, -184, -278, -375, -989 and -1175, while some were 280

upregulated such as let-7, miR-2, -13a, and -92a. Predicted interactions revealed that the miRNA 281

could potentially regulate genes involved in Imd, Toll, JNK, and JAK/STAT pathways, regulation 282

of AMPs and melanization. However, functional analysis and validation of these interactions 283

remain to be investigated. 284

285

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286

4. Role of miRNAs in regulation of insect immunity 287

Despite many reports demonstrating the role of miRNAs in vertebrate immunity, the information 288

pertinent to their role in insect immunity is scant and mostly based on bioinformatics predictions or 289

correlating miRNA abundance with that of mRNAs. For example, Fullaondo et al. identified over 290

70 miRNAs that could be involved in regulating immune related pathways such as Toll, Imd and 291

JAK/STAT, melanization and JNK pathways using bioinformatics approaches (Fullaond and Lee, 292

2012). Similarly, a computational approach was used to identify Anopheles gambiae miRNAs that 293

may be involved in regulating immune responses. Two miRNAs, aga-miR-2304 and aga-miR-2390, 294

were predicted to target the genes encoding suppressin and prophenoloxidase 295

(Thirugnanasambantham et al., 2013). However, these predictions await experimental confirmation 296

of miRNA-target interactions. In the flour beetle Tribolium castaneum, microbial challenge and 297

stress led to alteration of the beetle’s miRNA profile (Freitak et al., 2012). Induction of immunity 298

by injection of peptidoglycan into the beetles resulted in differential expression of 59 miRNAs from 299

which 21 were upregulated. Similarly, changes in the abundance of Manduca sexta miRNAs were 300

observed following immune challenge involved in pattern recognition, activation of 301

prophenoloxidase, synthesis of AMPs and conserved immune signal transduction pathways (Zhang 302

et al., 2014b). In An. gambiae, the possibility of involvement of miRNAs in defence against 303

Plasmodium berghei invasion was demonstrated as Dicer-1 and Ago1 depletion in the mosquitoes 304

led to increased sensitivity of the mosquitoes to P. berghei infection (Winter et al., 2007). However, 305

no specific miRNA was identified responsible for the effect observed. 306

One of the miRNAs whose role in insect immunity has experimentally been established is miR-8 307

which negatively regulates expression of the AMPs, such as Drosomycin and Diptericin, in D. 308

melanogaster (Choi and Hyun, 2012). This allows maintenance of low level of AMPs expression 309

under non-infection conditions, enabling the homeostasis of immunity. However, in miR-8 null 310

Drosophila, the transcript levels of the AMPs significantly increased; although it was not shown 311

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how miR-8 regulates AMPs. It was predicted that the miRNA could potentially target the transcripts 312

of Gram-negative binding protein 3 (GNBP3), an inducer of the Toll pathway upon fungal 313

infection, and Pvf, a modulator of the JNK pathway. Negative regulation of AMPs by miR-8 was 314

also demonstrated in the larvae of Plutella xylostella (Etebari and Asgari, 2013). This study further 315

showed that miR-8 positively regulates the transcript levels of serpin-27, inhibiting activation of the 316

Toll pathway and hence low level of production of AMPs when there is no infection/parasitization. 317

Upon infection, miR-8 levels are reduced leading to a decrease in serpin-27 levels and consequently 318

activation of the Toll pathway. 319

In Aedes albopictus (C6/36) cells, the abundance of the mosquito-specific aae-miR-2940 was 320

shown to be selectively reduced following WNVKUN infection, a flavivirus (Slonchak et al., 2014). 321

As indicated above, aae-miR-2940 positively regulates the metalloprotease ftsh in mosquitoes 322

(Hussain et al., 2011), and this protein was found to enhance WNVKUN replication (Slonchak et al., 323

2014). Reduced aae-miR-2940 levels led to lower metalloprotease levels and consequently reduced 324

virus titers. This suggested that reduction in the miRNA levels might be an anti-viral response to 325

WNV infection. Similarly, reduced levels of aae-miR-2940 were detected in Ae. albopictus Singh’s 326

cell line infected with Chikungunya virus, an alphavirus (Shrinet et al., 2014). These results suggest 327

that reduced levels of aae-miR-2940 might be a common response to viral infection. 328

In Ae. aegypti, two immune related genes, cactus and REL1, were found to be the target of a blood 329

meal induced miRNA, aae-miR-375 (Hussain et al., 2013b). Interestingly, cactus, which is an 330

inhibitor of the Toll pathway, was positively regulated by aae-miR-375, whereas REL1, which is an 331

activator of AMPs, was downregulated by the same miRNA. The increase in cactus and decrease in 332

REL1 was found to have a positive effect on DENV replication since AMPs have been shown to 333

negatively affect DENV (Luplertlop et al., 2012; Xia et al., 2008). Coincidence of negative 334

regulation of AMPs upon blood feeding may provide a fitness advantage for the virus. 335

Finally, it is well-known that immune related genes evolve faster than non-immune genes due to 336

constant evolutionarily pressures on hosts to neutralize pathogen novelties. Interestingly, when 337

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analysing 50 genes involved in the Drosophila Toll and Imd signalling pathways, Han et al realized 338

a strong negative correlation between the rate of evolution of the immune proteins and the number 339

of their regulatory miRNAs, suggesting that genes regulated by more miRNAs evolve slower due to 340

stronger functional constraints (Han et al., 2013). This suggests the role of miRNAs in the evolution 341

of immune related genes in insects. 342

5. Conclusions 343

In the last decade, we have witnessed major developments in our understanding of the biogenesis 344

and the function of miRNAs in various aspects of insect biology. Most of these efforts have focused 345

on the role of miRNAs in development with the main focus on D. melanogaster. Recently, there 346

have been a number of publications on the role of these small RNAs in host-pathogen interactions. 347

As an initial step towards understanding their role in the interactions, the majority of investigators 348

have focused on the effect of infection on the host miRNA profile with several being identified as 349

differentially abundant miRNAs. It is of great interest to see these efforts to be followed up by 350

empirical approaches to demonstrate the functional role of these differentially abundant miRNAs in 351

host-pathogen interactions and insect immunity. In addition, a number of virus-encoded miRNAs 352

have been characterized that either regulate virus replication or modulate host genes/miRNAs to 353

facilitate their replication. 354

Extending research on miRNAs beyond scientific curiosity and basic research to utilize them in 355

applied insect biology with the aim to reduce crop damage by agricultural pests, inhibit mortality in 356

beneficial insects and inhibit/limit transmission of vector-borne pathogens should be considered as 357

future prospects. However, these efforts may encounter regulatory limitations when their 358

applications are to extend to the field arena since genetic manipulations of insects or plants are most 359

likely inevitable. Some attempts have already been made in this area with promising results either in 360

cell culture or at the laboratory scale. miRNA research is certainly a very fast moving area of 361

research and we shall see a strong progress in this field in the near future. 362

363

16

364

Acknowledgements 365

The authors would like to acknowledge Dr Karyn Johnson from the University of Queensland for 366

critically reading this review. The authors would like to acknowledge the Australian Research 367

Council (DP110102112, DE120101512) and National Health and Medical Research (APP1062983, 368

APP1027110) for providing funding that contributed to some of the findings reported in this review 369

from our lab. 370

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20

Table 1. miRNAs encoded in viral genomes that target viral own or host transcripts. 548

549 Virus miRNA and its

origin Target in viral/host

genome Effect on virus

infection Reference

DNA virus Heliothis virescens

ascovirus (HvAV3e)

HvAV-miR-1 is derived from MCP

Downregulates DNA polymerase 1

limits viral DNA replication during late infection

(Hussain et al., 2008)

Heliothis zea nudivirus (HzNV-1)

hv-miR-246 and -2959 from pag-1 gene

Both miRNAs target hhi1 transcript

help virus to enter latent phase

(Wu et al., 2011)

Bombyx mori nucleopolyhedrovirus (BmNPV)

BmNPV-miR-1 downregulates Exportin-5 cofactor Ran resulting in the blockage of pre-miRNA export into the cytoplasm

(Singh et al., 2012)

BmNPV

bmnpv-miR-3 is derived from the plus strand early in infection

targets 3′UTR of BmNPV DNA binding protein (P6.9) mRNA that transcribes from the opposite strand at the same locus

helps virus to escape the early immune response from the host

(Singh et al., 2014)

Autographa californica nucleopolyhedrovirus (AcMNPV)

AcMNPV-miR-1 is located on the negative strand of the core gene ODV-E25

downregulates ODV-E25 mRNA on positive strand

results in a reduction of infectious budded virions and accelerates the formation of occlusion-derived virions

(Zhu et al., 2013)

RNA virus Dengue virus-2

(DENV-2) DENV-vsRNA-5 is produced from a stem-loop in the 3’UTR of DENV-2

NS1 gene region Autoregulates viral replication late in infection

(Hussain and Asgari, 2014)

West Nile virus (Kunjin strain, WNVKUN)

KUN-miR-1 is derived from the 3’SL located at the end of the 3’UTR

Targets and induces transcript level of cellular GATA-4 transcription factor

Enhances viral RNA replication

(Hussain et al., 2012)

550

21

Figure legend: 551

Figure 1: Canonical pathway of miRNA biogenesis. miRNA genes are transcribed in the nucleus 552

mostly by RNA polymerase II in the form of primary miRNA (pri-miRNA) that contains one or 553

more stem-loop structures, and similar to mRNAs it is 5’capped and polyadenylated. The stem-loop 554

is cleaved from pri-miRNA by Drosha in association with Pasha to form the precursor miRNA (pre-555

miRNA which is then transported into the cytoplasm facilitated by Exportin-5/Ran. The hairpin 556

head is removed either by Dicer-1 or Ago2 resulting in a miRNA duplex which initiates the 557

formation of the RNA induced silencing complex (RISC). One of the strands in usually degraded 558

(miRNA*) and the other strand guides the miR-RISC complex to target mRNA (posttranscriptional 559

regulation) or transported into the nucleus where it binds to a promoter region and regulate gene 560

transcription (transcriptional gene regulation, TGR). The miRNA* may also be loaded into one of 561

the Ago proteins and perform a function by interacting with target sequences. The outcome of 562

miRNA-target interaction may vary from mRNA degradation, translational repression to 563

upregulation of target transcript levels. 564

565

566

567

Graphical abstract

AAAAA

5’ cap

Ago2 TRBP

AAAA 5' cap pri-miRNA

Drosha

Pasha pre-miRNA

Exportin-5

miRNA duplex

Dicer-1

miR-RISC

miR*-RISC

TGR

RNA Pol II

AAAAA

5’ cap

nucleus

cytoplasm

24

Highlights 572

•••• MiRNAs are small non-coding RNAs that regulate gene expression. 573 •••• Following infection, miRNA profile of the host is altered either due to defence or manipulation by the 574

pathogen. 575 •••• Virus-encoded miRNAs regulate host or viral genes facilitating establishment of infection. 576 577

578