The immune response of the small interfering RNA pathway in the defense against bee viruses

The immune response of the small interfering RNApathway in the defense against bee virusesJinzhi Niu, Ivan Meeus, Kaat Cappelle, Niels Piot andGuy Smagghe

Available online at www.sciencedirect.com

ScienceDirect

Most bee viruses are RNA viruses belonging to two major

families of Dicistroviridae and Iflaviridae. During viral infection,

virus-derived double stranded RNAs activate a major host

innate immune pathway, namely the small interfering RNAs

pathway (siRNA pathway), which degrades the viral RNA or the

viral genome. This results in 21–22 nucleotide-long virus-

derived siRNAs (vsiRNAs). Recent studies showed that

vsiRNAs, matching to viruses from the family of Dicistroviridae

and Iflaviridae, were generated in infected bees. Moreover,

higher virus titers in honeybees also resulted in higher amounts

of vsiRNAs, demonstrating that the siRNA response is

proportional to the intensity of viral infection. Intriguingly, non-

specific dsRNA could also trigger an immune response, leading

to the restriction of the viral infection, however this mechanism

is still unclear. Other findings demonstrated that bees can be

protected through introducing virus specific-dsRNA to activate

the siRNA response against the target virus. The latter is

highlighting a new strategy to tackle bee viruses.

Addresses

Department of Crop Protection, Faculty of Bioscience Engineering,

Ghent University, Coupure Links 653, B-9000 Ghent, Belgium

Corresponding author: Smagghe, Guy ([email protected])

Current Opinion in Insect Science 2014, 6:22–27

This review comes from a themed issue on Pests and resistance

Edited by Guy Smagghe and Luc Swevers

For a complete overview see the Issue and the Editorial

Available online 28th September 2014

http://dx.doi.org/10.1016/j.cois.2014.09.014

2214-5745/# 2014 Elsevier Inc. All rights reserved.

IntroductionAs obligate intracellular parasites, the replication of

viruses depends on the host and this interplay leads to

a constant ‘arms-race’: on the one hand the host’s immune

system tries to eliminate viral infections, but on the other

hand viruses try to surpass the host’s immune system in an

attempt to successfully infect the host. In addition, the

host has to allocate resources for the immune response

during pathogen invasion, which has its trade-off against

other physiological functions [1,2]. After a virus has

breached the physical and chemical barriers, insects rely

Current Opinion in Insect Science 2014, 6:22–27

on their innate immunity responses, such as RNA inter-

ference (RNAi), Toll, Imd, Jak-Stat and autophagy

pathways to combat viruses (for reviews see [3–5]). In a

well-preserved mechanism, RNAi is activated by double-

stranded RNA (dsRNA) which leads to the down-regulation

of gene expression at a post-transcriptional level. The RNAi

mechanism can be divided into three major pathways based

on the type of the small RNAs produced: microRNAs

(miRNAs), small interfering RNAs (siRNAs) and Piwi-inter-

acting RNAs (piRNAs) [5,6]. During viral infection, the

siRNA pathway is triggered by virus-derived dsRNAs, which

finally results in cleavage of viral RNA.

Insect pollination is an indispensable component of glo-

bal food production, which is estimated to have an

economic value of s153 billion [7]. Recent declines in

bees raise the concerns about a pollination shortage and

the spreading of viral diseases is one of the main suspects

responsible for these losses [8,9]. Under natural con-

ditions, bee viruses are found in an array of wild and

domesticated pollinators, forming an intricate multi-host

network where the viruses can be transmitted among the

different pollinators [8,10,11]. The transmission predo-

minantly occurs due to common food sources, such as

pollen and nectar, shared by the pollinator community.

Moreover, multiple viruses are also present in bees; up to

3–4 viruses can infect the same bee [11,12]. These com-

plex characteristics of viral infections challenge the bee’s

innate immune system. In addition, stressors like insec-

ticides and Varroa mites (a viral vector), could also affect

the immune response of the bee, facilitating viral in-

fection [13��,14,15]. Here we focus on the current

research progress in the understanding of the siRNA

pathway of bees, its response during viral infection,

and its applications in the protection of pollinator health.

The molecular mechanism of the siRNApathway and its antiviral actionDuring viral infection, virus-related dsRNAs are gener-

ated, such as replication intermediates, viral genome

itself with dsRNA structure, virus-encoded siRNAs and

viral transcript-genome hybrids [5,16]. Those virus-

related dsRNAs are recognized by the host and processed

into 21–22 nucleotide-long vsiRNAs by a ribonuclease III

(RNase III) enzyme called Dicer-2; then the vsiRNAs are

loaded onto Argonaute (Ago-2), forming the RNA-

induced silencing complex (RISC). Then the passenger

strand of the vsiRNAs is degraded and the other strand

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siRNA pathway against bee viruses Niu et al. 23

(guide strand) serves as a viral sequence-specific guide to

degrade viral RNA by complementary binding (Figure 1)

[16].

To achieve an effective antiviral immunity, it is also crucial

to pass on this local siRNA antiviral immunity of infected

cells to uninfected cells. This normally requires uptake of

dsRNA by uninfected cells [17]. Unlike vertebrates,

insects lack an adaptive immune system but the uptake

of virus-related dsRNA by uninfected cells would prime

these cells for an effective immune response upon viral

infection. Currently, two dsRNA uptake mechanisms are

described in insects, transmembrane channel-mediated

uptake and endocytosis-mediated uptake [18,19]. Little

is known about dsRNA uptake or the spreading of RNAi

signals in bees, but it seems that honeybees are inefficient

in spreading RNAi signals such as siRNAs across tissues

[20]. Moreover, in most cases the silencing of genes

in honeybees or bumblebees requires high amounts of

Figure 1

Viral infection

Virus-relateddsRNA

VsiRNAs

Virus RNAclearance

Ago-2

RISC

Dic

er-2

...AAAA

71

53

2310

38

8998

0.5

0.5

100

85

Response of the siRNA pathway to viral infection and phylogenetic trees of D

The sequences from Bombus terrestris (XP_003394821.1), B. impatiens (XP

saltator (EFN79336.1), Apis florea (XP_003697097.1), A. dorsata (XP_006623

(EGI69620.1), Microplitis demolitor (EZA46212.1), Cerapachys biroi (EZA6155

for Dicer-2 based on RNase III and PAZ domains; the sequences from B. te

(XP_395048.4), A. dorsata (XP_006625010.1), A. dorsata (XP_006625011.1),

(XP_008214884.1), A. echinatior (EGI64275.1), Camponotus floridanus (EFN6

Piwi domains.

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dsRNA (Table 1), especially compared with that in the

desert locust Schistocerca gregaria [21]. The latter may

suggest a low dsRNA uptake efficiency in bees. SID-1, a

multispan transmembrane protein, is speculated to be an

important factor in systemic RNAi, however, its function in

bees is still unknown. Although it should be remarked that

after silencing the honeybee Toll-related receptor 18W by

a feeding-soaking delivery of dsRNA, the expression level

of the transmembrane protein SID-1 increased 3.4-fold,

while the target gene’s expression was decreased 60-fold

[22]. The latter indicates a role of SID-1 in dsRNA uptake,

but solid evidence is still lacking. Therefore, two questions

concerning dsRNA uptake in bees need to be addressed in

future studies: (i) What is the mechanism for dsRNA

uptake? and (ii) What is the contribution of virus-related

dsRNA uptake in controlling viral infection?

Silencing genes in honeybees and bumblebees has

been achieved by administrating gene-specific dsRNA

Apis dorsata Discer-2

Apis dorsata Ago-2-isoform 1

Apis dorsata Ago-2-isoform 2

Apis mellifera Ago-2

Bombus terrestris Ago-2

Bombus impatiens Ago-2

Nasonia vitripennis Ago-2

Acromyrmex echinatior Ago-2

Camponotus floridanus Ago-2

Cerapachys biroi Ago-2

Megachile rotundata Ago-2

Megachile rotundata Piwi-Ago

Apis mellifera Dicer-2

Apis florea Dicer-2

Bombus terrestris Dicer-2

Bombus impatiens Dicer-2

Megachile rotundata Dicer-2

Microplitis demolitor Dicer-2

Nasonia vitripennis Dicer-2

Harpegnathos saltator Dicer-2

Acromyrmex echinatior Dicer-2

Cerapachys biroi Dicer-2

Nasonia vitripennis Dicer-1

0

100100

92

49

4499

9571

Current Opinion in Insect Science

icer-2 and Ago-2. Phylogenetic trees were constructed by MEGA 6.0 [53].

_003485689.1), Megachile rotundata (XP_003703800.1), Harpegnathos

214.1), A. mellifera (XP_006571379.1), Acromyrmex echinatior

2.1), Nasonia vitripennis (XP_001605287.1, XP_001602524.2) were used

rrestris (XP_003398529.1), B. impatiens (XP_003492410.1), A. mellifera

M. rotundata (XP_003705687.1, XP_003708345.1), N. vitripennis

8927.1), C. biroi (EZA61145.1) were used for Ago-2 based on PAZ and

Current Opinion in Insect Science 2014, 6:22–27

24 Pests and resistance

Table 1

List of doses (per bee) for silencing target genes by dsRNA injection in adult bees.

Species Target genes DsRNA dose References

Bombus ignitus Ferritin heavy chain subunit/transferrin 20 mg [37]

Peptidoglycan recognition proteins 20 mg [38]

1-Cys peroxiredoxin/2-Cys peroxiredoxin 20 mg [39]

Bombus terrestris Defensin/abaecin/nautilus 1 mg [40]

Apis mellifera Octopamine receptor/dopamine receptor 500, 600 pg* [41,42]

Hypopharyngeal amylase gene 10 mg [43]

Relish 5 mg [44]

Vitellogenin 5, 10, 30 mg [45–50]

NMDA receptor subunit NR1 2.6 mg [51]

Insulin receptor substrate 30 mg [52]

Glycerol-3-phosphate dehydrogenase 5 mg [20]

Ultraspiracle 30 mg [50]

* Targeted genes and detection were in brain, which greatly reduces the amount of dsRNA required.

(Table 1), and titers of honeybee viruses can also be

reduced with virus-specific dsRNA (including siRNA)

[23�,24,25�]. These studies confirmed the conserved

function of the siRNA pathway. To better understand

the molecular aspects of this pathway, we searched for

available protein sequences of core components of the

siRNA pathway such as Dicer-2 and Ago-2 in bees, ants,

and wasps from Genbank, analyzed the predicted

domains, and then phylogenetic trees were constructed

(Figure 1). Analyzed by HMMER (http://hmmer.jane-

lia.org/), RNase III and PAZ domains in Dicer-2-like

proteins, PAZ and Piwi domains in Ago-2-like proteins

were found in bees, ants and wasps. All the sequences of

Dicer-2 and Ago-2 were clearly separated from their close

counterparts Dicer-1 (Nasonia vitripennis), and Piwi-Ago(Megachile rotundata), respectively (Figure 1). It is not

surprising that the sequences were clustered together

based on taxonomic kinship. Insect behavior (social vs.

solitary lifestyle) seemed to have no influence on the

clustering of the genes. To prove the relation of these

genes with the siRNA response, further study is required,

and here the RNAi-of-RNAi approach is proposed as a

useful technique to evaluate the involvement of these

core proteins in insects [21].

Responses of the siRNA pathway upon viralinfectionDeep-sequencing analysis of samples collected from

colonies suffering from colony collapse disorder (CCD)

revealed abundant siRNAs of 21–22 nucleotides perfectly

matching the Israeli acute paralysis virus (IAPV), Kashmir

bee virus (KBV) and Deformed wing virus (DWV) gen-

omes [26��]. To further confirm if these small RNAs were

derived from viruses, honeybees experimentally infected

with IAPV showed a high incidence of small RNAs

matching the IAPV genome [26��]. In addition, small

RNAs matching to Varroa destructor virus-1 (VDV-1)

and DWV genomic sequences were also found in field-

collected honeybees but not in bumblebees [27��]. Lack

of detection of these RNAs in these bumblebees without

Current Opinion in Insect Science 2014, 6:22–27

virus pre-screening could be caused by limited sample

collection, as DWV and VDV-1 can infect bumblebees

and other pollinators [8,10,11]. Therefore, it can be con-

cluded that the siRNA pathway in bees can generate

vsiRNAs from various viruses. The siRNA response in

multi-virus infections is still unclear since these two

studies used pooled samples for sequencing and the

infection status of the individual bees was not confirmed.

DWV, when transmitted by Varroa destructor mites, can

directly be delivered into the hemolymph of honeybees,

thereby giving DWV an advantage over its host, facilitat-

ing replication and spread, which can lead to high virus

titers [28]. Although the significant changes in expression

of Dicer-2 and Ago-2 were absent in bees, vsiRNAs match-

ing to DWV were detected by small RNA sequencing.

Moreover, the intensity of infection seemed to be corre-

lated with the amount of vsiRNAs, indicating that the

siRNA response is proportional to the intensity of the

viral infection [13��]. Although these high levels of vsiR-

NAs do not necessarily result in an RNAi-antiviral action

because virus-encoded suppressors of RNAi (VSR) may

inhibit downstream activity of RNAi, for instance, inhi-

biting slicer activity of Ago-2 [29]. Recently, it has been

suggested that IAPV encoded a VSR [25�]. However, the

data are not yet conclusive and need further analysis.

Beyond the siRNA pathway, also Toll, Imd and Jak-

STAT pathways may be activated during viral infection,

and the induction of antimicrobial peptides (AMPs) is

used as a proxy for activating these pathways [4]. How-

ever, honeybees infected with ABPV did not produce

elevated levels of specific AMPs, such as hymenoptaecin

and defensin, and they also did not show general anti-

microbial activities [30]. Intriguingly, the siRNA and the

Jak-STAT pathways perform cross-talk in mosquitoes in a

Dicer-2-dependent manner through the action of a

secreted signaling molecule, namely Vago, leading to an

antiviral defense state in uninfected cells [31,32]. The up-

regulation of the honeybee ortholog of Vago was also

observed in DWV-infected bees [13��]. By contrast, a

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siRNA pathway against bee viruses Niu et al. 25

core component from Jak-STAT pathway, namely Hops-cotch, and some Toll-related genes were down-regulated

in bees infected with DWV [13��]. However, it is still

unclear which factors could be responsible for reducing

the expression of the immune-related genes in honeybees

and the effect on viral infections.

Apart from virus-specific dsRNA generated during viral

infections, non-specific dsRNA also seems to mediate an

antiviral response in reducing viral titers. Co-injection of

non-specific dsRNA with a model virus, the recombinant

Sindbis virus with green fluorescent protein (SINV-GFP),

to honeybees, showed reduced SINV-GFP titers [33��].Both non-specific dsRNA and SINV-GFP significantly

decreased the expression of various AMPs in these bees,

but the majority of genes for which the transcription

levels increased were not canonical insect immune genes.

So far, three hypotheses can be drawn about the involve-

ment of a non-specific dsRNA in the induction of the

immune response: (i) non-specific dsRNA induces the

siRNA immune response in some extent, and this helps to

restrict viral infection; (ii) recognition of dsRNA by the

host triggers an unknown immune pathway; (iii) Non-

specific dsRNA is recognized by different immune path-

ways, including some unknown pathway, the antiviral

response is a combined effect from the interplay between

various pathways.

Using the siRNA pathway to control beevirusesThrough ingestion of IAPV-specific dsRNA or siRNA in

honeybees infected with IAPV, the IAPV titter were

reduced [24,25�]. Feeding larvae with DWV-specific

dsRNA in advance of inoculation with DWV reduced

the DWV viral titer and wing deformity, while feeding

adult workers with DWV-specific dsRNA in advance of

inoculation with DWV increased their longevity and

reduced DWV titers. In addition, direct feeding DWV-

specific dsRNA did not affect larval survival rates which

suggests that it is non-toxic to larvae [23�]. Also ingestion

of SBV-specific dsRNA could significantly reduce virus

titers in SBV-infected bees [34]. Beside laboratory con-

ditions, the large-scale field application of this strategy

also is able to reduce the IAPV disease in honeybees.

These studies together demonstrate the use of targeted

treatments for viral diseases in bees by using the innate

RNAi immune pathway [35]. Moreover, dsRNA ingested

by bees can be transferred to the Varroa mite and from the

mite onwards to a parasitized bee. This bidirectional

transfer of dsRNA between honeybee and V. destructorcan lead to an approach to use RNAi to control mites,

thereby reducing virus transmission [36].

Conclusion and perspectivesIn conclusion, during viral infection, the siRNA pathway

in bees is activated and thus leads to the degradation of

the viral RNA or its genome, therefore playing a major

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role in the defense against different viruses in bees. More-

over, the bees can be protected through the introduction of

virus specific-dsRNA in large scale field applications.

However, there are still some questions that need to be

addressed in the future: (i) What is the involvement of

the siRNA pathway in multi-virus infections? (ii) What is

the influence of pre-infection with a non-virulent virus

(or persistent infection) on the siRNA pathway, and

subsequent effect to the infection of other viruses? (iii)

What kind of factors can enhance the activity of siRNA

pathway? (iv) How does the host sustain the balance

between its siRNA immune investment to control virus

and other stressors presented, such as food shortage,

pesticides, parasite mites or other pathogen load.

AcknowledgementsThe authors acknowledge support of the Special Research Fund of GhentUniversity (BOF-UGent) and the Fund for Scientific Research-Flanders(FWO-Vlaanderen, Belgium). Jinzhi Niu is recipient of a doctoral grantfrom the China Scholarship Council (CSC:2011699012).

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