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
www.sciencedirect.com
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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