Science - 31 January 2020

replication in the hemolymph using a quan-
titative polymerase chain reaction assay. DWV
levels were lower, on average, in bees colonized
byS. alviwith any dsRNA-producing plasmid,
including the off-target pDS-GFP control (figs.
S6A and S7). Thedicergene was also up-
regulated in bees inoculated with most dsRNA-
producing plasmids after virus exposure (fig.
S6B). These results suggest some nonspecific
induction of an immune response in bees
colonized withS. alviexpressing dsRNA.
However, only the pDS-DWV2 plasmid sig-
nificantly increased survival in a separate ex-
periment in which bees were injected with
purified virus (fig. S6C).

To validate the latter finding, we performed

a larger experiment to assess whether dsRNA-

producing bacteria improved survival after

DWV injection. This procedure mimics the

natural route of DWV transmission viaVarroa

mites feeding on bees ( 2 ). We injected cohorts

of 7-day-old bees with DWV and monitored

their survival over 10 days (Fig. 3). After DWV

injection, bees with bacteria bearing pNR died

rapidly. Likewise, pDS-GFP provided no sig-

nificant protection. In contrast, pDS-DWV2

significantly improved survival of virus-injected

bees. Thus, symbiont-mediated RNAi can pro-

tect honey bees from DWV, and it does so in a

targeted, sequence-specific manner.

Finally, we tested whether symbiont-produced

dsRNA can protect bees againstVarroamites.

WhenVarroamites parasitize bees, they feed

on fat bodies ( 18 ) and ingest dsRNA present in

that tissue, triggering their own RNAi response.

Using mite RNAi to target essential mite genes

results in mite death or lowered reproduction

( 8 ). We designed a dsRNA-producing plasmid

with 14 concatenated sequences from essential

genes previously shown to killVarroa(pDS-

VAR) (Fig. 4A and fig. S8) ( 8 ). We inoculated

bees withS. alvibearing pNR, pDS-GFP, or

pDS-VAR; introduced adultVarroamites 5 days

later; and monitored mite survival for 10 days.

Mites that fed on bees colonized with pDS-VAR

Leonardet al.,Science 367 , 573–576 (2020) 31 January 2020 2of4

Fig. 1. EngineeredS. alvicolonizes and functions in bee guts.(A) Colonization of newly emerged honey bees by different inoculum sizes. The percentage of
bees colonized in each treatment is annotated above the inoculation dose.N= 53 bees from two hives. (B) Stability ofS. alvicolonization over time.N= 48 bees
from three hives. Colors in (A) and (B) correspond to different source hives. (C) Stability of GFP expression by engineeredS. alviover time. (D) Photograph of
dissected bee.S. alviresides in the ileum (gray box). (EandF) Ilea of bees 11 days after colonization with nonfluorescent (E) or fluorescent (F)S. alvi. E2-Crimson
fluorescence from engineeredS. alviis blue. Scale bars, 150mm. Error bars in (A) to (C) are 95% bootstrap confidence intervals.

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