A male-killing gene encoded by a symbiotic virus of Drosophila

Collection and maintenance of Drosophila biauraria

We used laboratory stocks of Drosophila biauraria (Diptera; Drosophilidae), which were originally collected at the Field Science Center for Northern Biosphere, Hokkaido University located at Tomakomai, Hokkaido in 2011 and 2015 using standard banana traps and sweeping22. Females were brought into the lab and iso-female lines (SP11-20, TM15-12, TM15-22, TM15-41, and TM15-47) were established using banana medium (270 g of bananas, 68 g of malt, 68 g of molasses, 40 g of dried yeast, 20 g of agar, and 2 liters of distilled water, supplemented with 9 ml of propionic acid and 0.036% (w/v) of butyl p-hydroxybenzoate). The all-female line (SP12F) was maintained by mating with males of a standard iso-female line (SP11-20). D. biauraria were maintained at 20 °C with banana medium under 16 h; 8 h light-dark regimen.

Estimate of egg hatch rates

Embryos were collected from grape-juice agar plates and dechorionated in 2.8% sodium hypochlorite solution. They were subsequently soaked in phosphate-buffered saline (PBS) containing 0.1% Triton X-100 (PBT), placed at 25 °C for 4 d, and counted under dissecting microscope. Hatched larvae (first-instar larvae) and unhatched but well-developed embryos were rinsed with 99% ethanol three times and stored in 99% ethanol.

Genetic sexing of embryos and hatchlings of D. biauraria

Y chromosome-linked markers for D. biauraria are unable to obtain by inferring the genome data of D. melanogaster because orthologs of Y chromosome-linked sequences of D. melanogaster are present in both males and females in the montium group, such as D. biauraria23. Therefore, by comparing the genomic data of male and female D. biauraria obtained by Illumina HiSeq, we isolated male-specific sequences. A pair of primers, DbY_c52202_F2 and DbY_c52202_R2 (Supplementary Table 1), designed from one of the sequences discriminated males and females unambiguously by polymerase chain reaction (PCR). A pair of primers, Db-actin5C-68-F and Db-actin5C-68-R (Supplementary Table 1), that amplify actin-5C was used to confirm that D. biauraria genomic DNA was properly extracted.

Each of the embryos and first-instar larvae, picked up from ethanol, was briefly air-dried and squashed in 20 μL of PrepMan™ Ultra Sample Preparation Reagent (ThermoFisher, Cat. No. 4318930). Samples were then incubated at 100 °C for 10 min, vortexed for 15 s and centrifuged at 20,000 × g for 2 min before being subjected to PCR.

Transinfection

After homogenizing 80 adults of SP12F in 300 μL of PBS, supernatant was sterilized using a 0.22-μm filter (MILLEX GV, Merck Millipore, Cat. No. SLGV033RS) and 0.2 μL was injected into the intersegmental membrane of thorax of mated females of D. biauraria SP11-20 line using a glass capillary needle (Drummond, Cat. No. 2-000-005) and an air pump Linicon LV-125 (Nitto Kohki Co., Ltd.). Injected females were individually placed in the vials with the banana medium to produce offspring. In subsequent generations, each female adult was placed with an SP11-20 male in a vial with the banana medium. An all-female line resulted from the transinfection, referred to as tr.SP11-20, was maintained by mating with males of SP11-20.

RNA sequencing

Total RNA was extracted from 30 individuals from each of the seven D. biauraria lines (30 females from SP12F and tr.SP11-20; 15 females and 15 males from SP11-20, TM15-12, TM15-22, TM15-41, and TM15-47) using RNeasy (Qiagen, Germany). Extracted RNA was subjected to RNA sequencing using Illumina HiSeq 2500 following the removal of ribosomal RNA using Ribo-Zero rRNA Depletion Kit (Illumina, Cat. No. MRZH11124) by Macrogen (South Korea). Generated raw RNA-seq reads of the seven lines were cleaned by Trimmomatic version 0.3624. A reference transcriptome assembly was constructed by de novo assembly of merged clean reads of three lines (SP12F, tr.SP11-20, and SP11-20) using Trinity version 2.2.025. The assembled contigs were annotated with descriptions of top-hit proteins in the NCBI non-redundant (NCBI-nr) protein database by blastx search (e-value <10−3). The coding DNA sequence (CDS) regions of the contigs were estimated using TransDecoder version 3.0.1 (github.com/TransDecoder/TransDecoder) and annotated with descriptions of hit protein domains in the Pfam database by HMMER version 3.1b226. Expression levels (transcripts per million (TPM) values) of the assembled contigs were calculated for each line by mapping the clean reads to the reference transcriptome assembly using “align_and_estimate_abundance.pl” script bundled with Trinity which uses Bowtie2 version 2.2.627 and RSEM version 1.2.3128. Contigs highly specifically expressed in the two all-female matrilines (SP12F and tr.SP11-20) were extracted by filtering with TPM value > 100 and fold change > 10 for the two lines when compared with other lines.

Rapid amplification of 3′-cDNA end (3′-RACE)

Total RNA extracted from D. biauraria SP12F was used as a template for RACE using ALL-TAIL™ Kit (Bioo Scientific Corporation, Cat. No. 5205). Following the manufacturer’s protocol, AIR™ Adenylated Linker C was ligated to the total RNA, followed by the reverse transcription using Linker C Universal RT-PCR Primer, a primer complementary to the AIR Adenylated Linker C. The products were subjected to PCR amplification using a primer specific to one of the four contigs (Supplementary Table 1) and Linker C Universal RT-PCR Primer. PCR products electrophoresed on agarose gel were excised, purified by Wizard® SV Gel and PCR Clean-Up System (Promega, Cat. No. A9281) and subjected to direct Sanger sequencing using ABI 3730XL sequencer (Applied Biosystems).

Phylogenetic analyses

One hundred thirteen putative amino acid sequences of RNA-dependent RNA polymerase (RdRp) of Partitiviridae, including an ORF1 (dsRNA1) sequence identified in Drosophila biauraria were aligned using the MAFFT version 7 using the “-auto” setting29. Multiple-sequence alignments were trimmed with the trimAL version 1.3 using setting “-automated1” to remove uninformative columns30. The phylogenetic analysis was performed using raxmlGUI 2.0 version 2.0.731. The BIC-based best model according to ModelTest-NG version 0.1.732 was used for phylogenetic tree reconstruction using ML + thorough bootstrap with a replication setting “autoMRE”33.

Fluorescent in situ hybridization (FISH)

Localization of the four dsRNAs of DbMKPV1 were visualized by whole-mount fluorescence in situ hybridization (FISH) using the midgut of female adults of 13 d after emergence as described previously34,35, using the fluorochrome-labeled oligonucleotide probes listed in Supplementary Table 1. AlexaFluor 488-labeled probes were used for the detection of dsRNA1 and dsRNA4. AlexaFluor 647-labeled probes were used for the detection of dsRNA2. AlexaFluor 555-labeled probes were used for the detection of dsRNA3. Designing probes were conducted by using Stellaris Probe Designer version 4.2 (www.biosearchtech.com/stellarisdesigner/, Biosearch Technologies) for picking up the plurality of candidate sequences. Then specificities of the probes were checked using ProbeCheck (131.130.66.200/cgi-bin/probecheck/probecheck.pl) and BLASTN 2.6.1 (blast.ncbi.nlm.nih.gov/Blast.cgi), and narrowed down to the 2 probes per ORF. Host cell nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI). Observations were made using a laser scanning confocal microscope LSM710 (Carl Zeiss, Germany) and analyzed using ZEN 2009 software (Carl Zeiss, Germany). The specificity of in situ hybridization was confirmed by the following control experiments: a no-probe control, an RNase digestion control as described36.

Drosophila melanogaster fly stocks and genetics

Laboratory stocks of D. melanogaster were maintained at 25 °C with banana medium. The following lines were obtained from the Bloomington Drosophila Stock Center (BDSC) at Indiana University and the Department of Drosophila Genomics and Genetic Resources (DGGR) at Kyoto Institute of Technology: Actin5C-GAL4 (act-GAL4; BDSC #4414), nanos-GAL4::VP16 (nos-GAL4; BDSC #4937), UASp-EGFP (DGGR #116072), and CyO, ActGFP (the green balancer; DGGR #107783). The four ORFs (ORF1–ORF4) encoded in the DbMKPV1 genome were expressed by the GAL4/UAS system37 in D. melanogaster. For the zygotic expression, Actin5C-GAL4/CyO flies were crossed to homozygous UAS transgenic flies. For maternal expression38, nanos-GAL4 females were crossed to UAS males, and the resultant female progeny were mated with Oregon-R (OR-NIG) males. For the GAL4/UAS expression in larval salivary glands (Fig. 3), we used a recombined Actin5C-GAL4, tubulin-GAL80ts line (generated by a combination of BDSC #4414 and BDSC #7108) balanced with CyO, ActGFP (DGRC #107783) to avoid male lethality and obtain wandering third-instar larvae. After maintained at 20 °C for 7–8 d, crosses were shifted to 29 °C and kept for 1 d before dissection. GFP on the balancer chromosome was utilized as a selection marker. For the expression of Spaid, UASp-Spaid-GFP transgenic flies were used.

Construction of transgenic D. melanogaster lines

First, total RNA was extracted from infected whole D. biauraria adult females (SP12F, n = 30) using the RNeasy Mini kit (Qiagen). Total RNA was used for cDNA synthesis by the PrimeScript RT-PCR Kit (Takara Bio, Japan). The four DbMKPV1 ORFs (without in-frame stop codons) were PCR amplified from the synthesized cDNA with specific primers. To generate two frameshift mutations of ORF4 (Δ1bp and Δ2bp), we designed and used two forward PCR primers with one/two-nucleotide deletion in the second codon of the coding sequence (5′-ATG GCG CAT-3′). Primers used for PCR amplification are listed in Supplementary Table 1. Each PCR fragment was cloned into the pENTR vector by the pENTR/D-TOPO cloning kit (Thermo Fisher Scientific). We utilized PrimeSTAR MAX DNA Polymerase (Takara Bio) for all PCR reactions above. The Gateway cassette containing the ORF fragments was transferred into the pPW destination vector (The Drosophila Genomics Resource Center #1130; The Drosophila Gateway Vector Collection by Terence Murphy) by the LR clonase II enzyme mix kit (Thermo Fisher Scientific) to construct pUASp-ORFs plasmids. Transgenic fly lines were generated by the standard microinjection method for P-element transformation (BestGene).

Reverse transcription quantitative polymerase chain reaction (RT-qPCR)

Total RNA was extracted from each of the three D. melanogaster adults from respective genotypes (act-Gal4.UAS-ORF4Δ1.M7, CyO.UAS-ORF4Δ1.M7, act-Gal4.UAS-ORF4Δ1.M3, CyO.UAS-ORF4Δ1.M3, act-Gal4.UAS-ORF4Δ2.M3, CyO.UAS-ORF4Δ2.M3, act-Gal4.UAS-ORF4F6, CyO.UAS-ORF4F6, act-Gal4.UAS-ORF4M1, and CyO.UAS-ORF4M1), using RNeasy Plus Mini kit (Qiagen). cDNA was synthesized from the total RNA by PrimeScript™ RT reagent Kit with gDNA Eraser (Perfect Real Time) (Takara Bio, Japan). qPCR was conducted by using KOD SYBR® qPCR Mix (TOYOBO, Japan) using two primer sets of ORF4_q_F1 and ORF4_q_R1 (Supplementary Table 1) for dsRNA4, and Dm_rp49_q_F1 and Dm_rp49_q_R1 (Supplementary Table 1) for DmRp49 as reference gene. Thermal conditions were managed by LightCycler® 96 Instrument (Roche, Switzerland) as 95 °C for 300 s followed by 40 cycles of 95 °C for 5 s and 60 °C for 20 s. The relative amounts of the RNA were calculated using quantification cycles (Cq) by ddCq analysis. The RNA levels of dsRNA4 were normalized by those of DmRp49, and the relative RNA levels of dsRNA4 were estimated by setting one of the act-Gal4.UAS-ORF4F6 as 1. Expression of GFP was checked by a fluorescent microscope MZ10F (Leica).

D. melanogaster staining and imaging

Larval salivary glands were dissected out from wandering third-instar larvae and fixed in 4% paraformaldehyde (EM Grade; Electron Microscopy Sciences, 15710) diluted in PBS for 20 min at room temperature. After washed in PBT and treated with a blocking buffer [PBT containing 1% bovine serum albumin (BSA, heat shock fraction; Sigma-Aldrich, A7906)] for 30 min, tissues were incubated with primary antibodies at 4 °C overnight, then washed in PBT and incubated with secondary antibodies at room temperature for 90 min. Antibodies were diluted in the blocking buffer. The following primary antibodies were used: rabbit anti-acetyl-histone H4 lysine 16 (H4K16ac; 1/2,000; Upstate, Sigma-Aldrich 07-329), mouse anti-phospho-histone H2Av (pH2Av; 1/500; DSHB, UNC93-5.2.1). Secondary antibodies (1:2,000; Alexa Fluor Plus 555/647 conjugate) were purchased from Thermo Fisher Scientific (A32773 and A32795). DNA staining was carried out with DAPI (0.5 µg/mL; Nacalai Tesque, 19178-91) together with secondary antibodies. Stained tissues were washed in PBT, mounted in ProLong Glass Antifade Mountant (Thermo Fisher Scientific, P36980), and observed under a confocal microscope (Olympus FLUOVIEW FV3000) equipped with a 40×/1.4 oil immersion objective (2×zoom scan; frame size: 1024 × 1024; 0.42 µm/slice with optimal intervals). The brightness and contrast of a single optical section were adjusted uniformly on the entire images and only black/white input levels were modified using EBImage package version 4.5.22 under R version 4.1.2 and Fiji ImageJ version 1.51r.

Statistics and reproducibility

The number of analyzed individuals and the number of biological replicates (at least thrice) are indicated in the figure legends. For each gene used for constructing transgenic flies, two independently established fly lines were examined.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

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