Insects
We used a laboratory strain (WT-C02 [34]) of the Mediterranean flour moth, Ephestia kuehniella, maintained on ground wheat with a small amount of dried yeast following the procedure described previously [67]. A laboratory culture of the Indian meal moth, Plodia interpunctella, was established in 2018 from larvae collected from nuts and other stored food products in České Budějovice, Czech Republic. The culture was maintained on a mixture of walnuts and raisins. Both species were reared under a 12-h light/12-h dark regime at 20–22°C. These rearing conditions were maintained in all experiments.
Identification of candidate feminizing small RNAs in Ephestia kuehniella and Plodia interpunctella
Transcript silencing by piRNAs relies on homology with their target RNA sequences [27]. Therefore, we screened the W chromosome reads of E. kuehniella generated by sequencing W chromosome DNA [45] for short sequences homologous to EkMasc and EkMascB [34]. We performed a BLASTn search with the full-length EkMasc and EkMascB sequences (GenBank Acc. No. MW505939 and MW505941, respectively) using low stringency settings (word size 7; expected e-value of 10) against the W chromosome sequence database (GenBank Acc. No. SRR926303). The hits obtained were mapped back to the respective sequence using Geneious 9.1.6 (https://www.geneious.com [68]) with default settings. The correct alignment of the reads was checked manually and corrected if necessary, and graphs were subsequently plotted using R version 3.5.2 [69] with the ggplot2 package [70]. To provide context about the location of reads that were mapped to the genes, exon maps and shading were added using Inkscape 0.92 (https://inkscape.org/). Based on the high coverage and short length, one putative feminizing piRNA sequence (EkMom piRNA) was identified and subsequently used in a BLASTn search against the E. kuehniella W chromosome database. We extracted the complete reads for all hits, aligned them and inferred a consensus sequence of EkMom (GenBank Acc. No. PP715506).
Primers EkMom_F1 and EkMom_R1 (Additional file 8: Table S1) were designed using Primer3 v2.3.4 [71] based on the EkMom consensus sequence to verify the presence of EkMom in the female genome. Genomic DNA (gDNA) was extracted from female and male pupae of E. kuehniella using the NucleoSpin DNA Insect kit (Macherey-Nagel, Düren, Germany) with slight modifications to the manufacturer’s protocol as previously described [17]. The PCR mixture consisted of 1× Ex Taq PCR buffer, 0.2 mM of each dNTP, 0.2 µM of each forward and reverse primer, 0.025 units of Ex Taq polymerase and 10 ng of gDNA. Amplification was performed using a thermocycling program with an initial denaturation at 94°C for 3 min, 35 cycles of denaturation at 94°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 2 min, and a final extension at 72°C for 5 min. Products were purified using the Wizard SV Gel and PCR Clean-Up System (Promega, Madison, WI, USA), cloned using the pGEM-T Easy Vector (Promega) and plasmids were isolated using the NucleoSpin Plasmid kit (Macherey-Nagel) and sequenced as described in our previous study [34].
In addition to the EkMom sequence, a large segment of EkMasc and EkMascB showed coverage on the W chromosome. In our previous study [34], a genetic sexing method using Masc-specific primers was described in which additional fragments were amplified in females, suggesting that the observed coverage on the W chromosome is indeed W chromosome-specific. We designed primers EkMascW_F1 and EkMascW_R1 (Additional file 8: Table S1) to amplify the largest possible fragment of this W-linked Masc (EkMascW) sequence, performed PCR, cloned the amplified fragments and sequenced four clones as described above (GenBank Acc. No. PP715507-PP715510).
To search for putative feminizing small RNAs in P. interpunctella, the complete PiMasc sequence (annotated as maker-scaffold92-augustus-gene-0.127-mRNA-1) obtained from LepBase (https://lepbase.org/ [72]) was used in a BLASTn search against three miRNA-seq databases derived from pooled eggs (GenBank Acc. Nos. SRX1539920–SRX1539922), using low stringency settings (see above) to detect homologous sequences. The sequences obtained were then mapped back to the PiMasc sequence, and the results were plotted using R. To identify the full PiMom sequence, we used a putative PiMom piRNA sequence in a BLASTn search against the unassembled genomic reads of a P. interpunctella female (GenBank Acc. No. ERX334127). The entire reads of the hits as well as the forward and reverse reads of the pair were aligned to create a consensus sequence of PiMom.
To test whether the assembled PiMom sequence was female-specific, primers PiMom_F1 and PiMom_R1 (Additional file 8: Table S1) were designed, and used in PCR. Genomic DNA was extracted from female and male pupae, and PCR and sequencing were performed as described above. The annealing temperature of the PCR was adjusted to 58°C. Two PiMom sequence types were identified (GenBank Acc. Nos. PP715511 and PP715512).
Evolutionary history of EkMascW
To determine the origin of EkMascW in E. kuehniella, we constructed a phylogenetic tree of EkMasc, EkMascB and four EkMascW sequences. The six sequences and the PiMasc sequence were aligned using MAFFT [73, 74]. The alignment was used to construct a phylogenetic tree using MrBayes [75] with default settings, with PiMasc selected as the outgroup.
Quantification of EkMom, EkMascW and PiMom copy numbers
The copy numbers of genes on the W chromosomes of E. kuehniella and P. interpunctella were determined by quantitative PCR (qPCR) as described in our previous study [34]. For the autosomal reference gene, the E. kuehniella ortholog of Acetylcholinesterase 2 (EkAce-2; GenBank Acc. No. MW505944), the primers Ek_Ace2_F × Ek_Ace2_R (Additional file 8: Table S1; [34]) were used. The P. interpunctella ortholog of Ace-2 (PiAce-2) was identified by a tBLASTn search in the available P. interpunctella transcriptome in Lepbase [72] using the EkAce-2 sequence [34]. Primers used for the amplification of EkMom, EkMascW, PiMom and PiAce-2 can be found in Additional file 8: Table S1. To estimate the copy number of EkMascW, primers were selected that amplified all EkMascW copies as well as EkMasc and EkMascB.
In both species, gDNA was extracted from female and male pupae using the NucleoSpin DNA Insect kit as described above. The experiments were performed using three biological and three technical replicates. The 10 µL qPCR mix consisted of 1× Xceed SG qPCR Mix Lo ROX (Institute of Applied Biotechnologies, Prague, Czech Republic), 400 nM of each forward and reverse primer and 10 ng of genomic DNA. For PiMom, primer concentrations were increased to 4 µM to increase qPCR efficiency. The thermocycling program included a combined annealing-extension step at 60°C for 20 s for E. kuehniella genes and 15 s for P. interpunctella genes to avoid amplification of multiple monomers. Copy number estimates were calculated by comparing the gene of interest to the autosomal reference gene as previously described [34]. For EkMascW, the female-to-male ratio was then calculated, and the female copy number estimate of EkMascW was obtained by multiplying the female-to-male ratio by the diploid Masc copy number in males (i.e. 4, two copies on each Z chromosome) and subtracting the haploid Masc copy number in males (i.e. 2, representing the single Z chromosome in females).
To obtain a second copy number estimate for EkMom and EkMascW in E. kuehniella, we used available genome sequencing data to compare the coverage of the two genes with the chromosome/genome sequencing depth. A single monomer of the consensus sequence of both genes was used in a BLASTn search against the unassembled W chromosome reads of E. kuehniella [45]. The reads obtained were then mapped against the respective sequences, and copy numbers were estimated by dividing the average coverage of the sequence of interest by the total chromosome sequencing depth estimated in the previous study [45].
To obtain a second copy number estimate for PiMom, we used a similar approach as described for E. kuehniella by using genomic reads from a female specimen of P. interpunctella (GenBank Acc. No. ERX334127). For P. interpunctella, the average coverage of PiMom was divided by the sequencing depth of the sequencing project. Since no genome size estimate is available for P. interpunctella, the genome size of E. kuehniella was used [76], as this is the closest relative with known genome size.
Chromosome preparations and fluorescence in situ hybridization (FISH)
Spread chromosome preparations were made from ovaries (for meiotic chromosomes) or wing imaginal discs (for mitotic chromosomes) of fifth instar female larvae of E. kuehniella and P. interpunctella, as described previously [77, 78].
To map the EkMascW and EkMom clusters on the W chromosome of E. kuehniella, we used two FISH techniques. First, we performed genomic in situ hybridization (GISH) to visualize the W chromosome, and then we re-probed the slides using tyramide signal amplification FISH (TSA-FISH) with two probes specific for EkMascW and EkMom. To map the PiMom cluster on the W chromosome of P. interpunctella, we used TSA-FISH with two probes, one for PiMom and one for the W-specific repeat PiSAT1 [46] to identify the W chromosome.
For GISH, high molecular weight DNA was isolated from individual female and male pupae using cetyltrimethylammonium bromide (CTAB) as previously described [79]. Female gDNA was fluorescently labelled using an improved nick translation protocol [80] with previously described modifications [17]. Approximately 1 µg of female gDNA was labelled with Cy3-dUTP (Jena Bioscience, Jena, Germany), the labelling reaction was incubated at 15°C for 2 h 15 min and inactivated by incubation at 70°C for 10 min. Male gDNA was sheared to act as a species-specific competitor in GISH, and therefore 12 µg DNA was incubated at 90°C for 20 min. The GISH procedure followed the protocol for CGH [81] with the previously described modifications [40] and some additional modifications (for details see Additional file 9: Methods S1).
For TSA-FISH in E. kuehniella, we prepared gene-specific probes for EkMom and EkMascW. EkMom was labelled with dinitrophenol-11-dUTP (DNP; PerkinElmer, Waltham, MA, USA), whereas EkMascW was labelled with fluorescein-dUTP (Jena Bioscience). For TSA-FISH in P. interpunctella, probes were prepared for PiMom and PiSAT1 labelled with DNP-11-dUTP and fluorescein-dUTP, respectively. Details on the preparation of the probes can be found in Additional file 10: Methods S2.
TSA-FISH was performed according to the published protocol [82] with some modifications. For E. kuehniella, the probes from the GISH experiment were removed from the slides and the slides were prepared for reprobing as previously described [49], with one additional step: after dehydration, the slides were incubated in 5× Denhardt’s solution at 37°C for 30 min. For P. interpunctella, the slides were pretreated as described for GISH. For both species, the probe mixture consisted of 20 ng of each probe (EkMom probe and three EkMascW probes for E. kuehniella; PiMom probe and PiSAT1 probe for P. interpunctella) in 50% deionized formamide, 10% dextran sulfate, 2× SSC in a total volume of 50 µL. The probe mixture was applied to the slides, covered with a coverslip and denatured at 70°C for 5 min. Hybridization was performed in a humid chamber at 37°C for 12–16 h. After hybridization, the coverslip was removed and the slides were washed three times in 50% formamide in 2× SSC at 46°C for 5 min each. The slides were then incubated three times in 2× SSC at 46°C for 5 min, three times in 0.1× SSC at 62°C for 5 min and once in TNT buffer (0.1 M Tri-HCl pH 7.5, 0.15 M NaCl, 0.05% Tween-20) at room temperature (RT) for 5 min. Slides were blocked with 200 µL of TNB buffer (0.1 M Tris-HCl pH 7.5, 0.15 M NaCl, 0.5% Blocking Reagent; PerkinElmer) at RT for 30 min in a humid chamber. Excess TNB buffer was poured off and the slides were incubated with 200 µL of antifluorescein-HRP conjugate (PerkinElmer), diluted 1:1000 with TNB buffer, in a humid chamber at RT for 1 h. Slides were washed three times in TNT buffer at RT for 5 min and tyramide amplification was performed using the TSA Plus Fluorescein kit (PerkinElmer) according to the manufacturer’s instructions. 100 µL of tyramide working solution was applied to each slide, the slides were covered with a coverslip and incubated in a humid chamber at RT for 8 min. Slides were washed three times in TNT buffer at RT for 5 min each and then incubated in 1% H2O2 in 1× PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4) to quench the antibody-conjugated peroxidase. Slides were washed three times in TNT buffer at RT for 5 min each and blocked in 200 µL of TNB buffer in a humid chamber at RT for 30 min. Excess TNB buffer was poured off again, but this time the slides were incubated with 200 µL of anti-DNP-HRP conjugate (PerkinElmer), diluted 1:1000 with TNB buffer, in a humid chamber at RT for 1 h. Slides were washed three times in TNT buffer at RT for 5 min, and tyramide amplification was performed using the TSA Plus Cyanine 3 (Cy3) detection kit (PerkinElmer) according to the manufacturer’s instructions. Again, 100 µL of the tyramide working solution was applied to each slide, the slides were covered with a coverslip and incubated in a humid chamber at RT for 8 min. The slides were washed three times in TNT buffer at RT for 5 min each and incubated in 1% Kodak PhotoFlo in H2O. Finally, the slides were mounted in VECTASHIELD antifade mounting medium with DAPI (Vector Laboratories, Burlingame, CA, USA). Slides were observed and images were captured as described for GISH (Additional file 9: Methods S1).
Structural analysis of EkMom and PiMom by Southern hybridization
The genomic structure of EkMom and PiMom repeats was analysed by Southern hybridization. EkMom and PiMom probes were prepared as described for TSA-FISH using the same primers and gDNA templates, but labelled with digoxigenin-11-dUTPs (Roche Diagnostics, Mannheim, Germany). For each species, gDNAs were isolated from individual female and male larvae (E. kuehniella) or pupae (P. interpunctella) using CTAB as previously described [79], and their concentrations were measured with an Invitrogen Qubit 3.0 Fluorometer using the dsDNA BR Assay Kit. Extracted gDNAs were either fully (one female and one male sample) or partially (approximately 25%; one female sample) digested with restriction enzymes that cut once per monomer but not in the sequence of the probe. Samples of E. kuehniella were digested with EcoT22I (TaKaRa, Otsu, Japan), and for partial digestion the enzyme was diluted 4× with nuclease-free water before use. Samples of P. interpunctella were digested with Eco32I (EcoRV) (Fermentas, Vilnius, Lithuania), and for partial digestion the enzyme was diluted 16× with nuclease-free water before use. Digestion reactions were performed according to the manufacturer’s instructions supplied with the restriction enzymes. All reactions were incubated at 37°C for 1 h, and enzymes were inactivated by addition of Gel Loading Dye, Purple (6×) (New England Biolabs, Ipswich, MA, USA). Hybridizations were performed as previously described [83] with some modifications [46].
Expression analysis of sex-determining genes during early embryogenesis of Ephestia kuehniella
To determine the time of onset of sexual differentiation in E. kuehniella, we analysed the expression of genes involved in sex determination during early embryogenesis. To this end, we analysed the expression of EkMom and the male-specific splice form of the E. kuehniella ortholog of the insulin-like growth factor II mRNA-binding protein (EkImpM) gene. As a control for successful amplification of cDNA, we used primers for the Ekrp49 gene (GenBank Acc. No. MW505943) designed in our previous study (Visser et al., 2021). The B. mori IMP protein sequence (GenBank Acc. No. LOC101745047) was used in a tBLASTn search against the male genome of E. kuehniella (GenBank Acc. No. PRJNA683200) to obtain partial exon sequences, after which primers were designed. Total RNA was isolated from approximately 50 pooled eggs collected within 24 hours post oviposition (hpo), a single male pupa and a single female pupa using the NucleoSpin RNA kit (Macherey-Nagel) according to the manufacturer’s instructions with some modifications. Briefly, samples were homogenized in the presence of lysis buffer, then β-mercaptoethanol was substituted for tris(2-carboxyethyl)phosphine (TCEP) during lysis and elution was performed with RNase-free H2O in a volume of 40 µL. Concentrations were measured with a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific), and 1 µg of RNA was converted to cDNA with the ImProm-II Reverse Transcription System kit (Promega) according to the manufacturer’s instructions using the Oligo(dT)15 primer supplied with the kit and a final concentration of 3 mM MgCl2. To isolate Imp in E. kuehniella, PCR was performed with primers EkImp_F1 and EkImp_R1 (Additional file 8: Table S1), using the PCR mixture and thermocycling program as described for the isolation of EkMom. The samples were then processed and sequenced in the same steps as described above. To identify the male-specific splice variant of EkImp, a set of primers (EkImp_3’RACE_F1 and EkImp_3’RACE_F2; Additional file 8: Table S1) was designed based on the identified sequence. Rapid Amplification of cDNA Ends PCR (RACE-PCR) was performed for the 3’-region of the cDNA as previously described (Visser et al., 2021) using RNA from the pooled sample of eggs. The male-specific splice variant, EkImpM, was identified (GenBank Acc. No. PP715505) and a reverse primer in the male-specific exon (Additional file 8: Table S1) was designed and used in RT-PCR to confirm male-specific expression of this splice variant.
For expression analysis during embryogenesis, DNA and RNA were isolated simultaneously from individual embryos using TRI reagent (Sigma-Aldrich, St. Louis, MO, USA) as previously described (Visser et al., 2021). Embryonic DNA was used for genetic sexing of embryos according to the previously described protocol (Visser et al., 2021). DNA contamination was removed from the total RNA fraction using the Invitrogen TURBO DNA-free kit (Thermo Fisher Scientific) according to the manufacturer’s instructions, and total RNA was converted to cDNA using the ImProm-II Reverse Transcription System kit according to the manufacturer’s instructions, using a mixture of Oligo(dT)15 primer and random primer (1:1) supplied with the kit and a final concentration of 3 mM MgCl2. In our previous study (Visser et al., 2021), the expression of EkMasc and EkMascB peaked in males around 16 hpo, so we used the same time series of 12–24 hpo with 2-h sampling intervals. The PCR mixtures for the Ekrp49, EkMom and EkImpM genes contained 1× Ex Taq PCR buffer, 0.2 mM of each dNTP, 0.2 µM of each forward and reverse primer (qrp49_F2 × qrp49_R2 or EkMom_F1 × EkMom_R1 or EkImpM_F1 × EkImpM_R1; Additional file 8: Table S1), 0.025 units of Ex Taq polymerase and 2 µL of cDNA in a total reaction volume of 10 µL. The thermocycling program consisted of an initial denaturation at 94°C for 3 min, 40 cycles of denaturation at 94°C for 30 s, annealing at 55°C for 30 s and extension at 72°C for 40 s, and a final extension at 72°C for 5 min. Amplified products were separated on a 1.5% agarose gel in 1× TAE buffer.
To study maternal provision of EkMom, RNA was isolated from pooled samples of mixed-sex embryos. We isolated RNA from two samples, each containing 30–40 pooled eggs collected less than 1 hpo, with TRI reagent according to the manufacturer’s protocol, using chloroform for phase separation and 20 µL of diethyl pyrocarbonate (DEPC)-treated water to dissolve the RNA pellets. RNA concentrations were measured using a NanoDrop 2000 spectrophotometer, and total RNA was treated with DNase using the TURBO DNA-free kit. For each sample, two cDNA conversion reactions were prepared with 500 ng of RNA each using the ImProm-II Reverse Transcription System kit. Reverse transcriptase was added to one of the two reactions per sample, while the other served as a negative control for downstream purposes. All cDNA reactions were prepared using a mixture of Oligo(dT)15 primer and random primer (1:1) and a final concentration of 3 mM MgCl2 as described above. PCR for EkMasc (targeting EkMasc and EkMascB simultaneously) with the primer pair Masc_F1 and Masc_R1 (Additional file 8: Table S1) was used to control for cDNA integrity. The primers EkMom_F1 and EkMom_R1 were used to amplify EkMom. In addition, a sample with gDNA was added to each PCR as a positive control to verify successful amplification. The same PCR mixture and thermocycling profile were used as described in the previous paragraph, but amplification was only performed for 35 cycles.
Expression of EkMom piRNA during embryogenesis of Ephestia kuehniella
To determine whether the putative EkMom piRNA is expressed during the onset of sex determination, we sequenced the small RNA fraction of eggs during early development. We chose two time periods for RNA extraction, one before sex-specific differential expression of EkMasc and EkMascB (Visser et al., 2021), i.e. 7–9 hpo, and one at the onset of sex-specific differential expression, i.e. 11–13 hpo. Since the expression of EkMasc and EkMascB decreases in females between 12 and 14 hpo, while it increases in males during the same period, we assumed that the expression in females must be reduced by EkMom piRNA. Total RNA was isolated from the two samples, each containing 200–300 pooled mixed-sex embryos, using TRI reagent according to the manufacturer’s protocol for tissue. To increase the purity of the RNA sample, two rounds of phase separation with chloroform and two rounds of ethanol washes were performed. The RNA pellets were dissolved in 30 µL RNase-free water, sample concentration and purity were assessed using NanoDrop 2000 spectrophotometer and RNA integrity was assessed using an RNase-free 1% agarose gel in 1× TBE buffer stained with ethidium bromide. Total RNA samples were sent to Novogene (HK) Co., Ltd. (Hong Kong, China) for small RNA library preparation (library type: 18–40 bp insert sRNA library) and sequencing of single-end 50 bp reads on an Illumina Novaseq 6000. Adapter trimming and quality control of reads were also performed by this company. Approximately 21–21.5 million reads were obtained for each sample (GenBank Acc. No. PRJNA1107079). Reads with high homology to EkMasc and EkMascB were identified using a BLASTn search in NCBI Genome Workbench (word size 7; expected e-value of 10), and all antisense reads were subsequently mapped back to the respective sequences using Geneious. For each peak, a representative sequence was extracted and compared to the EkMasc, EkMascB, EkMascW and EkMom sequences to determine their origin. Data for the graphs were exported and plotted using R.
Sliding window analysis of EkMom and PiMom
To compare the sequence homology between EkMom and PiMom, the consensus sequences of EkMom and PiMom monomers were aligned with MAFFT and the sequence identity between the two sequences was exported. These data were used to calculate the average sequence identity over a 10-nucleotide window corresponding to the size of the ping-pong signature. The window was shifted by one nucleotide until the end of the alignment. Data were plotted using R with the ggplot2 package and additional information was added using Inkscape 0.92.
Detection of maternal provision of PiMom and PiMasc in Plodia interpunctella
To test whether PiMom piRNA was present before transcription began, we isolated total RNA from two samples of pooled eggs. The first sample contained 28 eggs collected within 4 hpo, and the second sample contained 14 eggs collected within 1 hpo. Extraction of RNA using TRI reagent and all other procedures, including treatment with the TURBO DNA-free kit and conversion to cDNA, were performed in the same manner as described above for the study of maternal provision of EkMom. RT-PCR was performed in a volume of 10 µL consisting of 1× Ex Taq PCR buffer, 0.2 mM of each dNTP, 0.2 µM of each PiMom_F1 and PiMom_R1 primer, 0.025 units of Ex Taq polymerase and 1 µL of cDNA. The same thermocycling program was used as described above for testing the female specificity of PiMom. To verify correct cDNA conversion, P. interpunctella Masculinizer (PiMasc) was amplified with the primers Masc_F1 × Masc_R1 (Additional file 8: Table S1) using the same PCR mixture and thermocycling program as in the PCR for EkMasc (see above).