Mite colony and experimental units
Phytoseiulus persimilis used in the present study were obtained from the lab of Predatory Mites, Institute of Plant Protection, Chinese Academy of Agricultural Sciences (IPP-CAAS), Beijing, China. P. persimilis were reared using Tetranychus urticae (Acari: Tetranychidae), which in turn were reared on 2-week-old bean seedlings (Phaseolus vulgaris L.). Both mite colonies have been maintained for more than 10 years. The rearing and experiment conditions were 25 ± 1°C, 70% ± 5% RH and L:D = 16:8 h.
Arenas for individually rearing were prepared based on the methods in Zhang et al. (2015). Each rearing unit contained four layers from bottom to top: a rectangular glass plate, a small bean leaf disc (with prey on it), a central layer with a 1.5-cm-diameter hole in the center as the arena for mites, and a rectangular glass cover to seal the arena. The layers were tightly clipped together on both ends.
Protein prediction and systematic evolutionary analyses of Pptra-2
The Pptra-2 gene sequence was obtained from Bi et al. (2019). Its open reading frame (ORF) and nucleotide sequence were predicted using the ORFFinder (http://www.ncbi.nlm.nih.gov/gorf/gorf.html). Peptides and conserved domains were predicted using SMART (http://smart.embl-heidelberg.de/). The isoelectric point (pI) and molecular weight (MW) was calculated using DNAMAN v.6.0. Serine, threonine or tyrosine phosphorylation sites and N-glycosylation sites were predicted using NetPhos v.3.1 servers (http://www.cbs.dtu.dk/services/NetPhos/) and NetNglyc v.1.0 servers (http://www.cbs.dtu.dk/servi ces/NetNGlyc/).
We screened tra-2 orthologs of various arachnoid and insect species that have relative complete genomes in NCBI or Uniprot. A total of 20 arachnoid species and 45 insect species were selected (Supplementary document S1). With sequences alignment performed, a phylogenetic tree was built using MEGA-X with a bootstrap of 1000 replicates.
Expression of Pptra-2 during Phytoseiulus persimilis development
Approximately 1000 mated P. persimilis females were collected and placed on a bean leaf with abundant T. urticae, to obtain ca. 2000 synchronize eggs laid in 12 hrs. Each egg was reared individually in a rearing unit. During P. persimilis development, eggs, larvae, protonymphs, deutonymphs, and newly emerged female and males were collected for mRNA extraction and tra-2 expression analyses. Sexes of phytoseiids are hard to tell until adult emergence, so immature samples were mix sex. Other newly emerged female and male adults were paired and allowed for one complete mating. Mated females were reared individually, and were collected 1d, 3d, 5d, 7d, and 9d after mating. For each timing, 3 biological replicates were collected, each either containing 80 eggs, 60 larvae, 50 protonymphs, 30 deutonymphs, or 15 adults, respectively. For each sample, mRNA was extracted according to the instructions of MicroElute Total RNA Kit (R6831-01, Omega). Relative expression of Pptra-2 was determined using qPCR, using methods and primers in Bi et al. (2019). Three technical replicates were performed for each biological replicate. Relative expression of different stages, sexes, and at different timings during adult female life after mating were compared, respectively, using one-way ANOVA. Multiple comparisons were conducted using LSD (α = 0.05).
Impact of Pptra-2 on reproduction of Phytoseiulus persimilis
Interferences of Pptra-2 were performed to female adults using dsRNA synthesized and purified with MEGAscript RNAi kit (Invitrogen) produced by Thermo Fisher Scientific (Pomerantz and Hoy 2015b; Bi et al. 2019). Primers containing the T7 promoter were amplified using TAKARA R040Q Primer Star MIX, and recollected using the Omega kit.
About 2000 synchronized eggs (laid within 12 h) of P. persimilis were collected and reared individually to adulthood. Approximately 1500 newly emerged P. persimilis females were achieved, and starved for 24 h for the following experiments. Half of them were allowed to feed on 3 µl sucrose dsRNA solution (containing 128 ng/µl dsRNA of target gene, 20% sucrose, and 6% blue food dye (McCormick, MD, USA)) for 72 h for interference, while the other half were allowed to feed on 3 µl dyed sucrose solution without dsRNA as CK. For both groups, individuals with blue dyes observed in intestines were used, and fed with sufficient T. urticae for 24 h for following observations.
Treated females were allowed to mate with newly emerged males for a single complete mating. For each treatment, 25 females were used for biological features observation. They were reared individually, with rearing units changed once a day and sufficient T. urticae provided daily. Eggs laid per day were collected and reared to adults, with offspring sex identified at adult emergence. Biological parameters, including pre-oviposition period, oviposition period, cumulative fecundity, egg hatching rate, and offspring female ratio were estimated. On the 5th day, interfered individuals were further divided into 2 groups: individuals with offspring hatching rate higher than 80% or lower than 80%, termed as HHR (high hatching rate) and LHR (low hatching rate), respectively. Biological parameters were also estimated for HHR and LHR separately. These parameters were compared using t-test between the interfered group and the CK, and using one-way ANOVA among HHR, LRH, and the CK.
Other Females were sampled 0d, 3d and 5d (for HHR and LHR separately) after mating for RNA extraction and tra-2 expression analyses, using the same methods as described in the previous section. For each sampling timing, 3 biological replicates were created, each containing ca. 15 individuals. Expression of tra-2 were compared between treated and CK using t-test at 0d and 3d, and compared among HHR, LHR, and CK using one-way ANOVA at 5d after mating.
Potential genes functionally related to Pptra-2
Transcriptome analyses were performed to seek for potential genes that functionally related to Pptra-2. Females of CK, HHR and LHR were collected 5d after mating. For each treatment, four biological replicates were set, each containing ca. 20 females. Sequencing libraries were generated using TruSeq RNA Sample Preparation Kit (Illumina, SanDiego, CA, USA). Library preparations were sequenced on Illumina Hiseq 4000 platform. The sequencing mode was PE150: paired-end, 2×150 bp read length. The number of reads, Ploy-N or read low-quality reads containing sequencing joints were removed, and the contents of Q20, Q30, GC and sequence repeats were calculated.
For sequenced genes, their functions were annotated based on the reference genome of P. persimilis (submission numbers 2524607), and orthologs were screened using Blast n, Blast x, Blast 2 GO and InterPro Scan. The original expression of each gene was counted in HTSeq v.0.9.1. The fragments per kilobase of transcript per million fragments sequenced (FPKM) were used to standardize the expression. Differential expressions genes (DEGs) between each two of the three treatments were compared using the DESeq Rpackage v.1.10.1 based on the following criteria: expression difference multiple log2FoldChange > 1 and significant P-value < 0.05. Subsequently, R language Pheatmap software package was employed to perform the bi-directional clustering analysis of all different genes of different samples. The GO enrichment analysis was performed using TopGO v.2.40.0. Linked signaling pathways were predicted based on Kyoto Encyclopedia of Genes and Genomes (KEGG) to identify the major biological functions of DEGs in each treatment group.
Orthologs of insect reproduction related genes, such as transformer, doublesex and intersex genes that potentially associated with tra-2, and the ovarian tumor, virilizer, groucho, daughterless genes that potentially associated with gender development, were also screened based on transcriptome annotation.