Expression and functional analyses of transformer-2 in Phytoseiulus persimilis and other genes potentially participating in reproductive regulation

DOI: https://doi.org/10.21203/rs.3.rs-1473471/v1

Abstract

Transformer-2 is an important sex-determining gene in insects and also plays roles in reproduction of Phytoseiid mites. We performed bioinformatics analyses for the transformer-2 ortholog in Phytoseiulus persimilis, measured its expression in different stages, and quantitatively identified its function in reproduction. This gene, termed as Pptra-2, encodes 288 amino acids with a conserved RRM domain. Its peak expression was observed in female adults, especially ca. 5 days after mating. Besides, expression in eggs is higher than that in other stages and male adults. When Pptra-2 was interfered through oral delivery of dsRNA, 56% females had their egg hatching rates decreased in the first 5 days, from ca. 100% to ca. 20%, and maintained at low levels during the rest of its oviposition period. To detect other genes functionally related to Pptra-2, transcriptome analyses were performed on the 5th day after mating. We compared mRNA expressions among interfered females with significant egg hatching rate decrease, interfered females with no significant egg hatching rate decrease, and the CK. A total of 403 differential genes were identified, among which 42 functional genes regulating female reproduction and embryonic development were screened and discussed.

Introduction

The reproductive pattern of phytoseiid mites (Arachnida: Acaridae) is called paternal genome loss (Nagelkerke and Sabelis 1998; Toyoshima and Amano 1998). Both females and males developed from fertilized eggs, but males have their paternal genome eliminated during early embryo development and became haploids. They also have relative stable offspring sex ratios (Amano and Chant 1978; Toyoshima and Amano 1998; Lv et al. 2019). Up to now, molecular mechanisms of phytoseiid reproduction are still unclear.

Arthropods have various reproductive patterns and mechanisms (Bachtrog et al. 2014). But studies showed insect sex determination pathway downstream genes are relatively conservative, mainly including transformer (tra), transformer-2 (tra-2), and downstream doublesex genes (Hediger et al. 2004; Sarno et al. 2010; Verhulst et al. 2010). It is valuable to investigate whether key genes in insect reproductive regulation also play important roles in phytoseiids. Hoy et al. (2015) and Bi et al. (2019) blasted orthologs of tra and tra-2 in Galendromus occidentalis (Acari: Phytoseiidae) and Phytoseiulus persimilis (Acari: Phytoseiidae) genomes, respectively, based on these genes in Drosophila melanogaster (Diptera: Drosophilidae). Both of them reported orthologs of tra-2, but orthologs of tra was not found.

In Insecta, tra-2 generally contains a highly conserved RRM (RNA-recognition motif), which is an RNA recognition domain with polyarginine/serine-rich regions at both ends. It often participates in specific splicing of downstream sex determination genes. For example, in D. melanogaster, tra-2 acts as a cofactor of the tra gene to regulate the specific shearing of doublesex gene, resulting in production of a sex-specific protein that leads to sex differentiation (Amrein et al. 1990; Hoshijima et al. 1991). In Nasonia vitripennis (Hymenoptera: Chalcidoidea), interference of tra-2 led to sexual reversal of diploid female offspring (Geuverink et al. 2017). Impact of tra-2 on sex determination have also been found in Musca domestica (Diptera: Muscoidea) (Burghardt et al. 2005), Anastrepha suspensa (Diptera: Tephritidae) (Schetelig et al. 2012), and Nilaparvata lugens (Hemiptera: Delphacidae) (Zhuo et al. 2017), etc.

Other roles played by tra-2 in insect reproductive regulation have also been reported. Tra-2 regulates testis development in Bombyx mori (Suzuki et al. 2012). In Bemisia tabaci (Hemiptera: Aleyrodidae), interference of tra-2 resulted in male dysplasia (Guo et al. 2018). In Diaphorina citri (Hemiptera: Liviidae), interference of tra-2 led to decreased female fertility and lower egg hatching rate (Yu and Killiny 2018). Tribolium castaneum (Coleoptera: Tenebrionidae) decreased fecundity after interference of tra-2 (Shukla and Palli 2013).

A few studies investigated the impact of tra-2 orthologs in phytoseiids reproduction. In M. occidentalis, fecundity decreased significantly when tra-2 was interfered (Pomerantz and Hoy 2015b). Bi et al. (2019) interfered 4 genes that potentially related to reproduction in P. persimilis. While the interferences of the other 3 genes all led to reduced fecundity, tra-2 is the only one that led to reduced hatching rate instead.

To further investigate the structure, expression and function of the tra-2 ortholog in P. persimilis (termed as Pptra-2), we predicted its protein function, performed evolutionary analyses, and measured its relative expression in different immature stages, and in adults of both sexes. We interfered Pptra-2 with dsRNA in adult females, and observed daily changes in their reproduction. Transcriptome analyses were conducted to screen genes potentially functionally related to Pptra-2. Samples were collected at the timing when the most significant impact of RNA interferences on reproduction was observed. These analyses provided bases for further detangling complicated reproductive mechanisms in Phytoseiidae.

Materials And Methods

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.

Results

Protein prediction and systematic evolutionary analyses of Pptra-2

The length of the Pptra-2 is 870bp, which encodes a total of 288 amino acids, and contains the highly conserved RRM domain (RNA recognition motif) (Fig. 1). Based on the phylogenetic tree (Fig. 2), All Arachnida species are grouped together, among which Parasitiformes mites is an early branch, separated right after Arachinda separated from Insecta. Parasitiformes are in turn classified to two sub-branches. One contains phytoseiids and other mite species, while the other contains Ixodida species. Pptra-2 has the highest homology with tra-2 in M. occidentalis. In Insecta, Lepidopterans have the closest genetic relationship with Arachnida, while Dipterans are most distant.

Expression of Pptra-2 during Phytoseiulus persimilis development

Expression of Pptra-2 are observed in all stages and both sexes of P. persimilis. Relative expressions were standardized using that of newly emerged female adults (Fig. 3). Relative expressions of immatures and male adults are overall low, with that in eggs and virgin females slightly higher (Fig. 3a). Expression of this gene increased significantly in female adults after mating, with peak expression (ca. 10.5) observed 5d after mating (Fig. 3b). Later on, Pptra-2 expression maintained stable at high levels (> 9.0).

Impact of Pptra-2 on reproduction of Phytoseiulus persimilis

When Pptra-2 was interfered and in the CK, no significant difference was observed in cumulative fecundity, oviposition duration, and proportion of female offspring. However, egg hatching rate decreased to 0.52 when Pptra-2 was interfered, with a large number of shriveled eggs failed to hatch (Table 1, Fig. 4a-b). In CK, egg hatching rates of all individuals distributed from 0.8 to 1. When Pptra-2 was interfered, the egg hatching rates showed a bimodal distribution (Fig. 4c). Approximately 44% individuals had egg hatching rate higher than 80% (with the mean being 0.96), while the rest had egg hatching rate lower (with the mean being 0.33). Based on these results, we further classified the interfered individuals into two groups: high hatching rate (HHR) and low hatching rate (LHR), and compared their reproductive parameters to the CK separately. 

 
Table 1

Parameters on reproduction of fertile Phytoseiulus persimilis individuals in Pptra-2 interfered treatments and the control

 

Tra-2 interfered

CK

 

All

HHR

LHR

Cumulative fecundity

64.2 ± 2.23

67.7 ± 2.81a

62.6 ± 2.20a

63.3 ± 2.52a

Oviposition duration (d)

16.0 ± 2.21

16.0 ± 2.40a

15.9 ± 1.90a

16.1 ± 2.10a

Egg hatching rate

0.52 ± 0.07

0.96 ± 0.02a

0.33 ± 0.04b

0.99 ± 0.01a

Proportion of female offspring

0.71 ± 0.02

0.78 ± 0.01a

0.67 ± 0.04b

0.77 ± 0.01a

For each parameter, means followed by different letters are statistically different (Tukey’s LSD tests: p < 0.05), HHR: high hatching rate, and LHR: low hatching rate

In HHR, no significant difference in all measured parameters were detected comparing to the CK. In LHR, egg hatching rate was ca. 66% lower than the CK, and proportion of female offspring ca. 11% lower. Figure 5 showed the daily egg hatching rate in HHR, LHR, and CK. In CK, daily egg hatching rate maintained high during the whole oviposition duration. In HHR, there is ca. 20% decrease in daily egg hatching rate, ca. 3–7 d after mating. Later on, daily egg hatching rate recovered to ca. 1 and maintained at high level. In LHR, daily egg hatching rate started to decrease ca. 1d after mating, reached the lowest level (ca. 0.13) 5 d after mating, and retained at low levels afterwards.

No significant difference in Pptra-2 expression was observed comparing the interfered individuals and the CK immediately after interference. But expression of this gene was ca. 53% lower in interfered individuals than in CK 3d later (Fig. 6a). On the 5th day after interference, we separated HHR and LHR individuals, and measured their Pptra-2 expression separately. The expressions were ca. 63% and 81% lower than that in the CK, in HHR and LHR, respectively (Fig. 6b).

Potential genes functionally related to Pptra-2

The raw reads obtained from 12 cDNA libraries including the control group and the treatment group ranged from 5039 to 6743 million bp (Table S2). Subsequently, 4980–6680 million bp clean reads were obtained after filtering out low quality sequences, respectively. Besides, Q20 was higher than 97%, and Q30 was higher than 94%, indicating the high quality of RNA sequencing. Then, the clean reads were mapped to the reference genome of P. persimilis, among which 94.78% were uniquely mapped.

RNA-seq showed 403 DEGs (Differentially Expressed Genes) among the three groups. The maximum amount of DEGs (306) was observed comparing individuals LHR vs CK, among which 129 genes were upregulated and 177 genes were down regulated. Comparing individuals in HHR and CK, 39 genes were upregulated and 96 genes were down regulated. While comparing HHR and LHR, 31 genes were up regulated and 10 were down regulated (Fig. S3). Venn diagram (Fig. 7) revealed 61 genes in common between groups LHR vs CK and HHR vs CK, while there were no common DEGs between the three groups.

LHR has the highest number of genes with expression decreased. In both HHR and LHR, Pptra-2 is the gene showed the most significant differences in expression. Its expression decreased by 49% in HHR and 64% in LHR, showing similar patterns as in qPCR analyses. As well as tra-2, there are 19 other DEGs have their expressions changed gradually from CK to LHR to HHR (Table 2). Among these genes, 11 has their expressions the highest in CK, followed by HHR and the LHR, 6 has their expressions the lowest in CK, and 2 only expressed the CK. However, all these genes are not annotated. 

 
Table 2

DEGs with expression changed gradually among the 3 groups (HHR: interfered individuals with high egg hatching rates; LHR: interfered individuals with low egg hatching rates; and the CK)

 

GenBank accession number

CK

HHR

LHR

LHR vs CK P

HHR vs CK

P

 

Pptra-2

41.53 ± 1.97

21.10 ± 4.67

13.47 ± 2.49

5.12E-16

7.81E-07

1

MZ753714

3.37 ± 0.28

6.89 ± 0.98

7.76 ± 0.0

1.11E-07

8.49E-06

2

MZ753715

5.46 ± 0.50

11.26 ± 1.78

15.17 ± 1.47

2.68E-07

2.03E-05

3

MZ753716

3.30 ± 0.42

7.19 ± 1.39

9.95 ± 0.89

1.01E-10

2.07E-05

4

MZ753717

1.64 ± 0.35

3.87 ± 0.37

5.99 ± 0.59

1.30E-10

7.23E-05

5

MZ753718

5.27 ± 0.21

10.98 ± 1.19

18.96 ± 0.29

3.06E-06

2.38E-04

6

MZ753719

15.57 ± 2.61

7.4 ± 2.20

0.58 ± 0.37

3.94E-04

1.79E-02

7

MZ753720

7.90 ± 1.08

4.28 ± 0.96

2.95 ± 0.46

4.07E-03

4.40E-03

8

MZ753721

2.11 ± 0.10

0.79 ± 0.27

0.35 ± 0.18

6.49E-03

3.16E-02

9

MZ753722

1.95 ± 0.10

0.83 ± 0.26

0.32 ± 0.11

3.99E-03

3.18E-02

10

MZ753723

1.75 ± 0.32

0.75 ± 0.32

0.26 ± 0.08

2.53E-05

2.02E-02

11

MZ753724

1.56 ± 0.56

0.38 ± 0.20

0.08 ± 0.05

8.67E-04

4.79E-02

12

MZ753725

2.41 ± 0.62

0

0

7.63E-03

1.70E-02

13

MZ753726

0.43 ± 0.13

0

0

9.03E-03

1.09E-02

14

MZ753727

0.69 ± 0.08

1.54 ± 0.20

2.29 ± 0.07

2.77E-03

8.64E-03

15

MZ753728

0.23 ± 0.08

0.99 ± 0.24

1.26 ± 0.30

2.66E-03

1.21E-02

16

MZ753729

0.49 ± 0.09

1.06 ± 0.10

1.58 ± 0.21

1.24E-05

3.14E-03

17

MZ753730

0.29 ± 0.05

0.91 ± 0.07

1.47 ± 0.33

3.34E-04

2.27E-02

18

MZ753731

0.70 ± 0.12

1.90 ± 0.49

2.57 ± 0.47

2.09E-04

2.20E-02

19

MZ753732

0.05 ± 0.03

0.41 ± 0.15

0.98 ± 0.21

4.34E-04

6.57E-03

According to GO enrichment analyses, 1088, 769, and 41 DEGs were annotated to biological process, cellular component, and molecular function when comparing LHR vs CK, HHR vs CK, and LHR vs HHR (Fig. S4). DEGs were mainly enriched in DNA integration, Serine-type, carboxypeptidase activity, ribose phosphate diphosphokinase activity, diphosphotransferase activity, and nucleoside monophosphate metabolism process.

KEGG pathway enrichments were summarized in Fig. S5. In LHR vs CK, DEGs were mainly enriched in the phosphate pathway, biosynthesis of amino acids, glycocosphingolipid metabolism lacto and neolacto series, Wnt signaling pathway, and Ubiquitin mediated proteolysis. In HHR vs CK, DEGs were mainly enriched in the biosynthesis of unsaturated fatty acids, fatty acid metabolism, mTOR signaling pathway, metabolic pathway, and lysosome. Comparing HHR and LHR, no enrichment in DEGs was observed.

Among the enriched pathways obtained from GO terms and KEGG pathways, 14 genes with its annotation suggested potential relationship with fecundity and hatching rate regulation were summarized in Table 3. These genes are also expected to be functionally related to Pptra-2. 

 
Table 3

Differentially expressed genes with important functions screened by GO and KEGG

 

GenBank accession number

From

Functional

1

MZ753733

GO term, KEGG

Sexual reproduction

2

MZ753734

GO term, KEGG

Sexual reproduction, mTOR signaling pathway

3

MZ753735

GO term, KEGG

sexual reproduction, Wnt signaling pathway

4

MZ753736

GO term

Growth

5

MZ753737

GO term

Growth

6

MZ753738

GO term

Response to growth factor

7

MZ753739

GO

Developmental process

8

MZ753740

GO term, KEGG

Developmental process, Lysosome

9

MZ753741

KEGG

Pentose phosphate pathway

10

MZ753742

KEGG

Pentose phosphate pathway

11

MZ753743

KEGG

Metabolic pathways

12

MZ753744

KEGG

Wnt signaling pathway

13

MZ753745

KEGG

Autophagy - animal

14

MZ753733

KEGG

Arachidonic acid metabolism


Orthologs of insect reproduction related genes were also screened from the transcriptomes and their relative expression were listed in Table 4, including the target gene Pptra-2. In the transcriptome data, we found doublesex, doublesex transcription factor and intersex, which are expected to be related to tra-2, based on insect reproductive regulation studies (Table 4), but none of them was significantly different among the three groups.

 
Table 4

FPKM expression of sex determination and reproduction-related genes identified in the transcriptome between different treatments (HHR: interfered individuals with high egg hatching rates; LHR: interfered individuals with low egg hatching rates; and the CK)

 

GenBank accession number

Gene name

CK

HHR

LHR

LHR vs CK P

HHR vs CK

P

1

MZ753747

doublesex

10.79 ± 0.82

11.79 ± 1.2

13.34 ± 0.75

0.77

0.81

2

OM685104

doublesex transcription factor

1.36 ± 0.08

1.27 ± 0.06

1.22 ± 0.14

0.59

0.74

3

OM685105

doublesex transcription factor

0.57 ± 0.03

0.50 ± 0.08

0.48 ± 0.04

0.45

0.61

4

OM685106

doublesex transcription factor

0.95 ± 0.07

1.10 ± 0.09

1.31 ± 0.13

0.06

0.38

5

OM685107

doublesex transcription factor

1.96 ± 0.32

1.74 ± 0.42

1.94 ± 0.12

0.97

0.61

6

OM685108

doublesex transcription factor

3.06 ± 0.02

4.74 ± 0.07

5.67 ± 0.17

0.30

0.10

7

OM685109

doublesex transcription factor

0.19 ± 0.04

0.16 ± 0.05

0.10 ± 0.02

0.73

0.86

8

OM685110

intersex

24.11 ± 0.91

27.05 ± 1.32

25.57 ± 0.99

0.84

0.46

9

OM685111

intersex

33.65 ± 0.90

34.37 ± 1.68

36.24 ± 0.73

0.69

0.91

 

/

transformer

/

/

/

/

/


Discussion

Herein we predicted the protein sequence and conserved domains of Pptra-2 gene, and described its expression pattern in P. persimilis. When this gene was interfered, P. persimilis has significantly reduced egg hatching rate and marginal reduced proportion of female offspring. Approximately 5 days after mating is the key timing point when maximum impact of RNAi was observed. A total of 42 genes that might be related to reproduction were screened based on transcriptome analyses. Among these genes, 19 were achieved based on differential expression analysis, 14 from gene function enrichment analysis, and 9 from homologous gene blast. cDNA sequences of all these genes have been submitted to NCBI and listed in Table 2-4.

Pptra-2 has an RNA-recognition motif (RRM). In eukaryotes, the RRM usually consists of two highly conserved domains, either two ribonucleoprotein (RNP) domains or two RNA-binding domain (RBD). These structures usually assist RNA splicing and gene expression (Glisovic et al. 2008). Table 5 summarized tra-2 splicing and functions in multiple insect and mite species. Different number of spliceosomes were observed in different species. Even for species in the same order and belong to the same branch in the phylogenetic tree, the number of tra-2 spliceosomes might differ. No obvious correlation between number of spliceosomes and the function of tra-2 has been detected, suggesting although conservatively related to reproduction, this gene has its expression and function varies across species.  

Table 5 

Alternative splicing and functions of transformer-2 in arthropods

Order

Species

Number of variable splicings

Functions

References

Diptera

D. melanogaster

4

Participate in female-specific splicing and male development

(Amrein et al. 1990)

M. domestica

1

Participate in female-specific splicing and male mating

(Burghardt et al. 2005)

A. suspensa

1

Participate in female-specific splicing and female development

(Schetelig et al. 2012)

A. albopictus

2

Abnormal ovarian development in female when interfered

(Li et al. 2019b)

Hymenoptera

A.mellifera

6

Embryonic mortality when interfered

(Nissen et al. 2012)

N. vitripennis

4

Embryonic mortality when interfered

(Geuverink et al. 2017)

Coleoptera

T. castaneum

3

Reduction in female fecundity when interfered

(Shukla and Palli 2013)

Hemiptera

N. lugens

3

Male developmental abnormalities when interfered

(Zhuo et al. 2017)

B. tabaci

4

Male developmental abnormalities when interfered

(Guo et al. 2018)

Lepidoptera

B. mori

6

Abnormal development of female embryos and male testis when interfered

(Suzuki et al. 2012; Suzuki et al. 2017; Xu et al. 2017)

Parasitiformes

M. occidentalis

1

Reduction in female fecundity when interfered

(Pomerantz and Hoy 2015b)

In Phytoseiidae, information about tra-2 spliceosomes are only available in M. occidentalis (Pomerantz and Hoy 2015a; Pomerantz and Hoy 2015b) and P. persimilis. In each of the two species, there is only 1 spliceosome of tra-2. However, tra-2 interference mainly led to reduced fecundity in M. occidentalis (Pomerantz and Hoy 2015b). In contrast, Bi et al. (2019) and the present study both showed that when Pptra-2 was interfered, only egg hatching rate decreased significantly. Expression and function of tra-2 orthologs in more phytoseiids need to be investigated to further identify potential variations in function of tra-2 orthologs in this family. 

In A. mellifera (Nissen et al. 2012), Bradysia odoriphaga (Diptera: Sciaroidea) and D. citri (Yu and Killiny 2018), tra-2 interference also resulted in large numbers of dead embryos. In our study, impact of Pptra-2 on egg hatching rate increased gradually for 5d after mating (Fig. 5). Based on our observations, some eggs in the LHR group were shriveled when being laid. There were also eggs appeared to be normal when laid and shriveled later. All these results showed there was a lag phase of interference. In the LHR group, the proportion of female offspring also decreased slightly. However, this 11% decrease is not high enough to support that Pptra-2 regulate offspring sex determination directly. It is more reasonable to assume that slightly higher mortality was expected in female embryos than in male embryos, possibly due to higher developmental requirements. 

We also found that Pptra-2 was expressed at each developmental stage and in male adults. In M. occidentalis, tra-2 was also amplified from samples of each developmental stage, but its expression level was not estimated quantitatively (Pomerantz and Hoy 2015a). Except in female adults, Pptra-2 has the highest expression level in eggs. The impact of tra-2 on embryo development has been identified in some insects. For example, when interference was performed to eggs, embryo mortality increased in honeybee (Nissen et al. 2012; Geuverink et al. 2017), and embryo sex changed in M. domestica (Burghardt et al. 2005), etc. Also in some insects, interference of tra-2 in males led to abnormal testicular development (Suzuki et al. 2012). Functions of Pptra-2 in other stages and males also worth investigations. 

Transcriptome analyses showed the expressions of orthologs to some genes functionally correlated to tra-2 in Insecta, including doublesex and intersex, does not differ when Pptra-2 was interfered. Doublesex is a relative conservative switch to the sex determination pathway in Insecta. It is regulated by sex-lethal (sxl), tra, and tra-2, and produces sex-specific proteins (Geuverink and Beukeboom 2014). Intersex is the terminal gene in sex-cascading. In Drosophila spp., it synergizes with doublesex and impacts sex-specific differentiation (Waterbury et al. 1999; Siegal et al. 2005). Ortholog of another important gene in insect sex determination, transformer, has not been found in Phytoseiidae yet. Marginal significant difference was observed in only one orthologs of doublesex transcription factor. Most genes significantly differed among CK, HHR, and LHR were not annotated (Table 2). Functions of these genes need to be further estimated and verified, which might provide specific reproduction and sex regulation pathways in phytoseiids.

Go and KEGG enrichment analyses also provided candidate genes that might be involved in reproduction. In Go analyses, DEGs are mainly enriched in cell differentiation, growth and development, catalytic activity, and biological metabolism. These genes are widely involved in the transcription and synthesis of proteins related to cell proliferation, biological reproduction and ovarian development (Zhang et al. 2011; Liu et al. 2021). They also play important roles in development of ovaries and embryos. In KEGG pathways, DEGs are mainly enriched in metabolic pathways, with amino acid metabolism, lipid metabolism and endocrine-related genes also included. These pathways are also important in development and reproduction. Their products guarantee material and energy requirements for ovarian development and oogenesis in insects (Smykal and Raikhel 2015). Detailed functions of some of these genes have been studied in insects. Ubiquitin proteasomes participated in degradation of a variety of proteins, and affect sperm synthesis and the development of oocytes and ovaries (Bebington et al. 2000). Lysosomes are involved in digestion of blood nutrients by hydrolase and phospholipase, and play important roles in nutrient absorption. Arachidonic acid metabolism has the ability to regulate sex hormones synthesis in arthropods, as well as participating in metabolism of fatty acids (Tian et al. 2014; Xu et al. 2017). Previous studies showed that tra-2 participate in fat synthesis in Drosophila spp. (Mikoluk et al. 2018). In the present study, genes involved in this pathway are all down regulated in the LHR group, which might be responsible for embryo mortality. 

As well as metabolism, DEGs are also enriched in some important signaling pathways. Wnt signaling pathway is generally important in organ and gonadal differentiation signal transduction, and participates in regulation of cell differentiation and maturation, and early embryo development (Jeays-Ward et al. 2004). The mTOR signaling pathway is a central controller of cell growth and proliferation. It is recognized as a key regulator of cell metabolism, multiplication, and differentiation. It is involved in various process of oogenesis, and plays a vital role in regulating female reproduction (Meirow et al. 2010; Anderson et al. 2018). These genes are all down-regulated after interference, but limited research on their functions in arthropods and especially in mites were available.

Research on reproduction and sex determination regulation and mechanisms in phytoseiids are still at beginning steps. Their tiny small sizes increased difficulties in application of many molecular biological techniques. For example, a minimum of more than 10 female individuals are required for each qPCR sample. RNAi in phytoseiids are mainly performed through feeding. The bimodal distribution of egg hatching rate suggested some individuals might have the target genes more interfered than the others, possibly because the amount of dsRNA digested by each mite varies. Therefore, qPCR and transcriptome analyses of pooled samples might result in underestimated differences in expression of target gene and correlated genes between treated samples and the CK. That is why we separate interfered individuals based on biological parameters 5d after mating and performed transcriptome analyses for LHR and HHR separately. However, more accurate measurements will be expected if RNA extraction can be conducted at individual level, or even at tissue level. On the other hand, other RNAi method should be tried to improve interfere efficiency. In Acari, more molecular biological studies have been carried out in spider mites. RNAi in T. urticae can be performed through injection and soaking (Suzuki et al. 2017; Mondal et al. 2021). Due to their chitinous dorsal and ventral shields, our preliminary trials in RNAi through injection in Phytoseiids ended up with unacceptable levels of mortality due to physical injury. Soaking organisms into dsRNA concentrates is an easier way to be carried out in RNAi of tiny insects and mites. However, its efficiency is also not stable (Yan et al. 2020b; Yan et al. 2021). Recently, nanoparticle materials have been used to enhance dsRNA epidermal penetration efficiency, so higher RNAi efficiency through soaking are expected. Successes have been achieved in some insects, such as Aphis glycines (Homoptera: Aphididae) (Yan et al. 2020a) and Agrotis ypsilon (Lepidoptera: Noctuidae) (Li et al. 2019a), etc. Some early trials have also been taken in RNAi of Phytoseiids (Wang et al. 2022), and more applications of this technique are expected. 

Overall, we conducted detailed research on expression of Pptra-2 and its impact on the P. persimilis reproduction, and quantitatively estimated the key timing point when Pptra-2 played its role. Potential related functional genes were also screened, providing hints for further investigations of phytoseiid reproduction.

Declarations

Funding 

This study was supported by the National Key R & D Program of China (2017YFD0200400), the National Natural Science Foundation of China (3197170803) and co-innovation project of CAAS and SDAAS ‘Key technologies on regional green agricultural development and integrated demonstration’.

Conflict of interest 

There is no conflict of interest concerning the results provided in this manuscript. The authors declare no conflicts of interest.

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