The identification and expression pattern of the sex determination genes and their sex-specific variants in the egg parasitoid Trichogramma dendrolimi Matsumura (Hymenoptera: Trichogrammatidae)

Introduction: Trichogramma wasps are egg parasitoids of agricultural lepidopteran pests. The sex of Trichogramma is determined by its ploidy as well as certain sex ratio distorters, such as the endosymbiotic bacteria Wolbachia spp. and the paternal sex ratio (PSR) chromosome. The sex determination systems of hymenopterans, such as Trichogramma spp., involve cascades of the genes transformer (tra), transformer-2 (tra2), and doublesex (dsx) and are associated with sex-specific tra and dsx splicing. First, these genes and their sex-specific variants must be identified to elucidate the interactions between the sex ratio disorders and the sex determination mechanism of Trichogramma. Methods: Here, we characterized the sex determination genes tra, tra2, and dsx in Trichogramma dendrolimi. Sex-specific tra and dsx variants were detected in cDNA samples obtained from both male and female Trichogramma wasps. They were observed in the early embryos (1–10 h), late embryos (12–20 h), larvae (32 h and 48 h), pre-pupae (96 h), and pupae (144 h, 168 h, 192 h, and 216 h) of both male and female T. dendrolimi offspring. Results: We detected female-specific tra variants throughout the entire early female offspring stage. The male-specific variant began to express at 9–10 h as the egg was not fertilized. However, we did not find any maternally derived, female-specific tra variant in the early male embryo. This observation suggests that the female-specific tra variant expressed in the female embryo at 1–9 h may not have originated from the maternal female wasp. Discussion: The present study might be the first to identify the sex determination genes and sex-specific gene splicing in Trichogramma wasps. The findings of this study lay the foundation for investigating the sex determination mechanisms of Trichogramma and other wasps. They also facilitate sex identification in immature T. dendrolimi and the application of this important egg parasitoid in biological insect pest control programs.


Introduction
Trichogramma spp., the egg parasitoids, are efficacious biological control agents against lepidopteran pests in agriculture and forestry (Zhou et al., 2019a;Zang et al., 2020).Female Trichogramma wasps oviposit into the eggs of their insect pest hosts (Zhou et al., 2019b).The sex of the Trichogramma wasp is determined by its ploidy.Female and male offspring develop from fertilized diploid and unfertilized haploid eggs, respectively (Zhou et al., 2003;Heimpel and de Boer, 2008;Zhang et al., 2020).The sex determination systems of Trichogramma are also affected by certain sex ratio distorters, such as the endoparasitic bacteria Wolbachia spp.and the paternal sex ratio (PSR) chromosome (Stouthamer et al., 1999;van Vugt et al., 2003;Zhou et al., 2022a).The endosymbiotic bacteria Wolbachia spp.induce parthenogenesis in Trichogramma wasps by transforming haploid male-destined eggs into diploid embryos during the first mitotic division (Huigens et al., 2000;Zhou et al., 2022b;Zhang et al., 2022).The PSR chromosome causes the generation of male offspring and the loss of the paternal genome during the first mitotic division of the zygote (van Vugt et al., 2003;Dalla Benetta et al., 2020).However, the interactions between the preceding sex ratio disorders and the sex determination systems of Trichogramma have not been studied in depth.Therefore, identification and analysis of the sex determination genes in Trichogramma wasps will help clarify the interactions between these insects and their sex ratio distorters.
Sex determination systems are usually directed by genetic pathways comprising several sex-determining genes (Wilkins, 1995;Verhulst et al., 2010a;Simoni et al., 2020).Sex determination systems consist of a primary genetic signal, sexspecific signal cascades, and the expression of genes regulating sexual development (Peng et al., 2020).The primary sex-specific signals of hymenopterans are governed by paralog genes designated as transformer (tra) or feminizer (fem) (Verhulst et al., 2010a).The rapidly evolving tra encodes arginine/serine (SR)-rich proteins.Order-specific domains have been detected in the tra/fem orthologs such as the hymenopteran (HYM) domain in hymenopterans as well as the domains characteristic of other insect orders.The tra pre-mRNA can be spliced into femalespecific and male-specific variants (Verhulst et al., 2010a;Geuverink et al., 2017a).Only a female-specific tra variant produces a functional TRA protein with the Ceritatis-Apis-Musca (CAM) domain and complexes with the protein encoded by tra2 to regulate female-specific doublesex (dsx) splicing (Geuverink et al., 2017b).TRA2 protein is more conserved than TRA.Its RNAbinding domain (RBD) has two regions rich in Ser-Arg (Geuverink et al., 2017b).Dsx interprets the sexual traits of various invertebrates (Chikami et al., 2022).The conserved dsx contains the DNA-binding motif (DM) domain and is localized to the bottom of the tra-tra2-dsx cascade that determines sex in hymenopterans (Verhulst et al., 2010b;Baral et al., 2019).
The haplodiploid sex determination system in hymenopterans is described by the Maternal Effect Genomic Imprinting Sex Determination (MEGISD) model (Beukeboom et al., 2007).In MEGISD, the sex-specific signal involves maternal tra mRNA imprinting and the alternative tra splicing induced by the zygote (Beukeboom and Kamping, 2006;Verhulst et al., 2010a).In Nasonia vitripennis, female-specific tra splicing is initiated by timely expression of the paternal wasp overruler of masculinization (wom) allele upon fertilization (Zou et al., 2020).Sex-specific tra cascade tra2 splicing initiates sexual development by directing dsx.In other hymenopterans, offspring sex is determined according to the MEGISD model and is regulated by the complementary sex determination (CSD) mechanism (Matthey-Doret et al., 2019).In CSD, the heterozygotic csd locus initiates female development, while the homozygotic csd locus promotes diploid male formation (Hagan and Gloag, 2021).CSD proteins direct the sex-specific tra/fem variants, which, in turn, leads to female development (Matthey-Doret et al., 2019;Hagan and Gloag, 2021).Our previous study showed that the CSD mechanism is absent in Trichogramma dendrolimi (Liu et al., 2019).The parasitoid wasps Trichogramma spp.could be the most important egg parasitoid hosts in biological insect pest control programs (Zang et al., 2020;Zhou et al., 2020).To the best of our knowledge, the sex determination genes and their sexspecific splicing have never been previously identified in Trichogramma wasps.
The present study aimed to identify the sex determination genes tra, tra2, and dsx, their sex-specific variants, and their expression patterns at different developmental stages of male and female Trichogramma dendrolimi Matsumura.The results of this work could help elucidate the sex determination mechanisms in Trichogramma wasps.The output of this study also facilitates sex identification in immature T. dendrolimi as it shows how sex-specific tra and dsx variants may be detected.

Insects
The isofemale T. dendrolimi line was established using a single mating pair of wasps.The insects were reared over several generations on the eggs of the rice moth Corcyra cephalonica (Lepidoptera: Pyralidae) at 25 °C and 70% relative humidity (RH) under a photoperiod of L16:D8.The host eggs were grouped in lots of ~500, glued onto a white card, and allowed to parasitize the host eggs.The latter were irradiated with ultraviolet (UV) light (TUV 30W UV lamps; Philips, Amsterdam, The Netherlands) for 45 min.These host egg cards were used for parasitization by T. dendrolimi.

Sample collection
A group of 100 adult male or female T. dendrolimi wasps was collected.Male T. dendrolimi offspring were obtained from host eggs parasitized by virginal female wasps.Mixtures of male and female offspring were obtained from host eggs parasitized by mated female wasps.Immature T. dendrolimi cannot be distinguished by sex.They include early embryos (at 1 h, 2 h, 3 h, 4 h, 5 h, 6 h, 7 h, 8 h, 9 h, and 10 h), late embryos (at 12 h, 14 h, 16 h, 18 h, and 20 h), larvae (at 32 h and 48 h), pre-pupae (at 96 h), and pupae (at 144 h, 168 h, 192 h, and 216 h).All the foregoing stages were collected in lots of ~1,000.

RNA extraction and cDNA synthesis
The total RNA of each sample was extracted.Samples included embryos, larvae, pupae, and adults.Total RNA of the T. dendrolimi wasp was extracted with TRIzol reagent (Invitrogen, Carlsbad, CA, United States).Total RNA (1 µg) was reverse-transcribed using a PrimeScript RT Kit (TaKaRa, Dalian, China) according to the manufacturer's protocol.The cDNA product was then immediately stored at −80 °C until subsequent use.

Splicing of sex-specific tra and dsx
Alternative tra and dsx variants were amplified using the specific primers listed in Table 1.The PCR products of the alternative splices were confirmed, cloned, and sequenced as previously described.Sex-specific tra and dsx exons were characterized using the fragments amplified from the cDNA samples of male and female T. dendrolimi.The full-length sexspecific transcripts were then merged with the predicted tra or dsx models for T. dendrolimi using the sequenced sex-specific fragments.

Phylogenetic and molecular evolutionary analyses
Sequences of the homologs in ten insect species from the National Center for Biotechnology Information (NCBI) database (https://www.ncbi.nlm.nih.gov/) were collected to delineate the evolution of tra, tra2, and dsx.A phylogenetic tree was generated using PhyloSuite v. 1.2.2 (Zhang et al., 2020) with the IQ-TREE fitted by the JTT model (Nguyen et al., 2015).Orthologous relationships were identified using 1,000 bootstrap replicates.Conserved domains were identified using the Conserved Domain Database of NCBI.

Sex-specific splicing expression patterns in T. dendrolimi
The primers used for sex-specific tra and dsx splicing were selected based on the different sequences of female-and malespecific variants and designed using Primer Premier v. 5.0.Thus, the specific regions of female-and male-specific variants can be amplified to different sizes by the single pair of primers.Sexspecific tra and dsx splicing was validated based on the real-time (RT)-PCR products amplified using the male and female cDNA samples.The dynamic expression of sex-specific tra and dsx splicing was detected in the male and mixed male-female offspring samples.

FIGURE 5
Detection of sex-specific tra variants in pooled samples of male offspring deposited by unmated female wasps (A) and mixed male and female offspring deposited by mated female wasps (B) collected from early embryos (at 1 h, 2 h, 3 h, 4 h, 5 h, 6 h, 7 h, 8 h, 9 h, and 10 h), late embryos (at 12 h, 14 h, 16 h, 18 h, and 20 h), larvae (at 32 h and 48 h), pre-pupae (at 96 h), and pupae (at 144 h, 168 h, 192 h, and 216 h) of T. dendrolimi.Lane M is the DL2000 DNA marker.
The RT-PCR products of the sex-specific variants were then detected by 1.5% agarose gel electrophoresis.

Results
Identification of tra, tra2, and dsx The full-length tra sequence was 1,592 bp long and included an open reading frame (ORF) of 1,128 bp encoding 375 amino acids (aa) (Table 2).The tra aa sequence had the same structure as those of other known insect tra.The HYM domain was detected in all tra/fem orthologs in hymenopterans.All orthologs had a CAM, a proline (Pro)-rich, and an Arg/Ser domain.The phylogenetic tree showed that the aa sequence of T. dendrolimi tra was closely related to that of N. vitripennis tra (Figure 1; Figure 2; Supplementary Figure S1).Alignment of the aa sequences in T. dendrolimi and N. vitripennis tra revealed 41.12% identity.
The full-length sequence of tra2 was 2,932 bp long and included an ORF of 885 bp encoding 294 aa (Table 2).The RBD was detected in the tra2 orthologs in hymenopterans.The aa sequence of T. dendrolimi tra2 was similar to that of N. vitripennis tra2 (Figure 2).Both the T. dendrolimi and N. vitripennis orthologs had a glycine (Gly)-rich domain (Figure 1; Supplementary Figure S2).Alignment of the aa sequences in T. dendrolimi and N. vitripennis tra2 disclosed 76.92% identity.
The dsx ortholog was 3,719 bp long and included an ORF of 768 bp encoding 255 aa (Table 2).The aa sequences of the T. dendrolimi and other insect dsx orthologs all had the DM and dimer domains (Figure 1; Supplementary Figure S3).The aa sequence of T. dendrolimi dsx was similar to that of N. vitripennis dsx (Figure 2).Alignment of the aa sequences in T. dendrolimi and N. vitripennis dsx revealed 76.63% identity.

Sex-specific variants of tra and dsx
Expression of the sex-specific tra and dsx variants was determined using the cDNA samples obtained from male and female wasps (Figure 3).Both the female-specific variant (ID: OQ847082) and male-specific variant (ID: OQ847083) of tra were registered on the NCBI database.The female-specific tra variant was composed of nine exons.The male-specific tra variant encoded a truncated protein consisting of 172 aa and an Arg/Ser domain.The Pro-rich and CAM domains were absent in the male-specific tra variant.Hence, these domains are only implicated in female-specific signals.
Both the female-specific variant (ID: OQ847080) and malespecific variant (ID: OQ847081) of dsx were registered on the NCBI database.Dsx included five exons.Female-specific exclusion was observed at the fifth exon, while male-specific exclusions occurred at the fifth exon and the fourth intron (Figure 4).
We detected sex-specific tra and dsx variants in pooled male and mixed male-female progeny samples.Male-specific tra variant was observed in the male offspring after 9 h.Female-specific tra variant was found in the mixed male-female progeny samples at all developmental stages (Figure 5).Both female-specific and male-specific dsx variants were detected in the offspring after 9 h (Figure 6).

Discussion
The present study identified the genes tra, tra2, and dsx which determine the sex of the natural enemy wasp T. dendrolimi.Sexspecific tra and dsx variants were detected in the male and female wasps.To the best of our knowledge, the present study may be the first to conduct a systematic analysis of the genes that determine sex in Trichogramma wasps.
In hymenopterans, the tra/fem orthologs have widely diverged, whereas those of tra2 and dsx are relatively more conserved.These factors might partially explain the observed variation among different insect species in terms of their sex determination systems (Beye et al., 2003).For example, the sex determination of CSD mechanisms has been found in at least 60 species of Hymenoptera (van Wilgenburg et al., 2006).Sex-specific tra/fem splicing is governed by CSD proteins in hymenopterans (Hasselmann et al., 2008).In contrast, the CSD mechanism is virtually absent in parasitoid wasps such as Nasonia spp.In the members of this genus, the instructor wom directs tra splicing and is activated only in the early embryo after fertilization (Zou et al., 2020).Divergence of the tra/fem orthologs may account for the observed differences in the instructor signals among various MEGISD and CSD mechanisms.
Similar to the previously reported Nasonia wasps (Zou et al., 2020), the Trichogramma wasps presented with a female-specific tra variant in the early female embryo (1-20 h).The male-specific variant began to express its tra variant at 9-10 h as fertilization was somewhat delayed.Unlike the Nasonia wasps (Zou et al., 2020), the Trichogramma wasps presented no maternally provided female-specific tra variant at the early male embryo stage.Thus, the female-specific tra variant expressed in the female embryo at 1-9 h may not have originated from the maternal female wasp.In the Asobara tabida wasp, no femalespecific variant was detected in the early fertilized or unfertilized eggs (Geuverink et al., 2017a).A non-sex-specific tra variant was detected in early A. tabida embryos, and it contained a putative duplicate CAM domain.However, the role of this non-sexspecific tra variant in A. tabida remains to be investigated.According to the MEGISD model, sex determination is initiated by a paternal instructor wom.In Nasonia, wom may only be expressed in the early embryo within several hours (Zou et al., 2020).In Trichogramma, the male-specific tra and dsx variants were detected and began to express, respectively, in the male embryo after 9 h.Therefore, the sex-determination mechanisms may be similar for both Nasonia and Trichogramma wasps and lack the effects of the maternally provided female-specific tra variant observed in A. tabida wasps.Nevertheless, it remains to be determined whether early sex-specific tra splicing is initially governed by a paternal factor such as wom.
To the best of our knowledge, this study may be the first case for analyzing alternative splicing of the sex determination genes tra, tra2, and dsx in Trichogramma wasps.The dynamics of sex-specific tra and dsx variant expression indicated that these sex-specific splicing events may be initially governed by an early paternal instructor such as wom found in Nasonia wasps.However, primers for the sex-specific tra and dsx variants could efficiently determine the sex of immature Trichogramma.The results of this work provide an important reference for investigating the sex determination mechanisms of Trichogramma wasps and help control the sex ratio of these natural enemy insects as they are being reared on a large scale for use in biological insect pest control programs.

FIGURE 1
FIGURE 1Phylogenetic analysis of amino acid sequences of tra (A), tra2 (B), and dsx (C) in different insect species.

FIGURE 2
FIGURE 2 cDNA and amino acid sequences and domains of tra (A), tra2 (B), and dsx (C) in T. dendrolimi.(A) The blue box indicates the putative autoregulation domain; the black box indicates the Arg/Ser domain; and the fuchsia box indicates the proline (Pro)-rich region.(B) The cyan box indicates the RNA recognition motif (RRM) domain.(C) The red box indicates the DNA-binding motif (DM) domain; the cyan box indicates the dimer domain.

FIGURE 3
FIGURE 3Detection of sex-specific tra (A) and dsx (B) splices from pooled cDNA samples of male and female wasps.Lane M is the DL2000 DNA marker.Lanes 1-3 in (A) are female-specific tra splices.Lanes 4-6 in (A) are male-specific tra splices.Lanes 1-3 in (B) are female-specific dsx splices.Lanes 4-6 in (B) are male-specific dsx splices.

FIGURE 4
FIGURE 4 Genomic structures of sex-specific tra (A) and dsx splices (B).Boxes represent exons.White boxes represent protein-coding exons.Black boxes represent non-translated exons.Light gray boxes indicate the arginine/serine (RS)-rich domain.Dark gray boxes in (A) indicate the proline (Pro)-rich domain.Dark gray boxes in (B) indicate the dsx dimer.

TABLE 1
Sequences of the primers.

TABLE 2
Length of the full sequence, ORF, and protein of tra, tra2, and dsx genes in T. dendrolimi and N. vitripennis.