Splicing and Expression Regulation of fruitless Gene in Bemisia tabaci (Hemiptera: Aleyrodidae)

: The fruitless ( fru ) gene is a key factor in controlling sexual behavior in insects. The homolog of fru has been identiﬁed in most insect species and exhibits conservation in the transcript architecture and regulation of male sexual behavior. However, the structure of fru transcripts in Hemiptera remains unknown. Here, we identiﬁed and characterized two fru transcripts in Bemisia tabaci , named fru-a and fru-b . fru-a contained a 1263 bp open reading frame (ORF) encoding for 420 amino acids, while fru-b contained a 1143 bp ORF encoding for 380 amino acids. These two proteins start with the same BTB domain and end with two different zinc ﬁnger domains, belonging to ZnA and ZnG, respectively. The expression of fru-a and fru-b differed signiﬁcantly between females and males, and both were expressed at lower levels in males. We demonstrated the presence of multiple TRA/TRA-2 binding sites and alternative splicing in fru-a and fru-b . Moreover, the RNAi result provided evidence that transformer regulates the expression of fru-a and fru-b . These results promote the study of the sex determination cascade in B. tabaci and lay the foundation for the study of sexual behavior in this insect.


Introduction
The mechanisms of sex determination in insects are diverse. Even among closely related species, the mechanisms for determining sex are different. Its divergent model comprises four levels of sex-determining regulators: primary signal > key gene > doubleswitch gene > sex-differentiation gene. The primary signal is variable among insect species, resulting in repression or activation of the key gene. The active state of this key gene is conveyed by alternative splicing of a conserved double-switch gene. This switch gene then transmits similar splicing information to the sex differentiation genes, which ultimately translate the molecular signal into a specific sexual phenotype. In Drosophila melanogaster (Diptera: Drosophilidae), the model is a well-characterized genetic hierarchy X:A > Sxl > tra/tra2 > dsx and fru [1]. This cascade appears to control sex determination in all Drosophila species and is partially conserved in other insect species, especially the tra > dsx and fru regulatory modules [2].
Transformer (tra) is a master regulator of sex determination in many insects. It not only regulates its own sex-specific alternative splicing, but also controls the splicing of downstream target genes dsx and fru [3]. Doublesex (dsx) is a double-switch gene that controls sexually dimorphic characteristics at the bottom of the somatic sex determination cascade and is functionally conserved in many insect species [4]. Fruitless (fru) is the

Insect Strains
The Q biotype Bemisia tabaci (Mediterranean, MED) strain used in this study was originally collected in Beijing, China, in 2009. Since then, the population has been reared on cotton in a naturally lit greenhouse with an ambient temperature of 25 ± 1 • C and a humidity of 70 ± 5%. Mitochondrial cytochrome oxidase I (mtCOI) markers were used every 2-3 generations to confirm population purity.

Sample Collection
Samples of eggs, 1st-, 2nd-, 3rd-, and 4th-instar nymphs and newly emerged females and males of the whitefly were collected, snap frozen in liquid nitrogen, and stored at −80 • C for subsequent experiments.
Clean, insect-free cotton was placed in an insect cage, and the plants were removed after 3 days of egg-laying. Then, the eggs were collected on this as egg-stage samples. The remaining egg-bearing cotton seedlings were placed in a clean insect-free cage and raised in a greenhouse. Five days later, when the whitefly had hatched, it was collected as a first-instar sample. Eight days later, it was collected as a second-instar sample. Twelve days later, it was collected as a third-instar sample, and sixteen days later, it was collected as a fourth-instar sample. When collecting the newly emerged female and male adults, the original adults on the leaves were first sucked away, and then, the emerged adults were collected in microscopic tubes (5.0 × 0.5 cm) after 1 h. Each tube contained 1 adult, and the sex of the insects was distinguished under a stereomicroscope. A total of 3 biological replicates were collected for each group of samples.

RNA Extraction and cDNA Synthesis
Total RNA was extracted by TRIzol reagent (Life Technologies). The purity and quantity of RNA were measured using NanoDrop 2000. RNA integrity was detected by 1% Tris/Borate/EDTA (TBE) agarose gel electrophoresis. The first strand of cDNA was synthesized with PrimeScript ® RT Reagent Kit (TaKaRa Biotech, Kyoto, Japan), and the product was used immediately or stored at −20 • C for future use.

Gene Cloning and Splice-Variant Detection
The annotated sequence obtained previously was used to clone the fru gene in B. tabaci [21]. The full-length primers for the Btfru gene were designed with Primer Premier 5.0 software (Table 1). PCR reactions consisted of 12.5 µL Es-Taq MasterMix, 10.5 µL ddH 2 O, 1 µL cDNA template, and 0.5 µL of each primer (10 mM). The reaction procedure was as follows: denaturation at 95 • C for 5 min; amplification, 95 • C for 30 s, 56 • C or 59 • C (depending on the primers) for 30 s, and 72 • C for 1 min (35 cycles); extension, 72 • C for 10 min. PCR products were purified by a DNA Gel Extraction Kit (NEB, Beijing, China), cloned into the pEASY-T1 vector (TransGen, Beijing, China), and sequenced. The analysis of alternative splicing was consistent with previous methods [22]. Table 1. Primer sequences for full-length gene cloning, alternative splicing analysis, gene expression analysis, and RNA interference were used in this study.

Application of Primers Gene Name Primer Name Primer Sequence (5 -3 )
Cloning and AS analysis

Phylogenetic Analysis
To analyze the evolutionary placement of fru zinc finger domains, we firstly collected the nucleotide sequences of known fru zinc fingers. Secondly, we aligned these sequences by CLUSTALW [25]. The phylogenetic tree was constructed with MEGA 6 software, using the neighbor-joining method with a p-distance model and pairwise deletion of gaps [26].

Real-Time Quantitative PCR
To distinguish the spliceosomes of fru, we designed quantitative primers in the BTB domain, the common region of fru-a and fru-b, and the ZnA and ZnG regions, respectively. The specific primers are shown in Table 1. Relative transcript levels of all fru isoforms, fru-a and fru-b, and the reference gene (SDHA) were assayed by real-time qPCR with the conditions described as follows [27]: denaturation at 95 • C for 10 min; amplification, 95 • C for 15 s, 60 • C for 30 s, and 72 • C for 30 s (40 cycles). Amplified products were identified using SuperReal PreMix Plus (SYBR Green) (Tiangen, Beijing, China). Three independent biological replicates were included for each stage. The relative differences in transcript levels were analyzed by the 2 −∆∆Ct method [28].

RNA Interference
To amplify dsRNA targeting Bttra, primers containing a T7 promoter sequence were designed ( Table 1). The dsRNA for enhanced green fluorescent protein (EGFP) was used as the negative control. All dsRNAs were prepared using the T7 Ribomax™ Express RNAi system (Promega, Madison, WI, USA). RNAi was achieved by directly feeding a 0.20 mL drop of diet solution containing 5% yeast extract, 30% sucrose (wt/vol), and 100 ng dsRNA to B. tabaci adults in an incubated chamber [29,30]. Approximately 40 newly emerged mixed adults (the ratio of males to females was 1:1) were introduced into an environmental chamber at 25 • C under a photoperiod of L14:D10 and a relative humidity (RH) of 70%. Each sample was represented by three technical replicates. Mortality was recorded, and B. tabaci specimens were collected after two days of feeding.

Characterization of Btfru
Previously, we successfully identified a fru homolog in B. tabaci [21]. In this study, we found two fru transcripts in our transcriptome data, named fru-a and fru-b. fru-a contains a 1263 bp open reading frame (ORF) encoding 420 amino acids with a predicted molecular weight (Mw) of 46.67 kDa and an isoelectric point (pI) of 6.47. fru-b contains a 1143 bp ORF encoding for 380 amino acids with a predicted Mw of 42.33 kDa and a pI of 6.43. Genomic structural analysis showed that fru-a and fru-b were located in the same scaffold, with fru-a containing 7 exons and 6 introns and fru-b containing 6 exons and 5 introns (Figure 1). The BTB domain and C 2 H 2 zinc finger domain were identified in the predicted fru-a and fru-b amino acid sequences ( Figure 1). The two transcripts both begin with an exon linked to the BTB-coding exon and end with alternative C 2 H 2 zinc finger domain coding exons. We also searched for the presence of Tra/Tra2 binding sites in the Btfru gene and found two in fru-a and one in fru-b, respectively ( Figure 1).
Horticulturae 2023, 9, x FOR PEER REVIEW 5 of 12 coding exons. We also searched for the presence of Tra/Tra2 binding sites in the Btfru gene and found two in fru-a and one in fru-b, respectively ( Figure 1).

Phylogenetic Analysis of Btfru
Phylogenetic analysis showed that Fru proteins were clustered within each insect order, and two BtFru were closely related to that of Lygus hesperus ( Figure S1). In order to reveal the evolutionary origin of fru-a and fru-b, the zinc finger domains of both were analyzed in depth. By searching the literature, the zinc finger sequences of the reported insect fru gene were collected, and the phylogenetic tree was constructed with fru-a and frub. The result is shown in Figure 2. The zinc fingers of fru-a and fru-b belong to ZnA and ZnG, respectively ( Figure 2). Multi-alignment analysis of the ZnA and ZnG sequences of known insects revealed high similarity in each species (67% and 71%, respectively), as shown in Figure S2. From the evolutionary analysis, it is known that ZnF and ZnB, ZnA and ZnC are likely to be derived from the same ancestor. Phylogenetic analysis also showed that not all insect FRUs contain all known zinc finger domains; for example, Drosophila fru has only ZnA, ZnB, and ZnC; Nasonia fru has ZnA, ZnB, ZnC, ZnF, and ZnG; the whitefly fru has only ZnA and ZnG.
In addition, we found that the C2H2 zinc finger domains of fru have the same consistent sequence, CX2CX9HX3HX6CX2CX5RXDX4HX4H, where C and H are cysteines and histidines, while X represents an unconserved amino acid. If the H sequences of the zinc finger domains are excluded, there are three additional amino acid residues (underlined) that are conserved in all frus: CX2CXKXVX5HX3HX6CX2CX5RXDX4HX3KH (Figure S2). This should be a typical feature of the fru zinc finger domains.

Phylogenetic Analysis of Btfru
Phylogenetic analysis showed that Fru proteins were clustered within each insect order, and two BtFru were closely related to that of Lygus hesperus ( Figure S1). In order to reveal the evolutionary origin of fru-a and fru-b, the zinc finger domains of both were analyzed in depth. By searching the literature, the zinc finger sequences of the reported insect fru gene were collected, and the phylogenetic tree was constructed with fru-a and fru-b. The result is shown in Figure 2. The zinc fingers of fru-a and fru-b belong to ZnA and ZnG, respectively ( Figure 2). Multi-alignment analysis of the ZnA and ZnG sequences of known insects revealed high similarity in each species (67% and 71%, respectively), as shown in Figure S2. From the evolutionary analysis, it is known that ZnF and ZnB, ZnA and ZnC are likely to be derived from the same ancestor. Phylogenetic analysis also showed that not all insect FRUs contain all known zinc finger domains; for example, Drosophila fru has only ZnA, ZnB, and ZnC; Nasonia fru has ZnA, ZnB, ZnC, ZnF, and ZnG; the whitefly fru has only ZnA and ZnG.
In addition, we found that the C 2 H 2 zinc finger domains of fru have the same consistent sequence, CX2CX9HX3HX6CX2CX5RXDX4HX4H, where C and H are cysteines and histidines, while X represents an unconserved amino acid. If the H sequences of the zinc finger domains are excluded, there are three additional amino acid residues (underlined) that are conserved in all frus: CX2CXKXVX5HX3HX6CX2CX5RXDX4HX3KH ( Figure S2). This should be a typical feature of the fru zinc finger domains.

Developmental Expression of Btfru
In order to reveal the expression patterns of fru-a and fru-b in different developmental stages of B. tabaci, quantitative primers specific to the fru-a and fru-b zinc fingers were designed, and the cDNA samples of different developmental stages of B. tabaci were used as the templates for qRT-PCR. The results showed that both fru transcripts were highly expressed in the egg stage, followed by the third-instar larvae, with the lowest expression in males and significant differences between males and females ( Figure 3).

Developmental Expression of Btfru
In order to reveal the expression patterns of fru-a and fru-b in different developmental stages of B. tabaci, quantitative primers specific to the fru-a and fru-b zinc fingers were designed, and the cDNA samples of different developmental stages of B. tabaci were used as the templates for qRT-PCR. The results showed that both fru transcripts were highly expressed in the egg stage, followed by the third-instar larvae, with the lowest expression in males and significant differences between males and females ( Figure 3).

Developmental Expression of Btfru
In order to reveal the expression patterns of fru-a and fru-b in different developmen stages of B. tabaci, quantitative primers specific to the fru-a and fru-b zinc fingers w designed, and the cDNA samples of different developmental stages of B. tabaci were u as the templates for qRT-PCR. The results showed that both fru transcripts were hig expressed in the egg stage, followed by the third-instar larvae, with the lowest express in males and significant differences between males and females ( Figure 3).

Analyses of Alternative Splicing Variants of Btfru
To analyze the spliceosomes of Btfru, we performed PCR amplification with fulllength primers using newly emerged males and females as templates ( Figure S3). All Btfru isoforms obtained from this analysis are listed in Table 2. In our study, 21 isoforms were found in fru-a, and amongst them, only 3 contained both the BTB and Zn finger domains (Nos. 1,18,21). No. 1 occurred in both females and males; No. 18 occurred only in males; No. 21 occurred in females. In addition, six isoforms were found in fru-b. No. 1 and No. 6 contain both BTB and Zn finger domains. No. 6 occurred only in females. The number of fru isoforms obtained from PCR assays was numerous, and their differences between males and females were enormous. To verify that this result was real and not caused by sequencing, we designed quantitative primers in the BTB domain and the ZnA and ZnG regions, respectively. The results are shown in Figure S4. Quantitative primers stuck in the BTB domain reflected the expression of all fru isoforms, and there was no significant difference between male and female expression at this time, indicating that most isoforms retained the BTB domain, which was consistent with the PCR sequencing results. The quantitative primers stuck in the ZnA and ZnG regions responded to the expression levels of specific zinc finger domains, which showed highly significant differences between males and females, with the expression of females in ZnG reaching 8.7-times higher than that of males. This result suggests that it is the use of different zinc finger domain endings that leads to the creation of the fru sex-specific isoforms.
Although we know that frus have sex-specific alternative splicing in B. tabaci, we did not find any sex-specific exons or stable sex-specific variants in fru-a and fru-b. All of the sex-specific variants were found in only one biological replicate. We tried many times, but failed to find a marker that could mark the sex-specific fru spliceosome. This is probably because the spliceosome of fru is too complex and variable.
At the same time, we also performed the fru spliceosome analysis of a single whitefly, and the results were even more complex, which further indicated that the population of the whitefly is too heterozygous and has too much individual variation.

Analysis of the Interaction between Bttra and Btfru
Comparative analysis of fru sequences of N. vitripennis, Apis mellifera, and B. tabaci revealed the presence of a short repeat sequence (T/G/C)GAAGAT(T/A) in all three genes ( Figure 4A). These repeats are thought to be TRA/TRA2 binding sites in hymenopterans and are conserved in the dsx and fru genes of N. vitripennis and A. mellifera [9]. This finding indicates that the splicing of Btfru is also mediated by tra, as in other insect species.

Analysis of the Interaction between Bttra and Btfru
Comparative analysis of fru sequences of N. vitripennis, Apis mellifera, and B. tabaci revealed the presence of a short repeat sequence (T/G/C)GAAGAT(T/A) in all three genes ( Figure 4A). These repeats are thought to be TRA/TRA2 binding sites in hymenopterans and are conserved in the dsx and fru genes of N. vitripennis and A. mellifera [9]. This finding indicates that the splicing of Btfru is also mediated by tra, as in other insect species.
Previously, we successfully identified a tra homolog in B. tabaci, named Bttra [21]. After silencing Bttra in B. tabaci adults, both fru-a and fru-b expression decreased significantly compared with the dsEGFP control groups ( Figure 4B). This result suggests that Bttra regulates the expression of Btfru.

Discussion
The fruitless gene performs multiple functions during Drosophila development. In recent years, fru homologues have been identified in most insect species. These Frus are conserved both in transcript architecture and in the regulation of male sexual behavior. However, the status of Hemiptera fru remains unknown. Here, we conducted a detailed analysis of fru transcripts in B. tabaci.
The fru transcripts generally begin with a broad-complex, tramtrack, and bric-a-brac (BTB) domain and end with one alternative C2H2 zinc finger domain [31]. The BTB domain is involved in protein oligomerization and recruitment of transcriptional corepressors. It is the most-conserved feature of all Fru proteins [32,33]. The BTB domain found in B. tabaci fru transcripts is encoded by a single exon (Figure 1), which is the same as N. vitripennis, but different from that of D. melanogaster [9]. In Drosophila, the BTB domain is encoded by three different exons [34]. Multi-alignment analysis of the BTB domains revealed a high identity rate (80%) (Figure S5), further illustrating the high conservation of the BTB domain in insect Fru proteins. Previously, we successfully identified a tra homolog in B. tabaci, named Bttra [21]. After silencing Bttra in B. tabaci adults, both fru-a and fru-b expression decreased significantly compared with the dsEGFP control groups ( Figure 4B). This result suggests that Bttra regulates the expression of Btfru.

Discussion
The fruitless gene performs multiple functions during Drosophila development. In recent years, fru homologues have been identified in most insect species. These Frus are conserved both in transcript architecture and in the regulation of male sexual behavior. However, the status of Hemiptera fru remains unknown. Here, we conducted a detailed analysis of fru transcripts in B. tabaci.
The fru transcripts generally begin with a broad-complex, tramtrack, and bric-a-brac (BTB) domain and end with one alternative C 2 H 2 zinc finger domain [31]. The BTB domain is involved in protein oligomerization and recruitment of transcriptional corepressors. It is the most-conserved feature of all Fru proteins [32,33]. The BTB domain found in B. tabaci fru transcripts is encoded by a single exon (Figure 1), which is the same as N. vitripennis, but different from that of D. melanogaster [9]. In Drosophila, the BTB domain is encoded by three different exons [34]. Multi-alignment analysis of the BTB domains revealed a high identity rate (80%) (Figure S5), further illustrating the high conservation of the BTB domain in insect Fru proteins.
The zinc finger domains in insect fru genes are diverse and less conserved than those in the BTB domain. The fru gene in Diptera encodes three C 2 H 2 -type zinc finger isoforms identified as A, B, and C. However, in Nasonia, the fru gene encodes five C 2 H 2 -type zinc fingers termed the A, B, C, F, and G exons [8,9,[35][36][37]. In this study, we found two C 2 H 2type zinc finger isoforms (A and G) in B. tabaci (Figure 2). Of the species that have been reported so far, only N. vitripennis and B. tabaci possess both A and G, while other species Horticulturae 2023, 9, 962 9 of 12 have either A or G. The specific function of these two zinc fingers remains for further experimental study.
By alternative splicing, the fru gene encodes a set of transcription factors in D. melanogaster. The transcript begins with one of four promoters (P1-P4) and ends with one of four final exons (A-D). Only those transcripts produced by the most-terminal P1 promoter are spliced in a sex-specific way [31,38]. Nasonia fru transcripts derive from at least six different promoters. As in Drosophila, the transcripts common to both sexes arise from the promoters closest to the BTB exon (P2-P3-P4-P5-P6 promoters). In contrast, sexspecific transcripts are derived from promoters further upstream in the fru locus (P0-P1) [9]. In addition to Drosophila and Nasonia, sex-specific isoforms were observed in A. mellifera, A. gambiae, Ceratitis capitata, Aedes aegypti, Musca domestica, and Blatella germanica [10,[35][36][37]. In this paper, we did not find any stable sex-specific variants in two fru transcripts. This may be partly due to the fact that B. tabaci is a rapidly evolving complex species with large individual differences [39]. On the other hand, it may be because we used the whole body instead of the head to analyze the fru transcripts. After all, in both Nasonia and Drosophila, sex-specific fru transcripts driven by P1 promoters are expressed primarily in the head [9]. Furthermore, the male-specific Ceratitis fru is expressed only in male head samples [40]. Therefore, we may be able to obtain some new fru transcripts by collecting head tissues from both males and females for transcriptome sequencing.
In D. melanogaster, sex-specific splicing of fru is regulated by tra and its cofactor tra-2 [17,31,38]. The pattern of tra/tra2 co-regulation of fru splicing is widely conserved in Hymenoptera, Coleoptera, and Diptera [40,41]. For example, injection of tra dsRNA in the early embryonic stage of B. dorsalis caused fruM to appear in pseudomales [14]. Similarly, by embryonic injection of tra2 dsRNA in C. capitata, male-specific fru mRNA was detected in XX-pseudomales [40]. Recently, researchers found that the expression of Drosophila male-specific fru in the gonads is regulated by dsx and is independent of tra. This led to the inference of a novel mechanism regulating sex-specific splicing of fru. This mechanism is regulated by dsx at the transcriptional level and is not mediated by splicing of tra [42]. In this study, the presence of the TRA/TRA-2 binding sites in Btfru and the expression changes induced by Bttra RNAi suggest that, like most insects, fru is regulated by tra in B. tabaci (Figure 4).
Functional studies of the Btfru gene can be complex. This is because the number of fru splice variants is large and varies greatly between individuals. Thus far, we have not found stable fru sex-specific variants in B. tabaci. Both Btfru transcripts were found to be highly expressed in females (Figure 3), indicating that the two Btfru transcripts may function in females, but not in males. The function of fru in males has been widely reported. In D. melanogaster, fru mutant males were unable to complete mating and exhibited courtship behavior towards both females and males. This confirmed that FruM proteins determine male courtship and orientation [8,43]. In addition, the functions of Drosophila FruM include influencing the formation of sexual dimorphisms in neurons and the formation of malespecific MOL, as well as controlling the differentiation of imaginal discs [16,18]. In Bombyx, mating behavior is independently regulated by fru. Loss of Bmfru completely prevents mating, but males can still exhibit courtship behavior [15]. RNAi knockdown of fru in S. gregaria prevents successful mating and affects male fertility [11]. Silencing the cockroach fru, males no longer showed courtship behavior [10]. All these experiments confirmed the ancestral function of fru in male sexual behavior. Its function in females, however, is rarely reported. Actually, female mate choice plays a key role in species reproduction. It affects both sexual selection within species and reproductive isolation between species. In 2019, a non-sex-specifically spliced fru transcript was revealed to influence female rejection behavior. fru mutant females not only did not show acceptance of copulation, but also actively rejected courting males, as evidenced by extrusion of the ovipositor, kicking and/or wing clipping, and actively moving away from courting males [44]. It was the first implication of fru in female behavior. Therefore, we still have much work to perform in the functional verification of Btfru.
With the in-depth study of the mechanism of insect sex determination, people began to try to conduct genetic manipulation of sex in some insects, for example RNAi knockdown of tra and tra2 to generate male-only progeny in B. dorsalis [45]. CRISPR-Cas9-targeted A. gambiae dsxF did not affect male development or fertility, whereas females showed an intersex phenotype and complete sterility [46]. Disruption of Osp in B. mori and Spodoptera litura results in female sterility, while male fertility is not affected [47]. The success of these experiments is driving the process of genetic manipulation of gender. Likewise, the possibility of the manipulation of the sex determination pathway opens up a new opportunity for pest control. In this study, we identified multiple spliceosomes of the sex-differentiation gene fru. Later, we can target specific spliceosomes for interference or gene editing, which is expected to realize the genetic regulation of sex in the whitefly.

Conclusions
In the present study, we cloned and characterized the fru gene in B. tabaci and explored the alternative splicing of fru in B. tabaci adults. Furthermore, we confirmed by RNAi that fru expression is regulated by tra. Our results suggest that tra is functionally conserved in controlling downstream sex-differentiation gene expression. Meanwhile, the sex-differentiation gene fru is far more complex in whiteflies than we thought. Future indepth studies of the mechanism of sex determination in whiteflies may reveal the function of this gene in whiteflies.

Supplementary Materials:
The following Supporting Information can be downloaded at: https: //www.mdpi.com/article/10.3390/horticulturae9090962/s1, Figure S1: The phylogenetic tree of known fru genes in insects, Figure S2: Conservation of zinc finger domains among insect species, Figure S3: PCR cloning of fru-a and fru-b in B. tabaci females and males, Figure S4. Expression levels of fru transcripts in B. tabaci adults, Figure S5: Conservation of BTB domains among insect species.

Conflicts of Interest:
The authors declare no conflict of interest.