Functional Analysis of Forkhead Transcription Factor Fd59a in the Spermatogenesis of Drosophila melanogaster

Simple Summary Spermatogenesis, which is regulated by many different genes, is a conserved process across species to produce mature sperm for animal reproduction. Fox transcription factors can bind to DNA sequences in the promoters to regulate gene expression. FoxD subfamily members are mainly involved in metabolism and early organ development. In Drosophila melanogaster, FoxD subfamily member Fd59a may regulate the development of the nervous system and control the egg-laying behavior of females. However, the functions of insect FoxD members are still largely unknown. In this study, we investigated the role of Fd59a in the spermatogenesis of Drosophila. We found that mutations in Fd59a caused swelling of the apical region in the testis, resulting in fewer mature sperm in the seminal vesicle and significantly lower fertility of Fd59a mutant males compared to the control flies. We also found that the homeostasis of the testis stem cell niche in Fd59a mutant and RNAi flies was disrupted, causing increased apoptosis of sperm bundles. RNA sequencing and qRT-PCR results suggested that Fd59a can regulate the expression of genes related to reproductive process and cell death. Our collective results indicated that Fd59a plays a key role in Drosophila spermatogenesis, which will help to understand the role of FoxD members in insect spermatogenesis. Abstract Spermatogenesis is critical for insect reproduction and is regulated by many different genes. In this study, we found that Forkhead transcription factor Fd59a functions as a key factor in the spermatogenesis of Drosophila melanogaster. Fd59a contains a conversed Forkhead domain, and it is clustered to the FoxD subfamily with other FoxD members from some insect and vertebrate species. Mutations in Fd59a caused swelling in the apical region of the testis. More importantly, fewer mature sperm were present in the seminal vesicle of Fd59a mutant flies compared to the control flies, and the fertility of Fd59a2/2 mutant males was significantly lower than that of the control flies. Immunofluorescence staining showed that the homeostasis of the testis stem cell niche in Fd59a2/2 mutant and Fd59a RNAi flies was disrupted and the apoptosis of sperm bundles was increased. Furthermore, results from RNA sequencing and qRT-PCR suggested that Fd59a can regulate the expression of genes related to reproductive process and cell death. Taken together, our results indicated that Fd59a plays a key role in the spermatogenesis of Drosophila.


Introduction
The Forkhead box (Fox) transcription factor, which contains a highly conserved DNA binding domain of ~100 amino acids consisting of three α helices, three β folds, and two ring connections, plays critical roles in organ development, innate immunity, and other Insects 2024, 15, 480 2 of 14 processes [1].Based on phylogenetic analysis, Fox proteins are assigned to different subclasses and named "Fox, subclass N, member X" [2].FoxD subfamily members are mainly involved in metabolism and early organ development [3].In mammals, FoxD1 regulates human early embryonic development and is associated with various diseases.For example, FoxD1 can promote SLC2A1 (Solute carrier family 2 member 1) transcription and inhibit the degradation of SLC2A1 to facilitate the proliferation, invasion, and metastasis of pancreatic cancer cells [4][5][6].In planarian, FoxD gene expression was induced by wound signaling, and it was involved in head regeneration [7].In Drosophila melanogaster, Fd59a/FoxD may regulate the development of the nervous system and control the egg-laying behavior of females [8].However, the functions of insect FoxD subfamily members are still largely unknown.
Spermatogenesis is a process to produce mature sperm for reproduction.The process of spermatogenesis is conserved from insects to vertebrates; thus, insect testis is an ideal model for studying the mechanisms of spermatogenesis [9].In D. melanogaster, germ stem cells (GSCs) differentiate into goniablasts under the control of the stem cell niche; then, goniablasts develop into spermatids through mitosis and meiosis.After nuclear elongation and individualization processes, round spermatids finally become mature sperm [10].
Spermatogenesis is regulated by multiple signaling pathways, such as TGF-β (Transforming Growth Factor β), Notch, JAK-STAT (Janus kinase-signal transducer and activator of transcription), BMP (Bone morphogenetic protein), and Hedgehog (Hh) pathways [11], and by many genes [12,13].Recent studies showed that different genes are involved in the spermatogenesis of insects.For example, knockdown expression of ribosomal protein S3 (RpS3) strongly disrupted spermatid elongation and individualization processes in D. melanogaster [14].The knockdown or mutation of the cytochrome c1-like (cyt-c1L) gene in early germ cells resulted in male sterility of D. melanogaster [15].Moreover, BmHen1, a gene in Bombyx mori encoding methyltransferase that modifies piRNAs, was found to regulate eupyrene sperm development [16].These results indicate that the molecular mechanism of insect spermatogenesis is much more complicated than what we have already known about.
In our previous study, we showed that B. mori FoxA participated in the development of wing disc [17].Microarray data showed that Fox genes were expressed in B. mori testis, and BmFoxD was expressed at a high level [18].In Drosophila, the expression of Fd59a/FoxD was also about 2-fold higher in the testis than in the ovary [19], suggesting that Fd59a may play a role in testis development or spermatogenesis.In this study, we found that the mutation and knockdown expression of Fd59a caused swelling in the apical region of the testis and decreased male fertility.More importantly, the loss of function of Fd59a disrupted the homeostasis of the testis stem cell niche and induced the apoptosis of sperm bundles, resulting in fewer mature sperm in the seminal vesicle.By analyzing RNA sequencing from the testis of Fd59a 2/2 mutants, we found that Fd59a may regulate the expression of genes related to reproductive and metabolic processes.Our findings suggest that Fd59a plays a role in Drosophila spermatogenesis.
All flies were reared on a fresh cornmeal/yeast/brown sugar diet with p-hydroxybenzoic acid methylester as a mold inhibitor at 25 • C with a photoperiod of approximately 12 L/ 12 D (light/dark) [21].

Bioinformatics Analysis
The amino acid sequences of Fd59a and its homologous proteins were obtained using protein BLAST at the National Center for Biotechnology Information (NCBI, https://blast.ncbi.nlm.nih.gov,accessed on 29 February 2024).Sequence alignment was performed by Cluster W. The construction of a phylogenetic tree was achieved by using RAxML (Random Axelerated Maximum Likelihood), approached with 1000 bootstrap replications [22].The identification of functional protein domains in Fd59a and its homologous proteins was performed by SMART (https://smart.embl.de/,accessed on 29 February 2024).The prediction of potential Fox binding sites in the promoter sequences of selected genes was accomplished using the JASPAR program (https://jaspar.genereg.net/,accessed on 29 February 2024).

RNA Isolation and Quantitative RT-PCR
To investigate the expression profile of the Fd59a gene from the embryo to adult stages, approximately 50 adult w 1118 flies (male/female ratio ~2:1) were collected in a cage for mating.Then, the flies were relocated to a fresh cage at 2 h intervals.Embryos at 2,4,6,8,10,12,14,16,18,20,22, and 24 h after egg laying; the 1st, 2nd, and 3rd instar larvae; pupae at the early, middle, and late stages; and 1-, 3-, and 5-day-old adults were collected.All samples were collected in RNAex Pro reagent (1000 µL) (Accurate Biology, Changsha, China) and stored in a −80 • C freezer for subsequent RNA isolation.
Total RNA was isolated from the above samples using a method described previously [20].The first-strand complementary DNA (cDNA) was synthesized from 1 µg of total RNA using the HiFiScript gDNA removal cDNA Synthesis Kit (cwbiotech, Taizhou, China).To analyze the expression of target genes, gene-specific primers (Table 1) were designed based on the sequences available in the Flybase.All primers were synthesized by Tsingke Biotechnology (Beijing, China).

Name
Forward Primer (5 Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was conducted by using QuantStudio™ 6 Flex (Thermo Fisher Scientific, Waltham, MA, USA) and ChamQ SYBR qPCR Master Mix (Vazyme, Nanjing, China), following the manufacturer's instructions.The qPCR cycling program was set as 95 • C for 30 s, succeeded by 40 cycles of 95 • C for 10 s and 60 • C for 30 s. Relative gene expression was normalized to the endogenous reference gene Rp49 by using the comparative CT (2 −∆∆Ct ) method [23].

Male Fertility Test
To evaluate the reproductive ability of male Fd59a mutant flies, a male fertility test was conducted.Ten 1-day-old virgin w 1118 females were housed with fifteen 3-day-old w 1118 or Fd59a 2/2 males in vials containing egg collection plates to collect eggs.The egg collection plates were replaced every 24 h, and the number of embryos and larvae in the plates was counted as described previously [24].

Immunofluorescence Staining
Testes of 3-day-old adult flies were dissected in 10 mM phosphate-buffered saline (PBS) (137 mM NaCl, 2.7 mM KCl, 10 mM Na 2 HPO 4 , 2 mM KH 2 PO 4 , pH 7.4) and fixed in 4% paraformaldehyde (PFA) prepared in PBS for 40 min at room temperature.Testis samples were washed in 3‰ PBT (10 mM PBS with 0.3% Triton X-100) at least 3 times (each for 15 min) and treated with blocking buffer (3‰ PBT with 5% normal goat serum) for 1 h at room temperature.Then, samples were incubated at 4 • C overnight with primary antibodies diluted in dilution buffer (3‰ PBT with 3% normal goat serum), washed three times in 3‰ PBT to remove unbound antibodies, and subsequently incubated with secondary antibodies diluted in a dilution buffer at room temperature for 3 h in darkness.Samples were washed at least three times with 3‰ PBT and mounted using VECTASHIELD ® antifade Mounting Medium containing 1.5 µg/mL DAPI (Vector Laboratories, Newark, NJ, USA).

TUNEL Assay
To analyze whether loss of function of Fd59a could induce cell death in sperm bundles, testes from 3-day-old adults of Fd59a 2/2 and Fd59a RNAi flies were dissected.The collected testes were fixed in 4% paraformaldehyde and rinsed in 3‰ PBT, as described above.
A terminal deoxyribonucleotide transferase (TdT)-mediated dUTP nick end labeling (TUNEL) assay was conducted using the TUNEL assay kit (C1088 and C1090, Beyotime Biotechnology, Shanghai, China).Testis samples prepared as above were incubated with a TUNEL reaction mixture containing 5 µL of TdT enzyme, 45 µL of fluorescent labeling solution, and a moderate enzyme dilution buffer at 4 • C overnight and then washed three times in 3‰ PBT.DAPI staining was performed as described above.

Bioinformatics Analysis of RNA-seq Data
The transcript expression levels were quantified by Fragments Per Kilobase per Million mapped (FPKM) read values, and differentially expressed genes (DEGs) were identified based on a |log 2 fold-change| > 1.5 along with a p-value adjustment below 0.05 across three biological replicates.The gene ontology (GO) enrichment of DEGs was analyzed using the GOseq R package (version 3.9).

Statistical Analysis
The size of the apical region from Fd59a 2/2 and w 1118 testes was quantitated by ImageJ (version 1.54j).For each sample, three biological replicates and at least three technical replicates for each biological sample were performed.Data were presented as the mean ± S.E.(standard error); significant differences were analyzed by the Student's t-test (for comparison between two groups) or by one-way analysis of variance followed by a least significant difference test (for multiple comparisons among groups) using GraphPad Prism version 9.0.

Expression Profile of Fd59a in Drosophila
To understand the functions of Fd59a, we first determined the expression profile of Fd59a at different developmental stages of Drosophila by qRT-PCR.The results showed that the expression of Fd59a mRNA peaked twice during development from embryo to adult stages, with the first peak around the mid-embryonic stage and then a plateau with a relatively high level from the late embryonic stage to the first instar larval stage and the second peak just at the metamorphosis period, maintaining at a relatively high level until the mid-pupal stage (Figure 1A), indicating that Fd59a may be involved in Drosophila development.

Sequence and Phylogenetic Analyses of Fd59a
The Fd59a gene is in the second chromosome.The full-length cDNA of Fd59a is 13 bp long, encoding a protein of 456 amino acid residues, with a calculated molecu weight of 49.1 kDa and pI of 5.19.
Fd59a belongs to the FoxD subfamily.To reveal the evolutionary relationship Fd59a, Fd59a/FoxD homologous sequences from some insect and vertebrate species w blasted and downloaded from NCBI (Table 2), sequence alignment was performed, an phylogenetic tree was constructed.The results showed that all the selected Fd59a/Fo homologous proteins contained a conserved Forkhead box domain (Figure 2A).Drosop Fd59a was the closest to FoxD3-A of Papilio Xuthus and FoxD3-like of Blattella german (Figure 2B) and they contained similar functional domains (Figure 2C).These results s gest that Drosophila Fd59a is evolutionarily close to insect FoxD members.It has been reported that the loss of function of Fd59a affected the egg laying of Drosophila females [8].Interestingly, FlyAtlas anatomical expression data from the Flybase showed that the expression level of Fd59a was about 2-fold higher in the testis than in the ovary [19].We performed qRT-PCR and confirmed that mRNA levels of Fd59a were significantly higher in the testis than in the ovary of 3-day-old w 1118 flies (Figure 1B), suggesting that Fd59a may also have a function in the testis of Drosophila.

Sequence and Phylogenetic Analyses of Fd59a
The Fd59a gene is in the second chromosome.The full-length cDNA of Fd59a is 1371 bp long, encoding a protein of 456 amino acid residues, with a calculated molecular weight of 49.1 kDa and pI of 5.19.
Fd59a belongs to the FoxD subfamily.To reveal the evolutionary relationship of Fd59a, Fd59a/FoxD homologous sequences from some insect and vertebrate species were blasted and downloaded from NCBI (Table 2), sequence alignment was performed, and a phylogenetic tree was constructed.The results showed that all the selected Fd59a/FoxD homologous proteins contained a conserved Forkhead box domain (Figure 2A).Drosophila Fd59a was the closest to FoxD3-A of Papilio Xuthus and FoxD3-like of Blattella germanica (Figure 2B) and they contained similar functional domains (Figure 2C).These results suggest that Drosophila Fd59a is evolutionarily close to insect FoxD members.2.

Loss of Function of Fd59a Affects Testis Development and Male Fertility
To determine the role of Fd59a in testis development in D. melanogaster, testes from Fd59a 1/1 and Fd59a 2/2 loss-of-function mutant flies as well as w 1118 flies were dissected.Fd59a 1 and Fd59a 2 were two mutant types of Fd59a, with Fd59a 1 as a hypomorphic mutation and Fd59a 2 as a null allele [8].We found that the apical region of testes from the 1/1 and Fd59a 2/2 mutant flies was swelled (Figure 3A(a1,b1,c1),B), with fewer mature sperm in the seminal vesicle of Fd59a 2/2 mutant testis compared to many mature sperm in the w 1118 flies (Figure 3A(a2,a3,b2,b3,c2,c3)).As Fd59a 2 was derived from EMS-based genetic screening, it may have contained unmapped mutations in the second chromosome.To exclude the possibility that the observed phenotypes were derived from other mutations in the second chromosome, we generated the hemizygous flies by crossing Fd59a 2 flies with Df(2R) BSC864 flies, which contained a deletion encompassing the Fd59a locus, and similar results were observed (Figure 3A(d1-d3,e1-e3)).As the phenotype of Fa59a 2/2 flies was more significant than that of Fa59a 1/1 flies, we carried out the following studies in the Fa59a 2/2 mutant flies.When Fd59a 2/2 males were crossed with w 1118 females, the hatching rate of F1 flies was significantly decreased compared to the control (Figure 3C).Together, these results suggest that Fd59a plays a critical role in the development of the testis and/or spermatogenesis of D. melanogaster.Forkhead box protein D3 Rattus norvegicus NP_542952.1

Loss of Function of Fd59a Affects Testis Development and Male Fertility
To determine the role of Fd59a in testis development in D. melanogaster, testes from Fd59a 1/1 and Fd59a 2/2 loss-of-function mutant flies as well as w 1118 flies were dissected.Fd59a 1 and Fd59a 2 were two mutant types of Fd59a, with Fd59a 1 as a hypomorphic mutation and Fd59a 2 as a null allele [8].We found that the apical region of testes from the Fd59a 1/1 and Fd59a 2/2 mutant flies was swelled (Figure 3A(a1,b1,c1),B), with fewer mature sperm in the seminal vesicle of Fd59a 2/2 mutant testis compared to many mature sperm in the w 1118 flies (Figure 3A(a2,a3,b2,b3,c2,c3)).As Fd59a 2 was derived from EMS-based genetic screening, it may have contained unmapped mutations in the second chromosome.To exclude the possibility that the observed phenotypes were derived from other mutations in the second chromosome, we generated the hemizygous flies by crossing Fd59a 2 flies with Df(2R) BSC864 flies, which contained a deletion encompassing the Fd59a locus, and similar results were observed (Figure 3A(d1-d3,e1-e3)).As the phenotype of Fa59a 2/2 flies was more significant than that of Fa59a 1/1 flies, we carried out the following studies in the Fa59a 2/2 mutant flies.When Fd59a 2/2 males were crossed with w 1118 females, the hatching rate of F1 flies was significantly decreased compared to the control (Figure 3C).Together, these results suggest that Fd59a plays a critical role in the development of the testis and/or spermatogenesis of D. melanogaster.

Loss of Function of Fd59a Affects Spermatogenesis
At the apical region of testis, about 10 hub cells cluster together and are surrounded by GSCs and CySCs to form the stem cell niche, which governs the proliferation and differentiation of GSCs and CySCs.The disruption of niche homeostasis can lead to swelling of the testis and defects in spermatogenesis [25].To clarify the role of Fd59a in spermatogenesis, Fas III and Vasa antibodies were used to specifically mark the hub cells and germ cells, respectively, and αSpectrin antibody was employed to identify spectrosomes and fusomes, which are crucial for the early development of germ cells.As a result, the distribution of GSCs and CySCs was scattered in the Fd59a 2/2 testis compared to the control

Loss of Function of Fd59a Affects Spermatogenesis
At the apical region of testis, about 10 hub cells cluster together and are surrounded by GSCs and CySCs to form the stem cell niche, which governs the proliferation and differentiation of GSCs and CySCs.The disruption of niche homeostasis can lead to swelling of the testis and defects in spermatogenesis [25].To clarify the role of Fd59a in spermatogenesis, Fas III and Vasa antibodies were used to specifically mark the hub cells and germ cells, respectively, and αSpectrin antibody was employed to identify spectrosomes and fusomes, which are crucial for the early development of germ cells.As a result, the distribution of GSCs and CySCs was scattered in the Fd59a 2/2 testis compared to the control testis (Figure 4A(a4,b4)), and a strong pattern of spectrosome and fusome formation was displayed in the control testis, while fewer spectrosomes and fusomes were observed in the Fd59a 2/2 mutant testis (Figure 4A(a2,a3,b2,b3),B,C).These results suggest that Fd59a may play a role in maintaining the homeostasis of the testis stem cell niche.
Insects 2024, 15, x FOR PEER REVIEW 9 of 15 testis (Figure 4A(a4,b4)), and a strong pattern of spectrosome and fusome formation was displayed in the control testis, while fewer spectrosomes and fusomes were observed in the Fd59a 2/2 mutant testis (Figure 4A(a2,a3,b2,b3),B,C).These results suggest that Fd59a may play a role in maintaining the homeostasis of the testis stem cell niche.Mammalian FoxD1 is related to apoptosis, as the knockdown expression of FoxD1 facilitates apoptosis in HNSCC (head and neck squamous cell carcinoma) cells [26].To determine whether the loss of function of Fd59a could induce apoptosis in the testis, a TUNEL assay was performed.TUNEL positive signals were detected in sperm bundles of the Fd59a 2/2 mutant flies but not in the w 1118 flies (Figure 5A(a1-a4,b1-b4)), suggesting that the loss of function of Fd59a induced the apoptosis of spermatid.Mammalian FoxD1 is related to apoptosis, as the knockdown expression of FoxD1 facilitates apoptosis in HNSCC (head and neck squamous cell carcinoma) cells [26].To determine whether the loss of function of Fd59a could induce apoptosis in the testis, a TUNEL assay was performed.TUNEL positive signals were detected in sperm bundles of the Fd59a 2/2 mutant flies but not in the w 1118 flies (Figure 5A(a1-a4,b1-b4)), suggesting that the loss of function of Fd59a induced the apoptosis of spermatid.
To further confirm the role of Fd59a in the testis, the expression of Fd59a in GSCs was knocked down by Nos-Gal4 (Figure 4D), and similar phenotypes, such as swelling in the apical region of the testis, fewer mature sperm in the seminal vesicle, and the apoptosis of sperm bundles, were observed in the Nos-Gal4>Fd59a RNAi flies (Figures 4A(c1-c3,d1-d3),B,C and 5A(c1-c4,d1-d4)).The knockdown expression of Fd59a in the 4-16 stages of spermatogonia by Bam-Gal4 (Figure 5C) also induced the apoptosis of sperm bundles (Figure 5A(e1-e4,f1-f4)).Moreover, only a few mature sperm were observed in the seminal vesicles of the Nos-Gal4>Fd59a RNAi and Bam-Gal4>Fd59a RNAi flies, while the control flies were filled with mature sperm (Figure 5B).These combined results suggest that the loss of function of Fd59a in the testis resulted in the disruption of the stem cell niche during spermatogenesis and increased the apoptosis of sperm bundles, finally leading to fewer mature sperm in the seminal vesicle.To further confirm the role of Fd59a in the testis, the expression of Fd59a in GSCs knocked down by Nos-Gal4 (Figure 4D), and similar phenotypes, such as swelling in apical region of the testis, fewer mature sperm in the seminal vesicle, and the apoptos

Fd59a Regulates Gene Expression in the Testis
To further clarify the role of Fd59a in spermatogenesis, RNA sequencing (RNAseq) was performed with RNAs isolated from the testes of w 1118 (control) and Fd59a 2/2 males.In total, 1863 differentially expressed genes (DEGs) with at least a 1.5-fold change (p-adjust < 0.05) were identified by RNA-seq, with 854 genes upregulated and 1009 genes downregulated in the testis of Fd59a 2/2 flies (Figure 6A).This result suggests that Fd59a may function as a transcription factor in the testis.
flies were filled with mature sperm (Figure 5B).These combined results suggest loss of function of Fd59a in the testis resulted in the disruption of the stem cell nic ing spermatogenesis and increased the apoptosis of sperm bundles, finally lea fewer mature sperm in the seminal vesicle.

Fd59a Regulates Gene Expression in the Testis
To further clarify the role of Fd59a in spermatogenesis, RNA sequencing (RN was performed with RNAs isolated from the testes of w 1118 (control) and Fd59a 2/2 m total, 1863 differentially expressed genes (DEGs) with at least a 1.5-fold change ( < 0.05) were identified by RNA-seq, with 854 genes upregulated and 1009 genes do ulated in the testis of Fd59a 2/2 flies (Figure 6A).This result suggests that Fd59a ma tion as a transcription factor in the testis.Gene ontology (GO) analysis revealed that 120 differentially expressed genes are associated with the reproductive process and 475 DEGs engage in the metabo cess (Figure 6B).Within DEGs related to reproduction, several have already been r to contribute to gonad development and spermatogenesis, such as Fz2 and Zpg [2 addition, 57 DEGs were implicated in cell death, including Rnrs and Ptp52F [29,30 To confirm the RNA-seq data, 20 DEGs associated with reproductive process death were chosen for qRT-PCR validation (Table 3).The expression patterns o DEGs in the testis were consistent with those of the RNA-seq data (Figure 6C).The bp promoter sequences upstream of the transcriptional start sites of these selecte Gene ontology (GO) analysis revealed that 120 differentially expressed genes (DEGs) are associated with the reproductive process and 475 DEGs engage in the metabolic process (Figure 6B).Within DEGs related to reproduction, several have already been reported to contribute to gonad development and spermatogenesis, such as Fz2 and Zpg [27,28].In addition, 57 DEGs were implicated in cell death, including Rnrs and Ptp52F [29,30].
To confirm the RNA-seq data, 20 DEGs associated with reproductive process and cell death were chosen for qRT-PCR validation (Table 3).The expression patterns of these DEGs in the testis were consistent with those of the RNA-seq data (Figure 6C).Then, 2000 bp promoter sequences upstream of the transcriptional start sites of these selected genes were downloaded, and the potential Fox binding sites were predicted using the JASPAR database (the relative profile score threshold was set to 85%).Except for the CG32817 promoter, several Fox binding sites were predicted in the promoter of each selected gene, with more than 10 potential Fox binding sites in the promoters of the Spd-2, Cal1, Blanks, Ptp52F, Lola, and Debcl genes (Table 3).This result further supports that Fd59a is an upstream regulator of these genes.Taken together, our results suggest that Fd59a serves as a transcription factor to regulate the expression of genes involved in reproduction, cell growth, and cell death in the testis directly or indirectly.

Discussion
Drosophila testis is an ideal system for studying spermatogenesis.In this study, we found that FoxD transcription factor Fd59a contributes to the spermatogenesis of Drosophila.So far, little is known about the functions of Drosophila Fd59a/FoxD and other insect FoxD members.It was reported that the loss of function of Fd59a affects the female egglaying behavior of Drosophila [8].Moreover, Drosophila CHES-1-like/FoxN suppressed the differentiation of germline stem cells by upregulating Dpp expression, whereas ectopic expression of CHES-1-like led to a significant decrease in male fertility [31].In B. mori, Fox family genes were expressed in the testis, with BmFoxL2-2 and BmFoxD at a higher level than other BmFox genes [18].However, the function of BmFoxD in the testis is still unknown.
In this study, we showed that spermatogenesis was disrupted and the apoptosis of sperm bundles was induced in Fd59a mutant and RNAi flies.Spermatogenesis is a complex process regulated by multiple signaling pathways and many different genes.The over-activation of the JAK-STAT signaling pathway led to the overgrowth of the testis and the disrupted structure of the testis stem cell niche in Drosophila [25,32].The over-activation of the JNK or loss of the Notch signaling caused cell death in the testis of Drosophila [33,34].In mammals, the deletion of Stat3 in the Foxd1 cell lineage protected mice from kidney fibrosis [35].Hypoxia-inducible factors (HIFs) regulated genes related to oxygen homeostasis, and a lack of Hif-p4h-2 (HIF prolyl-4-hydroxylases) in the FoxD1 lineage led to the dysregulation of genes involved in the Notch signaling pathway [36].These results suggest that there is a genetic interaction between mammalian FoxD subfamily members and the JAK-STAT and Notch pathways.However, mRNA levels of genes related to the above three signaling pathways did not change significantly in the Fd59a 2/2 testis (RNA-seq data), indicating that Fd59a is not involved in the JAK-STAT, JNK, or Notch signaling pathway.Therefore, insect FoxD members may function differently from mammalian FoxD subfamily members.
It has been shown that Fd59a is expressed in octopaminergic neurons and that it regulates the egg-laying behavior of female Drosophila [8].In the Chinese mitten crab (Eriocheir sinensis), the expression of the octopamine receptor changed significantly in the androgenic gland (AG) between the proliferation and secretion phases [37].We showed that the loss of function of Fd59a caused defects in spermatogenesis.These combined results suggest that octopaminergic neurons and octopamine may play a role in spermatogenesis, which needs to be determined by further study.
The results from RNA-seq and qRT-PCR showed that many genes related to reproduction and cell death were differentially expressed in the testis of Fd59a 2/2 flies.Among the reproduction-related DEGs, Fz2 and Zpg have been reported to regulate germ stem cell development in Drosophila testis [27,28], while Blanks functioned in sperm individualization [38].Among the cell death-related DEGs, Ptp52F enhanced autophagy and apoptosis in the Drosophila midgut [30].In addition, several potential Fox binding sites were predicted in the promoters of selected DEGs.Thus, Fd59a acts as a transcription factor to regulate the expression of genes involved in spermatogenesis and maintain the survival of sperm cells.
It was reported that octopamine was essential for increasing GSCs in mating Drosophila females [39], and the β-adrenergic-like octopamine receptor (OctβR) was strongly expressed in adult testis [40].In Fd59a 2/2 adult testis, Octβ2R expression was downregulated; thus, it is possible that Fd59a regulates spermatogenesis partly through regulating the expression of Octβ2R, and Fd59a may be a key factor linking the nervous system to the male reproduction system.

Figure 1 .
Figure 1.Expression of Fd59a at different developmental stages and in adult ovary and testis.Expression of Fd59a at different developmental stages.Drosophila embryos at 2, 4, 6, 8, 10, 12, 14, 18, 20, 22, and 24 h after egg laying; first (L1), second (L2), and third (L3) instar larvae; early, mid and late pupae; and 1-, 3-, and 5-day-old adult flies were collected to prepare total RNAs for analysis of the transcriptional expression of Fd59a by qRT-PCR.(B) Expression of Fd59a in the te and ovary of 3-day-old adult flies.Data were presented as means ± S.E., and significant differen were determined by the Student's t-test and indicated by asterisks.** p < 0.01.

Figure 1 .
Figure 1.Expression of Fd59a at different developmental stages and in adult ovary and testis.(A) Expression of Fd59a at different developmental stages.Drosophila embryos at 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, and 24 h after egg laying; first (L1), second (L2), and third (L3) instar larvae; early, middle, and late pupae; and 1-, 3-, and 5-day-old adult flies were collected to prepare total RNAs for the analysis of the transcriptional expression of Fd59a by qRT-PCR.(B) Expression of Fd59a in the testis and ovary of 3-day-old adult flies.Data were presented as means ± S.E., and significant differences were determined by the Student's t-test and indicated by asterisks.** p < 0.01.

Figure 2 .
Figure 2. Sequence analysis of D. melanogaster Fd59a.Sequence alignment (A), Maximum Likelihood phylogenetic tree (B), and functional domains (C) of Fd59a with its homologous proteins from some insect and vertebrate species.For detailed information on the sequences, see Table2.

Figure 6 .
Figure 6.RNA-seq analysis of RNAs from the testes of Fd59a 2/2 and w 1118 flies.(A) Heatma ferentially expressed genes (DEGs) in the testis of Fd59a 2/2 flies relative to w 1118 flies.(B) GO of DEGs in the testes between Fd59a 2/2 and w 1118 flies.(C) qRT-PCR validation of selected DE RNA sequencing data.Data are presented as means ± S.E.Significant differences were det by the Student's t-test and are indicated by asterisks * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 6 .
Figure 6.RNA-seq analysis of RNAs from the testes of Fd59a 2/2 and w 1118 flies.(A) Heatmap of differentially expressed genes (DEGs) in the testis of Fd59a 2/2 flies relative to w 1118 flies.(B) GO analysis of DEGs in the testes between Fd59a 2/2 and w 1118 flies.(C) qRT-PCR validation of selected DEGs from RNA sequencing data.Data are presented as means ± S.E.Significant differences were determined by the Student's t-test and are indicated by asterisks * p < 0.05, ** p < 0.01, *** p < 0.001.

Table 1 .
Primers used in this study.

Table 2 .
Fd59a homologous protein sequences used in the phylogenetic tree.

Table 2 .
Fd59a homologous protein sequences used in the phylogenetic tree.

Table 3 .
Differentially expressed genes selected for qRT-PCR validation and the number of predicted Fox binding sites in the 2 kb promoter regions.