Open Access
How to translate text using browser tools
1 March 2017 Comparative Transcriptome Analysis of Three Bactrocera dorsalis (Diptera: Tephritidae) Organs to Identify Functional Genes in the Male Accessory Glands and Ejaculatory Duct
Chuan-Bei Tian, Dong Wei, Lin-Fan Xiao, Wei Dou, Huai Liu, Jin-Jun Wang
Author Affiliations +

The insect male accessory glands/ejaculatory duct (MAG/ED) are important tissues of the male reproductive system. The MAG/ED's functions in reproduction have been well studied in Drosophila (Diptera: Drosophilidae) but remain largely unknown in the important agricultural pest Bactrocera dorsalis (Hendel) (Diptera: Tephritidae). In the present study, we re-assembled the transcriptome datasets of B. dorsalis's fat body, testis, and MAG/ED and compared these tissue-specific transcriptomes. Clean reads from these transcriptome data sets were de novo re-assembled and clustered into 31,782 unigenes (average 922 bp). In total, 21,306 unigenes were functionally annotated by Blasting against online databases. Comparative transcriptomic analysis identified numerous genes that were identified with the expressed tissue-bias patterns. Some MAG/ED-specific genes potentially involved in spermatozoa motility and capacitation (e.g., perlucin, glucose dehydrogenase, lipase), mating regulation (pheromone-binding protein—related protein), and immunity (lectin) were identified in B. dorsalis. The expressions of some of these genes were further validated by realtime quantitative polymerase chain reaction at transcriptional level. All of these identifications will help us to explore the physiological regulation of mating and reproduction in B. dorsalis in the future.

The male accessory glands (MAGs) are the source of a variety of secreted proteins and peptides (accessory gland proteins, Acps) that belong to a number of different functional categories (Davies & Chapman 2006; Dottorini et al. 2007). The importance of these proteins/peptides in modulating reproductive progress has become more evident with the increased understanding of protein identities and functions (Wolfner 1997, 2002; Gillott 2003). Acps are major components of the seminal fluid proteins that are transferred from male to female together with sperm during copulation. In addition to improving the male's probability of paternity (Avila et al. 2011), they induce numerous post-mating physiological and behavioral changes in females (Gillott 2003), such as decreasing receptivity of remating (Miyatake et al. 1999; Radhakrishnan & Taylor 2008), affecting sperm storage parameters (Avila et al. 2010; King et al. 2011), increasing egg production and ovulation rate (Ram & Wolfner 2007; Xu & Wang 2011), modulating sperm competition by plug formation (Lung & Wolfner 2001), and changes in feeding behaviors (Lee & Klowden 1999) and longevity (Xu & Wang 2011). Most of these changes in females have been reported to result from gene expression changes induced by Acps in females (Rogers et al. 2008; Thailayil et al. 2011). The ejaculatory duct (ED) also secretes proteins that are mixed with Acps, and transferred to the female during mating (Lung et al. 2001).

Recently, various molecular and genetic tools coupled with bioinformatics have been used widely to identify and analyze Acps and the encoding genes in insects (Braswell et al. 2006; Avila et al. 2011), particularly in Drosophila species (Diptera: Drosophilidae). Transcriptome data are being used widely to analyze the transcripts in male reproductive organs of many arthropod species such as Aedes aegypti (L.) (Diptera: Culicidae), Dermacentor variabilis Say (Ixodida: Ixodidae), Lutzomyia longipalpis (Lutz & Neiva) (Diptera: Psychodidae), and Teleogryllus oceanicus (Le Guillou) (Orthoptera: Gryllidae) (Champagne & Brown 2007; Sonenshine et al. 2011; Azevedo et al. 2012; Bailey et al. 2013).

As one of most economically important insect pests, the oriental fruit fly, Bactrocera dorsalis (Hendel) (Diptera: Tephritidae), is distributed worldwide and has a powerful reproduction (Wan et al. 2012; Wei et al. 2015a). Many transcriptome sequencings were conducted in B. dorsalis to identify functional genes involved in development (Shen et al. 2011), reproduction (Zheng et al. 2012), and insecticide metabolism and resistance (Hsu et al. 2012). Tissue-specific transcriptomes of B. dorsalis midgut, fat body, and testis were characterized and studied in our previous studies (Shen et al. 2013; Yang et al. 2014; Wei et al. 2015b). It is difficult to identify individual Acps among the seminal fluid proteins due to their rapid evolution (Almeida & DeSalle 2008). Therefore, systematic gene expressions studies are limited, although several aspects of the reproductive biology in B. dorsalis have been studied (Ren et al. 2008; Wei et al. 2015a). Recently, the expression of some immune-related genes in MAGs was detected and studied (Lung et al. 2001; Belardinelli et al. 2005; Wong et al. 2008). In a previous transcriptome analysis of B. dorsalis MAGs, we identified many genes involved in immunity in the reproductive duct and several juvenile hormone—related genes (Wei et al. 2016). However, there is still an information gap in understanding the functional molecules and molecular genetics and evolution of Acps encoding genes in B. dorsalis.

In this study, we obtained the transcriptome data of B. dorsalis fat body, testis, and MAGs from the Sequence Read Archive in NCBI, and re-assembled the data. Systematic characterization and interpretation of the data allowed us to identify many MAG-specific genes potentially involved in sperm maturation, viability, mating regulation, and immunity that could advance our understanding of the reproductive biology of B. dorsalis.

Materials and Methods


The raw data of B. dorsalis fat body, testis, and MAG/ED transcriptomes were sequenced and can be downloaded from the Sequence Read Archive of the National Center of Biotechnology Information (NCBI). The accession numbers of the datasets were SRR1026844 (Yang et al. 2014), SRR1032039 (Wei et al. 2015b), and SRR1168415 (Wei et al. 2016). All the samples were collected from the same laboratorial strain under the same condition. The fat body sample was collected from different developmental stages, whereas testis and MAG/EG samples were collected from male adults.


The raw data of the MAG transcriptome were re-assembled together with the fat body and testis transcriptomes. Overlapping sequences of specific lengths with no base calls (N) were combined in contigs to form longer fragments. De novo re-assembly of these transcriptomes was carried out with the short read assembling program Trinity based on clean reads (Grabherr et al. 2011). The Trinity program consists of 3 independent software modules: Inchworm, Chrysalis, and Butterfly, which were applied sequentially to process large volumes of RNA-seq reads. All of this process was conducted in the same way as in our previous transcriptome sequencing (Wei et al. 2015b).

The resulting sequences were distinct singletons, and then the sequence clustering software Tgicl was used to splice and remove the redundancy to obtain long, non-redundant unigenes ( Then, all unigenes were divided into 2 classes, resulting in 1 cluster with the prefix CL followed by cluster ID with similarity more than 70%, and the 2nd cluster with the prefix Unigene followed by cluster ID. In the final step, Blastx alignment was performed between unigenes and proteins in databases such as NCBI nr, Swiss-Prot, and assigned to Kyoto encyclopedia of genes and genomes (KEGG) and clusters of orthologous groups (COG) with an E-value of 10−5, and the best alignment results were used to determine sequence direction of unigenes. The protein annotation was retrieved with the highest sequence similarity to unigenes. If results from different databases were conflicting, a priority order of NCBI nr, Swiss-Prot, KEGG, and COG was followed. If a unigene did not align to any of the above databases, the software of ESTScan was used to determine its sequence direction (Iseli et al. 1999). The direction from 5′ to 3′ ends was provided for unigenes with confirmed directions; for those without direction, sequences from the assembly software were provided.


Gene annotation was conducted after re-assembly, which provided the necessary information of gene expression and function, including protein functional annotation, COG functional annotation, and gene ontology (GO) functional annotation. Unigene sequences were compared using Blastx to proteins in databases, namely NCBI nr, Swiss-Prot, KEGG, and COG with an E-value <10−5. Using enzyme commission (EC) number terms, biochemical pathway information was collected by downloading relevant maps from the KEGG database, which contains a systematic analysis of inner cell metabolic pathways and functions of individual gene products (Kanehisa & Goto 2000). COG is a database in which orthologous gene products are classified. The functions of unigenes assembled from the 3 tissues were predicted and classified by aligning to COG database. Homology searches were then carried out by query of the NCBI nr database using the Blastx algorithm. After annotation, the Blast2Go program was used to obtain GO annotations (Conesa et al. 2005), and the software WEGO (Ye et al. 2006) was used to perform GO functional classification to understand the distribution of gene functions at the macro level.


To determine specific expression of genes, expression profiles were calculated using the reads per kilobase mapped (FPKM) method (Mortazavi et al. 2008), which does not include the length of different genes and sequencing level as parameters during calculation of gene expression. The false discovery rate (FDR) control statistical method was used to test multiple hypotheses to correct for P-values (P < 0.01) (Benjamini & Yekutieli 2001). The smaller the FDR, the larger the difference in the expression level between the 2 samples. To identify these differential genes expressed in each tissue in the current analysis, sequences were evaluated with FDR ≤⃒ 0.001. Genes with FPKM < 0.1 were filtered as no expression in tissue. The absolute value of log2Ratio ≥⃒ 1 was used to judge the significance of differences in the initial gene expression analysis. After the analysis, we identified many genes that were only expressed or detectable in MAG/ED. Such genes were thereafter aligned to GO, COG, and KEGG assignments.


To validate the expression of MAG-specific genes, different tagmata and tissues including head (5 males), thorax (5 males), abdomen (5 males), midgut (20 males), fat body (50 males), Malpighian tubules (50 males), testis (50 males), and MAG/ED (100 males) were cut off or dissected from 3-d-old adult B. dorsalis males. Total RNA was isolated from each sample by using the TRIzol reagent (Invitrogen, Carlsbad, California). After DNA digestion with DNase (Promega, Madison, Wisconsin), total RNA (1 µg for each) was reverse transcribed into 1st-strand cDNA by using the PrimeScript RT Reagent Kit (TaKaRa, Dalian, China). The quantitative real-time polymerase chain reaction (qRT-PCR) included 5.0 µL GoTaq qPCR Master Mix (Promega, Madison, Wisconsin), 3.5 µL nuclease-free water, 0.5 µL template cDNA, and 0.5 µL of each primer (10 µM). The relative expression was determined by the StepOne Plus Real-Time PCR System (Life Technologies, Woodlands, Singapore). The thermal program was 1 cycle of 95 °C for 2 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 30 s. At the end of each run, a melting curve was generated to rule out the possibility of primer-dimer formation. The sequences of the specific primer sets were designed using online software Primer3 (version 4.0.0) (Table S1). A fragment of the ribosomal protein subunit 3 from B. dorsalis was also amplified as the inner reference (Wei et al. 2015b). Three biological replicates were performed for each sample. The relative expression levels were calculated using the 2−DDCt method (Livak & Schmittgen 2001). Data were analyzed statistically by 1-way analysis of variance (ANOVA) for expression comparisons in the SPSS 16.0 software (SPSS Inc., Chicago, Illinois), and a value of P < 0.05 was considered to be statistically significant.



Re-assembly of transcriptome data produced 31,782 unigenes with a mean length of 922 bp, and the total length of nucleotides was 29,300,417 bp (Table 1). The results indicated that the re-assembly sequences were longer than those in each transcriptome. A greater number of distinct clusters (7,426) suggested more isoforms were assembled in this study because more raw data were used in the assembly program. Of these unigenes, 3,655 (11.50%) unigenes were longer than 2,000 bp, whereas 1,257 unigenes in fat body, 2,438 unigenes in testis and 1,705 unigenes in MAG tissues were longer than 2,000 bp (Figure S1).

Table 1.

Assembly summary of transcriptomes of fat body, testis, and male accessory glands and ejaculatory duct (MAG/ED) from Bactrocera dorsalis.



After Blastx against the NCBI nr, nt, Swiss-Prot, KEGG, COG, and GO databases, 21,306 distinct sequences (67.04%) were matched to known genes encoding functional proteins (Table 1). Most of these sequences (20,180) were annotated in the NCBI nr database, whereas 12,456 were annotated in NCBI nt, 15,657 in Swiss-Prot, 13,782 in KEGG, 7,168 in COG, and 13,577 in GO. All the sequences annotated to NABI nr database were thereafter deposited at DDBJ/EMBL/GenBank with the Transcriptome Shotgun Assembly project accession number of GEYS0000000. Taking annotation in the nr database for the following analysis, there were almost 83.42% (16,835 out of 20,180) unigenes that showed strong similarity to 36 Drosophila species with 12.99% of the sequences matching homologous sequences from D. virilis Sturtevant, followed by D. willistoni Sturtevant (12.05%), D. melanogaster Meigen (11.73%), D. mojavensis Patterson (10.86%), D. ananassae Doleschall (8.19%), D. grimshawi Oldenberg (7.82%), and D. pseudoobscura pseudoobscura Frolova (7.22%) (Figure S2A). Only 306 sequences (1.52%) were annotated to the genus Bactrocera. The most important reason was the availability of the genome sequences of many Drosophila species, but the genome sequence of B. dorsalis had not been released when we performing the annotation. For the predicted proteins, over half of the hits had over 60% similarity with known proteins in the nr database (Figure S2B).


Annotations of the unigenes provided information on their potential functions in each tissue of B. dorsalis. All of the re-assembled sequences were aligned to the COG database for functional prediction and classification. In total, 7,168 genes were assigned to 25 functional categories (Figure S3). Similar with each specific tissue transcriptome, the most common category was “general function prediction only,” which included 2,856 distinct sequences. The top 3 smallest groups were “RNA processing and modification” (1.20%), “extracellular structures” (1.00%), and “nuclear structure” (0.04%). For GO functional analysis, 13,577 sequences were categorized into 59 functional groups of 3 ontologies: biological process, cellular component, and molecular (Figure S4). There were 28 functional groups that contained more than 1,000 unigenes, including 17 biological processes, 8 cellular components, and 3 molecular functions. The most dominant groups in 3 ontologies were “cellular process” (9,880 unigenes) in biological process, “cell part” (7,839 unigenes) in cellular component, and “binding” (7,200 unigenes) in molecular function. KEGG annotation is usually used to categorize gene functions emphasizing biochemical pathways (Kanehisa & Goto 2000). In total, 13,782 sequences were remapped to 256 predicted pathways, of which 46 contained over 200 unigenes. Remarkably, the most abundant pathway was “metabolic pathways” including 1,919 sequences (Table S2).


Descriptive and quantitative transcriptome analysis is important to interpret the functional elements of genomes and reveal the molecular constituents of cells and tissues. To identify differentially expressed genes (DEGs) in MAG/ED, fat body, and testis tissues, we compared the transcriptomes from the 3 tissues and identified a number of genes highly presented in each transcriptome. Altogether, 25,054 unigenes were expressed in all the 3 tissues. In this study, transcriptome sequencings were based on a mixture of total RNA from different age/development stages of male flies. Genes with tissue-specific expression may be critical to the function of each tissue. Among all the unigenes, there were 458 unigenes that were expressed specifically in MAG/ED, 385 in fat body, and 2,238 unigenes in testis tissues (Fig. 1). Another 207 unigenes were expressed lowly in the 3 tissues. In testis tissue, mitosis and meiosis take place in the process of spermatogenesis. It is possible that many specific unigenes are involved, explaining the particularly large number of unigenes expressed specifically in testis tissue.

In this study, there were 182 unigenes that were 100-fold higher expressed in MAG/ED than both fat body and testis tissues (Table S3). These unigenes were considered as functional genes highly expressed in MAG/ED. The MAG/ED-specific unigenes (641) were classified into 20 categories (Fig. 2) after COG annotation. Interestingly, the most prominent category was “cell wall/membrane/envelope biogenesis.” Categories of “cell cycle control, cell division, chromosome partitioning” and “transcription” also made up a large proportion. All these MAG/ED-specific highly expressed genes were also classified into 3 main categories and assigned to 45 subcategories by using the GO assignment (Fig. 3). Different from each tissuespecific transcriptome, the predominant groups in biological process were “cellular process,” “single-organism process,” and “multi-organism process.” All the MAG/ED-specific highly expressed genes were thereafter mapped to 6 groups of KEGG pathways including 105 pathways (Fig. 4). Remarkably, “metabolic pathways” was the most important pathway, which contained 32 unigenes. In total, there were 51 unigenes that were involved in immunity, e g., “maturity onset diabetes of the young” involved in endocrine and metabolic diseases, “Vibrio cholerae infection” and “tuberculosis” involved in bacterial infection, “Herpes simplex infection” and “Epstein-Barr virus infection” involved in viral infection.

Fig. 1.

Statistics of sequences expressed specifically in each analyzed tissue of Bactrocera dorsalis.



To identify MAG/ED-specific genes in this study, we compared the MAG/ED transcriptomes with those of fat body and testis, and identified a number of genes highly expressed in the MAG/ED. Because genes in MAG/ED have a rapid evolution frequency, most of the unigenes were annotated with non-specific functional descriptions; 294 out of 641 unigenes were distinct singleton sequences, and only 53 of them were predicted genes (Table S3). Notable unigenes specific to MAG/ED were circadian clock—controlled protein (Unigene4743), C-type lectin (Unigene12723), vitellogenin-1 (Unigene12065), venom serine protease (Unigene8523), perlucin (Unigene5896), glucose dehydrogenase (Unigene9651), lipase 1 (Unigene5218), and pheromone-binding protein—related protein 5 (Unigene20808). These MAG/ED-specific genes may be involved directly in the male accessory proteins' functions.

The expression profiling of several annotated unigenes (>500 bp) were also validated at transcriptional level by qRT-PCR. The results showed that all of these genes had high expressions in MAG/ED tissue (Fig. 5). Besides, 4 unknown distinct unigenes (>500 bp) with the largest FPKM values were determined in tissues of male adults, and all of the 4 unigenes were highly expressed in MAG/ED tissue (Fig. 6). Other MAG/ED-specific genes sequences of <500 bp or less then 100-fold expressed compared with other tissues were also identified, such as oocyte maturation arresting factor (Unigene21945), antigen 5 precursor (Unigene21744), N-acetylgalactosaminyltransferase (Unigene22691), reverse transcriptase polyprotein (Unigene 21663), and potassium-dependent sodium—calcium exchanger (Unigene21750). These protein-coding genes also had high expression in MAG/ED tissue (Fig. S5). Regulatory mechanism determination of genes expressed differentially or specifically would provide new insights on the regulation of complex processes such as reproduction, development, and evolution.


There is an increase in the studies on insect MAG and testis tissues to investigate the mating response mechanism and the function of specific seminal proteins through transcriptome (Scolari et al. 2012; Bailey et al. 2013) and proteome analysis (Simmons et al. 2013; Xu et al. 2013). Although the comparative analysis of reproductive tissues was studied in the cricket T. oceanicus before (Bailey et al. 2013), the current study is the first report to compare 3 tissue-specific transcriptomes by re-assembling the raw data in Tephritidae insects. Not much is known about the genes involved in the reproduction of this species, particularly the rapidly evolving Acps (Swanson et al. 2001; Braswell et al. 2006). This knowledge gap was previously suggested as the likely reason for the high proportion of unknown proteins in the testis and MAG transcriptomes of T. oceanicus males (Bailey et al. 2013). In each transcriptome, the number of unigenes may not necessarily reflect the real transcriptome complexity, as many of the assembled sequences may represent distinct non-overlapping regions of the same transcripts. Thus, we re-assembled sequences of B. dorsalis MAG/ED, fat body, and testis transcriptomes for more molecular and genetic information, and the obtained results will help us in future research.

Fig. 2.

Clusters of orthologous groups (COG) functional classification of unigenes expressed highly and specifically in male accessory glands and ejaculatory duct tissue of Bactrocera dorsalis.


It is known that sperm function cannot be accomplished without Acps (La Vignera et al. 2011). This could explain why many functional genes involved in sperm motility, capacitation, and acrosome reaction were found highly expressed in MAG tissue. In insects, a remarkable array of proteins and peptides has been reported to occur in the male seminal fluids, several of which regulate critical female reproductive functions. Semen is particularly rich in fructose that provides nutrient energy for the spermatozoa and is secreted by the seminal vesicle. The positive correlation between the levels of fructose in the semen and the motility of sperms has previously been demonstrated (Patel et al. 1988). In the B. dorsalis MAG/ED transcriptome, aldose reductase unigenes, 6-phosphofructo kinase, fructose-1, 6-bisphosphatase, and fructose 1,6 bisphosphate-aldolase were identified (Wei et al. 2016). The transcripts of a glucose dehydrogenase (Unigene9651) and a lipase (Unigene5218) were highly detected in MAG/ED tissue, and because MAG/ED tissue actively secretes proteins and lipid, these organs may have a high demand for energy. In addition, sperms stay in the female genital tract for a long time, suggesting that some ATPases could be transported to females during mating. These ATPases, also identified as Acps (Sirot et al. 2011), are postulated to provide energy to sperms by hydrolyzing triglycerides (Walker et al. 2006). It has been reported that ATPase and UTPase amounts in MAGs are associated with the motility of sperms (Werner & Simmons 2008).

During the mating process, pathogens are introduced into the female reproductive tract (Lung et al. 2001). Therefore, to ensure successful fertilization, immunity-related proteins are likely to be present in the MAG/ED to protect seminal fluids from microbial infection. Lectins were mostly studied to investigate the immune response. In a previous study, 17 immuno- and C-type lectins transcripts were identified in the B. dorsalis fat body (Yang et al. 2014). Lectins were also identified in Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae), D. melanogaster, and Meligethes aeneus (F.) (Coleoptera: Nitidulidae) involved in immune response (Vogel et al. 2014). These types of genes can mediate the pathogen recognition in insects by phagocytosis (Pace & Baum 2002). Perlucin is a member of the C-type lectin family, which participates in the immune response to various stressors and defends against invading pathogens (Lin et al. 2013). Several immune functions have been proposed for C-type lectins, including cell adhesion, glycoprotein turnover, activation of prophenoloxidase cascade, hemocyte-mediated nodule formation, encapsulation, and opsonization (Zhang et al. 2011). Some of such genes were also identified in our previous study in MAGs of B. dorsalis, but only the genes involved in bacterial infection were identified (Wei et al. 2016). In this study, we identified a C-type lectin (Unigene12723) and a perlucin (Unigene5480 and Unigene5896) highly expressed in MAG/ED tissue. In addition, antigen 5 is a major allergen in the venom of vespids. This homologous gene or protein has been identified in many insect species (Hoffman 1993; Hawdon et al. 1996). This gene has also been investigated in mammal testis tissue and was found to be involved in spermatogenesis (Mizuki et al. 1992; King & Lu 1997), but the biological function of antigen 5 and its sequence-related proteins are still unknown. This study is the first to report the presence of antigen 5 in B. dorsalis. The gene's high-level expression in MAG/ED tissue suggests that it may exhibit an immune-related function in B. dorsalis reproductive tissues. Venom allergen and venom serine protease also were highly expressed in MAG/ED tissue of B. dorsalis.

Fig. 3.

Gene ontology (GO) classification of unigenes expressed highly in male accessory glands and ejaculatory duct tissue of Bactrocera dorsalis.


Fig. 4.

Kyoto encyclopedia of gene and genomes (KEGG) analysis of unigenes expressed highly in male accessory glands and ejaculatory duct tissue of Bactrocera dorsalis. Each category contains more than 1 unigene sequences.


Fig. 5.

Six examples of the tissue expression profiling of predicted distinct unigenes (>500 bp) expressed highly in male accessory glands and ejaculatory duct tissue of Bactrocera dorsalis. Relative expression levels were determined by qRT-PCR in head (HE), thorax (TH), abdomen (AB), midgut (MG), fat body (FB), Malpighian tubules (MT), testes (TE), and male accessory glands and ejaculatory duct (MAG) samples from B. dorsalis males. Relative expression levels were calculated based on the value in head, which was ascribed an arbitrary value of 1. Different letters above the bars indicate significant differences based on Tukey's test (P ≤⃒ 0.05).


Acps in insects evolve quickly and play an important role in ensuring the successful mating and fertilization. A comparative structural modeling approach indicates that major functional classes of mammalian and Drosophila seminal fluid proteins are conserved, suggesting a conserved function despite differences in reproductive strategies (Mueller et al. 2004). Evolutionary changes in these secretions may be driven by sexual selection and sexual conflict and have important implications in fertilization compatibility and evolution of reproductive isolation (Braswell et al. 2006). Indeed, in D. melanogaster, the evolutionary rate of many of these genes is so fast that they lack detectable orthologues even in other Drosophila species (Swanson et al. 2001; Mueller et al. 2005; Begun et al. 2006). Further investigation of the expression and regulation of these genes may provide new insights on the regulation of complex processes such as reproduction, development, and evolution.

In this comparative transcriptomic analysis, a number of functional genes possibly involved in immunity, sperm fertilization, and female mating regulation were identified in MAG/ED tissue. The identification of these unigenes could provide the foundation for further understanding the patterns and processes of mating regulation mechanisms and molecular evolution among reproductive proteins in MAG/ED tissues of Tephritidae insects. This transcriptome comparison will advance our understanding of the function of the reproductive proteins in MAG/ED tissue that may play roles in the regulation of reproductive processes in B. dorsalis.

Fig. 6.

Four examples of the tissue expression profiling of unknown distinct unigenes (>500 bp) expressed highly in male accessory glands and ejaculatory duct tissue of Bactrocera dorsalis. Relative expression levels were determined as described in Fig. 5.



This work was supported in part by the National Natural Science Foundation of China (Grant No. 31601640), the National Student Innovation Training Program (201410635069), the earmarked fund for Modern Agro-industry (Citrus) Technology Research System (CARS-27), the Fundamental Research Funds for the Central Universities (XDJK2016C081) of China, and the Project Funded by China Postdoctoral Science Foundation (2016T90828).

References Cited


Almeida FC, DeSalle R. 2008. Evidence of adaptive evolution of accessory gland proteins in closely related species of the Drosophila repleta group. Molecular Biology and Evolution 25: 2043–2053. Google Scholar


Avila FW, Ram KR, Qazi MCB, Wolfner MF. 2010. Sex peptide is required for the efficient release of stored sperm in mated Drosophila females. Genetics 186: 595–600. Google Scholar


Avila FW, Sirot LK, Laflamme BA, Rubinstein CD, Wolfner MF. 2011. Insect seminal fluid proteins: identification and function. Annual Review of Entomology 56: 21–40. Google Scholar


Azevedo RVDM, Dias DBS, Bretãs JAC, Mazzoni CJ, Souza NA, Albano RM, Wagner G, Davila AMR, Peixoto AA. 2012. The transcriptome of Lutzomyia longipalpis (Diptera: Psychodidae) male reproductive organs. PLoS One 7: e34495. Google Scholar


Bailey NW, Veltsos P, Tan YF, Millar AH, Ritchie MG, Simmons LW. 2013. Tissuespecific transcriptomics in the field cricket Teleogryllus oceanicus. G3-Genes Genomes Genetics 3: 225–230. Google Scholar


Begun DJ, Lindfors HA, Thompson ME, Holloway AK. 2006. Recently evolved genes identified from Drosophila yakuba and Drosophila erecta accessory gland expressed sequence tags. Genetics 172: 1675–1681. Google Scholar


Belardinelli M, Fausto AM, Guerra L, Buonocore F, Bongiorno G, Maroli M, Mazzini M. 2005. Lipase and antibacterial activities of a recombinant protein from the accessory glands of female Phlebotomus papatasi (Diptera: Psychodidae). Annals of Tropical Medicine and Parasitology 99: 673–682. Google Scholar


Benjamini Y, Yekutieli D. 2001. The control of the false discovery rate in multiple testing under dependency. Annals of Statistics 29: 1165–1188. Google Scholar


Braswell WE, Andrés JA, Maroja LS, Harrison RG, Howard DJ, Swanson WJ. 2006. Identification and comparative analysis of accessory gland proteins in Orthoptera. Genome 49: 1069–1080. Google Scholar


Champagne DE, Brown MR. 2007. Analysis of the transcriptome of Aedes aegypti male reproductive accessory glands. American Journal of Tropical Medicine and Hygiene 77: 270–271. Google Scholar


Conesa A, Götz S, García-Gómez JM, Terol J, Talón M, Robles M. 2005. Blast2GO: a universal tool for annotation, visualization and analysis in functional genomics research. Bioinformatics 21: 3674–3676. Google Scholar


Davies SJ, Chapman T. 2006. Identification of genes expressed in the accessory glands of male Mediterranean fruit flies (Ceratitis capitata). Insect Biochemistry and Molecular Biology 36: 846–856. Google Scholar


Dottorini T, Nicolaides L, Ranson H, Rogers DW, Crisanti A, Catteruccia F. 2007. A genome-wide analysis in Anopheles gambiae mosquitoes reveals 46 male accessory gland genes, possible modulators of female behavior. Proceedings of the National Academy of Sciences of the United States of America 104: 16215–16220. Google Scholar


Gillott C. 2003. Male accessory gland secretions: modulators of female reproductive physiology and behavior. Annual Review of Entomology 48: 163–184. Google Scholar


Grabherr MG, Haas BJ, Yassour M, Levin JZ, Thompson DA, Amit I, Adiconis X, Fan L, Raychowdhury R, Zeng Q. 2011. Full-length transcriptome assembly from RNA-Seq data without a reference genome. Nature Biotechnology 29: 644–652. Google Scholar


Hawdon JM, Jones BF, Hoffman DR, Hotez PJ. 1996. Cloning and characterization of Ancylostoma-secreted protein. A novel protein associated with the transition to parasitism by infective hookworm larvae. Journal of Biological Chemistry 271: 6672–6678. Google Scholar


Hoffman DR. 1993. Allergens in Hymenoptera venom XXIV: the amino acid sequences of imported fire ant venom allergens Soli II, Soli III, and Soli IV. Journal of Allergy and Clinical Immunology 91: 71–78. Google Scholar


Hsu JC, Chien TY, Hu CC, Chen MJM, Wu WJ, Feng HT, Haymer DS, Chen CY. 2012. Discovery of genes related to insecticide resistance in Bactrocera dorsalis by functional genomic analysis of a de novo assembled transcriptome. PLoS One 7: e40950. Google Scholar


Iseli C, Jongeneel CV, Bucher P. 1999. ESTScan: a program for detecting, evaluating, and reconstructing potential coding regions in EST sequences. International Conference on Intelligent Systems for Molecular Biology, 1999, pp. 138–148. Google Scholar


Kanehisa M, Goto S. 2000. KEGG: Kyoto encyclopedia of genes and genomes. Nucleic Acids Research 28: 27–30. Google Scholar


King M, Eubel H, Millar AH, Baer B. 2011. Proteins within the seminal fluid are crucial to keep sperm viable in the honeybee Apis mellifera. Journal of Insect Physiology 57: 409–414. Google Scholar


King TP, Lu G. 1997. Hornet venom allergen antigen 5, Dol m 5: its T-cell epitopes in mice and its antigenic cross-reactivity with a mammalian testis protein. Journal of Allergy and Clinical Immunology 99: 630–639. Google Scholar


La Vignera S, Vicari E, Condorelli R, D'Agata R, Calogero A. 2011. Male accessory gland infection and sperm parameters (review). International Journal of Andrology 34: e330–e347. Google Scholar


Lee JJ, Klowden MJ. 1999. A male accessory gland protein that modulates female mosquito (Diptera: Culicidae) host-seeking behavior. Journal of the American Mosquito Control Association 15: 4–7. Google Scholar


Lin J, Ma K, Bai Z, Li J. 2013. Molecular cloning and characterization of perlucin from the freshwater pearl mussel, Hyriopsis cumingii. Gene 526: 210–216. Google Scholar


Livak KJ, Schmittgen TD. 2001. Analysis of relative gene expression data using realtime quantitative PCR and the 2−△△Ct method. Methods 25: 402–408. Google Scholar


Lung O, Wolfner M. 2001. Identification and characterization of the major Drosophila melanogaster mating plug protein. Insect Biochemistry and Molecular Biology 31: 543–551. Google Scholar


Lung O, Kuo L, Wolfner MF. 2001. Drosophila males transfer antibacterial proteins from their accessory gland and ejaculatory duct to their mates. Journal of Insect Physiology 47: 617–622. Google Scholar


Miyatake T, Chapman T, Partridge L. 1999. Mating-induced inhibition of remating in female Mediterranean fruit flies Ceratitis capitata. Journal of Insect Physiology 45: 1021–1028. Google Scholar


Mizuki N, Sarapata DE, Garcia-Sanz JA, Kasahara M. 1992. The mouse male germ cell—specific gene Tpx-1: molecular structure, mode of expression in spermatogenesis, and sequence similarity to two non-mammalian genes. Mammalian Genome 3: 274–280. Google Scholar


Mortazavi A, Williams BA, McCue K, Schaeffer L, Wold B. 2008. Mapping and quantifying mammalian transcriptomes by RNA-Seq. Nature Methods 5: 621–628. Google Scholar


Mueller JL, Ripoll DR, Aquadro CF, Wolfner MF. 2004. Comparative structural modeling and inference of conserved protein classes in Drosophila seminal fluid. Proceedings of the National Academy of Sciences of the United States of America 101: 13542–13547. Google Scholar


Mueller JL, Ram KR, McGraw LA, Qazi MCB, Siggia ED, Clark AG, Aquadro CF, Wolfner MF. 2005. Cross-species comparison of Drosophila male accessory gland protein genes. Genetics 171: 131–143. Google Scholar


Pace KE, Baum LG. 2002. Insect galectins: roles in immunity and development. Glycoconjugate Journal 19: 607–614. Google Scholar


Patel S, Skandhan K, Mehta Y. 1988. Seminal plasma fructose and glucose in normal and pathological conditions. Acta Europaea Fertilitatis 19: 329–332. Google Scholar


Radhakrishnan P, Taylor PW. 2008. Ability of male Queensland fruit flies to inhibit receptivity in multiple mates, and the associated recovery of accessory glands. Journal of Insect Physiology 54: 421–428. Google Scholar


Ram KR, Wolfner MF. 2007. Seminal influences: Drosophila Acps and the molecular interplay between males and females during reproduction. Integrative and Comparative Biology 47: 427–445. Google Scholar


Ren LL, Qi LY, Jiang QG, Zhou SD, Dai HG. 2008. Oviposition preference of oriental fruit fly, Bactrocera dorsalis. Chinese Bulletin of Entomology 45: 593–597. Google Scholar


Rogers DW, Whitten MM, Thailayil J, Soichot J, Levashina EA, Catteruccia F. 2008. Molecular and cellular components of the mating machinery in Anopheles gambiae females. Proceedings of the National Academy of Sciences of the United States of America 105: 19390–19395. Google Scholar


Scolari F, Gomulski LM, Ribeiro JMC, Siciliano P, Meraldi A, Falchetto M, Bonomi A, Manni M, Gabrieli P, Malovini A, Bellazzi R, Aksoy S, Gasperi G, Malacrida AR. 2012. Transcriptional profiles of mating-responsive genes from testes and male accessory glands of the Mediterranean fruit fly, Ceratitis capitata. PLoS One 7: e46812. Google Scholar


Shen GM, Dou W, Niu JZ, Jiang HB, Yang WJ, Jia FX, Hu F, Cong L, Wang JJ. 2011. Transcriptome analysis of the oriental fruit fly (Bactrocera dorsalis). PLoS One 6: e29127. Google Scholar


Shen GM, Dou W, Huang Y, Jiang XZ, Smagghe G, Wang JJ. 2013. In silico cloning and annotation of genes involved in the digestion, detoxification and RNA interference mechanism in the midgut of Bactrocera dorsalis [Hendel (Diptera: Tephritidae)]. Insect Molecular Biology 22: 354–365. Google Scholar


Simmons L, Tan YF, Millar A. 2013. Sperm and seminal fluid proteomes of the field cricket Teleogryllus oceanicus: identification of novel proteins transferred to females at mating. Insect Molecular Biology 22: 115–130. Google Scholar


Sirot LK, Hardstone MC, Helinski ME, Ribeiro JM, Kimura M, Deewatthanawong P, Wolfner MF, Harrington LC. 2011. Towards a semen proteome of the dengue vector mosquito: protein identification and potential functions. PLoS Neglected Tropical Diseases 5: e989. Google Scholar


Sonenshine DE, Bissinger BW, Egekwu N, Donohue KV, Khalil SM, Roe RM. 2011. First transcriptome of the testis-vas deferens-male accessory gland and proteome of the spermatophore from Dermacentor variabilis (Acari: Ixodidae). PLoS One 6: e24711. Google Scholar


Swanson WJ, Clark AG, Waldrip-Dail HM, Wolfner MF, Aquadro CF. 2001. Evolutionary EST analysis identifies rapidly evolving male reproductive proteins in Drosophila. Proceedings of the National Academy of Sciences of the United States of America 98: 7375–7379. Google Scholar


Thailayil J, Magnusson K, Godfray HCJ, Crisanti A, Catteruccia F. 2011. Spermless males elicit large-scale female responses to mating in the malaria mosquito Anopheles gambiae. Proceedings of the National Academy of Sciences of the United States of America 108: 13677–13681. Google Scholar


Vogel H, Badapanda C, Knorr E, Vilcinskas A. 2014. RNA-sequencing analysis reveals abundant developmental stage—specific and immunity-related genes in the pollen beetle Meligethes aeneus. Insect Molecular Biology 23: 98–112. Google Scholar


Walker MJ, Rylett CM, Keen JN, Audsley N, Sajid M, Shirras AD, Isaac RE. 2006. Proteomic identification of Drosophila melanogaster male accessory gland proteins, including a pro-cathepsin and a soluble gamma-glutamyl transpeptidase. Proteome Science 4: 9. Google Scholar


Wan XW, Liu YH, Zhang B. 2012. Invasion history of the oriental fruit fly, Bactrocera dorsalis, in the Pacific-Asia region: two main invasion routes. PLoS One 7: e36176. Google Scholar


Wei D, Feng YC, Wei DD, Yuan GR, Dou W, Wang JJ. 2015a. Female remating inhibition and fitness of Bactrocera dorsalis (Diptera: Tephritidae) associated with male accessory glands. Florida Entomologist 98: 52–58. Google Scholar


Wei D, Li HM, Yang WJ, Wei DD, Dou W, Huang Y, Wang JJ. 2015b. Transcriptome profiling of the testis reveals genes involved in spermatogenesis and marker discovery in the oriental fruit fly, Bactrocera dorsalis. Insect Molecular Biology 24: 41–57. Google Scholar


Wei D, Tian CB, Liu SH, Wang T, Smagghe G, Jia FX, Dou W, Wang JJ. 2016. Transcriptome analysis to identify genes for peptides and proteins involved in immunity and reproduction from male accessory glands and ejaculatory duct of Bactrocera dorsalis. Peptides 80: 48–60. Google Scholar


Werner M, Simmons LW. 2008. Insect sperm motility. Biological Reviews 83: 191–208. Google Scholar


Wolfner MF. 1997. Tokens of love: functions and regulation of Drosophila male accessory gland products. Insect Biochemistry and Molecular Biology 27: 179–192. Google Scholar


Wolfner MF. 2002. The gifts that keep on giving: physiological functions and evolutionary dynamics of male seminal proteins in Drosophila. Heredity 88: 85–93. Google Scholar


Wong A, Albright SN, Giebel JD, Ram KR, Ji S, Fiumera AC, Wolfner MF. 2008. A role for Acp29AB, a predicted seminal fluid lectin, in female sperm storage in Drosophila melanogaster. Genetics 180: 921–931. Google Scholar


Xu J, Wang Q. 2011. Seminal fluid reduces female longevity and stimulates egg production and sperm trigger oviposition in a moth. Journal of Insect Physiology 57: 385–390. Google Scholar


Xu JJ, Baulding J, Palli SR. 2013. Proteomics of Tribolium castaneum seminal fluid proteins: identification of an angiotensin-converting enzyme as a key player in regulation of reproduction. Journal of Proteomics 78: 83–93. Google Scholar


Yang WJ, Yuan GR, Cong L, Xie YF, Wang JJ. 2014. De novo cloning and annotation of genes associated with immunity, detoxification and energy metabolism from the fat body of the oriental fruit fly, Bactrocera dorsalis. PLoS One 9: e94470. Google Scholar


Ye J, Fang L, Zheng H, Zhang Y, Chen J, Zhang Z, Wang J, Li S, Li R, Bolund L. 2006. WEGO: a web tool for plotting GO annotations. Nucleic Acids Research 34: W293–W297. Google Scholar


Zhang X, Wang X, Sun C, Wang J. 2011. C-type lectin from red swamp crayfish Procambarus clarkii participates in cellular immune response. Archives of Insect Biochemistry and Physiology 76: 168–184. Google Scholar


Zheng WW, Peng T, He W, Zhang HY. 2012. High-throughput sequencing to reveal genes involved in reproduction and development in Bactrocera dorsalis (Diptera: Tephritidae). PLoS One 7: e36463. Google Scholar


[1] Supplementary material in Florida Entomologist 100(1) (Mar 2017) is online at

Chuan-Bei Tian, Dong Wei, Lin-Fan Xiao, Wei Dou, Huai Liu, and Jin-Jun Wang "Comparative Transcriptome Analysis of Three Bactrocera dorsalis (Diptera: Tephritidae) Organs to Identify Functional Genes in the Male Accessory Glands and Ejaculatory Duct," Florida Entomologist 100(1), 42-51, (1 March 2017).
Published: 1 March 2017
accessory gland protein
comparative transcriptome
mosca de la fruta oriental
oriental fruit fly
proteína accesoria de las glándulas
proteína fluida seminal
Back to Top