| Multiple mating is a typically reproductive strategy in nature, which widely occurs in insects. Females will produce more eggs and offsprings because of the male accessory gland (MAGs) secreting proteins (Acps) and sperms from males by multiple matings. The ability of multiple mating is related with the two tissues, testis and MAGs. Mating success and the fertilization are also regulated by functional proteins in testis and MAGs. Previous study of reproductive proteins began when the biochemical fractionation of gametes and gonads led to the purification and characterization of specific proteins. Recently, various molecular and genetic tools, e.g. next-generation sequencing technologies coupled with bioinformatics, have been widely used to identify and analyze the reproductive proteins in many insects. With the help of high throughput technologies, we can get more information with less cost and less time. So there are more and more studies focus on the identification of reproductive proteins in many non-model insect species, which will pave the way for the further molecular characterization, functional and evolutionary research.As one of the most important agricultural pests worldwide, little research focus on the remating and multiple mating, and also reproductive protein identification in Bactrocera dorsalis. In current study, we investigated the multiple mating and its relationship with MAGs in B. dorsalis, as well as the influence on female fitness. Then we carried out the transcriptome sequencing of testis and MAGs to indentify the functional genes involved in spermatogenesis, sperm maturation, immunity and mating regulation. Several tissue-specific expressed genes were identified by quantitative real time PCR (qRT-PCR). Thereafter, we performed the proteomic analysis of different aged testis to identify highly presented proteins in mature testis. On the other hand, the proteomic analysis of MAG secretion was also conducted to identify the Acps, combined with the transcriptome data. The all results can help us understand the biological background of mating behavior of B. dorsalis. The identification of reproductive proteins will pave the way of the further functional and evolutionary research. The main result are as follows:1 Multiple mating and the influence in fitness of B. dorsalis1.1 Investigation of maturation and design of rearing cageIn this study, we designed a cage for single fly rearing and egg-collecting. This device was granted the national invention patent license with the accession number of ZL 2012 2 0529146.1. After investigation of the mating behavior and copulation duration, we found that there are no differences of mating success and courtship duration between 8-d and the following days’ flies. We concluded that B. dorsalis adults get mature on 8th day after emergence.1.2 The relationship of multiple mating and the MAGsIn this study, we investigated the morphology of MAGs by dissecting the mature male flies. The MAGs consists one pair of long mesodermal accessory glands (2.0-2.5 mm) and three pairs of convoluted ectodermal accessory glands which appear spongy and sometimes branched. Both glands adhere to the ejaculatory duct. Both length and area of MAG were significantly larger after mating by measuring the sizes. We also found that male B. dorsalis can mate successfully in consecutive days, but there was a significant difference in remating tendency between virgin females and mated females regardless of whether they mated with virgin or mated males. This remating inhibition had no relationship with males’ mating experience. In addition, there were no significant differences between courtship and copulation duration among different treatments.1.3 Effects of remating on female fitnessFemale longevities were significantly influenced by mating. Females housed alone lived longer than mated females, but no significant differences were found among the different matings groups of mated females. Among the groups, mated females laid more eggs than virgin females, while no significant differences were observed among females housed alone regardless of whether they mated once, twice or mated with previously mated males. More egg-laying was also found in sex-mixed females. Females that mated with previously mated males had the lowest hatchability among groups except the virgin females. Significant hatchability was not observed among females that mated once, twice or those housed with males at a sex ratio of 1:1. Females in high sex ratio had the highest hatchability. In addition, we found that most eggs laid by mated females emerged in the previous 20 weeks, which corresponded to the egg laying ratio. We also found that females housed with males produced more offsprings, and females housed with higher number of males (1♀:2♂) produced more offsprings which was consistent with the number of egg-laying. Intriguingly, there was no significant difference in the number of offspring produced by females mated to virgin or non-virgin males. Second mating of non-virgin females did not increase offspring number. We also observed that most of the offspring produced in the first 15 weeks.2 Transcriptome sequencing and functional genes analysis of testis2.1 Transcriptome sequencing and functional annotationIn order to investigate the gene expression profiling, we carried out the transcriptome sequencing and analysis of testis. The raw data was deposited in the Transcriptome Shotgun Assembly database with the accession number of SRR1032039. Illumina sequencing produced 52 016 732 clean reads representing 4 681 505 880 nucleotides. These short reads were assembled into 47 677 contigs with the mean length of 443 bp, and then clustered into 30 516 unigenes with the mean length of 756 bp.All the sequences were submitted to NCBI nr, nt, and Swiss-Prot, KEGG, COG, GO databases and/or websites for predication with a cut-off value of 10-5. Finally, a total of 20 921 unigenes were annotated, including 20 107,1 1673,15 498,13 705,6 588 and 14 325 in nr, nt, Swiss-Prot, KEGG, COG, and GO database/website, respectively. We also found that most of the hits (57%) had over 60% similarity with genes in nr database, and 5% of them had similarity score of more than 90%. Most of the unigenes (86.22%) showed the strong similarity to those of Drosophila genus. Only 316 unigenes annotated to Bactrocera genus.GO classification showed that 14 325 unigenes were assigned into three categories, including 59 functional groups. There were 28 groups contained more than 1 000 unigenes. In COG functional annotation, there were 6 588 unigenes were annotated and divided into 25 categories, and 2 507 unigenes belonged to "General function prediction only". In the biochemical pathway annotation,13 705 unigenes were mapped to 254 predicted pathways, in which there were 99 pathways contains more than 100 unigenes. The key pathways that are probably relevant to spermatogenesis and reproduction were analyzed.2.2 Functional gene identification and analysisBy BLAST in nr database, a total 181 distinct unigenes encoding serine/threonine-protein kinase were identified in testis transcriptome. The mean length was 1 140 bp. There were 19 unigene contained the open reading frame, two of which were testis-specific serine/threonine-protein kinase (TSSK), CL2209 and unigene14017. The lengths of the two TSSK were 1 497 and 1 819 bp, respectively, and the ORFs were 1 065 and 900 bp, respectively. Both of the two amino acid sequences contained the same functional sites. We identified almost 27 cyclin unigenes (average 1 183 bp) in B. dorsalis. Many of them function in the meiosis and mitosis, such as Cyclin A, Cyclin B, Cyclin E, Cyclin J and G2/M-specific cyclin, etc. Besides,15 Cdk unigenes (average 1 034 bp), including Cdk2, Cdk5, Cdk7, etc, were identified in testis transcriptome.Six unigenes encoding 4 Neprilysin encoding unigenes were identified in this study. All the deduced amino acid sequences contained the same active sites and Zn binding sites. Similar to Neprilysin, three Ferritin encoding unigenes were also identified in testis transcriptome, and all the three deduced anmino sequences contained the same Fe binding sites. These two kinds of ptroteins may function in testis development and sperm maturation. Heat shock proteins (HSPs) are important in the fertilization of insects. In B. dorsalis testis, we identified 55 HSPs encoding sequences,27 of which containing ORF with the average of 1 154 bp. Such Hsp60, Hsp70, Hsp90 and small Hsps were identified. The identification of all of these genes in B. dorsalis testis transcriptome will help us understand the development and spermatogenesis.In addition,33 actin and actin-related unigenes (average 1 286 bp) were identified in testis. Previous study demonstrated that ubiquitin in ubiquitin-dependent proteolytic system play the key role in male fertilization in insects. In this study, we identified 220 distinct unigenes (average 1 489 bp) in testis transcriptome, including ubiquitin-activating enzyme, ubiquitin conjugating enzyme etc.2.3 Tissue expression profiling of functional genes in tesitsWe performed the qRT-PCR to determine the tissue expression profiling of functional genes identified in this study. Finally, two TSSKs were found that highly expressed in testis. In addition to TSSKs, two cyclin genes, namely Cyclin B and Cyclin J, were also highly expressed in testis. Two metalloproteins named Nep4 and Fer3, and one HSPs encoding genes named Hsp70-2 were also determined highly expressed in testis. The tissue specific expression profiling will help us study their function in male fertilization in the future.3 Transcriptome sequencing and functional genes analysis of MAGs3.1 Transcriptome sequencing and functional annotationAfter the testis transcriptome analysis, we carried out the transcriptome analysis of MAGs. The raw data was deposited in the Transcriptome Shotgun Assembly database with the accession number of SRR1047924. Illumina sequencing produced 54 577 630 clean reads representing 4 911 986 700 nucleotides. These short reads were assembled into 47 514 contigs with the mean length of 399 bp. We ultimately obtained 30 669 unigenes (average 756 bp).All the sequences were submitted to NCBI nr, nt, and also swiss-Prot, KEGG, COG, GO databases or websites for predication. Finally,20 419 unigenes were annotated in databases and/or websites, including 19 366,11 623,14 779,13 283,6 072 and 14 042 in nr, nt, Swiss-Prot, KEGG, COG, and GO database/website, respectively. We also found that most of the hits (more than 60%) had over 60% similarity with genes in nr database, and 5% of them had similarity score of more than 90%. Most of the unigenes (85.1%) showed a strong similarity to those of Drosophila genus. Only 664 unigenes annotated to Bactrocera genus.GO classification showed that 14 042 unigenes were assigned into three categories, including 59 functional groups. There were 23 groups contained more than 2 000 unigenes. In COG functional annotation,6 072 unigenes were annotated and divided into 25 categories, and 2 341 unigenes (38.6%) belonged to "General function prediction only". In the pathway annotation,13 705 unigenes were mapped to 256 predicted pathways, in which there were 41 pathways contains more than 200 unigenes. "Metabilic pathways" is the most prominent pathway, including 1 727 unigenes (13.8%).3.2 Functional gene identification and analysisThe secretion contains many kinds of molecules involved in sperm capacitation and tissue development. We identified three aldose reductase, six 6-phosphofructo kinase, and one fructose-1,6-bisphosphatase in polyol pathway in MAGs. Moreover, many serpin that may influence the sperm capacitation were found in MAGs transcriptome, e.g. Serpin-4, Serpin 27, Serpin42Dc, Serpin 77 ba. Moreover, many genes involved in JH biosynthesis and metabolism were identified in testis, several genes involved in Ecdysone synthesis related genes were identified in MAGs, namely neverland, shroud, spook, disembodi.The male and female reproductive duct can be infested by microbial organism when copulation. We identified many functional genes that could function in recognizing the microbial organism in MAGs. Indeed, we identified 5 peptidoglycan receptor proteins (PGRPs), many Lectins and other immune-recognition related protein encoding genes in MAGs. In addition, we identified many antimicrobial peptides encoding genes in MAGs, including 4 attacin,3 cecropins,1 diptericin, and 21 cyclophilins encoding genes.3.3 Tissue expression profiling of functional genes in tesitsTissue expression profiling showed that two of five PGRPs (Unigene277 and Unigene372)weve highly expressed in MAGs, and the rest one highly was expressed in fat body. One of the Lectin genes(Unigene17466) with ORF was highly expressed in MAGs, while the other three PGRPs (Unigene7941, Unigene15169 and Unigene9) and three Lectins (Unigenel6864, Unigene17418 and Unigene17562) were highly expressed in midgut. Two Defensin genes(Unigenel3452 and Unigene16260) were highly expressed in fat body. Similarly, three Attacins (Unigene8670, Unigene8671 and Unigene14157) were highly expressed in fat body, while the last one (Unigenel6681) was expressed in most tissues. Interestingly, three Cecropins were highly expressed in midgut (UnigeneCL1332), fat body(Unigene10593) and Malpighian tubule (Unigenel6193), respectively. High expression of one Diptericin (Unigenel3875) was determined in fat body and testis. There were 16 cyclophilin homologous genes were identified in MAGs with only one of them (Unigene120) had a high expression in MAGs.After being challenged by three microbial inducible factors, five PGRPs expressions were un-regulated; expressions of two Lectins (Unigene17418 and Unigene17466) were down-regulated. In the response of AMPs, all the two Defensins, three Attacins and one Diptericin were up-regulated by three triggers, especially the PGN-EB; two Cecropins (CL1332 and Unigene10593) were up-regulated induced by three triggers, while the other one was down-regulated by PGN-EB and GEG; three of four Cyclophilins were up-regulated induced by PGN-EN and EGE, while the other one was down-regulated induced by PGN-EN.4 Proteomic analysis of testis from different days old male flies4.1 Protein identification in testisThe proteomes sequencing of three different aged testis (1-d,5-d and 9-d) samples were performed in this study. The chromatogram and the number of peptide of three runs showed the high sequencing quality. A total of 2 789 proteins were identified in all testis samples, including 1912 reliable proteins which were identified in as least two runs. There were 7 179 unique peptides identified from testis, in which 3 443 unique peptides were identified in all samples. The next analysis showed that 46.65% proteins were identified by more than 5 peptides in the present study, according to the mean number of peptides of 2.8. Sequence coverage analysis showed that 45.08% proteins has over 30% coverage. The average of sequence coverage was 30.71% in this study.4.2 Comparative proteomic analysisAfter comparative quantitative analysis of three developing testis samples,49,76 and 124 proteins were identified as specific expressed in 1-d,5-d and 9-d samples only, respectively. However, there were 1442 proteins in all three samples. We also conducted the analysis of quantitative changes of protein in three developing testis. All the proteins were clustered into four types of change, including 393,384,570 and 565 proteins in each type, respectively. There were 141 and 111 proteins screened out as the potential functional proteins which were highly presented in 9-d mature testis and 5-d testis, respectively. All of these 252 potential functional proteins were then analyzed for functional annotation such as COG, GO and KEGG pathway annotation. We found that some proteins highly presented in 5-d were involved in "cell cycle and cell division" and "motility", while more proteins highly presented in 5-d samples were involved in energy production and metabolism. Some pathways, e.g. "peroxisome", "RNA transport", "protein processing in endoplasmic reticulum", "MAPK signaling pathway", "focal adhesion" which function in spermatogenesis were highly presented in 9-d testis sample.5 Male accessory glands proteins identificationAll unigenes are BLASTX to protein databases to decide the coding region sequences, then translated into amino (Coding sequences, CDSs). Finally,19 438 unigene sequences were found to match with the known proteins. Most of the amino acid sequences were shorter than 500 residues. The SFPs secreted by MAGs were separated and identified by label-free LC-MS based on the CDSs dataset. After combining all data from the two runs, we identified 25 606 peptides that were presented in both samples. These peptides were matched to 3 942 protein groups, from which we were able to annotate 2 927 significant CDSs. Most of these proteins corresponded to 2-10 peptides. The average number of peptides per protein was 8.75, leading to an average sequence coverage of 29.2. There were 334 proteins (11.4%) with a sequence coverage of> 50%. Following ORF prediction,1 116 proteins (40.9%) containing ORFs were screened out for next signal peptide prediction. Using SignalP,90 of these ORFs contained predicted 5’signal peptides that were identified as putative Acps. Amongst these there were 27 known proteins and 63 proteins with no functional description (70%). The known proteins consisted of proteases, odorant binding proteins, metalloproteinase, ribosomal protein, serine protease inhibitor, and some immunity-related proteins.Comparing the relative abundance of Acps in MAGs. We knew that the function of the most abundant proteins were unknown. Using this determination, the most abundant protein was CG5867 (Unigene15698), a protein of unknown function, with a molar proportion of 4.38% of total protein. Several of most abundant proteins were previously-characterized SFPs, such as odorant binding protein (Unigenel5912) and the immune-related proteins, cyclophilins (CL728). However, several proteins not previously linked to MAGs secretions were also in the top quartile for abundance, including CG5867 (Unigene15698), which was a hemolymph juvenile hormone-binding protein, and GH20332 (Unigene120), a cyclophilin-type peptidyl-prolyl cis-trans isomerase. After combining InterProScan and Gene Ontology analysis, these predicted proteins were classified into 10 categories based on their molecular functions. Most proteins clustered into the same functional categories, including proteases, protease inhibitors, mediators of immune responses, and odorant binding proteins. There were also 32 (34.4%) proteins of unknown function. Several of the most abundant novel Acps (no functional description) were assayed for tissue-specific expression patterns by qRT-PCR. Of these, six novel Acps enconding genes were highly expressed in MAGs of B. dorsalis. Notably, CG5867 was predominantly expressed in MAGs, with a tens of thousands of times higher expression level than that in head.In summary, we investigated the morphology of male B. dorsalis accessory glands, and compared the size of MAGs after mating, and also investigated the fitness of females after multiple matings. Transcriptome sequencing of testis and MAGs tissues were carried out in this study. Through data analysis we understand the gene expression profiling in these tissues. Quantitative RT-PCR was performed to determined the gene expression among tissues. Finally, many genes were identified as potential functional genes. Moreover, the prominent Label-free quantitative proteome sequencing technology was used in this study to investigate the proteins presented in different aged testis. By compared proteomic analysis, we found many protein highly expressed in mature male testis, which may be involved in testis development and spermatogenesis. Combined transcriptomic and proteomic analysis, we identified 90 SFPs in MAGs secretion, providing much bioinformation of reproductive proteins in B. dorsalis. Overall our study paved the way for further study of functional study and evolutionary study of reproductive proteins. |
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