High-throughput sequencing to reveal genes involved in reproduction and development in Bactrocera dorsalis (Diptera: Tephritidae)

Abstract

Background: Tephritid fruit flies in the genus Bactrocera are of major economic significance in agriculture causing considerable loss to the fruit and vegetable industry. Currently, there is no ideal control program. Molecular means is an effective method for pest control at present, but genomic or transcriptomic data for members of this genus remains limited. To facilitate molecular research into reproduction and development mechanisms, and finally effective control on these pests, an extensive transcriptome for the oriental fruit fly Bactrocera dorsalis was produced using the Roche 454-FLX platform.

Results: We obtained over 350 million bases of cDNA derived from the whole body of B. dorsalis at different developmental stages. In a single run, 747,206 sequencing reads with a mean read length of 382 bp were obtained. These reads were assembled into 28,782 contigs and 169,966 singletons. The mean contig size was 750 bp and many nearly full-length transcripts were assembled. Additionally, we identified a great number of genes that are involved in reproduction and development as well as genes that represent nearly all major conserved metazoan signal transduction pathways, such as insulin signal transduction. Furthermore, transcriptome changes during development were analyzed. A total of 2,977 differentially expressed genes (DEGs) were detected between larvae and pupae libraries, while there were 1,621 DEGs between adults and larvae, and 2,002 between adults and pupae. These DEGs were functionally annotated with KEGG pathway annotation and 9 genes were validated by qRT-PCR.

Conclusion: Our data represent the extensive sequence resources available for B. dorsalis and provide for the first time access to the genetic architecture of reproduction and development as well as major signal transduction pathways in the Tephritid fruit fly pests, allowing us to elucidate the molecular mechanisms underlying courtship, ovipositing, development and detailed analyses of the signal transduction pathways.

Figure 1. Assembled contig length distribution of the B.dorsalis transcriptome.

The x-axis indicates contig size and the y-axis indicates the number of contigs of each size.

Figure 2. Orthologous genes shared between Bactrocera, Drosophila, Anopheles, and Bombyx.

The Venn diagram shows the number of orthologous groups of genes shared between the genomes/transcriptomes of these species.

Figure 3. Characteristic analysis of the homology search of ESTs against the nr database.

(A) Identity distribution of the top BLAST hits for each sequence. (B) Species distribution is shown as a percentage of the total homologous sequences with an E-value of at least 1.0E⁻⁵. The first hit of each sequence was used for analysis. Homo: Homo sapiens; Rat: Rattus norvegicus.

Figure 4. GO analyses of Bactrocera transcriptome data.

GO analysis of Bactrocera sequences corresponding to a total of 28,782 contigs that are predicted to be involved in biological processes (A) and molecular functions (B) and cellular component (C). Classified gene objects are depicted as percentages (in brackets) of the total number of gene objects with GO assignments.

Figure 5. Histogram of clusters of orthologous groups (COG) classification.

A total of 58,199 predicted proteins have a COG classification among the 25 categories.

Figure 6. Changes in gene expression profile during development.

The number of up-regulated and down-regulated genes between larvae and pupae, between adults and pupae, and between adults and larvae are summarized here.

Figure 7. qRT-PCR confirmation of the differentially expressed genes between each two stages (larvae vs. pupae, adults vs. larvae, and adults vs. pupae).

The transcript abundance from DEG data is shown above each gene. Relative transcript levels are calculated by real-time PCR using 16 s rRNA as the standard. 2F, feeding 2th instar larvae; 2M, molting 2th instar larvae; 3M, metamorphic molting larvae; P1, prepupa; P2, pupa. Three biological replicates were performed, and the data shown are typical results.