RNA Interference-Mediated Knockdown of Male Fertility Genes in the Queensland Fruit Fly Bactrocera tryoni (Diptera: Tephritidae)

Abstract

The Queensland fruit fly, Bactrocera tryoni, is Australia’s most important horticultural pest. The Sterile Insect Technique (SIT) has been used to control this species for decades, using radiation to sterilize males before field-release. This method of sterilization can potentially reduce the insects’ abilities to compete for mates. In this study, RNA interference (RNAi) techniques were examined for their potential to sterilize male B. tryoni without adversely affecting mating competitiveness. B. tryoni adults were injected or fed double-stranded RNAs (dsRNAs) targeting spermatogenesis genes (tssk1, topi and trxt); quantitative reverse-transcriptase PCR analyses confirmed that transcript levels were reduced 60–80% for all three genes following injections. Feeding produced a significant gene knockdown for tssk1 and trxt after three days, but interestingly, two genes (trxt and topi) produced an excess of transcripts after 10 days of feeding. Despite these fluctuations in transcript levels, all three dsRNAs impacted the fecundity of treated males, with tssk1- and topi-dsRNA-treated males producing 75% fewer viable offspring than the negative controls. Mating competition assays demonstrated that dsRNA-treated males can actively compete with untreated males. These findings suggest that RNAi technology could serve as an alternative to radiation as a means of sterilizing these insects in an SIT program.

RNAi-Mediated Knockdown of Tssk1 and Tektin1 Genes Impair Male Fertility in Bactrocera dorsalis

“…Highest expression of both genes was observed in testis of mature male adults of B. dorsalis . These results were consistent with the studies indicating that Tssk1 and Tektin1 are essential for male fertility [23,29]. We may suggest that these genes have important functions in spermatogenesis of target insect B. dorsalis .…”

“…ORF of these two genes encodes amino acids of 900 and 1266bp, respectively. Nucleotide sequence of Tssk1 gene shares more than 95% identity with B. tryoni [23]. Amino acid sequence of Tssk1 also aligned with other insects (Figure S1).…”

“…We have selected two genes, Tssk1 (XM_011212727.2) and Tektin1 (XM_011202998.1), which are related to male fertility based on previous studies [23,33]. Partial nucleotide sequences of Tssk1 and Tektin1 were 1761 and 1607 bp long.…”

“…Selection of two target genes was made based on previous studies [23,38]. Sequences of Tssk1 (GenBank accession number: XM_011212727.2) and Tektin1 (GenBank accession number: XM_011202998.1) were chosen as target genes.…”

“…Moreover, the expression of Tssk1 has been identified in the testis of the Bactrocera tryoni . Double-stranded RNA (dsRNA) feeding of Tssk1 in B. tryoni significantly silenced the gene and caused male sterility, suggesting its role in the male reproduction of tephritid insects [23].…”