miR-8-3p regulates mitoferrin in the testes of Bactrocera dorsalis to ensure normal spermatogenesis

Abstract

Genetics-enhanced sterile insect techniques (SIT) are promising novel approaches to control Bactrocera dorsalis, the most destructive horticultural pest in East Asia and the Pacific region. To identify novel genetic agents to alter male fertility of B. dorsalis, previous studies investigated miRNA expression in testes of B. dorsalis. One miRNA, miR-8-3p was predicted to bind the 3'UTR of putative B. dorsalis mitoferrin (bmfrn). The ortholog of bmfrn in D. melanogaster is essential for male fertility. Here we show that bmfrn has all conserved amino acid residues of known mitoferrins and is most abundantly expressed in B. dorsalis testes, making miR-8-3p and mitoferrin candidates for genetics-enhanced SIT. Furthermore, using a dual-luciferase reporter system, we show in HeLa cells that miR-8-3p interacts with the 3'UTR of bmfrn. Dietary treatments of adult male flies with miR-8-3p mimic, antagomiR, or bmfrn dsRNA, altered mitoferrin expression in the testes and resulted in reduced male reproductive capacity due to reduced numbers and viability of spermatozoa. We show for the first time that a mitoferrin is regulated by a miRNA and we demonstrate miR-8-3p as well as bmfrn dsRNA to be promising novel agents that could be used for genetics-enhanced SIT.

Figure 1. Mitoferrin is most abundantly expressed in testes and targeted by miR-8-3p.

(A) Expression pattern of the B. dorsalis gene mitoferrin in different tissues (whole body, head, thorax, gut, malpighian tube (Mt) and testis) of 12 d old adult males (n = 20). Mitoferrin expression in tissues is relative to whole body expression. The letters above the bars show significant differences (Least Significant Difference in one-way analysis of variance, P < 0.05) in bmfrn expression. The data represent the mean of three independent experiments. Error bars indicate SD. (B) Potential miRNA target sites of miR-279-3p, miR-8-3-p, miR-275-3p, miR-34-3p and miR-304-5pin the 3′-UTR of the mitoferrin as detected by RNAhybrid. Seed sequence of the miRNAs and their putative binding sites in the 3′-UTR are indicated by grey shading. mfe:match free energy. (C) Dual-luciferase assay in HeLa cells co-transfected with psiCHECK-2 -bmfrn 3′-UTR (100 ng) together with negative control miRNA (miR-NC) or miRNA mimics (50 nM) as indicated. Data represent means of three independent experiments, error bars indicate SD. ****P < 0.001, ANOVA with Bonferroni’s Multiple Comparison Test, testing selected pairs (miRNA vs respective miR-NC). (D) Expression pattern of miR-8-3p in different tissues (whole body, head, thorax, gut, malpighian tube (Mt) and testis) of 12 d old adult males (n = 3). Expression in tissues is relative to whole body expression. “a” above the bars indicates significant differences in miR-8-3p expression compared with whole fly homogenate. The data represent the means with SD. N = 3.

Figure 2. Dietary delivery of miR-8-3p mimics/antagomiRs and bmfrn dsRNA alter bmfrn expression in testes.

Relative abundances of miR-8-3p (A) and bmfrn (B,C) were determined by qRT-PCR of cDNA made from total RNA isolated from testes 1, 3, 7, and 12 days after the indicated dietary treatments of adult flies. Data represent the mean values ± SD of three independent experiments (20 flies per experiment and treatment). Treatments were compared with their respective controls using ANOVA (t-test, p < 0.05).*, ** and *** indicates P < 0.5, P < 0.01 and P < 0.005 respectively.

Figure 3. Increasing or decreasing miR-8-3p levels in testes, as well as reduction of bmfrn expression in the testis result in reduced reproductive capabilities due to reduced sperm production and viability.

Newly eclosed male flies, maintained for 1, 3, 7 or 12 days on indicated treatments were used to determine (A) reproductive capacity, (B) sperm counts and (C) sperm viability. (A) Single males were mated with three females, eggs were collected and the percentage of larvae eclosed from the eggs was determined. Data represent the mean + /− SD from 20 males per treatment. (B) Spermatozoa from the seminal vesicles of individual males were counted. Data represent mean + /− SEM from 30 males per treatment. (C) Average percentage of live sperms per male was determined for seminal vesicles of 20 males per treatment. The effect of treatments was analyzed using generalized linear models with a binomial error distribution and a logit-link function ***indicates P < 0.0001.