The characterization of the circadian clock in the olive fly Bactrocera oleae (Diptera: Tephritidae) reveals a Drosophila-like organization

Abstract

The olive fruit fly, Bactrocera oleae, is the single most important pest for the majority of olive plantations. Oxitec's self-limiting olive fly technology (OX3097D-Bol) offers an alternative management approach to this insect pest. Because of previously reported asynchrony in the mating time of wild and laboratory strains, we have characterized the olive fly circadian clock applying molecular, evolutionary, anatomical and behavioural approaches. Here we demonstrate that the olive fly clock relies on a Drosophila melanogaster-like organization and that OX3097D-Bol carries a functional clock similar to wild-type strains, confirming its suitability for operational use.

Figure 1

Schematic representation of the predicted structure of B. oleae PER, CLK, CYC and CRY. B. oleae sequences (top) are compared to their respective D. melanogaster counterparts (bottom). Coloured parts are the functional domains for which the percentage of identity between the two sequences is indicated. Rectangle length corresponds to sequence length; the entire length of the proteins is indicated at the right end. An identification key for the different domains is present in the figure. Multiple sequence alignment and a more detailed overview of proteins structure can be found in Supplementary Figures S1–S4.

Figure 2

Phylogenetic analysis of B. oleae PER and CYC. Gene trees of (A) CYC and (B) PER reconstructed from amino acids with Maximum Likelihood using RaxML. Values at the nodes represent bootstrap values, determined with 1000 replicates.

Figure 3

Temporal expression of the clock genes per, Clk, cyc and cry in B. oleae heads. In Argov male flies, per and Clk mRNA levels are strongly rhythmic and in antiphase to each other (per: H(7) = 20.9, p = 0.003; CircWave, p < 0.001 – Clk: H(7) = 21.2, p = 0.003; CircWave, p < 0.001). The gene cyc is constantly expressed (H(7) = 4.1, p = 0.771; CircWave, p > 0.05). cry mRNA shows weak oscillation (H(7) = 11.6, p = 0.115; CircWave, p < 0.001).

Figure 4

Comparison of clock genes expression between wild-type and transgenic B. oleae. Oscillations in the expression of per, Clk, cyc and cry under LD12:12 was investigated in three different genotypes: Demokritos, Argov (wild-type) and OX3097D-Bol. No significant interaction among timepoint and genotype was found using 2-way ANOVA (all p-values > 0.05, see Supplemetal Table 1), indicating no differences in amplitudes nor phases among strains.

Figure 5

The clock network of B. oleae. (a) The clock neuropeptide PDF (magenta) is expressed in 4 l-LN_v and 4 s-LN_v, as well as in 4 putative insulin producing cells (ipc-2) in the PL. ITP (cyan) is expressed in the 5^th s-LN_v and in one cell of the LN_d group, as well as in putative ipc-1 and ipc-2. (b) anti-PDP1 (green) co-localize in the nuclei of PDF positive cells (magenta) in the LN_v cluster. Antibody reveals also other putative clock clusters (LN_d, DN), and stains many other non-clock cells. (c,d) PDP1 protein level in the nuclei oscillates under LD12:12 in the s-LN_v (H(7) = 34.78, p < 0.001) and l-LN_v (H(7) = 19.259, p < 0.05). AME: accessory medulla; ME: medulla; CA: calyx; LO: lobula.

Figure 6

Locomotor activity of B. oleae under different conditions. Daily activity profile under LD12:12 (top) and one representative actogram, respectively under DD (middle) and LL (bottom) of males (right) and females (left) of Demokritos, Argov and OX3097D-Bol at 20 °C. Horizontal bars aside the actograms separate LD and DD/LL phases.

Figure 7

Average free running periods of the different strains of B. oleae. No significant differences in the free-running periods of males and females of Demokritos, Argov and OX3097D-Bol strains are found under DD (H(5) = 10.92, p > 0.05).