Susceptibility of Drosophila suzukii larvae to the combined administration of the entomopathogens Bacillus thuringiensis and Steinernema carpocapsae

Abstract

Non-native pests are often responsible for serious crop damage. Since Drosophila suzukii has invaded North America and Europe, the global production of soft, thin-skinned fruits has suffered severe losses. The control of this dipteran by pesticides, although commonly used, is not recommended because of the negative impact on the environment and human health. A possible alternative is the use of bio-insecticides, including Bacillus thuringiensis and entomopathogenic nematodes, such as Steinernema carpocapsae. These biological control agents have a fair effectiveness when used individually on D. suzukii, but both have limits related to different environmental, methodological, and physiological factors. In this work, we tested various concentrations of B. thuringiensis and S. carpocapsae to evaluate their efficacy on D. suzukii larvae, when administered individually or in combination by using agar traps. In the combined trials, we added the nematodes after 16 h or concurrently to the bacteria, and assessed larvae lethality from 16 to 48 h. The assays demonstrated a higher efficacy of the combined administration, both time-shifted and concurrent; the obtained data also showed a relevant decrease of the time needed to kill the larvae. Particularly, the maximum mortality rate, corresponding to 79% already at 16 h, was observed with the highest concentrations (0.564 µg/mL of B. thuringiensis and 8 × 10² IJs of S. carpocapsae) in the concurrent trials. This study, conducted by laboratory tests under controlled conditions, is a good starting point to develop a further application step through field studies for the control of D. suzukii.

Figure 1

Effects of various concentrations of B. thuringiensis (Bt) administered to L1 stage of D. suzukii larvae. Results were recorded 24 and 48 h post-treatment. A relevant mortality was observed, at 24 h, with the highest concentration (1.128 μg/mL), and at 48 h, also with 0.564 μg/mL. The trend in the graphs clearly indicates that the efficiency of the bacteria is dependent on the administered concentration. C control.

Figure 2

Bt lethal concentrations (LC₅₀ and LC₉₀), at 24 h (a) and 48 h (b), were calculated by Probit analysis with 95% confidence intervals.

Figure 3

Effects of various amounts of S. carpocapsae (Sc) administered to L1 stage of D. suzukii larvae. Mortality rates were recorded at 24 and 48 h after administration. A concentration-dependent efficacy was observed, though relevant only with the highest number of Sc (1.6 × 10³ IJs), at both 24 and 48 h. C control.

Figure 4

Sc lethal concentrations (LC₅₀ and LC₉₀), at 24 h (a) and 48 h (b), were calculated by Probit analysis with 95% confidence intervals.

Figure 5

Evaluation of the potential adverse effects of Bt (0.564 μg/mL) on Sc (5 × 10, 4 × 10² and 8 × 10² IJs) viability at 24 h (a) and 48 h (b). The mortality of Sc in all assays did not exceed about 3% (24 h) and 5% (48 h) and was comparable to the controls with Sc alone. These assays were carried out before the combined administration of the two bio-insecticides. C: control, Sc alone; T: treatment, Sc plus Bt.

Figure 6

Mortality of D. suzukii after time-shifted administration of Bt at t₀, plus Sc after 16 h. Graphs from (a) to (d) show the effects of different concentrations of the two entomopathogens at 16, 24, 32 and 48 h. Trials show an improvement in the effectiveness of the combination that mainly depends on the increase in Bt concentration. However, all results show a clear enhancement of the efficacy compared to assays with single administration of entomopathogens (Figs. 1 and 3). C control.

Figure 7

Simultaneous administration of Bt and Sc to D. suzukii larvae. Both Bt and Sc were added to the agar traps at t_0, and larvae mortality was recorded at 16, 24, 32, 48 h. The greater effectiveness of this treatment is clear compared to both individual (Figs. 1 and 3) and time-shifted (Fig. 6) administrations. In these assays, either the increase in Bt or Sc concentrations has a positive effect on the efficacy of the treatment, and, except for the lowest combination (a), a reduction in the time lapse between administration and mortality was observed. With the highest combination (d), a relevant mortality rate, about 80%, was already observed 16 h after the start of the trials and reached 100% at 48 h. C control.

Figure 8

Cross assessment of the results obtained in single and combined administrations at 24 and 48 h. The graphs allow a direct comparison of the data obtained in the different trials showing the general improvement in the effectiveness of bio-insecticides when used in combination against D. suzukii larvae. A significant increase in mortality induced by the concurrent administration of Bt and Sc is evident. Bt concentration is expressed in μg/mL, Sc administration is expressed as total number of IJs.

Figure 9

A possible model of the effects induced by the combined administration of B. thuringiensis (Bt) and S. carpocapsae (EPN) to D. suzukii larvae. The presence of B. thuringiensis, ingested during feeding by D. suzukii larvae, could facilitate and speed up the passage of S. carpocapsae from the intestine to the hemocoelic cavity. Bt toxins, produced as parasporal inclusions, are activated in the intestinal lumen of the larva. Active toxins interact with the membrane receptors of the intestinal epithelium and are responsible for the formation of pores that alter the physiology of the cells leading to their lysis. The resulting epithelial lesions could provide an easy access route for EPN to other body regions of the larvae.