Introduced through human activities, such as travels and transcontinental trade, and supported by climate change, several invasive species can settle in habitats where formerly they could not survive. The presence of allochthonous species can negatively affect native species reducing biodiversity, represent a threat to human health and, in the case of phytophagous insects, cause serious damage to the agroeconomic system29. Characterized by rapid spread and enormous impact, an invasion like that of D. suzukii has few precedents. This species is thus becoming a model of study in the biology of alien species and for the development of pest management techniques30.
The protection of crops from D. suzukii invasion is mainly conducted by means of insecticides31 which, as known, are unselective, in many cases remain in the environment and, if persistent on fruit, are harmful to human and animal health32. In addition, their efficiency can be reduced by weather events such as rainfall, and their prolonged use may induce resistance phenomena in target insects. Moreover, the preference of D. suzukii for ripening fruit requires that any treatment be carried out close to the harvesting, which inevitably means that insecticide residues can remain on the marketed fruit33. Alternatively to synthetic chemicals, natural compounds that can act as repellents, toxicants by contact or ingestion, and overall deterrents, have been tested mainly against D. suzukii adults34. All these control procedures have positive aspects, such as low cost, sometimes good effectiveness, and ease of use for farmers, but, as mentioned, their massive use affects environment and organisms at various levels. Considering the limits of the chemical control, several studies have addressed the problem of D. suzukii management through the development and improvement of biological control methods. However, given the recent introduction of this insect in Europe and America specific projects in these areas are still limited. Until now, only the main entomopathogens commonly used in biological control, have been tested against D. suzukii35.
Several serovars of Bt were tested at different concentrations on D. suzukii. Cossentine et al.27 described the results of the assays with 22 serotypes of Bt, and among them, only few are effective against the target dipteran. In particular, the serovars thuringiensis, kurstaki, thompsoni, pakistani, and bolivia show an effectiveness of more than 75% on first stage larvae. The mortality rates, however, are related to a dietary administration of at least 108 spores/mL, below this concentration the insecticidal activity is not relevant. Moreover, the efficacy on adult individuals is extremely low, only the serovar thuringiensis induces a mortality of about 44%. In the literature, data on the low effects of serovar israelensis on this fly are also reported36. The data obtained from our trials, carried out with the serovar kurstaki, agree with Cossentine et al.27, demonstrating that only high concentrations of the bacterium produce significant effects on larvae in a relatively short time (24–48 h).
As regards the insecticidal activity of EPN, the literature includes several studies carried out in laboratory or field with applications on plants and soil. The data collected using different species of nematodes, such as S. carpocapsae (Sc), S. feltiae, S. kraussei, H. bacteriophora25,37,38,39 and the rare species Oscheius onirici40, are quite promising but, as observed for Bt, also for EPN high concentrations are required, and their effectiveness is very variable between laboratory and field studies. In accordance with previous results29, our current data indicate a good efficacy of Sc: treatments with an amount of 1.6 × 103 IJs (corresponding to 80 IJ/cm2) resulted in a larvae mortality above 98%, 48 h post-treatment.
Since the reproduction of D. suzukii occurs by spawning in the mesocarp of the fruit, the technical limit of the application of bio-insecticides such as Bt or EPN cannot be disregarded. Larvae development is protected by the fruit pulp and this may be one of the causes of the low effectiveness of Bt in field applications. As known, to be effective, Bt must be ingested and should therefore reach the larva by penetrating inside the fruit; perhaps a timely spraying of the crops before oviposition could theoretically promote the penetration of microorganisms thanks to the drive of ovipositor. Based on these considerations, it is reasonable to assume that coupling Bt with EPN, which, being motile, can actively reach the targets, could significantly improve the effectiveness of D. suzukii control methods in the field.
The simultaneous application of two control agents, with different modes of action, may result in additive or complementary effects. In particular, the tissue damage inflicted by Bt to the intestinal epithelium could facilitate the access of nematodes in the passage from the gut to the hemocoel (Fig. 9). When in the hemolymph, the nematode and its symbionts implement strategies of immunoevasion and immunodepression of host immune responses4,41,42, leading to a drastic and more rapid physiological alteration that results in the death of the target insect in a shorter time.
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.
As previously reported, and supported by literature, it is desirable that studies on biological control of D. suzukii will be increased and possibly new strategies for the use of bio-insecticides will be tested. In this perspective, we started a project aimed at assessing whether the association of entomopathogenic organisms and microorganisms is a promising strategy, capable of making safer and faster measures to control the diffusion of insect pests. Besides this, a need to reduce Bt concentrations used in field application has arisen from recent observations on the possible insecticidal effect on non-target insect populations, caused by intensive use and consequent bioaccumulation of the bacterium in crop applications43. It is also known that high concentrations of Bt serovars which produce the cry1Ba1 and 1 beta-exotoxin toxins, are toxic not only to several dipterans but also to mammals44,45.
Even when using EPN, limitations have been highlighted: such as with Bt, they are susceptible to runoff in case of rainfall, have a significant sensitivity to drying at high temperatures, and, depending on the species, are more or less effective in certain environmental conditions as well as susceptible to the presence of pesticides46,47.
On the basis of these considerations and the relevant literature, and after excluding adverse effects on the nematode induced by the presence of Bt, we conducted the assays with combinations of Bt and Sc, administering them both simultaneously and time-shifted. According to our data, the mortality of D. suzukii larvae increases from < 45%, in single administration of Bt or Sc, to 65.8%, in the case of combined time-shifted administration (Bt: 0.564 μg/mL and Sc: 8 × 102 IJs) at 24 h. These effects are even more significant in the case of trials carried out by simultaneous administration; in this case the mortality within 24 h rises to 91.7%. At 48 h post-treatment, the data show a lethality that rises to 78.3% using the lowest concentrations of the two entomopathogens (Bt: 0.282 μg/mL and Sc: 4 × 102 IJs), until reaching the total mortality of the larvae with the highest concentrations (Bt: 0.564 μg/mL and Sc: 8 × 102 IJs).
The data at 24 h indicate a clear concentration-dependence of Bt, which at the highest concentrations is extremely effective in all the combined tests. Sc seems to be mainly influenced by the incubation time with the larvae. This could be explained by the different mechanism of action and intake of the two bio-insecticides: Bt is ingested rapidly by the larva during feeding, while Sc must actively seek and penetrate the host; thus, both time and concentration could contribute to a greater effectiveness of the nematodes. The administration of Bt at t0 in both combined trials is justified by the specific action at the intestinal level of this bacterial strain48 since the damage and possible lesions at the level of the intestinal epithelium can promote the rapid passage of Sc with its symbionts to the hemocoelic cavity (Fig. 9). Moreover, the altered physiology of the larva, induced by Bt, makes the insect more permissive to the action of the nematode. Conversely, anticipating the administration of Sc would not support the action of Bt, as the debilitated larvae may not actively feed, therefore not ingesting an enough number of bacteria.
Our data agree with the literature describing the increased efficacy of the use of entomopathogen combinations. Bt plus EPN combination was used against other insect species, such as the Coleoptera Cyclocephala hirta and Cyclocephala pasadenae49, the Lepidoptera Spodoptera exigua, Autographa gamma and Plutella xylostella50,51, and the Diptera Tipula paludosa52 and Culex pipiens53.
The results of the application of different combined formulations, such as entomopathogens with pesticides, or with products of natural origin, suggest that their concurrent effects improve the effectiveness compared to single uses54,55,56. Also, the combination of entomopathogens with pesticides could reduce the needed amount of the chosen pesticide. This kind of trials always requires checking for possible adverse effects of the chemical on the viability of organisms or microorganisms coupled in the formulation and during the administration57,58,59.
Our results obtained by the simultaneous administration of Bt and Sc to D. suzukii larvae show a significant increase in the efficacy and a substantial reduction in the time of killing of the larvae. A reduction of the time lapse between administration and effects is particularly important for the control of D. suzukii: since the larvae of this fly prefer fruit close to ripening, so just before harvesting and sale, the speed of neutralization is essential to avoid excessive damage to the harvest that would affect its marketing. Moreover, the combined use of Bt and Sc may allow to reduce the amount of both the bio-insecticides applied in the field, limiting possible ecological issues and maybe improving the cost–benefit ratio.
The trials performed in the laboratory are obviously not transposable in an immediate intervention in the field but provide an essential basis for the possible improvement of methods and technologies that can be used in the field. This is particularly stimulating when the test results, which prelude possible trials on crops, are unequivocally encouraging. However, it is important to consider that the variability of the protocols used by the investigators in laboratory, semi-field and field tests, and the consequent lack of standardized protocols in the experimentation, often make difficult a correct data comparison. Besides this, the great individual variability in the physiology of the organisms used, as both bio-insecticides and target organisms, should be accounted for.
To our knowledge, this is the first work carried out on D. suzukii that assesses the possible combination of B. thuringiensis and S. carpocapsae. The data obtained represent a good starting point to improve the methods of control of the spread of this pest and can stimulate further studies aimed at an extensive screening of the possible combined administrations, fundamental for a future conscious and safe use of biological control methods.
Source: Ecology - nature.com
