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INTRODUCTION
According to the World Health Organization, 17% of infectious diseases worldwide are caused by vector-borne microorganisms (OMS, 2017). Among the most important and in need of surveillance, prevention and control are dengue, yellow fever, Zika fever and chikungunya (OMS, 2017). These diseases are caused by arboviruses transmitted by the same vector, Aedes aegypti, a hematophagous dipteran belonging to the family Culicidae primarily associated with urban and suburban areas (Nelson, 1986).
The lack of vaccines for most viruses transmitted by A. aegypti means that efforts are concentrated on the control of vector populations. However, even with vaccine implementation, an integrated approach with vector control research should be pursued to achieve high coverage rates in high risk communities (Espinal et al., 2019). For more than 50 years, the use of chemical insecticides based on organophosphorus compounds has intensified to control A. aegypti larvae and adults (Jayaraj et al., 2016; Soborio et al., 2019). As a consequence, mosquito populations have been subjected to intense selection pressure. In 1999, Aedes populations resistant to organophosphorus compounds were reported throughout the Americas (Rodríguez et al., 2010). The same occurred with the use of pyrethroid compounds (Da-Cunha et al., 2005), with resistance to these insecticides observed in at least 21 Aedes species (Fonseca and Quiñones, 2005). Cross-resistance of these mosquitoes to other insecticides has also been reported (Rodríguez et al., 2003).
Due to an increase in the number of outbreaks of diseases caused by arboviruses transmitted by A. aegypti (OPS, 2017), and the loss of efficacy of chemical insecticides, whose compounds are highly toxic to the environment, non-target organisms and human health (Cárdenas et al., 2005), it is necessary to seek new strategies for the control of A. aegypti.
Biological control is proposed as an alternative to the use of chemical insecticides. One of the most widely used organisms for biological control is the bacterium Bacillus thuringiensis subsp. israeliensis (Bti). The mechanism of action of this microorganism is the production of Cry and Cyt toxins, specific for the larvae of these dipteran species, which cause lysis of the epithelial cells of the intestine, resulting in the death of the insect (Mendoza-Almanza et al., 2020; Ritchie et al., 2010). The active ingredient of commercial formulations of Bti are the toxins Cry4Aa, Cry4Bb, Cry11Aa and Cyt1Aa; these four toxins act together to reduce the occurrence of resistant insect populations under field conditions, However, in the laboratory, A. aegypti strains resistant to one of the Cry toxins has already been reported (Silva-Filha et al., 2021).
Plant extracts obtained from fruits of Ricinus communis, Sapindus saponaria, Melia azedarach, Carica papaya, Annona muricata and Gliricidia sepium species have also been evaluated, and larvicidal activity on A. aegypti has been demonstrated (Bobadilla et al., 2005; Corradine et al., 2014; Gomez, 2015; Prophiro et al., 2008; Rojas et al., 2015; Sharma et al., 1998).
The application of mixtures of compounds with insecticidal activity with different modes of action has been proposed as an alternative to minimize the generation of resistance and to improve the control of immature dipteran stages (Chang et al., 2014). Likewise, the active compounds of mixtures can interact, presenting additive and/or synergistic effects to enhance the insecticidal activity, or antagonistic effects causing a decrease in the efficacy of the compounds compared to the use of each one separately (Lemes et al., 2014).
In view of the above, the study of low-dose mixtures of biological insecticides that minimize the possibility of generating resistance in the populations of target organisms becomes more relevant. In this study, the individual effect of Bti and ethanolic extracts of A. muricata, R. communis and S. saponaria and that of binary combinations on A. aegypti larvae were evaluated in order to determine the most effective antivectorial treatment among them.
MATERIALS AND METHODS
Obtaining ethanolic extracts
We used 500 g of plant material of each species, which were selected for their availability in market places, where they were commercially acquired. This material corresponded to seeds of A. muricata and whole fruits of R. communis and S. saponaria. Each material was dried, crushed and subjected to exhaustive extraction by percolation with 95% ethanol. Subsequently, the extract was filtered and concentrated by distillation under reduced pressure in a rotary evaporator at a controlled temperature of 40 °C. Finally, the material obtained was stored in amber glass bottles for later use (Carrion and Garcia, 2010). The solubility of the plant extracts was evaluated in distilled water and Tween 80 at 0.05% and 0.1%.
Cultivation of Bacillus thuringiensis subsp. israelensis
A colony of Bti strain AM65-52 was seeded in nutrient broth and incubated at 30 ± 2 oC and 150 rpm for 16 h. Subsequently, we made a subculture in 30 ml NSYM (8 g nutrient broth, 0.05%, yeast extract, 5 x 10-5 M MnCl2, 1 x 10-3 M MgCl2,7 x 10-4 M CaCl2) which was incubated at 30 °C without shaking for seven days. 1 ml of culture was taken, centrifuged at 12,000 rpm for 5 minutes, and 1 ml of saline solution (0.85% NaCl) was added and centrifuged again under the same conditions. Subsequently, the supernatant was discarded and the cells were dried at 50 °C for 48 h to determine the dry weight.
Aedes aegypti colony maintenance
The larvae of A. aegypti were obtained from the insectary of the Department of Basic Sciences at La Salle University. The colony was maintained at 25-30 °C, 38-50% relative humidity and 12 h day:12 h night photoperiod. The larvae were placed in plastic containers with rested water and fed with pulverized purine.
Toxicity bioassays
For the determination of the individual larvicidal activity of Bti and plant extracts, five concentrations of each were evaluated as follows: 1) Bti strain AM65-52: 33 x 10-3 mg/ml, 33 x 10-4 mg/ml, 33 x 10-5 mg/ml, 33 x 10-6 mg/ml and 33 x 10-7 mg/ml, 2) A. muricata: 0.001 mg/ml, 0.005 mg/ml, 0.01 mg/ml, 0.1 mg/ml and 0.5 mg/ml, and 3) R. communis and S. saponaria: 0.05 mg/ml, 0.1 mg/ml, 0.8 mg/ml, 1.5 mg/ml and 3 mg/ml. For the binary mixtures, combinations of those concentrations that presented larval mortality values between 30% and 60% for each compound applied individually were used.
To rule out the larvicidal activity of the emulsifier, Tween 80 was added at 0.05% and 0.1% in containers with 100 ml of distilled water and 20 third instar larvae of A. aegypti. Distilled water was used as a negative control.
Three bioassays (replicates) were performed and three experimental units per treatment and concentration were used in each replicate. The experimental units consisted of autoclaved beakers, each containing 20 third instar A. aegypti larvae and a final volume of 100 ml of distilled water and each extract, Bti or a binary mixture. To make the mixtures, concentrations of both Bti and the three plant extracts were selected that were lower than the lethal concentration 50 (LC50) in order to evaluate whether the interaction between these components was synergistic, additive or antagonistic.
In each bioassay, three replicates were carried out for the controls, which were distilled water or the solvent used for each plant extract. The experimental units were randomly distributed in a room with controlled temperature at 25 °C and photoperiod of 12 h light and 12 h dark. At 24 and 48 h, larval mortality was recorded, considering that a larva was dead if it did not move when touched with a wooden stick.
Statistical analysis
The determination of the CL50 was performed from the number of live and dead larvae in each of the trials by using the TRAP program version 3.1a. Analysis of compound interactions in the mixtures was performed using the Independent Action Hypothesis, in which it is assumed that the biocontrollers in each mixture acts independently and, therefore, each has its own probability of causing larval death (Drescher and Boedeker, 1995; Lemes et al., 2014). The Independent Action Hypothesis was calculated according to the formula used by Lemes et al. (2014):
P1 and P2 represent the proportions of dead larvae for compounds 1 and 2 of the mixtures (Bti and plant extract). The difference between expected and observed mortality was estimated by means of the R program (R Core Team, 2020) version 3.6, based on Pearson's distribution test (chi2) with a significance level of p<0.05.
RESULTS
The ethanolic extract of S. saponaria fruit was soluble in distilled water, while the suitable solvent for the extract of A. muricata seeds was 0.05% Tween 80, and 0.1% Tween 80 for the extract of R. communis fruit. Tween 80 in both concentrations evaluated did not cause mortality in larvae.
The results obtained in the evaluation of the larvicidal activity of all the compounds individually demonstrate their effectiveness for the control of A. aegypti larvae (Table 1). The highest toxicity values were presented by Bti, followed by ethanolic extracts of A. muricata, S. saponaria and R. communis (Table 1).
Table 1 Mean lethal concentration (LC50) of B. thuringiensis subsp. israelensis and ethanolic extracts on A. aegypti larvae at 48 hours of exposure.
| Treatment | LC50 (mg/ml) | Confidence interval (mg/ml) |
|---|---|---|
| B. thuringiensis subsp. israelensis | 0.002 | 0.0005-0.0778 |
| A. muricata | 0.01 | 0.01-0.02 |
| R. communis | 0.5 | 0.45-1.58 |
| S. saponaria | 0.12 | 0.11-0.13 |
In the bioassays conducted with the mixture of Bti and the ethanolic extract of A. muricata seeds, higher A. aegypti larval mortality was observed when only the bacteria or only the plant extract was added (Table 2); when comparing the mortality observed with that expected with this combination, no statistical significance was observed (p>0.05), which indicates that in the binary mixture the compounds act independently.
Table 2 Interaction of mixtures of plant extracts and B. thuringiensis subsp. israeliensis for the control of A. aegypti larvae.
| Treatment | Concentration (mg/ml) | Mortality | Mortality | χ2 | p |
|---|---|---|---|---|---|
| expected (%) | observed (%) | ||||
| B. thuringiensis subsp. Israeliensis | 33 x 10-4 | - | 43 | - | - |
| A. muricata | 0.001 | - | 35 | - | - |
| R. communis | 0.1 | - | 43 | - | - |
| S. saponaria | 0.1 | - | 60 | - | - |
| Bti + A. muricata | 33 x 10-4 + 0.001 | 52.3 | 55 | 0.28 | 0.59 |
| Bti + R. communis | 33 x 10-4 + 0.1 | 43.8 | 8.3 | 51.14 | 8.6 x 10-13 |
| Bti + S. saponaria | 33 x 10-4 + 0.1 | 18.3 | 57.75 | 3 x 10-14 |
The results obtained from the mixtures of B. thuringiensis with R. communis and the bacterium with S. saponaria showed a significant difference between the observed and expected mortality percentages (with a p<0.05). However, the observed mortality was lower than expected, indicating the presence of an antagonistic interaction between the compounds (Table 2).
DISCUSSION
In this work, the larvicidal activity of B. thuringiensis subsp. israelensis and ethanolic extracts of A. muricata, R. communis and S. saponaria on a laboratory strain of A. aegypti was evaluated. In individual larvicidal activity assays, the LC50 obtained in this study indicate that the extracts were more effective for larval control than previously reported. Parra et al. (2007) obtained a LC50 of 0.9 mg/mL with A. muricata seed extract and a LC50 of 0.86 mg/mL with R. communis fruit extract. Bobadilla et al. (2005) reported a LC50 of 0.02 mg/mL with A. muricata seed extract, which is very close to that found in the present investigation. On the other hand, authors such as Amariles et al. (2013) obtained a CL50 of 0.5 mg/mL, with S. saponaria fruit extracts.
The LC50 observed in this study with Bti AM65-52, is similar to the LC50 of 0.004 mg/mL obtained with Bti strain BtMA-750 isolated from a Brazilian soil (Vieira-Neta et al., 2021) and lower than the LC50 of 0.0068 mg/mL and 0.018 mg/mL reported with the spore and crystal preparation of Bti LBIT-1250L and Bti IPS-82 (Garcia et al., 2021).
Considering the LC50 values obtained in this study, the three plant extracts and Bti strain AM65-52 are considered promising for the biological control of A. aegypti larvae. However, those with the highest individual larvicidal activity were the bacterium, followed by the ethanolic extract of A. muricata seeds, and therefore would be recommended for further studies with larvae collected in the field or under semi-controlled conditions.
The mixture of Bti and A. muricata seed extract did not present a statistical difference between observed and expected mortality, indicating that each biocontroller acts independently and the two present different sites or mechanisms of action (Abendroth et al., 2011; Drescher and Boedeker, 1995); this type of effect has also been called zero interaction or additive (Abendroth et al., 2011). It would be interesting to conduct additional studies in which the application of this binary mixture in a single formulation is analyzed, which could be advantageous because each one could be at sublethal concentrations and also could facilitate its application in the field, by using the two biocontrollers in a single product.
In addition to this, the use of the mixture between the AM65-52 strain of Bti and the ethanolic extract of A. muricata would be favorable, because the bacteria and the extract present different mechanisms of action. Bacillus thuringiensis subsp. israelensis presents the toxins Cry4Aa, Cry4Ba, Cry11Aa and Cyt1Aa, the first three toxins bind to membrane receptors of larval gut cells, while Cyt1Aa presents affinity for membrane unsaturated phospholipids (Ben, 2014). The most biologically active molecules of Annona plants in mosquito larvae are the bistetrahydrofuran and monotetrahydrofuran acetogenins, specifically muricatacin and anonnacin present in the seeds of A. muricata (Rieser et al., 1991; Rupprecht et al. 1990).
The antagonism observed in mixtures of B. thuringiensis with R. communis and S. saponaria has also been reported for other mixtures by authors such as Murugan et al. (2002), who found this interaction in mixtures of Bti and Pongamia pinnata oils for the control of Culex quinquefasciatus larvae. Another example is the results obtained by Chang et al. (2014), who also observed antagonism in mixtures of cinnamaldehydes with eugenol against larvae of Anopheles sinensis, as well as in mixtures of cinnamaldehydes with anethole and eugenol with anethole against larvae of A. albopictus and An. sinensis, the components of the mixtures being the main constituents of essential oils of Cinnamomum cassia, Syzygium aromaticum and Illicium verum.
To the best of our knowledge, the mechanism that causes the antagonistic effect observed in binary mixtures of Bti with R. communis and S. saponaria has not been reported, neither in antagonistic results obtained by Murugan et al. (2002), nor by Lemes et al. (2014). A possible explanation for the observed antagonism is that a molecule of the extracts interacts with a toxin of the bacterium, affecting the mode of action of one of these compounds or that they bind to the same receptor in the vector's gut. Such hypotheses will need to be tested with future experiments.
In this work, it was observed that B. thuringiensis subsp. israelensis presents differences in its interaction with ethanolic extracts of A. muricata, R. communis and S. saponaria. Similarly, Lozano and Dussán (2017) reported that the mixture between Lysinibacillus sphaericus S-layer protein and binary toxin can be additive or synergistic against C. quinquefasciatus larvae, depending on the strain from which these proteins were obtained. It was also observed that there is an additive or antagonistic effect between Beauveria bassiana and B. thuringiensis for the control of Bemisia tabaci nymphs, depending on the concentration of each biocontrol agent (Somoza-Vargas et al., 2018).
Since ethanolic extracts were evaluated in the bioassays conducted in this study, an alternative that could improve the larvicidal activity of mixtures of these extracts with Bti is the fractionation of these extracts or the direct purification of the metabolites responsible for the larvicidal activity. Some authors such as Chang et al. (2014), performed mixtures between B. thuringiensis and the main constituents of three essential oils for the control of An. sinensis and A. albopictus, obtaining a synergistic effect in the three mixtures. Similarly, Murugan et al. (2002), reported a synergistic effect between B. thuringiensis and Azadirachta indica oil, on larvae of C. quinquefasciatus. Another alternative would be the evaluation of the sequential application of Bti with the extracts evaluated in this study.
In conclusion, the mixture between the ethanolic extract of seeds of A. muricata and B. thuringiensis subsp. israelensis, presented an independent effect between the compounds, allowing it to be considered viable for the control of A. aegypti larvae, since it is composed of two different active principles. On the other hand, the mixtures made from ethanolic extracts of R. communis and S. saponaria combined with Bti produced an antagonistic effect between them. Therefore, it would be advisable to apply these extracts independently or sequentially. Likewise, it is important to continue conducting studies focused on the analysis of interactions between plant extracts and microorganisms for the control of mosquito vectors of pathogenic microorganisms, in order to obtain the most appropriate formulations for resistance management and, in the future, to develop new products that generate minimum toxicity to non-target organisms and the environment.
