Efficacy of 2 Commercial Formulations of Bacillus thuringiensis H-14 in Larvae of Anopheles albimanus W and Aedes aegypti L (Diptera: culicidae).

The bacterial genus and species of Bacillus thuringiensis var israelensis (Bti), is entomotoxic, used in the biological control of mosquito vectors of human diseases, such as malaria and dengue. Following the studies carried out in Mexico and Guatemala with ovillantas, in which the presence of larvae of both species of Anopheles albimanus W and Aedes aegypti L were observed, and with the intention of continuing to develop an ecologically friendly mosquito control, Bti was added to the ovillantas, to improve and already efficient method on the elimination of both types of mosquitoes. The objectives of this work were: to analyze the effectiveness of two commercial formulations of Bti, serovar H-14 (Bactimos) wettable powder, 3,500 ITU, from Biochem prods., and Vectobac, 2,000 ITU wettable powder, (Abbot Lab.) on second and third instars larvae of A. albimanus and Ae. aegypti (Diptera Culicidae). The two formulations were effective against A. albimanus W(higher concentrations), while Ae. aegypti L was very susceptible to Bti, therefore it is proposed for the best control of these genus and vector species of malaria and dengue at adequate concentrations.

permethrin and deltamethrin (15). Since 1970, the 23rd. World Health Assembly, due to the high toxicity, low selectivity of chemical pesticides and the increase resistance of the species, recommended to use new methods of vector control, such as biological (16). In this sense, vector control is no longer based only on applying chemical agents, for the above environmental reasons; a study with ovillantas in 2017 showed that the decrease in Aedes spp can be due to the implementation of a simple ovillanta (with no chemicals) and the active participation of the community in the affected area (17). In this study, according to preliminary calculations, the systematic elimination of larvae and eggs in the application area, caused a reduction of up to 77% of Aedes spp. The continued use of ovillantas in other sites on the Pacific coast of Mexico has led to the identification of other problems associated with community participation; in Troncones, Mexico a two-year (May 2016-April 2018) study on the use of the ovillantas, showed that in the high season of the second year, the amount of eggs and larvae of Aedes spp collected from the ovillantas did not rise to the level of the previous year, which indicated that the vector mosquito was effectively reduced in the region( Figure. 1) (18). This demonstrated that the constant destruction of the eggs and larvae of Aedes species in the region, induced an evident decrease in the number of eggs surviving from previous years (19,20,21). In Figure 2, it is observed that in July 2016 49,600 eggs were collected in the evaluated area, a quantity that decreased during the low season of mosquito reproduction. In July 2017, the same increase in the capture of Aedes spp eggs was not recorded, since only 9,200 eggs were detected in that period of the high season, which indicated that the existence of Aedes spp was considerably reduced compared to the previous year. However, in the absence of eggs and larvae of Aedes spp, larvae, other genera and species of vector mosquitoes such as Anopheles spp were found and identified in the ovillantas, therefore, it is necessary to find an effective way to control the different species of larvae born in the ovillantas, regardless of the gender and species of mosquito. surface of the water. Although the complete identification of the larvae was not carried out, it was assumed that they belonged to the species A. albimanus or A. pseudopuntipennis, known to exist in the state of Guerrero. Under this observation and with the aim to improving the efficiency of the ovillantas in the destruction of larvae of different species of mosquitoes, a quarter of an imported MosquitoDunks (Summit Chemical Co. Baltimore, USA) donut was installed inside the ovillantas, as means to obtain biological control of these vector mosquitoes. Surprisingly, the MosquitoDunks label does not state the variety of B. thuringiensis (Bt) used in its manufacture. At the end of several weeks of using Bt in the ovillantas, it was observed that in some of the ovillantas there were only dead larvae, while in others, there were living larvae. Our first though was that the concentration of Bt was different among the ovillantas, but it also was observed that the dead larvae were of the species Aedes, while the ones that survived were of the species Anopheles. These results made it necessary to carry out a study on commercial formulations of Bti to determine the minimum concentration necessary to achieve the maximum adequate larval control of all genera and species of vector mosquitoes arriving to the ovillantas. Based on the above, the objective of this work was to analyze the effectiveness of commercial Bti products for the larval control of vector mosquitoes of the genera and species of Aedes and Anopheles. Adult mosquitoes are generally fed with a 10% sugar solution in cotton balls previously sterilized with alcohol, or with a solution of corn syrup, sugar syrup or fresh cut fruit (grapes) or dried raisins.

Larvae of Anopheles albimanus W.
For the species A. Albimanus W, a strain collected in El Gancho, municipality of Suchiate, Chiapas, Mexico was used. The birth of larvae is monitored daily and transferred to different containers marked with the day. First instar larvae were fed with powdered fish, second to fourth instar larvae were fed with a mixture of vegetable protein (incaparin) or with vegetable rabbit food powder (Chowchow, Purina). For the present study, a certain number of second and third instar larvae of A. albimanus were transferred to pewter containers and fed exclusively with an incaparin solution.

Larvae of Aedes aegypti L
To create the Ae. aegypti colony, fourth instar larvae were initially collected from vases in the municipal cemetery of Tapachula, Chiapas, Mexico. The collected larvae were subjected to the temperature and humidity conditions described above for the CIP insectary. The larvae were placed in pewter trays inside collapsible cages and fed with incaparin until they pupated. Emerging adult females have been kept for several generations, fed rabbit blood for reproduction (under strict CIP rules of ethics on animal care) and fed corn syrup and raisins. For the present study, a certain number of second and third instar Ae. aegypti larvae were transferred to pewter containers and fed with an incaparin solution.

Formulations used for this study
This study aimed to determine the larvicidal activity of two commercial products available in Mexico, Bactimos (~ 3,000 UTI / mg) and Vectobac (~ 3,000 UTI / mg). Both products are made with

Preparation of bioassays
Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 28 October 2020 For each bioassay, a modified technique was used based on the models reported by Georghiou (28) and Van Essen (29). In each experiment 20 larvae of the desired age were placed in 10 ml of distilled water in a plastic cup with a total volume of 50 ml. In a second 150 ml wax paper container, 90 ml of distilled water and enough Bti (by weight) of the studied formulation were added to give the desired concentration in 100 ml of total solution. The larvae and 10 ml of solution were transferred with a rubber bulb pipet, to have at the end a total volume of 100 ml.      Each solution was stored for a maximum of 5 days at 5 ° C and was preheated before use in a water bath until reaching 28-29 ° C with cyclical stirring without water. All bioassays were carried out within an atmosphere of 70-75% relative humidity. A small amount of incaparin was added to each bioassay as food for the larvae. were close and are shown in Table 5.

Results
In no experiment was larval death observed in the absence (controls) of Bti-based biolarvicide To establish the normality of the mosquito population, Chi-square tests (2) and regression analysis were obtained for each experiment. A statistical analysis with a significant difference of 2 may indicate that the population of larvae did not respond independently, compared to the control, to the conditions of the experiment, or that the regression line between Probit vs log (dose) does not adequately describe the relationship dose-mortality of these experiments.

Discussion
The application of Bti as a larval biological control against vector mosquitoes of different genera and species is effective due to the selective toxicity for insects classified as: Diptera (34,35) The toxicity of Bti has been reported against a wide diversity of genera and mosquito species, with multiple formulations available in the international market (36,37). In this case, two commercial products were obtained in Mexico, Bactimos WG and Vectobac WDG, both recommended as biological larvicides for the control of mosquito vectors of human infectious diseases (38,39). Using lower but with a significant statistical difference, it was registered that both products showed satisfactory results against this genus and species of mosquito according to with the type of toxin that specifically damages the mosquito's midgut (25,26,34). The similarities that exists between the lethal concentrations (CL) registered in this site, with respect to other parts of the world, support the assumption for this genus and species (42). In addition, the similarity that exists between the CL of States of America, for example; the fight against the mosquito is done because they are annoying and represent a potential danger to the health of man and breeding animals; these are mosquitoes without diseases, therefore, the fight is not anti-vector but anti-mosquito. The resources for the control they apply are at the level of socioeconomic development and therefore the fight must be a complete integrated control. (26,41) The Vectobac formulation was more effective against Ae. aegypti and A. albimanus than Bactimos. For Ae. Aegypti, Vectobac is more recommended than Bactimos due to its efficacy, but with respect to A. albimanus this genus and species was totally resistant (27) at low concentrations of either formulation. So, it is recommended that in these cases, for practical purposes, insist on testing the susceptibility to biopesticides of mosquito vectors according to each zone(24,34)5.

Conclusions
Given the increase in resistance to pesticides, at the larval level (44) or in the adult stage (45), of the different disease vector species, several formulations of biopesticides have been developed in recent years (46). The type of formulations and support in these biopesticides is of utmost importance, both for their proper implementation and for their application in an economical and sustainable way.
Here we have shown that both formulations available in Mexico work against the larvae of both mosquitoes, but Vectobac works at a lower concentration than Bactimos against the larvae of Aedes spp.
Supplementary Materials: The following are available online at www.mdpi.com/xxx/s1,