Increased Attraction and Stability of Beauveria bassiana-Formulated Microgranules for Aedes aegypti Biocontrol

Aedes aegypti (Linn.) incidence has increased in recent years, causing human viral diseases such as dengue, which are often fatal. Beauveria bassiana (Bals., Vuillemin) efficacy for Ae. aegypti biological control has been evidenced but it relies on host susceptibility and strain virulence. We hypothesized that B. bassiana conidia microgranular formulations (MGF) with the additives acetone, lactic acid, and sugar increase Ae. aegypti adult exposure, thus improving their biocontrol effectiveness. Beauveria bassiana strain four (BBPTG4) conidia stability was assessed after 0 d, 5 d, and 30 d storage at 25 °C ± 2 °C with additives or in MGF after 91 d of storage at 25 °C ± 2 °C or 4 °C ± 1 °C, whereas mortality was evaluated after adult exposure to MGF + conidia, using home-made traps. Additives did not show toxicity to conidia. In addition, we observed that sugar in MGF increased Ae. aegypti adults’ attraction and their viability resulted in a 3-fold reduction after 5 d and 1- to 4-fold decrease after 30 d of storage, and formulations were less attractive (p < 0.05). Conidia stability was higher on MGF regardless of the storage temperature, losing up to 2.5-fold viability after 91 d. In conclusion, BBPTG4 infected and killed Ae. aegypti, whereas MGF attracting adults resulted in 42.2% mortality, increasing fungus auto dissemination potential among infected surviving adults. It is necessary to further evaluate MGF against Ae. aegypti in the field.


Introduction
Aedes aegypti populations and prevalence have recently increased. This mosquito is the vector agent of several arboviral diseases, including dengue, which is currently present in 128 countries [1,2]. In addition, zika virus has been reported in the Americas and the Pacific, causing outbreaks, and threatening public health due to its association with neurological complications [3]. It has been estimated that half of the world population has been exposed to diseases transmitted by this mosquito vector [4].
Ae. aegypti control includes integrated vector management, vector surveillance based on health information systems, and emergency preparedness. Unfortunately, evaluating these control components in Latin America and the Caribbean has proven to be unsuccessful, because they have not been properly addressed [5]. Insects' development of resistance by frequent exposure to chemical insecticides reduces arbovirus control [6], which leads to an increasing interest in other management tools for this dengue vector, including entomopathogenic fungi (EPF) application [7]. Selected Beauveria bassiana (Bals.) Vuillemin strains infect and kill adult mosquitos by direct exposure or by horizontal dissemination via copula [8].
After application, fungi-based bioinsecticides are threatened by environmental conditions, such as high temperature and direct solar radiation. Low humidity is also a limiting

Mosquito Source and Rearing Conditions
Ae. aegypti strain was provided by the Laboratorio de Entomología of Facultad de Ciencias Biológicas at Universidad Autónoma de Nuevo León, México. Ae. aegypti colony was kept inside of an insect breeding room at 25 • C ± 2 • C and 80% RH, following the protocol described in the Guide for the Installation and Maintenance of Aedes aegypti Linn. (Diptera: Culicidae) insectary (http://www.cenaprece.salud.gob.mx/programas/interior/ vectores/descargas/pdf/GuiaInstalacionMantenimientoInsectario.pdf) (website accessed on 5 June 2022) from the Ministry of Health of Mexico. Adults were kept in a 38.1 cm width × 60.0 cm height pop-up butterfly cage (Carolina Biological Supply Company, Burlington, NC, USA), placed inside an insect rearing room at 25 • C ± 2 • C, 60% ± 10% RH, and 14 h light: 10 h darkness photoperiod. Mosquitoes were fed on 5% sugar solutionsoaked cotton bolls in a 20 mL plastic cup, placed near a cage corner. Sugar solution was replenished daily using a 3 mL syringe, whereas females were also blood-fed by a human arm, following the Ministry of Health of México's protocol. For oviposition, 2 L plastic cylindrical containers with 700 mL of tap water and 0.5 g of fish flakes (Wardley ® , Grupo Acuático Lomas, S.A. de C.V., Cuajimalpa, México) were placed inside the adult cage. After emerged larvae was observed, the container top was covered with muslin mesh and the oviposition container was replaced for a new one. Larvae from neonate to fifth instar were fed with fish flakes. Pupae were transferred to cages for adult emergence and this cycle was repeated. The emerged 5-to 8-day-old Ae. aegypti adults were changed to a different release cage for bioassays. About 20 males and 20 females were kept untested for the maintenance of the mosquito colony under the rearing conditions described above [31].

B. bassiana Culture and Mass Production
Beauveria bassiana strain four (BBPTG4) (Genbank: KC759730), originally isolated from cockroaches and tested against Epilachna varivestis Mulsant (Coleoptera: Coccinellidae) larvae [32], was maintained in the Colección de Hongos Entomopatógenos of the Unidad de Formulación de Biológicos at Universidad Autónoma de Nuevo León, México. For conidia re-activation, BBPTG4 strain was grown on potato dextrose agar (PDA) (BD Difco, Ciudad de México, México) in Petri dishes (5 cm diameter × 1 cm depth) (Med Lab S.A. de C.V., Estado de México, México) from a monosporic stock. Fungus inoculation was made with 100 µL of conidial suspension. Inoculated Petri dishes were then incubated at 25 • C for 6 d to 8 d in darkness, until sporulation. Produced conidia were removed by adding 1 mL of 0.5% INEX-A ® (Cosmocel, Monterrey, N. L., México) as a dispersant to obtain a stock solution. We measured conidia viability from the stock suspension in all experiments. For this test, 20 µL of the suspension was set for germination counting on potato dextrose broth (PDB) (BD Difco) and incubated at 25 • C ± 2 • C. Conidia were considered viable by counting 100 conidia three times to determine germination percentage [33]. Next, the tested amount was adjusted to reach an initial concentration of 1 × 10 8 viable conidia/mL. Once fungus viability was evidenced, 200 µL of the conidia stock was inoculated into an Erlenmeyer flask with 200 mL of PDB and incubated at 25 • C ± 2 • C in an automatic rotary shaker at 120 rpm (Orbit 1900, Labnet, Ciudad de México, México) for 5 d, until blastopore structures were detected and adjusted to 1 × 10 8 blastospores/mL, using a Neubauer chamber under a phase-contrast microscope at 40×. This suspension was used to inoculate rice for semi-massive production by solid fermentation.
Solid fermentation for semi-massive BBPTG4 conidia production as an active ingredient (AI) was performed as reported elsewhere [34]. In brief, 100 g of pre-moistened sterile parboiled rice grains, used as solid substrate, was placed in 800 mL glass bottles, containing 30 mL of hydration sterile solution (0.97 g/L KH 2 PO 4 , 410 µL/L of H 2 SO 4 , and 0.31 g/L yeast extract). Solid fermentation was performed by inoculating 100 g of hydrated rice grains with 1 × 10 8 viable blastospores/mL and incubating at 25 • C ± 2 • C for 8 d to 14 d, in darkness. During incubation, rice solid culture in bottles was mixed daily with a spatula for aeration, under sterile conditions. Rice-cultured conidia were harvested using a standard testing No. 40 sieve (426 µm opening size). Produced conidia were quantified by taking 15 mg, suspended in 0.5% INEX-A ® (emulsifier agent), counted, and stored at 4 • C to prepare granular formulations.
To evaluate germination tube development percentage, conidia viability was determined on PDA medium in a Petri dish plate by colony forming units (CFU) count, selecting conidia dilutions (4.3 × 10 5 conidia/mL), based on viability percentage results. CFU number was multiplied by the dilution factor to obtain the viable conidia value. We analyzed CFU means of recorded data among treatments for each time kept at 25 • C ± 2 • C for 6 d, by one-way ANOVA (p < 0.05) and the honestly significant difference (HSD) Tukey test for post hoc multiple means comparison. All tests were performed using the SPSS version 21.0 [35].

Conidia Viability in Microgranular Formulations
B. bassiana-based microgranular formulations (MGFs) were prepared as previously reported [34,36]. In brief, a mixture containing 7.5 g of nixtamalized processed corn flour, 7.5 g of cornstarch, 0.075 g of sugar, and 5.4 mL of soybean oil constituted the dried granules total weight used as the matrix. Ingredients were mixed and homogenized by adding 10 mL of purified drinking water and MGFs without conidia (untreated) to be used as the negative control. Microgranules were obtained using a fine sieve N • 40 to pass through the dough. The estimated granule size was 0.1 mm.
For comparison, conidia viability of BBPTG4 mixed on MGF after storage was determined as described above, including three replicate determinations by formulation, storage time, and temperature. BBPTG4 was tested as an active ingredient at 1.0 × 10 8 viable conidia/g of MGF and viability was determined at 4 d, 11  Since additive combinations, such as lactic acid + acetone, showed to be effective for the attraction of Ae. aegypti and Anopheles sp., additives were evaluated using them alone [24] or the combination of lactic acid + acetone, which were also added to MGFs. Treatments included (a) MGF (sugar), (b) MGF + acetone at 1% (w/v) of MGF, (c) MGF + 100 µL lactic acid at 85% per gram of MGF, and (d) MGF (sugar) + 100 µL lactic acid at 85% + acetone at 1% (w/v) of MGF.
For attractiveness evaluation [37], triplicates of 30 Ae. aegypti adults were used [38]. For this, adults were transferred to a cage similar to that used for rearing but inside the cage we placed a conventional home mosquito trap (conventional CO 2 bioproducing system) and the plastic cup with cotton soaked in 5% sugar solution for feeding. The conventional home trap consisted of an empty 2 L plastic soda bottle, cutting one third of the top area to achieve a funnel-shaped container, which was placed upside down on the top side of the cut bottle. The bottom of each trap was filled with 700 mL of tap water and the external surface was covered with a black paint color.
The bottom of the upside-down funnel-shaped soda bottle mosquito trap was covered with muslin and placed in a container filled with 700 mL of tap water, after which 6 g of each treatment with microgranules were added, instead of the sugar and yeast extract, to test the attractiveness by using the mosquito trap system (BG CO 2 Mosquito Trap|Nixalite accessed on 30 June 2022).
One trap/treatment was placed inside the cage containing Ae. aegypti, allowing adults to reach the treatment by the narrow funnel area but avoiding the mosquitoes to reach the trap solution or to directly feed on the cotton soaked with 5% sugar solution. After 48 h, a muslin-mesh net was placed on the top of the funnel area of the trap and trap-attracted mosquitoes were immobilized by placing the whole trap inside of a refrigerator for 10 min. Next, adults were taken from the trap by removing the muslin-mesh net placed initially at the bottom area of the funnel from the trap top. We selected the MGF + additive treatment showing the highest adult attraction to be used with B. bassiana conidia as the active ingredient (AI). For this, conidia were added to MGF to reach a final concentration of 1.0 × 10 8 conidia/g, using a control without AI. Adult attractiveness evaluation was performed as described above. The mosquitoes that entered the traps and were in contact with the MGF were collected in a second cage to record mortality. For this, alive mosquitoes were individually collected from the funnel mesh as previously described but using sterile conditions. Adults were separately placed inside of a 1 L plastic container with a cup with 5% sugar solution-soaked cotton to ensure mosquito feeding and survival, using the same incubation conditions as those for the colony. Sugar solution was added, if necessary, as explained above.
For mortality evaluation, Ae. aegypti adult's survival was determined every third day for each treatment and bioassay in triplicate for up to 12 d. Dead mosquitoes were counted to register mortality percentage and placed inside of a humid chamber to visualize aerial mycelium development [39,40], using a stereoscope to confirm the presence of Bb mycelium. Ae. aegypti adult's attraction and mortality percentage means were analyzed by the Student t test (p < 0.05) for independent samples. Conidia viability in combination with MGF was evaluated every 15 d for three months after storage at 25 • C ± 2 • C or 4 • C ± 1 • C, testing the remaining B. bassiana conidia germination percentage as mentioned above. Since Spirulina algae provides stability to conidia due to their fat content [20] and increases attraction by producing high amounts of CO 2 [21], to improve Ae. aegypti adult's attraction to MGFs, dried Spirulina sp. was added to the formulation dough, testing ingredients at the same amounts as detailed above ( For this experiment, we used the formulation that showed the highest Ae. aegypti attraction in Section 2.4.1.1. The formulation consisting of B. bassiana-based MGFs + Sp showed the highest Ae. aegypti attraction. We used 2.6 × 10 8 conidia/g for treatments with an initial viability of 70% of the stock suspension and prepared replicate determinations for the storage temperatures 25 • C ± 2 • C and 4 • C ± 1 • C. Similarly, for shelf-life determination, 50 mg of each replicate were suspended in 0.05% INEX-A, after which 1/10 dilutions were made. We selected 4.3 × 10 5 conidia/mL to measure spore germination, based on viability percentage results. Two millimeters thick agar sections were placed on a series of glass slides. One drop of the conidia suspension was then placed on each agar disc and slides incubated at 25 • C ± 2 • C in darkness, after which percent germination was determined after 17 h, counting viable and non-viable conidia. Evaluations were determined at 0 d, 15 d, 30 d, and 45 d at experimental storage temperatures. Percentages means of recorded data for each treatment throughout the storage periods were analyzed by one-way ANOVA and the honestly significant difference (HSD) Tukey test (p < 0.05) for post hoc multiple means comparison. Germination percentage between treatments in each period was analyzed by the Student t test (p < 0.05) for independent samples [39].

Conidia Viability on MGFs with Spirulina sp. and Solid Formulation (SF) with Coco Fiber
To improve MGFs + Sp formulation, we performed treatments containing flour and water as mentioned above (Section 2.3.2, developing solid formulations by adding vegetable fat or ground coconut fiber (Table 1). MGFsSp, MGFs, SFSp, and SF treatments and controls (conidia suspended in 0.05% INEX-A ® or 0.05% INEX-A ® alone) were prepared under sterility, vacuum packed in triplicate, and stored at room and cold temperatures ( Table 2). In addition, B. bassiana conidia shelf life was evaluated at 0 h, 24 h, 15 d, 30 d, and 120 d after preparation and viability was measured as explained above. Data were analyzed by the one-way ANOVA (p < 0.05) and HSD Tukey for post hoc multiple means comparison tests. Negative control MGF = microgranular formulation, AI = B. bassiana conidia as active ingredient, see Table 1 for conidia per gram in the final formulation.

Aedes aegypti Adult Infection by Microgranular Formulations and Solid Formulations
Treatments were the same used for B. bassiana conidia shelf life, involving MGF + sugar + Spirulina, MGF + sugar, solid formulation + sugar + Spirulina, and solid formulation + sugar that were stored at room temperature ( Table 2). Ae. aegypti adult biocontrol efficacy was evaluated in triplicate, using 10 adults exposed for 48 h to each treatment. Treatments were replaced by cotton soaked in water solution with 5% sugar. Adult survival data were collected daily for up to 16 d. For mosquito handling, adults were immobilized for 5 min at 4 • C. The infection traps consisted of 1 L bottles and the container lid was covered with a fine mesh, where 5 g of each formulation and cotton with sugar was placed. Dead mosquitoes were then processed as explained in Section 2.3.3.2 [40].

B. bassiana Conidial Viability after Exposure to Additives
At time zero, we observed 90% to 100% conidia viability. Nevertheless, with all tested attractants, conidial viability experienced a 1000-fold decrease within 5 d to 30 d. Lactic acid treatments induced 3-to 4-fold viability decrease, whereas 1% and 5% acetone caused 1-fold viability reduction (5.7 × 10 5 viable conidia), compared with the resulting viability after 5 d of storage. After 30 d of storage, conidia viability reduction was variable among treatments. It decreased 1-to 4-fold, compared with the remaining viability after 5 d of storage. Moreover, the negative control conidia in 0.5% INEX-A lost 100% viability in both tested samples. Similarly, results showed high viability reduction among treatments, where additives were tested at the highest concentrations.

B. bassiana Conidial Viability after Exposure to MGF
BBPTG4 conidia remained viable after mixing in MGF. On this formulation, conidia lost less than 10-fold viability (from 1.5 × 10 7 conidia/mL to 1.6 × 10 6 conidia/mL at 25 • C and from 1.7 × 10 7 conidia/mL to 1.5 × 10 6 conidia/mL at 4 • C) after 30 d of storage, regardless of the storage temperature ( Figure 1A). After this, BBPTG4 conidia lost 15-fold (1.0 × 10 6 conidia/mL) viability but remained stable up to 91 d of storage, regardless of the storage temperature. Untreated MGF, used as a negative control, did not develop microbial growth as contamination (data not shown).

Ae. aegypti Attraction Efficacy by Microganule-Formulated Additives
Among tested treatments, the highest Ae. aegypti attraction efficacy (p < 0.05) was observed after using MGF ( Figure 1B). Treatment attractions were 18.9%, 23.3%, and 22.2% lower in MGF + acetone, MGF + lactic acid, and MGF + lactic acid + acetone treatments, respectively ( Figure 1B). In addition, when conidia were combined with all Conidia viability was determined in MGF at time zero (4 d) (1.5 × 10 7 conidia/mL as active ingredient (AI) with MGF) and up to 91 d storage at 25 • C ± 2 • C or 4 • C ± 1 • C, as explained in the text. (B) Percentages of Ae. aegypti adults attracted to different treatments. Ae. aegypti adults were exposed to MGF without additive, MGF + acetone (added at 1% (w/v) of MGF), MGF + 100 µL/g of lactic acid at 85%, and MGF + 100 µL/g of lactic acid at 85% + 1% acetone/g of MGF and attraction percentages determined, as explained in the text. Data represent mean + SEM of triplicate determinations from three independent experiments. Same letter on each column indicates that treatments are not significantly different (HSD Tukey test; p < 0.05).

Ae. aegypti Attraction and Mortality by MGFs with B. bassiana
After B. bassiana conidia (active ingredient) were mixed in MGF, named from now on as MGFs, for the sucrose use as additive, it was selected as the best treatment in Section 3.1. Results showed non-significant attraction (29.7%; t (4) = 1.778, p = 0.150) compared with untreated MGFs (Figure 2A). Mortality of attracted mosquitoes by conidia was lower than 40% but significantly (t (4) = 5.983, p = 0.04) higher than that of untreated MGFs (<5% mortality) ( Figure 2B).

Ae. aegypti Attraction and Mortality by MGFs with B. bassiana
After B. bassiana conidia (active ingredient) were mixed in MGF, named from now on as MGFs, for the sucrose use as additive, it was selected as the best treatment in Section 3.1. Results showed non-significant attraction (29.7%; t (4) = 1.778, p = 0.150) compared with untreated MGFs (Figure 2A). Mortality of attracted mosquitoes by conidia was lower than 40% but significantly (t (4) = 5.983, p = 0.04) higher than that of untreated MGFs (< 5% mortality) ( Figure 2B).

Ae. aegypti Attraction and Mortality by MGFs with B. bassiana
After B. bassiana conidia (active ingredient) were mixed in MGF, named from now on as MGFs, for the sucrose use as additive, it was selected as the best treatment in Section 3.1. Results showed non-significant attraction (29.7%; t (4) = 1.778, p = 0.150) compared with untreated MGFs (Figure 2A). Mortality of attracted mosquitoes by conidia was lower than 40% but significantly (t (4) = 5.983, p = 0.04) higher than that of untreated MGFs (< 5% mortality) ( Figure 2B).

Ae. aegypti Attraction and Mortality by MGFs with B. bassiana
After B. bassiana conidia (active ingredient) were mixed in MGF, named from now on as MGFs, for the sucrose use as additive, it was selected as the best treatment in Section 3.1. Results showed non-significant attraction (29.7%; t (4) = 1.778, p = 0.150) compared with untreated MGFs (Figure 2A). Mortality of attracted mosquitoes by conidia was lower than 40% but significantly (t (4) = 5.983, p = 0.04) higher than that of untreated MGFs (< 5% mortality) ( Figure 2B).
Furthermore, solid formulation + Spirulina stored at 4 °C treatment showed lower viability (2.92 × 10 7 CFU/mL) compared with all other treatments stored at the same
Results obtained after 30 d of storage at 4 • C or 25 • C, showed lower conidia viability among most formulations. However, MGF + sugar + Spirulina treatment had higher viability after storage at 25 • C temperature (1.17 × 10 8 CFU/mL). Similarly, MGF + sugar at 4 • C and MGF + sugar + Spirulina at 25 • C (1.1 × 10 8 and 1.2 × 10 8 CFU/mL, respectively), resulted in higher conidia viability compared with other treatments. In contrast, we did not observe viable conidia in solid formulation + sugar treatment stored at 4 • C temperature and solid formulation + sugar + Spirulina and solid formulation + sugar stored at 25 • C ( Figure 4C).

Aedes aegypti Biocontrol by MGF and FS with B. bassiana Conidia
We evaluated the insect survival of four formulations against Ae. aegypti adults. After 5 d of exposure, the positive control (B. bassiana suspension at 1 × 10 8 conidia/mL) showed a survival percentage of 6.25%, whereas MGF + sugar + Spirulina and solid formulation + sugar + Spirulina resulted in 40% and 20% survival, respectively. High survival was detected by solid formulation MGF + sugar + Spirulina and solid formulation MGF + sugar treatments, showing above 65%. After 9 d of exposure, insect survival was reduced in treatments without Spirulina; MGF + sugar and solid formulation + sugar showed 5% and 10% survival after 16 d, respectively ( Figure 5A).
After 30 d of storage, conidia treatments also affected mosquitoes. In this regard, MGF + sugar + Spirulina was the most effective, since after 7 d of exposure none exposed adult survived, whereas solid formulation + Spirulina and solid formulation showed 10% adult survival and MGFs and positive control resulted in 20% adult survival. The positive control, solid formulation + sugar, and solid formulation MGF + sugar + Spirulina treatments reduced the adults' survival to zero after 11 d, 14 d, and 15 d of exposure, respectively, unlike the negative control, which after day 16 maintained 33.3% survival ( Figure 5B). After 30 d of storage, conidia treatments also affected mosquitoes. In this regard, MGF + sugar + Spirulina was the most effective, since after 7 d of exposure none exposed adult survived, whereas solid formulation + Spirulina and solid formulation showed 10% adult survival and MGFs and positive control resulted in 20% adult survival. The positive control, solid formulation + sugar, and solid formulation MGF + sugar + Spirulina treatments reduced the adults' survival to zero after 11 d, 14 d, and 15 d of exposure, respectively, unlike the negative control, which after day 16 maintained 33.3% survival ( Figure 5B).

Discussion
For a successful vector control application, we aim to improve entomopathogenic fungi shelf-life storage, increase insect attraction, and maintain viability in open places [19]. In the present study, we developed an effective "attraction-infection-kill"

Discussion
For a successful vector control application, we aim to improve entomopathogenic fungi shelf-life storage, increase insect attraction, and maintain viability in open places [19]. In the present study, we developed an effective "attraction-infection-kill" formulation against Ae. aegypti by combining B. bassiana conidia, attractants, and a surfactant. We evaluated the active ingredient s stability during storage at room temperature in solution and in MGF. We first assessed the cytotoxicity of the previously reported attractants to Ae. aegypti acetone, lactic acid, and sugar against B. bassiana conidia [23]. Results showed the absence of cytotoxic effect, and conidia remained in suspension with the attractant and the surfactant (0.5% INEX-A). After 5 d of storage at 25 • C ± 2 • C, conidia significantly lost their viability (from 4.3 × 10 8 to 0.29 × 10 5 conidia/mL), regardless of the treatment.
This effect may be the result of conidia permeability changes, since they were stored in suspension at room temperature, allowing enzyme (proteases, peptidases, chitinases, lipases, and phospholipases) and other metabolite production [42]. After 30 d of storage, conidia in both controls (mixed in 0.5% INEX-A) lost 100% viability. This may be the effect of the surfactant by favoring conidia germination and eventually, their death due to oxygen and nutrients scarcity [43], since after 5 d of storage~6.0 × 10 5 conidia/mL remained viable. This adverse effect of attractants on conidia viability increases the need for formulations and attractants that do not alter it.
Regardless of the attractant, conidia significantly lost viability in a dose-response manner, mainly in the lactic acid and sugar treatments. Osmotic stress may have played an important role in conidia over the time, probably due to super-saturation and cell wall turgor lost [44]. However, the highest B. bassiana conidia viability after 30 d was observed in treatments with 1% and 5% acetone, plus 1% sugar.
In general, conidia survival is reduced in many conventional formulations [18]. Results of the present study demonstrated that MGF provided stability to B. bassiana conidia after storage at room temperature, where 80% viability remained after 32 d of storage. Moreover, MGF enhanced conidia viability and mosquito attraction. Furthermore, viability reduction was observed regardless of the storage temperature, demonstrating that temperature was not a determining factor of conidia stability.
According to our results with sugar in MGF, this formulation increased Ae. aegypti adult attraction, which may result in one commercial formulation for mosquito control in domestic traps with some modifications. Since these traps have been used for Ae. aegypti females to lay eggs, and acetone has shown up to 78% attraction to females despite its volatility [23], it may be important to evaluate its efficacy versus application over time, when conidia remain viable in the trap and CO 2 -bioproducing system (BG CO 2 Mosquito Trap | Nixalite accessed on 30 June 2022).
The most important aspect for any formulation based on a biocontrol agent is the product efficacy. Several studies reported that unformulated B. bassiana conidia suspensions at 1 × 10 8 and 6 × 10 8 conidia/mL, directly applied on a filter paper against Ae. aegypti adults, caused 89% to 90% mortality, where surviving infected adults increased the biocontrol rate by co-infection via copula [8]. Indeed, they reported Ae. aegypti adult mortality to up to 40%, after testing in a 123.8 cm 3 infection system [8]. In our laboratory, a domestic trap with MGF at 1 × 10 8 conidia/g, placed in an area almost 10 times higher (1205.1 cm 3 ), was used, reaching a similar mortality (42%). Therefore, the function of the formulation was fulfilled, not only due to the stability of the formulation but also to the possibility of increasing the doses generally used.
In fact, when a lower concentration (2.4 × 10 4 conidia/g) was used in the same infection system, using MGF as a base, the attraction was not significantly increased by 20%. When evaluating the MGF humid formulation, we found that it improves the attraction percentage by 20%, which may be due to the reactivation of the microorganisms (Spirulina and others). However, the difference was not significant compared with MGF + Spirulina dry. Although granule wetting did not enhance the attraction (although a trend was observed), this is consistent with a series of products on the market, whose application depends on wetting for the reactivation of the control agent and the volatilization of attractants.
Our results showed that the addition of a low concentration of Spirulina sp. (3.8%) enhanced the effectiveness of B. bassiana for Ae. aegypti biocontrol. Others have also shown that the addition of phage-stimulating additives, such as chitin in the formulation with B. thuringiensis, reversed the toxicity of the pathogen, perforating peritrophic membranes of the midgut in larvae and increasing the accessibility of toxins to epithelial cells [45]. However, in our study, we used inexpensive attractants such as sugar or Spirulina sp., which showed promising results.
Research on insect control, particularly culicids, focuses on effectivity. For instance, a pet trap covered with fabric impregnated by a synthetic bait (AtrAedes, Agrisense Ltd., Cardiff, UK) in combination with Metarhizium anisopliae (Metsh ® ) against Ae. aegypti females, under a controlled environment (intra-domicile conditions), reported up to 68% mortality [27,29]. Another reported trap system using black plastic flower pots (which have larvicidal and adulticidal activities, included a juvenile hormone analogue (pyriproxyfen) to attract gravid Ae. aegypti females. Experiments using this system included combinations with B. bassiana spores, and were performed under three blunt screens, resulting in lower Ae. aegypti adult survival percentages [46]. Nevertheless, the conidia shelf life on this fabric-based Ae. aegypti bait was rather low. Results showed that conidia viability was preserved for 45 d at 25 • C.
To develop a formulation that provides B. bassiana conidia stability, viability, and effectiveness, we tested one microgranular (MGF) and one solid formulation, which contained vegetable fat, Spirulina sp., or coconut fiber. One study reported a M. anisopliae formulation to control Rhipicephalus microplus (Canestrini) (Acari: Ixodidae). It contained 10% oil to produce a homogeneous emulsion and field dispersion, providing adhesion at the application time [47]. We also developed emulsions with B. bassiana conidia and after their exposure to Ae. aegypti adults we observed repellency, due to the viscosity it presented (data not shown). This demonstrated that, in addition to being careful about the technology for the preparation and ingredients used, we must consider the insect s ethology, when using formulations.
In addition, the use of coconut fiber, as the one used in our work, in formulations to improve Pseudomonas chlororaphis shelf life, maintained bacteria viability for up to 8 months [48]. In contrast, in the present study, we observed that solid formulation significantly decreased B. bassiana conidia viability from 15 d of storage and at 30 d we did not find viable conidia (p < 0.01). This loss of viability may suggest that this type of ground substrate does not generate a homogeneous mixture, allowing conidia exposure to abiotic factors (light, increased temperatures, and low humidity).
The most effective formulations in our study were MGF + sugar + Spirulina and MGF + sugar, at 4 • C which contained flour, corn starch, and vegetable oil. In these formulations, B. bassiana conidia viability was maintained at 1.4 × 10 8 CFU/mL and 1.6 × 10 8 CFU/mL after 120 d of storage. Viability increased probably because of Spirulina, which may have provided stability to conidia due to their fat content (11% of lipids) [20]. We also found the viability of 7.20 × 10 6 CFU/mL for MGF + sugar at 25 • C (an adequate concentration to reach mortalities greater than 40%), which was statistically different from the rest of the treatments stored at 25 • C. This may be due to different additives that provided protection, unlike solid formulation. In conclusion, the addition of Spirulina increases the attraction of Ae. aegypti, which correlated with its mortality. However, it does not provide any conservative benefit to the MGF, as it could be seen in this experiment with the treatments MGF + sugar + Spirulina at both temperatures and MGF + sugar at 4 • C.
MGF + sugar + Spirulina and MGF + sugar treatments showed values of 4.5 × 10 7 , 1.10 × 10 7 , 5.20 × 10 7 , and 1.46 × 10 8 CFU/mL and 3.89 × 10 7 , 7.49 × 10 7 , 1.08 × 10 8 , and 1.60 × 10 8 CFU/mL at 0 d, 15 d, 30 d, and 120 d, respectively, at 4 • C. The same trend was observed in samples stored at 25 • C for in the same treatments. It was evident that the best MGF formulations were those stored at 4 • C with conidia at a range of 1 × 10 8 CFU/mL after 120 d, whereas for the granular formulation treatments stored at 25 • C, the final viability values in MGF + sugar + Spirulina and MGF + sugar treatments at 25 • C were 6.67 × 10 5 CFU/mL and 7.20 × 10 6 CFU/mL, respectively, after 120 d of storage. After comparing these results with conidia viability in the positive controls, in general they showed decreasing and lower viability values. This confirms that when powdered vegetable oil was added to the formulations, conidia viability was higher after storage. In this regard, it has been emphasized that bioinsecticide storage induces a slow metabolism, especially when they are stored under refrigeration [48]. This agrees with our MGF results, which suggested a slower conidia metabolism but not dormancy, as observed among other microorganisms under −20 • C storage. This also suggested that the addition of powdered vegetable oil to our formulations reduced conidia loss of moisture when they are stored in microgranules. However, due to the physical conidia hydrophobicity, conidia in an oil-based emulsion may be kept viable for longer storage periods [49,50]. In this regard, Cordyceps fumosorosea (Wize) kept germination and viability after storage, which was associated with the addition of corn oil to the formulation [51]. Other authors have suggested that the adequate amount of oil for B. bassiana conidia formulations should be from 1% to 2% [52]. Nevertheless, the amount tested in our granular formulation was higher. This may be important since keeping any pesticide under freezing conditions is more expensive than at 4 • C. Our results suggest that formulations with ingredients such as powdered vegetable oil may help to improve B. bassiana conidia shelf life.
Regarding formulations effectiveness, it was observed that at time 0, the positive control was effective in terms of the survival percentage of Ae. aegypti, probably due to an increased conidia exposure to mosquitoes than in formulation. We also observed that the most effective treatments to decrease survival at time 0, were those formulated with Spirulina (MGF + sugar + Spirulina and solid formulation + sugar + Spirulina). However, the best treatment after 30 d was MGF + sugar + Spirulina, since at 7 d after exposure, it caused a mortality of 100%, unlike the other treatments, which agrees with the viability found in these treatments in the range of 2 × 10 7 to 1 × 10 8 CFU/mL. Other authors reported that the use of B. bassiana conidia in a liquid formulation with oil, reduced Ae. aegypti survival by 61% to 69%, after 10 d of exposure in a semi-field system with small cages [52]. Furthermore, after using black fabrics impregnated with B. bassiana conidia in PET plastic traps, an increase in Ae. aegypti attractiveness from 31% to 66% and a 52% decrease in survival was observed after 120 h [27].
An important aspect is to demonstrate that the system works after storage for a long time, by validating the effectiveness in the field, as well as identifying if the additives used in our study provide residual activity in the field. Previous reports have demonstrated that B. thuringiensis microencapsulation, developed for Choristoneura rosaceana (Harris) larvae control, caused up to 33% mortality immediately after application, which persisted for up to 14 d, where activity was affected due to biotic factors (precipitation, temperature, and solar radiation) [53]. These results confirm the importance of identifying the proper time and place of application. In the case of MGF, it appears that these types of formulations may have to be tested outside houses, under trees or bushes and perhaps garages with dark places, places for which flies have an affinity.
We consider that although a reduction in conidia viability in formulations developed in this study was observed, this commonly produces unfavorable results. However, it is acceptable for use in mosquito control and has appropriate characteristics to continue with the investigation. For example, its high attraction and mortality are characteristics that make this B. bassiana-based bioformulation, an alternative to reduce the use of chemical agents and increase public awareness about the benefits of the use of biological products, whose action does not compromise human health. In fact, according to the effectiveness data of previously mentioned species (C. fumosorosea, B. bassiana, and B. thuringiensis), this bioinsecticide still possesses functional viability.
The use of a formulation that attracts mosquitos in a trap, combined with conidia, may increase the target infection rate. Since a tested trap is easy to make and install, its use in the home or field seems feasible. There are many traps that catch mosquitos but the idea to attract and expose them to B. bassiana conidia is to increase the infection/mortality rate, knowing transmission feasibility via copula [8]. After testing an attractant device with M. anisopliae for the Ceratitis capitata (Wiedemann) biocontrol under field conditions, results showed a fruit fly population reduction and that the inoculation dishes needed mid-season replacing to provide protection for the entire season [54]. We believe that similar replacement over the time for the trap-fungi formulation described in this study may help to increase mosquito infection and their biocontrol.

Conclusions
In conclusion, the combination of formulated granules with B. bassiana as an active ingredient attracted and infected Ae. aegypti adults in a domestic trap, where conidia remained viable for up to one month at 25 • C ± 2 • C. Our results open a new strategy to develop a formulation as a vector control management tool. Formulation improvements in the manufacturing process generates knowledge on the ingredient's effective combina-tion for insect target attraction, mortality, and B. bassiana conidia viability, as well as the preparation and equipment use. Taken together, our results provide the elements to obtain a commercial product for Ae. aegypti biocontrol.