Encapsulation of Beauveria bassiana conidia as a new strategy for the biological control of Aedes aegypti larvae

Abstract

The virulence of encapsulated fungal conidia against Aedes aegypti larvae was investigated. Molecular studies confirmed that the fungal isolate used here was Beauveria bassiana. Different conidial concentrations were tested. A concentration of 1 × 10⁸ conidia mL^{- 1} was the most effective, resulting in 7% larval survival after 7 days. Next, alginate capsules (0.65%) containing conidia were prepared with different densities of calcium chloride (0.01 M, 0.009 M, and 0.008 M CaCl₂) and tested against larvae. Furthermore, groups of capsules were prepared with bird diet to act as an attractant. All capsule densities tested reduced larval survival (ranging from 22 to 67%). However, capsules with 0.008 M CaCl₂ were the most effective. Furthermore, fungus-only capsules were more efficient when compared to those containing bird diet. Laboratory and semi-field bioassays were conducted using mixtures of capsules with different densities. In the laboratory, survival ranged from 26 to 53%, whereas in semi-field conditions, 35%, and 80% survival was observed for groups exposed to fungus-only capsules or capsules containing diet, respectively. Histopathological studies of larvae exposed to capsules showed the presence of the fungus in the digestive tract and visible damage to enterocytes. These findings offer new insights into the biological control of Ae. aegypti larvae.

Keywords: alginate; biocontrol; capsules; dengue; enterocytes; entomopathogenic fungi.

Fig. 1

Aedes aegypti survival following exposure to Beauveria bassiana conidial suspensions at three different concentrations. Larvae were exposed to conidial suspension for two days. After 48 h, larvae were transferred to pure sterile tap water. The survival was monitored then daily for the next five days. Log-rank test provided the statistical comparison (P values ≤ 0.05), and chi-square values (χ²). Note: degrees of freedom (df); CTR = control; con/mL = conidia mL^− 1. ns = not significant.

Fig. 2

Survival curves of Aedes aegypti larvae exposed to encapsulated Beauveria bassiana conidia using 0.65% alginate and different concentrations of calcium chloride (0.01 M, 0.009 M, or 0.008 M). Each group of larvae was exposed to capsules produced using 0.01 M, 0.009 M, or 0.008 M CaCl₂ for seven days. Survival was monitored daily for seven days. The log-rank test provided statistical comparisons (P values ≤ 0.05), and chi-square values (χ²). Note: degrees of freedom (df), ns = not significant; F = capsules with conidia; F + B = capsules with conidia and bird food; A = capsules produced with pure alginate (control group); A + B = alginate and bird food capsules (extra control group).

Fig. 3

Amount of conidia released from alginate capsules into the surrounding water. Quantification was performed on the first and seventh days of the experiment. The same lowercase letters indicated no significant differences (P ≥ 0.05) when comparing different groups at the same time. Results followed by the same capital letters were not statistically different (P ≥ 0.05) over time (day 1 to day 7). F = capsules with conidia; F + B = capsules with conidia and bird food.

Fig. 4

Survival of Aedes aegypti larvae exposed to Beauveria bassiana encapsulated using 0.65% alginate with different concentrations of calcium chloride (0.01 M, 0.009 M, or 0.008 M). Each group of larvae was exposed to a mixture of capsules with 0.01 M, 0.009 M, or 0.008 M CaCl₂ for seven days. Survival was monitored daily for seven days. The log-rank test provided statistical comparisons (P ≤ 0.05), and chi-square (χ²) values. Note: degrees of freedom (df); ns = not significant; MF = capsules with conidia; MF + B = mixtures of capsules with conidia + bird food; MA = mixtures of capsules of pure alginate (control group); MA + B = mixtures of capsules of alginate + bird food (control group).

Fig. 5

Aedes aegypti larval survival rates following exposure to Beauveria bassiana capsules with 0.65% alginate and different concentrations of calcium chloride (0.01 M, 0.009 M, or 0.008 M). Each group of larvae was exposed to 20 g of capsules with 0.01 M, 0.009 M, and 0.008 M CaCl₂, totaling 60 g of capsules per liter of water in each container. Survival was monitored daily for seven days under semi-field conditions. The log-rank test was used for statistical comparison (P values ≤ 0.05), and calculating the chi-square value (χ²). Note: degrees of freedom (df); MF = mixtures of capsules with conidia; MF + B = mixtures of capsules with conidia and diet; MA = mixtures of pure alginate capsules (control group); MA + B = mixtures of capsules with alginate and diet (second control group).

Fig. 6

Aedes aegypti larval midgut following exposure to alginate capsules (A and A.1), or alginate combined with bird food (B and B.1). Regardless of the treatment, there were no abnormal observations. Specimens demonstrated healthy cuboidal epithelial cells (enterocytes) with normal brush borders (bb), nucleus (n) with no retraction or degenerated cytoplasm (c) ag = alginate.

Fig. 7

Aedes aegypti larvae midgut sections following exposure to capsules with conidia only (A; A.1; B and B.1). Abnormal morphology was observed with degenerated enterocytes (A.1) and disrupted brush borders (dbb). Peritrophic membrane (empty arrow), production of extracellular material (ecm), lipid droplets (LD), and the presence of Beauveria bassiana propagules in the midgut lumen were observed. ecp = ectoperitrophic space; ag = alginate.

Fig. 8

Aedes aegypti larval midguts following exposure to encapsulated conidia combined with bird food (A, B, C, and C.1). The midguts showed columnar enterocytes with homogenous cytoplasm (c) but with nuclear (n) retraction. Brush borders (bb) were observed without degeneration. Furthermore, the presence of peritrophic membranes (empty arrow), the production of extracellular material (ecm), and germinated conidia (arrows) were observed.