Beauveria bassiana interacts with gut and hemocytes to manipulate Aedes aegypti immunity.

BACKGROUND: Mosquito-borne diseases affect millions of people. Chemical insecticides are currently employed against mosquitoes. However, many cases of insecticide resistance have been reported. Entomopathogenic fungi (EPF) have demonstrated potential as a bioinsecticide. Here, we assessed the invasion of the EPF Beauveria bassiana into Aedes aegypti larvae and changes in the activity of phenoloxidase (PO) as a proxy for the general activation of the insect innate immune system. In addition, other cellular and humoral responses were evaluated. METHODS: Larvae were exposed to blastospores or conidia of B. bassiana CG 206. After 24 and 48 h, scanning electron microscopy (SEM) was conducted on the larvae. The hemolymph was collected to determine changes in total hemocyte concentration (THC), the dynamics of hemocytes, and to observe hemocyte-fungus interactions. In addition, the larvae were macerated to assess the activity of PO using L-DOPA conversion, and the expression of antimicrobial peptides (AMPs) was measured using quantitative Real-Time PCR. RESULTS: Propagules invaded mosquitoes through the midgut, and blastopores were detected inside the hemocoel. Both propagules decreased the THC regardless of the time. By 24 h after exposure to conidia the percentage of granulocytes and oenocytoids increased while the prohemocytes decreased. By 48 h, the oenocytoid percentage increased significantly (P < 0.05) in larvae exposed to blastospores; however, the other hemocyte types did not change significantly. Regardless of the time, SEM revealed hemocytes adhering to, and nodulating, blastospores. For the larvae exposed to conidia, these interactions were observed only at 48 h. Irrespective of the propagule, the PO activity increased only at 48 h. At 24 h, cathepsin B was upregulated by infection with conidia, whereas both propagules resulted in a downregulation of cecropin and defensin A. At 48 h, blastospores and conidia increased the expression of defensin A suggesting this may be an essential AMP against EPF. CONCLUSION: By 24 h, B. bassiana CG 206 occluded the midgut, reduced THC, did not stimulate PO activity, and downregulated AMP expression in larvae, all of which allowed the fungus to impair the larvae to facilitate infection. Our data reports a complex interplay between Ae. aegypti larvae and B. bassiana CG 206 demonstrating how this fungus can infect, affect, and kill Ae. aegypti larvae.

Entomopathogenic fungi and Schinus molle essential oil: The combination of two eco-friendly agents against Aedes aegypti larvae.

Aedes aegypti transmits arbovirus, which is a public health concern. Certain filamentous fungi have the potential to control the disease. Here, the effects of Metarhizium anisopliae s.l. CG 153, Beauveria bassiana s.l. CG 206 and Schinus molle L. were investigated against Aedes aegypti larvae. In addition, the effect of essential oil on fungal development was analyzed. Fungal germination was assessed after combination with essential oil at 0.0025 %, 0.0075 %, 0.005 %, or 0.01 %; all of the oil concentrations affected germination except 0.0025 % (v/v). Larvae were exposed to 0.0025 %, 0.0075 %, 0.005 %, or 0.01 % of the essential oil or Tween 80 at 0.01 %; however, only the essential oil at 0.0025 % achieved similar results as the control. Larvae were exposed to fungi at 107 conidia mL-1 alone or in combination with the essential oil at 0.0025 %. Regardless of the combination, M. anisopliae reduced the median survival time of mosquitoes more than B. bassiana. The cumulative survival of mosquitoes exposed to M. anisopliae alone or in combination with essential oil was 7.5 % and 2 %, respectively, and for B. bassiana, it was 75 % and 71 %, respectively. M. anisopliae + essential oil had a synergistic effect against larvae, whereas B. bassiana + essential oil was antagonistic. Scanning and transmission electron microscopy, and histopathology confirmed that the interaction of M. anisopliae was through the gut and hemocoel. In contrast, the mosquito's gut was the main route for invasion by B. bassiana. Results from gas chromatography studies demonstrated sabinene and bicyclogermacrene as the main compounds of S. molle, and the in-silico investigation found evidence that both compounds affect a wide range of biological activity. For the first time, we demonstrated the potential of S. molle and its interaction with both fungal strains against A. aegypti larvae. Moreover, for the first time, we reported that S. molle might be responsible for significant changes in larval physiology. This study provides new insights into host-pathogen interplay and contributes to a better understanding of pathogenesis in mosquitoes, which have significant consequences for biological control strategies.

Larvicidal activity, route of interaction and ultrastructural changes in Aedes aegypti exposed to entomopathogenic fungi.

Blastospores or conidia (formulated or not) of entomopathogenic fungi were assessed against Aedes aegypti larvae. Larvae (L2) were exposed to 105, 106, 107, and 108 propagules mL-1 water suspension. Mineral oil at 0.1%, 0.5%, or 1.0% (v/v) was employed to observe the effect on larval survival. The 0.1% mineral oil did not affect larval survival. Accordingly, 107 propagules mL-1 and 0.1% mineral oil were used to prepare all fungal emulsions. The fungal suspension or formulation was prepared as follows: 107 propagules mL-1 on 0.03% TweenⓇ 80 (v/v) aqueous solution or 107 propagules mL-1 on 0.03% TweenⓇ 80 plus 0.1% mineral oil; larval survival rates were evaluated for 7 days, and median survival time (S50) was also determined. The presence of fungi in larvae was examined both histologically and by scanning electron microscopy 24 h or 48 h after exposure. To evaluate the larval growth, larvae were exposed to 107 propagules mL-1 for 48 hours and their length measured using a digital caliper. Here, propagules had similar results in reducing the larvae survival rate and time. The treatment with Beauveria bassiana s.l. at 108 propagules mL-1 or with Metarhizium anisopliae s.l. at 108 blastopores mL-1 reduced the larval survival time to two days. M. anisopliae s.l. at 108 conidia mL-1 reduced the survival time to three days. The survival time of larvae submitted to the other treatments ranged from 6 days to over 7 days. M. anisopliae s.l. or B. bassiana s.l. oil-in-water emulsions at 107 propagules mL-1 yielded better results than the water suspensions, the larvae survival rate was 2 days for both propagules in oil-in-water emulsion. Larvae exposed to blastospores from both isolates or M. anisopliae conidia were longer than in the other treatments. Scanning electron microscopy and histology analyzes found fungi predominantly in the gut, mouthparts, and perispiracular lobes of larvae. Formulated fungus yielded better results than the aqueous suspensions for control of mosquito larvae. Thus, for the first time, the effect of mineral oil on the fungal interaction on A. aegypti larvae was observed as well as the effect of entomopathogenic fungi in the growth of larvae, supporting the search for strategies to control this arthropod.

In vitro acaricidal activity of Cymbopogon citratus, Cymbopogon nardus and Mentha arvensis against Rhipicephalus microplus (Acari: Ixodidae).

The chemical composition and acaricidal activity of plant-derived essential oils was assessed against Rhipicephalus microplus ticks. The essential oils of Mentha arvensis, Cymbopogon citratus and C. nardus were assessed for acaricidal activity against Rhipicephalus microplus. Essential oils (EO) of plants were separated by hydrodistillation (three times) and analyzed using gas chromatography - mass spectrometer (GC-MS). For bioassays, engorged females of R. microplus were exposed to C. citratus and C. nardus EO at 2%, 3%, 4% and 5% concentrations; and to M. arvensis EO at 1%, 3%, and 5% for 5 min. The weight egg mass, nutrient index (N.I), egg production index (E.P.I), hatching and control rate were evaluated. Non-feed larvae of R. microplus were exposed to essential oils with 0.25%, 0.5%; 1%; 1.5% and 2% concentrations; the mortality rate was measured after 48 h. Only engorged females presented reduced biological activities (oviposition, E.P.I) after exposure to M. arvensis at 3%, when in comparison to both positive and negative controls. The hatchability of R. microplus larvae ranged from 66.9% (after exposure to C. nardus EO at 5%) to 99.2% (positive control). The nutrition index was lower (46.6%) for the exposure to M. arvensis EO at 5%. M. arvensis at 3% and 5% concentrations was significantly efficient for engorged females when compared to control (53.7% and 47.5%, respectively). C. citratus EO at 1%, 1.5% and 2% concentrations yielded better results in the larval packet test, causing 100% mortality. Nonetheless, C. nardus and M. arvensis EO at 2% yielded 66% and 39% mortality, respectively. The study showed that M. arvensis presented potential for the control of R. microplus engorged females while C. citratus and C. nardus presented potential as a larvicide.