Effects of Beauveria bassiana (Hypocreales: Cordycipitaceae) on the midgut of the Chrysomya megacephala (Fabricius, 1794) (Diptera: Calliphoridae) maggots

Beauveria bassiana is an entomopathogenic fungus widely used in pest management. After contact with target organisms, fungal conidia germinate and colonize tissues and organs, causing death by starvation and/or septicemia. Chrysomya megacephala is an insect pest with worldwide distribution. Its larvae cause secondary myiasis in animals of interest, and adults are pathogen vectors. This study aimed to analyze the effects of the Ballvéria ® biopesticide on the midgut of C. megacephala third-instar maggots. Four concentrations (1, 1.5, 2, and 4%) of the biopesticide were applied to an artificial diet, followed by conditioning of the maggots. Mortality data and samples for histological and ultrastructural analysis were collected every 24 h, for 144 h. Mortality data were analyzed using SPSS 25.0, and lethal concentrations (LC 50 and LC 90 ) were calculated using Probit regression. Concentrations of 2 and 4% resulted in mortality rates of 26 and 36%, respectively. LC 50 and LC 90 were estimated at 5.3 and 10.9%, respectively. Observational, histological, and ultrastructural analyses revealed the presence of tegumentary melanizations, conidia in the midgut, spacing in the basal labyrinth, degeneration of microvilli, absence of the peritrophic membrane, fungal extrusion on the external surface of the midgut, and dispersion of hyphae, conidiophores, and conidia close to muscle fibers. Internally, hyphae are located on microvilli and cell projections. Our data confirm that the Ballvéria ® biopesticide causes cytotoxic effects in the midgut of C. megacephala maggots and can be used as a sustainable alternative in its biological control for Integrate Pest Management. Characteristics may be associated with the presence of autophagic vacuoles and mitochondria with different morphologies observed in TEM; the absence of electron density in mitochondria, becoming more electron lucent, indicates the process of cristolysis.


Introduction
The oriental latrine fly, Chrysomya megacephala (Fabricius, 1794) (Diptera: Calliphoridae), is an insect with distribution worldwide (Sontigun, et al., 2018). Its synanthropic behavior, added to your habit to the visitation of decaying organic matter, allows it to mechanically disperse pathogens, posing a risk to the health of animals and humans (Greenberg, 1973).
Studies that demonstrate the efficiency of chemical agents such as permethrin and deltamethrin in the control of C. megacephala are available in literature (Sukontason, et al., 2005;Oliveira, et al., 2021). However, the indiscriminate use of chemical controllers causes environmental damage and exposes living organisms to harmful molecules (Nascimento & Melnyk, 2016;Nicolopoulou-Stamati, et al., 2016), besides selecting populations of resistant insect pests (Sparks & Nauen, 2015).
The use of biological controllers can be as alternatives to reduce the use of chemical agents. Microbial entomopathogen-based biopesticides have high specificity for pest control (Caleffe, et al., 2019). Beauveria bassiana (Hypocreales: Cordycipitaceae) is a filamentous fungus with entomopathogenic properties (Eley, et al., 2007) indicated for its propriety biocontroller of various insect pests (Zimmermann, 2007), with for example, dipterans (White, et al., 2021a;2021b).
After contact with target organisms, fungal conidia germinate and colonize insect organs; in this process, mycotoxins and enzymes are released, leading to insect death (Bergamo, et al., 2019;Wang, et al., 2021). The midgut is the main target of biopesticides, and thus, it is a key organ to be investigated to analyze the effects of ingested biopesticides (Scudeler, et al., 2016). In addition, this organ has been useful for evaluating sublethal effects, which can compromise the physiological functions of insects (Scudeler, et al., 2013).
The midgut of C. megacephala maggots comprises a single layer of cuboidal epithelial cells, which project for the intestinal lumen from their basement membranes, and each possesses long microvilli covering their apical surfaces, being responsible for absorption of nutrients and secretion of different molecules; within its lumen, it is present the peritrophic membrane, a structure that acts as a mechanical barrier for cell protection (Boonsriwong, et al., 2011). Damage to this organ interferes with the homeostasis of the organism and causes death .
Considering the risks posed by C. megacephala to the health of animals and humans, we investigated potential biocontroller and possible morphological alterations in the midgut epithelium of C. megacephala maggots treated with the commercial product Ballvéria ® , a commercial biopesticide containing B. bassiana isolate IBCB-66 conidials. This study provides data that may help better understand entomopathogen action on susceptible insects and serve as a model for future studies involving target and non-target organisms.
The application of the product was based on the manufacturer's guidelines for dipteran control. Four concentrations, 1% (10 g/L), 1.5% (15 g/L), 2% (20 g/L), and 4% (40 g/L), were prepared by diluting the commercial product in sterile distilled water pH 7.0 at 25 °C. Aliquots (300 μL) of biopesticide solutions were added to the surface of polyethylene Petri dishes (90 × 15 cm) with 10 mL of the artificial diet. Sterile water (pH 7.0) was then added to the control group. Each Petri dish contained 10 maggots. Each bioassay treatment was performed in five replicates (n= 50 per group). The dishes were maintained in a climatized room at 25 ± 2 °C, a 12:12 h photoperiod, and 70 ± 10% RH. After bioassays, observations were made every 24 h using a Zeiss stereomicroscope (Carl Zeiss, Oberkochen, Germany). Mortality was measured every 24 h for 144 h.

Light microscopy
For light microscopy, control and treated maggots were collected 48 h after beginning the bioassays (n= 4 per group).

Scanning electron microscopy (SEM)
For SEM, maggots were quickly cryoanesthetized at -20 °C for 3 min and then their midguts were dissected in insect physiologic solution (0.1 M NaCl, 0.1 M KH2PO4, 0.1 M Na2HPO4) using a stereomicroscope (Carl Zeiss, Oberkochen, Germany). Control and treated maggots were collected 48 h after beginning the bioassays (n= 4 per group). Maggots and dissected midguts were fixed in alcoholic Bouin solution (7.5 mL formaldehyde, 2 mL picric acid, and 0.5 mL acetic acid) for 24 h at room temperature (25 °C). The samples were dehydrated in increasing concentrations of ethanol (70, 80, 90, and 100%, v/v). After dehydration, samples were subjected to critical point drying (Leica EM CPD030, Leica Biosystems, Wetzlar, Germany), and the maggots underwent fracture with a stainless-steel blade. All the samples were coated with a gold layer in an IC-50 metalizer (Shimadzu, Kyoto, Japan) and analyzed using a Quanta 250 scanning electron microscope (FEI Company, Eindhoven, Netherlands) at the Microscopy Center of the Complex of Research Support Centers of the State University of Maringá, Paraná, Brazil.

Transmission electron microscopy (TEM)
For TEM, control and treated maggots from the bioassays (n= 4 per group) were selected and dissected. Samples of the midgut were fixed in 2.5% (v/v) glutaraldehyde and 4% (v/v) paraformaldehyde in 0.1 M phosphate buffer (pH 7.3) for 24 h at room temperature (25 °C), and post-fixed in 1% (w/v) osmium tetroxide diluted in the same buffer for 2 h. The samples were washed in distilled water and a 0.5% (w/v) aqueous solution of uranyl acetate for 2 h, dehydrated in increasing concentrations of acetone (50, 60, 70, 80, 90, and 100% v/v), and embedded in Araldite ® resin (Huntsman Advanced Materials, Salt Lake City, UT, USA). Ultrathin sections (0.5 μm) were stained with toluidine blue for preliminary sample selection.
Ultrathin sections of selected samples were contrasted with uranyl acetate and lead citrate. The samples were analyzed using a Tecnai™ Spirit transmission electron microscope (FEI Company, Eindhoven, Netherlands) at the Electron Microscopy Center of the Institute of Biosciences of Botucatu, São Paulo State University, Botucatu, São Paulo, Brazil.

Statistical analyses
Normality and homogeneity were verified using the Kolmogorov-Smirnov and Bartlett tests, respectively. One-way analyses of variance by Kruskal-Wallis and post hoc Dunn tests were performed (IBM, 2017), with α= 0.05. SPSS (version 25.0; IBM, Armonk, NY, USA) was used for the Probit regression analyses. The results from the bioassays were used to estimate LC50 and LC90. GraphPad Prism 6 (GraphPad Software Inc., San Diego, CA, USA) was used to design mortality graphs.

Results
The control maggots had a vermiform format and cream color ( Figure 1A). Maggots treated with all concentrations of Ballvéria ® maintained the body shape seen in the control group but demonstrated a gradual reduction in mobility until they ceased to move; some regions showed color alterations in the tegument, initially brown, and black after 48 h ( Figure 1B).

Mortality of C. megacephala maggots
The Kruskal-Wallis test indicated significant differences in C. megacephala mortality and entomopathogen concentration (X 2 = 12.476, p= 0.005). Comparisons between the control and treatment groups indicated significant differences (Dunn's test, p < 0.05) (Figure 2). for death of 50% and 90% maggots. A concentration of 0% represents the control group.
In multiple comparison analysis, control, 1, and 1.5% concentrations did not differ significantly according to Dunn's test (1%, p= 0.863; 1.5%, p= 0.057). The other concentrations differed significantly from that of the control (2%, p= 0.007; 4%, p= 0.000). The mortality in the group exposed to a concentration of 1% did not differ between the groups exposed to concentrations of 1.5% (p= 0.324) and 2% (p= 0.057); however, differences were observed in the group exposed to a Research, Society and Development, v. 11, n. 14, e313111436389 2022 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v11i14.36389 6 concentration of 4% (p= 0.000). The mortality in the group exposed to a concentration of 1.5% was not different from that in the group exposed to a concentration of 2%; however, there were differences in the group exposed to a concentration of 4% (p= 0.020). The comparison between the 2 and 4% groups did not demonstrate differences (p= 0.145) ( Figure. 2).

Digestive system
C. megacephala maggots presented a tubular digestive system, varying at the anterior, medial, and posterior regions: foregut, midgut, and hindgut, respectively. The esophagus, cardia, and gastric cecae were present in the anterior region.

Light microscopy
Histologically, the midgut epithelium displayed a simple epithelium supported by muscle fiber bundles and predominantly cuboid cells (Figures 4A and C). In the control samples, the apical region of the cells presented striated border projections into the lumen, and secretion was observed at the cell apex ( Figure 4B). Cuboid cells presented acidophilic cytoplasm and basophilic nuclei, which demonstrated an oval shape and were localized in the apical region of the cells ( Figure   4B).
After 48 h of treatment with 2% Ballvéria ® , the presence of conidia was also recorded in the midgut lumen ( Figures   4C and D). The cells presented alterations in the nuclear region; in addition, cell injuries were observed, with cell lysis and extracellular spaces ( Figure 4E).

SEM
Ultrastructurally, the midgut of third-instar C. megacephala maggots presented a tubular form (Figures 5A and B). In the control group, we externally observed some muscle fiber bundles ( Figure 5C). Internally, cell projections and microvilli were observed ( Figure 5F). After 48 h, the midgut of maggots treated with a 2% solution of Ballvéria® presented extrusion of fungi, and we observed hyphae, conidiogenous cells, and conidia dispersed on the muscle fiber bundles (Figures 5D and E).
Internally, in the midgut lumen, it was possible to observe hyphae present over the microvilli and cell projections ( Figure 5G).

TEM
In control maggots, the midgut comprised cuboid cells with prolonged microvilli to the lumen ( Figures. 6A, C, and E).
Certain bundles of peritrophic membranes were present on microvilli, together with certain bacteria that formed the microbiota of this insect ( Figure 6A). The basal and infranuclear regions of the cuboid cells presented a well-developed basal labyrinth supported by a basement membrane that was externally surrounded by muscle fiber bundles (Figure.  After treatment with 2% Ballvéria ® , microvilli degenerate, different mitochondrial morphologies accumulate, with an electron-lucent aspect, cell projections occur in the apical region, and bundles of peritrophic membranes are absent ( Figures.  6B and D). Spherites and digestive vacuoles were present in the cytoplasm (Figs. 6B and D). Nuclear regions showed nucleolar disintegration in electron-dense small agglomerates (Figures 6B and D). In the basal region of the midgut epithelium, the basal labyrinth demonstrates extracellular space formation between the basement membrane and the basal region of epithelial cells ( Figure 6F). Research, Society andDevelopment, v. 11, n. 14, e313111436389 2022 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v11i14.36389

Discussion
A review of the biological control of Calliphoridae by Caleffe et al. (2019) showed that the major fungal species used to control this family were isolates of Metarhizium anisopliae and B. bassiana. In addition, studies utilizing different isolates of B. bassiana for the control of dipterans are available in literature (White, et al., 2021a;2021b;Quintero-Zapata, et al., 2022). However, to the best of our knowledge, there are no data on the effects of the B. bassiana isolate IBCB-66 on C. megacephala maggots.
In this study, the action of Ballvéria ® commercial product on maggots could have occurred in three ways: i) contact with a contaminated diet, ii) ingestion of the contaminated diet, or iii) both (contact and ingestion). However, we did not observe the presence of fungal structures in the tegumentary epithelium of treated maggots during the observation period, indicating that the mortality data for C. megacephala maggots are related to ingestion.
The absence of fungal structures in the integumentary epithelium may be associated with the locomotion of insects on the moist substrate, providing mechanical removal of conidia before germination, and the cuticle of dipterans can demonstrate physical characteristics different from those observed in other host insects, interfering with the adhesion of conidia in their body (White, et al., 2021a). Fungal conidia show greater interactions with hydrophobic and non-polar surfaces (Holder & Keyhani, 2005), but dipteran maggots show cuticles with polarized characteristics because of the elasticity of the cuticle during this stage of development (Hillerton & Vincent, 1983).
The pathogenicity effects of B. bassiana vary according to the toxins synthetized by fungal isolates and the host in question (Whang, et al., 2021), factors that may interfere with the biocontrol of different insects. The application of the Ballvéria ® (B. bassiana isolate IBCB-66) at concentrations of 2 and 4% resulted in higher mortality, controlling 26% and 36% of the maggots, respectively. These results were inferior to those described by Quintero-Zapata et al. (2022) in Aedes aegypti (Diptera: Culicidae) treated with solutions in 1.5 × 10 7 conidia/mL of the B. bassiana isolate NB3, and similar to isolate GHA, the authors observed that the application of the entomopathogenic fungi resulted in 63 and 30.7% mortality, respectively.
The melanization observed in this study is a process controlled by proteases (Kanost, et al., 2012), enzymes that trigger a cascade of serine proteases, culminating in the activation of phophenoloxidases, a melanogenesis-controlling enzyme, and phenoloxidase, which oxidizes tyrosine to dihydroxyphenylalanine, resulting in the production of dihydroxyphenylalanine and dopamine, precursors of melanin (Dubovskiy, et al., 2008;Nakhleh, et al., 2016). Melanization is related to the generation of reactive oxygen species (ROS) (Kumar, et al., 2003). Its occurrence in dipterans has been confirmed in some studies, for example, the sand fly Lutzomyia longipalpis (Diaz-Albiter, et al., 2012).
ROS originate from the mitochondria (Huang, et al., 2018;Wen, et al., 2016), and their accumulation results in oxidative stress (Felton & Summers, 1995), which in turn can cause cell death via apoptosis, autophagy, or necrosis (Radogna, et al., 2016;Luckhart, et al., 2013). Characteristics may be associated with the presence of autophagic vacuoles and mitochondria with different morphologies observed in TEM; the absence of electron density in mitochondria, becoming more electron lucent, indicates the process of cristolysis.
However, in vitro studies have shown that different mycotoxins significantly reduce cell viability (Bogus, et al., 2021), inhibiting immune responses. The B. bassiana isolate ARSEF 252 secretes oosporein, which is responsible for downregulating the synthesis of antimicrobial peptides and dual oxidase expression in the midgut, reducing immune responses (Wei, et al., 2017). In addition, fungi upregulate the expression and translation of oxidative stress-related genes (e.g., upregulation of superoxide dismutase accelerates the conversion of superoxide ions into molecular oxygen and hydrogen peroxide), elevating the tolerance of B. bassiana to oxidative stress (Xie, et al., 2010).
These changes, combined with the observed histopathological symptoms, were similar to those described in studies using plant-derived toxins and insecticides (Almehmadi, 2011;Ling & Zhang, 2011;Qi, et al., 2011;Scudeler & Santos, 2013;Scudeler, et al., 2016). The extracellular spaces in the basal labyrinth of midgut cells, observed by light microscopy and TEM, were also reported by Daquila et al. (2019), who studied the action of Bacillus thuringiensis on the midgut of Diatraea saccharalis (Lepidoptera: Crambidae) larvae. These alterations may be caused by toxins released by entomopathogenic microorganisms, resulting in their detachment from the basement membrane of epithelial cells.

Conclusion
Our data show that the Ballvéria ® has negative effects on the midgut of C. megacephala maggots. These changes in epithelial cells interfere with nutrient absorption and digestive system homeostasis, causing insect death. In addition, studies using different combinations of B. bassiana isolates with other entomopathogenic microorganisms or plant extracts are encouraged. Biopesticides containing the B. bassiana isolate IBCB-66 showed biocontrol effects in C. megacephala maggots, suggesting its potential for use in the Integrate Pest Management.