Histological Examination of Ae. aegypti Larvae

Third-instar Ae. aegypti larvae were treated with an LC₅₀ of honokiol (6.5 mg/L) or magnolol (25 mg/L) in all experiments as described above. At 24 h post-treatment, Ae. aegypti larvae untreated and treated with honokiol or magnolol were immediately fixed in Bouin’s fluid^{75 (link)} at 4 °C for 24 h. Larvae were then dehydrated in an ethanol-tetrahydrofuran-xylene series and embedded in Paraplast X-tra (Sigma-Aldrich). The embedded preparations of the larvae were sectioned at a 5 μm thickness using a Microm HM 340E rotary microtome (Thermo Scientific, Walldorf, Germany). The sections were dried at 40 °C overnight, subsequently deparaffinized with CitriSolv (Fisher Scientific, Fair Lawn, NJ, USA), and rehydrated with a series of ethanol in phosphate-buffered saline (PBS) solutions^{30 (link)}. The rehydrated sections were stained in Weigert’s iron hematoxylin for 30 s, followed by Carson’s trichrome staining procedure⁷⁶ . This staining protocol stained the columnar and goblet cells of the midgut blue and red, respectively. These sections were then dehydrated, cleared in xylene, and mounted in EMS Permount (Electron Microscopy Sciences, Hatfield, PA, USA). Images were observed and captured using a DMIL LED microscope (Leica Microsystems, Wetzlar, Germany) equipped with a Leica MC 170 HD. Observations were taken of 15 larvae under the microscope.