*2.6. Enzyme Assays*

The third and fourth instars of *Ae. aegypti* previously used in the larval bioassays were thoroughly washed with dechlorinated distilled water then rinsed using sterile tissue paper [25]. The prepared enzyme homogenates were prepared based on our previous research [15] and kept on ice for further enzyme assays. Furthermore, enzyme estimations of carboxylesterase (α and β), SOD, glutathione S-transferase (GST), and CYP450 were analyzed based on the adapted methodology of Thanigaivel et al. [26].

#### *2.7. Gut Histological and External Physiological Assay*

The My-It-treated (1 × 10<sup>5</sup> conidia/mL) and control larvae were fixed overnight in Bouin's solution and then de-hydrated and fixed in blocks using paraffin wax. Microtome (Model: Leica, Germany) larval tissue blocks were fixed on sterile microscopic glass slides and stained using hematoxylin and eosin for examination under a bright field microscope and images were captured under an Optika vision lite microscope (2.0 ML). The captured midgut images of both My-It-treated and control larvae were further compared for toxicological screening.

The photomicrography assay was performed with the previous adapted protocol of Coelho et al. [27] with slight modifications. The My-It-treated and control fourth instar larvae were sequentially stabilized in an ethanol dehydration range from 35–70% for 25 min at 27 ◦C and fixed on microscopic glass slides. Finally, the sections were observed at 40× magnification under a light microscope (Optika vision lite 2.0 ML).

#### *2.8. Non-Target Toxicity Assay*

The non-target toxicity assay of My-It against beneficial aquatic organisms was performed according to our previous procedure [15]. The non-target beneficial organism *Toxorhynchites splendens* Wiedemann (Culicidae: Diptera) was tested with lethal dosage of My-It (1 × 105, 1 × 1010, 1 × 10<sup>15</sup> and 1 × 10<sup>20</sup> conidia/mL) with three replications and each replication contained twenty larvae. Dechlorinated sterile water without the addition of My-It was kept as the negative control. For individual assay, ten replications followed. Finally, the mortality rate was recorded 24 h post-treatment.
