Treatment dosage (conidia/ml)

**Figure 4.** Oviposition deterrent index and fecundity evaluation of My-It against dengue mosquitoes. Means (± SE) followed by the same letters above bars indicated no significant difference (*p* ≤ 0.05) using ANOVA analysis. The different letters (a–d) indicate significant differences between the control and treatments.

#### *3.3. Fecundity of My-It*

The sub-lethal dosage significantly reduces the mean number of eggs laid by the female dengue mosquito in a dose dependent manner. At the maximum dosage of 1 × 104, My-It showed maximum reduction in fecundity rate (20.1-F4,20 = 14.22, *p* ≤ 0.001), followed by 1 × 10<sup>3</sup> (50.11-F4,20 = 14.22, *p* ≤ 0.001), 1 × 10<sup>2</sup> (66.1-F4,20 = 14.22, *p* ≤ 0.001), 1 × 10<sup>1</sup> (85.45-F4,20 = 14.22, *p* ≤ 0.001), and the control (120.45-F4,20 = 14.22, *p* ≤ 0.001) mean number of eggs (Figure 4).

#### *3.4. Enzyme Inhibition of My-It*

A sub-lethal dosage of My-It statistically regulates the major enzymes of both third and fourth instars of dengue larvae. The level of α- carboxylesterase was significantly reduced in a concentration dependent manner on both the larval instars. The level of α- carboxylesterase in the third instar reduced at the maximum rate of 0.3451 mg/protein at the maximum dosage of 1 × 10<sup>5</sup> (F4,20 = 18.99, *p* ≤ 0.001), and it was not significant with 1 × 10<sup>4</sup> (0.312 mg/protein-F4,20 = 18.99, *p* ≤ 0.001) and 1 × 10<sup>3</sup> (0.351 mg/protein-F4,20 = 18.99, *p* ≤ 0.001). However, there is no significant di fference between 1 × 10<sup>2</sup> (0.5110 mg/protein-F4,20 = 18.99, *p* ≤ 0.001) and 1 × 10<sup>1</sup> (0.6543 mg/protein-F4,20 = 18.99, *p* ≤ 0.001) (Figure 5A). Similar trends were observed in the α- carboxylesterase level in fourth instars with the maximum reduction rate observed in My-It 1 × 10<sup>5</sup> (0.4514 mg/protein-F4,20 = 20.12, *p* ≤ 0.001) which is significant, as is the case with with 1 × 10<sup>2</sup> and 1 × 10<sup>1</sup> dosages. However, there was no statistical significance observed with the 1 × 10<sup>4</sup> and 1 × 10<sup>3</sup> dosages (F4,20 = 20.12, *p* ≤ 0.001) (Figure 5A).

**Figure 5.** (**A**) α-carboxylestrase; (**B**) β-carboxylestrase; ( **C**) SOD; (**D)** GST; (**E**) CYP450 enzyme activity of third and fourth instar larvae of *Ae. aegypti* after treatment with My-It. The data were fitted on a polynomial (regression) model. Letters (a–d) mean (± SE) followed by the same letters above bars indicated no significant di fference (*p* ≤ 0.05) using ANOVA analysis.

*J. Fungi* **2020**, *6*, 196

The level of β- carboxylesterase statistically declined at the maximum sub-lethal dosage of My-It of 1 × 10<sup>5</sup> (0.6700 mg/protein-F4,20 = 18.25, *p* ≤ 0.001) and (0.823 mg/protein-F4,20 = 16.66, *p* ≤ 0.001) in third and fourth instars, respectively (Figure 5B). However, the level of β- carboxylesterase was 1.600 mg/protein and 1.9320 mg/protein in the third and fourth instars, respectively, at the minimal dosage of My-It (1 × 101) (Figure 5B).

Correspondingly, the level of SOD also declined in a concentration dependent manner with the maximum enzyme reduction observed in the My-It dosage of 1 × 10<sup>5</sup> conidia/mL with 9.76 U/mg (F4,20 = 12.45, *p* ≤ 0.001) and 11.32 U/mg (F4,20 = 17.77, *p* ≤ 0.001) in the third and fourth instars, respectively (Figure 5C). However, the level of SOD increased to 26.70 U/mg and 28.32 U/mg in the third and fourth instars, respectively.

The level of glutathione S-transferase uplifted steadily in both third and fourth instar larvae treated with My-It. The level increased to 0.589 mg/min (F4,20 =18.27, *p*≤0.001) and 0.4995 mg/min (F4,20 = 12.44, *p* ≤ 0.001) in the third and fourth instar larvae, respectively, treated with My-It 1 × 10<sup>5</sup> conidia/mL (Figure 5D). However, there is no significant difference between My-It 1 × 10<sup>5</sup> conidia/mL, My-It 1 × 10<sup>4</sup> conidia/mL, and My-It 1 × 10<sup>3</sup> conidia/mL in both of the treated larvae.

The enzyme activity of CYP450 increased in a dose dependent manner with the maximum enzyme rate in My-It 1 × 10<sup>5</sup> conidia/mL in third (8.3341 μmol 7-OH/mg larvae/min-F4,20 = 25.22, *p* ≤ 0.001) and fourth (8.1320 μmol 7-OH/mg larvae/min-F4,20 = 18.88, *p* ≤ 0.001) instar larvae, respectively (Figure 5E). In all the treatments, third instar larvae were slightly sensitive in the enzyme regulations of the sub-lethal dosage of My-It (Figure 5).

#### *3.5. The <sup>E</sup>*ffi*cacy of of My-It on Gut-Histology*

Treatment with a sublethal dosage of My-It significantly induced adverse effects on the gu<sup>t</sup> though uniformity in the epithelial layer (Epi), gu<sup>t</sup> lumen (Lu), and peritrophic membrane (pM ) were detected in the control larva (Figure 6A), whereas the cellular organelles were severely affected and cranked in the treatment with My-It (1 × 10<sup>5</sup> conidia/mL) (Figure 6B).

**Figure 6.** Cross-section through mid-gut of fourth instar *Ae. aegypti* treated with My-It. (**A**), control, compared with (**B**), treated. (Epi) vacuolated gu<sup>t</sup> epithelium; (Lu) gu<sup>t</sup> lumen; (pM) peritrophic membrane.

#### *3.6. The <sup>E</sup>*ffi*cacy of My-It on the External Physiology of Ae. aegypti Larvae*

The external physiological analysis of the fourth instar larvae showed that the sub-lethal dosage of My-It (1 × 10<sup>5</sup> conidia/mL) drastically affected the gu<sup>t</sup> lumen (GL), segments (S), epithelial layer (EL), and anal segments (AS) (Figure 7B), whereas in the control larvae, the gu<sup>t</sup> cells, including EL, AS, and GL, appeared to be normal (Figure 7A).

**Figure 7.** Photo-micrographic analysis of fourth instar larvae of *Ae. aegypti* (**A**) control larvae and (**B**) My-It-treated larvae. (GL)—gut lumen; (S)—segments; (AS)—anal segments; (EL)—epithelial layer.

#### *3.7. Non-Target Toxicity of My-It*

The non-target toxicity of the aquatic predator *Tx. Splendens*-discriminating dosage of My-It 1 × 10<sup>20</sup> (3 to 4 fold higher dosages used in larvicidal assay) displayed a lower mortality rate (45.43%-F4,20 = 25.66, *p* ≤ 0.001), followed by My-It 1 × 10<sup>15</sup> (32.14%-F4,20 = 25.66, *p* ≤ 0.001), My-It 1 × 10<sup>10</sup> (25.40%-F4,20 = 25.66, *p* ≤ 0.001), My-It 1 × 10<sup>5</sup> (14.43%-F4,20 = 25.66, *p* ≤ 0.001), and the control (3.20%-F4,20 = 25.66, *p* ≤ 0.001). There is significant difference between the My-It treatments and the control (Figure 8).

**Figure 8.** Impact of My-It on the non-target organism *Tx. splendens*. Letters (a–d) mean (± SE) followed by the same letters above bars indicate no significant difference (*p* ≤ 0.05) using Probit analysis.
