**4. Discussion**

Determining the mosquito resistance pattern against di fferent groups of synthetic chemicals displays a significant role in managing arthropod vectors [29]. Due to up-surging resistance observed in synthetic chemicals, there is an urgen<sup>t</sup> need for novel substitutes to managing blood-sucking pests [30,31]. Novel discovery of natural materials with diversified blends of active ingredients with potential insecticidal properties may provide a suitable remedy for synthetic chemical resistance [32–34]. Among di fferent biological insecticides, fungal strains have unique modes of action by penetrating the cuticle and blocking the development of pests [35]. Fungal strains generate a vast range of chemicals with an extensive band of actions against insect pests [36]. Amid fungi, the genus Isaria is an important fungal strain composing >100 di fferent species playing a significant role in conserving biodiversity and that are widely used in agriculture and medical treatment [18,37,38]. *I. tenuipes* is most common species of "*Isaria"* with a wide range of insecticidal activity, especially against agriculture pests belonging to the lepidopteran group [39].

The present study revealed that larval toxicity of My-It displayed a significant mortality rate at the maximum dosage of 1 × 10<sup>8</sup> conidia/mL, with a larval mortality rate of more than 94% recorded in all the treated larval instars. Similar to our results, mycotoxins derived from *Aspergillus flavus* also displayed prominent mortality rates of more than 90% at the maximum lethal dosage of 2 × 10<sup>8</sup> conidia/mL [15]. Generally, fungal strains enter the body of a mosquito and create a way to the hemocoel and deliver humoral and cellular immune defensive mechanism straddling by the host of the mosquito species as it tries to overawe the mycotoxin infections [40,41]. Similar to the above statements, My-It delivers acute toxicity to the di fferent instars of dengue mosquito vector.

In general, oviposition represents the vital position in the life cycle of any arthropods, as oviposition inhibition directly signifies the reduction rate in the growth and development of pest populations [42]. Similar to the larvicidal activity, the sub-lethal dosage of My-It significantly a ffects the reproduction stage of dengue mosquito in dose dependent manner. The sub-lethal dosage of My-It (1 × 104) delivered significant ODI percentage as compared to the control; likewise, fungal strains can significantly inhibit the oviposition of other agriculture pests [36]. Previously, the phyto-pathogenic fungal strains derived from *Botrytis cinerea* blocked the oviposition of a major European insect pest (grapevine moth), *Lobesia botrana* [43]. Likewise, the mean number of eggs laid by the gravid female mosquito (fecundity rate) was also reduced considerably due to the maximum sub-lethal dosage of My-It (1 × 104). The volatile and non-volatile metabolites of fungal strains play a significant role in blocking the fecundity rate of arthropods, especially blood-sucking pests [44]. Similar to the above statement, the major allelochemicals in My-It might cause the blockage of the egg-laying capability of female dengue vectors. Similar to our report, a previous in vitro assay revealed that fungal toxins marginally declined the fecundity rate [45]. Similarly, a previous review by Ondiaka et al. [46] stated that the entomo-toxin derived from *Metarhizium anisoplia* displayed a significant fecundity rate against di fferent insect pests.

Generally, insect resistance against any chemical toxins can primarily be accessed through investigating the level of key biomarker enzymes, including detoxifying and digestive enzymes, such as carboxylesterase, SOD, glutathione S-transferase, and, more importantly, the chief detoxifying enzyme cytochrome P-450 [47]. In the present investigation, the sub-lethal dosage of My-It (1 × 10<sup>5</sup> conidia/mL) heavily reduced carboxylesterase (both α and β) enzyme regulation ratios in a dose dependent manner. In support of our findings, the sub-lethal dosage of *A. flavus* heavily inhibited the level of both <sup>α</sup>-β-carboxylesterase and SOD activity [15]. Generally, upregulation of esterase activity will deliver substantial insect resistance against the specific chemicals tested. Resistance developed in esterase-related protein delivered regulatory alterations in the structural genes by modifying the loci of specific genes in insects and also amplified the DNA methylation genes in insect pests [48]. Likewise, Hemingway and Ranson [49] reported that the enzyme families esterase, CYP450s, GST, and SOD are the major four enzymes responsible for pest resistance against the chemical toxins. Backing the above statement, the mycotoxins derived from *I. tenuipes* delivered significant reduction in the carboxylesterase and SOD activity and also delivered a substantial increase in the rate of GST and CYP450 levels.

The gut-histology and physiological alterations results clearly evident that My-It heavily damaged the internal gu<sup>t</sup> cells and external physiology of dengue larvae compared to the control. Similarly, the sub-lethal dosage of *A. flavus* considerably injured the gu<sup>t</sup> epithelial and lumen cells of *Ae. aegypti* larvae [15]. Comparably, a previous review by Rudin and Hecker [50] stated that the pM (peritrophic membrane) stimulates parasite growth in mosquitoes by developing barriers. The above statement was well supported by our present study which shows that the sub-lethal dosage of My-It a ffected the gu<sup>t</sup> cells of dengue larvae.

It is essential to gage the primary and secondary impact of any forms of pesticides upon non-target species [26,31,51]. *Toxorhynchites* are an excellent predator against the dengue larvae '*Aedes*' and are measured to be not hurtful to their well-beings and well-fixed as they are non-blood feeders and considered to be a good biological predator for reducing the populations of blood-sucking mosquitoes [52]. Since they share the same ecological regions as dengue larvae, it is essential to investigate the non-toxicity screening of same chemicals tested against dengue larvae. The non-target screening of My-It against the giant mosquito (*Tx. splendens*) showed they are less at-risk (maximum 45% mortality rate), even if they are treated with the maximum dosage of My-It (1 × 1020) which is the highest dosage used in the larvicidal assays. It is evident that biologically-derived pesticides, especially mycotoxins and their related compounds, were target specific and harmless or less toxic to the beneficial species. Thus, the present toxicological investigation of My-It recommends that it is a highly favorable biological agen<sup>t</sup> in managing medically-challenging arthropods, especially the dengue mosquito, and its non-toxic activity against aquatic predators will add on to its biologically safe insecticides. Further investigation on the active allelochemicals of My-It and its specific mode of action against the dengue mosquito vector's biological activity needs to be intensely inspected.

**Author Contributions:** S.K., P.V.-S., S.S.-N. and P.K. designed the research plan and drafted the manuscript. S.K., P.V.-S., S.S.-N., V.R. and P.K. performed the experimental works and data compilation. R.G., V.R., H.F.K., N.R., K.A., T.-J.K., M.A.E.-S., P.K. and S.K. coordinated the work and discussed the results. S.K., S.S.-N. and P.K. revised the manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was partially funded by the Post-Doctoral Program, Chiang Mai University, Thailand. It was also financially supported by the D. S. Kothari Postdoctoral Fellowship (2019-20-BL/18-19/0168) program under University Grants Commission, New Delhi, India, M.A. El-Sheikh extends his appreciation to the Researchers Supporting Project Number (RSP-2020/182), King Saud University, Riyadh, Saudi Arabia.

**Acknowledgments:** This research was partially funded by the Post-Doctoral Program, Chiang Mai University, Thailand, and was also financially supported by the D. S. Kothari Postdoctoral Fellowship (2019-20-BL/18-19/0168) program under University Grants Commission, New Delhi, India. The gran<sup>t</sup> from the Researchers Supporting Project Number (RSP-2020/182), King Saud University, Riyadh, Saudi Arabia is gratefully acknowledged.

**Conflicts of Interest:** The authors declare no conflict of interest.
