*4.10. Statistical Analyses*

Proportion not biting (PNB) was calculated using the procedure described by Ali et al. [22]. As the K & D module bioassay system can handle only four treatments along with negative and positive controls to make direct comparisons among more than four test compounds and to compensate for variation in overall response among replicates, biting deterrent activity was quantified as biting deterrence index (BDI) [22]. The BDI's were calculated using the following formula:

$$
\begin{bmatrix}
BDI\_{i,j,k}
\end{bmatrix} = \begin{bmatrix}
PNB\_{i,j,k} & - & PNB\_{c,j,k} \\
\hline
PNB\_{d,j,k} & - & PNB\_{c,j,k}
\end{bmatrix}
$$

where PNB*i,j,k* denotes the proportion of females not biting when exposed to test compound *i* for replication *j* and day *k* (*i* = 1–4, *j* = 1–5, *k* = 1–2), PNB*c,j,k* denotes the proportion of females not biting the solvent control "*c*" for replication *j* and day *k* (*j* = 1–5, *k* = 1–2) and PNB*d,j,k* denotes the proportion of females not biting in response to DEET "*d"*(positive control) for replication *j* and day *k* (*j* = 1–5, *k* = 1–2). This formula adjusts for inter-day variation in response and incorporates information from the solvent control as well as the positive control.

A BDI value of 0 indicates an effect similar to ethanol, while any value greater than 0 indicates biting deterrent effect relative to ethanol. BDI values not significantly different from 1, are statistically similar to DEET. BDI values were analyzed using SAS Proc ANOVA [31]. To determine whether confidence intervals include the values of 0 or 1 for treatments, Scheffe's multiple comparison procedure with the option of CLM was used in SAS [31]. LC50 values for larvicidal data were calculated by using SAS, Proc Probit [31].

#### **5. Conclusions**

The essential oil of *M. grandiflora* seeds exhibited biting deterrent activity activity similar to DEET. All the major compounds (concentration >1%) except 1-decanol that were present only in seed essential oil were not active biting deterrents which indicated that the major activity of this essential oil might be due to 1-decanol. 1-Decanol also showed promising larvicidal activity. This high activity of 1-decanol indicated the potential of this compound to be developed as an effective mosquito population management tool. Further studies will be continued to evaluate these natural products in different formulations in large scale laboratory bioassays and field trials.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/1420-3049/25/6/1359/s1. The total ion current (TIC) chromatogram of essential oils of the leaves, flowers, immature fruits, mature fruits and seeds of *Magnolia grandiflora* are available as supporting information (Figure S1).

**Author Contributions:** Conceptualization, A.A. and I.A.K.; sample collection and identification, V.R.; methodology, A.A., N.T., B.D., J.M.B., K.H.C.B.; formal analysis, N.T. and A.A.; resources, J.M.B. and V.R.; writing—original draft preparation, A.A. and N.T.; writing—review and editing, A.A., N.T., and V.R.; funding acquisition, I.A.K. All authors have read and agreed to the published version of the manuscript.

**Funding:** This study was supported in part by USDA/ARS grant No. 58-6066-6-043.

**Acknowledgments:** This study was supported in part by USDA/ARS grant No. 58-6066-6-043. We thank Dan Kline, Mosquito and Fly Research Unit, Center for Medical, Agricultural and Veterinary Entomology, USDA-ARS, Gainesville, for supplying mosquito eggs.

**Conflicts of Interest:** The authors declare no conflict of interest. Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture. USDA is an equal opportunity provider and employer.
