1. Expression and Isolation of Target Proteins with a Baculovirus-mediated Insect Cell Expression System
<ol>
	<li>Directionally clone blunt-ended PCR products of target proteins from house flies.
	<ol>
		<li>Design PCR primers of green fluorescent protein (GFP) and the house fly Md&#945;E7 gene based on their sequences and the special requirements of the chosen vector (Table 1).</li>
		<li>Use a thermostable, proofreading DNA polymerase and primers from step 1.1.1 to perform a 150&#160;&#181;L PCR reaction (30&#160;&#181;L of reaction buffer, 3&#160;&#181;L of 10 mM dNTPs, 1.5&#160;&#181;L of DNA polymerase, 6&#160;&#181;L of house fly template DNA, 7.5&#160;&#181;L of forward primer, 7.5&#160;&#181;L of reverse primer, with water to a final volume 150&#160;&#181;L). Heat the PCR reaction to 98&#160;&#176;C for 30 s, followed by 35 cycles of 98&#160;&#176;C for 10 s, 53&#160;&#176;C for 30 s, and 72&#160;&#176;C for 60 s, and then a final extension at 72&#160;&#176;C for 2 min).
		<ol>
			<li>Run a 1% agarose gel with 150&#160;&#181;L of PCR product.</li>
		</ol>
		</li>
		<li>Excise the target DNA fragments from the agarose gel with a sharp, clean scalpel: 1317 bp for Md&#945;E7 and 858 bp for GFP. Purify the DNA using commercially available gel extraction kit following manufacturer&#8217;s protocol (Table of Materials). Dissolve the purified DNA in 15&#160;&#181;L of distilled water.</li>
		<li>Run a 1% agarose gel with 1&#160;&#181;L of purified DNA to check integrity. Use another 1&#160;&#181;L of purified DNA to measure concentrations with the spectrophotometer.</li>
	</ol>
	</li>
	<li>Construct an entry plasmid for target proteins
	<ol>
		<li>Set up the cloning reaction. Mix freshly purified DNA products from step 1.1.3 and commercially available entry vectors containing attL-sites (Table of Materials) at a molar ratio of 1:1 (0.5-2&#160;&#181;L of fresh PCR product at 70-200 ng/&#181;L concentrations: 0.5&#160;&#181;L vector). Then add 1&#160;&#181;L of commercially available salt solution (1.2 M NaCl2 and 0.06 M MgCl2) and add water to a final volume of 6&#160;&#181;L. Mix gently and incubate at room temperature for 1 h.</li>
		<li>Transfer 4&#160;&#181;L of cloning reaction products in step 1.2.1 into 50&#160;&#181;L of chemically competent E. coli cells. Incubate on ice for 30 min. Heat-shock the cells for 30 s in a 42&#160;&#176;C water bath without shaking.</li>
		<li>Put the tube back in ice for another 2 min. Add 250&#160;&#181;L of room temperature S.O.C. medium (2% tryptone; 0.5% yeast extract; 10 mM NaCl; 2.5 mM KCl; 10 mM MgCl2; 10 mM MgSO4; 20 mM glucose). Incubate at 37&#160;&#176;C for 1 h with gently shaking.</li>
		<li>Spread 50-200&#160;&#181;L of the bacterial culture in step 1.2.3 on the selective LB plates (1 g of tryptone; 1 g of NaCl; 0.5 g of yeast extract; 1.5 g of agar dissolved in 100 mL of distilled water. Autoclave and add 0.1% of 50 mg/mL kanamycin). Incubate LB plates for 16 h at 37&#160;&#176;C for the colony growth.</li>
		<li>Pick 5-10 colonies and re-suspend them individually into 5&#160;&#181;L of distilled water.</li>
		<li>Perform PCR by adding 5&#160;&#181;L of reaction buffer, 0.5&#160;&#181;L of 10 mM dNTPs, 0.25&#160;&#181;L of DNA polymerase, 1&#160;&#181;L of suspended colonies, 1.25&#160;&#181;L of 10&#160;&#181;M M13 forward primer, 1.25&#160;&#181;L of 10&#160;&#181;M reverse primer of the target gene, and water to final volume of 25&#160;&#181;L. Heat the PCR reaction to 98&#160;&#176;C for 30 s, followed by 35 cycles of 98&#176; C for 10 s, 53&#160;&#176;C for 30 s, and 72&#160;&#176;C for 60 s, and then a final extension at 72&#160;&#176;C for 2 min (Table 1).</li>
		<li>Re-culture 3&#160;&#181;L of suspended colonies from step 1.2.5 in TB media (100 mL of phosphate buffer containing 0.17 M KH2PO4 and 0.72 M K2HPO4 with 450 mL of base broth medium containing 6 g of tryptone, 12 g of yeast and 2 mL of glycerol, sterilized, containing 0.1% 50 mg/mL kanamycin) for 16 h. Extract ultra-pure plasmid DNA following manufacturer&#8217;s protocol.</li>
		<li>Use 200 ng/&#181;L ultra-pure plasmid from step 1.2.7 for commercial Sanger sequencing with M13 forward and reverse primers. Verify the correct insertion of the target gene in vector based on the sequence map provided by manufacturer.</li>
		<li>Store the sequence-verified ultra-pure plasmid DNA samples of Md&#945;E7 and GFP in -20&#160;&#176;C. 
		Note: These are the entry plasmid DNA of Md&#945;E7 and GFP.</li>
	</ol>
	</li>
	<li>Construct a recombinant baculovirus by performing Lambda recombination (LR) reaction.
	<ol>
		<li>Respectively, mix 1&#160;&#181;L of 300 ng/&#181;L entry plasmid DNA of GFP, Md&#945;E7 from step 1.2.7 or entry plasmid DNA of chloramphenicol acetyltransferase gene (CAT) provided by the cell transfection kit with 5&#160;&#181;L of commercially available (containing attL sites) C-term linear DNA (containing attR sites) in 250&#160;&#181;L PCR tubes. Add TE buffer to make a total volume of 8&#160;&#181;L. 
		Note: The reactions of GFP and CAT were used as control groups.</li>
		<li>Add 2&#160;&#181;L of commercially available Lambda recombination (LR) enzyme mix (Table of Materials) into each mixture of step 1.3.1 to catalyze the LR reaction. Gently mix and incubate at 25&#160;&#176;C overnight (&#8776;16 h). 
		Note: The LR reaction facilitates the recombination of an attL-containing entry plasmid DNA with an attR-containing C-term linear DNA to generate an attB-containing expressing virus. This step produces the recombinant baculovirus for each target protein.</li>
		<li>Dilute 1&#160;&#181;L of LR reaction products from step 1.3.2 20X. Perform PCR using a Polyhedrin forward primer and V5 reverse primer (Table 1). Use 5&#160;&#181;L of PCR products to run a 1% agarose gel to check the quality. Figure 1 shows an example of the LR reaction product from the Md&#945;E7 gene.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58106/58106fig1.jpg" /> 
Figure 1: Example results of Md&#945;E7 LR reaction by PCR analysis. Dilute 2&#160;&#181;L of LR reaction 200-fold and use 2&#160;&#181;L of dilution together with Polyhedrin forward primer and V5 reverse primer to perform a 25&#160;&#181;L PCR. Use 5&#160;&#181;L of PCR products to run 1% agarose gel to check the quality of LR reaction. a
<ol start="4">
	<li>Transfect insect Sf9 cells
	<ol>
		<li>Culture insect Sf9 cells with 5 mL of complete cell growth medium (serum-free medium with 10% fetal bovine serum (FBS)) in T25 treated flasks at 27 &#730;C.</li>
		<li>Remove the supernatant and flush bottom-attached cells with 3 mL of fresh complete cell growth medium. Transfer 0.5 mL of re-suspended cells into a new T25 treated flask. Add 4.5 mL of complete growth medium. Incubate at 27&#160;&#176;C for 3-4 days until the next transfer.</li>
		<li>Seed 2 mL of log-phase growth insect Sf9 cells culture (&#8776;3.0-5.0&#215;106 cells) evenly on the cell culture well. Allow cells to attach for at least 3 h at room temperature in the hood.</li>
		<li>Check the cell attachment by observing with an inverted phase microscope at 250X. Remove the cell culture medium and replace with 2 mL of Grace&#8217;s insect medium.</li>
		<li>Prepare transfection mixture A solution (8&#160;&#181;L of commercially available cell infection reagent (Table of Materials) with 100&#160;&#181;L of Grace&#8217;s insect medium) and transfection mixture B solution of each target gene (9&#160;&#181;L of LR reaction products from step 1.3 with 100&#160;&#181;L of unsupplemented Grace&#8217;s insect medium) in 1.5 mL centrifuge tubes, respectively.</li>
		<li>Gently mix transfection mixture A and B together by tapping the tubes. Incubate at room temperature for 35 min in the hood.</li>
		<li>Evenly add the mixture from step 1.4.6 dropwise onto the seeded cells from step 1.4.4. Seal wells with tapes and incubate at 27&#160;&#176;C overnight.</li>
		<li>Replace 2 mL of Grace&#8217;s insect medium with 2 mL of complete growth medium. Add 100&#160;&#181;M ganciclovir into each well to negatively select against non-recombinant baculovirus. Seal wells with tape and incubate at 27&#160;&#176;C for 72 h.</li>
		<li>Collect 72 h post infection cell culture medium from each well and transfer to 1.5 mL centrifuge tubes. Centrifuge at 1,500 x g for 5 min at 4&#160;&#176;C to remove cells or large debris.</li>
		<li>Transfer the supernatant into new 1.5 mL centrifuge tubes. Store them at 4&#160;&#176;C with protection from light. 
		Note: These are the P1 virus stock solutions for each target gene.</li>
		<li>Amplify the low-titer P1 viral stock (1&#215;105-1&#215;106 pfu/mL) to a high titer P2 viral stock (5&#215;107-1&#215;108 pfu/mL).</li>
		<li>Seed 2 mL of log-phase growth insect Sf9 cells culture (&#8776;3.0-5.0&#215;106 cells) evenly on the cell culture well. Allow cells to attach for at least 3 h at room temperature in the hood.</li>
		<li>Inoculate 5&#160;&#181;L of P1 virus stock obtained in step 1.4.10 in the cell seeded well of step 1.4.12, respectively. Then, add 100&#160;&#181;M ganciclovir to each well. Seal wells and incubate at 27&#160;&#176;C for 72 h.</li>
		<li>Collect P2 virus stock solutions of 72 h post infection. Store at 4&#160;&#176;C with protection from light. 
		Note: These are the P2 virus stock solutions for each target gene.</li>
		<li>(Optional) Repeat steps 1.4.12-1.4.14 with 5&#160;&#181;L of P2 virus stock to collect P3 virus stock solutions. 
		Note: These are the P3 virus stock solutions for each target gene.</li>
		<li>Store all the constructed baculovirus at 4&#160;&#176;C with protection from light. 
		Note: The GFP and CAT gene were served as control group. Figure 2 showed the signs of cells when infected by GFP-recombinant baculovirus at different amplification stages.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58106/58106fig2.jpg" /> 
Figure 2: Example of infected signs of Sf9 cells at different baculovirus amplification stages. The figure shows GFP-recombinant baculovirus cells under natural light and fluorescent light. At the P1 infection stage, the infection ratio is low. At the P2 infection stage, the infection ratio was significantly enhanced. At the P3 infection stage, almost all the cells showed symptoms of detachment from cell culture plate, increases of cell diameter, as well as the cessation of cell growth. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58106/58106fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="5">
	<li>Large-scale expression of target proteins in insect Sf9 cells
	<ol>
		<li>Culture 10 mL of log phase growth insect Sf9 cells with complete growth medium in T25 non-treated flasks.</li>
		<li>Add 200&#160;&#181;L of P2 virus stock solution (obtained in step 1.4.14) to infect cell cultures in step 1.5.1 for 72 h.</li>
		<li>Collect 72 h post infection cell culture medium. Centrifuge at 1,500 x g for 5 min at 4&#160;&#176;C.</li>
		<li>Discard supernatant and wash pellets twice with 1 mL of 0.1 M PBS buffer (pH 7.5).</li>
		<li>Fully dissolve cell pellets with 1 mL of insect cell protein extraction &#38; lysis buffer (Table of Materials). Add 10% glycerol in cell lysis. Immediately store at -80&#160;&#176;C.</li>
		<li>Repeat steps 1.5.1-1.5.5 with P2 virus stock solution of CAT proteins to serve as the control group. 
		Note: Repeat step 1.5 in triplicate for different protein preparations.</li>
	</ol>
	</li>
</ol>
2. A Cell-based MTT Cytotoxicity Assay
<ol>
	<li>Culture 5 mL of log phase growth (1.5-2.5&#215;106 cells/mL) insect Sf9 cells with complete growth medium in T25 non-treated flasks.</li>
	<li>Add 25&#160;&#181;L of P1 virus stock solution (obtained in step 1.4.14) to infect cell cultures in step 2.1 for 48 h with gentle shaking at 27 &#730;C.</li>
	<li>Prepare 100 mM permethrin standard stock solutions in acetonitrile. Use acetonitrile to dilute to 50 mM, 25 mM, 12.5 mM and 6.25 mM by adding 500&#160;&#181;L, 250&#160;&#181;L, 125&#160;&#181;L and 62.5&#160;&#181;L permethrin stock solution in a total volume of 1,000&#160;&#181;L, respectively.</li>
	<li>Evenly seed 200&#160;&#181;L of infected cell cultures from step 2.2 supplemented with 300&#160;&#181;L of complete growth medium into 24-well plate at a density of 2&#215;105 cells/mL.</li>
	<li>Add 4&#160;&#181;L of permethrin standard solutions (6.25 mM, 12.5 mM, 25 mM and 50 mM) into each well to make a final concentration 50&#160;&#181;M, 100&#160;&#181;M, 200&#160;&#181;M and 400&#160;&#181;M, respectively. Only add 4&#160;&#181;L of acetonitrile into wells for cell viability calculations (Figure 3).</li>
	<li>Seal plates with tapes and incubate at 27&#160;&#176;C for 48 h with protection from light.</li>
	<li>Prepare 5 mg/mL MTT (Thiazolyl Blue Tetrazolium Bromide) reagent dissolved in buffer (2.0 g of NaCl, 0.05 g of KCl, 0.36 g of NaH2PO4&#8729;2H2O, 0.28 g of NaH2PO4, 0.05 g of KH2PO4 dissolved in 200 mL of distilled water, pH 7.5).</li>
	<li>Remove cell culture medium from step 2.6 after 48 h incubation without touching the bottom-attached cell layer. Add 200&#160;&#181;L of MTT reagent in step 2.7 into each well. Incubate at 37&#160;&#176;C for 4 h until dark purple formazan precipitates formed in each well (Figure 3).</li>
	<li>Incubate at 37&#160;&#176;C for 4 h until dark purple formazan precipitates form in each well. Add 500&#160;&#181;L of DMSO to fully dissolve precipitates (Figure 3).</li>
</ol>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58106/58106fig3.jpg" /> 
Figure 3: Example of MTT results. After 48 h post infection with P1 virus stock solution of CAT recombinant baculovirus solution, evenly seed 500&#160;&#181;L cells expressing CAT gene in a 24-well cell culture plate. Respectively, add 4&#160;&#181;L permethrin at a different dose (6.25 mM, 12.5 mM, 25 mM and 50 mM) in each row to make the final concentration to 50&#160;&#181;M, 100&#160;&#181;M, 200&#160;&#181;M and 400&#160;&#181;M. The control row was treated with acetonitrile only and marked as CK in the plate. (A) After 48 h of incubation at 27&#160;&#176;C, discard the cell culture medium on the upper layer and replace with 200&#160;&#181;L of yellow colored MTT reagents. Then incubate at 37&#160;&#176;C for 48 h. Dark purple colored reduction precipitates form in each well indicating that the survival cells are capable of metabolizing the yellow color MTT reagents into the dark purple colored precipitate. (B) 500&#160;&#181;L of DMSO solvent was added into each well to dissolve the formed precipitate. The absorbance value of each well was detected by spectrophotometer at 540 nm. As permethrin concentrations increase , the color will gradually change from dark red to light red, suggesting that the cell viabilities were gradually decreased. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58106/58106fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="10">
	<li>Transfer 200&#160;&#181;L of dissolved solution into 96-well plate. Measure absorbance values at 540 nm using a microplate reader (Table of Materials).</li>
	<li>Calculate cell viability by comparing the absorbance values of permethrin treated cells with those of only acetonitrile treated cells.</li>
	<li>For the control group, repeat all steps with P2 virus stock solution of chloramphenicol acetyltransferase (CAT) recombinant baculovirus obtained in step 1.4.</li>
	<li>Repeat 4 times for different virus preparations.</li>
</ol>
3. In Vitro Metabolic Assay
<ol>
	<li>Prepare 100 mM permethrin standard stock solutions in acetonitrile. Use acetonitrile to dilute to 50 mM, 25 mM, 12.5 mM and 6.25 mM. Detect the corresponding peak area under each concentration using HPLC. Create and calculate the standard curve of permethrin based on the peak area with different permethrin concentrations.</li>
	<li>Measure the protein concentrations in step 1.5 with Bradford methods21.</li>
	<li>Prepare 700&#160;&#181;L of metabolic reaction with 40&#160;&#181;M permethrin standard and 1 mg of proteins obtained in step 1.5 dissolved in 0.2 M Tris-HCl buffer (pH 7.4). Incubate at 30&#160;&#176;C for 2 h with gentle shaking. Protect from light.</li>
	<li>Quench the reaction by adding 700&#160;&#181;L of ice-cold acetonitrile. Incubate at 30 &#730;C for another 30 min with gentle shaking. Protect from light.</li>
	<li>Centrifuge the reaction mixture at 16,000 x g for 2 min at room temperature. Collect the supernatant and filter through a 0.45&#160;&#181;m membrane. Transfer the filtration into ultraclean brown glass vials for HPLC analysis.</li>
	<li>Run the HPLC under the optimal chromatographic conditions (Mobile phase A: 90% acetonitrile and 10% water; Mobile phase B: 5% acetonitrile adjusted to pH 2.3 with 85% phosphoric acid). Gradient elute with a flow rate of 1 mL/min and measure at a wavelength of 232 nm.</li>
	<li>Calculate the depletion percentage of permethrin by comparing with reactions without protein sample added.</li>
	<li>Use CAT protein obtained in step 1.5.6 to serve as the control.</li>
	<li>Repeat all the steps with different protein preparations in step 1.5.7.</li>
</ol>

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<li>Nakamura, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+in+vitro+metabolism+of+a+pyrethroid+insecticide%2C+permethrin%2C+and+its+hydrolysis+products+in+rats%5BTitle%5D)+AND+%22Toxicology%22%5BJournal%5D)">The in vitro metabolism of a pyrethroid insecticide, permethrin, and its hydrolysis products in rats.</a> Toxicology. 235 (3), 176-184 (2007).</li>
<li>Kruger, N. J. The Bradford method for protein quantitation. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aKruger%2C+NJ+%22The+protein+protocols+handbook%22">The protein protocols handbook</a>. , Humana Press. 15-21 (2002).</li>
<li>Macauley-Patrick, S., Fazenda, M. L., McNeil, B., Harvey, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Heterologous+protein+production+using+the+Pichia+pastoris+expression+system%5BTitle%5D)+AND+%22Yeast%22%5BJournal%5D)">Heterologous protein production using the Pichia pastoris expression system.</a> Yeast. 22 (4), 249-270 (2005).</li>
<li>Berger, I., Fitzgerald, D. J., Richmond, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Baculovirus+expression+system+for+heterologous+multiprotein+complexes%5BTitle%5D)+AND+%22Nature+Biotechnology%22%5BJournal%5D)">Baculovirus expression system for heterologous multiprotein complexes.</a> Nature Biotechnology. 22 (12), 1583(2004).</li>
<li>Terpe, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Overview+of+bacterial+expression+systems+for+heterologous+protein+production%3A+from+molecular+and+biochemical+fundamentals+to+commercial+systems%5BTitle%5D)+AND+%22Applied+Microbiology+and+Biotechnology%22%5BJournal%5D)">Overview of bacterial expression systems for heterologous protein production: from molecular and biochemical fundamentals to commercial systems.</a> Applied Microbiology and Biotechnology. 72 (2), 211(2006).</li>
<li>Bulter, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Functional+expression+of+a+fungal+laccase+in+Saccharomyces+cerevisiae+by+directed+evolution%5BTitle%5D)+AND+%22Applied+Microbiology+and+Biotechnology%22%5BJournal%5D)">Functional expression of a fungal laccase in Saccharomyces cerevisiae by directed evolution.</a> Applied Microbiology and Biotechnology. 69 (2), 987-995 (2003).</li>
<li>Stepanenko, A. A., Dmitrenko, V. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Pitfalls+of+the+MTT+assay%3A+Direct+and+off-target+effects+of+inhibitors+can+result+in+over%2Funderestimation+of+cell+viability%5BTitle%5D)+AND+%22Gene%22%5BJournal%5D)">Pitfalls of the MTT assay: Direct and off-target effects of inhibitors can result in over/underestimation of cell viability.</a> Gene. 574 (2), 193-203 (2015).</li>
</ol>

In recent decades, heterologous expression systems have been widely used to express and isolate large amounts of proteins, thus allowing biochemical and functional determination and characterization of enzymes in vitro. To date, several different model systems including Escherichia coli, Pichia pastoris, Sacccharomyces cerevisiae, and Spodoptera frugiperda have been adapted for recombinant protein expression, and the choice of the in vitro system is crucial for large scale generation of interested proteins22,23,24,25. In our study, the baculovirus-mediated insect cell expression system to generate house fly carboxylesterases was chosen because it provides a similar intracellular environment to insect cells and maintains some important eukaryotic processing capabilities9. Despite the advantages of the baculovirus-mediated expressing system, its limitations should also be addressed. The baculovirus-mediated insect cell expression system can produce target proteins only if the insect cells were infected with constructed recombinant baculovirus. The current development of creating stably transformed insect cell lines has made it possible not only to constitutively produce interested proteins in the absence of baculovirus infection, but also to improve cell capabilities of glycosylating and folding generated proteins through engineering modifications9.
To better investigate the roles of carboxylesterases in metabolizing insecticides in cells and thus protecting cells from chemical damages, the MTT assay was conducted to measure the cell cytotoxicity against different permethrin treatments. During this process, a lot of experimental parameters associated with cell metabolism, such as cell medium compositions, cell growth rate, concentration and consumption of energy supply metabolites, can influence the MTT reduction and thereby lead to inaccurate results. To minimize the over/underestimation of the MTT assay, keep the cell culture medium, cell growth state, and the incubation conditions consistent among different experimental treatments26. In order to determine the cell viability effects caused by baculovirus infection rather than permethrin, we showed that the infection process of GFP-recombinant baculovirus to insect Sf9 cells, and found that at the P2 infection stage, the cells have the highest infection ratio with lowest cell death ratio compared with to other infection stages (Figure 2). This can better explain the reason of choosing cells at P2 infection stage to conduct the MTT assay.
By comparing the viabilities of cells when infected by Md&#945;E7- and CAT-recombinant baculovirus in an MTT study, we found an enhanced viability of cells when expressing Md&#945;E7 proteins, which can better support the metabolic roles of carboxylesterases to permethrin in insect cells and protect cells from chemical damages (Figure 4). The in vitro metabolic study, which is recognized as a direct method to reflect the metabolic capabilities of carboxylesterases toward insecticides, should also been explored to compensate the limitations of MTT assay in better characterizing protein metabolic functions.
In this study, the heterologous expression of carboxylesterases in insect Sf9 cells with a baculovirus-mediated expression system, the measurement of cell viabilities toward different permethrin treatments by the MTT assay, and the characterization of carboxylesterases through an in vitro metabolism assay all work together to provide a systematic, scientific and accurate strategy to investigate the roles of detoxification enzymes in insects, thus facilitating a better understanding of insecticide resistance mechanisms and thereby developing innovative strategies for pest management.

Insecticide resistance is currently a major issue of house fly control worldwide1,2. Efforts to determine the mechanism of insecticide resistance facilitates better understanding of this issue and thus provide novel strategies to effectively prevent or minimize the spread of resistance development3. Carboxylesterases, as one of the major detoxification enzymes, have attracted a lot of attention for their roles in sequestering and metabolizing insecticides in various insects4,5,6. Our previous study has identified multiple carboxylesterases in house flies and their expression levels were not only constitutively up-regulated in the resistant ALHF strain but also can be induced to higher levels in response to permethrin treatments7. However, the functional characterizations of these carboxylesterase genes in metabolizing insecticides remain to be explored.
Since the first report in early 1980s8, a baculovirus-mediated foreign gene expression system has been widely employed due to its high protein production efficiency and eukaryotic protein processing capabilities9. This binary system is composed of two essential elements: the constructed recombinant baculovirus delivering foreign genes into the host cells, and the large-scale expression of interested proteins by cells infected by recombinant baculovirus. Over the past decades, the baculovirus mediated cell expression system has been widely used to produce thousands of recombinant proteins, ranging from cytosolic enzymes to membrane-bound proteins in insect and mammal cells10. Our previous study has successfully expressed multiple CYP450 enzymes in insect Sf9 cells with this system11. In this study, we constructed a carboxylesterase-recombinant baculovirus to infect insect Sf9 cells, explored the cell tolerance to different permethrin treatments, and large-scale expressed carboxylesterase proteins in vitro for functional exploration. Instead of investigating multiple carboxylesterase isozyme mixtures from insect homogenates as adopted by previous studies12,13, this baculovirus-mediated insect cell expression system allows the specific expression and isolation of targeted proteins for better characterization of their biochemical and structural properties.
The tetrazolium salt-based assay (MTT) is a high-throughput colorimetric method developed and optimized to measure cell viability. This assay is based on the mechanism that only living cells are capable of metabolizing the yellow-colored MTT reagent to a dark purple colored formazan precipitate, which can be colorimetrically analyzed after dissolved in organic solvents14,15. Several more accurate but time-consuming methods, such as Trypan blue exclusion and the thymidine titration assay16,17, have been developed in recent years. However, the cell-based MTT assay is still currently recognized as the most rapid and easily-operated method to quickly detect cell viability. Here, we use the MTT assay to explore the cell tolerance against insecticide treatments. The enhanced tolerance of cells when infected with carboxylesterase recombinant baculovirus strongly supports the metabolic roles of carboxylesterases to insecticides, which in turn suggests their involvement in insecticide resistance.
Additionally, an in vitro metabolic assay was also conducted in this study. Compared with general carboxylesterase assays that use common substrates such as &#945;-napthyl acetate (&#945;-NA) and &#946;-naphthyl acetate (&#946;-NA) to reflect hydrolytic activities of carboxylesterases, the in vitro metabolic study is regarded as an accurate way to directly measure activities of carboxylesterases toward insecticides18. This method has been successfully employed in various insects to characterize multiple cytochrome P450s in association with insecticide resistance11,19,20. However, this method has not yet been applied in carboxylesterase studies. With the availability of carboxylesterase proteins produced by baculovirus-mediated expression system, we can perform an in vitro metabolic study of carboxylesterases toward permethrin, which can further provide strong evidence of the involvement of carboxylesterases in conferring pyrethroid resistance in house flies.

<table><tbody><tr><td>Q5 High-Fidelity DNA Polymerase</td><td>New England Biolabs inc.</td><td>M0491L</td><td></td></tr><tr><td>QIAquick Gel Extraction Kit</td><td>QIAGEN</td><td>28704</td><td></td></tr><tr><td>pENTR/D-TOPO Cloning Kit, with One Shot TOP10 Chemically Competent E. coli</td><td>Invitrogen by life technology</td><td>K240020</td><td>S.O.C medium and universal M13 sequence primers were included in this kit.</td></tr><tr><td>PureLink HiPure Plasmid Miniprep Kit</td><td>Invitrogen by life technology</td><td>K210002</td><td></td></tr><tr><td>Gateway LR Clonase II Enzyme mix for BaculoDirect&lt;sup&gt;TM&lt;/sup&gt; Kits</td><td>Invitrogen by life technology</td><td>11791-023</td><td></td></tr><tr><td>BaculoDirect C-Term Linear DNA Transfection Kit</td><td>Invitrogen by life technology</td><td>12562-019</td><td>Cellfectin transfection reagent and ganciclovir were included in this kit</td></tr><tr><td>pENTR-CAT plasmid</td><td>Invitrogen by life technology</td><td></td><td>Included in BaculoDirect C-Term Linear DNA Transfection Kit, concentration: 0.5 ug/uL</td></tr><tr><td>Heat inactivated Fetal Bovine Serum, Certified</td><td>Gibco by Life Technologies</td><td>10082-139</td><td></td></tr><tr><td>Sf9 cells in Sf-900 III SFM</td><td>Gibco by Life Technologies</td><td>12659017</td><td></td></tr><tr><td>Insect Cell-PE LB Insect Cell Protein Extraction &amp;amp; Lysis Buffer</td><td>G Biosciences by A Geno Technology Inc</td><td>786-411</td><td></td></tr><tr><td>Sf-900 III SFM (1&amp;times;) Serum Free Medium Complete</td><td>Gibco by Life Technologies</td><td>12658-019</td><td></td></tr><tr><td>Grace&#039;s Insect Medium, unsupplemented</td><td>Gibco by Life Technologies</td><td>11595030</td><td></td></tr><tr><td>Permethrin (isomers) analytical standard</td><td>SUPELCO by Solutions Within&lt;sup&gt;TM&lt;/sup&gt;</td><td>442748</td><td></td></tr><tr><td>Methanol (analytical graded)</td><td>Sigma-Aldrich</td><td>67-56-1</td><td></td></tr><tr><td>Acetonitrile (analytical graded)</td><td>Sigma-Aldrich</td><td>75-05-8</td><td></td></tr><tr><td>GHP Acrodisc 25 mm Syringe Filters with 0.45 &amp;mu;m GHP Membrane (HPLC Certified)</td><td>Pall Life Sciences</td><td>21890388</td><td></td></tr><tr><td>Alliance Waters 2695 HPLC System</td><td>Waters</td><td></td><td></td></tr><tr><td>T100 Thermal Cycle</td><td>Bio-Rad Laboratories Inc.</td><td>1861096</td><td></td></tr><tr><td>Nanodrop 2000/2000c Spectrophotometers</td><td>ThermoFisher Scientific</td><td>ND2000CLAPTOP</td><td></td></tr><tr><td>Cytation 5 Cell Imaging Multi-Mode Reader</td><td>BioTek</td><td></td><td></td></tr></tbody></table>

functional characterization of carboxylesterases in insecticide resistant house flies, musca domestica

Carboxylesterase-mediated metabolism is thought to play a major role in insecticide resistance in various insects. Several carboxylesterase genes were found up-regulated in the resistant house fly strain, whereas their roles in conferring insecticide resistance remained to be explored. Here, we designed a protocol for the functional characterization of carboxylesterases. Three example experiments are presented: (1) expression and isolation of carboxylesterase proteins through a baculovirus-mediated insect Spodoptera frugiperda (Sf9) cell expression system; (2) a cell-based MTT (3-[4, 5-dimethykthiazol-2-yl]-2, 5-diphenyltetrazolium bromide) cytotoxicity assay to measure the tolerance of insect cells to different permethrin treatments; and (3) in vitro metabolic studies to explore the metabolic capabilities of carboxylesterases toward permethrin. The carboxylesterase gene Md&#945;E7 was cloned from a resistant house fly strain ALHF and used to construct a recombinant baculovirus for Sf9 cells infection. The cell viabilities against different permethrin treatments were measured with the MTT assay. The enhanced cell tolerance of the experimental group (Md&#945;E7-recombinant baculovirus infected cells) compared with those of the control groups (CAT-recombinant baculovirus infected cells and GFP-recombinant baculovirus infected cells) to permethrin treatments suggested the capabilities of Md&#945;E7 in metabolizing insecticides, thereby protecting cells from chemical damages. Besides that, carboxylesterase proteins were expressed in insect Sf9 cells and isolated to conduct an in vitro metabolic study. Our results indicated a significant in vitro metabolic efficiency of Md&#945;E7 toward permethrin, directly indicating the involvement of carboxylesterases in metabolizing insecticides and thus conferring insecticide resistance in house flies.

The cell viability toward different permethrin treatments (MTT assay)
The cytotoxicity of permethrin was examined in Md&#945;E7-recombinant baculovirus infected Sf9 cells (experimental group) and CAT-recombinant baculovirus (provided by baculovirus infected kit) infected cells (control groups). The enhanced cell tolerances to permethrin in Md&#945;E7 expressing cells strongly support the metabolic roles of this carboxylesterase against insecticides and thus protecting cells from chemical damages. In our study, the cell viability against different permethrin concentrations (50, 100, 200 and 400 &#181;M) was calculated in comparison with cells treated with acetonitrile alone. Our results showed that the viability of Md&#945;E7 expressing cells was significantly higher (ranging from 86.00% to 101.92%) (Figure 4B) than that of control cells (ranging from 67.59% to 81.43%) (Figure 4A) when exposed to permethrin at different concentrations, indicating the important roles of Md&#945;E7 in metabolizing permethrin in insect cells.
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/58106/58106fig4.jpg" /> 
Figure 4: The viabilities of insect Sf9 cells under different permethrin treatments. (A) The viability of control cells infected by CAT recombinant baculovirus (B) The viability of experimental cells infected by Md&#945;E7 recombinant baculovirus. Four replications were conducted for each permethrin concentration. Data was shown as mean &#177; SEM. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58106/58106fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
In vitro metabolism of permethrin by Md&#945;E7
Permethrin metabolism was assayed by incubating a 40 &#181;M permethrin standard solution together with Md&#945;E7 proteins extracted from infected Sf9 cells. The reactions with CAT proteins served as controls. The depletion percentage of permethrin was calculated in comparison with reactions without enzyme added. Reactions were monitored by reverse-phase HPLC after a 120 min incubation period. Since the permethrin standard is actually a mixture of cis- and trans- isomers, two peaks were observed in the HPLC chromatographic profiles, with elution times of 10.67 min and 10.87 min for trans-permethrin and cis-permethrin, respectively (Figure 5). The depletion percentage of permethrin by Md&#945;E7 proteins (39.18&#177;3.78%) was significantly higher than that of CAT proteins (7.29&#177;0.81%) (Figure 6), which is not only be consistent with MTT results but also directly demonstrates the capabilities of Md&#945;E7 in metabolizing permethrin in vitro.
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/58106/58106fig5.jpg" /> 
Figure 5: The HPLC profile of permethrin standard. The permethrin standard used in this study is a mixture of trans-permethrin and cis-permethrin isomers. The red arrows indicate the peaks for the trans-permethrin isomer and cis-permethrin isomer, respectively. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58106/58106fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/58106/58106fig6.jpg" /> 
Figure 6: In vitro metabolism of permethrin by Md&#945;E7 and CAT recombinant proteins. The depletion percentages of permethrin by Md&#945;E7 and CAT proteins were calculated. Three replications were conducted for each permethrin concentration. Data was shown as mean &#177; SEM. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58106/58106fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Functional Characterization of Carboxylesterases in Insecticide Resistant House Flies, Musca Domestica at JoVE.com

Functional Characterization of Carboxylesterases in Insecticide Resistant House Flies, Musca Domestica

Here, we present a protocol to produce house fly carboxylesterase proteins in vitro with a baculovirus mediated insect cell expression system and later functionally characterize their roles in metabolizing permethrin, thereby, conferring pyrethroid resistance by conducting cell-based MTT assay and in vitro metabolic studies.

Functional Characterization of Carboxylesterases in Insecticide Resistant House Flies, Musca Domestica

Carboxylesterase-mediated metabolism is thought to play a major role in insecticide resistance in various insects. Several carboxylesterase genes were ...

functional-characterization-carboxylesterases-insecticide-resistant

Nanyang Technological University

Research

JoVE Journal

Biochemistry

7.5K Views.  Auburn University. Hier presenteren we een protocol om te produceren huis vliegen carboxylesterase eiwitten in vitro met een baculovirus gemedieerde insect cel expressie systeem en later functioneel karakteriseren van hun rollen in metaboliseringssysteem permethrin, daarmee overdracht van pyrethroïde weerstand door het uitvoeren van cel-gebaseerde MTT assay en in vitro metabole studies.

Video: Functional Characterization of Carboxylesterases in Insecticide Resistant House Flies, Musca Domestica

A Calcium Bioluminescence Assay for Functional Analysis of Mosquito (Aedes aegypti) and Tick (Rhipicephalus microplus) G Protein-coupled Receptors

Arthropod hormone receptors are potential targets for novel pesticides as they regulate many essential physiological and behavioral processes. The majority of them belong to the superfamily of G protein-coupled receptors (GPCRs). We have focused on characterizing arthropod kinin receptors from the tick and mosquito. Arthropod kinins are multifunctional neuropeptides with myotropic, diuretic, and neurotransmitter function. Here, a method for systematic analyses of structure-activity relationships of insect kinins on two heterologous kinin receptor-expressing systems is described. We provide important information relevant to the development of biostable kinin analogs with the potential to disrupt the diuretic, myotropic, and/or digestive processes in ticks and mosquitoes.
The kinin receptors from the southern cattle tick, Boophilus microplus (Canestrini), and the mosquito Aedes aegypti (Linnaeus), were stably expressed in the mammalian cell line CHO-K1. Functional analyses of these receptors were completed using a calcium bioluminescence plate assay that measures intracellular bioluminescence to determine cytoplasmic calcium levels upon peptide application to these recombinant cells. This method takes advantage of the aequorin protein, a photoprotein isolated from luminescent jellyfish. We transiently transfected the aequorin plasmid (mtAEQ/pcDNA1) in cell lines that stably expressed the kinin receptors. These cells were then treated with the cofactor coelenterazine, which complexes with intracellular aequorin. This bond breaks in the presence of calcium, emitting luminescence levels indicative of the calcium concentration. As the kinin receptor signals through the release of intracellular calcium, the intensity of the signal is related to the potency of the peptide.
This protocol is a synthesis of several previously described protocols with modifications; it presents step-by-step instructions for the stable expression of GPCRs in a mammalian cell line through functional plate assays (Staubly et al., 2002 and Stables et al., 1997). Using this methodology, we were able to establish stable cell lines expressing the mosquito and the tick kinin receptors, compare the potency of three mosquito kinins, identify critical amino acid positions for the ligand-receptor interaction, and perform semi-throughput screening of a peptide library. Because insect kinins are susceptible to fast enzymatic degradation by endogenous peptidases, they are severely limited in use as tools for pest control or endocrinological studies. Therefore, we also tested kinin analogs containing amino isobutyric acid (Aib) to enhance their potency and biostability. This peptidase-resistant analog represents an important lead in the development of biostable insect kinin analogs and may aid in the development of neuropeptide-based arthropod control strategies.

A Calcium Bioluminescence Assay for Functional Analysis of Mosquito Aedes aegypti and Tick Rhipicephalus microplus G Protein-coupled Receptors

This protocol provides instructions for clonal-cell line selection and a calcium bioluminescence assay to analyze the structure-activity relationships of synthesized arthropod neuropeptides on their cognate GPCRs. This assay can be used for receptor deorphanization and structure-activity relationship studies for synthetic analog design and peptide/drug-lead discovery.

Arthropod hormone receptors are potential targets for novel pesticides as they regulate many essential physiological and behavioral processes.  The ...

Immunology and Infection

A Method for Evaluating Insecticide Efficacy against Bed Bug, Cimex lectularius, Eggs and First Instars

Standard toxicity evaluations of insecticides against insect pests are primarily conducted on adult insects. Evaluations are based on a dose-response or concentration-response curve, where mortality increases as the dose or concentration of an insecticide is increased. Standard lethal concentration (LC50) and lethal dose (LD50) tests that result in 50% mortality of a test population can be challenging for evaluating toxicity of insecticides against non-adult insect life stages, such as eggs and early instar or nymphal stages. However, this information is essential for understanding insecticide efficacy in all bed bug life stages, which affects control and treatment efforts. This protocol uses a standard dipping bioassay modified for bed bug eggs and a contact insecticidal assay for treating nymphal first instars. These assays produce a concentration-response curve to further quantify LC50 values for insecticide evaluations.

A Method for Evaluating Insecticide Efficacy against Bed Bug,  Cimex lectularius, Eggs and First Instars

Insecticide evaluations are often targeted against adult insects, rather than immature stages. Here, we present a protocol for evaluating insecticides against bed bug eggs with a comparison to the first nymphal bed bug stage. These protocols could be adjusted for other insects to evaluate insecticide efficacy in non-adult life stages.

Standard toxicity evaluations of insecticides against insect pests are primarily conducted on adult insects. Evaluations are based on a dose-response or ...

Environment

Crystal Structure of the N-terminal Domain of Ryanodine Receptor from Plutella xylostella

Development of potent and efficient insecticides targeting insect ryanodine receptors (RyRs) has been of great interest in the area of agricultural pest control. To date, several diamide insecticides targeting pest RyRs have been commercialized, which generate annual revenue of 2 billion U.S. dollars. But comprehension of the mode of action of RyR-targeting insecticides is limited by the lack of structural information regarding insect RyR. This in turn restricts understanding of the development of insecticide resistance in pests. The diamondback moth (DBM) is a devastating pest destroying cruciferous crops worldwide, which has also been reported to show resistance to diamide insecticides. Therefore, it is of great practical importance to develop novel insecticides targeting the DBM RyR, especially targeting a region different from the traditional diamide binding site. Here, we present a protocol to structurally characterize the N-terminal domain of RyR from DBM. The x-ray crystal structure was solved by molecular replacement at a resolution of 2.84 &#197;, which shows a beta-trefoil folding motif and a flanking alpha helix. This protocol can be adapted for the expression, purification and structural characterization of other domains or proteins in general.

Crystal Structure of the N-terminal Domain of Ryanodine Receptor from Plutella xylostella

In this article, we describe the protocols of protein expression, purification, crystallization and structure determination of the N-terminal domain of&#160;ryanodine receptor from diamondback moth (Plutella xylostella).

Development of potent and efficient insecticides targeting insect ryanodine receptors (RyRs) has been of great interest in the area of agricultural pest ...

Reproducible dsRNA Microinjection and Oviposition Bioassay in Mosquitoes and House Flies

Synthetic dsRNAs, used to induce RNA interference, may have dose dependent phenotypic effects. These effects are difficult to define if the dsRNAs are delivered using a non-quantitative method. Accurate delivery of known quantities of nucleic acids or other chemicals is critical to measure the efficacy of the compound being tested and to allow reliable comparison between compounds.
Here we provide a reproducible, quantitative microinjection protocol that ensures accurate delivery of specific doses of dsRNA, reducing the mortality typically induced by injection injury. These modifications include the addition of Rhodamine B, a graduated injection needle, and an improved recovery method borrowed from Isoe and Collins. This method allows calculation of dose responses and facilitates comparisons between compounds. Versions of this method have been successfully used on three genera of mosquitoes as well as house flies to assess the reduction in fecundity resulting from gene silencing of ribosomal RNA transcripts.
This protocol provides strategies to reduce several challenges of small insect microinjection. Together, mechanical delivery of dsRNAs accompanied by visual verification, identification of effective locations for delivery, and inclusion of a post-injection recovery period ensure accurate dosing and low injury mortality. This protocol also describes an oviposition bioassay for uniform determination of effects on fecundity.

This protocol describes a microinjection methodology that we have standardized and used for several years to deliver specific quantities of nucleic acids directly to the hemolymph of mosquitoes and house flies. This protocol results in minimal injection mortality and allows dose correlated measurements of fecundity.

Synthetic dsRNAs, used to induce RNA interference, may have dose dependent phenotypic effects. These effects are difficult to define if the dsRNAs are ...

Embryo Microinjection and Knockout Mutant Identification of CRISPR/Cas9 Genome-Edited Helicoverpa Armigera (H&#252;bner)

The cotton bollworm, Helicoverpa armigera, is one of the most destructive pests in the world. A combination of molecular genetics, physiology, functional genomics, and behavioral studies has made H. armigera a model species in Lepidoptera Noctuidae. To study the in vivo functions of and interactions between different genes, clustered regularly interspaced short palindromic repeats (CRISPR)/ associated protein 9 (Cas9) genome editing technology is a convenient and effective method used for performing functional genomic studies. In this study, we provide a step-by-step systematic method to complete gene knockout in H. armigera using the CRISPR/Cas9 system. The design and synthesis of guide RNA (gRNA) are described in detail. Then, the subsequent steps consisting of gene-specific primer design for guide RNA (gRNA) creation, embryo collection, microinjection, insect rearing, and mutant detection are summarized. Finally, troubleshooting advice and notes are provided to improve the efficiency of gene editing. Our method will serve as a reference for the application of CRISPR/Cas9 genome editing in H. armigera as well as other Lepidopteran moths.

Embryo Microinjection and Knockout Mutant Identification of CRISPR/Cas9 Genome-Edited Helicoverpa Armigera H&#252;bner

Presented here is a protocol of Helicoverpa armigera (H&#252;bner) embryo microinjection and knockout mutant identification created by CRISPR/Cas9 genome editing. Mutant insects enable further research of gene function and interaction among different genes in vivo.

The cotton bollworm, Helicoverpa armigera, is one of the most destructive pests in the world. A combination of molecular genetics, physiology, functional ...

Behavior

Physiological Recordings and RNA Sequencing of the Gustatory Appendages of the Yellow-fever Mosquito Aedes aegypti

Electrophysiological recording of action potentials from sensory neurons of mosquitoes provides investigators a glimpse into the chemical perception of these disease vectors. We have recently identified a bitter sensing neuron in the labellum of female Aedes aegypti that responds to DEET and other repellents, as well as bitter quinine, through direct electrophysiological investigation. These gustatory receptor neuron responses prompted our sequencing of total mRNA from both male and female labella and tarsi samples to elucidate the putative chemoreception genes expressed in these contact chemoreception tissues. Samples of tarsi were divided into pro-, meso- and metathoracic subtypes for both sexes. We then validated our dataset by conducting qRT-PCR on the same tissue samples and used statistical methods to compare results between the two methods. Studies addressing molecular function may now target specific genes to determine those involved in repellent perception by mosquitoes. These receptor pathways may be used to screen novel repellents towards disruption of host-seeking behavior to curb the spread of harmful viruses.

Physiological Recordings and RNA Sequencing of the Gustatory Appendages of the Yellow-fever Mosquito Aedes aegypti

Using two methods to estimate gene expression in the major gustatory appendages of Aedes aegypti, we have identified the set of genes putatively underlying the neuronal responses to bitter and repulsive compounds, as determined by electrophysiological examination.

Electrophysiological recording of action potentials from sensory neurons of mosquitoes provides investigators a glimpse into the chemical perception of ...

Biology

Laboratory Rearing of Stable Flies and Other Muscoid Diptera

Stable flies, Stomoxys calcitrans, are serious pests of livestock, humans, companion animals and wildlife worldwide. During the last 20+ years, changes in agronomic practices resulted in serious outbreaks of stable flies in several countries. These outbreaks disrupted livestock production and human recreation resulting in public demands for increasing research and management efforts for this pest. A simple and inexpensive procedure for rearing stable flies for laboratory studies is presented. The procedure uses locally available diet components, equipment and supplies. The procedure can be adapted for rearing other muscoid flies including face fly (Musca autumnalis), horn fly (Haematobia irritans), and house fly (Musca domestica). The procedure produces stable fly puparia averaging 12.5 mg and ~35% egg to adult survival. Approximately 3000 flies are produced in each pan.

A procedure for rearing stable flies (Stomoxys calcitrans) is presented. The procedure uses locally available materials for diet components, equipment and supplies.

Stable flies, Stomoxys calcitrans, are serious pests of livestock, humans, companion animals and wildlife worldwide. During the last 20+ years, changes in ...

Topical Application Bioassay to Quantify Insecticide Toxicity for Mosquitoes and Fruit Flies

The continued use of insecticides for public health and agriculture has led to widespread insecticide resistance and hampering of control methods. Insecticide resistance surveillance of mosquito populations is typically done through Centers for Disease Control and Prevention (CDC) bottle bioassays or World Health Organization (WHO) tube tests. However, these methods can result in a high degree of variability in mortality data due to variable insecticide contact with&#160;the insect, the relatively small numbers of organisms tested, extensive variation in mass between populations, and constantly changing environmental conditions, leading to variable outcomes. This paper presents the topical application bioassay, adapted as a high-throughput phenotypic bioassay for both mosquitoes and fruit flies, to test large numbers of insects along&#160;a range of insecticide concentrations.
This assay 1) ensures consistent treatment and insecticide contact with every organism, 2) produces highly specific dose-response curves that account for differences in average mass between strains and sexes (which is particularly important for field-collected organisms), and 3) allows for the calculation of statistically rigorous median lethal doses (LD50), which are necessary for resistance ratio comparisons-an alternative surveillance approach from diagnostic dose mortality, which is also used for larvicide resistance surveillance. This assay will be a complementary tool for accurately phenotyping mosquito populations and, as illustrated using fruit flies, is easily adaptable for use with other insects. We argue that this assay will help fill the gap between genotypic and phenotypic insecticide resistance in multiple insect species.

We describe the methodology and importance of the topical application bioassay to measure insecticide susceptibility in mosquitoes and fruit flies. The presented assay is high-throughput, utilizes insect mass-thus allowing for calculating a mass-relativized lethal dose instead of concentration-and likely has lower variability than other similar methods.

The continued use of insecticides for public health and agriculture has led to widespread insecticide resistance and hampering of control methods. ...

Protocols for CRISPR/Cas9 Mutagenesis of the Oriental Fruit Fly Bactrocera dorsalis

The Oriental fruit fly, Bactrocera dorsalis, is a highly invasive and adaptive pest species that causes damage to citrus and over 150 other fruit crops worldwide. Since adult fruit flies have great flight capacity and females lay their eggs under the skins of fruit, insecticides requiring direct contact with the pest usually perform poorly in the field. With the development of molecular biological tools and high-throughput sequencing technology, many scientists are attempting to develop environmentally friendly pest management strategies. These include RNAi or gene editing-based pesticides that downregulate or silence genes (molecular targets), such as olfactory genes involved in searching behavior, in various insect pests. To adapt these strategies for Oriental fruit fly control, effective methods for functional gene research are needed. Genes with critical functions in the survival and reproduction of B. dorsalis serve as good molecular targets for gene knockdown and/or silencing. The CRISPR/Cas9 system is a reliable technique used for gene editing, especially in insects. This paper presents a systematic method for CRISPR/Cas9 mutagenesis of B. dorsalis, including the design and synthesis of guide RNAs, collecting embryos, embryo injection, insect rearing, and mutant screening. These protocols will serve as a useful guide for generating mutant flies for researchers interested in functional gene studies in B. dorsalis.

Protocols for CRISPR/Cas9 Mutagenesis of the Oriental Fruit Fly Bactrocera dorsalis

This paper presents the step-by-step protocols for CRISPR/Cas9 mutagenesis of the Oriental fruit fly Bactrocera dorsalis. Detailed steps provided by this standardized protocol will serve as a useful guide for generating mutant flies for functional gene studies in B. dorsalis.

The Oriental fruit fly, Bactrocera dorsalis, is a highly invasive and adaptive pest species that causes damage to citrus and over 150 other fruit crops ...

Assessment of Phytochemical Effects on Helicoverpa armigera Through Feeding Assay

Developing a Feeding Assay System for Evaluating the Insecticidal Effect of Phytochemicals on Helicoverpa armigera

Helicoverpa armigera, a lepidopteran insect, is a polyphagous pest with a worldwide distribution. This herbivorous insect is a threat to plants and agricultural productivity. In response, plants produce several phytochemicals that negatively impact the insect's growth and survival. This protocol demonstrates an obligate feeding assay method to evaluate the effect of a phytochemical (quercetin) on insect growth, development, and survival. Under controlled conditions, the neonates were maintained until the second instar on a pre-defined artificial diet. These second-instar larvae were allowed to feed on a control and quercetin-containing artificial diet for 10 days. The insects' body weight, developmental stage, frass weight, and mortality were recorded on alternate days. The change in body weight, the difference in feeding pattern, and developmental phenotypes were evaluated throughout the assay time. The described obligatory feeding assay simulates a natural mode of ingestion and can be scaled up to a large number of insects. It permits one to analyze phytochemicals' effect on the growth dynamics, developmental transition, and overall fitness of H. armigera. Furthermore, this setup can also be utilized to evaluate alterations in nutritional parameters and digestive physiology processes. This article provides a detailed methodology for feeding assay systems, which may have applications in toxicological studies, insecticidal molecule screening, and understanding chemical effects in plant-insect interactions.

Author Spotlight: Development and Challenges of Feeding Assays for Insect Pest Management 

This protocol describes the obligate feeding assay to evaluate the potentially toxic effect of a phytochemical on the lepidopteran insect larvae. This is a highly scalable insect bioassay, easy to optimize the sublethal and lethal dose, deterrent activity, and physiological effect. This could be used for screening eco-friendly insecticides.

Helicoverpa armigera, a lepidopteran insect, is a polyphagous pest with a worldwide distribution. This herbivorous insect is a threat to plants and ...

Functional Characterization of Carboxylesterases in Insecticide Resistant House Flies, Musca Domestica

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