Name: functional characterization of carboxylesterases in insecticide resistant house flies, musca domestica
Uploaded: 2018-08-23T14:30:00
Description: Watch this Scientific Journal Video about Functional Characterization of Carboxylesterases in Insecticide Resistant House Flies, Musca Domestica at JoVE.com

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 rea...

<ol>
	<li>Scott, J. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insecticide+resistance+in+house+flies+from+the+United+States:+Resistance+levels+and+frequency+of+pyrethroid+resistance+alleles.">Insecticide resistance in house flies from the United States: Resistance levels and frequency of pyrethroid resistance alleles.</a> Pesticide Biochemistry and Physiology. 107 (3), 377-384 (2013).</li><li>Li, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+whole+transcriptomal+linkage+analysis+of+gene+co-regulation+in+insecticide+resistant+house+flies,+Musca+domestica.">A whole transcriptomal linkage analysis of gene co-regulation in insecticide resistant house flies, Musca domestica.</a> BMC Genomics. 14, 803 (2013).</li><li>Liu, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insecticide+resistance+in+mosquitoes:+impact,+mechanisms,+and+research+directions.">Insecticide resistance in mosquitoes: impact, mechanisms, and research directions.</a> Annual Review of Entomology. 60, 537-559 (2015).</li><li>Grigoraki, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptome+profiling+and+genetic+study+reveal+amplified+carboxylesterase+genes+implicated+in+temephos+resistance,+in+the+Asian+tiger+mosquito+Aedes+albopictus.">Transcriptome profiling and genetic study reveal amplified carboxylesterase genes implicated in temephos resistance, in the Asian tiger mosquito Aedes albopictus.</a> e0003771. 9, e0003771 (2015).</li><li>Grigoraki, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Carboxylesterase+gene+amplifications+associated+with+insecticide+resistance+in+Aedes+albopictus:+Geographical+distribution+and+evolutionary+origin.">Carboxylesterase gene amplifications associated with insecticide resistance in Aedes albopictus: Geographical distribution and evolutionary origin.</a> PLOS Neglected Tropical Diseases. 11, e0005533 (2017).</li><li>Wheelock, C., Shan, G., Ottea, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overview+of+carboxylesterases+and+their+role+in+the+metabolism+of+insecticides.">Overview of carboxylesterases and their role in the metabolism of insecticides.</a> Journal of Pesticide Science. 30, 75-83 (2005).</li><li>Feng, X., Li, M., Liu, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Carboxylesterase+genes+in+pyrethroid+resistant+house+flies,+Musca+domestica.">Carboxylesterase genes in pyrethroid resistant house flies, Musca domestica.</a> Insect Biochemistry and Molecular Biology. 92, 30-39 (2018).</li><li>Mosmann, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+colorimetric+assay+for+cellular+growth+and+survival:+application+to+proliferation+and+cytotoxicity+assays.">Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays.</a> J Immunol Methods. 65 (1-2), 55-63 (1983).</li><li>Jarvis, D. 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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Baculovirus+expression+system+for+heterologous+multiprotein+complexes.">Baculovirus expression system for heterologous multiprotein complexes.</a> Nature Biotechnology. 22 (12), 1583 (2004).</li><li>Gong, Y., Li, T., Feng, Y., Liu, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+function+of+two+P450s,+CYP9M10+and+CYP6AA7,+in+the+permethrin+resistance+of+Culex+quinquefasciatus.">The function of two P450s, CYP9M10 and CYP6AA7, in the permethrin resistance of Culex quinquefasciatus.</a> Scientific Reports. 7 (1), 587 (2017).</li><li>Cao, C. W., Zhang, J., Gao, X. W., Liang, P., Guo, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overexpression+of+carboxylesterase+gene+associated+with+organophosphorous+insecticide+resistance+in+cotton+aphids,+Aphis+gossypii+(Glover).">Overexpression of carboxylesterase gene associated with organophosphorous insecticide resistance in cotton aphids, Aphis gossypii (Glover).</a> Pesticide Biochemistry and Physiology. 90 (3), 175-180 (2008).</li><li>Zhang, L., Gao, X., Liang, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beta-cypermethrin+resistance+associated+with+high+carboxylesterase+activities+in+a+strain+of+house+fly,+Musca+domestica+(Diptera:+Muscidae).">Beta-cypermethrin resistance associated with high carboxylesterase activities in a strain of house fly, Musca domestica (Diptera: Muscidae).</a> Pesticide Biochemistry and Physiology. 89, 65-72 (2007).</li><li>Van Meerloo, J., Kaspers, G. 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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 ...

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 BaculoDirectTM 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 &#38; 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&#215;) Serum Free Medium Complete</td>
			<td>Gibco by Life Technologies</td>
			<td>12658-019</td>
			<td></td>
		</tr>
		<tr>
			<td>Grace&#39;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 WithinTM</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 &#956;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 aga...

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 ...

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