Accepted Manuscript Title: Cytochrome p450s –their expression, regulation, and role in insecticide resistance Author: Nannan Liu, Ming Li, Youhui Gong, Feng Liu, Ting Li PII: DOI: Reference:
S0048-3575(15)00007-3 http://dx.doi.org/doi: 10.1016/j.pestbp.2015.01.006 YPEST 3779
To appear in:
Pesticide Biochemistry and Physiology
Received date: Accepted date:
1-11-2014 10-1-2015
Please cite this article as: Nannan Liu, Ming Li, Youhui Gong, Feng Liu, Ting Li, Cytochrome p450s –their expression, regulation, and role in insecticide resistance, Pesticide Biochemistry and Physiology (2015), http://dx.doi.org/doi: 10.1016/j.pestbp.2015.01.006. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1
Prepared for Publication in
2
Pesticide Biochemistry and Physiology
3 4 5 6 7 8
Cytochrome P450s –Their Expression, Regulation, and Role in Insecticide Resistance
9 10
Nannan Liu1*, Ming Li1, Youhui Gong1,2, Feng Liu1, Ting Li1
11 12
1
13
USA
14
2
Department of Entomology and Plant Pathology, Auburn University, Auburn, Alabama 36849,
Department of Entomology, China Agricultural University, Beijing, China
15 16
*To whom correspondence should be addressed: [email protected]; Phone: 334-844-2661
17 18 19
Highlights
20 21 22
1) The characterization of P450-mediated detoxification in insecticide resistance is key for controlling insect pests.
23
2) P450 gene expression studies have progressed from the analysis of single gene
24
sequences to whole genome sequence analysis and from characterizing the expression
1 Page 1 of 25
25
of individual genes to examining the expression of multiple genes simultaneously,
26
revealing that the overexpression of multiple P450 genes in insecticide resistant strains
27
is a common phenomenon that has now been observed in many resistant insect species
28
3) Many studies have support that the up-regulation of resistance P450 genes is regulated
29
by trans and/or cis factors in insecticide-resistant insects.
30
4) With the exciting discovery of the potential function of GPCR and GPCR-related genes
31
in insecticide resistance and their regulatory function in resistance-related P450 gene
32
expression, future research should focus on the downstream factors in the GPCR
33
regulatory pathway in order to characterize their involvement in insecticide resistance.
34 35 36
Graphical Abstract
37 38 39 40 2 Page 2 of 25
41 42
Abstract
43 44
P450s are known to be critical for the detoxification and/or activation of xenobiotics such as
45
drugs and pesticides and overexpression of P450 genes can significantly affect the disposition of
46
xenobiotics in the tissues of organisms, altering their pharmacological/toxicological effects. In
47
insects, P450s play an important role in detoxifying exogenous compounds such as insecticides
48
and plant toxins and their overexpression can result in increased levels of P450 proteins and
49
P450 activities. This has been associated with enhanced metabolic detoxification of insecticides
50
and has been implicated in the development of insecticide resistance in insects. Multiple P450
51
genes have been found to be co-overexpressed in individual insect species via several
52
constitutive overexpression and induction mechanisms, which in turn are co-responsible for high
53
levels of insecticide resistance. Many studies have also demonstrated that the transcriptional
54
overexpression of P450 genes in resistant insects is regulated by trans and/or cis regulatory
55
genes/factors. Taken together, these earlier findings suggest not only that insecticide resistance is
56
conferred via multi-resistance P450 genes, but also that it is mediated through the interaction of
57
regulatory genes/factors and resistance genes. This chapter reviews our current understanding of
58
how the molecular mechanisms of P450 interaction/gene regulation govern the development of
59
insecticide resistance in insects and our progress along the road to a comprehensive
60
characterization of P450 detoxification-mediated insecticide resistance.
61 62
Keywords
63
Insecticide resistance, cytochrome P450, interaction/regulation, induction, homology modeling
64 3 Page 3 of 25
65
Introduction
66 67
Cytochrome P450s have long been of particular interest to researchers because of their critical
68
biological function in the detoxification and/or activation of xenobiotics such as drugs,
69
pesticides, plant toxins, chemical carcinogens and mutagens, as well as their role in the
70
metabolism of endogenous compounds such as hormones, fatty acids, and steroids. Basal and/or
71
up-regulation of P450 gene expression is known to considerably affect the disposition of
72
xenobiotics or endogenous compounds in the tissues of organisms and is thus responsible for
73
altering their pharmacological/toxicological effects [1]. Insect cytochrome P450s are involved in
74
detoxifying exogenous compounds such as insecticides [2], [3] and [4] and plant toxins [5] and
75
[6]. They are also known to be an important component in the biosynthesis and degradation
76
pathways of endogenous compounds such as pheromones, 20-hydroxyecdysone, and juvenile
77
hormone (JH) in insects [7], [8], [9], [10] and [11] and thus perform important functions in insect
78
growth, development, and reproduction. While all insects probably have certain capacity to
79
detoxify insecticides and xenobiotics, the degree to which an insect can metabolize or detoxify
80
toxic substances is of critical for their survival in a chemically unfavorable situation [12], as is
81
the development of resistance. Whereas the constitutively increased expression and induction of
82
P450 gene expression are both thought to be directly linked to the degree that insects are capable
83
to enhance their metabolism and detoxification of insecticides, they have also been implicated in
84
the adaptation of insects to their environment [12], [13], [14] and [15] and their development of
85
resistance to insecticides. A number of studies have revealed that multiple P450 genes are co-
86
overexpressed/up-regulated in individual resistant organisms such as house flies and mosquitoes
87
[14], [15], [16], [17] and [18], demonstrating elevated expression levels of P450 genes in
4 Page 4 of 25
88
resistant insects. The transcription overexpression of P450 genes in resistant insects is also
89
known to be regulated by trans and/or cis regulatory genes/factors [19], [20] and [21].
90 91
Taken together, these findings suggest that insecticide resistance is conferred via multi-resistance
92
P450 gene overexpression and induction and is arbitrated through the interaction of regulatory
93
genes and resistance P450 genes. Characterization of the total number of P450 genes directly
94
involved in resistance in a single resistant insect and factors involved in regulation of resistance
95
P450 gene expression [22] and [23] is now the focus of attention for those working in the fields
96
of insecticide resistance and insect toxicology. The emergence of the “Genomics Era” during the
97
last decade, coupled with the availability of new functional study techniques such as double-
98
stranded RNA-mediated gene interference (RNAi) and transgenic gene expression, have led to
99
significant progress being made towards characterizing not only the number of P450 genes but
100
also identifying the functions of the genes that are involved in insecticide resistance at the whole
101
genome level. This review focuses on our journey towards the characterization of the P450
102
detoxification-mediated insecticide resistance of insects.
103 104
Synergism studies – the initial step in the characterization of P450 detoxification in
105
insecticide resistance
106 107
The initial characterization of the importance of metabolic detoxification in insecticide resistance
108
has primarily been documented in many insect species on the basis of synergistic studies. These
109
studies represent the initial step towards revealing the possible mechanisms involved in
110
insecticide resistance using chemical synergists such as piperonyl butoxide (PBO), S,S,S,-
5 Page 5 of 25
111
tributyl phosphorotrithioate (DEF), and diethyl maleate (DEM), which are inhibitors of
112
cytochrome P450 monooxygenases, hydrolases, and glutathione S-transferases (GSTs),
113
respectively. By comparing the resistance levels with and without the synergists, researchers can
114
begin to narrow down precisely which detoxification mechanisms are involved in the
115
development of insecticide resistance. Our laboratory has conducted a series of studies looking at
116
the mechanisms involved in pyrethroid resistance in the house fly Musca domestica, mosquito
117
Culex quinquefasciatus and German cockroach, Blattella germanica. We found that permethrin
118
resistance levels in all three were largely suppressed by PBO, indicating that P450
119
monooxygenase-mediated detoxification is one of the major mechanisms involved in the
120
development of pyrethroid resistance in these insect species [24], [25] and [26]. These findings
121
strongly support those reported in numerous similar studies on insecticide resistance in different
122
insect species, including those in other mosquito [27], [28] and [29] and house fly strains [30].
123
Although in many cases the initial evidence generated by synergistic studies designed to
124
determine the importance of detoxification proteins such as P450s in insecticide resistance is
125
undoubtedly reliable [2], other studies have produced evidence that suggests synergists are
126
deficient inhibitors for some of the detoxification enzymes responsible for resistance [2], [31]
127
and [32]. To resolve this disagreement, independent studies using approaches such as enzyme
128
activity assays, metabolism studies, gene expression analysis, and, if possible, functional studies,
129
are needed to confirm the precise role of detoxification proteins in resistance.
130 131
Characterization of gene expression – pinpointing the individual P450 genes involved in
132
insecticide resistance
133
6 Page 6 of 25
134
P450 constitutive overexpression
135
One noteworthy characteristic of insect P450s that is known to be associated with enhanced
136
metabolic detoxification of insecticides in insects is the increased protein production and
137
activities of these enzymes through the transcriptional up-regulation of the genes in insecticide
138
resistant insects. Gene expression studies have progressed from the analysis of single gene
139
sequences to whole genome sequence analysis and from characterizing the expression of
140
individual genes to examining the expression of multiple genes simultaneously, revealing that
141
the overexpression of multiple P450 genes in insecticide resistant strains is a common
142
phenomenon that has now been observed in many resistant insect species [2], [3], [14], [15],
143
[16], [17], [18], [19], [33], [34], [35], [36], [37], [38] and [39]. These studies have revealed that
144
the interaction of multiple insecticide resistance P450 genes may be responsible for the
145
development of resistance. Recently, our group conducted a systematic study to characterize the
146
expression profiles of 204 P450 genes in the mosquito Culex quinquefasciatus at a whole
147
genome level using real-time quantitative PCR analysis [34], identifying multiple P450 genes as
148
up-regulated in individual resistant mosquito strains and leading us to conclude that multiple
149
P450 genes are co-overexpressed, which in turn is responsible for the detoxification of
150
insecticides and the development of insecticide resistance in these mosquito strains. Further
151
evidence supporting this conclusion was provided by our whole transcriptome study using
152
RNAseq data analysis [40]. This P450 gene overexpression in insecticide resistance is not unique
153
to mosquitoes; multiple P450 gene co-overexpression has also been implicated in the
154
development of insecticide resistance in other insect species. An elegant study utilizing whole
155
transcriptome analysis clearly demonstrated that multiple P450 gene co-overexpression is also
7 Page 7 of 25
156
responsible for high levels of insecticide resistance in house flies. These findings suggest that
157
high levels of insecticide resistance are conferred via multi-resistance P450 gene overexpression.
158 159
A number of studies have revealed that the overexpression of resistance-related P450 genes is
160
controlled by various regulatory factors [40]. For example, the up-regulation of 2 P450 genes,
161
CYP6A1 and CYP6D1, which are located on autosomes 5 and 1, respectively, in insecticide
162
resistant house flies is known to be regulated by trans-regulatory factors on autosome 2 [19],
163
[20], [33] and [35], while the up-regulation of CYP6A2 and CYP6A8 in the insecticide resistant
164
fruit fly Drosophila melanogaster is transcriptionally regulated by trans-regulatory factors on
165
autosome 3 [21]. Genetic linkage analyses of house fly lines that compared different autosomal
166
combinations from resistant wide type strain(s) revealed that the up-regulation of P450 gene
167
overexpression expression occurred mainly through the co-regulation of factors among multiple
168
autosomes, especially between autosomes 2 and 5 [14] and [15]. Taken together, these findings
169
suggest not only that insecticide resistance is conferred via multiple resistance P450 genes, but
170
also that it is mediated through the interaction of several regulatory genes/factors and resistance
171
genes [22] and [23].
172 173
P450 induction
174 175
Another interesting characteristic of some insect P450 genes is their induced expression by
176
exogenous and endogenous compounds, a phenomenon known as induction. It has been
177
suggested that induction is in fact responsible for the enhanced metabolism of pharmaceutical
178
drugs, insecticides and endogenous compounds [2], [3] and [41]. While all insects probably
8 Page 8 of 25
179
possess some capacity to detoxify insecticides and xenobiotics, the degree to which individual
180
insects can metabolize and detoxify these highly toxic chemicals is of considerable importance
181
for their survival in a chemically unfriendly environment [12]. It is possible that the induction of
182
P450s and their activities in insects is involved in the adaptation of insects to their environment,
183
the detoxification of insecticides and the development of insecticide resistance [12], [13], [16],
184
[17], [35], [42], and [43]. It has also been suggested that induction associated with increased
185
tolerance of insects to insecticides may help the development of resistance [44]. Chemical
186
inducers could act as substrates for P450s, allowing the induction or modulation of P450s by
187
these substrates to in turn reduce the effects of the substrates by enhancing the substrate
188
metabolism [45]. Our group recently discovered that multiple P450 that are constitutively
189
overexpressed in resistant mosquitoes can be further induced in a clear concentration dose- and
190
time-dependent manner when the mosquitoes are exposed to further doses of the same
191
insecticide [18] and [46], increasing the overall expression levels of multiple P450 genes in the
192
resistant mosquitoes. This finding strongly supports the mechanism proposed by Okey [45]. We
193
also observed that P450 gene induction in mosquitoes follows a resistance-specific pattern, with
194
similar results being reported earlier in Drosophila melanogaster [17], where the expression of
195
CYP6g1 and CYP12d1 were induced in the DDT resistant strains post-exposure to DDT. Studies
196
by Zhu et al. [14] and [15] indicated that several P450 genes were up-regulated in insecticide
197
resistant house flies through a similar induction mechanism. Zhu et al. [14] and [15] also
198
reported that although some P450 genes, such as CYP4D4v2, CYP4G2, and CYP6A38 in
199
resistant house flies, are not constitutively overexpressed, their expression can be induced when
200
insects are under insecticide application pressure, suggesting that these genes may be uniquely
201
involved in the response to insecticide exposure through the induction of their expression, which
9 Page 9 of 25
202
in turn enhances their capacity to detoxify the insecticide and leads to enhanced insecticide
203
resistance [14] and [15].
204 205
Similar regulatory mechanisms may govern P450 constitutive overexpression and induction, and
206
it has been suggested that both contribute to the development of insecticide resistance [12]. It is
207
also possible that the higher inducible expression of transcripts is due primarily to mutations in
208
trans regulatory factors or their signaling cascades [47]. However, little is known about the trans
209
regulatory signaling components that mediate the expression of insect detoxificative P450s [47].
210
It has also been suggested that the induction of P450s is a complex phenomenon involving
211
multiple DNA-binding elements in each P450 promoter, transcriptional factors, and the overlap
212
of regulatory pathways [39]. It would therefore be particularly interesting to further characterize
213
the transcriptional elements in the promoter region of P450 genes and the regulatory factors
214
involved in the regulation of both the constitutive overexpression and induction of the P450
215
genes that have been implicated in the development of insecticide resistance in order to more
216
fully understand the molecular mechanisms involved.
217 218
In silico 3D P450 homology modeling and insecticide molecular docking
219 220
In silico homology modeling and molecular docking of protein, a technique that can be used to
221
build a three-dimensional model of proteins from their amino acid sequences on the basis of the
222
alignment with a similar protein with a known structure (the template), has become a very
223
effective tool for understanding the relationship between the structure of proteins such as P450s
224
and substrates such as insecticides to provide reasonable explanations of substrate specificities
10 Page 10 of 25
225
and metabolic specificities caused by allelic variations or mutations [47] and [48]. The
226
importance of in silico homology modeling in the post-genomic era is growing rapidly since it
227
provides a useful avenue for characterizing/understanding the biological functions of the millions
228
of protein sequences now being generated by high-throughput next generation sequencing
229
technology. The tertiary structure of P450s contains several conserved structures such as helices
230
A to L that act as the protein backbone, a hydrophobic N-terminal domain that acts as the
231
transmembrane anchor, and the active site, which is buried deep within the enzyme structure and
232
connects to the surrounding environment via a network of channels that serve as access/egress
233
paths. Six regions of a P450 have been designated as substrate recognition sites (SRSs, Figure 1)
234
based on studies of human P450s in the CYP2 family [49]. These SRSs contribute to the function
235
of a P450, with SRS1, SRS4, SRS5, and SRS6 known to be involved in catalytic site formation
236
and SRS2 and SRS3 to participate in substrate access channel formation [47].
237 238
In silico homology modeling and molecular docking have been used to predict and validate the
239
function of P450s in several studies in insects. With this new computer modeling system to help
240
them cope with the highly complex functional metabolism studies, researchers can now
241
confidently state that several insect P450 genes are important in the development of resistance to
242
insecticides, including CYP6Z1, CYP6Z2 and CYP6M2 in Anopheles gambiae [50], [51] and
243
[52], CYP6AA3 and CYP6P7 in Anopheles minumus [53], CYP6Z8 in Aedes aegypti [54],
244
CYP6QB23 in Meligethes aeneus [55], and CYP6BQ8, CUP6BQ9, CYP6BQ10 and CYP6BQ11
245
in Tribolium castaneum [56]. Utilizing in silico homology modeling and molecular docking
246
methodology, Liu [57] evaluated various models for binding permethrin to the active site(s) of
247
CYP6AA7, an insect P450 that is known to be overexpressed in Cx. quinquefasciatus [34] and
11 Page 11 of 25
248
[58], revealing that three permethrin binding models of CYP6AA7 were present in Culex
249
quinquefasciatus. All the residues involved in the permethrin binding were located in the SRSs
250
and the hydrophobic residues of phenylalanine (Phe), valine (Val) and proline (pro) were all
251
conserved in the catalytic sites of the three permethrin binding modes, indicating the importance
252
of these residues for permethrin binding and suggesting that these enzymes provide a favorable
253
chemical environment for hydrophobic insecticide compounds. The short distance between the
254
putative metabolic sites and the heme-oxygen in an orientation nearly perpendicular to the heme
255
plane (within 6.0 Å) [56] and [59] suggests that CYP6AA7 presents a favorable binding affinity
256
to permethrin and imparts a putative hydrolytic ability to permethrin.
257 258
Regulatory genes, factors, and pathways in insecticide resistance
259 260
The results of our previous studies [14], [15], [34], [60] and [61] as well as those of others [19],
261
[20], [21], [33] and [35] suggest that the interaction of multiple genes may be responsible for
262
insecticide resistance. Many studies have also reported that the up-regulation of resistance P450
263
genes is regulated by trans and/or cis factors in insecticide-resistant insects. Taken together,
264
these studies suggest that not only is insecticide resistance conferred via multiple resistance
265
genes, but it is mediated through the interaction of regulatory factors and resistance P450 genes.
266
The information above allowed us to propose a hypothetic model (Figure 2), in which trans
267
regulatory factors or cis regulatory elements may be crucial in the regulation of the resistance
268
P450 gene expression and, in turn, are involved in the detoxification of insecticides and the
269
evolution of insecticide resistance in insects. However, as yet none of the regulatory factors in
270
insecticide resistance have been identified and the regulation pathways have not been examined.
12 Page 12 of 25
271
Using the SSH/cDNA array technique, Liu et al [60] identified 22 unique genes that are over-
272
expressed in in pyrethroid resistant Cx. quinquefasciatus, in addition to two new P450 genes
273
known to be important in resistance; a further 20 ESTs, including a signaling transduction gene
274
that encodes a G-protein coupled receptor, were identified that had not previously been
275
implicated in insecticide resistance in any insect species, further suggesting the involvement of
276
multiple up-regulated gene interactions and signaling regulatory factor(s) in the insecticide
277
resistant mosquitoes [60].
278 279
Because of the important roles that GPCRs play in recognizing extracellular messengers,
280
transducing signals to the cytosol and mediating the cellular responses necessary for the normal
281
physiology of organisms [62], our group has taken significant steps towards characterizing the
282
GPCR s’ function in insecticide resistance. We were the first to explore the function of GPCRs
283
and GPCR-related genes in the insecticide resistance of mosquitoes, Culex quinquefasciatus
284
using quantitative RT-PCR and functional genomic methods [63]. By comparing the expression
285
of 115 GPCR-related genes at a whole genome level in resistant and susceptible Cx.
286
quinquefasciatus mosquitoes, we were able to identify several GPCR-related genes that were up-
287
regulated in the highly resistant Culex mosquito strains. Knockdown of these four GPCR-related
288
genes using the RNAi technique revealed significant decreases in the levels of resistance of the
289
mosquitoes to permethrin. More interestingly, knockdown of these four GPCR-related genes
290
directly affected the expression of the resistance P450 genes, suggesting that the role of GPCR-
291
related genes in resistance is related to the regulation of resistance P450 gene expression [63].
292
This new information clearly indicates the role of GPCR-regulated pathway in the regulation of
293
the resistance cytochrome P450-mediated detoxification of insecticides and the evolution of
13 Page 13 of 25
294
resistance in Cx. quinquefasciatus. Sun et al. [64] recently reported that overexpression of a
295
GPCR related arrestin gene in deltamethrin resistant Culex pipiens pallens regulated the
296
insecticide resistance-associated P450 gene, further supporting the regulatory role of GPCRs in
297
P450-mediated insecticide resistance. These exciting discoveries shed new light on the potential
298
function of GPCR and GPCR-related genes in insecticide resistance and their regulatory function
299
on resistance-related P450 gene expression. However, the complete regulatory pathway remains
300
largely unclear. Thus, future research needs to focus on the downstream factors in the GPCR
301
regulatory pathway in order to characterize their involvement in insecticide resistance and,
302
consequently, support the effort to identify new insecticide targets and strategies with which to
303
control mosquitoes and mosquito-borne diseases. This will also help to build a better
304
understanding of the regulatory pathway of insecticide detoxification and evolutionary
305
insecticide selection in mosquitoes [63].
306 307
Conclusions
308 309
Insect cytochrome P450s are known to play an important role in detoxifying insecticides and
310
plant toxins, resulting in the development of resistance to insecticides and facilitating the
311
adaptation of insects to their plant hosts. A significant characteristic of insect P450s that is
312
associated with enhanced metabolic detoxification of insecticides in insects is the increased
313
levels of P450 proteins and P450 activity that result from the constitutive transcriptional
314
overexpression of P450 genes in insecticide resistant insects. Another interesting characteristic of
315
insect P450 genes is that the expression of some P450 genes can be induced by exogenous and
316
endogenous compounds, a phenomenon known as induction. Given the close correlation between
14 Page 14 of 25
317
the production of these genes and the development of resistance and/or P450-mediated resistance
318
in ALHF, these observations provide overwhelming evidence that P450 induction and
319
constitutive overexpression are co-responsible for the detoxification of insecticides, evolutionary
320
insecticide selection, and the ability of insects to adapt to changing environments. Yet, compared
321
to the extensive knowledge accumulated regarding P450 gene expression, our understanding of
322
the molecular mechanisms involved in gene regulation and interactions in insecticide residence is
323
wholly inadequate. With the exciting discovery of the potential function of GPCR and GPCR-
324
related genes in insecticide resistance and their regulatory function in resistance-related P450
325
gene expression, future research should focus on the downstream factors in the GPCR regulatory
326
pathway in order to characterize their involvement in insecticide resistance. This is expected to
327
support the effort to identify new insecticide targets and strategies with which to control
328
mosquitoes and mosquito-borne diseases, as well as help us build a better understanding of the
329
regulatory pathway of insecticide detoxification and evolutionary insecticide selection in
330
mosquitoes [63].
331 332
Acknowledgments
333 334
The authors sincerely thanks all the people who have ever worked and are currently working in
335
their laboratory for their hard work and generating valuable data for understanding importance of
336
P450s in insecticide resistance and their dedication to the field of insecticide resistance research.
337 338 339 340 15 Page 15 of 25
341 342 343 344
References
345 346
[1] P. Pavek, Z. Dvorak, Xenobiotic-induced transcriptional regulation of xenobiotic
347
metabolizing enzymes of the cytochrome P450 superfamily in human extrahepatic tissues,
348
Curr. Drug Metab. 9 (2008) 129-143.
349
[2] R. Feyereisen, Insect CYP genes and P450 enzymes, in: L.I. Gilbert (Ed.), Insect Molecular
350
Biology and Biochemistry, Elsevier Academic Press, Oxford, 2012, pp. 236-316.
351
[3] R. Feyereisen, Arthropod CYPomes illustrate the tempo and mode in P450 evolution,
352 353 354
Biochem. Biophys. Arch. 1814 (2011) 19-28. [4] J.G. Scott, Cytochromes P450 and insecticide resistance, Insect Biochem. Mol. Biol. 29 (1999) 757-777.
355
[5] M.R. Berenbaum, Coumarins, in: G.A. Rosenthal, M.R. Berenbaum (Eds.), Herbivores: Their
356
Interaction with Secondary Plant Metabolites, Academic Press, New York, 1991, pp. 221-
357
249.
358 359 360
[6] M.A. Schuler, P450s in plant-insect interactions, Biochem. Biophys. Acta. 1814 (2011) 3645. [7] J.R. Reed, D. Vanderwel, S. Choi, J.G. Pomonis, R.C. Reitz, G.J. Blomquist, Unusual
361
mechanism of hydrocarbon formation in the housefly: cytochrome P450 converts aldehyde to
362
the sex pheromone component (Z)-9-tricosene and CO2, Proc. Natl. Acad. Sci. USA. 91
363
(1994) 10000-10004.
16 Page 16 of 25
364
[8] T.D. Sutherland, G.C. Unnithan, J.F. Andersen, P.H. Evans, M.B. Murataliev, L.Z. Szabo,
365
E.A. Mash, W.S. Bowers, R. Feyereisen, A cytochrome P450 terpenoid hydroxylase linked
366
to the suppression of insect juvenile hormone synthesis, Proc. Nat. Acad. Sci. USA. 95
367
(1998) 12884-12889.
368
[9] J. Winter, C. Eckerskorn, R. Waditschatka, H. Kayser, A microsomal ecdysone-binding
369
cytochrome P450 from the insect Locusta migratoria purified by sequential use of type-II
370
and type-I ligands, Bio. Chem. 382 (2001) 1541-1549.
371
[10] L.I. Gilbert, Halloween genes encode P450 enzymes that mediate steroid hormone
372
biosynthesis in Drosophila melanogaster, Mol. Cell. Endocrinol. 215 (2004) 1–10.
373
[11] R. Niwa, T. Matsuda, T. Yoshiyama, T. Namiki, K. Mita, Y. Fujimoto, H. Kataoka,
374
CYP306A1, a cytochrome P450 enzyme, is essential for ecdysteroid biosynthesis in the
375
prothoracic glands of Bombyx and Drosophila, J. Biol. Chem. 279 (2004) 35942–35949.
376
[12] L.C. Terriere, Induction of detoxication enzymes in insects, Ann. Rev. Entomol. 29 (1984)
377
71-88.
378
[13] L.C. Terriere, Enzyme induction, gene amplification, and insect resistance to insecticides,
379
in: G.P. Georghiou, T. Saito (Eds.), Pest Resistance to Pesticides, Plenum Press, New
380
York, 1983, pp. 265-297.
381 382 383
[14] F. Zhu, J.N. Feng, L. Zhang, N. Liu, Characterization of two novel cytochrome P450 genes in insecticide-resistant house-flies, Insect Mol. Biol. 17 (2008a) 27-37. [15] F. Zhu, T. Li, L. Zhang, N. Liu, Co-up-regulation of three P450 genes in response to
384
permethrin exposure in permethrin resistant house flies, Musca domestica, BMC Physiol.
385
8 (2008b) 18.
17 Page 17 of 25
386 387 388
[16] F. Zhu, N. Liu, Differential expression of CYP6A5 and CYP6A5v2 in pyrethroid-resistant house flies, Musca domestica, Arch. Insect Biochem. Physiol. 34 (2008) 147-161. [17] R.A. Festucci-Buselli, A.S. Carvalho-Dias, M. de Oliveira-Andrade, C. Caixeta-Nunes,
389
H.M. Li, J.J. Stuart, W. Muir, M.E. Scharf, B.R. Pittendrigh, Expression of Cyp6g1 and
390
Cyp12d1 in DDT resistant and susceptible strains of Drosophila Melanogaster, Insect
391
Mol. Biol. 14 (2005) 69–77.
392
[18] N. Liu, T. Li, W.R. Reid, T. Yang, L. Zhang, Multiple cytochrome P450 genes: Their
393
constitutive overexpression and permethrin induction in insecticide resistant mosquitoes,
394
Culex quinquefasciatus. PLoS ONE 6 (2011) e23403.
395
[19] F.A. Carino, J.F. Koener, F.W.Jr. Plapp, R. Feyereisen, Constitutive overexpression of the
396
cytochrome P450 gene CYP6A1 in a house fly strain with metabolic resistance to
397
insecticides, Insect Biochem. Mol. Biol. 24 (1994) 411-418.
398
[20] N. Liu, J.G. Scott, Genetic analysis of factors controlling high-level expression of
399
cytochrome P450, CYP6D1, cytochrome b5, P450 reductase, and monooxygenase activities
400
in LPR house flies, Musca domestica, Biochem. Genet. 34 (1996) 133-148.
401
[21] S. Maitra, S.M. Dombrowski, M. Basu, O. Raustol, L.C. Waters, R. Ganguly, Factors on
402
the third chromosome affect the level of Cyp6a2 and Cyp6a8 expression in Drosophila
403
melanogaster, Gene 248 (2000) 147-156.
404 405
[22] J.R. Misra, M.A. Horner, G. Lam, C.S. Thummel, Transcriptional regulation of xenobiotic detoxification in Drosophila, Genes Dev. 25 (2011) 1796–1806.
18 Page 18 of 25
406 407 408 409
[23] J.R. Misra, G. Lam, C.S. Thummel, Constitutive activation of the Nrf2/Keap1 pathway in insecticide-resistant strains of Drosophila, Insect Biochem. Mol. Biol. 43 (2013) 1116-1124. [24] N. Liu, X. Yue, Insecticide resistance and cross-resistance in the house fly (Diptera: Muscidae), J. Econ. Entomol. 93 (2000) 1269-1275.
410
[25] J.W. Pridgeon, A.G. Appel, W.J. Moar, N. Liu, Variability of resistance mechanisms in
411
pyrethroid resistant German cockroaches (Dictyoptera: Blattellidae), Pestic. Biochem.
412
Physiol. 73 (2002) 149-156.
413 414 415
[26] Q. Xu, H. Liu, L. Zhang, N. Liu, Resistance in the mosquito, Culex quinquefasciatus, and possible mechanisms for resistance, Pest Manag. Sci. 61 (2005) 1096-1102. [27] R.D. McAbee, K.D. Kang, M.A. Stanich, J.A. Christiansen, C.E. Wheelock, A.D. Inman,
416
B.D. Hammock, A.J. Cornel, Pyrethroid tolerance in Culex pipiens pipiens var molestus from
417
Marin County, California, Pest Manag. Sci. 60 (2004) 359–368.
418
[28] F. Chandre, F. Darriet, M. Darder, A. Cuany, J.M.C. Doannio, N. Pasteur, P. Cuillet,
419
Pyrethroid resistance in Culex quinquefasciatus from West Africa, Med. Vet. Entomol. 12
420
(1998) 359-366.
421
[29] S. Kasai, I.S. Weerashinghe, T. Shono, P450 monooxygenases are an important mechanism
422
of permethrin resistance in Culex quinquefasciatus Say larvae, Arch. Insect. Biochem.
423
Physiol. 37 (1998) 47-56.
424 425 426 427
[30] N. Liu, J.G. Scott, Genetics of resistance to pyrethroid insecticides in the house fly, Musca domestica, Pestic. Biochem. Physiol. 52 (1995) 116-124. [31] T.M. Brown, P.K. Bryson, G.T. Payne, Synergism by propynyl aryl ethers in permethrinresistant tobacco budworm larvae, Pestic. Sci. 46 (1996) 323–331.
19 Page 19 of 25
428 429 430 431
[32] L. Zhang, K. Harada, T. Shono, Genetic analysis of pyriproxifen resistance in the housefly, Musca domestica L, Appl. Ent. Zool. 32 (1997) 217–226. [33] N. Liu, J.G. Scott, Increased transcription of CYP6D1 causes cytochrome P450-mediated insecticide resistance in house fly, Insect Biochem. Mol. Biol. 28 (1998) 531-535.
432
[34] T. Yang, N. Liu, Genome analysis of cytochrome P450s and their expression profiles in
433
insecticide resistant mosquitoes, Culex quinquefasciatus, PLoS ONE 6 (2011) e29418.
434
[35] N. Liu, J.G. Scott, Phenobarbital induction of CYP6D1 is due to a trans acting factor on
435 436
autosome 2 in house flies, Musca domestica, Insect. Mol. Biol. 6 (1997) 77-81. [36] S. Kasai, I.S. Weerashinghe, T. Shono, M. Yamakawa, Molecular cloning, nucleotide
437
sequence, and gene expression of a cytochrome P450 (CYP6F1) from the pyrethroid-resistant
438
mosquito, Culex quinquefasciatus Say, Insect Biochem. Mol. Biol. 30 (2000) 163-171.
439
[37] K. Itokawa, O. Komagata, S. Kasai, Y. Okamura, M. Masada, T. Tomita, Genomic
440
structures of Cyp9m10 in pyrethroid resistant and susceptible strains of Culex
441
quinquefasciatus, Insect Biochem. Mol. Biol. 40 (2010) 631-640.
442
[38] M.C. Hardstone, O. Komagata, S. Kasai, T. Tomita, J.G. Scott, Use of isogenic strains
443
indicates CYP9M10 is linked to permethrin resistance in Culex pipiens quinquefasciatus,
444
Insect Mol. Biol. 19 (2010) 717-726.
445
[39] X. Li, M.R. Berenbaum, M.A. Schuler, Cytochrome P450 and actin genes expressed in
446
Helicoverpa zea and Helicoverpa armigera: paralogy/orthology identification, gene
447
conversion and evolution, Insect Biochem. Mol. Biol. 32 (2002) 311-320.
448
[40] M. Li, W.R. Reid, L. Zhang, J.G. Scott, X. Gao, M. Kristensen, N. Liu, A whole
449
transcriptomal linkage analysis of gene co-regulation in insecticide resistant house flies,
450
Musca domestica, BMC Genomics 14 (2013) 803.
20 Page 20 of 25
451 452
[41] L.M. Tompkins, D.W. Andrew, Mechanisms of cytochrome P450 induction, J. Biochem. Mol. Toxicol. 21 (2007) 176-181.
453
[42] R. Poupardin, S. Reynaud, C. Strode, H. Ranson, J. Vontas, J.-P. David, Cross-induction of
454
detoxification genes by environmental xenobiotics and insecticides in the mosquito Aedes
455
aegypti: Impact on larval tolerance to chemical insecticides, Insect Biochem. Mol. Biol. 38
456
(2008) 540-551.
457
[43] M.E. Scharf, S. Parimi, L.J. Meinke, L.D. Chandler, B.D. Siegfried. Expression and
458
induction of three family 4 cytochrome P450 (CYP4) genes identified from insecticide-
459
resistant and susceptible western corn rootworms, Diabrotica virgifera virgifera, Insect
460
Mol. Biol. 10 (2001) 139-146.
461
[44] L.B. Brattaten. Potential role of plant allelochemicals in the development of insecticide
462
resistance, P. Barbosa, D.K. Letourneau (eds.), Novel Aspects of Insect Plant Interaction,
463
John Wiley, New York (1988), pp. 313-348.
464 465 466
[45] A.B. Okey, Enzyme induction in the cytochrome P-450 system, Pharmacol. Ther. 45 (1990) 241-298. [46] Y. Gong, T. Li, L. Zhang, X. Gao, N. Liu, Permethrin induction of multiple cytochrome
467
P450 genes in insecticide resistant mosquitoes, Culex quinquefasciatus, Int. J. Biol. Sci. 9
468
(2013) 863-871.
469
[47] M.A. Schuler, M.R. Berenbaum, Structure and function of cytochrome P450S in insect
470
adaptation to natural and synthetic toxins: Insights gained from molecular modeling, J.
471
Chem. Ecol. 39 (2013) 1232-1245.
472 473
[48] M. Hiratsuka, In vitro assessment of the allelic variants of cytochrome P450, Drug Metab. Pharmacokinet. 27 (2011) 68-84.
21 Page 21 of 25
474
[49] O. Gotoh, Substrate recognition sites in cytochrome P450 family 2 (CYP2) proteins inferred
475
from comparative analyses of amino acid and coding nucleotide sequences, J. Biol. Chem.
476
267 (1992) 83-90.
477
[50] T.L. Chiu, Z. Wen, S.G. Rupasinghe, M.A. Schuler, Comparative molecular modeling of
478
Anopheles gambiae CYP6Z1, a mosquito P450 capable of metabolizing DDT, Proc. Natl.
479
Acad. Sci. USA. 105 (2008) 8855-8860.
480
[51] L. McLaughlin, U. Niazi, J. Bibby, J.P. David, J. Vontas, J. Hemingway, H. Ranson, M.
481
Sutcliffe, M. Paine, Characterization of inhibitors and substrates of Anopheles gambiae
482
CYP6Z2, Insect Mol. Biol. 17 (2008) 125-135.
483
[52] B.J. Stevenson, J. Bibby, P. Pignatelli, S. Muangnoicharoen, P.M. O’Neill, L.Y. Lian, P.
484
Müller, D. Nikou, A. Steven, J. Hemingway, M.J. Sutcliffe, M.J.I. Paine, Cytochrome P450
485
6M2 from the malaria vector Anopheles gambiae metabolizes pyrethroids: Sequential
486
metabolism of deltamethrin revealed, Insect Biochem. Mol. Biol. 41 (2011) 492-502.
487
[53] P. Lertkiatmongkol, E. Jenwitheesuk, P. Rongnoparut, Homology modeling of mosquito
488
cytochrome P450 enzymes involved in pyrethroid metabolism: insights into differences in
489
substrate selectivity, BMC research notes 4 (2011) 321.
490
[54] C.P. Alexia, B. Jaclyn, R.K. Myriam, R. Jessica, G.C. Emilie, P. Rodolphe, A.R.
491
Muhammad, P. Mark, D.V. Chantal, R. Stephane, The central role of mosquito cytochrome
492
P450 CYP6Zs in insecticide detoxification revealed by functional expression and structural
493
modelling, Biochem. J. 455 (2013) 75-85.
494 495
[55] C. Zimmer, C. Bass, M. Williamson, M. Kaussmann, K. Wölfel, O. Gutbrod, R. Nauen, Molecular and functional characterization of CYP6BQ23, a cytochrome P450 conferring
22 Page 22 of 25
496
resistance to pyrethroids in European populations of pollen beetle, Meligethes aeneus,
497
Insect Biochem. Mol. Biol. 45 (2014) 18-29.
498
[56] F. Zhu, T.W. Moural, K. Shah, S.R. Palli, Integrated analysis of cytochrome P450 gene
499
superfamily in the red flour beetle, Tribolium castaneum, BMC Genomics 14 (2013) 174.
500
[57] N. Liu, Insecticide resistance in mosquitoes: impact, mechanisms, and research directions,
501
Annu. Rev. Entomol. 60 (2015) 537-559.
502
[58] T. Yang, N. Liu, Permethrin resistance variation and susceptible reference line isolation in a
503
field population of the mosquito, Culex quinquefasciatus (Diptera: Culicidae), Insect Sci.
504
(2013) 1-8.
505
[59] K.A. Feenstra, E.B. Starikov, V.B. Urlacher, J.N. Commandeur, N.P. Vermeulen,
506
Combining substrate dynamics, binding statistics, and energy barriers to rationalize
507
regioselective hydroxylation of octane and lauric acid by CYP102A1 and mutants, Protein
508
Sci. 16 (2007) 420-431.
509 510 511 512 513 514 515
[60] N. Liu, H. Liu, F. Zhu, L. Zhang, Differential expression of genes in pyrethroid resistant and susceptible mosquitoes, Culex quinquefasciatus (S.), Gene 394 (2007) 62-68. [61] W.R. Reid, L. Zhang, F. Liu, N. Liu, The transcriptome profile of the mosquito Culex quinquefasciatus following permethrin selection, PLoS One 7 (2012) e47163. [62] M.C. Lagerström, H.B. Schiöth, Structural diversity of G protein-coupled receptors and significance for drug discovery, Nat. Rev. Drug Discov. 7 (2008) 339-357. [63] T. Li, L. Liu, L. Zhang, N. Liu, Role of G-protein-coupled receptor-related genes in
516
insecticide resistance of the mosquito, Culex quinquefasciatus, Scientific Reports 4 (2014)
517
6474.
23 Page 23 of 25
518
[64] S. Yan, P. Zou†, X.Y. Yu, C. Chen, J. Yu, L.N. Shi, S.C. Hong, D. Zhou, X.L. Chang, W.J.
519
Wang, B. Shen, D.H. Zhang, L. Ma, C.L. Zhu, Functional characterization of an arrestin
520
gene on insecticide resistance of Culex pipiens pallens, Parasit. Vec. 5 (2012) 134.
521 522 523
Figure Legends
524 525
Figure 1. Topology of P450s. The secondary structures of helices and sheets are labeled. Six
526
putative SRSs are colored and predicted according to predicted models [49]. SRSs 1 to 6 are
527
shown in red, white, yellow, blue, purple and orange, respectively. The heme group is
528
represented by sticks.
529 530
Figure 2. A hypothetical model of P450 gene regulation in insecticide resistance. This model is
531
based on our previous studies [14], [15], [34], [57] and [6] as well as those of others [19], [33]
532
and [21], suggesting the interaction of resistance P450 genes, basal transcriptional Factors (BTF)
533
and trans regulatory factors (TRF) or cis elements in P450 gene (CYP) expression in insecticide
534
resistance. The P450 gene(s) in resistance insects interacts with regulatory factors that
535
facilitate/regulate the overexpression of the resistanceP450 gene (s), whereas, this regulatory
536
pathway(s) in susceptible insects is obstructed due to unknown mechanisms.
537 538 539 540 541 24 Page 24 of 25
542 543 544 545 546 547 548 549 550 551 552 553
25 Page 25 of 25
S0048-3575(15)00007-3 http://dx.doi.org/doi: 10.1016/j.pestbp.2015.01.006 YPEST 3779
To appear in:
Pesticide Biochemistry and Physiology
Received date: Accepted date:
1-11-2014 10-1-2015
Please cite this article as: Nannan Liu, Ming Li, Youhui Gong, Feng Liu, Ting Li, Cytochrome p450s –their expression, regulation, and role in insecticide resistance, Pesticide Biochemistry and Physiology (2015), http://dx.doi.org/doi: 10.1016/j.pestbp.2015.01.006. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1
Prepared for Publication in
2
Pesticide Biochemistry and Physiology
3 4 5 6 7 8
Cytochrome P450s –Their Expression, Regulation, and Role in Insecticide Resistance
9 10
Nannan Liu1*, Ming Li1, Youhui Gong1,2, Feng Liu1, Ting Li1
11 12
1
13
USA
14
2
Department of Entomology and Plant Pathology, Auburn University, Auburn, Alabama 36849,
Department of Entomology, China Agricultural University, Beijing, China
15 16
*To whom correspondence should be addressed: [email protected]; Phone: 334-844-2661
17 18 19
Highlights
20 21 22
1) The characterization of P450-mediated detoxification in insecticide resistance is key for controlling insect pests.
23
2) P450 gene expression studies have progressed from the analysis of single gene
24
sequences to whole genome sequence analysis and from characterizing the expression
1 Page 1 of 25
25
of individual genes to examining the expression of multiple genes simultaneously,
26
revealing that the overexpression of multiple P450 genes in insecticide resistant strains
27
is a common phenomenon that has now been observed in many resistant insect species
28
3) Many studies have support that the up-regulation of resistance P450 genes is regulated
29
by trans and/or cis factors in insecticide-resistant insects.
30
4) With the exciting discovery of the potential function of GPCR and GPCR-related genes
31
in insecticide resistance and their regulatory function in resistance-related P450 gene
32
expression, future research should focus on the downstream factors in the GPCR
33
regulatory pathway in order to characterize their involvement in insecticide resistance.
34 35 36
Graphical Abstract
37 38 39 40 2 Page 2 of 25
41 42
Abstract
43 44
P450s are known to be critical for the detoxification and/or activation of xenobiotics such as
45
drugs and pesticides and overexpression of P450 genes can significantly affect the disposition of
46
xenobiotics in the tissues of organisms, altering their pharmacological/toxicological effects. In
47
insects, P450s play an important role in detoxifying exogenous compounds such as insecticides
48
and plant toxins and their overexpression can result in increased levels of P450 proteins and
49
P450 activities. This has been associated with enhanced metabolic detoxification of insecticides
50
and has been implicated in the development of insecticide resistance in insects. Multiple P450
51
genes have been found to be co-overexpressed in individual insect species via several
52
constitutive overexpression and induction mechanisms, which in turn are co-responsible for high
53
levels of insecticide resistance. Many studies have also demonstrated that the transcriptional
54
overexpression of P450 genes in resistant insects is regulated by trans and/or cis regulatory
55
genes/factors. Taken together, these earlier findings suggest not only that insecticide resistance is
56
conferred via multi-resistance P450 genes, but also that it is mediated through the interaction of
57
regulatory genes/factors and resistance genes. This chapter reviews our current understanding of
58
how the molecular mechanisms of P450 interaction/gene regulation govern the development of
59
insecticide resistance in insects and our progress along the road to a comprehensive
60
characterization of P450 detoxification-mediated insecticide resistance.
61 62
Keywords
63
Insecticide resistance, cytochrome P450, interaction/regulation, induction, homology modeling
64 3 Page 3 of 25
65
Introduction
66 67
Cytochrome P450s have long been of particular interest to researchers because of their critical
68
biological function in the detoxification and/or activation of xenobiotics such as drugs,
69
pesticides, plant toxins, chemical carcinogens and mutagens, as well as their role in the
70
metabolism of endogenous compounds such as hormones, fatty acids, and steroids. Basal and/or
71
up-regulation of P450 gene expression is known to considerably affect the disposition of
72
xenobiotics or endogenous compounds in the tissues of organisms and is thus responsible for
73
altering their pharmacological/toxicological effects [1]. Insect cytochrome P450s are involved in
74
detoxifying exogenous compounds such as insecticides [2], [3] and [4] and plant toxins [5] and
75
[6]. They are also known to be an important component in the biosynthesis and degradation
76
pathways of endogenous compounds such as pheromones, 20-hydroxyecdysone, and juvenile
77
hormone (JH) in insects [7], [8], [9], [10] and [11] and thus perform important functions in insect
78
growth, development, and reproduction. While all insects probably have certain capacity to
79
detoxify insecticides and xenobiotics, the degree to which an insect can metabolize or detoxify
80
toxic substances is of critical for their survival in a chemically unfavorable situation [12], as is
81
the development of resistance. Whereas the constitutively increased expression and induction of
82
P450 gene expression are both thought to be directly linked to the degree that insects are capable
83
to enhance their metabolism and detoxification of insecticides, they have also been implicated in
84
the adaptation of insects to their environment [12], [13], [14] and [15] and their development of
85
resistance to insecticides. A number of studies have revealed that multiple P450 genes are co-
86
overexpressed/up-regulated in individual resistant organisms such as house flies and mosquitoes
87
[14], [15], [16], [17] and [18], demonstrating elevated expression levels of P450 genes in
4 Page 4 of 25
88
resistant insects. The transcription overexpression of P450 genes in resistant insects is also
89
known to be regulated by trans and/or cis regulatory genes/factors [19], [20] and [21].
90 91
Taken together, these findings suggest that insecticide resistance is conferred via multi-resistance
92
P450 gene overexpression and induction and is arbitrated through the interaction of regulatory
93
genes and resistance P450 genes. Characterization of the total number of P450 genes directly
94
involved in resistance in a single resistant insect and factors involved in regulation of resistance
95
P450 gene expression [22] and [23] is now the focus of attention for those working in the fields
96
of insecticide resistance and insect toxicology. The emergence of the “Genomics Era” during the
97
last decade, coupled with the availability of new functional study techniques such as double-
98
stranded RNA-mediated gene interference (RNAi) and transgenic gene expression, have led to
99
significant progress being made towards characterizing not only the number of P450 genes but
100
also identifying the functions of the genes that are involved in insecticide resistance at the whole
101
genome level. This review focuses on our journey towards the characterization of the P450
102
detoxification-mediated insecticide resistance of insects.
103 104
Synergism studies – the initial step in the characterization of P450 detoxification in
105
insecticide resistance
106 107
The initial characterization of the importance of metabolic detoxification in insecticide resistance
108
has primarily been documented in many insect species on the basis of synergistic studies. These
109
studies represent the initial step towards revealing the possible mechanisms involved in
110
insecticide resistance using chemical synergists such as piperonyl butoxide (PBO), S,S,S,-
5 Page 5 of 25
111
tributyl phosphorotrithioate (DEF), and diethyl maleate (DEM), which are inhibitors of
112
cytochrome P450 monooxygenases, hydrolases, and glutathione S-transferases (GSTs),
113
respectively. By comparing the resistance levels with and without the synergists, researchers can
114
begin to narrow down precisely which detoxification mechanisms are involved in the
115
development of insecticide resistance. Our laboratory has conducted a series of studies looking at
116
the mechanisms involved in pyrethroid resistance in the house fly Musca domestica, mosquito
117
Culex quinquefasciatus and German cockroach, Blattella germanica. We found that permethrin
118
resistance levels in all three were largely suppressed by PBO, indicating that P450
119
monooxygenase-mediated detoxification is one of the major mechanisms involved in the
120
development of pyrethroid resistance in these insect species [24], [25] and [26]. These findings
121
strongly support those reported in numerous similar studies on insecticide resistance in different
122
insect species, including those in other mosquito [27], [28] and [29] and house fly strains [30].
123
Although in many cases the initial evidence generated by synergistic studies designed to
124
determine the importance of detoxification proteins such as P450s in insecticide resistance is
125
undoubtedly reliable [2], other studies have produced evidence that suggests synergists are
126
deficient inhibitors for some of the detoxification enzymes responsible for resistance [2], [31]
127
and [32]. To resolve this disagreement, independent studies using approaches such as enzyme
128
activity assays, metabolism studies, gene expression analysis, and, if possible, functional studies,
129
are needed to confirm the precise role of detoxification proteins in resistance.
130 131
Characterization of gene expression – pinpointing the individual P450 genes involved in
132
insecticide resistance
133
6 Page 6 of 25
134
P450 constitutive overexpression
135
One noteworthy characteristic of insect P450s that is known to be associated with enhanced
136
metabolic detoxification of insecticides in insects is the increased protein production and
137
activities of these enzymes through the transcriptional up-regulation of the genes in insecticide
138
resistant insects. Gene expression studies have progressed from the analysis of single gene
139
sequences to whole genome sequence analysis and from characterizing the expression of
140
individual genes to examining the expression of multiple genes simultaneously, revealing that
141
the overexpression of multiple P450 genes in insecticide resistant strains is a common
142
phenomenon that has now been observed in many resistant insect species [2], [3], [14], [15],
143
[16], [17], [18], [19], [33], [34], [35], [36], [37], [38] and [39]. These studies have revealed that
144
the interaction of multiple insecticide resistance P450 genes may be responsible for the
145
development of resistance. Recently, our group conducted a systematic study to characterize the
146
expression profiles of 204 P450 genes in the mosquito Culex quinquefasciatus at a whole
147
genome level using real-time quantitative PCR analysis [34], identifying multiple P450 genes as
148
up-regulated in individual resistant mosquito strains and leading us to conclude that multiple
149
P450 genes are co-overexpressed, which in turn is responsible for the detoxification of
150
insecticides and the development of insecticide resistance in these mosquito strains. Further
151
evidence supporting this conclusion was provided by our whole transcriptome study using
152
RNAseq data analysis [40]. This P450 gene overexpression in insecticide resistance is not unique
153
to mosquitoes; multiple P450 gene co-overexpression has also been implicated in the
154
development of insecticide resistance in other insect species. An elegant study utilizing whole
155
transcriptome analysis clearly demonstrated that multiple P450 gene co-overexpression is also
7 Page 7 of 25
156
responsible for high levels of insecticide resistance in house flies. These findings suggest that
157
high levels of insecticide resistance are conferred via multi-resistance P450 gene overexpression.
158 159
A number of studies have revealed that the overexpression of resistance-related P450 genes is
160
controlled by various regulatory factors [40]. For example, the up-regulation of 2 P450 genes,
161
CYP6A1 and CYP6D1, which are located on autosomes 5 and 1, respectively, in insecticide
162
resistant house flies is known to be regulated by trans-regulatory factors on autosome 2 [19],
163
[20], [33] and [35], while the up-regulation of CYP6A2 and CYP6A8 in the insecticide resistant
164
fruit fly Drosophila melanogaster is transcriptionally regulated by trans-regulatory factors on
165
autosome 3 [21]. Genetic linkage analyses of house fly lines that compared different autosomal
166
combinations from resistant wide type strain(s) revealed that the up-regulation of P450 gene
167
overexpression expression occurred mainly through the co-regulation of factors among multiple
168
autosomes, especially between autosomes 2 and 5 [14] and [15]. Taken together, these findings
169
suggest not only that insecticide resistance is conferred via multiple resistance P450 genes, but
170
also that it is mediated through the interaction of several regulatory genes/factors and resistance
171
genes [22] and [23].
172 173
P450 induction
174 175
Another interesting characteristic of some insect P450 genes is their induced expression by
176
exogenous and endogenous compounds, a phenomenon known as induction. It has been
177
suggested that induction is in fact responsible for the enhanced metabolism of pharmaceutical
178
drugs, insecticides and endogenous compounds [2], [3] and [41]. While all insects probably
8 Page 8 of 25
179
possess some capacity to detoxify insecticides and xenobiotics, the degree to which individual
180
insects can metabolize and detoxify these highly toxic chemicals is of considerable importance
181
for their survival in a chemically unfriendly environment [12]. It is possible that the induction of
182
P450s and their activities in insects is involved in the adaptation of insects to their environment,
183
the detoxification of insecticides and the development of insecticide resistance [12], [13], [16],
184
[17], [35], [42], and [43]. It has also been suggested that induction associated with increased
185
tolerance of insects to insecticides may help the development of resistance [44]. Chemical
186
inducers could act as substrates for P450s, allowing the induction or modulation of P450s by
187
these substrates to in turn reduce the effects of the substrates by enhancing the substrate
188
metabolism [45]. Our group recently discovered that multiple P450 that are constitutively
189
overexpressed in resistant mosquitoes can be further induced in a clear concentration dose- and
190
time-dependent manner when the mosquitoes are exposed to further doses of the same
191
insecticide [18] and [46], increasing the overall expression levels of multiple P450 genes in the
192
resistant mosquitoes. This finding strongly supports the mechanism proposed by Okey [45]. We
193
also observed that P450 gene induction in mosquitoes follows a resistance-specific pattern, with
194
similar results being reported earlier in Drosophila melanogaster [17], where the expression of
195
CYP6g1 and CYP12d1 were induced in the DDT resistant strains post-exposure to DDT. Studies
196
by Zhu et al. [14] and [15] indicated that several P450 genes were up-regulated in insecticide
197
resistant house flies through a similar induction mechanism. Zhu et al. [14] and [15] also
198
reported that although some P450 genes, such as CYP4D4v2, CYP4G2, and CYP6A38 in
199
resistant house flies, are not constitutively overexpressed, their expression can be induced when
200
insects are under insecticide application pressure, suggesting that these genes may be uniquely
201
involved in the response to insecticide exposure through the induction of their expression, which
9 Page 9 of 25
202
in turn enhances their capacity to detoxify the insecticide and leads to enhanced insecticide
203
resistance [14] and [15].
204 205
Similar regulatory mechanisms may govern P450 constitutive overexpression and induction, and
206
it has been suggested that both contribute to the development of insecticide resistance [12]. It is
207
also possible that the higher inducible expression of transcripts is due primarily to mutations in
208
trans regulatory factors or their signaling cascades [47]. However, little is known about the trans
209
regulatory signaling components that mediate the expression of insect detoxificative P450s [47].
210
It has also been suggested that the induction of P450s is a complex phenomenon involving
211
multiple DNA-binding elements in each P450 promoter, transcriptional factors, and the overlap
212
of regulatory pathways [39]. It would therefore be particularly interesting to further characterize
213
the transcriptional elements in the promoter region of P450 genes and the regulatory factors
214
involved in the regulation of both the constitutive overexpression and induction of the P450
215
genes that have been implicated in the development of insecticide resistance in order to more
216
fully understand the molecular mechanisms involved.
217 218
In silico 3D P450 homology modeling and insecticide molecular docking
219 220
In silico homology modeling and molecular docking of protein, a technique that can be used to
221
build a three-dimensional model of proteins from their amino acid sequences on the basis of the
222
alignment with a similar protein with a known structure (the template), has become a very
223
effective tool for understanding the relationship between the structure of proteins such as P450s
224
and substrates such as insecticides to provide reasonable explanations of substrate specificities
10 Page 10 of 25
225
and metabolic specificities caused by allelic variations or mutations [47] and [48]. The
226
importance of in silico homology modeling in the post-genomic era is growing rapidly since it
227
provides a useful avenue for characterizing/understanding the biological functions of the millions
228
of protein sequences now being generated by high-throughput next generation sequencing
229
technology. The tertiary structure of P450s contains several conserved structures such as helices
230
A to L that act as the protein backbone, a hydrophobic N-terminal domain that acts as the
231
transmembrane anchor, and the active site, which is buried deep within the enzyme structure and
232
connects to the surrounding environment via a network of channels that serve as access/egress
233
paths. Six regions of a P450 have been designated as substrate recognition sites (SRSs, Figure 1)
234
based on studies of human P450s in the CYP2 family [49]. These SRSs contribute to the function
235
of a P450, with SRS1, SRS4, SRS5, and SRS6 known to be involved in catalytic site formation
236
and SRS2 and SRS3 to participate in substrate access channel formation [47].
237 238
In silico homology modeling and molecular docking have been used to predict and validate the
239
function of P450s in several studies in insects. With this new computer modeling system to help
240
them cope with the highly complex functional metabolism studies, researchers can now
241
confidently state that several insect P450 genes are important in the development of resistance to
242
insecticides, including CYP6Z1, CYP6Z2 and CYP6M2 in Anopheles gambiae [50], [51] and
243
[52], CYP6AA3 and CYP6P7 in Anopheles minumus [53], CYP6Z8 in Aedes aegypti [54],
244
CYP6QB23 in Meligethes aeneus [55], and CYP6BQ8, CUP6BQ9, CYP6BQ10 and CYP6BQ11
245
in Tribolium castaneum [56]. Utilizing in silico homology modeling and molecular docking
246
methodology, Liu [57] evaluated various models for binding permethrin to the active site(s) of
247
CYP6AA7, an insect P450 that is known to be overexpressed in Cx. quinquefasciatus [34] and
11 Page 11 of 25
248
[58], revealing that three permethrin binding models of CYP6AA7 were present in Culex
249
quinquefasciatus. All the residues involved in the permethrin binding were located in the SRSs
250
and the hydrophobic residues of phenylalanine (Phe), valine (Val) and proline (pro) were all
251
conserved in the catalytic sites of the three permethrin binding modes, indicating the importance
252
of these residues for permethrin binding and suggesting that these enzymes provide a favorable
253
chemical environment for hydrophobic insecticide compounds. The short distance between the
254
putative metabolic sites and the heme-oxygen in an orientation nearly perpendicular to the heme
255
plane (within 6.0 Å) [56] and [59] suggests that CYP6AA7 presents a favorable binding affinity
256
to permethrin and imparts a putative hydrolytic ability to permethrin.
257 258
Regulatory genes, factors, and pathways in insecticide resistance
259 260
The results of our previous studies [14], [15], [34], [60] and [61] as well as those of others [19],
261
[20], [21], [33] and [35] suggest that the interaction of multiple genes may be responsible for
262
insecticide resistance. Many studies have also reported that the up-regulation of resistance P450
263
genes is regulated by trans and/or cis factors in insecticide-resistant insects. Taken together,
264
these studies suggest that not only is insecticide resistance conferred via multiple resistance
265
genes, but it is mediated through the interaction of regulatory factors and resistance P450 genes.
266
The information above allowed us to propose a hypothetic model (Figure 2), in which trans
267
regulatory factors or cis regulatory elements may be crucial in the regulation of the resistance
268
P450 gene expression and, in turn, are involved in the detoxification of insecticides and the
269
evolution of insecticide resistance in insects. However, as yet none of the regulatory factors in
270
insecticide resistance have been identified and the regulation pathways have not been examined.
12 Page 12 of 25
271
Using the SSH/cDNA array technique, Liu et al [60] identified 22 unique genes that are over-
272
expressed in in pyrethroid resistant Cx. quinquefasciatus, in addition to two new P450 genes
273
known to be important in resistance; a further 20 ESTs, including a signaling transduction gene
274
that encodes a G-protein coupled receptor, were identified that had not previously been
275
implicated in insecticide resistance in any insect species, further suggesting the involvement of
276
multiple up-regulated gene interactions and signaling regulatory factor(s) in the insecticide
277
resistant mosquitoes [60].
278 279
Because of the important roles that GPCRs play in recognizing extracellular messengers,
280
transducing signals to the cytosol and mediating the cellular responses necessary for the normal
281
physiology of organisms [62], our group has taken significant steps towards characterizing the
282
GPCR s’ function in insecticide resistance. We were the first to explore the function of GPCRs
283
and GPCR-related genes in the insecticide resistance of mosquitoes, Culex quinquefasciatus
284
using quantitative RT-PCR and functional genomic methods [63]. By comparing the expression
285
of 115 GPCR-related genes at a whole genome level in resistant and susceptible Cx.
286
quinquefasciatus mosquitoes, we were able to identify several GPCR-related genes that were up-
287
regulated in the highly resistant Culex mosquito strains. Knockdown of these four GPCR-related
288
genes using the RNAi technique revealed significant decreases in the levels of resistance of the
289
mosquitoes to permethrin. More interestingly, knockdown of these four GPCR-related genes
290
directly affected the expression of the resistance P450 genes, suggesting that the role of GPCR-
291
related genes in resistance is related to the regulation of resistance P450 gene expression [63].
292
This new information clearly indicates the role of GPCR-regulated pathway in the regulation of
293
the resistance cytochrome P450-mediated detoxification of insecticides and the evolution of
13 Page 13 of 25
294
resistance in Cx. quinquefasciatus. Sun et al. [64] recently reported that overexpression of a
295
GPCR related arrestin gene in deltamethrin resistant Culex pipiens pallens regulated the
296
insecticide resistance-associated P450 gene, further supporting the regulatory role of GPCRs in
297
P450-mediated insecticide resistance. These exciting discoveries shed new light on the potential
298
function of GPCR and GPCR-related genes in insecticide resistance and their regulatory function
299
on resistance-related P450 gene expression. However, the complete regulatory pathway remains
300
largely unclear. Thus, future research needs to focus on the downstream factors in the GPCR
301
regulatory pathway in order to characterize their involvement in insecticide resistance and,
302
consequently, support the effort to identify new insecticide targets and strategies with which to
303
control mosquitoes and mosquito-borne diseases. This will also help to build a better
304
understanding of the regulatory pathway of insecticide detoxification and evolutionary
305
insecticide selection in mosquitoes [63].
306 307
Conclusions
308 309
Insect cytochrome P450s are known to play an important role in detoxifying insecticides and
310
plant toxins, resulting in the development of resistance to insecticides and facilitating the
311
adaptation of insects to their plant hosts. A significant characteristic of insect P450s that is
312
associated with enhanced metabolic detoxification of insecticides in insects is the increased
313
levels of P450 proteins and P450 activity that result from the constitutive transcriptional
314
overexpression of P450 genes in insecticide resistant insects. Another interesting characteristic of
315
insect P450 genes is that the expression of some P450 genes can be induced by exogenous and
316
endogenous compounds, a phenomenon known as induction. Given the close correlation between
14 Page 14 of 25
317
the production of these genes and the development of resistance and/or P450-mediated resistance
318
in ALHF, these observations provide overwhelming evidence that P450 induction and
319
constitutive overexpression are co-responsible for the detoxification of insecticides, evolutionary
320
insecticide selection, and the ability of insects to adapt to changing environments. Yet, compared
321
to the extensive knowledge accumulated regarding P450 gene expression, our understanding of
322
the molecular mechanisms involved in gene regulation and interactions in insecticide residence is
323
wholly inadequate. With the exciting discovery of the potential function of GPCR and GPCR-
324
related genes in insecticide resistance and their regulatory function in resistance-related P450
325
gene expression, future research should focus on the downstream factors in the GPCR regulatory
326
pathway in order to characterize their involvement in insecticide resistance. This is expected to
327
support the effort to identify new insecticide targets and strategies with which to control
328
mosquitoes and mosquito-borne diseases, as well as help us build a better understanding of the
329
regulatory pathway of insecticide detoxification and evolutionary insecticide selection in
330
mosquitoes [63].
331 332
Acknowledgments
333 334
The authors sincerely thanks all the people who have ever worked and are currently working in
335
their laboratory for their hard work and generating valuable data for understanding importance of
336
P450s in insecticide resistance and their dedication to the field of insecticide resistance research.
337 338 339 340 15 Page 15 of 25
341 342 343 344
References
345 346
[1] P. Pavek, Z. Dvorak, Xenobiotic-induced transcriptional regulation of xenobiotic
347
metabolizing enzymes of the cytochrome P450 superfamily in human extrahepatic tissues,
348
Curr. Drug Metab. 9 (2008) 129-143.
349
[2] R. Feyereisen, Insect CYP genes and P450 enzymes, in: L.I. Gilbert (Ed.), Insect Molecular
350
Biology and Biochemistry, Elsevier Academic Press, Oxford, 2012, pp. 236-316.
351
[3] R. Feyereisen, Arthropod CYPomes illustrate the tempo and mode in P450 evolution,
352 353 354
Biochem. Biophys. Arch. 1814 (2011) 19-28. [4] J.G. Scott, Cytochromes P450 and insecticide resistance, Insect Biochem. Mol. Biol. 29 (1999) 757-777.
355
[5] M.R. Berenbaum, Coumarins, in: G.A. Rosenthal, M.R. Berenbaum (Eds.), Herbivores: Their
356
Interaction with Secondary Plant Metabolites, Academic Press, New York, 1991, pp. 221-
357
249.
358 359 360
[6] M.A. Schuler, P450s in plant-insect interactions, Biochem. Biophys. Acta. 1814 (2011) 3645. [7] J.R. Reed, D. Vanderwel, S. Choi, J.G. Pomonis, R.C. Reitz, G.J. Blomquist, Unusual
361
mechanism of hydrocarbon formation in the housefly: cytochrome P450 converts aldehyde to
362
the sex pheromone component (Z)-9-tricosene and CO2, Proc. Natl. Acad. Sci. USA. 91
363
(1994) 10000-10004.
16 Page 16 of 25
364
[8] T.D. Sutherland, G.C. Unnithan, J.F. Andersen, P.H. Evans, M.B. Murataliev, L.Z. Szabo,
365
E.A. Mash, W.S. Bowers, R. Feyereisen, A cytochrome P450 terpenoid hydroxylase linked
366
to the suppression of insect juvenile hormone synthesis, Proc. Nat. Acad. Sci. USA. 95
367
(1998) 12884-12889.
368
[9] J. Winter, C. Eckerskorn, R. Waditschatka, H. Kayser, A microsomal ecdysone-binding
369
cytochrome P450 from the insect Locusta migratoria purified by sequential use of type-II
370
and type-I ligands, Bio. Chem. 382 (2001) 1541-1549.
371
[10] L.I. Gilbert, Halloween genes encode P450 enzymes that mediate steroid hormone
372
biosynthesis in Drosophila melanogaster, Mol. Cell. Endocrinol. 215 (2004) 1–10.
373
[11] R. Niwa, T. Matsuda, T. Yoshiyama, T. Namiki, K. Mita, Y. Fujimoto, H. Kataoka,
374
CYP306A1, a cytochrome P450 enzyme, is essential for ecdysteroid biosynthesis in the
375
prothoracic glands of Bombyx and Drosophila, J. Biol. Chem. 279 (2004) 35942–35949.
376
[12] L.C. Terriere, Induction of detoxication enzymes in insects, Ann. Rev. Entomol. 29 (1984)
377
71-88.
378
[13] L.C. Terriere, Enzyme induction, gene amplification, and insect resistance to insecticides,
379
in: G.P. Georghiou, T. Saito (Eds.), Pest Resistance to Pesticides, Plenum Press, New
380
York, 1983, pp. 265-297.
381 382 383
[14] F. Zhu, J.N. Feng, L. Zhang, N. Liu, Characterization of two novel cytochrome P450 genes in insecticide-resistant house-flies, Insect Mol. Biol. 17 (2008a) 27-37. [15] F. Zhu, T. Li, L. Zhang, N. Liu, Co-up-regulation of three P450 genes in response to
384
permethrin exposure in permethrin resistant house flies, Musca domestica, BMC Physiol.
385
8 (2008b) 18.
17 Page 17 of 25
386 387 388
[16] F. Zhu, N. Liu, Differential expression of CYP6A5 and CYP6A5v2 in pyrethroid-resistant house flies, Musca domestica, Arch. Insect Biochem. Physiol. 34 (2008) 147-161. [17] R.A. Festucci-Buselli, A.S. Carvalho-Dias, M. de Oliveira-Andrade, C. Caixeta-Nunes,
389
H.M. Li, J.J. Stuart, W. Muir, M.E. Scharf, B.R. Pittendrigh, Expression of Cyp6g1 and
390
Cyp12d1 in DDT resistant and susceptible strains of Drosophila Melanogaster, Insect
391
Mol. Biol. 14 (2005) 69–77.
392
[18] N. Liu, T. Li, W.R. Reid, T. Yang, L. Zhang, Multiple cytochrome P450 genes: Their
393
constitutive overexpression and permethrin induction in insecticide resistant mosquitoes,
394
Culex quinquefasciatus. PLoS ONE 6 (2011) e23403.
395
[19] F.A. Carino, J.F. Koener, F.W.Jr. Plapp, R. Feyereisen, Constitutive overexpression of the
396
cytochrome P450 gene CYP6A1 in a house fly strain with metabolic resistance to
397
insecticides, Insect Biochem. Mol. Biol. 24 (1994) 411-418.
398
[20] N. Liu, J.G. Scott, Genetic analysis of factors controlling high-level expression of
399
cytochrome P450, CYP6D1, cytochrome b5, P450 reductase, and monooxygenase activities
400
in LPR house flies, Musca domestica, Biochem. Genet. 34 (1996) 133-148.
401
[21] S. Maitra, S.M. Dombrowski, M. Basu, O. Raustol, L.C. Waters, R. Ganguly, Factors on
402
the third chromosome affect the level of Cyp6a2 and Cyp6a8 expression in Drosophila
403
melanogaster, Gene 248 (2000) 147-156.
404 405
[22] J.R. Misra, M.A. Horner, G. Lam, C.S. Thummel, Transcriptional regulation of xenobiotic detoxification in Drosophila, Genes Dev. 25 (2011) 1796–1806.
18 Page 18 of 25
406 407 408 409
[23] J.R. Misra, G. Lam, C.S. Thummel, Constitutive activation of the Nrf2/Keap1 pathway in insecticide-resistant strains of Drosophila, Insect Biochem. Mol. Biol. 43 (2013) 1116-1124. [24] N. Liu, X. Yue, Insecticide resistance and cross-resistance in the house fly (Diptera: Muscidae), J. Econ. Entomol. 93 (2000) 1269-1275.
410
[25] J.W. Pridgeon, A.G. Appel, W.J. Moar, N. Liu, Variability of resistance mechanisms in
411
pyrethroid resistant German cockroaches (Dictyoptera: Blattellidae), Pestic. Biochem.
412
Physiol. 73 (2002) 149-156.
413 414 415
[26] Q. Xu, H. Liu, L. Zhang, N. Liu, Resistance in the mosquito, Culex quinquefasciatus, and possible mechanisms for resistance, Pest Manag. Sci. 61 (2005) 1096-1102. [27] R.D. McAbee, K.D. Kang, M.A. Stanich, J.A. Christiansen, C.E. Wheelock, A.D. Inman,
416
B.D. Hammock, A.J. Cornel, Pyrethroid tolerance in Culex pipiens pipiens var molestus from
417
Marin County, California, Pest Manag. Sci. 60 (2004) 359–368.
418
[28] F. Chandre, F. Darriet, M. Darder, A. Cuany, J.M.C. Doannio, N. Pasteur, P. Cuillet,
419
Pyrethroid resistance in Culex quinquefasciatus from West Africa, Med. Vet. Entomol. 12
420
(1998) 359-366.
421
[29] S. Kasai, I.S. Weerashinghe, T. Shono, P450 monooxygenases are an important mechanism
422
of permethrin resistance in Culex quinquefasciatus Say larvae, Arch. Insect. Biochem.
423
Physiol. 37 (1998) 47-56.
424 425 426 427
[30] N. Liu, J.G. Scott, Genetics of resistance to pyrethroid insecticides in the house fly, Musca domestica, Pestic. Biochem. Physiol. 52 (1995) 116-124. [31] T.M. Brown, P.K. Bryson, G.T. Payne, Synergism by propynyl aryl ethers in permethrinresistant tobacco budworm larvae, Pestic. Sci. 46 (1996) 323–331.
19 Page 19 of 25
428 429 430 431
[32] L. Zhang, K. Harada, T. Shono, Genetic analysis of pyriproxifen resistance in the housefly, Musca domestica L, Appl. Ent. Zool. 32 (1997) 217–226. [33] N. Liu, J.G. Scott, Increased transcription of CYP6D1 causes cytochrome P450-mediated insecticide resistance in house fly, Insect Biochem. Mol. Biol. 28 (1998) 531-535.
432
[34] T. Yang, N. Liu, Genome analysis of cytochrome P450s and their expression profiles in
433
insecticide resistant mosquitoes, Culex quinquefasciatus, PLoS ONE 6 (2011) e29418.
434
[35] N. Liu, J.G. Scott, Phenobarbital induction of CYP6D1 is due to a trans acting factor on
435 436
autosome 2 in house flies, Musca domestica, Insect. Mol. Biol. 6 (1997) 77-81. [36] S. Kasai, I.S. Weerashinghe, T. Shono, M. Yamakawa, Molecular cloning, nucleotide
437
sequence, and gene expression of a cytochrome P450 (CYP6F1) from the pyrethroid-resistant
438
mosquito, Culex quinquefasciatus Say, Insect Biochem. Mol. Biol. 30 (2000) 163-171.
439
[37] K. Itokawa, O. Komagata, S. Kasai, Y. Okamura, M. Masada, T. Tomita, Genomic
440
structures of Cyp9m10 in pyrethroid resistant and susceptible strains of Culex
441
quinquefasciatus, Insect Biochem. Mol. Biol. 40 (2010) 631-640.
442
[38] M.C. Hardstone, O. Komagata, S. Kasai, T. Tomita, J.G. Scott, Use of isogenic strains
443
indicates CYP9M10 is linked to permethrin resistance in Culex pipiens quinquefasciatus,
444
Insect Mol. Biol. 19 (2010) 717-726.
445
[39] X. Li, M.R. Berenbaum, M.A. Schuler, Cytochrome P450 and actin genes expressed in
446
Helicoverpa zea and Helicoverpa armigera: paralogy/orthology identification, gene
447
conversion and evolution, Insect Biochem. Mol. Biol. 32 (2002) 311-320.
448
[40] M. Li, W.R. Reid, L. Zhang, J.G. Scott, X. Gao, M. Kristensen, N. Liu, A whole
449
transcriptomal linkage analysis of gene co-regulation in insecticide resistant house flies,
450
Musca domestica, BMC Genomics 14 (2013) 803.
20 Page 20 of 25
451 452
[41] L.M. Tompkins, D.W. Andrew, Mechanisms of cytochrome P450 induction, J. Biochem. Mol. Toxicol. 21 (2007) 176-181.
453
[42] R. Poupardin, S. Reynaud, C. Strode, H. Ranson, J. Vontas, J.-P. David, Cross-induction of
454
detoxification genes by environmental xenobiotics and insecticides in the mosquito Aedes
455
aegypti: Impact on larval tolerance to chemical insecticides, Insect Biochem. Mol. Biol. 38
456
(2008) 540-551.
457
[43] M.E. Scharf, S. Parimi, L.J. Meinke, L.D. Chandler, B.D. Siegfried. Expression and
458
induction of three family 4 cytochrome P450 (CYP4) genes identified from insecticide-
459
resistant and susceptible western corn rootworms, Diabrotica virgifera virgifera, Insect
460
Mol. Biol. 10 (2001) 139-146.
461
[44] L.B. Brattaten. Potential role of plant allelochemicals in the development of insecticide
462
resistance, P. Barbosa, D.K. Letourneau (eds.), Novel Aspects of Insect Plant Interaction,
463
John Wiley, New York (1988), pp. 313-348.
464 465 466
[45] A.B. Okey, Enzyme induction in the cytochrome P-450 system, Pharmacol. Ther. 45 (1990) 241-298. [46] Y. Gong, T. Li, L. Zhang, X. Gao, N. Liu, Permethrin induction of multiple cytochrome
467
P450 genes in insecticide resistant mosquitoes, Culex quinquefasciatus, Int. J. Biol. Sci. 9
468
(2013) 863-871.
469
[47] M.A. Schuler, M.R. Berenbaum, Structure and function of cytochrome P450S in insect
470
adaptation to natural and synthetic toxins: Insights gained from molecular modeling, J.
471
Chem. Ecol. 39 (2013) 1232-1245.
472 473
[48] M. Hiratsuka, In vitro assessment of the allelic variants of cytochrome P450, Drug Metab. Pharmacokinet. 27 (2011) 68-84.
21 Page 21 of 25
474
[49] O. Gotoh, Substrate recognition sites in cytochrome P450 family 2 (CYP2) proteins inferred
475
from comparative analyses of amino acid and coding nucleotide sequences, J. Biol. Chem.
476
267 (1992) 83-90.
477
[50] T.L. Chiu, Z. Wen, S.G. Rupasinghe, M.A. Schuler, Comparative molecular modeling of
478
Anopheles gambiae CYP6Z1, a mosquito P450 capable of metabolizing DDT, Proc. Natl.
479
Acad. Sci. USA. 105 (2008) 8855-8860.
480
[51] L. McLaughlin, U. Niazi, J. Bibby, J.P. David, J. Vontas, J. Hemingway, H. Ranson, M.
481
Sutcliffe, M. Paine, Characterization of inhibitors and substrates of Anopheles gambiae
482
CYP6Z2, Insect Mol. Biol. 17 (2008) 125-135.
483
[52] B.J. Stevenson, J. Bibby, P. Pignatelli, S. Muangnoicharoen, P.M. O’Neill, L.Y. Lian, P.
484
Müller, D. Nikou, A. Steven, J. Hemingway, M.J. Sutcliffe, M.J.I. Paine, Cytochrome P450
485
6M2 from the malaria vector Anopheles gambiae metabolizes pyrethroids: Sequential
486
metabolism of deltamethrin revealed, Insect Biochem. Mol. Biol. 41 (2011) 492-502.
487
[53] P. Lertkiatmongkol, E. Jenwitheesuk, P. Rongnoparut, Homology modeling of mosquito
488
cytochrome P450 enzymes involved in pyrethroid metabolism: insights into differences in
489
substrate selectivity, BMC research notes 4 (2011) 321.
490
[54] C.P. Alexia, B. Jaclyn, R.K. Myriam, R. Jessica, G.C. Emilie, P. Rodolphe, A.R.
491
Muhammad, P. Mark, D.V. Chantal, R. Stephane, The central role of mosquito cytochrome
492
P450 CYP6Zs in insecticide detoxification revealed by functional expression and structural
493
modelling, Biochem. J. 455 (2013) 75-85.
494 495
[55] C. Zimmer, C. Bass, M. Williamson, M. Kaussmann, K. Wölfel, O. Gutbrod, R. Nauen, Molecular and functional characterization of CYP6BQ23, a cytochrome P450 conferring
22 Page 22 of 25
496
resistance to pyrethroids in European populations of pollen beetle, Meligethes aeneus,
497
Insect Biochem. Mol. Biol. 45 (2014) 18-29.
498
[56] F. Zhu, T.W. Moural, K. Shah, S.R. Palli, Integrated analysis of cytochrome P450 gene
499
superfamily in the red flour beetle, Tribolium castaneum, BMC Genomics 14 (2013) 174.
500
[57] N. Liu, Insecticide resistance in mosquitoes: impact, mechanisms, and research directions,
501
Annu. Rev. Entomol. 60 (2015) 537-559.
502
[58] T. Yang, N. Liu, Permethrin resistance variation and susceptible reference line isolation in a
503
field population of the mosquito, Culex quinquefasciatus (Diptera: Culicidae), Insect Sci.
504
(2013) 1-8.
505
[59] K.A. Feenstra, E.B. Starikov, V.B. Urlacher, J.N. Commandeur, N.P. Vermeulen,
506
Combining substrate dynamics, binding statistics, and energy barriers to rationalize
507
regioselective hydroxylation of octane and lauric acid by CYP102A1 and mutants, Protein
508
Sci. 16 (2007) 420-431.
509 510 511 512 513 514 515
[60] N. Liu, H. Liu, F. Zhu, L. Zhang, Differential expression of genes in pyrethroid resistant and susceptible mosquitoes, Culex quinquefasciatus (S.), Gene 394 (2007) 62-68. [61] W.R. Reid, L. Zhang, F. Liu, N. Liu, The transcriptome profile of the mosquito Culex quinquefasciatus following permethrin selection, PLoS One 7 (2012) e47163. [62] M.C. Lagerström, H.B. Schiöth, Structural diversity of G protein-coupled receptors and significance for drug discovery, Nat. Rev. Drug Discov. 7 (2008) 339-357. [63] T. Li, L. Liu, L. Zhang, N. Liu, Role of G-protein-coupled receptor-related genes in
516
insecticide resistance of the mosquito, Culex quinquefasciatus, Scientific Reports 4 (2014)
517
6474.
23 Page 23 of 25
518
[64] S. Yan, P. Zou†, X.Y. Yu, C. Chen, J. Yu, L.N. Shi, S.C. Hong, D. Zhou, X.L. Chang, W.J.
519
Wang, B. Shen, D.H. Zhang, L. Ma, C.L. Zhu, Functional characterization of an arrestin
520
gene on insecticide resistance of Culex pipiens pallens, Parasit. Vec. 5 (2012) 134.
521 522 523
Figure Legends
524 525
Figure 1. Topology of P450s. The secondary structures of helices and sheets are labeled. Six
526
putative SRSs are colored and predicted according to predicted models [49]. SRSs 1 to 6 are
527
shown in red, white, yellow, blue, purple and orange, respectively. The heme group is
528
represented by sticks.
529 530
Figure 2. A hypothetical model of P450 gene regulation in insecticide resistance. This model is
531
based on our previous studies [14], [15], [34], [57] and [6] as well as those of others [19], [33]
532
and [21], suggesting the interaction of resistance P450 genes, basal transcriptional Factors (BTF)
533
and trans regulatory factors (TRF) or cis elements in P450 gene (CYP) expression in insecticide
534
resistance. The P450 gene(s) in resistance insects interacts with regulatory factors that
535
facilitate/regulate the overexpression of the resistanceP450 gene (s), whereas, this regulatory
536
pathway(s) in susceptible insects is obstructed due to unknown mechanisms.
537 538 539 540 541 24 Page 24 of 25
542 543 544 545 546 547 548 549 550 551 552 553
25 Page 25 of 25
