Gene identification and proteomic analysis of the esterases of the cotton bollworm, Helicoverpa armigera

Abstract

Some of the resistance of Helicoverpa armigera to conventional insecticides such as organophosphates and synthetic pyrethroids appears to be due to metabolic detoxification by carboxylesterases. To investigate the H. armigera carboxyl/cholinesterases, we created a data set of 39 putative paralogous H. armigera carboxyl/cholinesterase sequences from cDNA libraries and other sources. Phylogenetic analysis revealed a close relationship between these sequences and 70 carboxyl/cholinesterases from the recently sequenced genome of the silkworm, Bombyx mori, including several conserved clades of non-catalytic proteins. A juvenile hormone esterase candidate from H. armigera was identified, and B. mori orthologues were proposed for 31% of the sequences examined, however low similarity was found between lepidopteran sequences and esterases previously associated with insecticide resistance from other insect orders. A proteomic analysis of larval esterases then enabled us to match seven of the H. armigera carboxyl/cholinesterase sequences to specific esterase isozymes. All identified sequences were predicted to encode catalytically active carboxylesterases, including six proteins with N-terminal signal peptides and N-glycans, with two also containing C-terminal signals for glycosylphosphatidylinositol anchor attachment. Five of these sequences were matched to zones of activity on native PAGE at relative mobility values previously associated with insecticide resistance in this species.

Introduction

The cotton bollworm, Helicoverpa armigera, is a major crop pest in Africa, Asia and Australasia and has evolved resistance to many of the classes of chemical insecticides used for its control, such as organophosphates (OPs) and synthetic pyrethroids (SPs). Enhanced metabolism of the insecticide by detoxifying enzymes such as cytochrome P450-dependent monooxygenases, glutathione-S-transferases and carboxylesterases is responsible for a significant proportion of this resistance (Bues et al., 2005, Yang et al., 2005, Ahmad et al., 2006, Ugurlu et al., 2007).

Most insect carboxylesterases characterised thus far are members of the carboxyl/cholinesterase (CCE) superfamily (Oakeshott et al., 2005). CCEs are α/β hydrolase fold containing proteins that use a catalytic mechanism relying on a catalytic triad featuring a serine nucleophile. Previous analyses of insect genomes sequenced to date have found 24–54 CCE genes in dipteran and hymenopteran species (various Drosophila spp., the mosquitoes Anopheles gambiae and Aedes aegypti, and the honey bee Apis mellifera; Oakeshott et al., 1993, Ranson et al., 2002, Claudianos et al., 2006, Strode et al., 2008). Some evidence suggests that lepidopteran species may have an even greater diversity of CCEs than these orders, with at least 24 larval carboxylesterase isozymes visible after non-denaturing polyacrylamide gel electrophoresis (native PAGE) and staining for carboxylesterase activity (Campbell, 2001, Usmani and Knowles, 2001). A few insect CCEs have been implicated in very specific functions, for example acetylcholinesterase (AChE) (Quinn, 1987), juvenile hormone esterase (JHE) (Kamita et al., 2003), and certain pheromone and odorant-degrading esterases (Maibeche-Coisne et al., 2004, Ishida and Leal, 2005, Merlin et al., 2007). Several others are proposed to have digestive or detoxification functions, based on their expression in the insect midgut (Oakeshott et al., 1993). However the physiological role of the vast majority of insect CCEs is unknown.

Studies of insects and acarids frequently implicate carboxylesterases in metabolic resistance to OPs and SPs. Two mechanisms for such resistance have been elucidated in detail, both involving OPs. The first involves the substitution of a single amino acid at one of two positions within the active site that enhances the enzyme's ability to hydrolyse OPs to less toxic products (Newcomb et al., 1997, Campbell et al., 1998). This mechanism has been shown in four higher dipterans and one hymenopteran, all proving to possess essentially equivalent substitutions in the active site of the enzymes (Campbell et al., 1997, Claudianos et al., 1999, Zhu et al., 1999, de Carvalho et al., 2006, Hartley et al., 2006). The second mechanism involves overexpression of esterases that bind but do not hydrolyse the OPs, effectively sequestering the insecticide away from its target site. Higher gene copy number in the genome has been shown to cause overexpression of such CCEs in some mosquitoes, aphids, and the brown planthopper, Nilaparvata lugens, with 100-fold or more amplification of the CCE sequence reported in some cases Devonshire 1977, Field et al., 1999, Small and Hemingway, 2000, Cui et al., 2007). In other cases an increase in transcription and/or translation of particular genes gives more modest levels of overexpression (Hawkes and Hemingway, 2002, Oakeshott et al., 2005).

The hydrophobicity of SPs adversely affects enzyme assays, complicating the study of metabolic resistance to these insecticides. Nevertheless, significant SP hydrolysis has been shown for several insect CCEs (Byrne et al., 2000, Huang and Ottea, 2004, Heidari et al., 2005). There is also increasing evidence that CCEs contribute to SP resistance, with higher levels of esterase activity found in SP resistant strains of some species (Park and Kamble, 1998), and observations that resistance can be mitigated by co-exposure to esterase inhibitors such as S,S,S-tri-n-butyl phosphorotrithioate (DEF) (Gunning et al., 1997, Gunning et al., 1999, Usmani and Knowles, 2001). There are also some reports that piperonyl butoxide, a known inhibitor of cytochrome P450s, might inhibit esterases as well (Young et al., 2006), suggesting that some of the resistance found in various insect species attributed to P450 oxidases might be due to esterases.

In H. armigera and its sibling species in the Americas, Helicoverpa zea, strains and individual insects resistant to OPs or SPs have been found with higher carboxylesterase activity compared to susceptible controls (Gunning et al., 1996, Campbell, 2001, Srinivas et al., 2004, Bues et al., 2005; Yidong Wu, pers. comm.; Keshav Kranthi, pers. comm.). In some of these cases, particular zones of activity after native PAGE have been implicated, and other esterase isozymes have been proposed to play a role in resistance based on their susceptibility to esterase inhibitors that decrease SP hydrolysis (Gunning et al., 1999, Usmani and Knowles, 2001). None of the CCE gene sequences encoding these enzymes have been identified, and without a H. armigera genome sequence available, the full diversity of CCEs in this species is yet unknown. The only publicly released genome sequence from a lepidopteran, the silkworm Bombyx mori (The International Silkworm Genome Consortium, 2008), has not yet been analysed in respect to CCEs, and it is currently uncertain whether lepidopteran genomes contain close orthologues of the resistance-associated esterases identified in other insect orders.

In the current study, we have investigated the CCEs of H. armigera through transcriptomics, comparative genomics and proteomics. We present 51 new H. armigera CCE sequences from cDNA libraries and other sources. After combining these sequences with those already available from public databases, we estimate that we have identified at least 39 non-allelic CCE sequences. We also identified 70 CCE sequences in the genome of B. mori. A phylogenetic analysis of these H. armigera and B. mori sequences was then carried out to determine which of them were closely related to other insect CCEs with proposed functions (Claudianos et al., 2006). Finally, we report a proteomic analysis of H. armigera esterases which links seven of its CCE sequences with larval carboxylesterase isozymes, five of which migrate with mobilities corresponding to zones of activity implicated in OP or SP resistance in this species.