A deficit of detoxification enzymes: pesticide sensitivity and environmental response in the honeybee

Abstract

The honeybee genome has substantially fewer protein coding genes ( approximately 11 000 genes) than Drosophila melanogaster ( approximately 13 500) and Anopheles gambiae ( approximately 14 000). Some of the most marked differences occur in three superfamilies encoding xenobiotic detoxifying enzymes. Specifically there are only about half as many glutathione-S-transferases (GSTs), cytochrome P450 monooxygenases (P450s) and carboxyl/cholinesterases (CCEs) in the honeybee. This includes 10-fold or greater shortfalls in the numbers of Delta and Epsilon GSTs and CYP4 P450s, members of which clades have been recurrently associated with insecticide resistance in other species. These shortfalls may contribute to the sensitivity of the honeybee to insecticides. On the other hand there are some recent radiations in CYP6, CYP9 and certain CCE clades in A. mellifera that could be associated with the evolution of the hormonal and chemosensory processes underpinning its highly organized eusociality.

Figure 1

Unrooted distance neighbour-joining tree showing phylogenetic relationships of the predicted GST proteins of A. mellifera (shown in red), D. melanogaster (green) and An. gambiae (blue). Radiations corresponding to the recognized Delta, Epsilon, Omega, Sigma, Theta and Zeta classes of GST are indicated. Alternative splice variants of the An. gambiae GSTS1 and GSTD1 sequences are designated by a hyphenated Arabic number. Distance bootstrap values of greater than 70% (500 replicates) are indicated at the relevant nodes by an asterisk (*).

Figure 2

Neighbour-joining phylogeny of the P450 sequences from the honeybee (in red) with selected P450s from D. melanogaster (Dm – green) and An. gambiae (Ag – blue). The outgroup (P450cam, the camphor hydroxylase from Pseudomonas putida), human CYP2J2 and CYP3A4, and two other P450s mentioned in the text (CYP4S4 from Mamestra brassicae and CYP4AW1 fom Phyllopertha diversa, both possibly involved in odour or pheromone clearance) are marked in blue. Orthologues of the D. melanogaster Halloween gene products involved in ecdysteroid biosynthesis (phm, spo, dib, shd, sad) and of the nompH gene product are marked on the right, as well as the CYP15 sequences orthologous to the juvenile hormone epoxidase identified in the cockroach. Two additional series of orthologues (CYP4AA1 and CYP301A1) of unknown function are marked by a hatch (#). The great diversity of the fruit fly and mosquito CYP3 and CYP4 clades and mitochondrial CYP12 family (see Table 1) is represented by just a few members for clarity of the tree. Inclusion of the other members does not alter the overall topology of the tree. Distance bootstrap values of greater than 50% (1000 replicates) are indicated at the relevant nodes by an asterisk (*).

Figure 3

Genomic regions encoding clusters of P450 genes in A. mellifera. Boxes correspond to exons, lines represent introns, and arrows indicate gene orientation. (A) Nine of 17 CYP6AS subfamily genes map to chromosome 13 on three adjacent scaffolds and six are clustered with at least one other CYP6AS subfamily member on two unassigned scaffolds. (B) All eight intronless CYP9 family genes map to a 16 kb region on chromosome 14. (C) The tight linkage of CYP18A1 and CYP306A1 on chromosome 13 is a case of possible microsynteny (albeit a different gene orientation) with the region of the D. melanogaster genome containing their putative orthologues.

Figure 4

Unrooted distance neighbour-joining tree showing a phylogeny of carboxyl/cholinesterase (CCE) proteins. Sequences from A. mellifera, D. melanogaster and An. gambiae genomes (red, green and blue, respectively) are shown with other previously characterized CCE sequences, NCBI accession number provided (see also Tables 1 and 2). Sequences were aligned using Clustal W followed by some minor corrections to conform to known structural features of CCEs and did not include N- and C-terminal extensions typical of many neuro/developmental members of this family. The resultant ≈ 530 amino acid alignment, spans a region equivalent to residues 65–558 of D. melanogaster AChE (CG17907), was analysed with MEGA3.1 (Kumar et al., 2004) using the neighbour-joining method with pair-wise deletion of gaps/missing data and the PAM 001 matrix substitution model to construct a phylogenetic tree. The tree (represented as a cladogram) was split into two connecting subtrees corresponding to metabolic enzymes (part A) and neuro/developmental proteins (part B) for ease of viewing. An asterisk (*) indicates nodes with greater than 50% resampling frequency (1000 bootstrap replications). Boundaries for the three major classes and 13 major clades are indicated on the right. Clade boundaries generally agree with those shown in Oakeshott et al. (2005a) although the addition of the A. mellifera data enhanced resolution among the dietary/detoxification clades A–C and one uncharacterized neuro/developmental clade (I).

Figure 4

Unrooted distance neighbour-joining tree showing a phylogeny of carboxyl/cholinesterase (CCE) proteins. Sequences from A. mellifera, D. melanogaster and An. gambiae genomes (red, green and blue, respectively) are shown with other previously characterized CCE sequences, NCBI accession number provided (see also Tables 1 and 2). Sequences were aligned using Clustal W followed by some minor corrections to conform to known structural features of CCEs and did not include N- and C-terminal extensions typical of many neuro/developmental members of this family. The resultant ≈ 530 amino acid alignment, spans a region equivalent to residues 65–558 of D. melanogaster AChE (CG17907), was analysed with MEGA3.1 (Kumar et al., 2004) using the neighbour-joining method with pair-wise deletion of gaps/missing data and the PAM 001 matrix substitution model to construct a phylogenetic tree. The tree (represented as a cladogram) was split into two connecting subtrees corresponding to metabolic enzymes (part A) and neuro/developmental proteins (part B) for ease of viewing. An asterisk (*) indicates nodes with greater than 50% resampling frequency (1000 bootstrap replications). Boundaries for the three major classes and 13 major clades are indicated on the right. Clade boundaries generally agree with those shown in Oakeshott et al. (2005a) although the addition of the A. mellifera data enhanced resolution among the dietary/detoxification clades A–C and one uncharacterized neuro/developmental clade (I).

Figure 5

Comparison of the physical locations of honeybee neuroligin genes predicted from the Version 3.0 assembly of the Honeybee Genome Project with orthologues from D. melanogaster and An. gambiae showing high levels of microsynteny.