Identification of a metallothionein gene in honey bee Apis mellifera and its expression profile in response to Cd, Cu and Pb exposure

Abstract

Metallothioneins are ubiquitous proteins important in metal homeostasis and detoxification. However, they have not previously been identified in honey bees or other Hymenoptera, where metallothioneins could be of ecophysiological and ecotoxicological significance. Better understanding of the molecular responses to stress induced by toxic metals could contribute to honey bee conservation. In addition, honey bee metallothionein could represent a biomarker for monitoring environmental quality. Here we identify and characterize a metallothionein gene in Apis mellifera (AmMT). AmMT is 1,680 bp long and encodes a 48 amino acids protein with 15 cysteines and no aromatic residues. A metal response element upstream of the start codon, coupled with numerous cis‐regulatory elements indicate the functional context of AmMT. Molecular modelling predicts several transition metal binding sites, and comparative phylogenetic analysis revealed five putative metallothionein proteins in three other hymenoptera species. AmMT was characterized by cloning the full‐length coding sequence of the putative metallothionein. Recombinant AmMT was found to increase metal tolerance upon overexpression in Escherichia coli supplemented with Cd, Cu or Pb. Finally, in laboratory tests on honey bees, gene expression profiles showed a dose‐dependant relationship between Cd, Cu and Pb concentrations present in food and AmMT expression, while field experiments showed induction of AmMT in bees from an industrial site compared to those from an urban area. These studies suggest that AmMT has metal binding properties in agreement with a possible role in metal homeostasis. Further functional and structural characterization of metallothionein in honey bees and other Hymenoptera are necessary.

Essential (Mg, Fe, Zn and Cu) and Non-Essential (Cd and Pb) Elements in Predatory Insects (Vespa crabro and Vespa velutina): A Molecular Perspective

“…The Cu-containing peak at fractions 20-22 might therefore have been related to Mt. A gene encoding a putative Mt (AmMT) has recently been identified in A. mellifera [39], suggesting a role in metal binding and detoxification, also in Hymenoptera, for these proteins. The genome of D. melanogaster contains at least five Mt loci (MtnA, MtnB, MtnC, MtnD and MtnE) [40], and these five Mts show low affinity for Zn and high affinity for Cu and Cd [41,42].…”

The evolution of insect metallothioneins

“…Outside the Drosophila genus [21], only 14 MT genes have been published in insects (electronic supplementary material, table S1). There is only one MT in the honeybee Apis mellifera [12], two MTs in the Chinese grasshopper Oxya chinensis [22], but five MTs in sequenced Drosophila species genomes [21]. These studies suggest that the number of MTs in species is dynamic and evolves rapidly across insects.…”

“…Another reason that makes phylogenetic analyses of MTs difficult is the scarcity of annotated MT sequences in sequenced genomes. Indeed, in silico gene predictions often fail to identify MT-encoding genes that contain very small exons separated by large introns [11,12].…”

The Evolution of Insect Metallothioneins

“…Another reason that makes phylogenetic analyses of MTs difficult is the scarcity of annotated MT sequences in sequenced genomes. Indeed, in silico gene predictions often fail to identify MT-encoding genes that contain very small exons punctuated by large introns (Ragusa et al 2017;Purać et al 2019).…”

“…Outside the Drosophila genus (Guirola et al 2011), only 14 MT genes have been published in insects (Suppl Table 1). There is only one MT in the honey bee Apis mellifera (Purać et al 2019), two MTs in the Chinese grasshopper Oxya chinensis ), but five MTs in sequenced Drosophila species genomes (Guirola et al 2011). The MT content is dynamic and evolves rapidly across insects.…”