Research reportMolecular profiling of behavioural development: differential expression of mRNAs for inositol 1,4,5-trisphosphate 3-kinase isoforms in naive and experienced honeybees (Apis mellifera)☆
Introduction
As a result of the extended adult development young honeybees exhibit a variety of behavioural and biochemical characteristics that distinguish them from mature 2–3-week-old foragers. In contrast to older bees, which are excellent learners and forage outside the hive with strong daily rhythms, younger workers perform within hive duties with no daily rhythms [51] and somewhat surprisingly, cannot be trained reliably in laboratory olfactory associative tasks until they are 6–7 days old [36], [39]. At the biochemical level, foragers have higher brain levels of biogenic amines and higher lymph titers of juvenile hormone [15], [48]. Recent studies have reported a number of changes occurring in the honeybee brain during the first 1–2 weeks of behavioural maturation. These include significant volumetric expansions of the mushroom body neuropil [16], [54], changes in the expression patterns of two ‘candidate’ genes [25], [51] and alterations in cholinergic neurotransmission system [18]. These findings prompted us to apply a more systematic approach to unravel the innate factors that may contribute to behavioural development in this insect. We employed differential display RT-PCR technology to identify brain transcripts that are differentially expressed in naive, newly born honeybees and experienced foragers. We found that one of the differentially expressed genes encodes d-myo-inositol 1,4,5-trisphosphate 3-kinase (IP3K), a core enzyme in the IP3-mediated phosphoinositides transduction cascade.
IP3 and other second messengers are key components of signalling cascades linking diverse environmental stimuli with a narrow range of cellular and molecular events that enable the expression of complex biological functions. The phosphoinositide system is particularly well developed in the brain and a large body of evidence indicates that many metabotropic receptors use phosphoinositide-derived signals to modulate both neural activity and the neural plasticity responsible for higher brain functions [1], [2], [29]. IP3 is generated from membrane phosphatidylinositol 1,4-bisphosphate following a receptor-mediated activation of phospholipase C [1]. IP3 couples IP3-receptor activation of calcium channels on the endoplasmic reticulum (ER) causing rapid mobilization of calcium ions from internal stores. The spatio-temporal characteristics of the resulting calcium waves and oscillations are believed to underlie many important processes including neuronal and sensory signaling, development, proliferation, and ageing [1], [3]. Calcium oscillations have also been shown to increase the efficiency and specificity of gene expression [12]. To terminate the initial calcium response IP3 is either dephosphorylated to IP2, which is recycled back to the plasma membrane phosphatidylinositols, or is phosphorylated to yield another second messenger, inositol 1,3,4,5-tetrakisphosphate (IP4) that may act as a modulator or controller of the IP3 receptor’s interaction with the plasma membrane [21], [22]. The conversion of IP3 to IP4 is catalyzed by d-myo-inositol 1,4,5-trisphosphate 3-kinase (IP3K).
Although many biochemical aspects of IP3K involvement in calcium signaling are well understood, much less is known about the genetic and/or molecular controls that regulate this important step in signal transduction. Sequencing of several IP3K cDNAs in the rat and human revealed the existence of at least three major transcripts encoding a highly conserved C-terminal domain containing binding sites for calmodulin, IP3 and ATP, and non-conserved, distinct N-terminal domains encoding putative regulatory motifs [11], [44], [45], [46], [47], [49]. The putative mammalian isoforms of IP3K are produced from separate genes and differ in their regulatory properties such as responses to calmodulin and phosphorylation by protein kinases [9], [10], [11], [42], [55]. They also show differences in tissue distribution and subcellular localisation [11], [30], [31], [32], [33], [34], [43], [53]. The functional significance of different isoforms and their spatio-temporal patterns of expression are, however, poorly understood. To date only one invertebrate gene encoding IP3K, that in a simple nematode C. elegans, has been studied in some detail [8]. In contrast to vertebrates, the nematode gene is single copy and its expression is regulated by alternative splicing.
Here we describe the cloning of the IP3K gene in the honeybee, the characterization of its transcripts and a detailed analysis of the expression patterns of the IP3K mRNAs in the brain and other body compartments during behavioural development.
Section snippets
Organism and tissue preparation
Foraging honeybee workers were captured near the hive entrance and snap-frozen in liquid nitrogen (LN). Only those that carried pollen or nectar were selected. We estimate their age to vary from 20 to 35 days. To obtain younger honeybees of known age a single brood frame was removed from the hive and incubated at 32 °C (80% humidity). Individual insects were collected at desired times (0–1, 24, 72 and 96 h) and snap-frozen in LN. The male bees (drones) were collected from the same hive as worker
The IP3K locus in the honeybee
We determined the sequences of several cDNAs isolated from the honeybee brain cDNA library and a number of RT-PCR products corresponding to 5′ and 3′ regions of different transcripts. The underlying 21,538 bp of genomic DNA was also determined by assembling sequences of a genomic clone and PCR products overlapping the 5′ region (GenBank AF388659). Our cloning strategy is illustrated in Fig. 1. We found that the IP3K locus in the honeybee spans over 16 kb of genomic DNA and the gene has eight
Discussion
We carried out a detailed study to determine the molecular basis for the differential expression of IP3K in brains of naive and experienced honeybees. A large 21.5-kb segment of the honeybee genome spanning the IP3K locus was isolated on overlapping genomic and PCR fragments and the exon–intron junctions were determined by examining several cDNA clones. The overall structure of the honeybee IP3K gene shows both similarities and differences with the known IP3K genes in other species. The
Acknowledgements
We thank Paul Helliwell for excellent technical assistance. We also thank Gene Robinson and Paul Ebert for the honeybee libraries. This work was partly supported by Human Frontier Science Program grant no. RGO134/1999-B.
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Sequences reported in this paper were submitted to GenBank with accession number: AF388659.