Kevin Heylen

The effects of four crop protection products on the morphology and ultrastructure of the hypopharyngeal gland of the European honeybee, Apis mellifera

This study describes the impact of sublethal doses of 4 pesticides on size and morphology of the honeybee worker’s hypopharyngeal glands. This gland plays an essential role in brood care by young workers, and thus colony growth. Contaminating 7 day old caged bees, we sampled after 1 day and 1 week for Captan-, Imidacloprid- and Indoxacarb-treated bees and after 1, 2, 3, 4, 5 and 7 days for Fenoxycarb-treated bees. The glands’ acini 1 week post-treatment were all significantly smaller in treated bees. However, lightand electron microscopy showed hardly any difference between controls and gland cells, treated with Captan, Imidacloprid or Indoxacarb. Yet upon Fenoxycarb-treatment, acini showed a decrease in size, a granular texture and unorganized cytoplasm more quickly than normal. Indeed, after only 7 days, Fenoxycarb-treated glands displayed features typical of the onset of foraging in older bees.

Cloning and expression of PKG, a candidate foraging regulating gene in Vespula vulgaris

In honey bees, enhancement of cGMP-dependent protein kinase (PKG) expression accompanies a behavioural transition from in-hive working nursing bees towards outdoors foraging worker bees. Accordingly this gene was named amfor or Apis mellifera foraging gene. In the red harvester ant Pogonomyrmex barbatus a gene homologue aff ected food seeking behaviour as well, but in this species PKG expression decreased from the onset of foraging behaviour. Since the wasp Vespula vulgaris is phylogenetically positioned between ants and bees, in this paper we tried to elucidate whether the involvement of PKG in foraging behaviour can be extended to this species and if so, whether its expression is enhanced or decreased by the transition from nursing to foraging. To enable this candidate gene approach, we fi rst had to clone the PKG homologue from the common wasp. QPCR indicated a relevantly higher expression of Vvfor in nursing versus foraging wasps although interpretation of the results was hampered by a remarkable degree of variation as could be predicted from wasps captured in the wild as a source for mRNA extraction and quantifi cation.

Amfor expression in the honeybee brain : A trigger mechanism for nurse-forager transition

The honeybees colony fitness relies on an optimized age-dependent division of labor. Transition from nursing activities to foraging activities is associated with an increase in the expression of the Amfor gene. Ben-Shahar et al. [Ben-Shahar, Y., Robichon, A., Sokolowski, M.B., Robinson, G.E., 2002. Influence of gene action across different time scales on behavior. Science 296, 741-744] showed that the Amfor transcripts and their gene products are involved in regulating the transition from one task to the next. In this study, we investigated the trajectory of the expression of this gene in the brain over time. The expression pattern could contribute to our understanding of the involvement of Amfor in the transition process. Is there a gradual increase in transcript or a peak in expression triggering a downstream path of multiple differential gene expression? Hereto, bees were sampled from colonies containing marked 1-day-old bees every 2 or 3 days around the expected time of transition from nurse to forager, from day 13 to 25. To quantify Amfor transcript in the brain, we developed a real-time RT-PCR assay, based on Taqman technology, using fluorescent probes. Results revealed a trigger mechanism rather than a continued elevation of Amfor expression. The appearance of an Amfor expression peak suggests that under normal physiological conditions foraging behavior is, at least in part, due to a trigger-effect of Amfor.

Recovery from Retinal Lesions: Molecular Plasticity Mechanisms in Visual Cortex Far beyond the Deprived Zone

In cats with central retinal lesions, deprivation of the lesion projection zone (LPZ) in primary visual cortex (area 17) induces remapping of the cortical topography. Recovery of visually driven cortical activity in the LPZ involves distinct changes in protein expression. Recent observations, about molecular activity changes throughout area 17, challenge the view that its remote nondeprived parts would not be involved in this recovery process. We here investigated the dynamics of the protein expression pattern of remote nondeprived area 17 triggered by central retinal lesions to explore to what extent far peripheral area 17 would contribute to the topographic map reorganization inside the visual cortex. Using functional proteomics, we identified 40 proteins specifically differentially expressed between far peripheral area 17 of control and experimental animals 14 days to 8 months postlesion. Our results demonstrate that far peripheral area 17 is implicated in the functional adaptation to the visual deprivation, involving a meshwork of interacting proteins, operating in diverse pathways. In particular, endocytosis/exocytosis processes appeared to be essential via their intimate correlation with long-term potentiation and neurite outgrowth mechanisms.

Aedes aegypti juvenile hormone acid methyl transferase, the ultimate enzyme in the biosynthetic pathway of juvenile hormone III, exhibits substrate control

We report on the cloning, sequencing, characterization, 3D modeling and docking of Aedes aegypti juvenile hormone acid methyl transferase (AeaJHAMT), the enzyme that converts juvenile hormone acid (JHA) into juvenile hormone (JH). Purified recombinant AeaJHAMT was extensively characterized for enzymatic activity and the Michaelis Menten kinetic parameters Km, Vmax, k(cat) (turn over number) and k(cat)/Km (catalytic efficiency) using JHA and its analogues as substrates. AeaJHAMT methylates JHA III 5-fold faster than farnesoic acid (FA). Significant differences in lower methyl transferase (MT) activities towards the cis/trans/trans, cis/trans/cis and the trans/cis/cis isomers of JHA I (1.32, 4.71 and 156-fold, respectively) indicate that substrate chirality is important for proper alignment at the catalytic cavity and for efficient methyl transfer by S-adenosyl methionine (SAM). Our 3D model shows a potential binding site below the main catalytic cavity for JHA analogues causing conformational change and steric hindrance in the transfer of the methyl group to JHA III. These, in silico, observations were corroborated by, in vitro, studies showing that several JHA analogues are potent inhibitors of AeaJHAMT. In vitro, and in vivo studies using [(3)H-methyl]SAM show that the enzyme is present and active throughout the adult life stage of A. aegypti. Tissue specific expressions of the JHAMT gene of A. aegypti (jmtA) transcript during the life cycle of A. aegypti show that AeaJHAMT is a constitutive enzyme and jmtA transcript is expressed in the corpora allata (CA), and the ovary before and after the blood meal. These results indicate that JH III can be synthesized from JHA III by the mosquito ovary, suggesting that ovarian JH III may play an important physiological role in ovarian development and reproduction. Incubating AeaJHAMT with highly pure synthetic substrates indicates that JHA III is the enzymes preferred substrate, suggesting that AeaJHAMT is the ultimate enzyme in the biosynthetic pathway of JH III.