Sensing the Underground - Ultrastructure and Function of Sensory Organs in Root-Feeding Melolontha melolontha (Coleoptera: Scarabaeinae) Larvae

Introduction

Below ground orientation in insects relies mainly on olfaction and taste. The economic impact of plant root feeding scarab beetle larvae gave rise to numerous phylogenetic and ecological studies. Detailed knowledge of the sensory capacities of these larvae is nevertheless lacking. Here, we present an atlas of the sensory organs on larval head appendages of Melolontha melolontha. Our ultrastructural and electrophysiological investigations allow annotation of functions to various sensory structures.

Results

Three out of 17 ascertained sensillum types have olfactory, and 7 gustatory function. These sensillum types are unevenly distributed between antennae and palps. The most prominent chemosensory organs are antennal pore plates that in total are innervated by approximately one thousand olfactory sensory neurons grouped into functional units of three-to-four. In contrast, only two olfactory sensory neurons innervate one sensillum basiconicum on each of the palps. Gustatory sensilla chaetica dominate the apices of all head appendages, while only the palps bear thermo-/hygroreceptors. Electrophysiological responses to CO₂, an attractant for many root feeders, are exclusively observed in the antennae. Out of 54 relevant volatile compounds, various alcohols, acids, amines, esters, aldehydes, ketones and monoterpenes elicit responses in antennae and palps. All head appendages are characterized by distinct olfactory response profiles that are even enantiomer specific for some compounds.

Conclusions

Chemosensory capacities in M. melolontha larvae are as highly developed as in many adult insects. We interpret the functional sensory units underneath the antennal pore plates as cryptic sensilla placodea and suggest that these perceive a broad range of secondary plant metabolites together with CO₂. Responses to olfactory stimulation of the labial and maxillary palps indicate that typical contact chemo-sensilla have a dual gustatory and olfactory function.     

HLA-DP/DR interaction in early onset pauciarticular juvenile chronic arthritis

We investigated the polymorphic second exon of the HLA-DPB1 and HLA-DRB1 genes, using in vitro DNA amplification by polymerase chain reaction (PCR) and oligonucleotide hybridization in 136 patients with early onset pauciarticular juvenile chronic arthritis (EOPA-JCA) and 199 healthy controls. The analysis of the HLA-DRB1 system revealed that most of the DRB1 alleles are not indifferent with respect to susceptibility to EOPA-JCA. There is a hierarchy of susceptible (DRB1*08, DR5), permissive (DRB1*01), moderately protective (DR2, DRB1*04), and protective (DRB1*07) alleles. In contrast, no hierarchy could be shown for the HLA-DPB1 system. DPB1*0201 was found to be susceptible. The relatively frequent alleles DPB1*0402 and DPB1*0401 seem to be indifferent. The associations with DPB1*0201, DR5, and DRB1*08 are independent of each other: that is to say they, are not brought about by linkage disequilibrium. The susceptible alleles DPB1*0201 and DR5 show evidence for interaction in the pathogenesis of EOPA-JCA. Interaction seems likely between DPB1*0201 and DRB1*08, DR5 and DRB1*08, or between DR6 and DRB1*08. The strongest interaction exists between DPB1*0201 and a common DQ factor associated with both DR5 and DRB1*08. Finally, we observed a hierarchy among the various marker combinations, where the risk of developing EOPA-JCA increases with the number of associated markers present in an individual.This work was supported by SFB217.The data presented here are part of the doctoral thesis of C. Paul.     

SFTG international collaborative study on in vitro micronucleus test III. Using CHO cells

In this report, results are presented from an international study of the in vitro micronucleus assay using Chinese hamster ovary cells. This study was coordinated by an organizing committee supported by the SFTG (the French branch of the European Environmental Mutagen Society). Test chemicals included mannitol, bleomycin, cytosine arabinoside, urethane and diethylstilboestrol. Mitomycin C was used as a positive control. Each chemical was evaluated in at least two laboratories following a variety of different protocols (short and long exposures, varying recovery times, with and without cytochalasin B) in order to help determine a standard protocol for routine testing in Chinese hamster ovary cells. Mannitol and urethane were negative, while bleomycin, cytosine arabinoside and diethylstilboestrol induced a dose dependent increase in micronucleated cells. In the presence of cytochalasin B, increases in micronuclei were observed in binucleated as well as mononucleated cells in cultures treated with bleomycin, cytosine arabinoside or diethylstilboestrol. Importantly, all three of these chemicals were detected in each of the different treatment/recovery regimens. No differences were seen in the sensitivity or accuracy of the responses in the presence of absence of cytochalasin B. Overall, these results demonstrate the suitability of Chinese hamster ovary cells for the in vitro micronucleus assay.     

Investigation of the proteolipid protein promoter activity during demyelination and repair

The transgenic plp-GFP mouse line expressing the green fluorescent protein (GFP) driven by the mouse myelin proteolipid protein (plp) gene promoter has been previously used to study the contribution of the plp lineage to oligodendrocyte development in the embryonic brain. Here, we show that the GFP fluorescence reflects the developmental expression of proteolipid protein during the postnatal development until adulthood in brain slices and in primary cultures of plp-GFP⁺ cells derived from postnatal animals. In the adult brain, plp-GFP-expressing cells are mature oligodendrocytes but not oligodendroglial progenitors. In the model of focal demyelination induced by lysolecithin (LPC) in the corpus callosum of adult plp-GFP animals, we observed an up-regulation of the morphogen Sonic Hedgehog (Shh) in the LPC-induced lesion but not in the control animals. Moreover, we show that the adenovirus-mediated transfer of Shh in the lesion results in the attenuation of the demyelination extent as evidenced by GFP fluorescence analysis in Shh-treated and control animals. Altogether these data show how plp-GFP fluorescence can be monitored to follow the oligodendrocyte lineage during demyelination and identify Shh morphogen as an important factor during repair.     

Effects of medium composition on the morphology and function of rat hepatocytes cultured as spheroids and monolayers

Primary hepatocytes cultured as monolayers or as spheroids were studied to compare the effects of four different culture media (Williams' E, Chee's, Sigma Hepatocyte, and HepatoZYME medium). Rat hepatocytes were cultured as conventional monolayers for 3 d or as spheroids for 2 wk. For spheroid formation a method was emplOyed that combined the use of a nonadherent substratum with rotation of cultures. Hepatocyte integrity and morphology were assessed by light and electron microscopy and by reduced glutathione content. Hepatocyte function was measured by albumin secretion and 7-ethoxycoumarin metabolism. Chee's medium was found to be optimal for maintenance of hepatocyte viability and function in monolayers, but it failed to support spheroid formation. For spheroid formation and for the maintenance of spheroid morphology and function, Sigma HM was found to be optimal. These results demonstrate that the medium requirements of hepatocytes differ markedly depending on the culture model employed. Spheroid culture allowed better preservation of morphology and function of hepatocytes compared with conventional monolayer culture. Hepatocytes in spheroids formed bile canaliculi. and expressed an actin distribution resembling that found in hepatocytes in vivo. Albumin secretion was maintained at the same level as that found during the first d in primary culture, and 7-ethoxycoumarin metabolism was maintained over 2 wk in culture at approximately 30% of the levels found in freshly isolated hepatocytes. The improved morphology and function of hepatocyte cultures as spheroids may provide a more appropriate in vitro model for certain applications where the maintenance of liver-specific functions in long-term culture is crucial.     

Impaired expression of the plastidic ferrochelatase by antisense RNA synthesis leads to a necrotic phenotype of transformed tobacco plants

Protoporphyrin IX is the last common intermediate of tetrapyrrole biosynthesis. The chelation of a Mg2+ ion by magnesium chelatase and of a ferrous ion by ferrochelatase directs protoporphyrin IX towards the formation of chlorophyll and heme, respectively. A full length cDNA clone encoding a ferrochelatase was identified from a Nicotiana tabacum cDNA library. The encoded protein consists of 497 amino acid residues with a molecular weight of 55.4 kDa. In vitro import of the protein into chloroplasts and its location in stroma and thylakoids confirm its close relationship to the previously described Arabidopsis thaliana plastid-located ferrochelatase (FeChII). A 1700-bp tobacco FeCh cDNA sequence was expressed in Nicotiana tabacum cv. Samsun NN under the control of the CaMV 35S promoter in antisense orientation allowing investigation into the consequences of selective reduction of the plastidic ferrochelatase activity for protoporphyrin IX channeling in chloroplasts and for interactions between plastidic and mitochondrial heme synthesis. Leaves of several transformants showed a reduced chlorophyll content and, during development, a light intensity-dependent formation of necrotic leaf lesions. In comparison with wild-type plants the total ferrochelatase activity was decreased in transgenic lines leading to an accumulation of photosensitizing protoporphyrin IX. Ferrochelatase activity was reduced only in plastids but not in mitochondria of transgenic plants. By means of the specifically diminished ferrochelatase activity consequences of the selective inhibition of protoheme formation for the intracellular supply of heme can be investigated in the future.     

Establishment of a chimeric, replication-deficient influenza A virus vector by modulation of splicing efficiency

Segment 8 of the influenza A virus codes for two proteins (NS1 and NS2/NEP) via splicing. Here, we developed a viral vector expressing a cytokine or chemokine instead of the interferon antagonist NS1. To achieve both the desired genetic stability and high transgene expression levels, NS2/NEP mRNA splicing efficacy had to be fine-tuned by modification of splicing elements. Expression levels of secreted foreign proteins could be further enhanced by fusing the N-terminal 13 amino acids of NS1 with an IgK-derived secretion signal peptide. Thus, the first start codon was used for translation initiation of both NS2/NEP and the foreign protein.     

The role of phosphatidylinositol 3-kinase in neural cell adhesion molecule-mediated neuronal differentiation and survival

The neural cell adhesion molecule, NCAM, is known to stimulate neurite outgrowth from primary neurones and PC12 cells presumably through signalling pathways involving the fibroblast growth factor receptor (FGFR), protein kinase A (PKA), protein kinase C (PKC), the Ras-mitogen activated protein kinase (MAPK) pathway and an increase in intracellular Ca2+ levels. Stimulation of neurones with the synthetic NCAM-ligand, C3, induces neurite outgrowth through signalling pathways similar to the pathways activated through physiological, homophilic NCAM-stimulation. We present here data indicating that phosphatidylinositol 3-kinase (PI3K) is required for NCAM-mediated neurite outgrowth from PC12-E2 cells and from cerebellar and dopaminergic neurones in primary culture, and that the thr/ser kinase Akt/protein kinase B (PKB) is phosphorylated downstream of PI3K after stimulation with C3. Moreover, we present data indicating a survival-promoting effect of NCAM-stimulation by C3 on cerebellar and dopaminergic neurones induced to undergo apoptosis. This protective effect of C3 included an inhibition of both DNA-fragmentation and caspase-3 activation. The survival-promoting effect of NCAM-stimulation was also shown to be dependent on PI3K.     

Nitrogen metabolism responses to water deficit act through both abscisic acid (ABA)-dependent and independent pathways in Medicago truncatula during post-germination

The modulation of primary nitrogen metabolism by water deficit through ABA-dependent and ABA-independent pathways was investigated in the model legume Medicago truncatula. Growth and glutamate metabolism were followed in young seedlings growing for short periods in darkness and submitted to a moderate water deficit (simulated by polyethylene glycol; PEG) or treated with ABA. Water deficit induced an ABA accumulation, a reduction of axis length in an ABA-dependent manner, and an inhibition of water uptake/retention in an ABA-independent manner. The PEG-induced accumulation of free amino acids (AA), principally asparagine and proline, was mimicked by exogenous ABA treatment. This suggests that AA accumulation under water deficit may be an ABA-induced osmolyte accumulation contributing to osmotic adjustment. Alternatively, this accumulation could be just a consequence of a decreased nitrogen demand caused by reduced extension, which was triggered by water deficit and exogenous ABA treatment. Several enzyme activities involved in glutamate metabolism and genes encoding cytosolic glutamine synthetase (GS1b; EC 6.3.1.2.), glutamate dehydrogenase (GDH3; EC 1.4.1.1.), and asparagine synthetase (AS; EC 6.3.1.1.) were up-regulated by water deficit but not by ABA, except for a gene encoding Δ(1)-pyrroline-5-carboxylate synthetase (P5CS; EC not assigned). Thus, ABA-dependent and ABA-independent regulatory systems would seem to exist, differentially controlling development, water content, and nitrogen metabolism under water deficit.     

Tuning of Pectin Methylesterification: PECTIN METHYLESTERASE INHIBITOR 7 MODULATES THE PROCESSIVE ACTIVITY OF CO-EXPRESSED PECTIN METHYLESTERASE 3 IN A pH-DEPENDENT MANNER*

Pectin methylesterases (PMEs) catalyze the demethylesterification of homogalacturonan domains of pectin in plant cell walls and are regulated by endogenous pectin methylesterase inhibitors (PMEIs). In Arabidopsis dark-grown hypocotyls, one PME (AtPME3) and one PMEI (AtPMEI7) were identified as potential interacting proteins. Using RT-quantitative PCR analysis and gene promoter::GUS fusions, we first showed that AtPME3 and AtPMEI7 genes had overlapping patterns of expression in etiolated hypocotyls. The two proteins were identified in hypocotyl cell wall extracts by proteomics. To investigate the potential interaction between AtPME3 and AtPMEI7, both proteins were expressed in a heterologous system and purified by affinity chromatography. The activity of recombinant AtPME3 was characterized on homogalacturonans (HGs) with distinct degrees/patterns of methylesterification. AtPME3 showed the highest activity at pH 7.5 on HG substrates with a degree of methylesterification between 60 and 80% and a random distribution of methyl esters. On the best HG substrate, AtPME3 generates long non-methylesterified stretches and leaves short highly methylesterified zones, indicating that it acts as a processive enzyme. The recombinant AtPMEI7 and AtPME3 interaction reduces the level of demethylesterification of the HG substrate but does not inhibit the processivity of the enzyme. These data suggest that the AtPME3·AtPMEI7 complex is not covalently linked and could, depending on the pH, be alternately formed and dissociated. Docking analysis indicated that the inhibition of AtPME3 could occur via the interaction of AtPMEI7 with a PME ligand-binding cleft structure. All of these data indicate that AtPME3 and AtPMEI7 could be partners involved in the fine tuning of HG methylesterification during plant development.