A novel fatty acid binding protein produced by teratocytes of the aphid parasitoid Aphidius ervi.

Aphidius ervi is an endophagous braconid, parasitoid of the pea aphid, Acyrthosiphon pisum. A. ervi teratocytes, deriving from the dissociation of the embryonic serosa, synthesize and release two major proteins into the host haemocoel. The gene of one of these proteins has been cloned and characterized. This gene codes for a 15.8 kDa protein belonging to the fatty acid binding protein (FABP) family, named Ae-FABP (A. ervi-FABP). It is abundantly present in the host haemolymph when the parasitoid larva attains its maximum growth rate. The recombinant Ae-FABP binds to fatty acids in vitro, showing a high affinity to C14-C18 saturated fatty acids and to oleic and arachidonic acid. The possible nutritional role for the parasitoid larva of Ae-FABP is discussed.

Physiological and molecular interaction in the host-parasitoid system Heliothis virescens-Toxoneuron nigriceps: current status and future perspectives.

Toxoneuron nigriceps (Viereck) (Hymenoptera, Braconidae) is an endophagous parasitoid of the tobacco budworm Heliothis virescens (F.) (Lepidoptera, Noctuidae). Parasitized H. virescens larvae are developmentally arrested and show a complex array of pathological symptoms ranging from the suppression of the immune response to an alteration of ecdysone biosynthesis and metabolism. Most of these pathological syndromes are induced by the polydnavirus associated with T. nigriceps (TnBV). An overview of our recent research work on this system is described herein. The mechanisms involved in the disruption of the host hormonal balance have been further investigated, allowing to better define the physiological model previously proposed. A functional genomic approach has been undertaken to identify TnBV genes expressed in the host and to assess their role in the major parasitoid-induced pathologies. Some TnBV genes cloned so far are novel and do not show any similarity with genes already available in genomic databases, while others code for proteins having conserved domains, such as aspartic proteases and tyrosine phosphatases. Sequencing of the entire TnBV genome is in progress and will considerably contribute to the understanding of the molecular bases of parasitoid-induced host alterations.

Female sterile mutations and egg chamber development in Drosophila melanogaster.

Drosophila oogenesis provides an excellent opportunity to study fundamental aspects of developmental biology and to learn the importance of multiple signalling pathways in the regulation of cellular morphogenesis. Taking advantage of the genetic and molecular approaches extremely powerful in this organism, over the years an enormous collection of data has accumulated on the genes involved in important steps of egg chamber development, such as germline and somatic stem cell maintenance, division and differentiation; oocyte determination and positioning; establishment of follicle cell fate and axes formation. These different processes are mediated by a reciprocal cross-talk between germline and somatic follicle cells. Here, in a schematic and simplified form, we point out what we believe are the main recent results on the molecular and cellular mechanisms underlying ovarian development and outline our recent contribution to this field.

Cardiochiles nigriceps polydnavirus: molecular characterization and gene expression in parasitized Heliothis virescens larvae.

Cardiochiles nigriceps Viereck is an endophagous parasitoid of larval stages of the tobacco budworm, Heliothis virescens (F.). This hymenopteran parasitoid, belonging to the family Braconidae, is associated with a polydnavirus (CnPDV), injected at ovi-position along with the egg. The infection of various tissues by CnPDV determines the suppression of the host immune system and the developmental arrest of mature host larvae. In this study, CnPDV has been characterized at the structural and molecular level. The negatively stained nucleocapsids show evident 'end structures' and a tail-like appendage. The CnPDV genome is typically segmented, with circular dsDNA molecules, ranging in size from 2.5 kb to more than 23 kb. The early expression pattern of CnPDV in parasitized hosts has been analysed and viral clones, genomic and cDNAs, identifying genes expressed within 48 h after parasitization have been isolated. The molecular organization of one of these genes, named CnPDV1, and its putative protein product have been determined. Significant sequence homologies with other known proteins were not detected. In situ hybridization experiments indicated that this gene is expressed in the prothoracic glands of parasitized host mature larvae. A functional analysis of CnPDV1 gene product is required to assess its possible role in the regulation of parasitoid-induced alterations of host larvae.

Nup154, a new Drosophila gene essential for male and female gametogenesis is related to the nup155 vertebrate nucleoporin gene.

The Nup154 gene of Drosophila encodes a protein showing similarity with known nucleoporins: rat Nup155 and yeast Nup170 and Nup157. Hypomorphic mutant alleles of Nup154 affected female and male fertility, allowing investigation of the gene function in various steps of oogenesis and spermatogenesis. Nup154 was required in testes for cyst formation, control of spermatocyte proliferation and meiotic progression. In ovaries, Nup154 was essential for egg chamber development and oocyte growth. In both the male and female germ line, as well as in several other cell types, the Nup154 protein was detected at the nuclear membrane, but was also present inside the nucleus. Intranuclear localization has not previously been described for rat Nup155 or yeast Nup170 and Nup157. In mutant egg chambers the Nup154 protein accumulated in the cytoplasm, while it was only barely detected at the nuclear envelopes. FG repeats containing nucleoporins detected with mAb414 antibody were also mislocalized to a certain extent in Nup154 mutant alleles. This suggests that Nup154 could be required for localizing other nucleoporins within the nuclear pore complex, as previously demonstrated for the yeast Nup170. On the other hand, no evident defects in lamin localization were observed, indicating that Nup155 mutations did not affect the overall integrity of the nuclear envelope. However, ultrastructural analyses revealed that in mutant cells the morphology of the nuclear envelope was altered near the nuclear pore complexes. Finally, the multiplicity of phenotypes observed in Nup154 mutant alleles suggests that this gene plays a crucial role in cell physiology.

The Drosophila melanogaster lipase homologs: a gene family with tissue and developmental specific expression.

We report the molecular cloning of Drosophila genes encoding putative lipase homologs, Dm lip1, lip2 and lip3, the definition of their structure and the expression patterns during development. These Drosophila lipases are related to acid lipases, with a common GHSQG motif, within a more general consensus GXSXG, identified as the active site shared by all the members of lipase superfamily. The lip1 and lip3 genes are transcribed in different tissues and developmental stages, suggesting that they have different functions. The lip1 gene, coding for a protein similar to digestive lipases, is expressed in ovaries and early embryos and, with a different sized transcript, in all the other developmental stages. The lip3 gene, whose translation product is more similar to lysosomal acid lipases, is expressed only during the larval period. The lip2 gene seems non-functional. The Drosophila putative lipases do not show similarity with the Drosophila yolk proteins that are reported to have sequence similarity with lipoprotein lipases, but share a consistent similarity with lepidopteran proteins reported as egg specific or yolk proteins, probably corresponding to lipase homologs. The results reported here are discussed in relation to the evolution and functions of lipases within the between species.

hold up is required for establishment of oocyte positioning, follicle cell fate and egg polarity and cooperates with Egfr during Drosophila oogenesis.

In Drosophila the posterior positioning of the oocyte within the germline cluster defines the initial asymmetry during oogenesis. From this early event, specification of both body axes is controlled through reciprocal signaling between germline and soma. Here it is shown that the mutation hold up (hup) affects oocyte positioning in the egg chamber, follicle cell fate and localization of different markers in the growing oocytes. This occurs not only in dicephalic egg chambers, but also in oocytes normally located at the posterior. Generation of mosaic egg chambers indicates that hup has to be at least somatically required. Possible interactions of hup with Egfr, the Drosophila epidermal growth factor receptor homolog, have been investigated in homozygous double mutants constructed by recombination. Stronger new ovarian phenotypes have been obtained, the most striking being accumulation of follicle cells in multiple layers posteriorly to the oocyte. It is proposed that the hup gene product is a component of the molecular machinery that leads to the establishment of polarity both in follicle cell layer and oocyte, acting in the same or in a parallel pathway of Egfr.

Prothoracic gland inactivation in Heliothis virescens (F.) (Lepidoptera:Noctuidae) larvae parasitized by Cardiochiles nigriceps Viereck (Hymenoptera:Braconidae).

Heliothis virescens (F.) last instar larvae parasitized by the endophagous braconid Cardiochiles nigriceps Viereck fail to attain the pupal stage, due to a parasitoid-induced alteration of ecdysteroid biosynthesis and metabolism. Currently available information on host prothoracic gland inactivation in this host-parasitoid system is reported here. Prothoracic glands of H. virescens mature larvae show a depressed biosynthetic activity, without undergoing gross morphological disruption. The ultrastructure of gland cells is characterized by minor parasitoid-induced changes, with the rough endoplasmic reticulum appearing more developed and electrondense than in nonparasitized controls. Eventually, the cells of prothoracic glands of parasitized host last instar larvae die but maintain their structural integrity. The inactivation of pupally committed host prothoracic glands is achieved through the disruption of the PTTH signal transduction pathway. The second messenger cAMP appears to be normally produced in response to PTTH stimulation of glands explanted from parasitized host larvae, however the downstream activation of the cAMP-dependent protein kinase does not appear to occur. In fact, a marked underphosphorylation of regulatory target proteins is observed. This underphosphorylation is associated with a significant reduction in general protein synthesis, which appears to be blocked at the translational level, to a redirection of specific protein synthesis and to a drastic suppression of ecdysteroidogenesis. These parameters appeared to be correlated in a kinetic time-course study, confirming their functional link. C. nigriceps polydnavirus (CnPDV) plays a major role in the inactivation of pupally committed host prothoracic glands, while putative factors occurring in the host haemolymph do not seem to be of particular importance at that developmental stage. Southern blot hybridization indicates the occurrence of PKI(protein kinase inhibitor)-like genes in the C. nigriceps genome, which, in contrast, are undetectable in H. virescens.

Organisation of regulatory elements in two closely spaced Drosophila genes with common expression characteristics.

Sperm tail proteins that are components of a specific structure formed late during spermatid elongation have been found to be encoded by the Mst(3)CGP gene family. These genes have been demonstrated to be regulated both at the transcriptional as well as at the translational level. We report here on the dissection of the regulatory regions for two members of the gene family, Mst84Da and Mst84Db. While high level transcription and negative translational control of Mst84Da is mediated by a short gene segment of 205 nt (-152/+53), Mst84Db expression is controlled by a number of distinct regulatory elements with different effects that all reside within the gene itself. We identify a transcriptional control element between +154 and +216, a translational repression element around +216 to +275 and an RNA stability element within the 3'UTR. Irrespective of the final common expression characteristics, correct regulation for any individual member of the gene family seems to be achieved by very different means. This confirms earlier observations that did not detect any other sequence elements in common apart from the TCE (translational control element).

Synthesis and expression of genes encoding putative insect neuropeptide precursors in tobacco.

Neuropeptides are the key molecules in a multiplicity of physiological processes and their use in pest control has recently been suggested. Most neuropeptides are produced in the form of a precursor that is cleaved by proteolysis to yield various biologically active peptides. To mimic this structure, a method has been developed for synthesizing genes that code for putative polyneuropeptide precursors. As a model neuropeptide, the 5-amino-acid proctolin, one of the best studied invertebrate neuropeptides, functioning both as a visceral and a skeletal neuromuscular transmitter, was chosen. The synthetic gene was introduced into bacteria and tobacco plants, where it was efficiently transcribed. We present our results as a possible approach for the expression, in a variety of organisms, of synthetic genes coding for a wide repertoire of insect neuropeptides.

Characterization of Aphidius ervi (Hymenoptera, Braconidae) ribosomal genes and identification of site-specific insertion elements belonging to the non-LTR retrotransposon family.

We performed a molecular analysis of the Aphidius ervi ribosomal gene structure. This insect belongs to a set of closely related Aphidiinae species of the genus Aphidius Nees, of relevant interest in biological control. We constructed A. ervi genomic libraries, cloned and characterized several rDNA repeating units and sequenced different regions of the rDNA cistrons. We have found that insertion sequences interrupt the A. ervi 28S rDNA genes: the sequences of the two 5' and 3' insertion-28S junctions show that the elements are present at the position where R1 elements have been found in various insect species. In addition, the insertion of the element produces a duplication of the 14 nt target region. The sequence analysis indicates that the A. ervi elements belong to the R1 retrotransposon family with a highly conserved reverse transcriptase domain.

Cell functions in Drosophila oogenesis.

We are studying Drosophila oogenesis by analysing at genetic and molecular levels several female-sterile mutations. Some (hold up, wavoid-like and abnormal oocyte) have been isolated by L. Sandler in region 32 of the second chromosome; others have been isolated by us and their phenotype is presented for the first time in this paper. We performed chromosome walking in 32D-32E-F(250 Kb) and 32A-B(100 Kb) and in the last years we molecularly identified several genes with specific maternal expression patterns. We will review here our studies on two of these genes: the Vitelline Membrane Protein gene 32E and the gene coding for a receptor form of Guanylate Cyclase.

A membrane guanylate cyclase Drosophila homolog gene exhibits maternal and zygotic expression.

A putative membrane form of guanylate cyclase gene was identified in region 32 of the second chromosome of Drosophila melanogaster. The Drosophila protein has a single hydrophobic sequence that divides the protein into a putative extracellular region and a cytoplasmic catalytic region which contains tyrosine kinase and cyclase domains showing varying degrees of conservation with other guanylate and adenylate cyclases. The gene shows a very interrupted organization with small exons separated by small introns and a low level of expression. Transcripts have been localized in situ during oogenesis in the germarium and later in stages 10-14 of egg chamber development. The putative maternal transcript can be seen in very early embryos and reappears later as a product of zygotic gene expression.

Capture errors and sequencing after frontal brain lesions.

This study addresses the effect of strong routine associations (capture errors) in hindering the control of on-line serial or sequencing tasks. Patients with focal frontal lobe lesions were significantly inferior to normal control subjects and patients with posterior brain lesions, when conditions that may lead to capture errors were present. The results suggest that the primary dysfunction exhibited by patients with frontal lobe lesions on capture error tasks may lie not in the disengagement from the invalid associations but in focusing attention to alternative strategies of response.

Complete reversion of the abo phenotype in D. melanogaster occurs only when the blood transposon is lost from region 32E.

The abnormal oocyte phenotype is characterized by instability, as shown by the loss and reappearance of the abo maternal effect under specific genetic conditions. Our previous finding that a correlation exists between the abo phenotype and the presence of a blood transposon in region 32E, led us to perform an extensive genetic and molecular analysis of the most significant aspects of the abo phenotype in different genetic backgrounds. The results of these experiments can be summarized as follows: Complete reversion occurs only when the blood trnasposon is lost, thus definitively demonstrating that the insertion of the blood transposon in region 32E is the molecular event that causes the pleiotropic abo phenotype. Partial reversion can also occur without loss of the transposon indicating that different molecular pathways may be involved in the loss of the abo phenotype. Reappearance of the full abo phenotype can occur only in heterozygous lines constructed from partially revertant abo homozygous lines that have not lost the blood transposon.

Cellular specificity of expression and regulation of Drosophila vitelline membrane protein 32E gene in the follicular epithelium: identification of cis-acting elements.

In this paper we analyze the expression in follicular cells and regulation of the vitelline membrane protein gene we identified in region 32E of the second chromosome of D. melanogaster (VMP32E). We report germ line transformation results obtained with different kinds of gene fusion leading to the identification of a follicular cell subpopulation involved in the expression of the VMP32E. We have characterized two 5' non-transcribed regions (-465/-249; -135/-39) where the cis-acting transcriptional regulatory sequences, directing tissue and temporal specificity, are contained. The region between -465 and -249, which appears to control transcriptional high efficiency, does not behave as an enhancer as it is incapable of conferring any expression to a reporter gene. The region between -135 and -39 can confer temporal specificity of expression of the VMP32E gene, albeit at a very low level. Most interestingly, sequence similarities to ecdysone response elements raise the possibility of hormonal control also for VMP gene expression.

The abnormal oocyte phenotype is correlated with the presence of blood transposon in Drosophila melanogaster.

The abnormal oocyte mutation (2;44) originates in the wild: it confers no visible phenotype on homozygous abo males or females, but homozygous abo females produce defective eggs and the probability of their developing into adults is much lower than that of heterozygous sister females. We isolated by chromosome walking 200 kb of DNA from region 32. This paper reports that a restriction enzyme site polymorphism analysis in wild type and mutant stocks allowed us to identify a DNA rearrangement present only in stocks carrying the abo mutation. The rearrangement is caused by a DNA insert on the abo chromosome in region 32E which, by restriction map and sequence analysis, was identified as copia-like blood transposon. The transposon, in strains that had remained in abo homozygous conditions for several generations and had lost the abo maternal-effect, was no longer present in region 32E. Certain features of the abo mutation, discussed in the light of this finding, may be ascribed to the nature of the particular allele studied.

Sex-, tissue-, and stage-specific expression of a vitelline membrane protein gene from region 32 of the second chromosome of Drosophila melanogaster.

This study isolated cDNA clones from egg-chamber and adult female Drosophila cDNA libraries using as probe a DNA fragment from a 200-kb "chromosome walk" in region 32E of the second chromosome of D. melanogaster. The present authors believe that these clones correspond to a new vitelline membrane protein (VMP) gene because 1) cDNA clones in Northern blots identify a transcript expressed in a tissue- and stage-specific manner: stage 10 egg-chambers; 2) the sequence of cDNAs and of the genomic subclone shows homology with the other VMP genes that have been identified to date; 3) the amino acid composition of the translational product has the high content of proline and alanine characteristic of VMPs. Two aspects emerging from this study are worth stressing: 1) the presence of a hydrophobic domain that is highly conserved in all the VMP genes; and 2) the particularly narrow period of expression of the isolated gene, which could be related to the mechanism of vitelline membrane assembly.