Stage and cell-specific expression and intracellular localization of the small heat shock protein Hsp27 during oogenesis and spermatogenesis in the Mediterranean fruit fly, Ceratitis capitata.

The cell-specific expression and intracellular distribution of the small heat protein Hsp27 was investigated in the ovaries and testes of the Mediterranean fruit fly, Ceratitis capitata (medfly), under both normal and heat shock conditions. For this study, a gfp-hsp27 strain was used to detect the chimeric protein by confocal microscopy. In unstressed ovaries, the protein was expressed throughout egg development in a stage and cell-specific pattern. In germarium, the protein was detected in the cytoplasm of the somatic cells in both unstressed and heat-shocked ovaries. In the early stages of oogenesis of unstressed ovaries, the protein was mainly located in the perinuclear region of the germ cells and in the cytoplasm of the follicle cells, while in later stages (9-10) it was distributed in the cytoplasm of the germ cells. In late stages (12-14), the protein changed localization pattern and was exclusively associated with the nuclei of the somatic cells. In heat shocked ovaries, the protein was mainly located in the nuclei of the somatic cells throughout egg chamber's development. In unstressed testes, the chimeric protein was detected in the nuclei of primary spermatocytes and in the filamentous structures of spermatid bundles, called actin cones. Interestingly, after a heat shock, the protein presented the same cell-specific localization pattern as in unstressed testes. Furthermore, the protein was also detected in the nuclei of the epithelial cells of the deferent duct, the accessory glands and the ejaculatory bulb. Our data suggest that medfly Hsp27 may have cell-specific functions, especially in the nucleus. Moreover, the association of this protein to actin cones during spermatid individualization, suggests a possible role of the protein in the formation and stabilization of actin cones.

Thermotolerance and HSP70 expression in the Mediterranean fruit fly Ceratitis capitata.

The relationship between Hsp70 expression and thermotolerance has been well documented in Drosophila melanogaster. However, there is limited information on this relationship in other insect species. In this report we describe the Hsp70-thermotolerance relationship in one of the major fruit fly pests, Ceratitis capitata (medfly). Hsp70 expression and thermotolerance were assayed at a range of temperatures in several stages of medfly development. The most thermotolerant stage was found to be the late larval stage (100% survival at 41 degrees C) followed by adult flies and late embryos (100% survival at 39 degrees C). These three stages showed a positive relationship between Hsp70 expression and thermotolerance. Mid-larval and mid-embryonic stages were found less thermotolerant and the Hsp70-thermotolerance relationship was not evident. Early embryos did not express Hsp70 at any temperature and exhibited the lowest thermotolerance. The relationship between Hsp70 and inducible thermotolerance was also studied in late larvae. A pretreatment at 37-39 degrees C increased thermotolerance at higher temperatures by approximately 1 degrees C. In parallel, the pretreatment increased Hsp70 expression suggesting a close link between Hsp70 expression and inducible thermotolerance. The increased Hsp70 levels after pretreatment were found to be due to the increased levels of the hsp70 RNA.

cDNA cloning, characterization, and developmental expression of the 20S proteasome alpha5 subunit in the Mediterranean fruit fly Ceratitis capitata.

In the present study, we report the cDNA cloning, characterization, and developmental expression of the 20S proteasome alpha5 subunit from the Mediterranean fruit fly Ceratitis capitata (medfly). Using an RT-PCR fragment that corresponds to the amino-terminal region of the Drosophila melanogaster 20S proteasome alpha5 subunit, we isolated a 987-bp cDNA that encodes the complete coding region of the medfly ortholog, which was named CcPSMA5. CcPSMA5 consists of 241 amino acids and has a predicted molecular weight of 26.4 kDa and pI 4.75. Comparison of the CcPSMA5 amino acid sequence with the sequences of all known 20S proteasome alpha5 subunits from different organisms indicated that the medfly 20S proteasome alpha5 subunit has the strongest homology to that of Drosophila. In situ hybridization showed that the CcPSMA5 gene is mapped in the region 44B of chromosome 4. Northern blot hybridization analysis showed that the CcPSMA5 mRNA has a size of approximately 1.2 kb. High levels of the CcPSMA5 mRNA were detected in freshly laid eggs, indicating that they were maternally deposited. The mRNA expression pattern during medfly development suggests that the CcPSMA5 gene is upregulated before mid-embryogenesis and at the onset of metamorphosis.

Structural characterization of the medfly hsp83 gene and functional analysis of its proximal promoter region in vivo by germ-line transformation.

In order to define the regulatory elements responsible for the expression of the medfly hsp83 (Cchsp83) gene, we determined the sequence of a genomic region of the gene that included 3,536 bp upstream of the transcription initiation site, the first untranslated exon of 144 bp, a 275-bp intron, and 516 bp of the second coding exon. Structural analysis of the 5' flanking region revealed the presence of a typical TATA box, 28 bp upstream of the transcription start site, and seven putative heat shock elements (HSEs) further upstream. The 5' untranslated region of the Cchsp83 mRNA was found to contain extensive secondary structure in the first 126 nucleotides. We carried out deletion functional analysis of the proximal promoter region (-380/+139) in vivo by germ line transformation using the lacZ as a reporter gene. We found that sequences in the -380/-86 region are essential for the constitutive expression of the Cchsp83 gene. Under normal conditions, the -380/+139 region was able to drive significant levels of transgene expression in all developmental stages of the medfly as well as in the ovaries and testis. In most stages, the temporal expression pattern of the reporter gene was similar to the respective pattern of the endogenous Cchsp83 gene. Although the -380/+139 promoter region contained two putative HSEs, it was found unable to confer any heat-induced expression in the reporter gene.

Cloning, characterization, and developmental expression of the ribosomal protein S21 gene of the Mediterranean fruit fly Ceratitis capitata.

Ribosomal protein S21 (RpS21) belongs to a small group of ribosomal or ribosome-associated proteins. Mutations in the RpS21 gene cause dominant Minute and recessive lethal tumorous phenotypes in Drosophila melanogaster. Studies in several organisms suggest that RpS21 is involved in the regulation of protein synthesis and cell growth. In this report, we used an RT-PCR fragment of D. melanogaster RpS21 mRNA to clone a RpS21 cDNA from the Mediterranean fruit fly, Ceratitis capitata. The isolated cDNA contained both 5' and 3' untranslated regions, and encoded a polypeptide of 83 amino acids with a predicted molecular mass of 9.1 kDa. The deduced protein sequence showed 91% amino acid identity to D. melanogaster RpS21 and strong homology with all known ribosomal S21 proteins. DNA blot hybridization indicated the existence of a single RpS21 gene in the Ceratitis capitata genome. Analysis of the 5' untranslated region revealed the occurrence of a major oligopyrimidine tract at the 5' end, which characterizes most mRNAs undergoing a growth-dependent translational control. Study of the mRNA patterns during development suggested that the expression of Ceratitis RpS21 is temporally regulated at the level of transcription.

Medfly promoters relevant to the sterile insect technique.

This review summarizes structural and functional studies on medfly promoters and regulatory elements that can be used for driving sex-specific, conditional and constitutive gene expression in this species. Sex-specific and conditional promoters are important for generating transgenic sexing strains that could increase the performance of the Sterile Insect Technique while strong constitutive promoters are necessary for developing sensitive transgenic marker systems. The review focuses on the functional analysis of the promoters of two male-specific and heat shock medfly genes. A special emphasis is put on the potential utility of these promoters for developing transgenic sexing strains.

Biosynthesis and regulation of two vitellogenins in the fat body and ovaries of Ceratitis capitata (Diptera).

In adult female Ceratitis capitata both fat body and ovaries synthesize two vitellogenins (Vg-1 and Vg-2) with the same molecular masses as the respective vitellins of the eggs. Furthermore, both tissues contain two abundant mRNAs which yield, in a cell-free system, two previtellogenin polypeptides with molecular masses approximately 1,000 daltons higher than the mature Vgs. In vivo and in vitro studies during development suggested co-ordinate synthesis of Vg-1 and Vg-2 in each tissue. Also, at least in the fat body, Vg synthesis appears to be regulated at the level of transcription. Although both Vg-1 and Vg-2 are synthesized in the fat body and ovaries synchronously, their relative synthetic rates differ in the two tissues. The ratio of Vg-1 to Vg-2 synthesis in the fat body is approximately twice the respective ratio in the ovaries. Adult C. capitata males do not synthesize Vgs; however, they do so after treatment with 20-hydroxyecdysone.