Male-specific serum proteins (MSSPs) are low molecular weight proteins which accumulate in high amounts in the haemolymph of adult males of the medfly Ceratitis capitata. By screening an expression library with anti-MSSP antibodies, we have isolated and determined the nucleotide sequence of a cDNA clone coding for one of the male-specific polypeptides (MSSP-alpha). The MSSP-alpha mRNA encodes a polypeptide of 144 amino acids with a secretory signal sequence of sixteen amino acids. Southern analysis indicated that there are multiple copies of MSSP genes in the medfly genome. Northern analysis showed that the MSSP mRNAs are synthesized only in adult males. The accumulation pattern of these mRNAs during development suggests that the expression of the MSSP genes is developmentally regulated at both transcriptional and translational levels. The predicted peptide sequence of MSSP-alpha shows significant similarity to a group of pheromone- and general odourant-binding proteins of insects.

In the present study, a genomic DNA clone encoding the medfly homolog of Drosophila melanogaster hsp27 gene, named Cchsp27, was isolated. We sequenced a part of the clone containing the coding region, the 5' untranslated region and approximately 2.8 Kb of the 5' flanking region of the gene. Phylogenetic analysis of several insect small heat shock proteins, suggested that CcHsp27 is orthologous to Drosophila Hsp27 and Sarcophaga crassipalpis Hsp25. The Cchsp27 gene was mapped at the 81A division of the sixth chromosome which coincides with one of the major heat shock puffs of medfly. Structural analysis of the 5' flanking region of the Cchsp27 gene revealed the presence of five putative heat shock elements and one putative ecdysone response element. In addition to heat induction, the Cchsp27 gene was expressed at several stages of normal medfly development. In general, the developmental expression pattern of the Cchsp27 gene was similar to the respective pattern of Drosophila hsp27 gene. However, there were some important differences in certain developmental stages suggesting differential regulation of the hsp27 gene in the two dipterans species. Salivary gland culture experiments showed that the Cchsp27 gene is regulated by 20-hydroxyecdysone.

In order to understand the role that 20-hydroxyecdysone plays during development of the Mediterranean fruit fly Ceratitis capitata (medfly), a major agricultural pest, we have cloned a Ceratitis ecdysone receptor (CcEcR) and studied its expression and its binding properties to an ecdysone response element. Using the conserved DNA binding region of the Drosophila melanogaster ecdysone receptor (DmEcR) B1 cDNA as a probe, we isolated a medfly cDNA clone containing the coding region, a part of the 5 H -untranslated region and the complete 3 H -untranslated region of a CcEcR. The deduced CcEcR polypeptide contained all five domains typical of a nuclear receptor. Alignment comparisons and phylogenetic analyses indicated that CcEcR most closely resembled the B1 isoform of DmEcR and Lucilia cuprina EcR homolog (LcEcR) relative to all other known ecdysone receptors. In situ hybridization analysis showed that the CcEcR gene is mapped in the region 53B of the 4R chromosome arm, while Northern hybridization analysis showed that CcEcR transcripts have a size of approximately 8 kb. Significant levels of CcEcR transcripts were detected in eggs, middle and late embryos, late third instar larvae and middle prepupae. The levels of the CcEcR transcripts during the other larval stages as well as during pupal and adult stages were much lower, while during the early stages of embryogenesis were very low. Electrophoretic mobility shift assays indicated that CcEcR binds specifically to the Drosophila hsp27 ecdysone response element as a heterodimer with Drosophila USP, the product of the ultraspiracle gene. Our structural and biochemical data suggest that CcEcR is the functional homolog of the B1 isoform of DmEcR.

In the present study, we characterized a 3320-bp genomic DNA fragment encoding two medfly (Ceratitis capitata) homologues of the Drosophila melanogaster heat shock protein 23 (hsp23) gene, named Cchsp23-alphaand -beta. The two medfly hsp23 genes are transcribed in opposite directions and encode two almost identical proteins. Furthermore, the two genes exhibit a very high degree of similarity in their 5' untranslated and proximal promoter regions. Phylogenetic analysis indicated that the CcHsp23 proteins are orthologous to Drosophila Hsp23 and Sarcophaga crassipalpis Hsp23. Structural analysis of the 5' flanking regions of the Cchsp23 genes revealed the presence of several putative heat shock elements. Both CcHsp23 genes are induced by heat in a similar manner. In addition to heat-induction, the Cchsp23 genes are expressed at several stages of normal development as well as in ovaries and testes. In general, the developmental expression patterns of the medfly genes are similar, suggesting that they are under similar regulatory mechanisms. However, the expression of the Cchsp23 genes differs significantly from the expression of the Drosophila hsp23 gene in certain embryonic and larval stages, suggesting differential regulation of the hsp23 genes in the two dipteran species. The expression of both Cchsp23 genes in adult flies is increased with age, especially in males.

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