Anastassios C. Mintzas

cDNA cloning, heat shock regulation and developmental expression of the hsp83 gene in the Mediterranean fruit fly Ceratitis capitata

This report presents the cDNA cloning, heat shock regulation and developmental expression of the hsp90 gene homologue of the Mediterranean fruit fly Ceratitis capitata (medfly). The isolated cDNA contained the coding region, the 3′UTR and most of the 5′UTR of the medfly hsp90 homologue, which was named Cchsp83. The deduced CcHSP83 polypeptide contained all the highly conserved amino acid segments that characterize the cytosolic members of the HSP90 family. Genomic analysis showed that the Cchsp83 gene is unique and was mapped at the 94C division of the sixth polytene chromosome. The size of the Cchsp83 mRNA was found to be approximately 2.7 kb. The predicted molecular mass of the CcHSP83 protein was 81.4 kDa, while the apparent molecular weight estimated by SDS‐PAGE was approximately 90 kDa. Phylogenetic analysis based on 14 insect HSP90 amino acid sequences was consistent with the known phylogeny at low taxonomic level. The Cchsp83 gene is constitutively expressed in all stages of medfly development and is induced from a low level to several‐fold by heat, depending on the developmental stage. Heat shock induction begins at 30 °C, reaching a maximum between 35 and 41 °C. Cchsp83 RNA expression is highly regulated during embryonic development; however, the temporal fluctuations in RNA levels during embryogenesis were not followed by similar fluctuations in the levels of the protein.

Cloning and characterization of a cDNA encoding a male-specific serum protein of the Mediterranean fruit fly, Ceratitis capitata, with sequence similarity to odourant–binding proteins

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‐α). The MSSP‐α 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‐α shows significant similarity to a group of pheromone‐ and general odourant‐binding proteins of insects.

Organization, evolution and expression of a multigene family encoding putative members of the odourant binding protein family in the medfly Ceratitis capitata

A multigene family encoding male specific serum polypeptides (MSSPs) that show significant structural similarity to the family of insect odourant binding proteins, has been characterized in the medfly Ceratitis capitata. This family comprises seven members classified in three subgroups, MSSP‐α, MSSP‐β and MSSP‐γ. The genes of subgroups α and β are clustered in tandem in a 35‐kb genomic region, and present an exceptionally high degree of similarity not only in their coding but also in the surrounding regions, while the genes of the γ subgroup are drastically divergent. Although MSSPs are predominantly expressed in the male fat body, detailed expression studies suggest that individual members of this family are expressed in a distinct sex‐ and tissue‐specific manner.

Polytene chromosomes as tools in the genetic analysis of the Mediterranean fruit fly, Ceratitis capitata.

Polytene chromosomes have been an essential tool in genetic analysis since their rediscovery in larval tissues of Drosophila and other Diptera (Heitz & Bauer, 1933; Painter, 1933) and the reproducible banding patterns of Drosophila polytene chromosomes enabled the first maps to be drawn (Painter, 1933; Bridges, 1935, 1938; Bridges & Bridges, 1939). These maps together with the photographic examples published by Lefevre (1976) and those based on electron microscopy (Sorsa, 1988) are still in use in Drosophila. Today, polytene chromosome maps are available for about 270 species of Drosophilids and for more than 250 other Diptera (Ashburner, 1992). However, only a small proportion of them is of sufficient quality to be used as genetic tools. The importance of polytene chromosomes as genetic tools can be found in their following uses:

The hsp27 gene of the Mediterranean fruit fly, Ceratitis capitata: structural characterization, regulation and developmental expression

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.

Developmental profiles and ecdysone regulation of the mRNAs for two ecdysone receptor isoforms in the Mediterranean fruit fly Ceratitis capitata

Using 5′ RACE with specific primers for the ecdysone receptor B1 isoform of the Mediterranean fruit fly (medfly), Ceratitis capitata, we isolated a cDNA clone encoding the specific region of the medfly ecdysone receptor A isoform (CcEcR‐A). The CcEcR‐A‐specific region was very similar to the EcR‐A‐specific region of Drosophila melanogaster and less similar to the EcR‐A‐specific regions of Lepidoptera. The developmental expression of both CcEcR‐A and CcEcR‐B1 mRNAs was studied in whole animals, salivary glands and ovaries by RT‐PCR, using isoform‐specific primers. Both CcEcR mRNAs are present in very early embryos, decrease to very low levels during the first hours of embryogenesis and are highly expressed in all consequent embryonic stages. During metamorphosis both isoforms are present showing two peaks; the first at the larval–prepupal transition and the second during the second half of prepupal development. These peaks are correlated with the two puffing cycles and the two major 20‐hydroxyecdysone (20E) increases that occur during medfly metamorphosis. CcEcR‐B1 mRNA was directly induced in larval salivary glands in vitro by 20E, even at very low concentrations of the hormone, while CcEcR‐A mRNA was slightly induced only by high 20E concentrations and in the absence of a protein synthesis inhibitor. During oogenesis, the CcEcR mRNAs were expressed synchronously, peaking at the beginning of both previtellogenic and vitellogenic phases.

Biosynthesis and tissue distribution of four major larval serum proteins during development of Ceratitis capitata (Diptera)

Abstract Antibodies to the four major larval serum proteins (MLSPs) of Ceratitis capitata were used to study the biosynthesis and tissue distribution of these proteins during larval, pupal and adult development of this species. MLSP-1,2 and 3 are synthesized only during the last larval instar and are accumulated in the haemolymph. During metamorphosis, these proteins are reabsorbed by the fat body and a fraction of them is deposited in the cuticle. MLSP-4 gives a completely different biosynthetic and accumulation pattern. The synthesis of this protein takes place at three different stages during development. The first peak of synthesis occurs at the wandering stage, about one day after the MLSP-1,2 and 3 peak, the second, about one day before adult eclosion and the third one day after adult eclosion. Furthermore, the synthesis of this protein in newly emerged adults can be stimulated by 20-hydroxyecdysone. MLSP-4 is accumulated in the haemolymph where it remains during all stages of development and is not incorporated by other tissues.

Expression and function of the Drosophila melanogaster ADH in male Ceratitis capitata adults: a potential strategy for medfly genetic sexing based on gene-transfer technology

The aim of development of a Mediterranean fruit fly Ceratitis capitata genetic sexing strain derives from the large scale SIT programmes being carried out to control this pest. Toward this direction, we present here the male‐specific expression of the Drosophila melanogaster alcohol dehydrogenase (ADH) in medfly transgenic adults generated by Minos‐mediated germ line transformation. This expression pattern is obtained by using a promoter fragment of the male‐specific gene MSSP‐α2 of the medfly. We show that the heterologous enzyme is functional in the medfly oxidizing both ethanol and 2‐propanol. Although leading to an approximately twofold increase of total ADH activity in male compared to female transgenic adults, these expression levels are not enough for performing genetic sexing when high doses of environmental alcohol are applied. This could be achieved either by further enhancement of the transgene expression or by generating an Adh− line to host the Minos insertions.

Evaluation of the activities of the medfly and Drosophila hsp70 promoters in vivo in germ-line transformed medflies.

The promoter of the hsp70 gene of Drosophila melanogaster has been widely used for the expression of foreign genes in other insects. It has been generally assumed that because this gene is highly conserved, its promoter will function efficiently in other species. We report the results of a quantitative comparison of the activities of the medfly and D. melanogaster hsp70 promoters in vivo in transformed medflies. We constructed transformed lines containing the lacZ reporter gene under the control of the two promoters by using Minos‐mediated germ‐line transformation. The activity of each promoter was evaluated in 15 transformed lines by β‐galactosidase quantitative assays. The heat‐inducible activity of the medfly promoter was found several times higher than the respective activity of the heterologous D. melanogaster promoter. These results were confirmed by northern blot analysis and indicate that the D. melanogaster promoter does not work efficiently in medfly. The −263/+105 medfly promoter region that was used in this study was found able to drive heat shock expression of the lacZ reporter gene in all stages of medfly, except early embryonic stages, in a similar fashion to the endogenous hsp70 genes. However the heat inducible RNA levels driven from this promoter region were significantly lower than the endogenous hsp70 RNA levels, suggesting that additional upstream and/or downstream sequences to the −263/+105 region may be necessary for optimum function of the medfly hsp70 promoter in vivo.

Two hsp23 genes in the Mediterranean fruit fly, Ceratitis capitata: structural characterization, heat shock regulation and developmental expression.

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‐αand ‐β. 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.