Hermes-mediated germ-line transformation of the Mediterranean fruit fly Ceratitis capitata

We report the use of the Hermes transposable element for germ‐line transformation of the Mediterranean fruit fly, Ceratitis capitata. Hermes was able to genetically transform this insect at an estimated frequency between 0.6 and 1.1%, which is comparable to the transformation frequencies obtained for this species when using other transposable elements. Hermes integrates into the medfly genome by a cut‐and‐paste mechanism and the sequences integrated into the genome are delimited by the terminal nucleotides of the Hermes inverted terminal repeats. Integration resulted in the generation of 8 bp target site duplications, the sequences of which conformed to the target site duplications generated by hAT element transposition in insects. The Hermes element is one additional genetic tool that can be deployed in manipulating and characterizing the medfly genome.     

Microsatellite polymorphism in the Mediterranean fruit fly, Ceratitis capitata

A total of forty-three simple sequence repeats (SSRs) were identified in the Mediterranean fruit fly (medfly) Ceratitis capitata. The most common SSR was the dinucleotide (TG)n/(CA)n occurring in thirty of the forty-three microsatellite loci. Polymorphism at ten dinucleotide markers was investigated in 122 flies from six natural populations sampled in the native and colonized areas. A very high level of allelic diversity was detected in the species range. An average of 13.6 alleles was found over all the ten loci indicating the informativeness of SSRs as genetic markers for the medfly. The distribution of microsatellite polymorphism in the species range reflects the medfly colonization history.     

Analysis of mitochondrial DNA and development of PCR-based diagnostic molecular markers for Mediterranean fruit fly (Ceratitis capitata) populations

A 2.99 kb mtDNA fragment containing two variable restriction endonuclease sites ( EcoRV and Xbal ) was subcloned and sequenced from the Mediterranean fruit fly (Ceratitis capitata). This fragment represents approximately one-fifth of the entire mitochondrial sequence. The sequence was aligned with the comparable region from Drosophila yakuba and Anopheles gambiae , resulting in 81.8% and 76.7% identity at the nucleotide level, and 77% and 67.7% identity, respectively, at the amino acid level. The sequenced region includes the complete genes for NADH dehydrogenase 4, NADH dehydrogenase 4L, NADH dehydrogenase 6, and transfer RNAs for proline, threonine and histidine, and part of the genes for NADH dehydrogenase 5 and cytochrome b. Oligonucleotide primers were designed to asymmetrically bracket each of two variable restriction endonuclease sites to allow PCR amplification and subsequent restriction endonuclease analysis of individual fly samples.     

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 degrees C, reaching a maximum between 35 and 41 degrees 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.     

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.     

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-α and -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.     

Isolation of cDNAs encoding 6-phosphogluconate dehydrogenase and glucose-6-phosphate dehydrogenase from the mediterranean fruit fly Ceratitis capitata: correlating genetic and physical maps of chromosome 5

We have isolated and determined the nucleotide sequences for cDNA clones encoding glucose-6-phosphate dehydrogenase (G6PD) and 6-phosphogluconate dehydrogenase (6PGD) from the medfly Ceratitis capitata. The derived amino acid sequences for G6PD and 6PGD are presented and compared with G6PDs and 6PGDs from other species. The codon usage of the cDNA clones has little bias with the notable exceptions of arginine, glycine and leucine. The chromosomal location of the genes for 6PGD and G6PD were determined by in situ hybridization to salivary gland polytene chromosomes. This localization orients a genetic map of enzymatic loci and illustrates a remarkable similarity in the intra chromosomal order of homologous genes between Drosophila melanogaster and medfly.     

Cloning and characterization of the ribosomal protein CcP0 of the medfly Ceratitis capitata

The gene of the ribosomal protein CcP0, the third member of the ribosomal P-protein family of the medfly Ceratitis capitata, was identified by genomic and cDNA sequence analysis. It codes for a polypeptide of 317 amino acids and its predicted amino acid sequence shows great similarity to the P0 proteins of other eukaryotic organisms. The CcP0 gene was expressed in Escherichia coli and the 34-kDa recombinant protein was identical to the P0 protein of purified medfly ribosomes. Both proteins reacted positively with a specific monoclonal antibody against the highly conserved C terminus of eukaryotic ribosomal P proteins. Interestingly, the medfly CcP0 seems to be the only P0 protein of higher eukaryotic organisms with basic character (pI 8.5), as shown by electrofocusing of purified ribosomes.     

The mitochondrial genome of the mediterranean fruit fly, Ceratitis capitata

The complete sequence of the mitochondrial genome of Ceratitis capitata has been determined. The circular genome is 15 980 bp long and contains a standard gene complement, i.e. the large and small ribosomal RNA subunits, twenty-two transfer RNA (tRNA) genes and thirteen genes encoding mitochondrial proteins. When comparing the sequence to fragments previously sequenced from other isolates it becomes apparent that interstrain polymorphisms are not rare. These differences are potentially useful for the development of diagnostic tools for population analysis applications, such as determining the source of recent introductions. Moreover, they could help obtain a solution to the long-lasting controversy on the possible eradication of the Medfly from certain locations.     

A new Minos vector for eye-specific expression of white+ marker in Ceratitis capitata and in distantly related dipteran species

The genetic transformation of insects by transposable elements is based on the use of selectable genetic markers required to identify transgenic individuals. Conserved regulatory sequences can be used to develop single constructs capable of adequate expression of a marker, across a range of different species. We present evidence that the Drosophila GBS regulatory element (Glass-binding site), derived from the Rh1 rhodopsin gene, is able to drive in vivo eye-specific expression of a Ccwhite+ transgene in the Mediterranean fruitfly Ceratitis capitata. The Ceratitis lineage diverged from that of Drosophila approximately 120 Myr ago. As the GBS regulatory sequence seems to be partially conserved in the more distantly related dipteran species Anopheles gambiae (250 Myr), we propose that the GBS may be widely useful for driving eye-specific expression in a wide range of dipteran species.     

Ccmar1, a full-length mariner element from the Mediterranean fruit fly, Ceratitis capitata

Using a PCR primer specific to the ITR sequence of a deleted mariner element we amplified a fragment of ∼1300 bp from the genome of Ceratitis capitata . Analysis of four clones showed that they differed by ∼4.6% in nucleotide sequence and exhibited high homology to mariner elements of the mellifera subfamily. One clone in particular, Ccmar1.18 , was found to possess an ORF of 338 amino acids together with many of the features typical of mariner elements. The consensus sequence, Ccmar1 , derived from these clones is presented. Maximum parsimony phylogenetic analysis of the Ccmar1 element confirms its position at the periphery of the mariner mellifera subfamily. The Ccmar1 element is estimated to be present in about 500 copies in the genome. The evolutionary history of the element in relation to the colonization history of the medfly is discussed.     

Diversity of expressed cytochrome P450 genes in the adult Mediterranean Fruit Fly, Ceratitis capitata

The cytochrome P450s comprise a superfamily of mostly microsomal haemoproteins which play a dominant role in the metabolism of a variety of endogenous and foreign compounds. The use of a degenerate PCR primer targeted to the haem-binding decapeptide unique to the cytochrome P450 superfamily resulted in the identification of 14 novel cytochrome P450s in the Mediterranean Fruit Fly, Ceratitis capitata. Analysis of the relative frequency of individual isoforms within the pool of isolated sequences suggests that the CYP4 and CYP6 P450 families contain the most highly expressed isoforms in adult C. capitata. Phylogenetic analyses of the conceptual amino acid translations of PCR-amplified cDNAs provides evidence that one of isolated sequences may represent a new P450 family.