Medfly Ceratitis Capitata Research Articles

CD and NMR structural characterization of ceratotoxins, natural peptides with antimicrobial activity.

Antibacterial properties of the secretion from the female reproductive accessory glands of medfly Ceratitis capitata are mostly ascribed to the presence of two peptides, ceratotoxin A and B, which exhibit a strong activity against gram-positive and gram-negative bacterial strains, and show sequence and function homology with cecropins, melittin, and magainins. CD experiments performed in different solvents indicate the presence of a significant content of helical structures in organic solvent. Two-dimensional nmr results for ceratotoxin A in methanol show a helical behavior for the 8-25 region of the peptide. A ramachandran classification of each residue for the structures obtained from distance geometry calculations lead to the definition of four structural families in which the central segment 10-19 is always helical and differences refer to residues 8-9 and 19-23. A sequence analysis of the two ceratotoxins and a systematic search on the protein data bank revealed the occurrence of a KX-hydrophobic-hydrophobic-P motif that seems to be important for helix stabilization.

Removal of malathion residues on lettuce by washing.

Malathion (S-1,2-bis(ethoxycarbonyl)ethyl O,O-dimethyl phosphorodithioate), a broad-spectrum insecticide, was used in California to eradicate infestations of the Mediterranean fruitfly (Medfly-Ceratitis capitata) (Segawa 1991). Under the eradication program, malathion in a protein-sugar bait was applied by helicopters over extensive agricultural and residential areas where many of the over 260 host plants susceptible to Medfly infestation could be found (Foote 1993). Homeowners were concerned about consuming fruits and vegetables grown in home gardens that were exposed to the bait sprays. Water rinsing or washing has been reported to remove malathion residues from vegetables (Smith et al. 1955, Wallis et al. 1957). The extent to which pesticide residues could be removed from treated produce by washing may be influenced by a variety of factors such as the chemical properties and formulation type of the pesticide, the nature of the commodity, the length of time that the residue has been on the commodity’s surface, and the rinsing time and rinsing agents used (Smith et al. 1955).

Erratic evolution of glycerol-3-phosphate dehydrogenase in Drosophila, Chymomyza, and Ceratitis.

We have studied the evolution of Gpdh in 18 fruitfly species by sequencing 1,077 nucleotides per species on average. The region sequenced includes four exons coding for 277 amino acids and three variable-length introns. Phylogenies derived by a variety of methods confirm that the nominal genus Zaprionus belongs within the genus Drosophila, whereas Scaptodrosophila and Chymomyza are outside. The rate of GPDH evolution is erratic. The rate of amino acid replacements in a lineage appears to be 1.0 x 10(-10)/site/year when Drosophila species are considered (diverged up to 55 million years ago), but becomes 2.3 x 10(-10) when they are compared to Chymomyza species (divergence around 60 My ago), and 4.6 x 10(-10) when species of those two genera are compared with the medfly Ceratitis capitata (divergence around 100 My ago). In order to account for these observations, the rate of amino acid replacement must have been 15 or more times greater in some lineages and at some times than in others. At the nucleotide level, however, Gpdh evolves in a fairly clockwise fashion.

Molecular characterization of ceratotoxin C, a novel antibacterial female-specific peptide of the ceratotoxin family from the medfly Ceratitis capitata.

Ceratotoxins A and B are antibacterial peptides produced by the sexually mature females of Ceratitis capitata. The gene expression is restricted to the female reproductive accessory glands, and is not affected by bacterial infection, but is enhanced by mating. We report here the purification and the amino acid sequence of ceratotoxin C, a novel member of the ceratotoxin family, the cloning of its cDNA and the analysis of its expression. Ceratotoxin C is coordinately expressed with the other members of the ceratotoxin family. Its antibacterial activity is directed against both Gram-negative and Gram-positive bacterial strains but it is lower than that of ceratotoxin A. We demonstrate in the genome of C. capitata the presence of at least three ceratotoxin genes which express, in the female accessory glands, a set of peptides presumably involved in the protection of the genital tract during fertilization.

Identification of the sex-determining region of the Ceratitis capitata Y chromosome by deletion mapping.

In the medfly Ceratitis capitata, the Y chromosome is responsible for determining the male sex. We have mapped the region containing the relevant factor through the analysis of Y-autosome translocations using fluorescence in situ hybridization with two different probes. One probe, the clone pY114, contains repetitive, Y-specific DNA sequences from C. capitata, while the second clone, pDh2-H8, consists of ribosomal DNA sequences from Drosophila hydei. Clone pY114 labeled most of the long arm and pDh2-H8 hybridizes to the short arm and the centromeric region of the long arm. In 12 of the analyzed 19 Y-autosome translocation strains, adjacent-1 segregation products survive to the late pupal or even adult stage and can, therefore, be sexed. This was correlated with the length of the Y fragment still present in these aberrant individuals and allowed us to map the male-determining factor to a region of the long arm representing approximately 15% of the entire Y chromosome. No additional factors, affecting for example fertility, were detected outside the male-determining region.

Water loss during cuticle sclerotization in the medfly Ceratitis capitata is independent of catecholamines

Insect cuticles undergo a striking water loss during the process of sclerotization. It has been considered that such dehydration is due to a physico-chemical displacement of the water due to incorporation of catecholamines. The dehydration of the puparium in the Medfly Ceratitis capitata throughout pupariation was studied. Water loss in the white pupa mutant, that fails to export catecholamines to the puparium, was not significantly different from that of the wild type or a melanic mutant strain, both with high content of cuticular catecholamines. These results are in clear contradiction to the catecholamine-displacement model for cuticle dehydration. An alternative model explaining the mechanism of water loss during sclerotization is proposed.

Box thorn, key early season host of the Mediterranean fruit fly

Abstract In the Mediterranean region, from November to early June, Mediterranean fruit fly or medfly (Ceratitis capitata Wiedemann) populations decrease drastically and in some places disappear. For area‐wide control or eradication programmes, it is important to know on which wild hosts a medfly population can establish itself and subsequently cause later in the season important losses in commercial crops. Trapping of medfly in a box thorn (Lycium europaeum L.) hedge of the oasis of Tozeur, Tunisia, indicates that despite this plant being largely neglected by medfly later in the year, it is a principal early‐spring host of the pest. Fruit infestation rate in May was c. 292 pupae/kg of fresh fruits and an average of two larvae per infested fruit. The trap capture rate was c. 26 flies/trap/h. The importance of this non‐commercial key‐host in the development and distribution of medfly population during the early season when no other host is available is discussed. The diurnal bimodal flight activity rhythm o...

CDNA Sequence and Expression of the Ceratotoxin Gene Encoding an Antibacterial Sex-specific Peptide from the Medfly Ceratitis capitata (diptera)

Ceratotoxins are antibacterial 3-kDa molecular mass amphiphilic peptides isolated from the female reproductive accessory glands of the medfly Ceratitis capitata. They are physiologically related to bee melittin and show amino acid sequence homology with magainin peptides. In this paper, we report the complete sequence of cDNA coding for ceratotoxin A and the expression of the gene during the life cycle of the insect. Experimental data show that the ceratotoxin is a gene expressed exclusively in the imaginal stages and that it is female-specific, related to sexual maturity, and stimulated by mating. Differently from most antibacterial insect hemolymph peptides, it is not induced by microbial infection. Western blot analysis using an anti-ceratotoxin antibody indicates the female accessory glands as the only site where the production of the ceratotoxin peptide occurs.

Use of a temperature-sensitive lethal mutation strain of medfly (Ceratitis capitata) for the suppression of pest populations

Before the Sterile Insect Technique can be applied successfully, the size of the target population has to be reduced to a manageable level. At present this reduction is achieved by the use of insecticides. Computer simulations have been performed to examine the possibility of achieving this initial population suppression by genetic control strategies; in particular, the effect of releasing fertile males carrying a recessive temperature-sensitive lethal mutation and a Y-autosome translocation has been simulated. The results show that the release of such males is most effective when applied under permissive conditions, i.e. those which allow flies homozygous for the temperature-sensitive lethal mutation to survive and spread the mutation through the population. However, combining this population replacement with a population-suppression strategy is even more effective. If the released males are partially sterile, e.g. due to the presence of a Y-autosome translocation, the population size is reduced before the restrictive conditions for the temperature-sensitive lethal mutation are reached, i.e. before the increase of temperatures in the target area eliminates all flies homozygous for this mutation. By combining these two strategies the resulting population should be low enough to apply the Sterile Insect Technique for eradication.

Expression of the major tyrosyl phosphoprotein of 54 KDa in the integument of the medfly Ceratitis capitata.

Expression of a 54 kDa tyrosyl phosphorylated protein in epidermal cells during the third instar larval stage was followed. It was demonstrated that the 54 kDa protein moiety and its phosphorylated counterpart follow the same developmental profile. The system seems to be regulated only at the onset of the second moult, by an initial signal which regulates both the synthesis and phosphorylation of a 54 kDa protein. The continuous presence this protein in epidermal cells during the third instar stage, as well as during apolysis and histolysis, suggests that it might participate in cell activities taking place during this developmental period. However, the 54 kDa protein could no be involved in specific epidermal cell activities such as histolysis, melanization and sclerotization, since these activities occur only at specific times during the third instar stage.

Synthesis and deposition of PCG‐100, the main pupal cuticle glycoprotein of the medfly Ceratitis capitata

AbstractWe have studied the developmental expression of the main pupal cuticle glycoprotein, PCG‐100, in the Medfly Ceratitis capitata. A polyclonal antiserum was raised against this protein. Western blotting analysis showed that this glycoprotein is integument‐ and stage‐specific. No PCG‐100 or immunologically related polypeptides were detected in other tissues or instars. As studied by microinjection of [35S]methionine in individual flies, in vivo synthesis and deposition of PCG‐100 begins approximately 48 h after the onset of pupariation, shortly after the time of head eversion. Synthesis is maximal at 54–64 h, decreases at 72 h, and practically ceases in fully shaped 4‐day‐old pupae. The time required for PCG‐100 deposition into the cuticle was found to be less than 10 min after its synthesis. This is the first time such in vivo analysis has been performed. © 1994 Wiley‐Liss, Inc.

Isolation and partial characterization of two alcohol dehydrogenase isozymes from the medfly Ceratitis capitata

Two alcohol dehydrogenase isozymes, namely ADH-1 and ADH-2 from Ceratitis capitata were purified to homogeneity and further characterized. After ammonium sulphate precipitation from an extract of whole third instar larvae, the two isozymes were separated by ion exchange chromatography on Q-Sepharose. A combination of affinity chromatography, gel filtration and ion exchange chromatography was then used to purify each isozyme (50 and 57 times with 53 and 58% yields, for ADH-1 and -2 respectively). A crucial step for obtaining homogeneous enzyme preparations was affinity chromatography on Cibacron Blue Sepharose coupled with specific elution with NAD. Each of the isozymes is a dimer with subunit molecular weight of ∼27 kDa. Both isozymes show a pH optimum of 9.6. ADH-1 proved to be immunochemically similar to ADH-2 when tested by Western blot analysis using polyclonal antibodies raised against ADH-1. While crude extracts of Dacus oleae ADH cross-react with these antibodies, no cross reactivity was observed with Drosophila melanogaster extracts. The sequence of a 22-residue peptide from ADH-1 was determined and showed 36% identity with residues 26–47 of the Drosophila melanogaster ADH sequence. Both the sizes of the purified proteins and the observed sequence similarity between ADH-1 and Drosophila ADH strongly suggest that the medfly ADH isozymes belong to the family of short chain dehydrogenases.

Expression of the major tyrosyl phosphoprotein of 54 KDa in the integument of the medflyCeratitis capitata

Expression of a 54 kDa tyrosyl phosphorylated protein in epidermal cells during the third instar larval stage was followed. It was demonstrated that the 54 kDa protein moiety and its phosphorylated counterpart follow the same developmental profile. The system seems to be regulated only at the onset of the second moult, by an initial signal which regulates both the synthesis and phosphorylation of a 54 kDa protein. The continuous presence this protein in epidermal cells during the third instar stage, as well as during apolysis and histolysis, suggests that it might participate in cell activities taking place during this developmental period. However, the 54 kDa protein could no be involved in specific epidermal cell activities such as histolysis, melanization and sclerotization, since these activities occur only at specific times during the third instar stage.

Sequences of two cDNA clones from the medfly Ceratitis capitata encoding antibacterial peptides of the cecropin family

Using a backtranslated oligodeoxyribonucleotide probe, encoding a conserved motif in insect antibacterial peptides, we have isolated two cDNA clones from the medfly, Ceratitis capitata. Sequence determination shows that the cDNAs encode two closely related peptides which are members of the cecropin family.

Elements Controlling Follicular Expression of the s36 Chorion Gene during Drosophila Oogenesis

An 84-bp proximal regulatory protein (PRR) of the Drosophila melanogaster s36 chorion gene is sufficient for directing proper temporal and spatial expression of a reporter gene in three domains of the follicle: anterior, posterior, and main body. Here we show that the fidelity of PRR-directed s36 expression is dependent on the proper dorsal-ventral differentiation of the follicular epithelium, which requires the Drosophila epidermal growth factor receptor homolog. Transgenic analysis of site-directed mutants of the PRR suggests that s36 expression is regulated by the concerted action of multiple positive activators. Several cis-acting transcriptional elements have been identified: some appear to function in a quantitative manner, while others either are essential or appear to regulate expression in particular spatial domains. The approximate locations of these regulatory elements have been defined; some map within sequences that are strongly conserved in widely divergent dipteran species. In fact, the PRR analog of the medfly Ceratitis capitata Ccs36 gene directs expression in a manner similar to the D. melanogaster s36 PRR. We propose a model for transcriptional regulation of s36 based on the prechoriogenic polarization of the follicular epithelium that surrounds the developing egg chamber.

Elements controlling follicular expression of the s36 chorion gene during Drosophila oogenesis.

An 84-bp proximal regulatory protein (PRR) of the Drosophila melanogaster s36 chorion gene is sufficient for directing proper temporal and spatial expression of a reporter gene in three domains of the follicle: anterior, posterior, and main body. Here we show that the fidelity of PRR-directed s36 expression is dependent on the proper dorsal-ventral differentiation of the follicular epithelium, which requires the Drosophila epidermal growth factor receptor homolog. Transgenic analysis of site-directed mutants of the PRR suggests that s36 expression is regulated by the concerted action of multiple positive activators. Several cis-acting transcriptional elements have been identified: some appear to function in a quantitative manner, while others either are essential or appear to regulate expression in particular spatial domains. The approximate locations of these regulatory elements have been defined; some map within sequences that are strongly conserved in widely divergent dipteran species. In fact, the PRR analog of the medfly Ceratitis capitata Ccs36 gene directs expression in a manner similar to the D. melanogaster s36 PRR. We propose a model for transcriptional regulation of s36 based on the prechoriogenic polarization of the follicular epithelium that surrounds the developing egg chamber.

Purification and primary structure of ceratotoxin A and B, two antibacterial peptides from the female reproductive accessory glands of the medfly Ceratitis capitata (insecta:Diptera)

In the present article we report the purification and the amino acid sequence of two antibacterial peptides present in the secretion of the female reproductive accessory glands of the dipteran insect Ceratitis capitata. Both peptides consist of 29 amino acids residues, and heat stable, strongly basic and differ from each other for the substitution of two amino acids. Their primary sequence and predicted secondary structure are related to other families of peptides known to have lytic and/or antibacterial activity. We propose the name ceratotoxins (from Ceratitis) for these antibacterial peptides.

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.

A biochemical genetic study of alcohol dehydrogenase isozymes of the medfly, Ceratitis capitata Wied.

A concerted effort is under way to analyze, at the genetic, biochemical, and molecular level, the Adh gene system in the medfly Ceratitis capitata, an important agricultural pest. The isoelectric focusing (IEF) pattern of alcohol dehydrogenase (ADH) of the medfly demonstrates the presence of two well-differentiated, genetically independent dimeric proteins, called ADH-1 and ADH-2. These proteins do not exhibit interlocus heterodimeric isozymes, and the genes are not controlled coordinately during development, Adh1 and Adh2 being expressed mainly in muscle or in fat body and ovary, respectively. From the intensity of the IEF isozyme patterns, primary alcohols are judged to be better substrates than secondary alcohols, in contrast with Drosophila melanogaster ADH, and ethanol is probably the most efficient substrate for both sets of isozymes. The isoelectric points of ADH-1 (pI = 5.4) and ADH-2 (pI = 8.6) are different from D. melanogaster ADH (pI = 7.6), but the medfly ADH-1 has a native molecular weight (approx. 58 kD) close to that of D. melanogaster. A population survey of samples both from laboratory strains and from wild geographically different populations showed that the Adh1 locus is more polymorphic than Adh2. The most variable populations are from Africa, the supposed source area of the species. Further, a case of selection at the Adh1 locus under laboratory conditions is reported. The hypothesis of Adh gene duplication and the degree of similarity between medfly and Drosophila ADH are also discussed.

Evidence for a genetic duplication involving alcohol dehydrogenase genes in Ceratitis capitata.

An Adh duplication is described in the medfly Ceratitis capitata. Evidence is presented for two separate Adh1 and Adh2 structural loci mapping at a distance of 0.49 recombination unit from each other. By deletion mapping the Adh region has been cytologically located near the free end of the left arm of the second chromosome within an area between 2C;3A segments of the polytene chromosome. The genetic analysis of the region around Adh has identified seven neighboring genes (Acon1, Mpi, Est6, Aox, Xdh, Mdh2, Lsp1) which identify the linkage group D. The orientation of loci with regard to the centromere sets the origin of the map of the left arm of the second chromosome close to the two Adh loci.