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Development of a diagnostic DNA probe for the fruit flies Ceratitis capitata and Ceratitis rosa (Diptera: Tephritidae) using amplified fragment-length polymorphism.

The AFLP technique (amplified fragment-length polymorphism) was employed to identify and isolate species specific markers in tephritids. We have found that the technique has good potential for this purpose, with the only difficult part being the reamplification of AFLP fragments from silver stained gels. Cloning of putative species-specific markers and genomic dot blot hybridizations resulted in the development of diagnostic probes for tephritid identification. A repetitive DNA sequence from the genome of Ceratitis capitata (Wiedemann) was isolated. This sequence rapidly and reliably identified C. capitata and C. rosa Karsch in a collection of closely related and outgroup species tested in this study. Although this probe has been developed for C. capitata and C. rosa, the proposed methodology can be applied to any group of organisms.

Animals↗

The ceratotoxin gene family in the medfly Ceratitis capitata and the Natal fruit fly Ceratitis rosa (Diptera: Tephritidae).

Ceratotoxins (Ctxs) are a family of antibacterial sex-specific peptides expressed in the female reproductive accessory glands of the Mediterranean fruit fly Ceratitis capitata. As a first step in the study of molecular evolution of Ctx genes in Ceratitis, partial genomic sequences encoding four distinct Ctx precursors have been determined. In addition, anti-Escherichia coli activity very similar to that of the accessory gland secretion from C. capitata was found in the accessory gland secretion from Ceratitis (Pterandrus) rosa. SDS-PAGE analysis of the female reproductive accessory glands from C. rosa showed a band with a molecular mass (3 kDa) compatible with that of Ctx peptides, also slightly reacting with an anti-Ctx serum. Four nucleotide sequences encoding Ctx-like precursors in C. rosa were determined. Sequence and phylogenetic analyses show that Ctxs from C. rosa fall into different groups as C. capitata Ctxs. Our results suggest that the evolution of the ceratotoxin gene family might be viewed as a combination of duplication events that occurred prior to and following the split between C. capitata and C. rosa. Genomic hybridization demonstrated the presence of multiple Ctx-like sequences in C. rosa, but low-stringency Southern blot analyses failed to recover members of this gene family in other tephritid flies.

Biological Evolution↗

cAMP-dependent protein kinases from the insect Ceratitis capitata.

Among the components of the two cyclic nucleotide system of Ceratitis capitata pharate adults, two cAMP-dependent protein kinase activities have been identified and purified through a sequence of chromatographic procedures. The properties of both protein kinases, A-1 and A-2, were studied and characterized in comparison with those of other sources. Protein kinase A-2 from Ceratitis capitata corresponds to type I from mammals mainly concerning about the dissociating effect of histones. Protein kinase A-2 exhibited a molecular weight of 39,000 in the presence of cAMP, whereas in the absence of the cyclic nucleotide two components of 80,000 and 159,000 were present and attributed to the forms RC and R2C2, respectively. Protein kinase activities A-1 and A-2 were markedly inhibited by increasing ionic strength whereas the activity (-cAMP/+cAMP) ratio for protein kinase A-2 increased versus NaCl concentration. Histones H1 and H2B were the best substrates for both A-1 and A-2 activities; the high mobility group of insect proteins (HMG) were also notably phosphorylated by A-2 preparation. Among the cyclic nucleotides assayed for the protein kinase activity A-2, cAMP induced a high activation at the lowest concentrations although high cAMP concentrations decreased the protein kinase activity, possibly through binding to the catalytic site. The protein kinase A-2 preparations exhibited a complex kinetics due to the presence of two forms with different affinity for ATP; these forms may be related to the aggregation properties of the enzyme.

Animals↗

Establishment of continuously in vitro growing cell lines of med-fly (Ceratitis capitata wied.).

The Mediterranean fruit fly, Ceratitis capitata Wiedemann, represents an extremely harmful insect pest of considerable agricultural and economic interest. As part of the study of this species, two diploid cell lines (2n = 12) of the Med-fly were established in vitro on two different culture media. The two lines were obtained from embryonic eggs 24 (+/- 1) hours old and were called CEC CC 128 and CEC CC 130. Detailed descriptions are given of the techniques used for the preparation of the primary cultures, their development and characterization, the choice and composition of the culture media and the intrinsic characteristics of the lines obtained. Indications of other elements are given, such as different cells, non cellular vesicles and glands containing symbiotic bacteria, observable in the first phase of development of the culture, before obtaining continuously growing cell lines. The doubling time of the population for both cell lines were estimated at 22 hours and the interval between successive sub-cultures at 7 days.

Animals↗

Genetic variability and gene flow in geographical populations of Ceratitis capitata (Wied.) (medfly).

Two African populations of Ceratitis capitata (Kenya and Réunion Isl.) and two Mediterranean ones (Sardinia and Procida Isl.) have been studied for genetic variability at 25 loci by electrophoresis. Wright's FST, Slatkin's Nm* gene flow estimator, Nei's distance (D) together with measures of variability such as H, P, A have been used to compare the population from Kenya with the other three. Parameters using gene frequencies (FST, D, Nm*) indicate the presence of substantial geographic heterogeneity, largely attributable to genetic drift and correlated with dispersion of the medfly from its source area (Subsaharan Africa) to the periphery. The Kenyan population has high genetic variability (assessed by H, P and A), as might be expected given its native status. Significant gene flow estimates between Kenya and the derived Mediterranean populations supports the hypothesis of recent colonization. Part of the geographic heterogeneity is related to the presence of fixed alleles in the more differentiated Réunion population although it maintains the genetic attributes of the ancestral population. Selection or other forces may have played an important role in the differentiation of this population.

Alleles↗

Conformational stability of fructose-1, 6-biphosphate aldolase from Ceratitis capitata.

Fructose 1, 6-biphosphate aldolase from Ceratitis capitata is a tetramer of identical subunits with 34% alpha-helix, 22% beta structure and 44% of aperiodic order. Increase of urea concentration up to 4.0 M results in non-cooperative reversible dissociation of the enzyme. Sodium dodecylsulphate 0.06% (w/v) dissociates the tetramer cooperatively with retention of the helical content. Thermal denaturation was a non-reversible cooperative process with a midpoint for the transition at 55 degrees. Cysteine residues are involved in this process and 2-mercaptoethanol preserves partially the enzyme activity. The acidic dissociation of the enzyme is a non-reversible process in contrast to the reversible basic dissociation. Increase of ionic strength results in a more ordered secondary structure for the monomer after acidic dissociation.

Animals↗

Presence of antibacterial peptides on the laid egg chorion of the medfly Ceratitis capitata.

Female reproductive accessory glands of the medfly Ceratitis capitata produce a secretion with antibacterial activity mainly ascribed to ceratotoxin peptides. To study whether the secretion from the accessory glands of the female protects the eggs and early larva from microbes, we examined whether ceratotoxins and other accessory gland components could be found on the egg surface. This was found to be the case; a water-soluble material with the same protein and antibacterial pattern as that of the accessory gland secretion was recovered from the laid egg surface and was observed as electrondense, clustered droplets over the outer exochorion. Such material showed the same electrophoretic pattern in both mated and virgin females. These findings indicate that the accessory gland secretion is spread, at oviposition, onto the eggs producing an antibacterial coating, irrespective of fertilization. This is the first report of antimicrobial components recovered from a material layered on insect laid eggs.

Animals↗

Fatty acid synthetase complex from the insect Ceratitis capitata.

Fatty acid synthesis capacity of the insect Ceratitis capitata has been investigated in vitro from [1-14C]acetyl-CoA using homogenates at different stages of development. A maximum activity was observed after 5--6 days of larval development. But homogenates of the pharate adult insect did not show synthetic capacity of fatty acids. Fatty acid synthetase complex has been isolated from the particle-free supernatant fraction of homogenates from the 6-day C. capitata larvae. The enzyme complex was purified 182-fold with respect to the protein contained in the crude extract. The complex was homogeneous when analysed by gel filtration and by polyacrylamide-gel electrophoresis. The molecular weight was 5.2X10(5). The enzyme was dissociated into half-molecular subunits. Amino acid analysis, general properties, stability and kinetic constants (V and Km) for the substrates are reported. The fatty acid synthetase complex from the insect contains 42+/-1-SH residues and one phosphopatetheine moiety per 5.2X10(5). Activity was dependent on the presence of NADPH; FMN strongly inhibited the enzyme activity promoted by NADPH. The enzyme complex synthesized a range of fatty acid (10:0--18:0), palmitate being the predominant end product. The proportions of fatty acids synthesized varied with substrate concentrations. Fatty acids released from the complex were almost completely in the free form.

Amino Acids↗

Cell death may regulate visual functionality in the retina of adults of the dipteran Ceratitis capitata.

The white eye mutation in the medfly Ceratitis capitata, like the homologous mutation in Drosophila melanogaster, was shown to impair visual function. Light and electron microscopy, combined with the DNA-end labelling histochemistry (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) technique), were used to investigate whether programmed cell death may contribute to the morpho-functional differences between the retina of adults from the white eye and wild type strains. Several photoreceptor nuclei in mature white eye flies appeared smaller and showed intensely Toluidine Blue-stained chromatin masses. At the ultrastructural level, they showed different stages of degeneration, resembling apoptotic figures. Positive TUNEL labelling in the white eye retina indicates that apoptosis may be a candidate mechanism for retinal cell degeneration in adult flies, where visual functionality is altered, to achieve the proper cell number. Apoptosis also appears to occur in the wild type retina in early adult life during normal tissue development.

Aging↗

Biochemical and genetic studies on alkaline phosphatase of Ceratitis capitata.

Two forms of alkaline phosphatase exist in the integument of the "white pupae" (wp) and dark pupae (dp) mutant strains of Ceratitis capitata, during transition from larvae to pupae. They were separated by DEAE-cellulose chromatography. Both isoenzymes have a molecular weight of approximately 180,000 and two pH optima, at 9.4 and at 11.0. The isoenzymes of the "dark pupae" mutant catalyze the hydrolysis of phosphotyrosine and beta-glycerophosphate but not phosphoserine, phosphothreonine, ATP, and AMP. In contrast, the isoenzymes of the white pupae mutant hydrolyze all the substrates tested. The ALPase 1 of the dark pupae mutant was inhibited by L-tyrosine, but L-phenylalanine had no effect on either isoenzyme. The effects of divalent cations, EDTA, temperature, urea, and 2-mercaptoethanol were also investigated. Electrophoretic analysis did not reveal any variants of the larval and pupal isoenzymes, but ALPase A, an adult stage-specific isoenzyme, was found to be polymorphic. The electrophoretic variants were shown to be controlled by three codominant alleles located on the third chromosome of Ceratitis capitata. Since we found no hybrid enzyme, we conclude that ALPase A is monomeric.

Alkaline Phosphatase↗

cDNA cloning of L-dopa decarboxylase from the eclosion stage of the insect Ceratitis capitata. Evolutionary relationship to other species decarboxylases.

The cDNA encoding the L-dopa decarboxylase (ddc) from the eclosion stage of the insect Ceratitis capitata was isolated by PCR and a molecular cloning strategy. The isolated cDNA clone encoded a protein of 431 amino acids with a calculated molecular weight of 47,843 Da. Northern blot analysis of poly(A)+ RNA showed an approximately 2 kb transcript. The deduced protein sequence shares a high percentage of homology with Ddc protein sequences of other species. Furthermore, the molecular weight of the deduced protein agreed well with that of the purified Ddc from the same insect. Data base search revealed significant and extensive sequence similarities among prokaryotic and eukaryotic PLP-dependent decarboxylases including Ceratitis capitata and bacterial histidine decarboxylase (HDC), strongly suggesting an ancient and common origin for all PLP-dependent decarboxylases.

Amino Acid Sequence↗

Genetic differentiation, gene flow and the origin of infestations of the medfly, Ceratitis capitata.

The genetic structure of natural populations of the economically important dipteran species Ceratitis capitata was analysed using both biochemical and molecular markers. This revealed considerable genetic variation in populations from different geographic regions. The nature of this variation suggests that the evolutionary history of the species involved the spread of individuals from the ancestral African populations through Europe and, more recently, to Latin America, Hawaii and Australia. The observed variation can be explained by various evolutionary forces acting differentially in the different geographic areas, including genetic drift, bottleneck effects, selection and gene flow. The analysis of the intrinsic variability of the medfly's genome and the genetic relationships among populations of this pest is a prerequisite for any control programme.

Africa↗

Genetic stability of sexing strains based on the locus sw of Ceratitis capitata.

This report deals with the process of improving the stability of medfly, Ceratitis capitata, genetic sexing strains (GSS) based on the sw mutation on chromosome 2. This gene affects the rate of development as well as the eye colour and iridescence. The improved sexing strains were produced by mapping sw with deletions and then inducing and screening for new translocations with breakpoints close to the marker. The stability was assessed in large populations over many generations. Twenty-two new Y-2 translocations were identified and polytene chromosome analysis was performed to locate breakpoints. The translocation strains were ranked according to the distance of their breakpoints from sw. The map position of sw is region 20D on 2R. As data on the stability of the 22 strains accumulated, Cast 191 was shown to be the most promising as no recombination between sw and the male sex was found. After rearing the strain for 22 generations under semi-mass rearing conditions, with a population size of 15,000 adults and scoring 1000 flies per generation, only one such event was detected (estimated frequency = 3.1 x 10(-6)). Further tests are being carried out with this strain to assess its suitability as a genetic sexing strain for medfly Sterile insect technique (SIT).

Animals↗

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.

Alcohol Dehydrogenase↗

Mitochondrial DNA restriction map for the Mediterranean fruit fly, Ceratitis capitata.

Molecular genetic research on the Mediterranean fruit fly, Ceratitis capitata, will provide tools to permit determination of source populations for new pest infestations. Restriction fragment length polymorphism (RFLP) of mitochondrial DNA provides some interpopulation discrimination. A restriction map, including the informative variable EcoRV and XbaI restriction sites, is constructed for the Mediterranean fruit fly, and several restriction sites are associated with specific gene regions based on polymerase chain reaction-RFLP and sequence analyses. A partial sequence of the mitochondrial 16S ribosomal RNA gene is reported.

Animals↗

In vivo biosynthesis of a stage-specific cuticle glycoprotein during early metamorphosis of the medfly Ceratitis capitata.

Cuticle proteins of an insect pest, the Medfly Ceratitis capitata, were resolved in polyacrylamide gels and partially characterized. The pupal cuticle was found to be different from cuticles of other insects since more than 80% w/w of the protein is a single mannose-containing polypeptide (PCG-100). The temporally-regulated in vivo biosynthesis and deposition of cuticle proteins was studied by microinjection of [35S]methionine followed by hand dissection of pupal cuticles. The major pupal glycoprotein, PCG-100, is cuticle- and stage-specific and was the earliest to be labeled and deposited. Its synthesis was maximal at around 46 hours after pupariation and then it decreased. The deposited PCG-100 and other minor pupal cuticle proteins become non-extractable at the end of the instar (7 days after pupariation) probably by sclerotization phenomena. These results provide insight into the temporal control of gene expression programs involved in cuticle deposition during medfly metamorphosis.

Animals↗