Medfly Genome Research Articles

Effects of lipopolysaccharide and juvenile hormone III treatments on cell growth and gene expression in the Ceratitis capitata (Diptera: Tephritidae) CCE/CC128 cell line.

The Mediterranean fruit fly Ceratitis capitata is one of the most important insect pest species in the world. It has a high colonization capacity and population variety, giving it considerable genetic diversity. Strategies for its control have included the sterile insect technique and insect growth regulators. Many studies have analyzed the medfly transcriptome, and along with the medfly genome sequence, the sequences of multiple genes related to sex determination, mating, development, pheromone detection, immunity, or stress have been identified. In this study, the medfly CCE/CC128 cell line was used to assess cell growth variation and changes in the expression of genes covering different functions, after lipopolysaccharide (LPS) and juvenile hormone III (JHIII) treatments. No significant effects on cell growth and gene expression were observed in the cells treated with LPS. In the cells treated with JHIII, the results showed significant effects on cell growth, and an overexpression was found of the Shade gene, one of the Halloween gene members of the cytochrome p450 family, which is involved in development and the synthesis of 20-hydroxyecdysone. This study shows preliminary results on the insect cell line in combination with whole-genome sequencing, which can facilitate studies regarding growth, toxicity, immunity, and transcriptome regulations as a response to different compounds and environmental alterations.

Delivery of Nucleic Acids through Embryo Microinjection in the Worldwide Agricultural Pest Insect, Ceratitis capitata.

The Mediterranean fruit fly (medfly) Ceratitis capitata (Wiedemann) (Diptera: Tephritidae) is a pest species with extremely high agricultural relevance. This is due to its reproductive behavior: females damage the external surface of fruits and vegetables when they lay eggs and the hatched larvae feed on their pulp. Wild C. capitata populations are traditionally controlled through insecticide spraying and/or eco-friendly approaches, the most successful being the Sterile Insect Technique (SIT). The SIT relies on mass-rearing, radiation-based sterilization and field release of males that retain their capacity to mate but are not able to generate fertile progeny. The advent and the subsequent rapid development of biotechnological tools, together with the availability of the medfly genome sequence, has greatly boosted our understanding of the biology of this species. This favored the proliferation of new strategies for genome manipulation, which can be applied to population control. In this context, embryo microinjection plays a dual role in expanding the toolbox for medfly control. The ability to interfere with the function of genes that regulate key biological processes, indeed, expands our understanding of the molecular machinery underlying medfly invasiveness. Furthermore, the ability to achieve germ-line transformation facilitates the production of multiple transgenic strains that can be tested for future field applications in novel SIT settings. Indeed, genetic manipulation can be used to confer desirable traits that can, for example, be used to monitor sterile male performance in the field, or that can result in early life-stage lethality. Here we describe a method to microinject nucleic acids into medfly embryos to achieve these two main goals.

Globalization and fruitfly invasion and expansion: the medfly paradigm

The phytophagous insects of the Tephritidae family commonly referred to as "true fruit flies" offer different case histories of successful invasions. Mankind has played an important role in altering the distributions of some of the more polyphagous and oligophagous species. However, the question arises why only a few species have become major invaders. The understanding of traits underlying adaptation in different environments is a major topic in invasion biology. Being generalists or specialists, along the K-r gradient of the growth curve, make a difference in term of food resources exploitation and interspecies competition and displacement. The species of the genus Ceratitis are good examples of r-strategists. The genetic and biological data of the most notorious Ceratitis species, the Mediterranean fruit fly Ceratitis capitata (medfly), are reviewed to investigate the traits and behaviours that make the medfly an important invader. It can be learnt from medfly, that invasions in a modern global trade network tend to be due to multiple introductions. This fact allows a maintenance or enhancement of genetic variability in the adventive populations, which in turn increases their potential invasiveness. Our current knowledge of the medfly genome opens the way for future studies on functional genomics.

Cchobo, a hobo-related sequence in Ceratitis capitata

A hobo-related sequence, Cchobo, with high similarity to the Drosophila melanogaster HFL1 and hobo108 elements was isolated from the medfly. Thirteen PCR-derived clones, which share 97.9-100% DNA identity, were sequenced, seven of which do not show frame-shift or stop codon mutations in their conceptual translations. The consensus sequence has 99.7% DNA identity with the D. melanogaster hobo element HFLI. In a phylogenetic analysis with other hobo-related elements, Cchobo clusters with the HFL1 and hobo108 elements from D. melanogaster and hobo-related elements from D. simulans, D. mauritiana and Mamestra brassicae. These elements may have undergone horizontal transfer in the recent past. The genomic distribution of Cchobo was studied by FISH to mitotic and polytene chromosomes, which revealed that Cchobo is distributed within both the heterochromatin and euchromatin. Intra- and interstrain polymorphisms were detected both at euchromatic and heterochromatic sites. These findings suggest that active copies of the element may be present in the medfly genome.

Medfly transposable elements: diversity, evolution, genomic impact and possible applications

The medfly genome has been shown to contain a rich assortment of transposable elements from the mariner, Tc1, hAT and gypsy/Ty3 families. These elements display different levels of diversity, abundance and distribution in the genome. The presence of actively transposing elements in the medfly genome is revealed by hybrid dysgenesis phenomena, insertion site polymorphisms and other genetic instabilities. The medfly has been a target of transformation studies involving the exogenous elements Minos, Hermes and piggyBac from three families. The presence of active endogenous homologous elements can have important implications for the stability of such transgenic lines. The potential applications of endogenous elements for medfly population analysis and control are discussed.

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.

Evolution of different subfamilies of mariner elements within the medfly genome inferred from abundance and chromosomal distribution.

The abundance and distribution pattern of eight mariner elements from three different subfamilies in the genome of the medfly Ceratitis capitata were determined. The copy numbers, as determined by slot-blot analysis, were very different for these elements. Their abundance did not change significantly within the native, the ancient or the newly derived populations, indicating that the rapid colonization process of the medfly had not affected the copy number of mariner elements. The distribution of the mariner elements was analyzed using fluorescent in situ hybridization (FISH) with charge-coupled device (CCD) camera analysis. The pattern of distribution in euchromatin and heterochromatin varied greatly and was distinctive and specific for each element. The implications of these findings are discussed and it is concluded that they generally support the hypothesis of a transposition/selection model in which the abundance and distribution patterns of these elements are regulated primarily by selection against deleterious effects due to meiotic ectopic recombination, while genetic drift would have played a minor role.

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-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.

Gene transfer into the medfly, Ceratitis capitata, with a Drosophila hydei transposable element.

Exogenous functional DNA was introduced into the germline chromosomes of the Mediterranean fruit fly (medfly) Ceratitis capitata with a germline transformation system based on the transposable element Minos from Drosophila hydei. Transformants were identified as phenotypic revertants of a white-eyed mutation carried by the recipient strain. Clusters of transformants were detected among the progeny of 390 individuals screened for germline transformation. Five independent and phenotypically active integration events were identified, in each of which a single copy of the transposon was inserted into a different site of the medfly genome. Molecular analysis indicates that they represent transposase-mediated insertions of the transposon into medfly chromosomes.

Genetic variation between strains of the Mediterranean fruit fly, Ceratitis capitata, detected by DNA fingerprinting.

DNA fingerprinting has been used to detect genetic variation in the Mediterranean fruit fly, Ceratitis capitata. Three different probes have been identified that can be used to detect DNA restriction fragment length polymorphisms between strains of this species. The strains used in this study differ only in terms of their geographic origin or genetic background. One of the probes used is the bacteriophage vector M13, and the other two are repetitive sequences derived from the medfly genome based on a weak homology to M13. Within a strain, each probe produces a consistent restriction fragment profile that is not affected by the method or timing of DNA extraction. Between strains, when M13 is used as a probe, an average of 10% of the observable bands are polymorphic. Use of the medfly genomic sequences as a probe increases the proportion of polymorphic bands between strains up to 30%. The fact that genetic differences between even such closely related strains can be reliably detected by this method holds great promise for studies of insect pests including the ability to monitor the movements of pest species, determining the extent of genetic variation in pest populations, and in making identifications from otherwise unidentifiable material.

The chorion genes of the medfly, Ceratitis capitata

We have isolated three new chorion cDNA clones from a Ceratitis capitata ovarian library. Their isolation was accomplished by differential screening of the library using as probes 32P-labeled poly(A)+ mRNAs obtained from hand-staged medfly choriogenic versus prechoriogenic follicles. RNA blot hybridization analysis revealed that the genes corresponding to these clones have unique temporal profiles of mRNA accumulation, restricted to specific choriogenic stages. In addition, in vitro translation products encoded by these cDNAs approximately comigrated with polypeptides synthesized de novo in culture by choriogenic follicles. All three genes are located in regions of the medfly genome that are specifically amplified in female ovaries. DNA sequence analysis has revealed that one of these clones is derived from a homolog of the Drosophila melanogaster s38 chorion gene. It appears that, although D. melanogaster and C. capitata are separated by at least 120 million years of evolution, the mechanisms by which chorion genes are expressed and regulated during development have been well maintained. We suggest that the regulatory elements controlling the expression of sex-specific (e.g., chorion) genes may be isolated and used to construct transgenic medfly strains from which females could be eliminated by negative selection; such strains could be used as part of an effort to control this agricultural pest.