Highly efficient genome editing by homology-directed repair using Cas9 protein in Ceratitis capitata.

The Mediterranean fruit fly Ceratitis capitata is a highly polyphagous and invasive insect pest, causing enormous economic damage in horticultural systems. A successful and environment-friendly control strategy is the sterile insect technique (SIT) that reduces pest populations through infertile matings with mass-released, sterilized insects. However, the SIT is not readily applicable to each pest species. While transgenic approaches hold great promise to improve critical aspects of the SIT to transfer it to new species, they are suspect to strict or even prohibitive legislation regarding the release of genetically modified (GM) organisms. In contrast, specific mutations created via CRISPR-Cas genome editing are not regulated as GM in the US, and might thus allow creating optimal strains for SIT. Here, we describe highly efficient homology-directed repair genome editing in C. capitata by injecting pre-assembled CRISPR-Cas9 ribonucleoprotein complexes using different guide RNAs and a short single-stranded oligodeoxynucleotide donor to convert an enhanced green fluorescent protein in C. capitata into a blue fluorescent protein. Six out of seven fertile and individually backcrossed G0 individuals generated 57-90% knock-in rate within their total offspring and 70-96% knock-in rate within their phenotypically mutant offspring. Based on the achieved efficiency, this approach could also be used to introduce mutations which do not produce a screenable phenotype and identify positive mutants with a reasonable workload. Furthermore, CRISPR-Cas HDR would allow to recreate mutations formerly identified in classical mutagenesis screens and to transfer them to related species to establish new (SIT-like) pest control systems. Considering the potential that CRISPR-induced alterations in organisms could be classified as non-GM in additional countries, such new strains could potentially be used for pest control applications without the need to struggle with GMO directives.

Larval Diet Affects Male Pheromone Blend in a Laboratory Strain of the Medfly, Ceratitis capitata (Diptera: Tephritidae).

The Mediterranean fruit fly (medfly) Ceratitis capitata is a polyphagous pest of fruits and crops with a worldwide distribution. Its ability to use different larval hosts may have multiple effects, including impacts on adult reproductive biology. The male sex pheromone, which plays a key role in attracting both other males to lekking arenas and females for mating, is a mixture of chemical compounds including esters, acids, alkanes and terpenes known to differ between laboratory strains and wild-type populations. The relationship between larval diet and adult pheromone composition remains unexplored. Here, we investigated the effect of larval diet, including laboratory media and fresh fruits, on the composition of the male pheromone mixture. Using Headspace Solid Phase Microextraction we collected the pheromone emitted by males reared as larvae on different substrates and found both qualitative and quantitative differences. A number of alkanes appeared to be typical of the pheromone of males reared on wheat bran-based larval medium, and these may be cuticular hydrocarbons involved in chemical communication. We also detected differences in pheromone composition related to adult male age, suggesting that variations in hormonal levels and/or adult diet could also play a role in determining the chemical profile emitted. Our findings highlight the plasticity of dietary responses of C. capitata, which may be important in determining the interactions of this pest with the environment and with conspecifics. These results also have applied relevance to increase the mating competitiveness of mass-reared C. capitata used in Sterile Insect Technique programs.

Apoptosis in medfly hemocytes is regulated during pupariation through FAK, Src, ERK, PI-3K p85a, and Akt survival signaling.

Focal adhesion kinase (FAK) and its downstream signaling targets are implicated in the process of apoptosis induced by external stimuli, in several mammalian systems. In this report, we demonstrate, that medfly (Ceratitis capitata) hemocytes do undergo apoptosis during larval development. In particular, we show using Western blot, ELISA and flow cytometry analysis, that FAK expression silencing in transfected by FAK double-stranded RNA (dsRNA) hemocytes, enhances twofold hemocyte apoptosis, by signaling through Src, MEK/ERK, and PI-3K/Akt signaling pathways. FAK expression silencing, in response to FAK dsRNA treatment, blocks partially the phosphorylation of its downstream targets. Pre-incubation of hemocytes, with specific inhibitors of FAK downstream signaling molecules, demonstrated that all these inhibitors reduced hemocyte viability and enhanced the magnitude of apoptosis about threefold. This data suggest that these pathways contribute to hemocyte survival and/or death during development. The expression and phosphorylation of FAK, Src, PI-3K p85a, Akt, and ERK signaling molecules appear to be dependent upon developmental stages. The expression and phosphorylation of the above signaling molecules, in annexin-positive and annexin-negative hemocytes is also distinct. The maximum expression and phosphorylation of FAK, Src, PI-3K p85a, Akt, and ERK appeared in annexin-positive hemocytes, in both early and late apoptotic hemocytes. The novel aspect of this report is based on the fact that hemocytes attempt to suppress apoptosis, by increasing the expression/phosphorylation of FAK and, hence its downstream targets signaling molecules Src, ERK, PI-3K p85a, and Akt. Evidently, the basic survival pathways among insects and mammals appear to remain unchanged, during evolution.

Identification of genes expressed in the accessory glands of male Mediterranean Fruit Flies (Ceratitis capitata).

Genes expressed in the male reproductive system exhibit rapid evolutionary change and encode products that underlie striking, fitness-related phenotypes. Despite this, they have been characterised in detail in relatively few species. We report here an initial characterisation of the genes expressed in the male reproductive accessory glands of the Mediterranean Fruit Fly (Ceratitis capitata). We describe 13 independent expressed sequence tags (ESTs), of which 9 showed significant homology to known sequences and of which 4 represented novel sequences. The evidence suggests that our transcripts are not homologues of genes encoding known accessory gland proteins (Acps) in Drosophila melanogaster, but that they do encode proteins that fall into known functional categories for Acps (e.g. proteases, lipases, cysteine-rich secretory proteins [CRISPs]). Our results are consistent with the finding that among Acps there is considerable evolutionary lability at the sequence level, but evolutionary constraint at the functional level. The results highlight the extraordinary diversity of male reproductive genes.

Cloning, characterization, and developmental expression of the ribosomal protein S21 gene of the Mediterranean fruit fly Ceratitis capitata.

Ribosomal protein S21 (RpS21) belongs to a small group of ribosomal or ribosome-associated proteins. Mutations in the RpS21 gene cause dominant Minute and recessive lethal tumorous phenotypes in Drosophila melanogaster. Studies in several organisms suggest that RpS21 is involved in the regulation of protein synthesis and cell growth. In this report, we used an RT-PCR fragment of D. melanogaster RpS21 mRNA to clone a RpS21 cDNA from the Mediterranean fruit fly, Ceratitis capitata. The isolated cDNA contained both 5' and 3' untranslated regions, and encoded a polypeptide of 83 amino acids with a predicted molecular mass of 9.1 kDa. The deduced protein sequence showed 91% amino acid identity to D. melanogaster RpS21 and strong homology with all known ribosomal S21 proteins. DNA blot hybridization indicated the existence of a single RpS21 gene in the Ceratitis capitata genome. Analysis of the 5' untranslated region revealed the occurrence of a major oligopyrimidine tract at the 5' end, which characterizes most mRNAs undergoing a growth-dependent translational control. Study of the mRNA patterns during development suggested that the expression of Ceratitis RpS21 is temporally regulated at the level of transcription.

Biosynthetic maturation of the corpus allatum of the female adult medfly, Ceratitis capitata, and its putative control.

The major juvenile hormone (JH) homolog synthesized in vitro by the adult female Medfly (Ceratitis capitata) corpus allatum (CA) is JHB(3), with JH-III the minor homolog. Methyl-incorporation in vitro in post-eclosion virgin females is age-dependent. Basal activity occurs during the first four days post-eclosion and increases significantly thereafter, peaking at five days. Biosynthetic maturation of the mated female CA is delayed by one day and reduced considerably. The delayed response may be due to direct cerebral or neural inhibition. Synthetic Drosophila melanogaster sex peptide depresses JH biosynthesis by the Medfly female CA in vitro. Male-derived accessory gland peptides of the Medfly are transferred to the female during mating and a Medfly SP-analog may be responsible for down-regulation of JH synthesis by the CA in mated Medfly females. Mevinolin, an inhibitor of the mevalonate pathway, significantly reduces the biosynthesis of JHB(3), while farnesoic acid, a proximate precursor of JHIII, significantly stimulates the biosynthesis of both JHB(3) and JHIII in vitro.