Biology subjects

Meccariello, A.

Publications and source records attributed to Meccariello, A..

9 recordsLinked to original sources

bioRxiv2024

A self-limiting Sterile Insect Technique alternative for Ceratitis capitata

Genetic biocontrol systems have broad applications in population control of insects implicated in both disease spread and food security. In this study we establish and characterise a novel split-CRISPR/Cas9 system we term Sex Conversion Induced by CRISPR (SCIC) in Ceratitis capitata (the Mediterranean fruit fly), a major agricultural pest with a global distribution. Using the white eye gene for toolkit selection, we achieved up to 100% CRISPR/Cas9 efficiency, displaying the feasibility of C. capitata split-CRISPR/Cas9 systems using constitutive promoters. We then induce sex-conversion by targeting the transformer gene in a SCIC approach aimed for SIT-mediated releases upon radiation-based sterilisation. Knock-out of transformer induced partial to full female-to-male sex-conversion, with remaining individuals all being intersex and sterile. SCIC population modelling shows superior performance to traditional population control strategies, allowing for faster population elimination with fewer released sterile males. Our results build the foundation for further genetic pest control methods of C. capitata and related tephritid agricultural pests. Significance statementAgricultural industry faces increasing threat from a multitude of pests including the domineering tephritid fruit flies. Genetic engineering of these pests has been recently tested to develop more efficient and affordable population control strategies. Here, we develop a new approach to improve existing population control measures by testing it in one of the most famous and dangerous tephritids, the Mediterranean fruit fly. Through optimisation, we achieved desired outcomes: female fly absence achieved via semi and full female-to-male sex conversion by CRISPR-mediated genome editing through gene mutations. For the first time in this insect, we used a split, and thus inducible, approach for such genome editing. Our work holds the potential to significantly improve tephritid population control strategies.

genetics↗

bioRxiv2023

Next-generation genetic sexing strain establishment in the agricultural pest Ceratitis capitata

Tephritid fruit fly pests pose an increasing threat to the agricultural industry due to their global dispersion and a highly invasive nature. Here we showcase the feasibility of an early-detection SEPARATOR sex sorting approach through using the non-model Tephritid pest, Ceratitis capitata. This system relies on female-only fluorescent marker expression, accomplished through the use of a sex-specific intron of the highly-conserved transformer gene from C. capitata and Anastrepha ludens. The herein characterized strains have 100% desired phenotype outcomes, allowing accurate male-female separation during early development. Overall, we describe an antibiotic and temperature-independent sex-sorting system in C. capitata, which, moving forward, may be implemented in other non-model Tephritid pest species. This strategy can facilitate the establishment of genetic sexing systems with endogenous elements exclusively, which, on a wider scale, can improve pest population control strategies like sterile insect technique.

bioengineering↗

bioRxiv2023

Gene drive and genetic sex conversion in the global agricultural pest Ceratitis capitata

Homing-based gene drives are novel interventions promising the area-wide, species-specific genetic control of harmful insect populations. Here we characterise a first set of gene drives in a tephritid agricultural pest species, the Mediterranean fruit fly Ceratitis capitata (medfly). Our results show that the medfly is highly amenable to homing-based gene drive strategies. By targeting the medfly transformer gene, we also demonstrate two different mechanisms by which CRISPR-Cas9 gene drive can be coupled to sex conversion, whereby genetic females are transformed into fertile and harmless XX males. Given this unique malleability of sex determination, we modelled gene drive interventions that couple sex conversion and female sterility and found that such approaches could be effective and tolerant of resistant allele selection in the target population. Our results open the door for developing gene drive strains for the population suppression of the medfly and related tephritid pests by co-targeting female reproduction and shifting the reproductive sex ratio towards males. They demonstrate the untapped potential for gene drives to tackle agricultural pests in an environmentally friendly and economical way.

synthetic biology↗

bioRxiv2020

Engineered sex distortion in the global agricultural pest Ceratitis capitata

Genetic sex ratio distorters have potential for the area-wide control of harmful insect populations. Endonucleases targeting the X-chromosome and whose activity is restricted to male gametogenesis have recently been pioneered as a means to engineer such traits. Here we enabled endogenous CRISPR/Cas9 and CRISPR/Cas12a activity during spermatogenesis of the Mediterranean fruit fly Ceratitis capitata, a worldwide agricultural pest of extensive economic significance. In the absence of a chromosome-level assembly, we analysed long and short-read genome sequencing data from males and females to identify two clusters of abundant and X-chromosome specific sequence repeats. When targeted by gRNAs in conjunction with Cas9 they yielded a significant and consistent distortion of the sex ratio in independent transgenic strains and a combination of distorters induced a strong bias towards males (~80%). Our results demonstrate the design of sex distorters in a non-model organism and suggest that strains with characteristics suitable for field application could be developed for a range of medically or agriculturally relevant insect species.

synthetic biology↗

bioRxiv2019

A fly model establishes distinct mechanisms for synthetic CRISPR/Cas9 sex distorters

Synthetic sex distorters have recently been developed in the malaria mosquito, relying on endonucleases that target the X-chromosome during spermatogenesis. Although inspired by naturally-occurring traits, it has remained unclear how they function and, given their potential for genetic control, how portable this strategy is across species. We established Drosophila models for two distinct mechanisms for CRISPR/Cas9 sex-ratio distortion - "X-shredding" and "X-meddling" - and dissected their target-site requirements and repair dynamics. X-shredding relies on sufficient meiotic activity of the endonuclease to overpower DNA repair and can operate on a single repeat cluster of non-essential sequences. X-meddling by contrast, i.e. targeting putative haplolethal X-linked genes, induced a bias towards males that is coupled to a loss in reproductive output, although a dominant-negative effect may drive the mechanism of female lethality. Our model system will guide the study and the application of sex distorters to medically or agriculturally important insect target species.

synthetic biology↗

bioRxiv2019

Maleness-on-the-Y (MoY) orchestrates male sex determination in major agricultural fruit fly pests

In insects, rapidly evolving primary sex-determining signals are transduced by a conserved regulatory module producing sex-specific proteins that direct sex determination and sexual differentiation1-4. In the agricultural pest Ceratitis capitata (medfly), a Y-linked maleness factor (M) is thought to repress the autoregulatory splicing of transformer (Cctra), which is required in XX individuals to establish and maintain female sex determination5,6. Despite previous attempts of isolating Y-linked genes using the medfly whole genome, the M factor has remained elusive7. Here, we report the identification of a Y-linked gene, Maleness-on the-Y (MoY), and show that it encodes a small novel protein which is both necessary and sufficient for medfly male sex determination. Transient silencing of MoY in XY individuals leads to the development of fertile females while transient expression of MoY in XX individuals results in fertile males. Notably, a cross between these sex reverted individuals gives rise to both fertile males and females indicating that a functional MoY can be maternally transmitted. In contrast to the diversity of M factors found in dipteran species8-11, we discovered MoY orthologues in seven other Tephritid species spanning [~]111 millions of years of evolution (Mya). We confirmed their male determining function in the olive fly (Bactrocera oleae) and the oriental fruit fly (Bactrocera dorsalis). This unexpected conservation of the primary MoY signal in a large number of important agricultural pests12 will facilitate the development of transferable genetic control strategies in these species, for example sterile male releases or sex-ratio-distorting gene drives.

genetics↗

bioRxiv2017

Molluscs community associated with the brown algae of the genus Cystoseira in the Gulf of Naples (South Tyrrhenian Sea)

The brown macroalgae of the genus Cystoseira are important habitat forming species along the rocky coasts all over the Mediterranean Sea. However, their decline at basin and local scale has been documented in many studies. We have characterized malacofauna associated with Cystoseira amentacea, C. compressa and C. crinita along the coasts of Ischia Island (Gulf of Naples). Samples were collected by snorkeling in the infralittoral belt. The surface within 20 x 20 cm frames was scraped off and collected in three replicates each sites. The diversity and structure of community were described by number of species, the exponential Shannon and the reciprocal Simpsons indexes of diversity. The patterns of diversity at spatial scale were assessed by alpha, beta and gamma diversity. A total of 53 species of molluscs were identified in those associations. Gastropoda were the most species-rich class followed by Bivalvia and Polyplacophora. Bivalves were dominant in terms of number of individuals because of the mussel Mytilus galloprovincialis. The species M. galloprovincialis was the most frequent and top dominant one inhabiting Cystoseira associations along the coasts of Ischia Island (96.6 % of the total abundance). Most of the identified molluscs species belonged to two feeding guilds: micrograzers and filter feeders (29 and 13 species respectively). Only juveniles were found providing the importance of Cystoseira associations as nursery for molluscs recruitment. Differences in composition and structure of molluscs assemblages were found within the three algal associations and seem to correspond both to different morphology and habitat in which these algal species live.

ecology↗

bioRxiv2017

Highly efficient DNA-free gene disruption in the agricultural pest Ceratitis capitata by CRISPR-Cas9 RNPs

The Mediterranean fruitfly Ceratitis capitata (medfly) is an invasive agricultural pest of high economical impact and has become an emerging model for developing new genetic control strategies as alternative to insecticides. Here, we report the successful adaptation of CRISPR-Cas9-based gene disruption in the medfly by injecting in vitro pre-assembled, solubilized Cas9 ribonucleoprotein complexes (RNPs) loaded with gene-specific sgRNAs into early embryos. When targeting the eye pigmentation gene white eye (we), we observed a high rate of somatic mosaicism in surviving G0 adults. Germline transmission of mutated we alleles by G0 animals was on average above 70%, with individual cases achieving a transmission rate of nearly 100%. We further recovered large deletions in the we gene when two sites were simultaneously targeted by two sgRNAs. CRISPR-Cas9 targeting of the Ceratitis ortholog of the Drosophila segmentation paired gene (Ccprd) caused segmental malformations in late embryos and in hatched larvae. Mutant phenotypes correlate with repair by non-homologous end joining (NHEJ) lesions in the two targeted genes. This simple and highly effective Cas9 RNP-based gene editing to introduce mutations in Ceratitis capitata will significantly advance the design and development of new effective strategies for pest control management.

genetics↗

bioRxiv2017

CRISPR-Cas9 targeted disruption of the yellow ortholog in the housefly identifies the brown body locus

The classic brown body (bwb) mutation in the housefly Musca domestica impairs normal melanization of the adult cuticle. In Drosophila melanogaster, a reminiscent pigmentation defect results from mutations in the yellow gene encoding dopachrome conversion enzyme (DCE). Here, we demonstrate that the bwb locus structurally and functionally represents the yellow ortholog of Musca domestica, MdY. In bwb Musca strains, we identified two mutant MdY alleles that contain lesions predicted to result in premature truncation of the MdY open reading frame. We targeted wildtype MdY by CRISPR-Cas9 RNPs and generated new mutant alleles that fail to complement existing MdY alleles, genetically confirming that MdY is the bwb locus. We further found evidence for Cas9-mediated interchromosomal recombination between wildtype and mutant bwb alleles. Our work resolves the molecular identity of the classic bwb mutation in Musca domestica and establishes the feasibility of Cas9-mediated genome editing in the Musca model.

genetics↗