Tephritid fruit fly transgenesis and applications

This chapter focuses on the first transformation experiments for a particular vector system for tephritid species in which it has been tested. Additional experiments are discussed as they relate to new marker systems and vectors developed for particular applications. These include vector stabilization systems, site-specific genomic targeting and the development of tephritid transgenic strains for control applications

CRISPR/Cas-mediated gene editing using purified protein in Drosophila suzukii

The spotted wing drosophila, Drosophila suzukii (Matsumura) (Diptera: Drosophilidae), is an invasive pest of stone fruits and berries currently without effective control management. The sterile insect technique (SIT) is an environmentally friendly and effective pest control method that releases sterile males to mate with wild females resulting in the suppression or eradication of targeted pest populations. New molecular technologies and genome editing methods are paving the way to bring this technology to invasive species like D. suzukii. The CRISPR/Cas technology is emerging as a versatile option for efficient tailored gene manipulation. Components of this system, the guide RNA (gRNA) and Cas9 protein, can be delivered into the organism in the form of DNA, RNA, or protein. We report CRISPR/Cas-mediated site-specific white gene editing using purified Cas9 protein delivered directly into D. suzukii embryos. Mutant flies with 13, 3, and 1 nt deletions within the cutting s ite of the Cas9 protein were retrieved. Moreover, the two independent G1 deletion events (13 and 3 nt) derived from the same G0 female. Our results show that the recombinant Cas9 protein could be a method of choice for generating heritable gene modifications in D. suzukii

Fitness Cost Implications of PhiC31-Mediated Site-Specific Integrations in Target-Site Strains of the Mexican Fruit Fly, Anastrepha ludens (Diptera: Tephritidae)

Site-specific recombination technologies are powerful new tools for the manipulation of genomic DNA in insects that can improve transgenesis strategies such as targeting transgene insertions, allowing transgene cassette exchange and DNA mobilization for transgene stabilization. However, understanding the fitness cost implications of these manipulations for transgenic strain applications is critical. In this study independent piggyBac-mediated attP target-sites marked with DsRed were created in several genomic positions in the Mexican fruit fly, Anastrepha ludens. Two of these strains, one having an autosomal (attP F7) and the other a Y-linked (attP 2-M6y) integration, exhibited fitness parameters (dynamic demography and sexual competitiveness) similar to wild type flies. These strains were thus selected for targeted insertion using, for the first time in mexfly, the phiC31-integrase recombination system to insert an additional EGFP-marked transgene to determine its effect on host strain fitness. Fitness tests showed that the integration event in the int 2-M6y recombinant strain had no significant effect, while the int F7 recombinant strain exhibited significantly lower fitness relative to the original attP F7 target-site host strain. These results indicate that while targeted transgene integrations can be achieved without an additional fitness cost, at some genomic positions insertion of additional DNA into a previously integrated transgene can have a significant negative effect. Thus, for targeted transgene insertions fitness costs must be evaluated both previous to and subsequent to new site-specific insertions in the target-site strain

Evaluation of Hydrogen Peroxide Fumigation and Heat Treatment for Standard Emergency Arthropod Inactivation in BSL-3 Insectaries

Climate change and global movements of people and goods have accelerated the spread of invasive species, including insects that vector infectious diseases, which threaten the health of more than half of the world’s population. Increasing research efforts to control these diseases include the study of vector – pathogen interactions, involving the handling of pathogen-infected vector insects under biosafety level (BSL) 2 and 3 conditions. Like microbiology BSL-3 laboratories, BSL-3 insectaries are usually subjected to fixed-term or emergency room decontamination using recognized methods such as hydrogen peroxide (H2O2) or formaldehyde fumigation. While these procedures have been standardized and approved for the inactivation of diverse pathogens on surfaces, to date, there are no current standards for effective room-wide inactivation of insects in BSL-3 facilities in case of an emergency such as the accidental release of a large number of infected vectors. As H2O2 is often used for standard room decontamination in BSL-3 facilities, we evaluated H2O2 fumigation as a potential standard method for the safe, room-wide deactivation of insects in BSL-3 insectaries in comparison to heat treatment. To account for physiological diversity in vector insect species, six species from three different orders were tested. For the H2O2 fumigation we observed a strong but also varying resilience across all species. Lethal exposure time for the tested dipterans ranged from nine to more than 24 h. Furthermore, the coleopteran, Tribolium castaneum, did not respond to continuous H2O2 exposure for 48 h under standard room decontamination conditions. In contrast, temperatures of 50°C effectively killed all the tested species within 2 to 10 min. The response to lower temperatures (40–48°C) again showed a strong variation between species. In summary, results suggest that H2O2 fumigation, especially in cases where a gas generator is part of the laboratory equipment, may be used for the inactivation of selected species but is not suitable as a general emergency insect inactivation method under normal room decontamination conditions. In contrast, heat treatment at 48 to 50°C has the potential to be developed as an approved standard procedure for the effective inactivation of insects in BSL-3 facilities. © Copyright © 2020 Häcker, Koller, Eichner, Martin, Liapi, Rühl, Rehling and Schetelig

Insect transgenesis applied to tephritid pest control

Tephritid fruit fly species cause major economical losses in crops worldwide. Genetic transformation of insect pests, which are targets of the Sterile Insect Technique (SIT), a key component of area-wide pest management, has been achieved for several years. For the medfly Ceratitis capitata as well as several Bactrocera and Anastrepha species, germline transformation can now be used to bioengineer strains that should increase the efficacy and cost-effectiveness of the SIT. Novel transformation vectors, robust genetic markers and diverse promoters to drive stage- and tissue-specific gene expression provide powerful tools to test the contribution that these technologies can make to current SIT programmes

Polyandry in the medfly - shifts in paternity mediated by sperm stratification and mixing

Background In the Mediterranean fruit fly (medfly), Ceratitis capitata, a highly invasive agricultural pest species, polyandry, associated with sperm precedence, is a recurrent behaviour in the wild. The absence of tools for the unambiguous discrimination between competing sperm from different males in the complex female reproductive tract has strongly limited the understanding of mechanisms controlling sperm dynamics and use. Results Here we use transgenic medfly lines expressing green or red fluorescent proteins in the spermatozoa, which can be easily observed and unambiguously differentiated within the female fertilization chamber. In twice-mated females, one day after the second mating, sperm from the first male appeared to be homogenously distributed all over the distal portion of each alveolus within the fertilization chamber, whereas sperm from the second male were clearly concentrated in the central portion of each alveolus. This distinct stratified sperm distribution was not maintained over time, as green and red sperm appeared homogeneously mixed seven days after the second mating. This dynamic sperm storage pattern is mirrored by the paternal contribution in the progeny of twice-mated females. Conclusions Polyandrous medfly females, unlike Drosophila, conserve sperm from two different mates to fertilize their eggs. From an evolutionary point of view, the storage of sperm in a stratified pattern by medfly females may initially favour the fresher ejaculate from the second male. However, as the second male's sperm gradually becomes depleted, the sperm from the first male becomes increasingly available for fertilization. The accumulation of sperm from different males will increase the overall genetic variability of the offspring and will ultimately affect the effective population size. From an applicative point of view, the dynamics of sperm storage and their temporal use by a polyandrous female may have an impact on the Sterile Insect Technique (SIT). Indeed, even if the female's last mate is sterile, an increasing proportion of sperm from a previous mating with a fertile male may contribute to sire viable progeny

Erratum: "The whole genome sequence of the Mediterranean fruit fly, Ceratitis capitata (Wiedemann), reveals insights into the biology and adaptive evolution of a highly invasive pest species", [Genome Biol. (2016), 17, (192)]

After publication of our recent article [1] we noticed that Monica Munoz-Torres had been omitted from the author list. We have now added her, and the updated Funding and Authors' contributions sections are below. © The Author(s). 2017