Anti-CRISPR Anopheles mosquitoes inhibit gene drive spread under challenging behavioural conditions in large cages.

CRISPR-based gene drives have the potential to spread within populations and are considered as promising vector control tools. A doublesex-targeting gene drive was able to suppress laboratory Anopheles mosquito populations in small and large cages, and it is considered for field application. Challenges related to the field-use of gene drives and the evolving regulatory framework suggest that systems able to modulate or revert the action of gene drives, could be part of post-release risk-mitigation plans. In this study, we challenge an AcrIIA4-based anti-drive to inhibit gene drive spread in age-structured Anopheles gambiae population under complex feeding and behavioural conditions. A stochastic model predicts the experimentally-observed genotype dynamics in age-structured populations in medium-sized cages and highlights the necessity of large-sized cage trials. These experiments and experimental-modelling framework demonstrate the effectiveness of the anti-drive in different scenarios, providing further corroboration for its use in controlling the spread of gene drive in Anopheles.

Introgression of a synthetic sex ratio distortion transgene into different genetic backgrounds of Anopheles coluzzii.

The development of genetically modified mosquitoes (GMM) and their subsequent field release offers innovative approaches for vector control of malaria. A non-gene drive self-limiting male-bias Ag(PMB)1 strain has been developed in a 47-year-old laboratory G3 strain of Anopheles gambiae s.l. When Ag(PMB)1 males are crossed to wild-type females, expression of the endonuclease I-PpoI during spermatogenesis causes the meiotic cleavage of the X chromosome in sperm cells, leading to fertile offspring with a 95% male bias. However, World Health Organization states that the functionality of the transgene could differ when inserted in different genetic backgrounds of Anopheles coluzzii which is currently a predominant species in several West-African countries and thus a likely recipient for a potential release of self-limiting GMMs. In this study, we introgressed the transgene from the donor Ag(PMB)1 by six serial backcrosses into two recipient colonies of An. coluzzii that had been isolated in Mali and Burkina Faso. Scans of informative Single Nucleotide Polymorphism (SNP) markers and whole-genome sequencing analysis revealed a nearly complete introgression of chromosomes 3 and X, but a remarkable genomic divergence in a large region of chromosome 2 between the later backcrossed (BC6) transgenic offspring and the recipient paternal strains. These findings suggested to extend the backcrossing breeding strategy beyond BC6 generation and increasing the introgression efficiency of critical regions that have ecological and epidemiological implications through the targeted selection of specific markers. Disregarding differential introgression efficiency, we concluded that the phenotype of the sex ratio distorter is stable in the BC6 introgressed An. coluzzii strains.

Gene-drive suppression of mosquito populations in large cages as a bridge between lab and field.

CRISPR-based gene-drives targeting the gene doublesex in the malaria vector Anopheles gambiae effectively suppressed the reproductive capability of mosquito populations reared in small laboratory cages. To bridge the gap between laboratory and the field, this gene-drive technology must be challenged with vector ecology.Here we report the suppressive activity of the gene-drive in age-structured An. gambiae populations in large indoor cages that permit complex feeding and reproductive behaviours.The gene-drive element spreads rapidly through the populations, fully supresses the population within one year and without selecting for resistance to the gene drive. Approximate Bayesian computation allowed retrospective inference of life-history parameters from the large cages and a more accurate prediction of gene-drive behaviour under more ecologically-relevant settings.Generating data to bridge laboratory and field studies for invasive technologies is challenging. Our study represents a paradigm for the stepwise and sound development of vector control tools based on gene-drive.

Detecting the population dynamics of an autosomal sex ratio distorter transgene in malaria vector mosquitoes.

The development of genetically modified (GM) mosquitoes and their subsequent field release offers innovative and cost-effective approaches to reduce mosquito-borne diseases, such as malaria. A sex-distorting autosomal transgene has been developed recently in G3 mosquitoes, a laboratory strain of the malaria vector Anopheles gambiae s.l. The transgene expresses an endonuclease called I-PpoI during spermatogenesis, which selectively cleaves the X chromosome to result in ~95% male progeny. Following the World Health Organization guidance framework for the testing of GM mosquitoes, we assessed the dynamics of this transgene in large cages using a joint experimental modelling approach.We performed a 4-month experiment in large, indoor cages to study the population genetics of the transgene. The cages were set up to mimic a simple tropical environment with a diurnal light-cycle, constant temperature and constant humidity. We allowed the generations to overlap to engender a stable age structure in the populations. We constructed a model to mimic the experiments, and used the experimental data to infer the key model parameters.We identified two fitness costs associated with the transgene. First, transgenic adult males have reduced fertility and, second, their female progeny have reduced pupal survival rates. Our results demonstrate that the transgene is likely to disappear in <3 years under our confined conditions. Model predictions suggest this will be true over a wide range of background population sizes and transgene introduction rates. Synthesis and applications. Our study is in line with the World Health Organization guidance recommendations in regard to the development and testing of GM mosquitoes. Since the transgenic sex ratio distorter strain (Ag(PMB)1) has been considered for genetic vector control of malaria, we recorded the dynamics of this transgene in indoor-large cage populations and modelled its post-release persistence under different scenarios. We provide a demonstration of the self-limiting nature of the transgene, and identified new fitness costs that will further reduce the longevity of the transgene after its release. Finally, our study has showcased an alternative and effective statistical method for characterizing the phenotypic expression of a transgene in an insect pest population.

Lures for red palm weevil trapping systems: aggregation pheromone and synthetic kairomone.

BACKGROUND: The optimisation of the lure is essential for the implementation of trapping systems to control insect pests. In this work, the response of the red palm weevil (RPW), Rhynchophorus ferrugineus Olivier, to increasing emission rates of its aggregation pheromone (ferrugineol) and the efficacy of a convenient synthetic kairomone based on fermentation odours (ethyl acetate and ethanol) have been evaluated in different years and locations along the Mediterranean basin. RESULTS: In general, although capture data and emission had noticeable variability among locations, significantly fewer RPW were captured in pyramidal Picusan® traps with the lowest ferrugineol emission rates tested (0.6-3.8 mg day-1 ). Captures increased rapidly with ferrugineol emission up to 4-5 mg day-1 ; then, higher emission rates did not improve or reduce captures, up to the highest emission rate tested of 50.9 mg day-1 . Thus, there is no evidence of an optimum release rate corresponding to a maximum of RPW catches. Traps baited with the synthetic kairomone (1:3 ethyl acetate/ethanol) captured 1.4-2.2 times more total weevils than traps baited only with ferrugineol. Moreover, in most of the locations, the synthetic blend was at least as effective as the local coattractants used (plant material + molasses). CONCLUSIONS: Ferrugineol emission rate can vary in a wide range without significantly affecting RPW response. Coattractants based on fermenting compounds, ethyl acetate and ethanol, are able to improve the attractant level of ferrugineol and could be employed to replace non-standardised natural kairomones in RPW trapping systems after further optimisation of their proportions and doses. © 2016 Society of Chemical Industry.

Anatomy of the antennal dorsal organ in female of Neodryinus typhlocybae (Hymenoptera: Dryinidae): A peculiar sensory structure possibly involved in perception of host vibration.

Neodryinus typhlocybae (Hymenoptera: Dryinidae) is a natural enemy of the planthopper Metcalfa pruinosa, which was introduced from North America into Europe and has become established in various regions as a pest species. Vibrational signals play a crucial role in the communication of M. pruinosa, which appears to be exploited by N. typhlocybae. Scanning and transmission electron microscopy have shown that the antennae of N. typhlocybae females have peculiar and complex sensory structures: deep longitudinal grooves that house long sensilla trichodea, termed here "Antennal Dorsal Organs." Such structures were not present on male antennae. These sensilla extend for the length of the grooves, without contact with the groove cuticle. Their hair shaft is empty and aporous, and inserted into a specialized socket, underneath which there is a cuticular ampulla-like chamber. Each sensillum is associated with two sensory neurons: one terminates at the proximal end of the dendritic sheath; the other continues into the sensillum sinus and is enclosed in the dendritic sheath. This second sensory neuron then enters the ampulla-like chamber through the circular opening, and then terminates with a conspicuous tubular body at the shaft base. The possible involvement of this peculiar structure in the context of host recognition mechanism is discussed.

Biomonitoring with honeybees of heavy metals and pesticides in nature reserves of the Marche Region (Italy).

The aim of this study was to carry out biomonitoring with honeybees (Apis mellifera L.) to assess the presence of pesticides and heavy metals (cadmium, chromium, nickel, lead) in all of the ten nature reserves of the Marche Region (central­eastern Italy). The study was carried out during the spring and summer seasons when the honeybees were active, over 3 years (2008­2010). Twenty-two colonies of honeybees bred in hives were used. Samples of live and dead honeybees and of honey were collected from 11 sampling stations from May to October in each year. No pesticide pollution was found. Significant differences in heavy metal concentrations were found among years, months and sites, and in particular situations. The analysis reveals that high heavy-metal concentrations occurred exclusively in live honeybees. For the seasonal averages, the most detected heavy metal was chromium, which exceeded the threshold more often than for the other elements, followed by cadmium and lead; nickel never exceeded the threshold. The data are discussed with an evaluation of the natural and anthropic sources taken from the literature and from local situations that were likely to involve heavy metal pollution.

Perception of host plant volatiles in Hyalesthes obsoletus: behavior, morphology, and electrophysiology.

The Palearctic planthopper Hyalesthes obsoletus is the natural vector of the grapevine yellow disease Bois noir. Grapevine is an occasional host plant of this polyphagous planthopper. To deepen our knowledge of the role of plant volatile organic compounds for H. obsoletus host plant searching, we carried out behavioral, morphological, and electrophysiological studies. We tested the attraction of H. obsoletus to nettle, field bindweed, hedge bindweed, chaste tree, and grapevine by using a Y-shaped olfactometer. The results showed a significant attraction of male H. obsoletus to chaste tree, and of the females to nettle. Male H. obsoletus were repelled by odor from hedge bindweed. Ultrastructural studies of the antennae showed at least two types of olfactory sensilla at the antennal pedicel: plaque organs and trichoid sensilla. Volatile organic compounds from nettle and chaste tree were collected, and the extracts were analyzed by coupling gas-chromatography to both mass-spectrometry and electroantennography. The volatile organic compounds that elicited electrophysiological responses in male and female antennae were identified. These findings are discussed with respect to behavior of H. obsoletus males and females in the field.