Prospects and Pitfalls: Next-Generation Tools to Control Mosquito-Transmitted Disease.

Mosquito-transmitted diseases, including malaria and dengue, are a major threat to human health around the globe, affecting millions each year. A diverse array of next-generation tools has been designed to eliminate mosquito populations or to replace them with mosquitoes that are less capable of transmitting key pathogens. Many of these new approaches have been built on recent advances in CRISPR/Cas9-based genome editing. These initiatives have driven the development of pathogen-resistant lines, new genetics-based sexing methods, and new methods of driving desirable genetic traits into mosquito populations. Many other emerging tools involve microorganisms, including two strategies involving Wolbachia that are achieving great success in the field. At the same time, other mosquito-associated bacteria, fungi, and even viruses represent untapped sources of new mosquitocidal or antipathogen compounds. Although there are still hurdles to be overcome, the prospect that such approaches will reduce the impact of these diseases is highly encouraging.

Deep orange gene editing triggers temperature-sensitive lethal phenotypes in Ceratitis capitata.

BACKGROUND: The Mediterranean fruit fly, Ceratitis capitata, is a significant agricultural pest managed through area-wide integrated pest management (AW-IPM) including a sterile insect technique (SIT) component. Male-only releases increase the efficiency and cost-effectiveness of SIT programs, which can be achieved through the development of genetic sexing strains (GSS). The most successful GSS developed to date is the C. capitata VIENNA 8 GSS, constructed using classical genetic approaches and an irradiation-induced translocation with two selectable markers: the white pupae (wp) and temperature-sensitive lethal (tsl) genes. However, currently used methods for selecting suitable markers and inducing translocations are stochastic and non-specific, resulting in a laborious and time-consuming process. Recent efforts have focused on identifying the gene(s) and the causal mutation(s) for suitable phenotypes, such as wp and tsl, which could be used as selectable markers for developing a generic approach for constructing GSS. The wp gene was recently identified, and efforts have been initiated to identify the tsl gene. This study investigates Ceratitis capitata deep orange (Ccdor) as a tsl candidate gene and its potential to induce tsl phenotypes. RESULTS: An integrated approach based on cytogenetics, genomics, bioinformatics, and gene editing was used to characterize the Ccdor. Its location was confirmed on the right arm of chromosome 5 in the putative tsl genomic region. Knock-out of Ccdor using CRISPR/Cas9-NHEJ and targeting the fourth exon resulted in lethality at mid- and late-pupal stage, while the successful application of CRISPR HDR introducing a point mutation on the sixth exon resulted in the establishment of the desired strain and two additional strains (dor 12del and dor 51dup), all of them expressing tsl phenotypes and presenting no (or minimal) fitness cost when reared at 25 °C. One of the strains exhibited complete lethality when embryos were exposed at 36 °C. CONCLUSIONS: Gene editing of the deep orange gene in Ceratitis capitata resulted in the establishment of temperature-sensitive lethal mutant strains. The induced mutations did not significantly affect the rearing efficiency of the strains. As deep orange is a highly conserved gene, these data suggest that it can be considered a target for the development of tsl mutations which could potentially be used to develop novel genetic sexing strains in insect pests and disease vectors.

Transgenic cotton and sterile insect releases synergize eradication of pink bollworm a century after it invaded the United States.

Invasive organisms pose a global threat and are exceptionally difficult to eradicate after they become abundant in their new habitats. We report a successful multitactic strategy for combating the pink bollworm (Pectinophora gossypiella), one of the world's most invasive pests. A coordinated program in the southwestern United States and northern Mexico included releases of billions of sterile pink bollworm moths from airplanes and planting of cotton engineered to produce insecticidal proteins from the bacterium Bacillus thuringiensis (Bt). An analysis of computer simulations and 21 y of field data from Arizona demonstrate that the transgenic Bt cotton and sterile insect releases interacted synergistically to reduce the pest's population size. In Arizona, the program started in 2006 and decreased the pest's estimated statewide population size from over 2 billion in 2005 to zero in 2013. Complementary regional efforts eradicated this pest throughout the cotton-growing areas of the continental United States and northern Mexico a century after it had invaded both countries. The removal of this pest saved farmers in the United States $192 million from 2014 to 2019. It also eliminated the environmental and safety hazards associated with insecticide sprays that had previously targeted the pink bollworm and facilitated an 82% reduction in insecticides used against all cotton pests in Arizona. The economic and social benefits achieved demonstrate the advantages of using agricultural biotechnology in concert with classical pest control tactics.

Comparative transcriptome analysis reveals the molecular mechanism of sterility induced by irradiation of Plutella xylostella (Linnaeus).

Plutella xylostella (Linnaeus) is an important pest of cruciferous plants, which is harmful all over the world, causing serious economic losses, and its drug resistance is increasing rapidly. The sterile insect technique (SIT) is a green control method and does not cause resistance. In this study, transcriptomics and bioinformatics were used to explore the effects of irradiation on the reproductive function of Plutella xylostella, and the response mechanism of sterility under irradiation was initially revealed. We identified 3342 (1682 up-regulated, 1660 down-regulated), 1963 (1042 up-regulated, 921 down-regulated) and 1531 (721 up-regulated, 810 down-regulated) differentially expressed genes (DEGs) in the 200 Gy vs CK (Control Check), 400 Gy vs CK and 400 Gy vs 200 Gy groups, respectively. GO and KEGG analyses were performed for DEGs in each group. The results showed that 200 Gy activated the downstream phosphorylation pathway and inhibited the cytochrome p450 immune response mechanism. 400 Gy promoted protein decomposition and absorption pathways, autophagy pathways, etc. Down-regulated genes were concentrated in the transformation process of energy metabolizing substances such as ATP, phosphorylation signaling pathway, and insulin, while up-regulated genes were concentrated in biological regulation and metabolic processes. Eight genes in the phosphorylation pathway were selected for qRT-PCR verification, and the results showed that the phosphorylation of different dose groups was regulated in different ways. 400 Gy used positive feedback regulation, while the phosphorylation of F1 used negative feedback regulation.

Different mechanisms of X-ray irradiation-induced male and female sterility in Aedes aegypti.

BACKGROUND: Aedes aegypti (Ae. aegypti) is the major vector that transmits many diseases including dengue, Zika, and filariasis in tropical and subtropical regions. Due to the growing resistance to chemical-based insecticides, biological control methods have become an emerging direction to control mosquito populations. The sterile insect technique (SIT) deploys high doses of ionizing radiation to sterilize male mosquitoes before the release. The Wolbachia-based population suppression method of the incompatible insect technique (IIT) involves the release of Wolbachia-infected males to sterilize uninfected field females. Due to the lack of perfect sex separation tools, a low percentage of female contamination is detected in the male population. To prevent the unintentional release of these Wolbachia-infected females which might result in population replacement, a low dose of X-ray irradiation is deployed to sterilize any female escapees. However, it remains unclear whether these irradiation-induced male and female sterilizations share common mechanisms. RESULTS: In this work, we set out to define the minimum dose of X-ray radiation required for complete female sterilization in Ae. aegypti (NEA-EHI strain). Further results showed that this minimum dose of X-ray irradiation for female sterilization significantly reduced male fertility. Similar results have been reported previously in several operational trials. By addressing the underlying causes of the sterility, our results showed that male sterility is likely due to chromosomal damage in the germ cells induced by irradiation. In contrast, female sterility appears to differ and is likely initiated by the elimination of the somatic supporting cells, which results in the blockage of the ovariole maturation. Building upon these findings, we identified the minimum dose of X-ray irradiation on the Wolbachia-infected NEA-EHI (wAlbB-SG) strain, which is currently being used in the IIT-SIT field trial. Compared to the uninfected parental strain, a lower irradiation dose could fully sterilize wAlbB-SG females. This suggests that Wolbachia-carrying mosquitoes are more sensitive to irradiation, consistent with a previous report showing that a lower irradiation dose fully sterilized Wolbachia-infected Ae. aegypti females (Brazil and Mexican strains) compared to those uninfected controls. CONCLUSIONS: Our findings thus reveal the distinct mechanisms of ionizing X-ray irradiation-induced male or female sterility in Ae. aegypti mosquitoes, which may help the design of X-ray irradiation-based vector control methods.

Postcopulatory Behavior of Tephritid Flies.

Mating produces profound changes in the behavior of female flies, such as an increase in oviposition, reduction in sexual receptivity, increase in feeding, and even excretion. Many of these changes are produced by copulation, sperm, and accessory gland products that males transfer to females during mating. Our knowledge on the function of the male ejaculate and its effect on female insects is still incipient. In this article, we review peri- and postcopulatory behaviors in tephritid flies. We address the effects of male copulatory behavior; copula duration; and the male ejaculate, sperm, and accessory gland products on female remating behavior. Many species from these families are pests of economic importance; thus, understanding male mating effects on female behavior contributes to both developing more effective environmentally friendly control methods and furthering our understanding of evolutionary implications of intersexual competition and sexual conflict.

Development of transgenic corn planthopper Peregrinus maidis that express the tetracycline transactivator.

The corn planthopper, Peregrinus maidis, is a vector of several maize viruses and is consequently a significant agricultural pest in many tropical and subtropical regions. As P. maidis has developed resistance to insecticides, the aim of this study was to develop transgenic P. maidis strains that could be used for future genetic biocontrol programs. To facilitate the identification of transgenic P. maidis, we isolated and characterized the promoters for the P. maidis ubiquitin-like and profilin genes. Transient expression assays with P. maidis embryos showed that both promoters were active. Transgenic lines were established using piggyBac vectors and fluorescent protein marker genes. The lines carried an auto-regulated tetracycline transactivator (tTA) gene, which has been widely used to establish conditional lethal strains in other insect species. The transgenic lines showed low levels of tTA expression but were viable on diet with or without doxycycline, which inhibits the binding of tTA to DNA. We discuss possible modifications to the tTA overexpression system that could lead to the successful development of conditional lethal strains. To our knowledge, this is the first report of a transgenic Hemiptera. The approach we have taken could potentially be applied to other Hemiptera and, for P. maidis, the technology will facilitate future functional genomics studies.

Sequence and expression analysis of the spermatogenesis-specific gene cognates, wampa and Prosα6T, in Drosophila suzukii.

The sterile insect technique (SIT) is a highly effective biologically-based method for the population suppression of highly invasive insect pests of medical and agricultural importance. The efficacy of SIT could be significantly enhanced, however, by improved methods of male sterilization that avoid the fitness costs of irradiation. An alternative sterilization method is possible by gene-editing that targets genes essential for sperm maturation and motility, rendering them nonfunctional, similar to the CRISPR-Cas9 targeting of ß2-tubulin in the genetic model system, Drosophila melanogaster. However, since genetic strategies for sterility are susceptible to breakdown or resistance in mass-reared populations, alternative targets for sterility are important for redundancy or strain replacement. Here we have identified and characterized the sequence and transcriptional expression of two genes in a Florida strain of Drosophila suzukii, that are cognates of the D. melanogaster spermatocyte-specific genes wampa and Prosalpha6T. Wampa encodes a coiled-coil dynein subunit required for axonemal assembly, and the proteasome subunit gene, Prosalpha6T, is required for spermatid individualization and nuclear maturation. The reading frames of these genes differed from their NCBI database entries derived from a D. suzukii California strain by 44 and 8 nucleotide substitutions/polymorphisms, respectively, though all substitutions were synonymous resulting in identical peptide sequences. Expression of both genes is predominant in the male testis, and they share similar transcriptional profiles in adult males with ß2-tubulin. Their amino acid sequences are highly conserved in dipteran species, including pest species subject to SIT control, supporting their potential use in targeted male sterilization strategies.

Sterility of Cydia pomonella by X ray irradiation as an alternative to gamma radiation for the sterile insect technique.

The codling moth Cydia pomonella is a major pest of global significance impacting pome fruits and walnuts. It threatens the apple industry in the Loess Plateau and Bohai Bay in China. Sterile insect technique (SIT) could overcome the limitations set by environmentally compatible area-wide integrated pest management (AW-IPM) approaches such as mating disruption and attract-kill that are difficult to suppress in a high-density pest population, as well as the development of insecticide resistance. In this study, we investigated the effects of X-ray irradiation (183, 366, 549 Gy) on the fecundity and fertility of a laboratory strain of C. pomonella, using a newly developed irradiator, to evaluate the possibility of X-rays as a replacement for Cobalt60 (60Co-γ) and the expanded future role of this approach in codling moth control. Results show that the 8th-day is the optimal age for irradiation of male pupae. The fecundity decreased significantly as the dosage of radiation increased. The mating ratio and mating number were not influenced. However, treated females were sub-sterile at a radiation dose of 183 Gy (20.93%), and were almost 100% sterile at a radiation dose of 366 Gy or higher. Although exposure to a radiation dose of 366 Gy resulted in a significant reduction in the mating competitiveness of male moths, our radiation biology results suggest that this new generation of X-ray irradiator has potential applications in SIT programs for future codling moth control.

CRISPR/Cas9-induced Mutation of Sex Peptide Receptor Gene Bdspr Affects Ovary, Egg Laying, and Female Fecundity in Bactrocera dorsalis (Hendel) (Diptera: Tephritidae).

The oriental fruit fly, Bactrocera dorsalis (Hendel) (Diptera: Tephritidae), is an invasive and polyphagous pest of horticultural crops, and it can cause huge economic losses in agricultural production. The rapid development of CRISPR/Cas9 gene editing technology has provided new opportunities for the scientific control of agricultural pests. Here, we explore the applicability of the B. dorsalis sex peptide receptor (Bdspr) as a target gene for the CRISPR/Cas9-based sterile insect technique (SIT) in B. dorsalis. We screened two high-efficient single guide RNAs (sgRNAs) for gene editing. The results showed that both mutation efficiency and germline transmission rate were 100% in the surviving G0 females (8/8) from injected embryos, and that 75% of mosaically mutated G0 females (6/8) were sterile. The 50% of heterozygous G1 females (4/8) could not lay eggs; 100% of eggs laid by them could not survive; and 62.5% of individual females (5/8) had abnormal ovaries. These results indicate that Bdspr plays an important role in regulating fertility, egg viability, and ovary development in female B. dorsalis, suggesting that the spr gene can be used for CRISPR/Cas9-based SIT in B. dorsalis.

Molecular Mechanism of Male Sterility Induced by 60Co γ-Rays on Plutella xylostella (Linnaeus).

Plutella xylostella (Linnaeus) is one of the notorious pests causing substantial loses to numerous cruciferous vegetables across many nations. The sterile insect technique (SIT) is a safe and effective pest control method, which does not pollute the environment and does not produce drug resistance. We used proteomics technology and bioinformatics analysis to investigate the molecular mechanisms responsible for the effects of different doses of radiation treatment on the reproductive ability of male P. xylostella. A total of 606 differentially expressed proteins (DEPs) were identified in the 200 Gy/CK group, 1843 DEPs were identified in the 400 Gy/CK group, and 2057 DEPs were identified in the 400 Gy/200 Gy group. The results showed that after 200 Gy irradiation, the testes resisted radiation damage by increasing energy supply, amino acid metabolism and transport, and protein synthesis, while transcription-related pathways were inhibited. After 400 Gy irradiation, the mitochondria and DNA in the testis tissue of P. xylostella were damaged, which caused cell autophagy and apoptosis, affected the normal life activities of sperm cells, and greatly weakened sperm motility and insemination ability. Meanwhile, Western blotting showed that irradiation affects tyrosine phosphorylation levels, which gradually decrease with increasing irradiation dose.

The gut microbiome analysis of Anastrepha obliqua reveals inter-kingdom diversity: bacteria, fungi, and archaea.

The fruit fly Anastrepha obliqua is an economically important pest. The sterile insect technique to control it involves mass production and release of sterile flies to reduce the reproduction of the wild population. As noted in different Tephritidae, the performance of sterile males may be affected by the assimilation of nutrients under mass-rearing conditions. In the wild, the fly's life cycle suggests the acquisition of different organisms that could modulate its fitness and physiology. For A. obliqua, there is no information regarding microorganisms other than bacteria. This study analyzed bacteria, fungal, and archaea communities in the A. obliqua gut through denaturing gradient gel electrophoresis (DGGE) profiles of 16S (using a different set of primers for bacteria and archaea) and 18S ribosomal DNA markers. We found that wild flies presented higher microbial diversity related to fructose assimilation than laboratory species, suggesting that microorganisms have led to a specialized metabolism to process nutrients associated with an artificial diet. We identified species that have not been previously described in this fruit fly, especially actinobacteria and archaea, by employing different primer sets aimed at the same molecular marker but targeting diverse hypervariable regions of 16S rDNA. The possibility that Archaea affect fly fitness should not be ignored. This report on the intestinal microbial (bacteria, archaea, and fungi) composition of A. obliqua contributes to our understanding of the role of microorganisms in the development and physiology of the flies.

Harnessing thermal plasticity to enhance the performance of mass-reared insects: opportunities and challenges.

Insects are mass-reared for release for biocontrol including the sterile insect technique. Insects are usually reared at temperatures that maximize the number of animals produced, are chilled for handling and transport, and released into the field, where temperatures may be considerably different to those experienced previously. Insect thermal biology is phenotypically plastic (i.e. flexible), which means that there may exist opportunities to increase the performance of these programmes by modifying the temperature regimes during rearing, handling, and release. Here we synthesize the literature on thermal plasticity in relation to the opportunities to reduce temperature-related damage and increase the performance of released insects. We summarize how and why temperature affects insect biology, and the types of plasticity shown by insects. We specifically identify aspects of the production chain that might lead to mismatches between the thermal acclimation of the insect and the temperatures it is exposed to, and identify ways to harness physiological plasticity to reduce that potential mismatch. We address some of the practical (especially engineering) challenges to implementing some of the best-supported thermal regimes to maximize performance (e.g. fluctuating thermal regimes), and acknowledge that a focus only on thermal performance may lead to unwanted trade-offs with other traits that contribute to the success of the programme. Together, it appears that thermal physiological plasticity is well-enough understood to allow its implementation in release programmes.

Effects of four chemosterilants on Bactrocera tau.

Bactrocera tau (Walker) is a fly pest species mainly distributed in Southeast Asia and the South Pacific; it causes substantial ecological and economic issues because of its destructiveness and rapid reproduction. Chemical sterilization technology can reduce the use of insecticides and is widely applied for insect pest control. In this study, the sterilization efficacy of varying concentrations of four chemosterilants, namely, hexamethylphosphoramide (HMPA), CSII Aqua, 5-fluorouracil (5-FU), and colchicine, on adult pumpkin flies was investigated. The results indicated that a solution of 0.03% HMPA had the highest sterilization efficacy. When the number of sterile males was equal to or exceeded 20 times that of untreated males, the hatching rate of offspring eggs was less than 10%. Chemosterilant treatment significantly altered the levels of acid phosphatase (ACP), alkaline phosphatase (AKP), and B. tau vitellogenin (BtVg); these substances have an important impact on reproductive development. The treatment also decreased the size of the reproductive organs (i.e., testes and ovaries). Our results suggest that 0.03% HMPA has unique sterilization properties and may represent a new chemical agent for the control of B. tau populations in agricultural settings.

The sterile insect technique: an international framework to facilitate transboundary shipments of sterile insects.

The sterile insect technique (SIT) has been successfully used since the 1950s as part of an integrated pest management approach in large-scale programmes to prevent, contain, suppress and eradicate key insect pests in many countries throughout the world. During this period, over one trillion live sterile insects have been shipped across borders. The very few adverse incidents from this significant trade were managed and resulted in no significant impacts. The phytosanitary and zoosanitary requirements by importing countries have been simple, facilitating the transboundary shipment of sterile insects, which is carried out mostly under the framework of cooperative agreements between the governments of the countries involved, and under technical cooperation projects of the United Nations. However, the shipment of sterile insects from sources outside this governmental framework, including public-private facilities, has been complicated, despite the availability of harmonised international guidelines in some cases, such as those for fruit flies. The SIT has great potential for the control of endemic pests or against the growing threat of invasive pests that can affect whole regions and even continents. Since SIT is species-specific, with negligible risk of introducing unwanted invasive species to the environment, and with the advantage of reducing insecticide use, a harmonised framework that recognises the low risk of SIT would facilitate shipments of sterile insects across borders and help to expand the use of this effective and environmentally friendly technology. The scope of this paper is limited to insects that have been sterilised using ionising radiation.

La technique de l'insecte stérile (TIS) est utilisée avec succès depuis les années 1950, en tant que composante d'une approche intégrée de gestion des ravageurs mise en place par des programmes de grande envergure appliqués dans de nombreux pays pour prévenir, maîtriser, supprimer et éliminer les principales espèces d'insectes nuisibles. Depuis lors, plus d'un trillion d'insectes stériles vivants ont été transportés au-delà des frontières nationales. Les très rares incidents indésirables survenus en marge de ces volumineux échanges ont été maîtrisés et n'ont eu aucun impact significatif. La simplicité des exigences phytosanitaires et de santé animale requises par les pays importateurs a facilité le transport transfrontalier des insectes stériles, le plus souvent effectué dans le cadre d'accords de coopération conclus entre les gouvernements des pays concernés et de projets de coopération technique des Nations Unies. En revanche, il est plus compliqué de transporter des insectes stériles issus de sources extérieures aux cadres gouvernementaux, en particulier des sites relevant d'une gestion publique-privée, et ce malgré l'existence de directives internationales harmonisées, par exemple celles s'appliquant aux différentes mouches de fruits. La TIS présente un grand potentiel pour lutter contre les ravageurs endémiques ou contre la menace croissante associée aux ravageurs envahissants, qui peuvent affecter des régions entières, voire des continents. Étant donné que la TIS est spécifique pour chaque espèce, qu'elle présente un risque négligeable d'introduire des espèces envahissantes non désirées et qu'elle offre l'avantage de réduire le recours aux insecticides, il conviendrait de disposer d'un cadre harmonisé qui reconnaisse le faible niveau de risque associé à la TIS et facilite ainsi les transports transfrontaliers d'insectes stériles, afin d'aider au déploiement plus large de cette technologie efficace et sans risque pour l'environnement. La portée de cet article se limite aux insectes stérilisés par une exposition aux radiations ionisantes.

Desde el decenio de 1950 se viene empleando con éxito la técnica del insecto estéril (sterile insect technique, SIT), aplicada en el manejo integrado de plagas, como parte de programas que se despliegan a gran escala para prevenir, contener, suprimir y erradicar importantes plagas de insectos en muchos países del mundo. En todos estos años, más de un billón de insectos estériles vivos han sido transportados en cargamentos transfronterizos. Los contados incidentes adversos ocasionados por este voluminoso comercio fueron debidamente manejados y se saldaron sin repercusiones de importancia. Los requisitos fitosanitarios y zoosanitarios impuestos por los países importadores han sido sencillos, cosa que ha facilitado el envío transfronterizo de insectos estériles, que mayoritariamente se inscribe en acuerdos de cooperación suscritos entre los gobiernos de los países en cuestión y en proyectos de cooperación técnica de las Naciones Unidas. Sin embargo, el transporte de insectos estériles procedentes de fuentes no sujetas a estos regímenes oficiales, incluidas instalaciones publico-privadas, ha sido complicado, pese a la existencia de directrices internacionales armonizadas aplicables en ciertos casos, como el de la mosca de la fruta. La técnica del insecto estéril puede ser sumamente útil para luchar contra plagas endémicas o contra la creciente amenaza de plagas invasoras capaces de afectar a regiones enteras o incluso a continentes enteros. Teniendo en cuenta que esta técnica utiliza en cada caso la liberación de insectos estériles para controlar insectos plaga de la misma especie, que entraña por ello un riesgo insignificante de introducir en el medio especies invasoras indeseadas y que además presenta la ventaja de reducir el uso de insecticidas, la existencia de un ordenamiento armonizado que reconozca el escaso nivel de riesgo que presenta la SIT facilitaría el envío transfronterizo de insectos estériles y ayudaría a extender el uso de esta técnica tan eficaz como ambientalmente respetuosa. Las consideraciones que aquí hacen los autores se aplican únicamente al caso de los insectos esterilizados con radiación ionizante.

Managing stakeholder concerns associated with releases of imported stock in insect control programmes.

A commitment to reducing pesticide use and the development of novel technologies are driving a renewed interest in insect-mediated pest and vector control programmes. Such programmes, along with conservation and pollination applications, lead to an increased transport volume of live insect stock. At release sites, concerns surrounding imported insects can be reduced by using local genotypes that have been mass-produced elsewhere. Remaining plausible concerns are likely to be centred on human factors (vector behaviour or capacity) and ecological factors (interacting species) and should be anticipated in the design of communication materials. Well-designed, locally relevant communication and engagement material is an important part of programme success. Stakeholder engagement is thus critical to reducing risks of perceived and plausible concerns affecting programme outcomes in an increasingly electronically connected world. Experience at release sites can help inform the design of accessible information useful at all stages of the transportation pathway. For transnationally transported insects, providing such information to specific stakeholders (e.g. courier companies and border authorities) will reduce the likelihood of delays, which can, in turn, affect the quality and mortality of the transported insects.

Les engagements pris en matière de réduction de l'utilisation de pesticides et les nouvelles technologies disponibles ont donné un nouvel essor aux programmes de contrôle des parasites et des vecteurs axés sur les insectes. Ces programmes, auxquels s'ajoutent d'autres applications destinées à favoriser la conservation et la pollinisation, se traduisent par un accroissement du volume de stocks d'insectes vivants transportés dans le monde. Dans les sites de destination des insectes transportés, les inquiétudes que peuvent susciter ces importations peuvent être tempérées en faisant appel à des génotypes locaux, produits en masse ailleurs puis réintroduits. D'autres préoccupations peuvent subsister, portant sur des facteurs humains (liées aux effets du comportement des vecteurs ou à leur capacité vectorielle) et écologiques (liées aux espèces en interaction) ; il convient d'anticiper ces inquiétudes lors de la conception des supports de communication. Des matériels de communication et de mobilisation bien conçus et prenant en compte le contexte local sont une composante importante de la réussite d'un programme. La participation des parties prenantes se révèle donc cruciale pour réduire le risque que des inquiétudes perçues et plausibles viennent affecter les résultats d'un programme dans un monde de plus en plus interconnecté par voie électronique. L'expérience acquise dans les sites de lâchers d'insectes peut contribuer à documenter la conception d'une information accessible, qui sera utile à chaque étape de la procédure de transport. Lorsqu'il s'agit d'insectes transportés au-delà des frontières nationales, la diffusion de ce type d'information à des intervenants spécifiques (par exemple, les sociétés de transport express et les autorités frontalières) permettra de limiter les circonstances entraînant des retards d'acheminement, car ceux-ci peuvent à leur tour affecter la qualité et la viabilité des insectes transportés.

El empeño de reducir el uso de plaguicidas y la aparición de tecnologías novedosas están alimentando un renovado interés por los programas de control de plagas y vectores mediante insectos. Este tipo de programas, junto con las aplicaciones de los insectos con fines de polinización o de protección ambiental, se traducen en el transporte de volúmenes crecientes de poblaciones de insectos vivos. Para apaciguar los recelos que despiertan los insectos importados en los sitios de suelta cabe recurrir al empleo de genotipos locales criados a gran escala en otro lugar. A la hora de concebir el material de comunicación es conveniente prever de antemano y tener en cuenta las suspicacias plausibles que aún puedan subsistir, que seguramente tendrán que ver con factores humanos (por ejemplo, preocupación por los posibles efectos del comportamiento o la capacidad vectorial del insecto introducido) o ecológicos (interacción con otras especies). El uso de material de comunicación y participación bien concebido y adaptado al contexto local es un ingrediente importante para el éxito de todo programa. La participación de las partes interesadas es pues fundamental para reducir los riesgos (ya sean imaginados o plausibles) que puedan afectar los resultados del programa, en un mundo que está cada vez más interconectado electrónicamente. La experiencia adquirida en los sitios de suelta puede ayudar a concebir material informativo accesible que sea de utilidad en todas las etapas del proceso de transporte. En el caso de los insectos transportados de un país a otro, el hecho de facilitar esta información a determinadas partes (como las empresas de transporte o las autoridades aduaneras) reducirá la probabilidad de demoras, cosa que a su vez incidirá en la calidad y la viabilidad de los insectos transportados.

Effects of Irradiated Sterile Male and Mating Sequence on the Fertility of Drosophila suzukii (Diptera: Drosophilidae).

The sterile insect technique has been explored in the laboratory to control populations of Drosophila suzukii (Matsumura) (Diptera: Drosophilidae), a globally invasive pest. We studied the reproductive behavior of D. suzukii including mating frequency, time between matings, and mating duration among non-irradiated flies. Irradiation doses were tested at 0, 60, 90, 110, 120, 150, and 180 Gy to select the optimal dose for producing sterile males. In addition, we examined the effects of mating sequence on offspring production where females were presented with irradiated males first and then wild males, or the reverse. Female D. suzukii were found to mate twice on average through their lifespan, with 16.53 ±â€…12.05 d between matings. The first mating duration was 24.64 ±â€…1.52 min shorter than the second mating. A dose of 90 Gy was suitable where irradiated males lived as long as non-irradiated males, and few eggs hatched from matings. The mating sequence experiment revealed first-male parentage preference. Wild females that mated with a wild male and then irradiated male produced more offspring than females mated with an irradiated and then wild male. Overall, the influence of mating sequence should be taken into consideration when applying the sterile insect technique (SIT) to control D. suzukii populations.

Competitive Sperm-Marked Beetles for Monitoring Approaches in Genetic Biocontrol and Studies in Reproductive Biology.

Sperm marking provides a key tool for reproductive biology studies, but it also represents a valuable monitoring tool for genetic pest control strategies such as the sterile insect technique. Sperm-marked lines can be generated by introducing transgenes that mediate the expression of fluorescent proteins during spermatogenesis. The homozygous lines established by transgenesis approaches are going through a genetic bottleneck that can lead to reduced fitness. Transgenic SIT approaches have mostly focused on Dipteran and Lepidopteran pests so far. With this study, we provide sperm-marked lines for the Coleopteran pest model organism, the red flour beetle Tribolium castaneum, based on the ß2-tubulin promoter/enhancer driving red (DsRed) or green (EGFP) fluorescence. The obtained lines are reasonably competitive and were thus used for our studies on reproductive biology, confirming the phenomenon of 'last-male sperm precedence' and that the spermathecae are deployed for long-term sperm storage, enabling the use of sperm from first mating events even after secondary mating events for a long period of time. The homozygosity and competitiveness of the lines will enable future studies to analyze the controlled process of sperm movement into the long-term storage organ as part of a post-mating cryptic female choice mechanism of this extremely promiscuous species.

The concomitant effects of self-limiting insect releases and behavioural interference on patterns of coexistence and exclusion of competing mosquitoes.

Aedes aegypti is the dominant vector of dengue, a potentially fatal virus whose incidence has increased eightfold in the last two decades. As dengue has no widely available vaccine, vector control is key to reducing the global public health burden. A promising method is the release of self-limiting Ae. aegypti, which mate with wild Ae. aegypti and produce non-viable offspring. The resultant decrease in Ae. aegypti population size may impact coexistence with Ae. albopictus, another vector of dengue. A behavioural mechanism influencing coexistence between these species is reproductive interference, where incomplete species recognition results in heterospecifics engaging in mating activities. We develop a theoretical framework to investigate the interaction between self-limiting Ae. aegypti releases and reproductive interference between Ae. aegypti and Ae. albopictus on patterns of coexistence. In the absence of self-limiting Ae. aegypti release, coexistence can occur when the strength of reproductive interference experienced by both species is low. Results show that substantial overflooding with self-limiting Ae. aegypti prevents coexistence. For lower release ratios, as the release ratio increases, coexistence can occur when the strength of reproductive interference is increasingly high for Ae. albopictus and increasingly low for Ae. aegypti. This emphasizes the importance of including behavioural ecological processes into population models to evaluate the efficacy of vector control.

"Maskandi experience": exploring the use of a cultural song for community engagement in preparation for a pilot Sterile Insect Technique release programme for malaria vector control in KwaZulu-Natal Province, South Africa 2019.

BACKGROUND: An assessment of the Sterile Insect Technique (SIT) as a complementary malaria vector control tool, is at an advanced stage in South Africa. The technique involves the release of laboratory-reared sterilized male mosquitoes of the major malaria vector Anopheles arabiensis, raising social, ethical and regulatory concerns. Therefore, its implementation largely depends on community participation and acceptance. Against this background, it is critical that robust and effective community strategies are developed. This study describes the development of a cultural song to engage the community and increase awareness on SIT and malaria control in KwaZulu-Natal, South Africa. METHODS: An exploratory concurrent mixed-methods study was conducted to get opinions about the effectiveness of a cultural song developed to engage communities and increase acceptability of the SIT technology. Two self-administered surveys (expert and community) were conducted. Additionally, more in depth opinions of the song and its effectiveness in conveying the intended information were investigated through three community dialogue sessions with community members in the study area. RESULTS: A total of 40 experts and 54 community members participated in the survey. Four themes were identified in relation to the appropriateness and effectiveness of the song, with a fifth theme focused on recommendations for adaptations. Overall, the song was well received with the audience finding it entertaining and informative. Responses to unstructured questions posed after the song showed an increase in the knowledge on malaria transmission and SIT technology. In particular, the explanation that male mosquitoes do not bite allayed anxiety and fears about the SIT technology. CONCLUSION: The song was deemed both culturally appropriate and informative in engaging community members about the SIT technology. It proved useful in promoting health messages and conveying SIT technology as a complementary malaria vector control tool. With minor adaptations, the song has potential as an area-wide community engagement tool in areas targeted for sterile male releases.