Sterile Insect Technique Research Articles

Qualitative analysis of a Filippov wild-sterile mosquito population model with immigration.

Effectively combating mosquito-borne diseases necessitates innovative strategies beyond traditional methods like insecticide spraying and bed nets. Among these strategies, the sterile insect technique (SIT) emerges as a promising approach. Previous studies have utilized ordinary differential equations to simulate the release of sterile mosquitoes, aiming to reduce or eradicate wild mosquito populations. However, these models assume immediate release, leading to escalated costs. Inspired by this, we propose a non-smooth Filippov model that examines the interaction between wild and sterile mosquitoes. In our model, the release of sterile mosquitoes occurs when the population density of wild mosquitoes surpasses a specified threshold. We incorporate a density-dependent birth rate for wild mosquitoes and consider the impact of immigration. This paper unveils the complex dynamics exhibited by the proposed model, encompassing local sliding bifurcation and the presence of bistability, which entails the coexistence of regular equilibria and pseudo-equilibria, as crucial model parameters, including the threshold value, are varied. Moreover, the system exhibits hysteresis phenomena when manipulating the rate of sterile mosquito release. The existence of three types of limit cycles in the Filippov system is ruled out. Our main findings indicate that reducing the threshold value to an appropriate level can enhance the effectiveness of controlling wild insects. This highlights the economic benefits of employing SIT with a threshold policy control to impede the spread of disease-carrying insects while bolstering economic outcomes.

A classic screening marker does not affect antennal electrophysiology but strongly regulates reproductive behaviours in Bactrocera dorsalis.

The key phenotype white eye (white) has been used for decades to selectively remove females before release in sterile insect technique programs and as an effective screening marker in genetic engineering. Bactrocera dorsalis is a representative tephritid pest causing damage to more than 150 fruit crops. Yet, the function of white in important biological processes remains unclear in B. dorsalis. In this study, the impacts of the white gene on electrophysiology and reproductive behaviour in B. dorsalis were tested. The results indicated that knocking out Bdwhite disrupted eye pigmentation in adults, consistent with previous reports. Bdwhite did not affect the antennal electrophysiology response to 63 chemical components with various structures. However, reproductive behaviours in both males and females were significantly reduced in Bdwhite-/- . Both pre-copulatory and copulation behaviours were significantly reduced in Bdwhite-/- , and the effect was male-specific. Mutant females significantly delayed their oviposition towards γ-octalactone, and the peak of oviposition behaviour towards orange juice was lost. These results show that Bdwhite might not be an ideal screening marker in functional gene research aiming to identify molecular targets for behaviour-modifying chemicals. Instead, owing to its strong effect on B. dorsalis sexual behaviours, the downstream genes regulated by Bdwhite or the genes from white-linked areas could be alternate molecular targets that promote the development of better behavioural modifying chemical-based pest management techniques.

X-rays are as effective as gamma-rays for the sterilization of Glossina palpalis gambiensis Vanderplank, 1911 (Diptera: Glossinidae) for use in the sterile insect technique

An area-wide integrated pest management strategy with a sterile insect technique (SIT) component requires a radiation source for the sterilisation of male insects. Self-contained gamma irradiators, which were exclusively used in past SIT programmes, are now facing increasing constraints and challenges due to stringent regulations. As a potential alternative, new generation high output X-ray irradiators have been proposed. The feasibility of using X-ray irradiators was assessed by comparing the effects of both gamma- and X-ray irradiators on biological parameters of Glossina palpalis gambiensis (Vanderplank, 1911), that are important for SIT applications. The gamma irradiator Foss Model 812 and two X-ray irradiators, the Rad Source 2400 and the blood irradiator Raycell Mk2 were used. Glossina palpalis gambiensis males were exposed to radiation as pupae. A radiation dose of 110 Gy or above induced more than 97% sterility in females that mated with the irradiated males for all the irradiators. Adult emergence rate, flight propensity, survival and mating performance did not differ between gamma- and X-rays irradiators. These results suggest that irradiating pupae with a dose of 110 Gy is optimal for both gamma-and X-ray irradiators used in this study, to achieve a sterility of approximately 99%. Similar research on other tsetse species could gradually phase out the use of gamma-ray irradiators in favour of X-rays irradiators, especially for smaller SIT programmes.

Cyclin B3 plays pleiotropic roles in female reproductive organogenesis and early embryogenesis in the silkworm, Bombyx mori.

The reproductive system plays a crucial role in insect survival, reproduction and species specificity. Understanding the molecular mechanisms underlying reproductive organogenesis contributes to improving the efficiency of sterile insect technique marked by an eco-friendly pest management strategy. Lepidoptera is one of the largest orders of insects, most of which are major pests in agriculture and forestry. Our study aimed to screen the genes responsible for reproductive organogenesis and unravel the mechanism underlying female reproductive organ defects. Morphological investigation of female reproductive organs showed a defective connection between oviductus geminus and oviductus communis on the second day of pupa (P2) in Speckled mutant silkworm. RNA_Seq identified a total of 18 049 transcripts that were expressed in the P2 female internal reproductive organs without ovary in Spc/+ compared to +Spc /+Spc . Differential expression analysis identified 312 up-regulated genes and 221 down-regulated genes in Spc/+. KEGG analysis identified 44 significantly enriched pathways. The results of qRT-PCR performed on 33 genes significantly matched the outcomes of the RNA_Seq. Dysfunction of Cyclin B3 resulted in a defective connection of the oviductus communis with the ovariole, dysfunction of oogenesis, and a petite body. Moreover, homozygous recessive lethality of Cyclin B3/Cyclin B3 occurred during early embryogenesis. Our results suggest that Cyclin B3 is a pleiotropic functional gene that regulates early embryogenesis, oogenesis, development, and female reproductive organogenesis. These results showed that Cyclin B3 has significant effects on lepidopteran mortality, growth, and reproductive physiology, which might be considered a novel and potentially eco-friendly target for lepidopteran pest management. © 2023 Society of Chemical Industry.

Embryonic microinjection of ribonucleoprotein complex (Cas9+sgRNA) of whitegene in melon fly, Zeugodacuscucurbitae (Coquillett) (Diptera: Tephritidae) produced whiteeye phenotype.

Melon fly, Zeugodacus cucurbitae (Coquillett) is a major pest of cucurbitaceous crops, and causes substantial yield losses and economic costs. CRISPR/Cas9 is a rapid and effective site-specific genome editing tool for the generation of genetic changes that are stable and heritable. The CRISPR/Cas9 tool uses synthetically designed single guide RNA (sgRNA) that is complementary to the target gene and guides the Cas9 enzyme to perform nuclease activity by making double-strand breaks in the target DNA sequences. This tool can be effectively exploited to improve traits critical for the management of insect pests by targeting specific genes encoding these traits without the need of extensive genetic information. The white gene is an important gene responsible for the transport of body pigment precursor molecules. In this study, we produced effective mutagenesis of the white gene of Z. cucurbitae using the CRISPR/Cas9 tool with double sgRNAto target multiple sites of white to increase the efficiency in the generation of frame-shift mutations resulting in the white eye phenotype in adults. This was achieved through embryonic microinjection of the ribonucleoprotein (RNP) complex in the pre-blastoderm embryo stage 1 h after embryo laying. Our success with the production of a white eye mutant fly by CRISPR/Cas9 mutagenesis is important for the research on gene function and protein-level modifications in melon fly and forms the basis for the development of new genetic control strategies such as precision guided sterile insect technique (pgSIT) for this pest of economic significance.

Mosquito Embryo Microinjection.

Genetically modified (GM) mosquitoes are an important tool in the fight against mosquito-borne disease, both indirectly through their use in research investigating host-pathogen interaction, mosquito olfaction, and anthropomorphic behavior and in future direct uses for suppression and possibly eradication through sterile insect technique (SIT) and/or gene-drive programs. Successful creation of GM mosquitoes depends on microinjection procedures that precisely deliver injection materials while causing as little damage to mosquito embryos as possible. Genetic modification reagents, such as transposon system components (vector plasmids, helper plasmids, and helper mRNA), and CRISPR-Cas9 components (guide RNAs, Cas9 protein, plasmids expressing Cas9 and/or guide RNAs, and donor plasmids used in homology-directed repair [HDR]), must be delivered into the preblastoderm embryo at the posterior end where the pole cells will form before cellularization occurs. Sharp needles that pierce the embryo easily are important tools in this procedure and work best when the embryos are not desiccated. The two main procedures for mosquito embryo microinjection involve injecting embryos under halocarbon oil or under aqueous solution.

The sterile insect technique can efficiently reduce the reproduction of the spotted wing drosophila (Drosophila suzukii) in strawberry

The sterile insect technique can efficiently reduce the reproduction of the spotted wing drosophila (<i>Drosophila suzukii</i>) in strawberry

Development of a novel genetic sexing strain of Ceratitis capitata based on an X-autosome translocation

Genetic sexing strains (GSS), such as the Ceratitiscapitata (medfly) VIENNA 8 strain, facilitate male-only releases and improve the efficiency and cost-effectiveness of sterile insect technique (SIT) applications. Laboratory domestication may reduce their genetic diversity and mating behaviour and hence, refreshment with wild genetic material is frequently needed. As wild males do not carry the T(Y;A) translocation, and wild females do not easily conform to artificial oviposition, the genetic refreshment of this GSS is a challenging and time-consuming process. In the present study, we report the development of a novel medfly GSS, which is based on a viable homozygous T(XX;AA) translocation using the same selectable markers, the whitepupae and temperature-sensitivelethal genes. This allows the enmasse cross of T(XX;AA) females with wild males, and the backcrossing of F1 males with the T(XX;AA) females thus facilitating the re-establishment of the GSS as well as its genetic refreshment. The rearing efficiency and mating competitiveness of the novel GSS are similar to those of the T(Y;A)-based VIENNA 8 GSS. However, its advantage to easily allow the genetic refreshment is of great importance as it can ensure the mass production of high-quality males and enhanced efficacy of operational SIT programs.

Aedes albopictus microbiota: Differences between wild and mass-reared immatures do not suggest negative impacts from a diet based on black soldier fly larvae and fish food.

The "Sterile Insect Technique" (SIT), a promising method to control Aedes albopictus, the Asian tiger mosquito, is gaining increasing interest. Recently, the role of microbiota in mosquito fitness received attention, but the link between microbiota and larval diet in mass rearing programs for SIT remains largely unexplored. We characterized the microbiota of four larval instars, pupae and eggs of non-wild (NW) lab-reared Ae. albopictus fed with a diet based on Black soldier fly (Hermetia illucens) larvae powder and fish food KOI pellets. We compared it with wild (W) field-collected individuals and the bacterial community occurring in rearing water-diet (DIET). A total of 18 bacterial classes with > 0.10% abundance were found overall in the samples, with seven classes being especially abundant. Overall, the microbiota profile significantly differed among NW, W and DIET. Verrucomicrobiae were significantly more abundant in W and DIET, Bacteroidia were more abundant in NW and DIET, and Gammaproteobacteria were only more abundant in W than in DIET. W-eggs microbiota differed from all the other groups. Large differences also appeared at the bacterial genus-level, with the abundance of 14 genera differing among groups. Three ASVs of Acinetobacter, known to have positive effects on tiger mosquitoes, were more abundant in NW than in W, while Serratia, known to have negative or neutral effects on another Aedes species, was less abundant in NW than in W. The bacterial community of W-eggs was the richest in species, while dominance and diversity did not differ among groups. Our data show that the diet based on Black soldier fly powder and fish food KOI influences the microbiota of NW tiger mosquito immature stages, but not in a way that may suggest a negative impact on their quality in SIT programs.

Effects of different diets on Aedes aegypti adults: improving rearing techniques for sterile insect technique.

The aim was to evaluate the effect of different energy diets available in adulthood on the longevity, dispersal capacity and sexual performance of Aedes aegypti produced under a mass-rearing system. To evaluate the effects of diets in relation to the survival of the adult male insects of Ae. aegypti, six treatments were used: sucrose at a concentration of 10%, as a positive control (sack10); starvation, as a negative control (starvation); sucrose at a concentration of 20% associated with 1 g/l of ascorbic acid (sac20vitC); wild honey in a concentration of 10% (honey10); demerara sugar in a 10% concentration (demerara10); and sucrose at a concentration of 20% associated with 1 g/l of ascorbic acid and 0.5 g/l of amino acid proline (sac20vitCPr). Each treatment had 16 cages containing 50 adult males. For the tests of flight ability and propensity to copulation, five treatments were used (saca10; sac20vitC; mel10; demerara10; and sac20vitCPr), with males each for flight ability and females copulated by a single male for copulation propensity. The diet composed of sucrose at a concentration of 20% associated with ascorbic acid, as an antioxidant, improved the survival, flight ability and propensity to copulate in Ae. aegypti males under mass-rearing conditions, and may be useful to enhance the performance of sterile males, thus improving the success of sterile insect technique programmes.

Identification and functional analysis of Cochliomyia hominivorax U6 gene promoters.

The New World screwworm, Cochliomyia hominivorax, is an obligate parasite, which is a major pest of livestock. While the sterile insect technique was used very successfully to eradicate C. hominivorax from North and Central America, more cost-effective genetic methods will likely be needed in South America. The recent development of CRISPR/Cas9-based genetic approaches, such as homing gene drive, could provide a very efficient means for the suppression of C. hominivorax populations. One component of a drive system is the guide RNA(s) driven by a U6 gene promoter. Here, we have developed an in vivo assay to evaluate the activity of the promoters from seven C. hominivorax U6 genes. Embryos from the related blowfly Lucilia cuprina were injected with plasmid DNA containing a U6-promoter-guide RNA construct and a source of Cas9, either protein or plasmid DNA. Activity was assessed by the number of site-specific mutations in the targeted gene in hatched larvae. One promoter, Chom U6_b, showed the highest activity. These U6 gene promoters could be used to build CRISPR/Cas9-based genetic systems for the control of C. hominivorax.

Techniques for Identifying and Sorting Transgenic Mosquito Larvae.

Transgenic mosquitoes are used in many aspects of mosquito research. First, they can help answer biological questions to advance scientific knowledge-for example, in the fields of mosquito-pathogen interactions, insect immunity, or olfaction. Second, transgenic technologies may be used to develop much needed novel vector control strategies, such as mosquitoes that are unable to transmit disease or transgenes that sterilize mosquito females to suppress vector populations. Here, we introduce how researchers use various selection markers to screen for transgenic mosquito larvae following a transgenesis experiment. Common procedures include using a binocular fluorescence microscope for initial screening. For higher-throughput screening, a flow cytometer known as Complex Object Parametric Analyzer and Sorter (COPAS) can be used to stabilize transgenic lines through the purification of homozygous individuals or to manage transgene frequency in established transgenic lines. In particular, COPAS sorting allows the production of mosquito larval cultures composed of a mixture of genotypes (control and genetically modified larvae) with the goal of raising both groups of mosquitoes under the same environmental conditions in preparation for a controlled phenotype assessment. It can also be used to produce large populations of male mosquitoes, which should facilitate the development of mosquito control intervention strategies similar to the sterile insect technique (SIT), which aims to release large numbers of sterile males that will mate with and sterilize wild females to suppress mosquito populations. Finally, the utilization of a puromycin resistance marker cassette to screen for transgenic Anopheles larvae is also introduced.

A novel protein‐based fruit fly trap in melon flies Bactrocera cucurbitae for effective pest control management

AbstractAgriculture remains a major source of subsistence for local communities in India. However, agricultural yield can be strongly affected by agricultural pest outbreaks. This can result in economic losses for small‐scale farmers who already experience socio‐economic challenges, such as lack of appropriate infrastructure and subsides. Sophisticated pest management techniques (e.g. sterile insect technique) are less accessible to small farmers in developing countries, and therefore, alternative cost‐effective approaches for pest management are needed. Here, we report our findings of a 3‐year field trial (2018–2020) in India, which was designed to test for the potential effectiveness of a novel, slow‐release formulation protein‐based trap, compared to standard Cuelure traps against melon flies Bactrocera cucurbitae (Diptera: Tephritidae). Protein‐bait traps can attract flies from both sexes (as opposed to male‐only, chemical traps), bearing the potential to have a greater long‐term impact on pest populations by decreasing future reproductive potential of trapped individuals. We found that, despite uneven sampling efforts, Cuelure had overall higher trapping performance, while protein‐bait traps, despite trapping at lower efficiency, were equally effective for trapping males and females. Simulations with our field data revealed that protein‐bait traps can have an ‘inclusive’ advantage by trapping females and thereby preventing future individuals. Overall, our study highlights the potential benefits of using this alternative trapping technique to supplement pest management in developing countries.

Understanding the Invasion, Ecological Adaptations, and Management Strategies of Bactrocera dorsalis in China: A Review

Bactrocera dorsalis (Hendel, 1912) (Diptera: Tephritidae), commonly known as the oriental fruit fly, is a highly destructive pest that globally infests fruits and vegetables, resulting in significant annual economic losses. Initially detected in Taiwan Island, it has rapidly expanded its distribution range to various regions in mainland China since the 1980s, with a continuous northward spread. To mitigate the damage caused by this pest, extensive efforts have been undertaken to comprehend its ecological and physiological adaptations and develop management strategies. This review article provides an overview of the invasion history of B. dorsalis in China, its ecological and physiological mechanisms facilitating its invasion, and the progress made in understanding its major biological characteristics. Moreover, the key approaches for managing B. dorsalis that have been or are likely to be implemented in China are presented, including quarantine measures, monitoring procedures, physical controls, biological controls, the sterile insect technique, RNA interference, and CRISPR-Cas-9. Finally, some suggestions for future research directions are provided.

Effect of 2 sex-sorting time schedules on SIT facility management.

Improvements are needed in mosquito mass-rearing to effectively implement the sterile insect technique (SIT). However, managing this technique is challenging and resource intensive. SIT relies on mass rearing, sterilization, and release of adult males to reduce field populations. Maintaining an acceptable level of female presence, who can transmit viruses through biting, is crucial. Females are also essential for facility sustainability. Sex sorting plays a vital role in the production process, and our current mechanical sorting approach aims to obtain a high number of adult males with minimal female contamination within 24 h of pupation. Utilizing protandry helps control female contamination. While the 24-h sorting period achieves desired contamination levels, it may not yield enough females to sustain breeding lines, leading to increased labor costs that impact project sustainability. By delaying the sorting procedure to 48 h, we obtained sufficient females to sustain breeding lines, achieving a balance between male production and female contamination using the automatic version of the Fay-Morlan device as the sorting tool.

Influência da recópula na técnica do inseto estéril em Ceratitis capitata (Diptera: Tephritidae): uma abordagem molecular

ABSTRACT The Mediterranean fruit fly, Ceratitis capitata (Diptera: Tephritidae), is one of the most harmful species to world horticulture, thus requiring suppression or eradication programs. The sterile insect technique is widely used for autocidal control of nuisance insects because it neither releases exotic agents into the environment nor introduces new genetic material into existing populations. In 2005, the Moscamed Brasil biofactory imported the tsl Vienna 8 C. capitata strain from the International Atomic Energy Agency (IAEA); this action required genetic testing of the strain’s biological characteristics. The objective of this study was to discriminate males of wild populations from tsl Vienna 8 using molecular markers, and to conclude which male line dominated in a remating condition. Four crosses, each using 100 males and 100 females, were performed: wild female × wild male; wild female × tsl Vienna 8 male; wild female × wild male, followed by remating with tsl Vienna 8 males after 48 h, and wild female × tsl Vienna 8 male, followed by remating with wild males after 48 h. The results showed that the tsl Vienna 8 strain is compatible with wild females from the São Francisco Valley region and that these males can successfully transfer their sperm to the female spermathecae. Regarding remating, the sperm of the second male dominated over that of the first one. Based on these findings, the sterile insect technique success may be reduced, since the progeny of a female that remates with a wild male could be viable.

Influence of environmental conditions on mass rearing parameters of tsetse flies at the Bobo-Dioulasso insectary (Burkina Faso): Retrospective study

With the goal of eradicating tsetse flies and trypanosomiasis in Africa, several control methods have been developed. One of this is the biological control through the application of the sterile insect technique (SIT) as a part of an area-wide integrated pest management. This method required the mass production of sterile males with a high competitiveness. The success of the tsetse mass rearing is strongly linked to the rearing conditions. The objective of this study was to do a retrospective evaluation of the impact of ambient conditions on the production performances of Glossina palpalis gambiensis at Insectary of Bobo Dioulasso. Data of productivity, fecundity, adult emergence and environmental conditions (temperature and relative humidity) of the rearing rooms were recorded from March to June 2020 and were analysed. The results showed a reduction of the normal pupae produced and the increase of soft pupae during this period. In addition, the relative humidity has shown a significant positive correlation with the production of soft pupae, whereas it had a significant negative correlation with the number of pupae produced per female per 10 days. However, the temperature variation during the period of the data recording didn’t have any impact on the production parameters. Rearing rooms need a better management to avoid large variations in environmental parameters.

Effect of the Sterile Insect Technique and Augmentative Parasitoid Releases in a Fruit Fly Suppression Program in Mango-Producing Areas of Southeast Mexico.

The Sterile Insect Technique (SIT), by means of sterile male releases of Anastrepha ludens (Loew), coupled with Augmentative Biological Control (ABC), by releasing the parasitoid Diachasmimorpha longicaudata (Ashmead), was evaluated in a commercial mango production area for one year. The obtained results were compared with mean fruit fly population values from two previous years without the combined use of both techniques. The treatments were: SIT + ABC, SIT, ABC, and Control, and each treatment was established in blocks of 5000 Ha separated by distances of 5-10 km. The evaluations were carried out through fruit sampling to assess percent parasitism and trapping of adult flies to obtain Flies per Trap per Day (FTD) values. The mean percentage of parasitism increased from 0.59% in the control treatment to 19.38% in the block with ABC. The FTD values decreased from ~0.129 and ~0.012 in the control block to 0.0021 in the block with SIT and ABC, representing a 98% suppression. The difference between the two periods in the control block was not significant. We conclude that the integration of both techniques resulted in an additive suppression of the pest population, supporting the use of both control techniques in an area-wide pest management context.

Male exposure to guava volatiles affects female remating behavior in Anastrepha fraterculus

AbstractPlant‐derived volatiles can have positive or negative effects on insects. Exposure to volatiles of guava (Psidium guajava L.) (Myrtaceae) enhances male mating success in the South American fruit fly, Anastrepha fraterculus (Wiedemann) (Diptera: Tephritidae), an important pest of various fruits. These volatiles can therefore be used as a post‐teneral, pre‐release supplement in sterile insect technique (SIT) programs. In SIT, mass‐reared sterile males compete for and mate with wild females, in order to reduce populations of insect pests. However, the success of SIT will be diminished if males are not able to inhibit females from remating with other (potentially wild) males. Here, we investigated the remating propensity of A. fraterculus females mated with males exposed to guava volatiles or non‐exposed (control) in choice and no‐choice tests. In addition, we tested whether exposure to guava volatiles affected male reproductive organ size (testes, accessory glands, and seminal vesicles) and cuticular hydrocarbon (CHC) profile. Females mated with guava‐exposed males were more likely to remate than females mated with control males, but there was no difference in the length of the refractory period (i.e., the time between the first and second mating event). Reproductive organ size did not differ between exposed and non‐exposed males, but exposed males had a higher quantity of waxes than non‐exposed males, and CHC profiles differed. The results are discussed in the context of SIT programs for the control of this insect pest.

Raspberry ketone feeding makes Queensland fruit fly, Bactrocera tryoni (Froggatt) more vulnerable to desiccation but not starvation.

Queensland fruit fly (Qfly) males exhibit accelerated sexual maturation when their diet is supplemented with raspberry ketone (RK) for 48 hours following emergence which is beneficial for Sterile Insect Technique (SIT) operation. The present study tests whether RK supplementation makes Qfly more vulnerable to starvation or desiccation. Flies were fed for 48 hours with yeast hydrolysate and sugar diet (1:3) that contained 0% RK (control), 1.25% RK (low dose) or 5% RK (high dose) to test subsequent vulnerability to starvation and desiccation. RK feeding decreased body weight and water content in males and increased lipid levels in both sexes before exposure to any stress treatment. Under nutritional stress, flies fed the low RK dose, but not the high RK dose, had higher survival than controls. Under desiccation stress, flies fed both the low and high RK doses had lower survival than the controls. Body weight, water content and lipid reserves at death were all affected by RK dose when under nutritional stress, but not when under desiccation stress. In the absence of stress, body weight at death was higher than controls in flies provided the high RK dose and lipids were lower than controls in flies provided the low RK dose. Feeding with RK makes Qflies more vulnerable to desiccation but not starvation. In most conditions, it is expected that the disadvantage of increased desiccation vulnerability would be outweighed by the benefits of accelerated sexual maturation in RK-fed young adult Qflies. This article is protected by copyright. All rights reserved.