Control Of Mosquito-borne Diseases Research Articles

Transgene removal using an in cis programmed homing endonuclease via single-strand annealing in the mosquito Aedes aegypti

While gene drive strategies have been proposed to aid in the control of mosquito-borne diseases, additional genome engineering technologies may be required to establish a defined end-of-product-life timeline. We previously demonstrated that single-strand annealing (SSA) was sufficient to program the scarless elimination of a transgene while restoring a disrupted gene in the disease vector mosquito Aedes aegypti. Here, we extend these findings by establishing that complete transgene removal (four gene cassettes comprising ~8-kb) can be programmed in cis. Reducing the length of the direct repeat from 700-bp to 200-bp reduces, but does not eliminate, SSA activity. In contrast, increasing direct repeat length to 1.5-kb does not increase SSA rates, suggesting diminishing returns above a certain threshold size. Finally, we show that while the homing endonuclease Y2-I-AniI triggered both SSA and NHEJ at significantly higher rates than I-SceI at one genomic locus (P5-EGFP), repair events are heavily skewed towards NHEJ at another locus (kmo), suggesting the nuclease used and the genomic region targeted have a substantial influence on repair outcomes. Taken together, this work establishes the feasibility of engineering temporary transgenes in disease vector mosquitoes, while providing critical details concerning important operational parameters.

Long non-coding RNAs as promising targets for controlling disease vector mosquitoes.

Hematophagous female mosquitoes are important vectors of numerous devastating human diseases, posing a major public health threat. Effective prevention and control of mosquito-borne diseases rely considerably on progress in understanding the molecular mechanisms of various life activities, and accordingly, the molecules that regulate the various life activities of mosquitoes are potential targets for implementing future vector control strategies. Many long non-coding RNAs (lncRNAs) have been identified in mosquitoes and significant progress has been made in determining their functions. Here, we present a comprehensive overview of the research advances on mosquito lncRNAs, including their molecular identification, function, and interaction with other non-coding RNAs, as well as their synergistic regulatory roles in mosquito life activities. We also highlight the potential roles of competitive endogenous RNAs in mosquito growth and development, as well as in insecticide resistance and virus-host interactions. Insights into the biological functions and mechanisms of lncRNAs in mosquito life activities, viral replication, pathogenesis, and transmission will contribute to the development of novel drugs and safe vaccines.

Entomological And Parasitological Indices Of Malaria Parasite Transmission In Imo State Polytechnic Omuma, Oru East, Nigeria

The study conducted between May and September of 2023 examined entomological and parasitological indicators of malaria parasite transmission in order to identify the predominant mosquito species and establish the malaria endemicity in the vicinity of Imo State Polytechnic Omuma. The Pyrethrum Spray Catch (PSC) was utilized to collect adult mosquitoes. Various mosquito species were gathered, categorized, and recognized. To detect the presence of a malaria parasite in the blood film stained with Giemsa stain, blood samples were taken from the same students whose rooms were utilized for the entomological survey during the parasitological study. Both the T-test and the Chi-square were used to assess the data. In all, 297 mosquitoes were detected in the research region (table 1), of which 230 Culex quinquefasciatus represented (77.4%) and 67 Anopheles gambiae (22.6%). The study was conducted at eight (8) sites. Comparing the results with the Anopheles gambiae recorded from the same site, Table 2b demonstrates that Culex quinquefasciatus had the highest prevalence and was substantially different at p<0.05. According to the results of the parasitological investigation, when 21 (42.0%) of the students residing in the hostel had their malaria parasite infection checked, 18 (85.7%) tested positive and 3 (14.3%) tested negative. When 29 students (58.0%) who were staying in the lodges had their malaria parasite infection checked, 23 (79.3%) of them tested positive, and 6 (20.7%) tested negative. Among the 50 learners randomly selected for parasitological analysis from the study’s school hostels and lodges, there was no significant difference (p>0.05) in the number of students who tested positive. The percentage of mosquito abundance and falciparum malaria presence found in this study are significant for public health. In order to develop strategies for the control of mosquito-borne diseases in Imo State Polytechnic Omuma and its surroundings, this study offers helpful baseline data.

Harnessing the power of mosquito gut microbiome in disease control

Mosquito-borne diseases pose an escalating threat to global public health, necessitating more effective strategies for vector control and disease prevention. Recent investigations into the mosquito gut microbiome unveil new avenues for symbiont-based control of mosquito-borne diseases, as it has been revealed that the mosquito gut microbiome has a profound impact on disease transmission. Accumulating evidence demonstrates that the mosquito gut microbiome plays a significant role in modulating infection by generating pathogen-targeting molecules, influencing the midgut physical barrier, shaping the biochemical environment, and regulating immune responses, and in mosquito ecology which affects disease transmission dynamics. Several proof-of-principle studies have explored some microbiome-based control measures, including supplementing the mosquitoes with microorganisms possessing anti-pathogen activities, immunizing hosts against pro-pathogen bacteria, and limiting mosquito larval development using bacteriophages. These strategies present promising efficacy, yet their practical implementation demands further scrutiny and assessment.

Seasonal Diversity in Mosquito Larval Ecology and Its Public Health Implications in Urban Slums of Lagos, Nigeria.

Mosquito-borne diseases (MBDs) are major public health burdens in tropical nations, including Nigeria. This study assessed mosquito larval species composition, abundance, and abiotic factors influencing mosquito breeding in slum communities of Lagos with the goal of informing MBD control measures. Three slum communities-Bariga, Makoko, and Ajegunle-were selected along with the nonslum community of Ikeja, which served as a control site. Larval sampling was done using the standard dipping technique between December 2021 and July 2022 across the dry and wet seasons. Mosquito larvae were raised to adults and identified using morphological keys and molecular assays. A total of 57,753 immature mosquitoes were collected from all study sites, with a significantly (P < 0.05) greater abundance in the dry season than the wet season. The majority (98.1%) of the mosquitoes collected belonged to Culex pipiens s.l., the only species found during the dry season in almost all locations. In the wet season, species identified from all sites combined were Anopheles gambiae ss., Anopheles arabiensis, Aedes aegypti, Aedes albopictus, Cx. pipiens s.l., and Lutzia tigripes, with Ajegunle having the greatest species diversity (H = 0.593). Among physicochemical parameters measured, only water temperature had a significant positive correlation (r = 0.934, P = 0.020) with larval densities in Ajegunle only. Permanent mosquito-breeding habitats had significantly greater larval densities than temporary habitats within the slum communities. These findings could inform the development of integrated vector control strategies that address the different species of mosquitoes in the fight against MBDs in urban slums.

The Imperative of a Comprehensive One Health Approach for Mosquito-Borne Disease Control in Indonesia

This review provides information on the importance of a comprehensive one-health approach for controlling mosquito-borne diseases in Indonesia. Despite the use of various control methods, Indonesia still faces numerous challenges and threats to its public health. The One Health paradigm offers a comprehensive framework for understanding and addressing the complexity of mosquito-borne diseases in Indonesia. Understanding the linkages between human, animal, and environmental health enables a deeper understanding of disease dynamics and facilitates the development of comprehensive strategies for disease prevention, control, and eradication. Important points that must be implemented immediately and receive attention from policymakers include strengthening vector surveillance and monitoring systems, strengthening vector control strategies in various sectors, increasing human health and behavioral capacity, effective communication and collaboration, and increasing infrastructure capacity and resource allocation. The sooner these issues are resolved, the more likely the goal of a mosquito-borne disease-free Indonesia can be achieved.

Detection of Wuchereria bancrofti through Molecular Xenomonitoring of Putative Mosquito Vectors in the Philippines

The advancement of molecular methods resulted in the application of molecular xenomonitoring (MX) as a complementary tool to detect the presence of circulating LF parasites in mosquito vectors. In the Philippines, a number of endemic areas are still undergoing evaluations by the World Health Organization (WHO) and Department of Health – Philippines (DOH). This study investigated the presence of filarial parasites in Oriental Mindoro (Region 4B) and Sultan Kudarat (Region 12), Philippines through MX. All collected and identified mosquito vectors were subjected to a real-time PCR assay targeting the Wuchereria bancrofti long-dispersed repeat Wb-LDR gene. This study detected the presence of W. bancrofti parasites in two species of mosquito vectors: (1) Aedes poicilius collected in Barangay Sta. Clara, Kalamansig, Sultan Kudarat (n=10 pools; 10.00%); and (2) Culex quinquefasciatus from Barangay Poblacion II, Victoria, Oriental Mindoro (n=344 pools; 0.29%) and Barangay Sta. Maria, Kalamansig, Sultan Kudarat (n=96 pools; 1.04%). These results support transmission assessment survey findings of continuous presence of LF parasites in the two endemic provinces of Oriental Mindoro and Sultan Kudarat. This study also suggests that further refinement of MX may produce broader applicability in the control and elimination of mosquito-borne diseases.

Classifying stages in the gonotrophic cycle of mosquitoes from images using computer vision techniques

The ability to distinguish between the abdominal conditions of adult female mosquitoes has important utility for the surveillance and control of mosquito-borne diseases. However, doing so requires entomological training and time-consuming manual effort. Here, we design computer vision techniques to determine stages in the gonotrophic cycle of female mosquitoes from images. Our dataset was collected from 139 adult female mosquitoes across three medically important species—Aedes aegypti, Anopheles stephensi, and Culex quinquefasciatus—and all four gonotrophic stages of the cycle (unfed, fully fed, semi-gravid, and gravid). From these mosquitoes and stages, a total of 1959 images were captured on a plain background via multiple smartphones. Subsequently, we trained four distinct AI model architectures (ResNet50, MobileNetV2, EfficientNet-B0, and ConvNeXtTiny), validated them using unseen data, and compared their overall classification accuracies. Additionally, we analyzed t-SNE plots to visualize the formation of decision boundaries in a lower-dimensional space. Notably, ResNet50 and EfficientNet-B0 demonstrated outstanding performance with an overall accuracy of 97.44% and 93.59%, respectively. EfficientNet-B0 demonstrated the best overall performance considering computational efficiency, model size, training speed, and t-SNE decision boundaries. We also assessed the explainability of this EfficientNet-B0 model, by implementing Grad-CAMs—a technique that highlights pixels in an image that were prioritized for classification. We observed that the highest weight was for those pixels representing the mosquito abdomen, demonstrating that our AI model has indeed learned correctly. Our work has significant practical impact. First, image datasets for gonotrophic stages of mosquitoes are not yet available. Second, our algorithms can be integrated with existing citizen science platforms that enable the public to record and upload biological observations. With such integration, our algorithms will enable the public to contribute to mosquito surveillance and gonotrophic stage identification. Finally, we are aware of work today that uses computer vision techniques for automated mosquito species identification, and our algorithms in this paper can augment these efforts by enabling the automated detection of gonotrophic stages of mosquitoes as well.

Application and prospects of gene editing in mosquito-borne disease control

Mosquito-borne diseases are a threat to about half of the worlds population, the harm caused by it has remained serious in recent years. However, traditional methods of insecticide and environmental control have not completely solved the mosquito problem, and many times they come with certain side effects on environment. At the biological and genetic level, it may be possible to find safe and sustainable ways to alleviate the burden of mosquito-borne pathogens. This study focuses on gene editing technology, introducing CRISPR technology and its development, reviewing recent advancements and their applications on kinds of illness like Dengue fever and COVID-19, discussing the future development of mosquito-borne disease control. Moreover, there are several limitations of CRISPR, to solve them, this review presents ideas that the CRISPR system can combine with Digital twin and achieve a better result in experiment. Against the backdrop of rapid developments in artificial intelligence, many cutting-edge technologies are expected to be applied to gene editing in the future.

Mosquito larvae exposed to a sublethal dose of photosensitive insecticides have altered juvenile development but unaffected adult life history traits

BackgroundLarvicides are critical for the control of mosquito-borne diseases. However, even sublethal exposure to a larvicide can alter development and life history traits, which can then affect population density and disease transmission dynamics. Photosensitive insecticides (PSIs) are a promising class of larvicide that are toxic when ingested and activated by light. We investigated whether the time of day when exposure occurs, or the process of pupation, affects larval susceptibility to PSI phototoxicity in the mosquito Anopheles gambiae, and whether sublethal exposure to PSIs alters life history traits.MethodsLarvae were treated with lethal concentrations of the PSIs methylene blue (MB) and rose bengal (RB), and larval survival was measured at various times of day. Additionally, larvae were exposed to two concentrations of each PSI that resulted in low and medium mortality, and the life history traits of the surviving larvae were measured.ResultsPupation, which predominantly occurs in the evening, protected larvae from PSI toxicity, but the toxicity of PSIs against larvae that had yet to pupate was unaffected by time of day. Larval exposure to a sublethal concentration of MB, but not RB, shortened the time to pupation. However, larval exposure to a sublethal concentration of RB, but not MB, increased pupal mortality. Neither PSI had a meaningful effect on the time to eclosion, adult longevity, or adult melanization potential.ConclusionsPSIs are lethal larvicides. Sublethal PSI exposure alters mosquito development, but does not affect adult life history traits.Graphical

Characterization of Anopheles funestus Larval Habitats in Fiyoni, Kwale County, Kenya: Insights into Malaria Vector Ecology and Control

The breeding of malaria-spreading vectors such as Anopheles funestus is influenced by various environmental factors that contribute indirectly to the transmission of the Plasmodium parasite. However, there is limited knowledge of larval habitat ecology that hinder prevention and control of mosquito-borne diseases. This study aimed to characterize larval habitats based on physicochemical and habitat characteristics, considering the abundance of A. funestus. A cross-sectional survey method was used to collect data on the established transects. Physical parameters (water temperature, pH, conductivity, and total dissolved solids) were measured using a 5-in-1 meter probe. Levels of chemical parameters (sulphate, COD, and BOD) were determined in the laboratory using standard methods. Observations were also made on habitat characteristics (including watercolor, habitat size, and canopy). There was significant effect (P&lt;0.05) of conductivity, pH, sulphate, COD, and BOD on the number of A. funestus larvae. Water samples with a high population of A. funestus larvae were found to have higher conductivity (Me of 470.5), TDS (Me = 235), and pH levels (Me of 6.71). Conversely, water samples with a high population of non-Anopheles funestus larvae were found to have higher COD (Me of 843.20), BOD (Me of 367.2), and SO4 levels (Me of 11.3). A significant correlation (p&lt;0.5) existed between A. funestus larvae and physical water parameters. For instance, Anopheles funestus larvae was high (Me of 36.85) in stagnant water and in semi-permanent water (Me of 47.37). The study demonstrates that both physicochemical and habitat parameters significantly influence the abundance of Anopheles funestus larvae in larval habitats. Parameters such as conductivity, pH, total dissolved solids, sulphate, COD, BOD, watercolor, depth, distance from the homestead, and habitat size were found to be important in determining the presence of A. funestus larvae. Therefore, vector control strategies should include larval source management by targeting rivers and other water bodies to prevent the emergence of Anopheles funestus.

Evaluation of the efficacy of commercially formulated aerosol based pyrethroid insecticides against &lt;i&gt;Culex&lt;/i&gt; species (Diptera: Culicidae)

Efforts to control mosquito borne diseases of serious public concern rely largely on the control of vectors using pyrethroids. Emergence of insecticide resistance makes control as well as choice of ideal insecticide difficult. Following standard guidelines effectiveness of twelve (12) commercially formulated aerosol insecticides against Culex species in Kano metropolis, Nigeria was evaluated for knockdown and mortality effect in a Peet-Grady chamber. Highest knockdown efficacy was observed with aerosol insecticide coded K11 containing; transfluthrin 0.15% and permethrin 0.23%, having KT50 of 3.7 ± 0.1 and KT95 of 10.0 ± 0.2 mins, which differ significantly (P&lt;0.05) with aerosol F6, aerosol H8 and aerosol J10. All insecticides with the exception of A1 expressed acceptable limit of mortalities (≥90) for effective aerosol insecticide. This study revealed that; number of active ingredients did not affect formulation performance. Permethrin and Transfluthrin were identified as the most effective pyrethroid isomers in the control of Culex mosquitoes. This study further revealed the susceptibility of Culex species of Kano metropolis to aerosol based pyrethroid insecticides. However, significantly low knockdown effect observed in some of the insecticides, suggests knockdown resistance (kdr) in the population of the tested mosquitoes. Constant monitoring of susceptibility status of other mosquito genera is recommended to address challenges that may face the control of mosquito borne diseases.

Expansions to the MGDrivE suite for simulating the efficacy of novel gene-drive constructs in the control of mosquito-borne diseases

ObjectiveThe MGDrivE (MGDrivE 1 and MGDrivE 2) modeling framework provides a flexible and expansive environment for testing the efficacy of novel gene-drive constructs for the control of mosquito-borne diseases. However, the existing model framework did not previously support several features necessary to simulate some types of intervention strategies. Namely, current MGDrivE versions do not permit modeling of small molecule inducible systems for controlling gene expression in gene drive designs or the inheritance patterns of self-eliminating gene drive mechanisms.ResultsHere, we demonstrate a new MGDrivE 2 module that permits the simulation of gene drive strategies incorporating small molecule-inducible systems and self-eliminating gene drive mechanisms. Additionally, we also implemented novel sparsity-aware sampling algorithms for improved computational efficiency in MGDrivE 2 and supplied an analysis and plotting function applicable to the outputs of MGDrivE 1 and MGDrivE 2.

Classification and identification of mosquitoes in China based on rDNA 28S D5 region

Accurate classification and identification of mosquitoes are essential for the prevention and control of mosquito-borne diseases. In this study, adult mosquitoes were collected from 15 cities across 14 provinces in China. They were identified morphologically with the dominant species determined. Furthermore, representative samples were identified at the molecular level based on rDNA 28S D5. In total, 880 adult mosquitoes were collected belonging to Culex (266), Aedes (473), Armigeres (13), and Anopheles (5). Aedes albopictus and “C. pipiens subgroup” were the dominant species. A total of 140 sequences of 28S D5 region (68 for “C. pipiens subgroup”, 51 for Ae. albopictus, 18 for Ar. subalbatus, and three for An. sinensis) ranging from 148 to 161 bp were obtained, with 100 % success of amplification and sequencing. Molecular identification were consistent with morphological classification. Sequence analysis showed that “C. pipiens subgroup” was identified into three clades: the traditional C. pipiens subgroup (Clade I), the newly discovered C. cf. perexiguus (Clade II), and C. new sp. (Clade III). Clade I contained the most abundant haplotypes (16) widely distributed without geographical differences. Clade II included six haplotypes that were aggregately distributed south of the Yangtze River. Only three sequences in Clade III showed two haplotypes with no geographical differences. Further morphological comparisons demonstrated differences in body color, beaks, and abdomens among the three clades. In conclusion, the rDNA 28S D5 region could effectively distinguish Culex, Aedes, Armigeres, and Anopheles species at the lower category level, demonstrating its potential as a mini-DNA barcode for mosquito identification.

The dynamics of deltamethrin resistance evolution in Aedes albopictus has an impact on fitness and dengue virus type-2 vectorial capacity

BackgroundWorldwide invasion and expansion of Aedes albopictus, an important vector of dengue, chikungunya, and Zika viruses, has become a serious concern in global public health. Chemical insecticides are the primary means currently available to control the mosquito populations. However, long-term and large-scale use of insecticides has selected for resistance in the mosquito that is accompanied by a genetic load that impacts fitness.ResultsA number of laboratory strains representing different resistance mechanisms were isolated and identified from laboratory-derived, deltamethrin-resistant Ae. albopictus recovered in previous work. Resistance levels and fitness costs of the strains were evaluated and compared to characterize the evolution of the resistance genotypes and phenotypes. The heterozygous F1534S mutation (1534F/S) in the voltage gated sodium channel (vgsc) gene product (VGSC), first detected in early stages of resistance evolution, not only confers high-level resistance, but also produces no significant fitness costs, leading to the rapid spread of resistance in the population. This is followed by the increase in frequency of homozygous F1534S (1534S/S) mosquitoes that have significant fitness disadvantages, prompting the emergence of an unlinked I1532T mutation with fewer side effects and a mating advantage better adapted to the selection and reproductive pressures imposed in the experiments. Metabolic resistance with no significant fitness cost and mediating a high-tolerance resistance phenotype may play a dominant role in the subsequent evolution of resistance. The different resistant strains had similar vector competence for dengue virus type-2 (DENV-2). Furthermore, a comparative analysis of vectorial capacity revealed that increased survival due to deltamethrin resistance balanced the negative fitness cost effects and contributed to the risk of dengue virus (DENV) transmission by resistant populations. The progressive evolution of resistance results in mosquitoes with both target-site insensitivity and metabolic resistance with lower fitness costs, which further leads to resistant populations with both high resistance levels and vectorial capacity.ConclusionsThis study reveals a possible mechanism for the evolution of deltamethrin resistance in Aedes albopictus. These findings will help guide practical strategies for insecticide use, resistance management and the prevention and control of mosquito-borne disease.

Prevention and control of mosquito-borne diseases across Toronto and Brasília: A document-based descriptive analysis

The present study analyzed documents by health departments in Toronto, Ontario, Canada and Brasília, Federal District, Brazil that address strategies for prevention and control of mosquito-borne diseases (MBDs) between 2012 and 2022. Nineteen documents were fully read and analyzed. Results suggest both Toronto and Brasília base their strategies on Integrated Vector Management, performing entomological, environmental, vector and epidemiological surveillance; source reduction; public education and mobilization; and chemical control as an ultimate resource. In addition, results indicate many documents lacked references lists with scientific evidence, although the most recent ones usually follow international protocols and national experiments results. Most do not teach workers on how to verify the effectiveness of the executed measures. Further research investigating people’s perceptions and behaviors towards MBDs can provide a more solid ground for effective health policy development, especially in cases of major public health concern, such as dengue and other neglected tropical diseases epidemics.

Enhancement of VGG16 model with multi-view and spatial dropout for classification of mosquito vectors.

Mosquitoes transmit pathogens that can cause numerous significant infectious diseases in humans and animals such as malaria, dengue fever, chikungunya fever, and encephalitis. Although the VGG16 model is not one of the most advanced CNN networks, it is reported that a fine-tuned VGG16 model achieves accuracy over 90% when applied to the classification of mosquitoes. The present study sets out to improve the accuracy and robustness of the VGG16 network by incorporating spatial dropout layers to regularize the network and by modifying its structure to incorporate multi-view inputs. Herein, four models are implemented: (A) early-combined, (B) middle-combined, (C) late-combined, and (D) ensemble model. Moreover, a structure for combining Models (A), (B), (C), and (D), known as the classifier, is developed. Two image datasets, including a reference dataset of mosquitoes in South Korea and a newly generated dataset of mosquitoes in Thailand, are used to evaluate our models. Regards the reference dataset, the average accuracy of ten runs improved from 83.26% to 99.77%, while the standard deviation decreased from 2.60% to 0.12%. When tested on the new dataset, the classifier's accuracy was also over 99% with a standard deviation of less than 2%. This indicates that the algorithm achieves high accuracy with low variation and is independent of a particular dataset. To evaluate the robustness of the classifier, it was applied to a small dataset consisting of mosquito images captured under various conditions. Its accuracy dropped to 86.14%, but after retraining with the small dataset, it regained its previous level of precision. This demonstrates that the classifier is resilient to variation in the dataset and can be retrained to adapt to the variation. The classifier and the new mosquito dataset could be utilized to develop an application for efficient and rapid entomological surveillance for the prevention and control of mosquito-borne diseases.

Tripartite interactions comprising yeast-endobacteria systems in the gut of vector mosquitoes.

It is shown that bacteria use yeast as a niche for survival in stressful conditions, therefore yeasts may act as temporary or permanent bacterial reservoirs. Endobacteria colonise the fungal vacuole of various osmotolerant yeasts which survive and multiply in sugar-rich sources such as plant nectars. Nectar-associated yeasts are present even in the digestive system of insects and often establish mutualistic symbioses with both hosts. Research on insect microbial symbioses is increasing but bacterial-fungal interactions are yet unexplored. Here, we have focused on the endobacteria of Wickerhamomyces anomalus (formerly Pichia anomala and Candida pelliculosa), an osmotolerant yeast associated with sugar sources and the insect gut. Symbiotic strains of W. anomalus influence larval development and contribute digestive processes in adults, in addition to exerting wide antimicrobial properties for host defence in diverse insects including mosquitoes. Antiplasmodial effects of W. anomalus have been shown in the gut of the female malaria vector mosquito Anopheles stephensi. This discovery highlights the potential of utilizing yeast as a promising tool for symbiotic control of mosquito-borne diseases. In the present study, we have carried out a large Next Generation Sequencing (NGS) metagenomics analysis including W. anomalus strains associated with vector mosquitoes Anopheles, Aedes and Culex, which has highlighted wide and heterogeneous EB communities in yeast. Furthermore, we have disclosed a Matryoshka-like association in the gut of A stephensi that comprises different EB in the strain of W. anomalus WaF17.12. Our investigations started with the localization of fast-moving bacteria-like bodies within the yeast vacuole of WaF17.12. Additional microscopy analyses have validated the presence of alive intravacuolar bacteria and 16S rDNA libraries from WaF17.12 have identified a few bacterial targets. Some of these EB have been isolated and tested for lytic properties and capability to re-infect the yeast cell. Moreover, a selective competence to enter yeast cell has been shown comparing different bacteria. We suggested possible tripartite interactions among EB, W. anomalus and the host, opening new knowledge on the vector biology.

Requirements for market entry of gene drive-modified mosquitoes for control of vector-borne diseases: analogies to other biologic and biotechnology products.

Gene drive-modified mosquitoes (GDMMs) are proposed as new tools for control and elimination of malaria and other mosquito-borne diseases, and promising results have been observed from testing conducted in containment. Although still at an early stage of development, it is important to begin now to consider approval procedures and market entry strategies for the eventual implementation of GDMMs in the context of disease control programs, as these could impact future research plans. It is expected that, as for other types of new products, those seeking to bring GDMMs to market will be required to provide sufficient information to allow the regulator(s) to determine whether the product is safe and effective for its proposed use. There already has been much emphasis on developing requirements for the biosafety components of the "safe and effective" benchmark, largely concerned with their regulation as genetically modified organisms. Other potential approval requirements have received little attention, however. Although GDMMs are expected to be implemented primarily in the context of public health programs, any regulatory analogies to other public health products, such as pharmaceuticals, vaccines, or chemical pesticides, must take into account the characteristics of live mosquito products. Typical manufacturing standards related to product identity, potency or quality will need to be adapted to GDMMs. Valuable lessons can be drawn from the regulatory approval processes for other whole organism and genetically modified (GM) organism products. Supply chain requirements, such as scale of production, location and design of production facilities, and methods of distribution and delivery, will be dependent upon the characteristics of the particular GDMM product, the conditions of use, and the region to be served. Plans for fulfilling supply chain needs can build upon experience in the development of other live insect products for use in public health and agriculture. Implementation of GDMMs would benefit from additional research on enabling technologies for long-term storage of mosquito life stages, efficient mass production, and area-wide delivery of GDMMs. Early consideration of these practical requirements for market entry will help to mitigate downstream delays in the development of these promising new technologies.

Larvicidal Activities of Allium sativum L. and Zingiber officinale Rosc. Extracts against Filariasis Vectors in Hadiya Zone, Ethiopia.

Mosquitoes present an immense threat to millions of people worldwide and act as vectors for filariasis disease. The objective of the study was to determine the effect of Allium sativum and Zingiber officinale extracts against filariasis vectors. The larvae were collected from the breeding site by using standard procedures for identification and larvicidal activities. Twenty grams (20 g) from each (Allium sativum and Zingiber officinale) were extracted separately by aqueous, ethanol, and methanol solvents. The phytochemical analysis was determined in the crude sample by using standard methods. Then, larvicidal effects were determined by introducing 10 larvae of the vectors to the concentrations of 250 ppm, 500 ppm, and 750 ppm of the crude sample, and data were subjected to probit analysis to determine the LC50 and Chi-squared test to check the significance of the mortality by R software. Anopheles funestus, Anopheles gambiae s.l., Anopheles pharoensis, Culex antennatus, and Culex quinquefasciatus were the filariasis vectors identified during the study period. The presence of phytochemical tests such as anthraquinones, flavonoids, glycosides, phenol, saponin, steroids, tannin, and terpenes was obtained. The larvicidal effects of the selected plant extracts ranged from 0%-100%. The lowest LC50 (53 ppm) was observed for A. sativum methanol test extract against Cx. quinquefasciatus. Ethanol extracts of A. sativum have a significant effect on An. funestus (X2 = 7.5, p = 0.02352) and Cx. quinquefasciatus (X2 = 10.833, p = 0.0.0044), whereas aqueous extracts have a significant effect only on An. gambiae s.l. (X2 = 7.0807, p = 0.029. Ethanol extracts of Z. officinale have a significant effect only on the mortality of An. pharoensis (X2 = 7.0807, p = 0.029), but methanol and aqueous extracts have no significant effect against filariasis vectors. In conclusion, A. sativum have a high toxic effect than Z. officinale extract against filariasis vectors in all type of solvents. So using those plant extracts is the best to reduce the risk of the synthetic chemical on nontarget organisms and the environment, in addition to the control of mosquito-borne diseases, but further studies will be conducted to evaluate the toxicity at different stages of the vectors.