Vector Signals

Briefing Document: Nanobody-Mediated Blocking of Malaria Transmission Targeting PfPIMMS43

Source: Excerpts from "s42003-025-08033-8.pdf" (A Nature Portfolio journal; https://doi.org/10.1038/s42003-025-08033-8)
Authors: Chiamaka Valerie Ukegbu, et al.
Date: Received - 04 December 2024 | Accepted - 02 April 2025 | Published - 30 April 2025

Executive Summary:

This study explores a novel strategy to block malaria transmission by targeting the Plasmodium falciparum protein PfPIMMS43 using single-domain VHH antibodies, also known as nanobodies. PfPIMMS43 is a critical surface protein for the parasite's development within the mosquito, specifically during the transition from ookinete to oocyst, and aids in evading the mosquito's immune response. Building on previous research demonstrating the potential of polyclonal antibodies against PfPIMMS43, this study successfully developed and characterized high-affinity nanobodies derived from llamas. These nanobodies were shown to significantly reduce both the intensity and prevalence of P. falciparum infection in Anopheles mosquitoes using both laboratory and field strains of the parasite. The study mapped the binding epitopes of the nanobodies to conserved regions in the second half of PfPIMMS43, confirming epitope accessibility. These findings establish PfPIMMS43 as a promising target for malaria transmission-blocking interventions and propose an innovative strategy utilizing genetically modified mosquitoes expressing these nanobodies in conjunction with gene drive technology for enhanced malaria control and elimination efforts.

Key Themes and Important Ideas:

1. Malaria Transmission as a Target: The study emphasizes the importance of targeting the parasite's development within the mosquito vector to interrupt the human-to-mosquito and mosquito-to-human transmission cycle. This is presented as a crucial approach to complement existing malaria control measures, especially in the face of challenges like insecticide failure, climate change, and funding limitations. The transition from ookinete to oocyst in the mosquito midgut is identified as a "key developmental bottleneck" for the parasite.
2. PfPIMMS43 as a Critical Transmission Target: The research highlights PfPIMMS43 as an "indispensable" surface protein for P. falciparum ookinetes and sporozoites. It is crucial for the ookinete-to-oocyst transition and plays a role in the parasite's ability to "evade the mosquito immune responses," specifically the complement-like system in the hemolymph. Previous studies, including those by the authors, had already indicated the potential of polyclonal antibodies targeting this protein in reducing transmission.
3. Nanobodies as a Promising Intervention Tool: The study focuses on the development and application of VHH domain nanobodies as an alternative and potentially superior approach to conventional antibodies for transmission blocking. Nanobodies, derived from camelids and sharks, are described as "smaller, more easily produced monoclonal, heavy-chain variable (VHH) domain antibodies." Their advantages include:

• "small size (~15 kDa)"
• "structural simplicity"
• "strong binding affinity"
• Easily bioengineered for targeting parasite antigens in mosquito vectors.

1. Development and Characterization of PfPIMMS43 Nanobodies: High-affinity nanobodies targeting PfPIMMS43 were successfully generated by immunizing llamas with recombinant PfPIMMS43. Nine nanobodies were selected based on variations in their antigen-binding regions (CDR1-3). Four nanobodies (G9, E5, C12, and E2) exhibited high nanomolar binding affinities to recombinant PfPIMMS43 (3, 5, 6, and 8 nM, respectively). These four nanobodies were also able to detect endogenous PfPIMMS43 protein expressed by P. falciparum ookinetes in infected mosquito midguts.
2. Significant Transmission Blocking Activity (TRA): The developed nanobodies demonstrated significant transmission-reducing activity in mosquito feeding assays.

• In standard membrane feeding assays (SMFAs) using laboratory P. falciparum NF54 and An. coluzzii mosquitoes, the four high-affinity nanobodies (G9, E5, C12, and E2) significantly reduced oocyst numbers at a concentration of 100 µg/ml, with reductions ranging from 83% to 99%. Oocyst reduction was concentration-dependent.
• In direct membrane feeding assays (DMFAs) using natural P. falciparum isolates from gametocytaemic children in Tanzania and local An. gambiae mosquitoes, G9 and E5 (the two nanobodies with the highest affinities to recombinant PfPIMMS43) also showed significant TRA, with reductions of 99% and 79% at 100 µg/ml, respectively. Both nanobodies significantly reduced mosquito infection prevalence in field conditions.

1. Epitope Mapping and Structural Insights: Epitope mapping revealed that the four nanobodies bind to "conserved regions in the second half of PfPIMMS43," specifically beyond amino acid residue 258. This suggests the C-terminal half of the protein is more immunogenic. G9 and E5 appear to recognize similar conformational epitopes, while C12 and E2 bind to distinct linear epitopes closer to the C-terminus. Homology modeling of the G9-PfPIMMS43 interaction suggests that G9 binds primarily through its CDR2 and CDR3 domains, interacting with residues in a β-sheet and an adjacent α-helix in the PfPIMMS43 structure. The model also supports the hypothesis that PfPIMMS43 is largely intrinsically disordered, potentially facilitating immune evasion.
2. Innovative Future Strategy: The study proposes a "novel approach" for malaria control: "genetically modifying mosquitoes to express nanobodies targeting key post-fertilization proteins such as PfPIMMS43, with the possibility of spreading these traits in wild populations via gene drive technologies." This strategy aims to overcome limitations of vaccine-induced antibodies, such as the challenge of achieving high and sustained titers against mosquito-stage antigens that are not naturally boosted in humans.
3. Conservation and Potential Challenges of PfPIMMS43: While PfPIMMS43 is largely conserved across P. falciparum isolates from Africa, the study notes the presence of single nucleotide polymorphisms (SNPs) in the epitope recognized by G9 and E5. These SNPs (M307I, L311I, L355H, L355P) have "high fixation indices and signatures of positive selection," suggesting potential evolutionary adaptations of the parasite to evade immune detection. Further investigation into the impact of these alleles on nanobody recognition is deemed necessary for field applications.

Supporting Quotes:

• "The transition from ookinete to oocyst is a critical step in the Plasmodium falciparum lifecycle and an important target for malaria transmission-blocking strategies."
• "PfPIMMS43, a surface protein of P. falciparum ookinetes and sporozoites, is critical for this transition and aids the parasite in evading mosquito immune responses."
• "Here, building on these findings, we have developed high-affinity single-domain VHH antibodies (nanobodies) derived from llama heavy-chain-only antibodies."
• "Importantly, they significantly reduce infection intensity and prevalence of laboratory and field strains of P. falciparum in An. coluzzii and An. gambiae, respectively."
• "These findings establish PfPIMMS43 as a promising transmission-blocking target."
• "To enhance mala...

Briefing Document: Nanofiber Encapsulation of Pseudomonas aeruginosa for Sustained Mosquito Larvicide Release

Date: Received - 13 December 2024 | Accepted - 04 April 2025 | Published - 21 April 2025
Source: Excerpts from "Nanofiber encapsulation of Pseudomonas aeruginosa for the sustained release of mosquito larvicides" https://doi.org/10.1038/s41598-025-97400-w

1. Executive Summary:

This study investigates a novel approach for mosquito vector control using nanofiber encapsulation of the bacterium Pseudomonas aeruginosa. The research addresses the inadequacy of current vector control strategies in eliminating mosquito-borne diseases by developing a method for the sustained release of bacterial larvicides. P. aeruginosa was selected for its potent larvicide production compared to other tested Pseudomonas species. The study demonstrates that encapsulating P. aeruginosa in electrospun nanofibers protects the bacteria, mimicking natural biofilms, enhances their survival in aquatic environments, and allows for prolonged larvicide production without harming non-target organisms (guppy fish). This nanotechnology-based method shows promise for controlling mosquito larvae in various breeding habitats over extended periods, potentially reducing application frequency and costs.

2. Background and Problem Statement:

• Mosquito-borne diseases (malaria, dengue, chikungunya, Zika, etc.) pose a significant global health threat, affecting hundreds of millions annually.
• Existing vector control strategies, primarily chemical insecticides and environmental management, are often insufficient for complete vector elimination.
• Increased insecticide resistance and environmental concerns associated with chemical methods necessitate the development of novel, sustainable approaches.
• Biological control using bacteria like Bacillus thuringiensis var. israelensis and Bacillus sphaericus offers a safer alternative, but their efficacy depends on persistence in the environment.
• Sustained-release formulations of microbial larvicides are highly desirable to reduce application frequency and costs.
• Conventional immobilization techniques for sustained release often suffer from limitations like low diffusion and reduced microbial viability.

"Despite the rising global incidence of vector-borne diseases such as malaria, dengue, chikungunya, and Zika, existing vector control strategies remain inadequate for completely eliminating vectors from their breeding sites."

3. Key Findings and Concepts:

• Superiority of Pseudomonas aeruginosa: Among the tested Pseudomonas species (P. fluorescens and P. putida), P. aeruginosa demonstrated the most potent larvicidal activity against four major mosquito vectors: Aedes aegypti, Culex quinquefasciatus, Cx. tritaeniorhynchus, and Anopheles stephensi.
• "During the initial screening, Pseudomonas aeruginosa proved to be more effective than the other two tested species, P. fluorescens and P. putida, in producing potent larvicides and was therefore selected for nanofiber encapsulation studies."
• Nanofiber Encapsulation Technique: Electrospinning was used to create a thin fibrous material at the nanoscale (1 nm - 1 µm) from Pluronic F127 dimethacrylate (F127-DM) and polyethylene oxide (PEO) to encapsulate and immobilize live P. aeruginosa bacteria.
• "In the present study, we rectified the shortcomings of conventional immobilization by developing a thin fibrous material at the nanoscale level (typically between 1 nm and 1 µm) using electrospinning to encapsulate and immobilize live bacteria."
• Protection and Sustained Release: Nanofiber encapsulation shields the bacterial cells from environmental stress, mimicking natural biofilms, thereby enhancing their survival and prolonging larvicide production. The cross-linking of the nanofibers prevents their rapid dissolution in water.
• "This study aimed to encapsulate larvicide-producing bacteria in nanofibers designed to shield bacterial cells from environmental stress—mimicking natural biofilms—thereby enhancing their survival in aquatic habitats and prolonging larvicide production."
• Efficacy in Batch Systems (Container Breeding Habitats): Nanofiber-encapsulated P. aeruginosa demonstrated sustained larvicidal activity in batch systems (simulating stagnant water bodies). The spent water containing released metabolites remained lethal to all four tested mosquito species for at least 8 days.
• "In the batch system, the spent water with metabolites of P. aeruginosa was lethal to all the tested species of larvae, such as Ae. aegypti, An. stephensi, Cx. tritaeniorhynchus, and Cx. quinquefasciatus, to varying degrees... The larvicidal potency of the spent water either remained the same as observed on the first day or increased during the subsequent days of incubation."
• Reduced Efficacy in Continuous Systems (Flowing Water Habitats): In continuous flow systems (simulating paddy fields or tanks with water inflow), the larvicidal efficacy of the released metabolites declined over subsequent days, suggesting this method might be less effective in such environments.
• "However, in a continuous system, although the 100% mortality of the tested larvae, Ae. aegypti and An. stephensi was recorded on the first day, but the efficacy declined on subsequent days."
• Optimal Nanofiber Quantity: 1 gram of P. aeruginosa-encapsulated nanofiber per 1 liter of water was sufficient to produce larvicidal metabolites capable of causing 100% mortality in Ae. aegypti larvae within 24 hours.
• "Our present study showed that even 1 g of P. aeruginosa-encapsulated nanofiber was sufficient to produce larvicidal exotoxin metabolites that can kill all the available mosquito larvae in 1 L of medium or water."
• Viability and Cell Washout: The encapsulated bacteria remained viable within the nanofibers, and no significant release or washout of free bacterial cells into the surrounding water was detected.
• "As there were no bacterial colonies appeared in the agar plates inoculated with the spent water of the nanofiber encapsulated P. aeruginosa, it is confirmed that there were no free bacteria released from the encapsulated fiber into the spent water..."
• Storage Stability: Nanofiber-encapsulated P. aeruginosa retained its larvicidal efficacy for up to one month when stored at 3°C. However, efficacy declined with longer storage periods.
• "The larvicidal efficacy of the spent water collected from the nanofibers stored for up to 1 month had killed 100% of the larvae of Ae. aegypti at 100% concentration... However, storage of bacteria-encapsulated fiber beyond 1 month shows declining efficacy..."
• Non-Target Organism Safety: The metabolites released by the nanofiber-encapsulated P. aeruginosa showed no mortality in guppy fish (Poecilia reticulata) at concentrations effective against mosquito larvae, indicating target-specificity.
• "No mortality was observed in the guppy fishes (P. reticulata) used as non-target organisms during the three days of the experiment..."
• Identified Larvicidal Compounds: GC-MS analysis of the spent water identified several compounds released by the encapsulated bacteria, with oleic acid, octadecanoic acid, and 2-methyl-Z,Z-3, 13-Octadecadienol being the major constituents. These compounds, including fatty acids, have been previously reported to hav...

Population Dependent Diffusion Model for Aedes Albopictus Spread in Europe

Source: Barman et al., "A climate and population dependent diffusion model forecasts the spread of Aedes Albopictus mosquitoes in Europe," Nature Portfolio journal, 2025, https://doi.org/10.1038/s43247-025-02199-z

Date: Received - 25 November 2024 | Accepted - 07 March 2025 | Published - 09 April 2025

Key Themes and Important Ideas/Facts:

This paper presents a novel spatio-temporal diffusion model that accurately forecasts the spread of Aedes albopictus mosquitoes in Europe by simultaneously considering climate suitability and human population factors. Ae. albopictus is a crucial vector for several arboviruses, including Dengue, Chikungunya, Zika, and Yellow Fever. The study highlights the increasing risk of autochthonous (local) transmission of these diseases in Europe due to the mosquito's expanding range, driven by environmental changes and global interconnectedness.

1. Predictable Spread of Ae. albopictus:

• The core finding is that the expansion of Ae. albopictus in Europe is predictable by integrating climate suitability and human population predictors within a single spatio-temporal diffusion model.
• The model demonstrates high accuracy in predicting areas of presence and absence (99% and 79% respectively).
• This predictability allows for anticipating future outbreaks by understanding the interplay between vector suitability and introduction.
• Quote: "These results show that the expansion of Ae. albopictus in Europe is predictable and provide a basis for anticipating future outbreaks in situations of dependent interacting co-drivers."

2. Drivers of Ae. albopictus Expansion:

• The study confirms that climate change (suitable climatic conditions), urbanization, and human population mobility are key factors facilitating the invasion of new habitats by this species.
• Quote: "Suitable climatic conditions favoured by climate change, urbanisation, and human populationmobility, seems to have facilitated the expansion of this invasive mosquito species into novel habitats."
• The passive transport of eggs through global travel and trade (e.g., used tires, lucky bamboo) and ground vehicles contributes significantly to its spread along transportation corridors.
• The mosquito's ecological and physiological plasticity (e.g., adaptation to cold, desiccation-resistant eggs, domestic container-breeding) enables its rapid and widespread expansion.

3. Model Development and Performance:

• The researchers developed a "highly predictive spatio-temporal vector diffusion model" that integrates climate suitability (temperature, humidity) and human population data.
• The model is a generalized additive mixed (GAM) model fitted within a Bayesian framework (INLA).
• It accounts for both short-range spread (geographical proximity) and potential long-range spread influenced by human population.
• The model demonstrates good overall performance, with AUC values around 0.80 for predicting new establishments in previously uncolonized areas.
• Quote: "Notably, model evaluation reveals that new introduction of Ae. albopictus into naïve areas, are very well predicted, which has not been achieved before with this type of model."
• Two versions of the model were calibrated: one using raw climate and population covariates, and another using a mechanistic mosquito life cycle model output as a covariate. Both showed similar predictive performance.

4. Key Covariates and Their Influence:

• Temperature: Median temperature (up to 24°C) shows a strong positive correlation with Ae. albopictus presence, decreasing at higher temperatures. Minimum temperature is positively correlated when median temperatures are high.
• Relative Humidity: Low relative humidity is negatively correlated with Ae. albopictus presence.
• Proximity: Geographical proximity to already established areas has a substantial impact on the spread, modeled through a spatio-temporal diffusion process.
• Human Population: Higher population density is associated with a higher likelihood of Ae. albopictus presence, likely reflecting increased introduction opportunities via human mobility, although the measured effect size was relatively small compared to climate factors.
• Human mobility modeled explicitly using a radiation model did not significantly improve model fit, suggesting that local diffusion and the human population covariate together can effectively capture its impact.

5. Implications for Public Health:

• The model can be a valuable tool for preparedness and response to Aedes-borne infections by identifying high-risk areas for new introductions.
• Quote: "This model can be integrated into early warning systems and help delineate areas at risk for the introduction and establishment of Ae. albo-pictus."
• Predictions can help target awareness and prevention messages to susceptible populations and guide vector control efforts.
• The model can also inform healthcare system preparedness for potential epidemics and the strategic deployment of available arboviral vaccines (Dengue and Chikungunya).
• The framework could be adapted for other regions and vector-borne diseases.

6. Limitations:

• Regional differences in Ae. albopictus surveillance efforts and data quality across Europe might influence the model's inferences.
• The use of yearly presence/absence data at a relatively coarse spatial resolution (NUTS3/GAUL) limits the granularity of the inference.
• The model showed slightly reduced predictive performance in the south-west geographical region (Iberian Peninsula), potentially due to a high number of regions with "no data."
• Quote: "Limitations of inferences drawn from the model come partly from Ae. albopictus surveillance, where we have regional differences in surveillance efforts across countries..."

7. Future Directions and Solutions:

• Addressing data gaps, particularly in regions with poor data quality, could improve model performance.
• Integrating data from Citizen Science initiatives like MosquitoAlert could complement traditional surveillance data and enhance model validation.
• The model framework can be further developed into operational early warning systems and climate services to enhance public health preparedness.

In Conclusion:

This research provides a significant advancement in our ability to predict the spread of Aedes albopictus in Europe. By simultaneously modeling climate suitability and human population dynamics within a diffusion framework, the developed model offers a robust and accurate tool for public health authorities to anticipate new establishments of this key vector and implement targeted interventions to mitigate the risk of arbovirus outbreaks. The findings underscore the importance of considering both environmental and human factors in understanding and predicting the spread of invasive disease vectors.

Parasite and Vector Circadian Clocks Mediate Efficient Malaria Transmission

Source: Bento et al., "Parasite and vector circadian clocks mediate efficient malaria transmission," Nature Microbiology, Published online 31 March 2025, https://doi.org/10.1038/s41564-025-01949-1

Date: Received: 03 September 2024 | Accepted: 26 March 2025 | Published: 04 April 2025

Key Themes:

This study uncovers a critical tripartite relationship between the Anopheles mosquito vector, the Plasmodium malaria parasite, and the mammalian host, highlighting the significant role of their respective circadian clocks in mediating efficient malaria transmission. The research demonstrates that both the mosquito salivary glands and the resident sporozoite parasite exhibit substantial circadian transcriptional activity, preparing them for the nocturnal blood-feeding behavior of the mosquito and subsequent host infection. The alignment of these circadian rhythms, particularly during nighttime, is shown to be crucial for maximizing transmission efficiency.

Most Important Ideas and Facts:

1. Circadian Rhythms in Mosquito Salivary Glands:

• Approximately half of the Anopheles stephensi mosquito salivary gland transcriptome displays circadian expression.
• Genes essential for efficient bloodmeals, such as those encoding anti-blood clotting factors (e.g., anophelin/cEF, aegyptin), vasodilators (Peroxidase 5B), and anti-inflammatory proteins (D7 long form L2), exhibit rhythmic expression, peaking around the times when mosquitoes prefer to feed.
• The study found that mosquitoes prefer to feed and ingest more blood at nighttime, as demonstrated by higher hemoglobin levels in their midguts after nocturnal feeding ("measuring haemoglobin levels, we demonstrate that mosquitos prefer to feed and ingest more blood at nighttime").
• Circadian clock genes (Clock, Cycle, Period, Vrille) are also rhythmically expressed in the salivary glands, suggesting an internal clock regulating these transcriptional changes.
• This rhythmic expression in salivary glands occurs independently of light/dark cycles (LD vs. constant dark DD), indicating genuine circadian control ("Genes in the salivary glands of infected mosquitos cycled independently of the mosquito light/dark schedule (LD versus DD), suggesting that they are under circadian control and that the time of day (rather than light) is the main driver of transcriptional fluctuations in mosquito salivary glands").
• Proteomics analysis confirmed rhythmic abundance of some salivary gland proteins, including those involved in glycolysis.

1. Circadian Rhythms in Salivary-Gland Sporozoites:

• A substantial subset (12-20%) of the Plasmodium berghei sporozoite transcriptome within the mosquito salivary glands also exhibits circadian cycling.
• This finding challenges the traditional view of salivary-gland sporozoites as transcriptionally quiescent ("Notably, we show a substantial subset of the salivary-gland-resident parasite transcriptome cycling throughout the day, indicating that this stage is not transcriptionally quiescent.").
• Sporozoite genes involved in motility, such as myosin A and thrombospondin-related sporozoite protein (TRSP), show rhythmic expression, potentially modulating their ability to initiate infection at different times of day ("Among the sporozoite genes undergoing rhythmic expression are those involved in parasite motility, potentially modulating the ability to initiate infection at different times of day.").
• Other cycling sporozoite genes include apical membrane antigen 1 (AMA1), circumsporozoite protein (CSP), and plasmepsin X, all implicated in invasion and host interaction.
• The rhythmic gene expression in sporozoites is not due to cell division within the salivary glands, as confirmed by EdU incorporation assays ("Taken together, our results show that the transcriptional daily rhythms identified in sporozoites are not a consequence of cell divi-sion but instead resemble a robust circadian rhythm.").

1. Alignment of Rhythms for Efficient Transmission:

• The study proposes a "circadian tripartite relationship" between the vector, parasite, and mammalian host that modulates malaria transmission efficiency.
• Increased mosquito biting and blood ingestion occur at nighttime, aligning with the peak expression of bloodmeal-related genes in the salivary glands.
• Sporozoite motility-associated genes peak in the early morning, potentially preparing them for transmission during the mosquito's nighttime feeding.
• Experimental infections in mice demonstrated that parasite load in the liver was significantly higher when infection was initiated during the nighttime (using both nighttime sporozoites and mice) compared to daytime infections. This effect was abolished when the rhythms of sporozoites and mice were mismatched ("We observed a reduced parasite load in the livers of mice when infection was initiated during the daytime (using daytime sporozoites and mice) compared with nighttime infections (using nighttime sporozoites and mice; Fig. 4f). By contrast, this increase in parasite load was abolished when the biological timing of the sporozoites and that of the mice were mismatched...").
• In vitro experiments with hepatocytes showed that parasite infection was significantly increased at night when the host cell circadian rhythms were synchronized with the time of sporozoite addition ("When the hepatocyte population was synchronous and matched the time with sporozoites, there was a significant increase in infection at night...").

1. Implications for Disease Intervention:

• Understanding this intricate circadian interplay could lead to novel strategies for combating malaria transmission, potentially by disrupting the synchronized rhythms.
• The findings may have broader implications for other vector-borne diseases like Zika and dengue, which might also exhibit daily rhythms in transmission.
• The timing of mosquito biting and the establishment of host infections are highlighted as important factors in malaria transmission biology.

Quotes Highlighting Key Findings:

• "Here we show that approximately half of the mosquito salivary gland transcriptome, particularly genes essential for efficient bloodmeals such as anti-blood clotting factors, exhibits circadian expression."
• "Notably, we show a substantial subset of the salivary-gland-resident parasite transcriptome cycling throughout the day, indicating that this stage is not transcriptionally quiescent."
• "Our findings suggest a circadian tripartite relationship between the vector, parasite and mammalian host that together modulates malaria transmission."
• "Together, our results suggest that the circadian regulation in both the mammalian host and the Anopheles mosquito vector has driven Plasmodium to evolve rhythms that align with both hosts, ensuring successful transmission."

Further Research Directions Identified:

• Dissecting the specific contribution of each player's physiological rhythms and clock mechanisms to the observed phenomenon.
• Investigating other layers of regulation (e.g., central brain-driven regulation) that shape the mosquito's feeding rhythm and influence salivary gland biology.
• Characterizing mosquito circadian rhythms across the lifespan and across multiple bloodmeals.
• Exploring the potential for tar...

Briefing Document: Radiation-Induced Alternative Splicing in Aedes aegypti Mosquitoes

Source: Bendzus-Mendoza, H., Rodriguez, A., Debnath, T., Bailey, C. D., Luker, H. A., & Hansen, I. A. (2025). Radiation exposure induces genome-wide alternative splicing events in Aedes aegypti mosquitoes. Scientific Reports, 15, 5885.

Date of Publication: Received - 19 June 2024 | Accepted - 14 March 2025 | Published - 24 March 2025

Key Themes and Important Ideas/Facts:

This study investigates the impact of ionizing radiation on alternative splicing events (ASEs) in male Aedes aegypti mosquitoes, a crucial aspect for improving the sterile insect technique (SIT). The researchers compared RNA sequencing data from male mosquitoes irradiated with a standard dose of 50 Grey (Gy) of X-rays to that of un-irradiated control mosquitoes. Their findings reveal that radiation exposure induces significant changes in alternative splicing patterns across the mosquito genome, affecting genes involved in key biological processes.

1. Background and Motivation:

• Aedes aegypti is a major vector for several deadly diseases, posing a significant public health threat. Controlling their populations is crucial.
• "Aedes aegypti, the yellow fever mosquito, is the primary vector of several deadly diseases, including yellow fever, dengue fever, chikungunya, and Zika[1]. These insects are a major public health threat in many parts of the world, especially in tropical and subtropical regions[2,3]."
• SIT, which involves releasing sterile males to mate with wild females, is an environmentally friendly method for controlling pest populations.
• "An alternative method for controlling Ae. aegypti populations is the sterile insect technique (SIT)."
• "SIT has been proven safe, effective, and environmentally friendly since it does not involve the use of pesticides. It is species-specific and does not make use of genetically modified organisms[10]."
• Ionizing radiation is the preferred method for sterilizing male insects in SIT programs, typically using doses between 5 and 300 Gy depending on the species. The optimal dose for Ae. aegypti is 50 Gy.
• "The use of ionizing radiation to sterilize male insects is the method of choice in many SIT programs[11]."
• "The optimal radiation dosage for sterilizing male Ae. aegypti has been previously determined to be 50 Gy[16]."
• A significant challenge with SIT is that while 50 Gy sterilizes males, it also reduces their fitness and lifespan, hindering their ability to compete with wild males.
• "While irradiating mosquitoes with 50 Gy causes complete sterility, it still reduces male fitness and lifespan[17]. This is a problem because sterile males must successfully compete with wild males for SIT to be effective."
• Previous research by the same group identified significant changes in gene expression (upregulation of DNA repair genes) in irradiated Ae. aegypti. This study focuses on the impact of radiation on alternative splicing.
• "To better understand the impact of radiation exposure on mosquitoes, we previously investigated changes in the transcriptome of irradiated male Ae. aegypti. We found dramatic changes in the transcriptome, specifically a robust up-regulation of the transcription of DNA repair genes..."

2. Key Findings on Alternative Splicing Events (ASEs):

• Radiation exposure significantly induced alternative splicing events in male Ae. aegypti.
• "The results of our analysis of our dataset confirmed our hypothesis that radiation-exposure triggers specific and significant ASEs in mosquitoes."
• The study identified 289 significantly differentially alternatively spliced events (FDR < 0.05 and |Δ PSI| > 0.1) in 181 unique genes after irradiation.
• "In total, we detected 15,016 ASEs among the transcriptomes of irradiated and non-irradiated mosquitoes... 289 of these ASEs were significantly differentially alternatively spliced between both groups... with 181 unique genes undergoing ASEs..."
• Five main types of ASEs were observed: alternative 3’ splice site (A3SS), alternative 5’ splice site (A5SS), mutually exclusive exon (MXE), retained intron (RI), and skipped exon (SE).
• "Specifically, 88 events were classified as alternative 3’ splice site events (A3SS), 104 as alternative 5’ splice site events (A5SS), 17 as mutually exclusive exon events (MXE), 12 as retained intron (RI) events, and 68 as skipped exon events (SE)..."
• A3SS, A5SS, and SE were the most prevalent types of ASEs induced by radiation. RI was the least common.
• "Of these five, A3SS, A5SS, and SE are by far the most prevalent after irradiation treatment... In support of this notion, we found that RI is indeed the rarest type of ASE in our Ae. aegypti dataset."
• These radiation-induced ASEs occurred genome-wide, affecting genes on all three chromosomes of Ae. aegypti.
• "The genes that undergo significant ASEs after irradiation were mapped to the three chromosomes of Ae. aegypti... We found that all five ASEs occurred genome wide."

3. Functional Analysis of Alternatively Spliced Genes (ASGs):

• Functional analysis of the 181 ASGs revealed their involvement in diverse biological processes, including:
• Actin filament binding and other cytoskeletal-related processes.
• Ubiquitin signaling processes (thiol-dependent deubiquitinase, deubiquitinase activity).
• Calcium ion binding.
• Phosphotransferase, kinase, and transferase activity.
• Nucleotide and nucleoside phosphate biosynthetic processes.
• Intracellular signal transduction, response to stimulus, and regulation of cellular processes.
• KEGG pathway analysis highlighted significant enrichment in the:
• Hippo signaling pathway: Involved in cell proliferation and apoptosis, and linked to radiation resistance in tumors. One identified ASG in this pathway is PP2A.
• "Among the identified pathways, the HIPPO signaling pathway... emerged as the most abundant. These pathways play crucial roles in various cellular processes such as signal transduction, response to stimuli, and regulation of cellular activities[44–46]... One of the alternatively spliced genes that we identified in this pathway is PP2A, which is part of a regulatory complex within the Hippo pathway[53]."
• Purine metabolism: Regulates DNA repair and has been implicated in promoting radiation resistance.
• "The purine metabolism pathway regulates DNA repair, has been associated with cancer therapy, and implicated to promote radiation resistance[54,55]."
• Notch signaling pathway: Involved in cell fate and associated with tumor cell proliferation and radiation resistance. NCSTN, an important regulator protein in this pathway, was found to be alternatively spliced.
• "The Notch signaling pathway, another conserved pathway involved in cell fate, has been associated with tumor cell proliferation, and radiation resistance[56,57]. An important regulator protein, NCSTN[58], is also alternatively spliced upon radiation exposure."

4. Overlap with Differentially Expressed Genes (DEGs):

• A cross-analysis with a previous study that identified DEGs after irradiation revealed that only a small number of genes (16) were both differentially expressed and alternatively spliced.
• "As previously described by Pinch et al.[18], 149 genes were significantly differentially expressed between the non-irradiated and irradiated Ae. aegypti...

Briefing Document: Targeting the Mosquito Prefoldin–Chaperonin Complex to Block Plasmodium Transmission

Citation: Dong, Y., Kang, S., Sandiford, S.L. et al. Targeting the mosquito prefoldin–chaperonin complex blocks Plasmodium transmission. Nat Microbiol (2025). https://doi.org/10.1038/s41564-025-01947-3

Date: Received - 22 November 2024 | Accepted - 27 January 2025 | Published - 06 March 2025

Overview:

This study investigates the role of the conserved Anopheles mosquito prefoldin (PFDN)–chaperonin (CCT/TRiC) system as a potential target for blocking the transmission of multiple Plasmodium species. The researchers demonstrate that disrupting this protein folding complex in mosquitoes, either through gene silencing or antibody inhibition, significantly reduces Plasmodium infection intensity and prevalence. The mechanism of action involves compromising the integrity of the mosquito midgut, leading to immune activation and the disruption of the parasite's immune evasion strategies. The findings suggest that the PFDN–chaperonin complex, particularly the PFDN6 subunit, holds promise as a multispecies transmission-blocking vaccine (TBV) target.

Main Themes and Important Ideas/Facts:

• The Mosquito PFDN–Chaperonin Complex is Essential for Plasmodium Infection:
• The Plasmodium infection cycle in mosquitoes relies on various host factors in the midgut.
• The mosquito prefoldin complex is crucial for the proper folding of proteins and macromolecular complexes, including actin and tubulin, which are essential for cell division, motility, cytoskeletal stability, and signal transduction – all of which influence Plasmodium infection.
• Silencing any of the six PFDN subunits (Pfdn1-6) or the CCT4 subunit via RNA interference significantly reduced Plasmodium falciparum oocyst loads in the Anopheles gambiae midgut.
• "Silencing any prefoldin subunit or its CCT/TRiC partner via RNA interference reduces Plasmodium falciparum oocyst loads in the mosquito midgut..."
• Co-silencing of different PFDN subunits did not have an additive effect, confirming that the complex functions as a unit in supporting parasite development.
• Targeting PFDN6 with Antibodies Blocks Plasmodium Transmission:
• Co-feeding mosquitoes with a PFDN6-specific antibody along with P. falciparum gametocytes resulted in a potent suppression of parasite infection at both the oocyst and sporozoite stages.
• "Ingestion of purified anti-PFDN6 polyclonal antibody (IgG) resulted in a significant decrease in parasite loads (either at the oocyst or sporozoite stage) compared with control cohorts fed on rabbit anti-GFP antibody..."
• The level of inhibition achieved with anti-PFDN6 antibodies was comparable to that of leading TBV candidates like Pfs230 and Pfs25, as well as antibodies targeting mosquito proteins AgAPN1 and FREP1.
• Anti-PFDN6 antibody also effectively blocked P. falciparum transmission in Anopheles stephensi and Plasmodium vivax transmission in Anopheles dirus, indicating a broad-spectrum effect across different mosquito and parasite species.
• Active immunization of mice with recombinant PFDN6 protein resulted in antibodies that, when mosquitoes fed on the immunized, infected mice, significantly reduced Plasmodium berghei oocyst infection intensity and prevalence, supporting its potential as a TBV target.
• PFDN Supports Plasmodium Development After Ookinetes Invade the Midgut Epithelium:
• Antibody blocking assays showed no significant difference in ookinete numbers in the midgut lumen at 24 hours post-infection, but a significant decrease in oocyst loads was observed at 36 hours and 8 days.
• Injection of anti-PFDN6 antibody into the mosquito haemolymph also reduced oocyst numbers, suggesting an effect on the basal side of the midgut epithelium where oocysts develop.
• "These results indicate that PFDN6 host factor function is exerted upon ookinete egress and oocyst formation on the basal side of the epithelium beneath the basal lamina."
• PFDN6 distribution largely overlapped with actin in the midgut epithelium, but it did not co-localize directly with the parasites, suggesting an indirect role in parasite development.
• Disruption of PFDN Compromises Midgut Integrity and Triggers Anti-Plasmodium Immunity:
• Attempting to create a Pfdn6 knockout mosquito line resulted in pre-adult lethality, likely due to cytoskeletal and gut integrity issues.
• Co-immunoprecipitation assays identified interactions between PFDN6 and actin, tubulin, and several extracellular matrix proteins, supporting its role in maintaining cellular and matrix integrity.
• Silencing Pfdn6 or co-feeding with anti-PFDN6 antibodies led to a "leaky gut," characterized by increased permeability and bacterial leakage from the midgut lumen into the haemolymph.
• "Interfering with the PFDN–CCT/TriC chaperonin complex results in a cascade of events, including compromised gut integrity and disrupted extracellular matrix organization. The increased gut permeability leads to bacterial leakage and systemic infection, ultimately augmenting antiplasmodial defences..."
• Bacterial leakage triggered a stronger anti-Plasmodium immune response, with upregulation of key immune factors like Tep1, FBN9, and LRRD7.
• The leaky gut condition also shortened mosquito lifespan in the presence of natural microbiota.
• PFDN Facilitates Parasite Immune Evasion Through Laminin:
• PFDN appears to be involved in maintaining the laminin coating on ookinetes and early oocysts, a basal lamina component previously shown to be important for parasite development and immune evasion.
• Silencing laminin resulted in a significant decrease in oocyst numbers, similar to Pfdn6 silencing.
• In Pfdn6-silenced mosquitoes, the laminin coating on parasites was compromised, leading to increased co-localization with the anti-Plasmodium factor Tep1.
• Co-silencing of Pfdn6 and Tep1 negated the individual effects on parasite infection, suggesting that PFDN's host factor role involves limiting parasite accessibility to Tep1.
• "These processes also compromise the parasite’s laminin-based immune evasion mechanism, enabling the primed immune system to attack it effectively."
• PFDN as a Potential Transmission-Blocking Vaccine Target:
• The high conservation of PFDN6 across mosquito species and its role as a host factor for multiple Plasmodium species make it a promising TBV target.
• In silico analysis identified a potential unique B cell epitope on A. gambiae PFDN6 with low homology to human PFDN6, which could be explored to minimize cross-reactivity concerns.
• The authors suggest that a multivalent vaccine targeting several PFDN–CCT/TriC complex epitopes could be a strategy to mitigate potential selective pressure.
• Conditional genetic inactivation of the PFDN–CCT/TriC complex via gene-drive mechanisms is also proposed as a potential malaria control strategy.

Quotes Highlighting Key Findings:

• "Here we show that the PFDN–CCT/TriC (T-complex protein ring complex) chaperonin complex serves as a host factor system for multiple Plasmodium species in all tested Anopheles species."
• "In summary, we show here that the role of the PFDN–CCT/TriC chaperonin complex Plasmodium host factor is based on maintaining midgut epithelial and extracellular matrix and basal lamina integrity that (1) limits the leakage of midgut microbiota into the haemolymph, where it can prime anti-Plasmodium immunity; (...

Briefing Document: Engineered Symbionts for Concurrent Malaria and Arbovirus Transmission Control

Citation: Hu, W., Gao, H., Cui, C. et al. Harnessing engineered symbionts to combat concurrent malaria and arboviruses transmission. Nat Commun 16, 2104 (2025). https://doi.org/10.1038/s41467-025-57343-2

Dates: Received - 27 July 2024 | Accepted - 19 February 2025 | Published - 01 March 2025

Prepared for: Saleh Lab

Executive Summary:

This research presents a novel paratransgenesis strategy utilizing engineered symbiotic bacteria (Serratia AS1) in mosquitoes to simultaneously combat the transmission of malaria parasites (transmitted by Anopheles mosquitoes) and arboviruses like dengue and Zika (transmitted by Aedes mosquitoes). The study demonstrates the successful engineering of Serratia AS1 to express anti-Plasmodium and anti-arbovirus effector proteins under the control of a blood-induced promoter. Both laboratory and semi-natural field-cage experiments show that these engineered bacteria effectively spread through mosquito populations and significantly inhibit pathogen infections in both Anopheles and Aedes mosquitoes, including reducing co-infection rates of dengue and Zika viruses. This work lays the groundwork for a promising tool to address the growing challenge of concurrent mosquito-borne disease outbreaks.

Main Themes and Important Ideas/Facts:

1. The Growing Threat of Concurrent Mosquito-Borne Diseases:

• More than half of the global population lives in areas at risk of contracting two or more major mosquito-borne diseases.
• The geographic distributions of Anopheles (malaria) and Aedes (arboviruses) mosquitoes are increasingly overlapping, leading to co-existence and potential co-infection scenarios.
• Co-infections of malaria and arboviruses (dengue, Zika, chikungunya) have been reported in various regions.
• Co-circulation and simultaneous co-infection of multiple arboviruses are also prevalent.
• These concurrent infections pose complex challenges for disease surveillance, diagnosis, and treatment.
• "The overlapping distributions highlight the inevitable co-existence and potential co-infection of malaria and arboviruses, or multiple arboviruses, in single host. Such scenarios pose complex and multifaceted public health challenges."

1. Limitations of Current Mosquito Control Strategies:

• Reliance on mosquito management is hampered by widespread insecticide resistance.
• Behavioral changes in Anopheles mosquitoes (e.g., outdoor biting) and antimalarial drug resistance have stalled progress in malaria control.
• Current global outbreaks highlight the inadequacies of existing control tools.
• "This situation underscores the urgent need for innovative intervention strategies to tackle the concurrent transmission of malaria and arbovirus diseases."

1. Paratransgenesis as a Promising Innovative Strategy:

• Paratransgenesis, utilizing genetically manipulated symbionts to inhibit pathogens, is an attractive approach.
• While promising for malaria, its effectiveness against arboviruses and its potential to simultaneously target both requires further exploration.
• "Here, we explore the potential of parastransgenesis strategy to concurrently inhibit the transmission of Plasmodium and arboviruses by Anopheles and Aedes mosquitoes, respectively."

1. Identification and Engineering of Serratia AS1 as a Multifunctional Symbiont:

• The symbiotic bacterium Serratia AS1 efficiently spreads through both Anopheles and Aedes mosquito populations (horizontally and vertically).
• Laboratory cage experiments confirmed efficient spread in An. stephensi and Ae. aegypti.
• Serratia AS1 proliferates in the mosquito midgut after a blood meal without significant fitness costs to the mosquitoes.
• Wild-type Serratia AS1 did not significantly affect dengue virus infection in Ae. aegypti, making it a suitable chassis for engineering.
• "...indicating that Serratia AS1 can efficiently spread throughout these two mosquito populations."
• "...without causing obvious negative impact on fitness costs in both the mosquito species..."

1. Development of Engineered Serratia AS1 Strains Expressing Anti-Pathogen Effectors:

• The study engineered Serratia AS1 to co-express anti-Plasmodium peptides (Shiva1, scorpine) and anti-arbovirus peptides (DN59, Z2) using the efficient HlyA secretion system.
• The initial construct (AS1-DK) with constitutive expression (using pnptII promoter) showed strong inhibition of P. berghei in Anopheles and DENV2 in Aedes.
• "We found that AS1-DK strongly inhibited P. berghei ANKA (Pb ANKA) development in An. stephensi mosquitoes...and DENV2 infection in Ae. aegypti mosquitoes."

1. Importance and Identification of a Blood-Induced Promoter (LipA):

• Constitutive expression of effectors can impose fitness burdens on bacteria and potentially affect mosquitoes or lead to resistance.
• Blood-inducible promoters are advantageous as effector expression is activated only upon blood meal ingestion, coinciding with pathogen entry.
• A comprehensive analysis of Serratia transcriptional and proteomic data identified several blood-induced promoters.
• The LipA promoter demonstrated the strongest inducible activity and the highest stringency for blood-induced expression.
• "Notably, the LipA promoter showed the strongest activity, coupled with the highest stringency for blood-induced expression."

1. Enhanced Pathogen Inhibition with LipA-Controlled Effector Expression (AS1-TK):

• An engineered strain (AS1-TK) utilizing the LipA promoter to drive the expression of scorpine, Shiva1, DN59 (two copies), and Z2 (two copies) was developed.
• AS1-TK strongly inhibited P. falciparum and P. berghei infection in Anopheles.
• AS1-TK effectively inhibited both DENV2 and Zika virus infections in Ae. aegypti.
• AS1-TK did not impose obvious fitness costs in either mosquito species.
• "The recombinant strain AS1-TK strongly inhibit development of the human malaria parasite P. falciparum NF54 infection...and rodent malaria parasite P. berghei ANKA...in An. stephensi mosquitoes. Moreover, AS1-TK effectively inhibit both DENV2...and Zika virus infections...in Ae. aegypti mosquitoes..."

1. Efficacy of AS1-TK in Semi-Natural Field-Cage Conditions:

• Outdoor field-cage experiments demonstrated that AS1-TK efficiently colonized both An. stephensi and Ae. aegypti mosquitoes under semi-natural conditions.
• Colonized female mosquitoes showed strong inhibition of P. berghei infection.
• AS1-TK effectively inhibited all four serotypes of dengue viruses and Zika virus infection in Ae. aegypti in the field-cage setting.
• "The outdoor field-cage experiments showed that the recombinant AS1-TK strain efficiently colonized both An. stephensi and Ae. aegypti mosquitoes..."

Briefing Document: Heat Exposure and Mosquito-Virus Interaction

Source: Perdomo, H. D., Khorramnejad, A., Cham, N. M., Kropf, A., Sogliani, D., & Bonizzoni, M. (2025). Prolonged exposure to heat enhances mosquito tolerance to viral infection. Communications Biology, 8(1), 761. https://doi.org/10.1038/s42003-025-07617-8

Dates: Received - 27 September 2024 | Accepted - 28 January 2025 | Published - 04 February 2025

Prepared for: Saleh Lab

Executive Summary:

This study investigates the impact of increased environmental temperature on the interaction between the mosquito species Aedes albopictus and the cell fusing agent virus (CFAV), an insect-specific virus. The researchers examined how short-term (one generation) and long-term (ten generations) exposure to a higher temperature (32°C/26°C day/night cycle) influences mosquito tolerance and resistance to CFAV infection, as well as their overall fitness. The key findings reveal that prolonged heat exposure leads to increased viral tolerance in mosquitoes without significant fitness costs, while short-term heat exposure results in increased resistance but at the expense of mosquito fitness. These findings have significant implications for understanding the effects of climate change on arbovirus transmission dynamics and the evolution of both mosquito vectors and the viruses they carry.

Main Themes and Important Ideas/Facts:

• Climate Change and Arbovirus Transmission:
• The study highlights the complex interplay between climate change, mosquito biology, and virus interactions, emphasizing the relevance of this research to the prevalence and transmission dynamics of arboviruses like dengue, Zika, and chikungunya, which threaten billions globally.
• "Current anthropogenic climate change has profound and complex impli-cations for the prevalence and the transmission dynamics of arboviruses such as dengue, Zika and chikungunya, which are an impending risk for 3.9 billion people in tropical and subtropical areas of the world."
• Rising temperatures are expected to shift the distribution and phenology of key arboviral vectors, Aedes aegypti and Aedes albopictus, further expanding disease risk.
• While increased temperature is known to accelerate viral replication in mosquitoes, the impact of temperature on the mosquito's response to viruses has been largely unexplored.
• Resistance vs. Tolerance in Mosquito Immunity:
• The study distinguishes between two key immunological strategies in mosquitoes: resistance (limiting viral replication) and tolerance (controlling the cost of infection without reducing viral load).
• The researchers employed a framework developed in plant pathology using reaction norms (measuring host longevity across pathogen doses) to differentiate between resistance and tolerance.
• Resistance exerts strong selective pressure on viruses, while tolerance has neutral or even positive effects on them. Understanding the shift between these responses due to climate change is crucial for predicting viral transmission dynamics and long-term evolutionary impacts.
• Experimental Design: Thermal Acclimation vs. Evolution:
• The study mimicked both acute thermal fluctuations (heat waves) and gradual, prolonged warming by exposing Aedes albopictus mosquitoes to a hot thermal regime (32°C for 14 hours, 26°C for 10 hours) for one generation (warm-acclimated) or ten generations (warm-evolved).
• These groups were compared to a control group maintained under standard laboratory conditions (28°C). All infected mosquitoes were kept at their rearing temperature post-infection.
• Prolonged Heat Exposure Enhances Viral Tolerance:
• Warm-evolved mosquitoes demonstrated increased tolerance to CFAV infection, meaning they could withstand higher viral loads without significant reductions in longevity.
• "We show that the length of the thermal challenge influences the outcome of the infection with warm-evolvedmosquitoes beingmore tolerant to CFAV infection..."
• Tolerance curves showed that warm-evolved mosquitoes had comparable vigour (survival time of uninfected hosts) to the standard group and the lowest severity (longevity at the highest viral load).
• "Multi-generational exposure to heat imposes no cost on CFAV infected mosquitoes."
• Short-Term Heat Exposure Enhances Viral Resistance but with Fitness Costs:
• Warm-acclimated mosquitoes exhibited higher resistance to CFAV, showing lower viral prevalence and load at 3 days post-infection compared to both warm-evolved and standard mosquitoes.
• "...warm-acclimatedmosquitoes being more resistant..."
• However, this increased resistance came at a cost, as warm-acclimated mosquitoes displayed reduced longevity and fecundity, and increased sterility and infertility, even in the absence of viral infection, suggesting the thermal challenge itself is stressful.
• "One generational exposure to heat...results in fitness costs and increased resistance to viral infection."
• Implications for Arbovirus Transmission:
• The shift towards tolerance in warm-evolved mosquitoes has significant implications for arbovirus transmission. While they may control viral replication less effectively (lower resistance), their ability to survive infection with higher viral loads could increase the likelihood of transmission over a longer period.
• "Increased viral tolerance raises the likelihood of viral trans-mission in warm-evolved mosquitoes."
• The study suggests that the duration of heat exposure is a critical factor in determining mosquito immune response and, consequently, arbovirus transmission dynamics.
• Implications for Biological Control using Insect-Specific Viruses (ISVs):
• The findings that temperature modulates CFAV prevalence and load have implications for the potential use of CFAV as a biological control agent against arboviruses.
• Short-term heat fluctuations could negatively impact CFAV prevalence in mosquito populations, while prolonged warming might favor its persistence.
• "Considering the potential application of CFAV as a biological control agent, our results support the conclusion that global warming could favour CFAV persistence, but unexpected thermal fluctuations could negatively impact its prevalence in mosquito populations."
• Evolutionary Consequences of Resistance and Tolerance:
• The study discusses the contrasting evolutionary pressures exerted by resistance and tolerance mechanisms on both mosquitoes and viruses.
• Resistance can drive an "arms race" leading to rapid molecular evolution.
• Tolerance, by minimizing the cost of infection, may reduce selective pressure on viruses, leading to different evolutionary trajectories.
• The interplay and potential trade-offs between resistance and tolerance mechanisms are crucial for understanding the long-term co-evolution of vectors and viruses.

Quotes:

• "These results highlight the importance of considering fluctuations in vector immunity in relation to the length of a thermal challenge to understand natural variation in vector response to viruses and frame realistic transmission models."
• "Exposure to heat increases overall Ae. albopictus tolerance to CFAV infection, but the length of the thermal challenge alters mosquito fitness and influences the immunological response to CFAV."
• "Thermal adaptation includesprofoundand...

Briefing Document: NSm Protein's Critical Role in Bunyavirus Transmission

Citation: Terhzaz, S., Kerrigan, D., Almire, F. et al. NSm is a critical determinant for bunyavirus transmission between vertebrate and mosquito hosts. Nat Commun 16, 1214 (2025). https://doi.org/10.1038/s41467-024-54809-7

Date: Received - 03 May 2024 | Accepted - 21 November 2024 | Published - 31 January 2025

Prepared for: Saleh Lab

Key Themes:

• NSm as a Unique Feature of Vector-Borne Bunyaviruses: The study establishes a strong correlation between the presence of the non-structural protein NSm and the ability of bunyaviruses to be transmitted by arthropod vectors (arboviruses). Non-arboviral bunyaviruses do not encode NSm.
• NSm's Dispensability in Monolayer Cell Cultures: While essential for in vivo transmission, NSm is shown to be largely dispensable for viral replication in both mammalian and mosquito cell lines in vitro.
• NSm's Crucial Role in Mosquito Midgut Infection and Dissemination: The research demonstrates that NSm is absolutely required for Bunyamwera virus (BUNV) to successfully infect the female Aedes aegypti mosquito following a blood meal. Specifically, it is critical for cell-to-cell spread and egress from the mosquito midgut, a major barrier to viral infection.
• Midgut-Specific Requirement: The necessity of NSm is specific to the midgut. When this barrier is bypassed through intrathoracic injection, the NSm deletion mutant (BUN-ΔNSm) can effectively infect other mosquito tissues, including the salivary glands, and be transmitted in saliva.
• NSm Facilitates Infection Foci Formation: In the midgut, BUNV-wt infection leads to the formation of large clusters of infected cells (infection foci), while BUN-ΔNSm remains confined to initially infected cells and fails to spread.
• NSm Acts Early and Transiently at the Periphery of Infection Foci: Immunocytochemistry reveals that NSm protein is localized mainly at the periphery of infection foci in the midgut, suggesting an early and transient role in spreading to neighboring cells. It is also found on the surface of infected cells and at cell junctions.
• Convergent Evolution of NSm: Phylogenetic analysis suggests that NSm has likely been acquired independently multiple times during the evolution of different bunyavirus families, indicating convergent evolution driven by the selective pressure of vector-borne transmission.
• Implications for Vaccine Development: The identification of NSm as a critical determinant for mosquito infection and transmission suggests that viruses lacking NSm could be promising candidates for live-attenuated vaccines against mosquito-borne bunyaviruses, as they would have a reduced risk of spillover in vectors.

Most Important Ideas and Facts:

• Exclusive Presence in Arboviruses: "Interestingly, this revealed a strong correlation between the presence of NSm and bunyaviruses vectored by arthropods (Fig. 1). Indeed, NSm is present in arbo-bunyaviruses only and all bunyaviruses transmitted by arthropods encode an NSm protein..." This highlights NSm as a potential signature for vector-borne transmission within the Bunyavirales order.
• Dispensable for In Vitro Replication: "Across all the cell lines tested (Fig. S2b), the growth curves of BUNV wild type (BUNV-wt) and BUN-ΔNSm were not significantly different, showing that NSm is not required for viral repli-cation in mosquito cells." This indicates that NSm's function is specific to the in vivo environment of the mosquito.
• Essential for Midgut Infection and Dissemination: "Taken together, these results revealed the crucial role of BUNV NSm in vivo as the deletion of NSm alone was sufficient to nearly abolish midgut infection and dissemination in the Ae. aegypti mosquito." This is the central finding of the study, establishing NSm's role in overcoming midgut barriers.
• Midgut Bypass Eliminates NSm Requirement: "...we did not observe differences in the salivary glands at 3 and 9dpi, indicating that both BUNV-wt and BUN-ΔNSm equally disseminated to and infected the salivary glands, when bypassing the midgut barrier through intra-thoracic inoculation." This confirms that NSm's primary function in vivo is related to traversing the midgut.
• Failure of Cell-to-Cell Spread in ΔNSm Virus: "By contrast, only a small number of individual midgut cells were positive for the viral N antigen at 72 h pbm after BUN-ΔNSm infection, showing that BUN-ΔNSm stays confined in the initially infected cells and does not spread to neighbouring cells to form large foci." This pinpoints the specific stage of infection where NSm is required.
• Rescue of ΔNSm Phenotype by NSm Expression: "Taken together, our data show that the ectopic expression of the NSm gene significantly res-cued BUN-ΔNSm infection in Ae. aegypti, demonstrating that BUNV NSm is a crucial viral determinant necessary for the successful spread of infection in the mosquito midgut." This provides strong evidence that the observed defects are directly attributable to the absence of NSm.
• NSm Localization at Infection Foci Periphery: "...the NSm protein was detected mainly at the periphery of foci (Fig. 7c–f, Fig. S5a, b), showing higher levels of expression in newly infected cells in these clusters." This suggests a dynamic role for NSm during the spread of infection.
• Convergent Evolution Implication: "As NSm is present in bunyaviruses belonging to only a few viral genera, and which are dispersed across phylogenetically distinct families12, the 'law of parsimony' supports a convergent evolution with independent and repeated acquisitions of NSm in bunyaviruses." This highlights the evolutionary importance of NSm for arboviral transmission.

Quotes:

• "Here we show that only vector-borne bunyaviruses express a non-structural protein, NSm, whose function has so far remained largely elusive."
• "Using as experimental system Bunyamwera virus (BUNV) and its invertebrate host, Aedes aegypti, we show that NSm is dis-pensable for viral replication in mosquito cells in vitro but is absolutely required for successful infection in the female mosquito following a blood meal."
• "More specifically, NSm is required for cell-to-cell spread and egress from the mosquito midgut, a known barrier to viral infection."
• "Notably, the requirement for NSm is specific to the midgut; bypassing this barrier by experimental intrathoracic infection of the mosquito eliminates the necessity of NSm for virus spread in other tissues, including the salivary glands."
• "Overall, we unveiled a key evolutionary process that allows the transmission of vector-borne bunyaviruses between arthropod and vertebrate hosts."
• "Our study reveals that NSm was acquired during the evolution of vector-borne bunyaviruses, and it is a critical determinant allowing virus transmission between vertebrate and arthropod hosts."
• "Thus, our findings demonstrate that the NSm protein is involved in cell-to-cell spread specifically in themidgut and it is therefore a key viral determinant for successful infection of bunya-viruses transmitted via a blood meal."
• "Since live-attenuated vaccine strains against arboviruses should not spillover in the vectors... the elucidation of the function of NSm as a crucial viral determinant for mosquito...

Detailed Briefing Document: Zika Virus Modulation of Human Fibroblasts Enhances Transmission Success

Source: Mozūraitis, R., Cirksena, K., Raftari, M., et al. (2025). Zika virus modulates human fibroblasts to enhance transmission success in a controlled lab-setting. Communications Biology, 8(1), 754. https://doi.org/10.1038/s42003-025-07543-9

Date of Briefing: Received - 02 July 2024 | Accepted - 14 January 2025 | Published - 30 January 2025

1. Executive Summary:

This study investigates how Zika virus (ZIKV) infection of human dermal fibroblasts alters the release of volatile organic compounds (VOCs) and how these changes impact the host-seeking and feeding behavior of its mosquito vector, Aedes aegypti. The researchers demonstrate that ZIKV infection leads to significant alterations in the VOC profile of infected fibroblasts at both the invasion and transmission stages. These altered VOCs enhance the attraction of Ae. aegypti mosquitoes, increase their blood meal size, and even positively influence mosquito fecundity and survival. Furthermore, the study utilizes transcriptomic and proteomic meta-analyses to reveal that ZIKV infection modulates the expression of genes and proteins involved in lipid metabolism and transport in human host cells, potentially driving the observed changes in VOC production. The findings suggest that ZIKV actively manipulates its vertebrate host to promote its transmission by enhancing the attractiveness and feeding efficiency of its mosquito vector.

2. Main Themes and Important Ideas/Facts:

• ZIKV alters the VOC profile of infected human dermal fibroblasts:
• The study found significant differences in the VOCs released by human dermal fibroblasts when infected with ZIKV compared to uninfected cells.
• At the invasion stage of ZIKV infection, fourteen VOCs were significantly increased, with the largest increase observed for 2,4-dimethyl-1-heptane. Five VOCs were significantly decreased.
• At the transmission stage, all eight significantly altered VOCs were increased compared to healthy cells. The largest increases were seen in sulcatone, decanal, dodecanal, and 2-methyl-1-pentene.
• Quote: "We found that ZIKV infection altered the volatome of human dermal fibroblasts (Supplementary Fig. 1a, c, SupplementaryTable 1, and SupplementaryData file 1)."
• ZIKV-induced VOCs enhance Ae. aegypti host-seeking behavior:
• Using a Y-tube olfactometer, the researchers demonstrated that synthetic blends of VOCs mimicking those emitted by ZIKV-infected fibroblasts at both invasion and transmission stages significantly attracted Ae. aegypti females.
• Notably, Anopheles gambiae (a mosquito species that does not transmit ZIKV) did not show a preference for these blends.
• Wind tunnel experiments with individual synthetic VOCs and blends confirmed the increased attractiveness of ZIKV-related odors to Ae. aegypti. Dodecanal, 2-methyl-1-pentene, and 4-methylheptane showed the highest single-compound efficiency.
• Quote: "Using a Y-tube olfactometer, we demonstrated that olfactory active volatiles tested in the blends representing emissions from fibroblasts infected with ZIKV at invasive (invasion blend) and transmission (transmission blend) stages significantly attracted Ae. aegypti females."
• ZIKV-induced VOCs increase Ae. aegypti blood-feeding behavior:
• Mosquitoes exposed to ZIKV-related odor blends (both invasion and transmission) took significantly larger blood meals compared to those exposed to blood alone. Blood meal size was more than doubled with the invasion blend and tripled with the transmission blend.
• A higher proportion of mosquitoes exhibited persistent feeding behavior (landing and feeding) when ZIKV-related odors were present near the blood source.
• Quote: "The invasion and transmission blends modified the blood-feeding behaviour of mated five to sevendays old Ae. aegypti females, as the amount of blood meal obtained by females were more than doubled and tripled when fed on blood with nearby located invasion and transmission blends, respectively, compared to control blood in the absence of ZIKV related-odour."
• ZIKV-induced VOCs enhance mosquito fitness (fecundity and survival):
• The study found that exposure to ZIKV-related odors not only increased blood meal size but also positively impacted mosquito fecundity (number of eggs laid) and survival.
• Quote: "Interestingly, both mosquito fecundity and survival were also enhanced by the ZIKV induced odour (Fig. 3f, g)."
• ZIKV manipulates host cell's transcriptome and proteome:
• RNA sequencing analysis of infected fibroblasts revealed significant alterations in the expression of 401 transcripts, with the largest changes observed at 24 hours post-infection (transmission stage). Enrichment analysis pointed towards antiviral defense mechanisms and changes in lipoprotein metabolic processes.
• Meta-proteome analysis of multiple published studies identified 2,801 enzymes affected by ZIKV infection, with enrichment observed in enzymes involved in response to oxidative stress and fatty acid derivative metabolic processes.
• Specifically, enzymes associated with the oxidative and reductive degradation of lipids and fatty acids, potentially leading to the production of the identified VOCs, were upregulated upon ZIKV infection.
• Quote: "In sum, -omics meta-analyses reveal ZIKV-induced upregulation of lipoprotein and lipid metabolism enzymes that potentially are linked to Virus Induced Mosquito Attractant (VIMA)-VOCs release."
• Potential mechanisms for VOC production:
• The researchers suggest that the increased emission of oxidized compounds (alcohols and carbonyls) is likely associated with induced oxidative stress during viral infection.
• Hydrocarbons are likely produced by oxidative fragmentation of lipids.
• The study identifies potential enzymes involved in fatty acid metabolism and modification, including those responsible for methyl branching and the formation of alkanes and alkenes, although the complete biosynthetic pathway remains to be fully elucidated in human fibroblasts.
• The increased expression of apolipoprotein L genes during the transmission stage might indirectly contribute to the enhanced feeding persistency of mosquitoes by increasing lipid transport and fragmentation.
• Implications for arbovirus transmission and control:
• The study highlights a novel mechanism by which an arbovirus manipulates its vertebrate host to enhance its transmission success by making the host more attractive to the vector and increasing the likelihood of a successful blood meal.
• The findings suggest potential for developing novel vector control strategies, such as using the identified attractive VOCs for baiting and lethal trapping of Ae. aegypti mosquitoes.
• Quote: "There are potentially profound implications of this previously unknown set of arboviral manipulations of its vertebrate host in manners that provoke transmission-enhancing behaviours in its invertebrate host. Among these are exploitation against arboviral transmission success by efficient baiting and lethal trapping as a more effective, sustainable, and environmentally benign approach to the very difficult problem of mosquito control."

3. Methodology:

The study employed a multi-faceted approach, including:

• In vitro...