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BACKGROUND: The global temperature has significantly risen in the past century. Studies have indicated that higher temperature intensifies malaria transmission in tropical and temperate countries. Temperature fluctuations will have a potential impact on parasite development in the vector Anopheles mosquito. METHODS: Year-long microclimate temperatures were recorded from a malaria-endemic area, Chennai, India, from September 2021 to August 2022. HOBO data loggers were placed in different vector resting sites including indoor and outdoor roof types. Downloaded temperatures were categorised by season, and the mean temperature was compared with data from the same study area recorded from November 2012 to October 2013. The extrinsic incubation period for Plasmodium falciparum and P. vivax was calculated from longitudinal temperatures recorded during both periods. Vector surveillance was also carried out in the area during the summer season. RESULTS: In general, temperature and daily temperature range (DTR) have increased significantly compared to the 2012-2013 data, especially the DTR of indoor asbestos structures, from 4.30 â„ƒ to 12.62 â„ƒ in 2021-2022, unlike the marginal increase observed in thatched and concrete structures. Likewise, the average DTR of outdoor asbestos structures increased from 5.02 â„ƒ (2012-2013) to 8.76 â„ƒ (2021-2022) although the increase was marginal in thatched structures and, surprisingly, showed no such changes in concrete structures. The key finding of the extrinsic incubation period (EIP) is that a decreasing trend was observed in 2021-2022 compared to 2012-2013, mainly in indoor asbestos structures from 7.01 to 6.35 days, which negatively correlated with the current observation of an increase in temperature. Vector surveillance undertaken in the summer season revealed the presence of Anopheles breeding in various habitats. Anopheles stephensi could be collected using CDC light traps along with other mosquito species. CONCLUSION: The microclimate temperature has increased significantly over the years, and mosquitoes are gradually adapting to this rising temperature. Temperature negatively correlates with the extrinsic incubation period of the parasite. As the temperature increases, the development of the parasite in An. stephensi will be faster because of a decrease in EIP, thus requiring relatively fewer days, posing a risk for disease transmission and a hindrance to malaria elimination efforts.

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Dirofilaria immitis is the causative agent of canine heartworm disease. We used the established heartworm development unit (HDU) principle to map the extrinsic incubation period (EIP) of D. immitis in Australia using historical weather data from 2013-2022. We found weather conditions suitable for EIP completion showed substantial seasonality and geographical variability. Whilst a considerable percentage of the Australian territory showed suitable weather conditions to always support EIP completion (17%), only 2.7% of the 2021 Australian human population lived in this region. Therefore, 97% of the population lived in an area that changed its EIP suitability within the study period. EIP completion is required prior to D. immitis transmission, meaning that infection risk of D. immitis is seasonal and location-dependent, being disrupted each year for most of the human population's dogs. We developed an online, open access tool allowing us to visualise EIP completion across Australia historically and in near real-time. We aim to support veterinarians to make risk-based recommendations for dirofilariosis prevention by using the tool, available at https://heartworm-mapping.adelaide.edu.au/shiny/.

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Temperature is a well-known effector of several transmission factors of mosquito-borne viruses, including within mosquito dynamics. These dynamics are often characterized by vector competence and the extrinsic incubation period (EIP). Vector competence is the intrinsic ability of a mosquito population to become infected with and transmit a virus, while EIP is the time it takes for the virus to reach the salivary glands and be expectorated following an infectious bloodmeal. Temperatures outside the optimal range act on life traits, decreasing transmission potential, while increasing temperature within the optimal range correlates to increasing vector competence and a decreased EIP. These relatively well-studied effects of other Aedes borne viruses (dengue and Zika) are used to make predictions about transmission efficiency, including the challenges presented by urban heat islands and climate change. However, the knowledge of temperature and chikungunya (CHIKV) dynamics within its two primary vectors-Ae. aegypti and Ae. albopictus-remains less characterized, even though CHIKV remains a virus of public-health importance. Here, we review the literature and summarize the state of the literature on CHIKV and temperature dependence of vector competence and EIP and use these data to demonstrate how the remaining knowledge gap might confound the ability to adequately predict and, thus, prepare for future outbreaks.

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BACKGROUND: The extrinsic incubation period (EIP), defined as the time it takes for malaria parasites in a mosquito to become infectious to a vertebrate host, is one of the most influential parameters for malaria transmission but remains poorly understood. The EIP is usually estimated by quantifying salivary gland sporozoites in subsets of mosquitoes, which requires terminal sampling. However, assays that allow repeated sampling of individual mosquitoes over time could provide better resolution of the EIP. METHODS: We tested a non-destructive assay to quantify sporozoites of two rodent malaria species, Plasmodium chabaudi and Plasmodium berghei, expelled throughout 24-h windows, from sugar-soaked feeding substrates using quantitative-PCR. RESULTS: The assay is able to quantify sporozoites from sugar-soaked feeding substrates, but the prevalence of parasite-positive substrates was low. Various methods were attempted to increase the detection of expelled parasites (e.g. running additional technical replicates; using groups rather than individual mosquitoes), but these did not increase the detection rate, suggesting that expulsion of sporozoites is variable and infrequent. CONCLUSIONS: We reveal successful detection of expelled sporozoites from sugar-soaked feeding substrates. However, investigations of the biological causes underlying the low detection rate of sporozoites (e.g. mosquito feeding behaviour, frequency of sporozoite expulsion or sporozoite clumping) are needed to maximise the utility of using non-destructive assays to quantify sporozoite dynamics. Increasing detection rates will facilitate the detailed investigation on infection dynamics within mosquitoes, which is necessary to explain the highly variable EIP of Plasmodium and to improve understanding of malaria transmission dynamics.

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Ingestion of an additional blood meal(s) by a hematophagic insect can accelerate development of several vector-borne parasites and pathogens. Most studies, however, offer blood from the same vertebrate host species as the original challenge (for e.g., human for primary and additional blood meals). Here, we show a second blood meal from bovine and canine hosts can also enhance sporozoite migration in Anopheles stephensi mosquitoes infected with the human- and rodent-restricted Plasmodium falciparum and P. berghei, respectively. The extrinsic incubation period (time to sporozoite appearance in salivary glands) showed more consistent reductions with blood from human and bovine donors than canine blood, although the latter's effect may be confounded by the toxicity, albeit non-specific, associated with the anticoagulant used to collect whole blood from donors. The complex patterns of enhancement highlight the limitations of a laboratory system but are nonetheless reminiscent of parasite host-specificity and mosquito adaptations, and the genetic predisposition of An. stephensi for bovine blood. We suggest that in natural settings, a blood meal from any vertebrate host could accentuate the risk of human infections by P. falciparum: targeting vectors that also feed on animals, via endectocides for instance, may reduce the number of malaria-infected mosquitoes and thus directly lower residual transmission. Since endectocides also benefit animal health, our results underscore the utility of the One Health framework, which postulates that human health and well-being is interconnected with that of animals. We posit this framework will be further validated if our observations also apply to other vector-borne diseases which together are responsible for some of the highest rates of morbidity and mortality in socio-economically disadvantaged populations.

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Dengue transmission is determined by a complex set of interactions between the environment, Aedes aegypti mosquitoes, dengue viruses, and humans. Emergence in new geographic areas can be unpredictable, with some regions having established mosquito populations for decades without locally acquired transmission. Key factors such as mosquito longevity, temperature-driven extrinsic incubation period (EIP), and vector-human contact can strongly influence the potential for disease transmission. To assess how these factors interact at the edge of the geographical range of dengue virus transmission, we conducted mosquito sampling in multiple urban areas located throughout the Arizona-Sonora desert region during the summer rainy seasons from 2013 to 2015. Mosquito population age structure, reflecting mosquito survivorship, was measured using a combination of parity analysis and relative gene expression of an age-related gene, SCP-1. Bloodmeal analysis was conducted on field collected blood-fed mosquitoes. Site-specific temperature was used to estimate the EIP, and this predicted EIP combined with mosquito age were combined to estimate the abundance of "potential" vectors (i.e., mosquitoes old enough to survive the EIP). Comparisons were made across cities by month and year. The dengue endemic cities Hermosillo and Ciudad Obregon, both in the state of Sonora, Mexico, had higher abundance of potential vectors than non-endemic Nogales, Sonora, Mexico. Interestingly, Tucson, Arizona consistently had a higher estimated abundance of potential vectors than dengue endemic regions of Sonora, Mexico. There were no observed city-level differences in species composition of blood meals. Combined, these data offer insights into the critical factors required for dengue transmission at the ecological edge of the mosquito's range. However, further research is needed to integrate an understanding of how social and additional environmental factors constrain and enhance dengue transmission in emerging regions.

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BACKGROUND: Environmental temperature is a key driver for the transmission risk of mosquito-borne pathogens. Epidemiological models usually relate to temperature data from standardized weather stations, but these data may not capture the relevant scale where mosquitoes experience environmental temperatures. As mosquitoes are assumed to spend most of their lifetime in resting sites, we analysed mosquito resting site patterns and the associated temperatures in dependence on the resting site type, resting site height and the surrounding land use. METHODS: The study was conducted in 20 areas in near-natural habitats in Germany. Ten areas were studied in 2017, and another 10 in 2018. Each study area consisted of three sampling sites, where we collected mosquitoes and microclimatic data in artificial (= garden pop-up bags) and natural resting sites at three height levels between 0 and 6 m. Land use of the study sites was characterized as forest and meadows based on reclassified information of the CORINE (Coordination of Information on the Environment) Land Cover categories. The hourly resting site temperatures and the data from the nearest weather station of the German meteorological service were used to model the duration of the extrinsic incubation period (EIP) of mosquito-borne pathogens. RESULTS: Anopheles, Culex and Culiseta preferred artificial resting sites, while Aedes were predominantly collect in natural resting sites. Around 90% of the mosquitoes were collected from resting sites below 2 m. The mosquito species composition did not differ significantly between forest and meadow sites. Mean resting site temperatures near the ground were approximately 0.8 °C lower than at a height of 4-6 m, which changed the predicted mean EIP up to 5 days at meadow and 2 days at forest sites. Compared with temperature data from standardized weather stations, the resting site temperatures near the ground would prolong the mean estimated EIP 4 days at forest sites and 2 days at meadow sites. CONCLUSIONS: The microclimate of mosquito resting sites differs from standardized meteorological data, which can influence the transmission of mosquito-borne pathogens. In a near-natural environment, colder temperatures at mosquitoes' preferred resting sites near the ground would prolong the EIP of mosquito-borne pathogens relative to data from weather stations.

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BACKGROUND: Sporozoites isolated from the salivary glands of Plasmodium-infected mosquitoes are a prerequisite for several basic and pre-clinical applications. Although salivary glands are pooled to maximize sporozoite recovery, insufficient yields pose logistical and analytical hurdles; thus, predicting yields prior to isolation would be valuable. Preceding oocyst densities in the midgut is an obvious candidate. However, it is unclear whether current understanding of its relationship with sporozoite densities can be used to maximize yields, or whether it can capture the potential density-dependence in rates of sporozoite invasion of the salivary glands. METHODS: This study presents a retrospective analysis of Anopheles stephensi mosquitoes infected with two strains of the rodent-specific Plasmodium berghei. Mean oocyst densities were estimated in the midguts earlier in the infection (11-15 days post-blood meal), with sporozoites pooled from the salivary glands later in the infection (17-29 days). Generalized linear mixed effects models were used to determine if (1) mean oocyst densities can predict sporozoite yields from pooled salivary glands, (2) whether these densities can capture differences in rates of sporozoite invasion of salivary glands, and (3), if the interaction between oocyst densities and time could be leveraged to boost overall yields. RESULTS: The non-linear effect of mean oocyst densities confirmed the role of density-dependent constraints in limiting yields beyond certain oocyst densities. Irrespective of oocyst densities however, the continued invasion of salivary glands by the sporozoites boosted recoveries over time (17-29 days post-blood meal) for either parasite strain. CONCLUSIONS: Sporozoite invasion of the salivary glands over time can be leveraged to maximize yields for P. berghei. In general, however, invasion of the salivary glands over time is a critical fitness determinant for all Plasmodium species (extrinsic incubation period, EIP). Thus, delaying sporozoite collection could, in principle, substantially reduce dissection effort for any parasite within the genus, with the results also alluding to the potential for changes in sporozoites densities over time to modify infectivity for the next host.

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Environmental temperature is a key driver of malaria transmission dynamics. Using detailed temperature records from four sites: low elevation (1800), mid elevation (2200 m), and high elevation (2600-3200 m) in the western Himalaya, we model how temperature regulates parasite development rate (the inverse of the extrinsic incubation period, EIP) in the wild. Using a Briére parametrization of the EIP, combined with Bayesian parameter inference, we study the thermal limits of transmission for avian (Plasmodium relictum) and human Plasmodium parasites (P. vivax and P. falciparum) as well as for two malaria-like avian parasites, Haemoproteus and Leucocytozoon. We demonstrate that temperature conditions can substantially alter the incubation period of parasites at high elevation sites (2600-3200 m) leading to restricted parasite development or long transmission windows. The thermal limits (optimal temperature) for Plasmodium parasites were 15.62-34.92°C (30.04°C) for P. falciparum, 13.51-34.08°C (29.02°C) for P. vivax, 12.56-34.46°C (29.16°C) for P. relictum and for two malaria-like parasites, 12.01-29.48°C (25.16°C) for Haemoproteus spp. and 11.92-29.95°C (25.51°C) for Leucocytozoon spp. We then compare estimates of EIP based on measures of mean temperature versus hourly temperatures to show that EIP days vary in cold versus warm environments. We found that human Plasmodium parasites experience a limited transmission window at 2600 m. In contrast, for avian Plasmodium transmission was not possible between September and March at 2600 m. In addition, temperature conditions suitable for both Haemoproteus and Leucocytozoon transmission were obtained from June to August and in April, at 2600 m. Finally, we use temperature projections from a suite of climate models to predict that by 2040, high elevation sites (~2600 m) will have a temperature range conducive for malaria transmission, albeit with a limited transmission window. Our study highlights the importance of accounting for fine-scale thermal effects in the expansion of the range of the malaria parasite with global climate change.

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Insect vectors are responsible for spreading many infectious diseases, yet interactions between pathogens/parasites and insect vectors remain poorly understood. Filling this knowledge gap matters because vectors are evolving in response to the deployment of vector control tools (VCTs). Yet, whilst the evolutionary responses of vectors to VCTs are being carefully monitored, the knock-on consequences for parasite evolution have been overlooked. By examining how mosquito responses to VCTs impact upon malaria parasite ecology, we derive a framework for predicting parasite responses. Understanding how VCTs affect the selection pressures imposed on parasites could help to mitigate against parasite evolution that leads to unfavourable epidemiological outcomes. Furthermore, anticipating parasite evolution will inform monitoring strategies for VCT programmes as well as uncovering novel VCT strategies.

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Microbiome research has gained considerable interest due to the emerging evidence of its impact on human and animal health. As in other animals, the gut-associated microbiota of mosquitoes affect host fitness and other phenotypes. It is now well established that microbes can alter pathogen transmission in mosquitoes, either positively or negatively, and avenues are being explored to exploit microbes for vector control. However, less attention has been paid to how microbiota affect phenotypes that impact vectorial capacity. Several mosquito and pathogen components, such as vector density, biting rate, survival, vector competence, and the pathogen extrinsic incubation period all influence pathogen transmission. Recent studies also indicate that mosquito gut-associated microbes can impact each of these components, and therefore ultimately modulate vectorial capacity. Promisingly, this expands the options available to exploit microbes for vector control by also targeting parameters that affect vectorial capacity. However, there are still many knowledge gaps regarding  mosquito-microbe interactions  that need to be addressed in order to exploit them efficiently. Here, we review current evidence of impacts of the microbiome on aspects of vectorial capacity, and we highlight likely opportunities for novel vector control strategies and areas where further studies are required. Video abstract.

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Mosquito-borne viruses are the cause of significant morbidity and mortality worldwide, especially in low- and middle-income countries. Assessing risk for viral transmission often involves characterization of the vector competence of vector-virus pairings. The most common determination of vector competence uses discreet, terminal time points, which cannot be used to investigate variation in transmission aspects, such as biting behavior, over time. Here, we present a novel method to longitudinally measure individual biting behavior and Zika virus (ZIKV) transmission. Individual mosquitoes were exposed to ZIKV, and from 9 to 24 days post-exposure, individuals were each offered a 180 µL bloodmeal every other day. Biting behavior was observed and characterized as either active probing, feeding, or no bite. The bloodmeal was then collected, spun down, serum collected, and tested for ZIKV RNA via qRT-PCR to determine individuals' vector competence over time. This included whether transmission to the bloodmeal was successful and the titer of expectorated virus. Additionally, serum was inoculated onto Vero cells in order to determine infectiousness of positive recovered sera. Results demonstrate heterogeneity in not only biting patterns but expectorated viral titers among individual mosquitoes over time. These findings demonstrate that the act of transmission is a complex process governed by mosquito behavior and mosquito-virus interaction, and herein we offer a method to investigate this phenomenon.

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Chikungunya virus (CHIKV) is a reemerging and rapidly spreading pathogen transmitted by mosquitoes. The emergence of new epidemic variants of the virus is associated with genetic evolutionary traits, including duplication of repeated RNA elements in the 3' untranslated region (UTR) that seemingly favor transmission by mosquitoes. The transmission potential of a given variant results from a complex interplay between virus populations and anatomical tissue barriers in the mosquito. Here, we used the wild-type CHIKV Caribbean strain and an engineered mutant harboring a deletion in the 3' UTR to dissect the interactions of virus variants with the anatomical barriers that impede transmission during the replication cycle of the virus in Aedes mosquitoes. Compared to the 3'-UTR mutant, we observed that the wild-type virus had a short extrinsic incubation period (EIP) after an infectious blood meal and was expectorated into mosquito saliva much more efficiently. We found that high viral titers in the midgut are not sufficient to escape the midgut escape barrier. Rather, viral replication kinetics play a crucial role in determining midgut escape and the transmission ability of CHIKV. Finally, competition tests in mosquitoes coinfected with wild-type and mutant viruses revealed that both viruses successfully colonized the midgut, but wild-type viruses effectively displaced mutant viruses during systemic infection due to their greater efficiency of escaping from the midgut into secondary tissues. Overall, our results uncover a link between CHIKV replication kinetics and the effect of bottlenecks on population diversity, as slowly replicating variants are less able to overcome the midgut escape barrier.IMPORTANCE It is well established that selective pressures in mosquito vectors impose population bottlenecks for arboviruses. Here, we used a CHIKV Caribbean lineage mutant carrying a deletion in the 3' UTR to study host-virus interactions in vivo in the epidemic mosquito vector Aedes aegypti We found that the mutant virus had a delayed replication rate in mosquitoes, which lengthened the extrinsic incubation period (EIP) and reduced fitness relative to the wild-type virus. As a result, the mutant virus displayed a reduced capacity to cross anatomical barriers during the infection cycle in mosquitoes, thus reducing the virus transmission rate. Our findings show how selective pressures act on CHIKV noncoding regions to select variants with shorter EIPs that are preferentially transmitted by the mosquito vector.

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Parasite drug resistance presents a major obstacle to controlling and eliminating vector-borne diseases affecting humans and animals. While vector-borne disease dynamics are affected by factors related to parasite, vertebrate host and vector, research on drug resistance in filarial parasites has primarily focused on the parasite and vertebrate host, rather than the mosquito. However, we expect that the physiological costs associated with drug resistance would reduce the fitness of drug-resistant vs. drug-susceptible parasites in the mosquito wherein parasites are not exposed to drugs. Here we test this hypothesis using four isolates of the dog heartworm (Dirofilaria immitis)-two drug susceptible and two drug resistant-and two vectors-the yellow fever mosquito (Aedes aegypti) and the Asian tiger mosquito (Ae. albopictus)-as our model system. Our data indicated that while vector species had a significant effect on vectorial capacity, there was no significant difference in the vectorial capacity of mosquitoes infected with drug-resistant vs. drug-susceptible parasites. Consequently, contrary to expectations, our data indicate that drug resistance in D. immitis does not appear to reduce the transmission efficiency of these parasites, and thus the spread of drug-resistant parasites in the vertebrate population is unlikely to be mitigated by reduced fitness in the mosquito vector.

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Mosquito-borne diseases, in particular malaria, have a significant burden worldwide leading to nearly half a million deaths each year. The malaria parasite requires a vertebrate host, such as a human, and a vector host, the Anopheles mosquito, to complete its full life cycle. Here, we focus on the parasite dynamics within the vector to examine the first appearance of sporozoites in the salivary glands, which indicates a first time of infectiousness of mosquitoes. The timing of this period of pathogen development in the mosquito until transmissibility, known as the extrinsic incubation period, remains poorly understood. We develop compartmental models of within-mosquito parasite dynamics fitted with experimental data on oocyst and sporozoite counts. We find that only a fraction of oocysts burst to release sporozoites and bursting must be delayed either via a time-dependent function or a gamma-distributed set of compartments. We use Bayesian inference to estimate distributions of parameters and determine that bursting rate is a key epidemiological parameter. A better understanding of the factors impacting the extrinsic incubation period will aid in the development of interventions to slow or stop the spread of malaria.

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Kerteszia cruzii is a sylvatic mosquito and the primary vector of Plasmodium spp., which can cause malaria in humans in areas outside the Amazon River basin in Brazil. Anthropic changes in the natural environments are the major drivers of massive deforestation and local climate change, with serious impacts on the dynamics of mosquito communities and on the risk of acquiring malaria. Considering the lack of information on the dynamics of malaria transmission in areas across the Atlantic Forest biome, where Ke. cruzii is the dominant vector, and the impact of climate drivers of malaria, the present study aimed to: (i) investigate the occurrence and survival rate of Ke. cruzii based on the distinct vegetation profiles found in areas across the coastal region of the Brazilian Atlantic Forest biome; (ii) estimate the extrinsic incubation period (EIP) and survival rates of P. vivax and P. falciparum parasites in Ke. cruzii under current and future scenarios. The potential distribution of Plasmodium spp. was estimated using simulation analyses under distinct scenarios of average temperature increases from 1 °C to 3.7 °C. Our results showed that two conditions are necessary to explain the occurrence and survival of Ke. cruzii: warm temperature and presence of the Atlantic Forest biome. Moreover, both Plasmodium species showed a tendency to decrease their EIP and increase their estimated survival rates in a scenario of higher temperature. Our findings support that the high-risk malaria areas may include the southern region of the distribution range of the Atlantic Forest biome in the coming years. Despite its limitations and assumptions, the present study provides robust evidence of areas with potential to be impacted by malaria incidence in a future scenario. These areas should be monitored in the next decades regarding the occurrence of the mosquito vector and the potential for malaria persistence and increased occurrence.

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BACKGROUND: Transmission dynamics of arboviruses like Zika virus are often evaluated by vector competence (the proportion of infectious vectors given exposure) and the extrinsic incubation period (EIP, the time it takes for a vector to become infectious), but vector age is another critical driver of transmission dynamics. Vectorial capacity (VC) is a measure of transmission potential of a vector-pathogen system, but how these three components, EIP, vector competence and vector age, affect VC in concert still needs study. METHODS: The interaction of vector competence, EIP, and mosquito age at the time of infection acquisition (Ageacquisition) was experimentally measured in an Aedes aegypti-ZIKV model system, as well as the age-dependence of probability of survival and the willingness to bite. An age-structured vectorial capacity framework (VCage) was then developed using both EIPMin and EIPMax, defined as the time to first observed minimum proportion of transmitting mosquitoes and the time to observed maximum proportion of transmitting mosquitoes. RESULTS: The within-mosquito dynamics of vector competence/EIP were not significant among treatments where mosquitoes were exposed at different ages. However, VCage revealed: (i) age-dependence in vector-virus interactions is important for transmission success; (ii) lower vector competence but at shorter EIPs was sufficient for transmission perpetuation; and (iii) R0 may be overestimated by using non-age-structured VC. CONCLUSIONS: The results indicate that ultimately the temporal component of the virus-vector dynamics is most critical, especially when exposure occurred at advanced mosquito age. While our study is limited to a single virus-vector system, and a multitude of other factors affect both vector competence and mosquito mortality, our methods can be extrapolated to these other scenarios. Results indicate that how 'highly' or 'negligibly' competent vectors are categorized may need adjustment.

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Aedes albopictus is an important vector of chikungunya virus (CHIKV). In Australia, Ae. albopictus is currently only known to be present on the islands of the Torres Strait but, should it invade the mainland, it is projected to spread to temperate regions. The ability of Australian Ae. albopictus to transmit CHIKV at the lower temperatures typical of temperate areas has not been assessed. Ae. albopictus mosquitoes were orally challenged with a CHIKV strain from either Asian or East/Central/South African (ECSA) genotypes (107 pfu/mL), and maintained at a constant temperature of either 18 °C or 28 °C. At 3- and 7-days post-infection (dpi), CHIKV RNA copies were quantified in mosquito bodies, and wings and legs using real time polymerase chain reaction (qRT-PCR), while the detection of virus in saliva (a proxy for transmission) was performed by amplification in cell culture followed by observation of cytopathic effect in Vero cells. Of the ≥95% of Ae. albopictus that survived to 7 dpi, all mosquitoes became infected and showed body dissemination of CHIKV at both temperatures and time points. Both the Asian and ECSA CHIKV genotypes were potentially transmissible by Australian Ae. albopictus at 28 °C within 3 days of oral challenge. In contrast, at 18 °C none of the mosquitoes showed evidence of ability to transmit either genotype of CHIKV at 3 dpi. Further, at 18 °C only Ae. albopictus infected with the ECSA genotype showed evidence of virus in saliva at 7 dpi. Overall, infection with the ECSA CHIKV genotype produced higher virus loads in mosquitoes compared to infection with the Asian CHIKV genotype. Our results suggest that lower ambient temperatures may impede transmission of some CHIKV strains by Ae. albopictus at early time points post infection.

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The rate of malaria transmission is strongly determined by parasite development time in the mosquito, known as the extrinsic incubation period (EIP), since the quicker parasites develop, the greater the chance that the vector will survive long enough for the parasite to complete development and be transmitted. EIP is known to be temperature-dependent but this relationship is surprisingly poorly characterized. There is a single degree-day model for EIP of Plasmodium falciparum that derives from a limited number of poorly controlled studies conducted almost a century ago. Here, we show that the established degree-day model greatly underestimates the rate of development of P. falciparum in both Anopheles stephensi and An. gambiae mosquitoes at temperatures in the range of 17-20°C. We also show that realistic daily temperature fluctuation further speeds parasite development. These novel results challenge one of the longest standing models in malaria biology and have potentially important implications for understanding the impacts of future climate change.

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Transmission of malaria parasites occurs when a female Anopheles mosquito feeds on an infected host to acquire nutrients for egg development. How parasites are affected by oogenetic processes, principally orchestrated by the steroid hormone 20-hydroxyecdysone (20E), remains largely unknown. Here we show that Plasmodium falciparum development is intimately but not competitively linked to processes shaping Anopheles gambiae reproduction. We unveil a 20E-mediated positive correlation between egg and oocyst numbers; impairing oogenesis by multiple 20E manipulations decreases parasite intensities. These manipulations, however, accelerate Plasmodium growth rates, allowing sporozoites to become infectious sooner. Parasites exploit mosquito lipids for faster growth, but they do so without further affecting egg development. These results suggest that P. falciparum has adopted a non-competitive evolutionary strategy of resource exploitation to optimize transmission while minimizing fitness costs to its mosquito vector. Our findings have profound implications for currently proposed control strategies aimed at suppressing mosquito populations.

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