Mosquito immunity, physiology, and the pathogen interaction

 

Over the past three decades, we have gained substantial insights into the mosquito proteins and cellular machinery mediating mosquito-pathogen interactions. Yet, we are just beginning to understand the complexity underlying mosquito-pathogen interactions. Mosquito infection is a dynamic phenotype, which is dependent upon both the specific mosquito-pathogen pairing, as well as in variation in key environmental factors. This is especially relevant for understanding how specific pathogens emerge, environmental conditions that favor emergence, and the biological constraints on the distributions of emerging mosquito-borne diseases. For example, there is overwhelming evidence that temperature markedly affects diverse aspects of mosquito physiology, life history, and pathogen replication within the mosquito because mosquitoes are small, cold-blooded organisms. Yet, the extent to which temperature shapes transmission directly, through effects on pathogen biology, or indirectly, through effects on mosquito immunity and physiology, remain largely unexplored.

Aedes aegypti - Zika virus

Anopheles stephensi - human malaria

Aedes and Anopheles photos are credited to iStock (Getty images) and Dreamstime.com, respectively. Human malaria gametocyte images taken from Coatney GR, Collins WE, Warren M, Contacos PG. The Primate Malarias. Bethesda: U.S. Department of Health, Education and Welfare; 1971. RNA-seq illustration taken from Oxford Gene Technology.

We currently have two ongoing projects that utilize RNA sequencing and bioinformatic analysis to identify panels of differentially expressed genes that are important in the physiological response of mosquitoes to temperature, to infection, and how temperature alters mosquito responses to infection in the yellow fever mosquito (Aedes aegypti) - Zika virus and the Anopheles stephensi (Indian malaria mosquito) - human malaria (Plasmodium falciparum) systems. 

Environmental drivers of mosquito life history and disease transmission

 

There are several key knowledge gaps that affect our ability to predict and, ultimately, mitigate the factors influencing vector-borne disease transmission. This is particularly important in systems where we lack fundamental knowledge on the relationships between key environmental variables and transmission. A large component of the research conducted in our group focuses on the effects of abiotic and biotic variation on mosquito life history and overall transmission.

1.) Temperature effects on Zika virus transmission

 

Mosquito-borne viruses are an emerging threat impacting human health and well-being. Diseases such as Zika, dengue, and chikungunya, which were once considered tropical and sub-tropical diseases, are now threatening temperate regions of the world due to climate change, globalization, and increasing urbanization. It is estimated that 3.9 billion people in 120 countries around the globe are at risk of contracting an arboviral disease. In spite of increasing efforts to develop vaccines and therapeutics, vector control is still the only way to mitigate the disease spread. Therefore, it is crucial to try to predict how these viruses might spread seasonally, geographically, and with climate change. We used empirical and mathematical modeling approaches to predict how Zika virus transmission changes with temperature.

Zika virus (ZIKV, dark blue) transmission has a non-linear relationship with temperature, with a similar temperature optimum where transmission is maximized and temperature maximum where temperature is minimized as other mosquito-borne viruses like dengue (DENV, light blue). Thus, as environmental temperatures warm and seasons extend with climate change and urbanization, environmental conditions at higher latitudes will become more favorable for Zika virus transmission. However, because the minimum temperature for Zika virus transmission is 5 C higher than for dengue virus,  the expansion of transmission risk at higher latitudes will be less dramatic than for dengue virus (Tesla et al. 2018 Temperature drives Zika virus transmission: evidence from empirical and mathematical models. Proceedings of the Royal Society Series B).

A melanized sephadex bead

expression of DEFCEC, and NOS

with heat-killed E. coli

2.) Variation in viremia on mosquito infection and the transmission of Zika virus

 

The number of people at risk for contracting Zika virus cannot be accurately estimated, as most infected hosts are asymptomatic, there is wide variation in the amount of virus mosquitoes are exposed to when feeding across human hosts, and the relationship between variation in host viremia and transmission to local mosquitoes is unclear. We used a combination of experimental and mathematical approaches to characterize the relationship between variation in the amount of virus mosquitoes are exposed to, the susceptibility of mosquitoes to Zika virus infection, and overall risk of transmission. 

Mosquito immune responses do not scale simply, as predicted, with realistic increases in ambient temperature.  Most responses studied actually ran faster at temperatures well below the thermal optimum for the mosquito vector (27 C; standard rearing temperature), while others ran faster at warmer than optimum temperatures.  The immune phenotype characterized in the lab is likely not the same as mosquito immunity described across the range of relevant field temperatures for transmission.  Temperature effects on mosquito immunity may in part explain variation in resistance across mosquito populations in the field.

 

Future Work will focus on characterizing how thermal variation influences mosquito immune markers relevant to the human malaria parasite (Plasmodium falciparum).  We will  use RNA sequencing to examine how changes in mean temperature affect the global transcriptional profiles of both the  mosquito and the parasite across various stages of parasite infection.  This work will begin exploring the mechanisms underpinning the effects of thermal variation on the  mosquito-malaria interaction by identifying candidate genes involved in response of the mosquito and the parasite to changes in temperature, as well as in the interaction.  In order to functionally characterize these candidate genes, we will also quantify how mosquito fitness (e.g. longevity) and parasite fitness (parasite prevalence and intensity) change across temperatures, as well as in response to loss of function of these genes.

Environment, Body Condition, & Vectorial Capacity - Highlight # 2

 

Vectorial capacity is a measure of the transmission potential of a mosquito population, is defined by the following equation, and is comprised of both mosquito and parasite traits:

 

 

The proportion of the mosquito population to become infectious (A), the rate at which mosquitoes become infectious (B), and the overall risk of transmission (RoC) increases with the concentration of virus mosquitoes imbibe in the blood meal. Further, the overall force of infection (the number of infectious mosquito bites a human host is predicted to experience, purple), which is a function of the number of mosquitoes alive (light blue) and the proportion of the mosquito population that is infectious (pink) on a given day, also increases with the concentration of virus mosquitoes are exposed to (D) (Tesla et al. 2018 Estimating the effects of variation in viremia on mosquito susceptibility, infectiousness, and Ro of Zika in Aedes aegypti PLoS Neglected Tropical Diseases).

3.) Variation in microclimate in urban centers and context-dependent interactions

 

The global distribution of mosquito species is strongly determined by climatic factors such as temperature and rainfall. Similarly, at a finer spatial scale, microclimate can significantly influence the demographics and transmission potential of mosquito species. Our lab examines the effect of microclimate on Aedes mosquitoes in urban areas, especially spatial heterogeneity due to anthropogenic causes such as the urban heat island. Our 2015 and 2016 studies in Athens, GA found that the microclimate mosquito larvae experience impacts larval survival, development rate, and the number of adult mosquitoes that emerge from larval habitats. Further, larval microclimate can carry-over to affect adult potential to transmit dengue virus.

The climate conditions that the invasive Asian tiger mosquito (Aedes albopictus) experiences vary across an urban landscape, like Athens GA, due to variation in the amount of impervious surfaces and the built environment. In general, mosquitoes reared on sites with high impervious surface (red) experienced lower survival in the larval environment, faster larval development rates, and lower susceptibility to dengue virus infection than mosquitoes reared on sites with intermediate (blue) and low (white) impervious surface cover. Further, mathematical models that do not incorporate carry-over effects of larval microclimate will under- or overestimate overall risk of disease transmission depending on the time of year (Murdock et al. 2017. Fine-scale variation in microclimate across an urban landscape shapes variation in mosquito population dynamics and the potential of Aedes albopictus to transmit arboviral disease. PLoS Neglected Tropical Diseases; Evans et al. 2018. Carry-over effects of urban larval environments on the transmission potential of dengue-2 virus. Parasites & Vectors).

Hugh Sturrock

Preliminary work suggests that changes in mosquito body condition can have profound effects on vectorial capacity through changes in both mosquito and parasite traits. Effects on mosquito traits are the following:

More recent work considers the interaction between microclimate, species interactions, and intra-specific competition on mosquito population dynamics and mosquito-borne disease transmission. One project in urban centers in India focuses specifically on competition between Aedes aegypti (the dengue vector) and Anopheles stephensi (the malaria vector). Both species are container breeders that occupy urban environments. We find the existence of strong asymmetric competition between these two species, the strength of which is dependent on temperature. Future work will explore whether these results match fine-scale patterns of species coexistence in the field.

 

Another project in the Athens, GA field system explores how microbial communities in mosquito larval habitats vary across an urban landscape and seasonally, and how these communities influence the microbial organisms that colonize mosquitoes, mosquito life history traits relevant for transmission, and the ability of mosquitoes to transmit human pathogens. Finally, we are also interested in exploring if fine-scale variation in microclimate across the built environment and season modifies competition for resources in the larval environment and in turn, mosquito population dynamics and arbovirus transmission potential.

An ongoing project in Athens, GA is exploring how microbial communities vary across common mosquito larval habitats (e.g. bird baths, gutters, and tires) with land use and season, how this variation determine microbial communities inside of the mosquito vector across different life stages (e.g. larvae, adult), and eventually will explore how variation in mosquito microbiota affects arbovirus transmission. This project is in collaboration with Dr. Mike Strand in the Department of Entomology at the University of Georgia. Pictures are credited to Kerri Miazgowicz.

Effects on parasite associated traits are the following:

4.) Resolving uncertainty and refining transmission models of malaria

 

The deadliest organism on the planet is the Anopheles mosquito, the insect that transmits malaria to humans. Human malaria is the leading killer among infectious diseases, resulting in approximately 216 million cases and 500,000 deaths, primarily in children under the age of 5. Dramatic reductions in disease burdens of human malaria in the last 20 years has led to ambitious calls for eradicating the disease by 2030. However, eradication hinges on our ability to eliminate transmission 1) from both symptomatic and asymptomatic hosts, 2) across highly heterogeneous landscapes created by biotic and abiotic factors, and 3) with the emergence of multi-drug resistance to the last remaining anti-malarials, artemisinin-combination therapies. Despite growing research efforts to develop new therapeutics and vaccines, mitigating malaria transmission still largely depends on conventional mosquito control methods (e.g. bed nets, indoor residual spraying). Developing tools that will allow us to successfully predict outbreaks and efficiently target current and future interventions to specific times and locations will aid effective mosquito and disease control.

 

Current work revolves around experimentally validating common assumptions made by mathematical models that predict transmission, identifying additional sources of ecological variation these models should incorporate, and building new models to improve prediction.  This includes assessing how transmission models perform when key mosquito life history traits (e.g. daily per capita mortality rate, egg production, and biting rate) change across the lifespan of the mosquito vector, in thermally variable environments, across different species of Anopheles mosquitoes, and with parasite infection. Additionally, we are interested in exploring the impact of individual variation in these life history traits and the presence of life history trade-offs across these traits on malaria transmission.

The predicted temperature optimum that maximizes malaria transmission and the temperature maximum that minimizes malaria transmission differ when mosquito life history traits (e.g. lifespan, lifetime egg production, and daily biting rate) are assumed to vary across realistic environments that incorporate daily temperature ranges (dtr) of 9 C and 12 C relative to environments that are set to constant average temperature (dtr 0 C). Further, overall malaria transmission risk is predicted to differ across the distribution of Anopheles stephensi (the Indian malaria mosquito) depending on whether models estimate key parameters across multiple mosquito species (J estimated, peach color) or a single species (An. stephensi; MSP models, red and purple). Less differences in overall malaria transmission risk were noted between models that incorporate trait data estimated across the lifespan of the mosquito (MSP lifetime, purple) relative to models that estimate these traits (MSP estimated, red). Figures developed by Kerri Miazgowicz and Dr. Sadie Ryan (University of Florida).

Assessing the effectiveness of novel mosquito control tools

Many vector-borne diseases currently lack effective therapeutics and vaccines (e.g. the arboviruses), and global malaria elimination efforts are being threatened by the evolution of mosquito and parasite resistance to current interventions. Thus, there is a pressing need to develop novel intervention strategies to control these diseases. Engineered traits that render mosquitoes ineffective at transmitting parasites and pathogens (e.g. life shortening, parasite or pathogen blocking, upregulated immunity) are a promising new avenue of research in vector-borne disease elimination efforts. The success of many of these technologies relies on mosquito release in the field and subsequent invasion of transgenic / transinfected traits in wildtype mosquito populations. These technologies often fail in the field either due to a poor understanding of how environmental variation shapes transgenic / transinfected traits or what female mosquitoes find sexy in their male mates (i.e. mating biology).

1.) Exploring implications of mosquito love songs and mate choice on fitness

This project is a series of exploratory studies investigating whether female mosquitoes utilize acoustic signals in mate choice and if these signals serve as reliable indicators of male fitness and offspring viability. We, in collaboration with Drs. Laura Cator (Imperial College) and Laura Harrington (Cornell University), are defining the relationship between newly identified acoustic courtship signals and the fitness of both males and their offspring in the Aedes aegypti - dengue virus system. At the University of Georgia, we are investigating the effects of female mate choice on offspring immune performance, a key fitness and transmission metric. We are interested in identifying if harmonic convergence is a reliable cue for male offspring immune performance and whether life history trade-offs exist between immunity and reproductive effort. The potential findings of this collaborative research will serve as a basis for a research program designed to facilitate application of acoustics in the assessment and improvement of proposed release lines.

C
humoral melanization 
bacterial killing
ability to transmit dengue virus

The buzz of a flying female mosquito acts as a mating signal, attracting males. The important behavioral component of the buzz is the fundamental frequency of the mosquito wing beat. This fundamental frequency typically occurs between 300-600 Hz depending on the mosquito species. For Aedes aegypti, the fundamental frequency for females is approximately 400 Hz and 600 Hz for males. Prior to mating, Ae. aegypti males and females modulate their flight tones when brought within a few centimeters of each other. Further, this modulation does not match the fundamental wing beat frequency of the male or female, but represents a shared harmonic of around 1200 Hz (A, Cator et al. 2009. Harmonic convergence in the love songs of the dengue vector mosquito. Science).  Pairs that harmonically converge are more likely to successfully mate (B, 1). Additionally, female mosquitoes also display a variety of rejection behaviors towards males that are deemed unsuitable as mates. These include actively kicking (B, 2) or holding (B, 3) the male away from her as he attempts to mate (Cator et al. 2011. The harmonic convergence of fathers predicts the mating success of sons in Aedes aegypti. Animal Behaviour). We are currently investigating whether or not offspring from parental pairs that harmonically converge differ in humoral (e.g. melanization neutrally charged sephadex beads) and cellular (e.g. ability to kill Escherichia coli) immune responses, as well as their susceptibility and resistance to dengue-2 virus (C, photos are provided by Courtney Murdock, and the dengue virus image was taken from the Protein Data Bank in Europe website).

2.) The effect of environmental variation on malaria control strategies

Artemisinin is the last line of anti-malarials against human malaria (Plasmodium falciparum) where resistance is characterized by delayed recovery times and increased number of parasites in patients infected with resistant strains. Although parasite strains vary widely in their ability to produce transmission stages of the parasite, numerous laboratory and field studies investigating the relationship between parasite numbers and probability of infection in the mosquito vector have collectively led to the description of a positive, albeit loose, log-linear relationship. This, in turn, has led to the general view that hosts with higher numbers of parasites contribute more to transmission, and the concern that the elevated parasite numbers observed in patients infected with artemisinin-resistant parasites could be contributing to the rapid proliferation of artemisinin-resistance in response to intense drug pressure. As a result, the WHO recommends treating patients with primaquine, the only known gametocide to date, in addition to artemisinin. 

 

However, the significant amount of variation around the positive relationship between parasite density and mosquito transmission suggests 1) low-density carriers introduce significant heterogeneity in transmission, 2) the relationship reaches an eventual asymptote where higher parasite numbers offer no apparent, additional benefits, and 3) there are still many unexplained factors influencing successful transmission of parasites to mosquitoes. In addition to biotic factors, such as variation in host immuno-physiology that allow for certain hosts to be highly infectious regardless of parasite densities, abiotic factors could influence this relationship. We are interested in exploring the relative effects of environmental temperature on this relationship, as well as the implications of environmental variation for the spread of artemisinin resistant parasites and the effectiveness of tools that target transmission stages of the parasite (e.g. primaquine and transmission blocking vaccines).

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