Pathogenic bacteria, viruses, fungi, and parasitic worms are often transmitted by vectors, commonly biting insects such as mosquitoes. Environmental stressors acting on mosquito larvae, including high density, food shortage, and extreme temperatures, can have lifelong impacts on adult biology; common manifestations are reduced survival, longevity, and body condition. Stressors may also enhance the physiological ability of adult mosquitoes to harbor and spread pathogens. This Doctoral Dissertation Improvement project pursues the mechanisms underlying the surprising result that stress during the larval stages of yellow fever mosquitoes actually enhances some aspects of general immunity, and produces adults with lower probability of spreading pathogens due to lower infection intensity and impaired body condition. The researchers will initiate laboratory experiments that manipulate larval density in order to quantify adult immune function, as red blood cell abundance and differentiation, vector susceptibility to parasite infection, and pathogen success. Results will elucidate general mechanisms by which stress alters insect immune systems and modifies interactions between insects and parasites.
Diseases transmitted by mosquitoes include some of the most important human health threats in the world. Understanding the effects of environmental stress on larvae may be of practical importance, as the container mosquitoes in this study are vectors of human disease and are widely distributed in urban environments where the probability of contact with humans is high. This project will contribute to ongoing dissertation work that relies heavily on both undergraduate and graduate-level research assistance; undergraduates will gain valuable experience and may contribute to published research. Further, in-service high school math and science teachers from the Professional MA program in Environmental Science Education at Bradley University (Peoria, IL) participate in this body of work as a summer research immersion component of their degree program.
Our previous research showed that density stress during the larval stageof Aedes aegypti can increase transcription of some immune-related genes in the adult, and produces adults that incur higher mortality due to parasite infection. However, parasite establishment and success is apparently lower in stressed individuals. These unexpected results suggested new hypotheses that were not part of the original dissertation proposal of candidate Jennifer Breaux. We proposed two hypotheses for this phenomenon: 1) Because larger mosquitoes take larger blood meals, they may imbibe more microfilaria (=mf) compared to smaller individuals, leading to a higher rate of parasite establishment and success in larger (i.e., non-stressed) mosquitoes. 2) Small-bodied (stressed) individuals experience higher mortality due to reduced mass and higher fitness costs associated with filarial infection, but those that survive to nematode maturity somehow limit parasitism better than do non-stressed individuals. We tested predictions of these two hypotheses: 1) Mosquito size and blood meal volume are positively related to the number of ingested mf and thus, parasite establishment and success and 2) Larval stress modifies trade-offs between growth and investment into cellular immunity, specifically number and differentiation of hemocytes and production of antimicrobial peptides. Specifically, we predict that relative abundances of hemocyte types (prohemocytes, granulocytes, oenocytoids) will be different for stressed vs. non-stressed adults, either initially (when non-stressed individuals should have more and a different distribution of hemocyte types) or following ingestion of a nematode-infected blood meal. We have found in this research that blood meal volume and number of ingested mf are, as predicted strongly positively related, but that larger females do NOT generally take larger blood meals, and on average, nonstressed mosquitoes do not ingest greater numbers of mf. Thus our first hypothesis does not account for the observed differences in nematode success in the the stressed vs. non-stressed adults. We have found in this research that numbers of hemocytes (total) appear to be greater for non-stressed females, and that this difference remains following both non-infectious and infectious blood meals. There was a significant synergistic effect (i.e. statistical interaction; Table 1) between larval density stress and blood meal type (pre-bloodmeal, non-infectious, infectious), such that the difference between stressed and non-stressed females decreased following an infectious blood meal (figure 1). Total hemocytes decreased from adult emergence to post-infectious blood meal approximately 6 days later, but this decrease was absent when an infectious blood meal was taken (figure 1). Thus it appears that the challenge of ingesting nematodes enhances abundance of hemocytes in this species. Because hemocyte abundance does not appear to be greater for stressed vs. non-stressed females following the infectious blood meal (figure 1), it is not clear that hemocyte abundances can account for the greater success of worms in the non-stressed females. We also found complex interactions of density and infectious blood meal impacting transcription of antimicrobial peptides (AMPs, particularly Cecropin and Transferrin - Fig. 2). The combination of high density and infectious blood meal leads to down regulation of Cecropin and Transferrin (Fig. 2). This effect may contribute to enahance mortality of density-stressed females challenged with mf ingestion. The interaction of female mosquitos' immune system with the nematode and with lingering effects of poor larval rearing conditioins thus appears to be complex. Our work had some limitations, most notably our inability to get expected numbers of hemocytes and some hemocyte subtypes (particularly prohemocytes) from our mosquitoes and this complicates the interpretation of our hemocyte results. Thus we continue to develop improved ways to collect hemocytes and to assay frequencies of different cell types. Broader impacts of this project include: 1. Training of Jennifer Breaux, who received her PhD from ISU in 2013. Dr. Breaux is now a Post Doc at Universidade Federal de Vicosa, Brazil, and continues to work on vector mosquitoes. Thus this grant has enhanced the career of a young scientist. 2. Training of three undergraduates in research. Liz Quinn worked on the project and gained valuable research experience that will enable her to convey knowledge about biological research to high school students as she pursues her career in secondary education in biology. She is now student teaching. Adam Wiggins worked on the project for one summer and as a result has shifted his emphasis as an undergraduate biology major from pre medical studies to research-oriented pursuit of a degree in biology. He continues to work in PI Juliano's lab. Joe Oremus worked on the project and obtained his first experience in research and continues to work in PI Juliano's lab on the ecoimmunology of mosquito-borne illnesses. 3. Our research incrementally improves our understanding of the interaction of environment and mosquito life history. This knowledge may yield new insights into ways of impeding disease transmission by mosquitoes and improves our understanding of the process of vector-borne disease transmission.
