Elizabeth A. McGraw

Wolbachia is an intracellular bacterium that has been stably transinfected into the mosquito vector of dengue, Aedes aegypti. This inherited infection causes a range of metabolic and phenotypic alterations in the mosquito, which might be related to neuronal abnormalities. In order to determine if these alterations were caused by the manipulation of neuroamines by this bacterium, we studied the expression of genes involved in the dopamine biosynthetic pathway and also measured the amount of dopamine in infected and uninfected mosquitoes of different ages. Wolbachia-infected mosquitoes exhibit greater expression of some genes related to the melanization pathway, but not for those directly linked to dopamine production. Although dopamine levels were higher in Wolbachia-positive mosquitoes this was not consistent across all insect ages nor was it related to the previously described Wolbachia induced "bendy" and "shaky" phenotypes.

Transcriptional profiling is an effective method of predicting age in the mosquito Aedes aegypti in the laboratory, however, its effectiveness is limited to younger mosquitoes. To address this we used a microarray to identify new gene candidates that show significant expression changes in older mosquitoes. These genes were then used to create a revised model, which upon evaluation in both laboratory and semi-field conditions, proved to have improved accuracy overall and for older mosquitoes. In association with the development of symbiont-based control strategies for Ae. aegypti, we also tested the model's accuracy for Wolbachia-infected mosquitoes and found no decline in performance. Our findings suggest that the new model is a robust and powerful tool for age determination in Australian Ae. aegypti populations.

Mosquitoes are vectors of many serious pathogens in tropical and sub-tropical countries. Current control strategies almost entirely rely upon insecticides, which increasingly face the problems of high cost, increasing mosquito resistance and negative effects on non-target organisms. Alternative strategies include the proposed use of inherited life-shortening agents, such as the Wolbachia bacterium. By shortening mosquito vector lifespan, Wolbachia could potentially reduce the vectorial capacity of mosquito populations. We have recently been able to stably transinfect Aedes aegypti mosquitoes with the life-shortening Wolbachia strain wMelPop, and are assessing various aspects of its interaction with the mosquito host to determine its likely impact on pathogen transmission as well as its potential ability to invade A. aegypti populations. Here we have examined the probing behavior of Wolbachia-infected mosquitoes in an attempt to understand both the broader impact of Wolbachia infection on mosquito biology and, in particular, vectorial capacity. The probing behavior of wMelPop-infected mosquitoes at four adult ages was examined and compared to uninfected controls during video-recorded feeding trials on a human hand. Wolbachia-positive insects, from 15 days of age, showed a drastic increase in the time spent pre-probing and probing relative to uninfected controls. Two other important features for blood feeding, saliva volume and apyrase content of saliva, were also studied. As A. aegypti infected with wMelPop age, they show increasing difficulty in completing the process of blood feeding effectively and efficiently. Wolbachia-infected mosquitoes on average produced smaller volumes of saliva that still contained the same amount of apyrase activity as uninfected mosquitoes. These effects on blood feeding behavior may reduce vectorial capacity and point to underlying physiological changes in Wolbachia-infected mosquitoes. Mosquitoes transmit diseases when they are actively searching for a source of blood. This so called probing behavior comprises the “searching” time, the beginning of the feeding process until the first sign of blood can be seen within the insect body. The manipulation of this behavior can have important consequences for the mosquito's ability to transmit pathogens, such as dengue virus or Plasmodium. In this study we examined the probing behavior of the main vector of dengue viruses, Aedes aegypti, when infected with an intracellular bacterium, Wolbachia pipientis. This bacterium alters the probing behavior of older mosquitoes such that they take longer to find a feeding site and longer to imbibe blood, which may make them more susceptible to human defense responses. The bacterium appears to reduce mosquito feeding success by preventing the mosquito from successfully inserting its stylet into human skin. The old age onset of reduced mosquito feeding success due to Wolbachia could selectively promote a reduction in dengue transmission.

The mosquito Aedes aegypti was recently transinfected with a life-shortening strain of the endosymbiont Wolbachia pipientis (wMelPop) as the first step in developing a biocontrol strategy for dengue virus transmission. In addition to life-shortening, the wMelPop-infected mosquitoes also exhibit increased daytime activity and metabolic rates. Here we sought to quantify the blood-feeding behaviour of Wolbachia-infected females as an indicator of any virulence or energetic drain associated with Wolbachia infection. In a series of blood-feeding trials in response to humans, we have shown that Wolbachia-infected mosquitoes do not differ in their response time to humans, but that as they age they obtain fewer and smaller blood meals than Wolbachia-uninfected controls. Lastly, we observed a behavioural characteristic in the Wolbachia infected mosquitoes best described as a “bendy” proboscis that may explain the decreased biting success. Taken together the evidence suggests that wMelPop infection may be causing tissue damage in a manner that intensifies with mosquito age and that leads to reduced blood-feeding success. These behavioural changes require further investigation with respect to a possible physiological mechanism and their role in vectorial capacity of the insect. The selective decrease of feeding success in older mosquitoes may act synergistically with other Wolbachia-associated traits including life-shortening and viral protection in biocontrol strategies. The primary mosquito vector of dengue virus, Aedes aegypti, has recently been artificially infected with a symbiotic bacterium called Wolbachia pipientis. This bacterium occurs naturally inside the cells of ∼66% of insect species. The Wolbachia used to infect A. aegypti shortens the insect's lifespan. Because only old mosquitoes are capable of transmitting dengue virus, the Wolbachia infection could theoretically reduce dengue virus transmission if infected mosquitoes were released into the wild. Here we have examined the effects of this Wolbachia infection on the mosquito's ability to obtain blood meals from human hosts. Blood is required for females to produce eggs, and so successful completion of this behaviour is necessary if Wolbachia-infected mosquitoes are to be competitive in the wild. Blood feeding on humans is also the time when viruses like dengue are transmitted, so changes in this behaviour can have consequences for the transmission rate of viruses. We show that Wolbachia-infected mosquitoes are less able to obtain blood meals, but only in old age. The reduced feeding success may be explained by a defect in the insect's proboscis. The finding is exciting as it may allow young mosquitoes to breed as normal but help reduce the lifespan and success of old mosquitoes, which are the primary transmitters of virus.

A virulent strain of the obligate intracellular bacterium Wolbachia pipientis that shortens insect lifespan has recently been transinfected into the primary mosquito vector of dengue virus, Aedes aegypti L. The microbe's ability to shorten lifespan and spread through host populations under the action of cytoplasmic incompatibility means it has the potential to be used as a biocontrol agent to reduce dengue virus transmission. Wolbachia is present in many host tissues and may have local effects on diverse biological processes. In other insects, Wolbachiainfections have been shown to alter locomotor activity and response time to food cues. In mosquitoes, locomotor performance relates to the location of mates, human hosts, resting sites and oviposition sites. We have therefore examined the effect of the virulent, life-shortening Wolbachia strain wMelPop on the locomotion of Ae. aegypti as they age and as the pathogenicity of the infection increases. In parallel experiments we also examined CO₂ production as a proxy for metabolic rate, to investigate a potential mechanistic explanation for any changes in locomotion. Contrary to expectation, we found that the infection increased activity and metabolic rate and that these effects were relatively consistent over the insect's lifespan. The results do not fit a standard model of bacterial pathogenesis in insects, and instead may reveal additional physiological changes induced by infection, such as either increased hunger or defects in the nervous system.

Drosophila harbor substantial genetic variation for antibacterial defense, and investment in immunity is thought to involve a costly trade-off with life history traits, including development, life span, and reproduction. To understand the way in which insects invest in fighting bacterial infection, we selected for survival following systemic infection with the opportunistic pathogen Pseudomonas aeruginosa in wild-caught Drosophila melanogaster over 10 generations. We then examined genome-wide changes in expression in the selected flies relative to unselected controls, both of which had been infected with the pathogen. This powerful combination of techniques allowed us to specifically identify the genetic basis of the evolved immune response. In response to selection, population-level survivorship to infection increased from 15% to 70%. The evolved capacity for defense was costly, however, as evidenced by reduced longevity and larval viability and a rapid loss of the trait once selection pressure was removed. Counter to expectation, we observed more rapid developmental rates in the selected flies. Selection-associated changes in expression of genes with dual involvement in developmental and immune pathways suggest pleiotropy as a possible mechanism for the positive correlation. We also found that both the Toll and the Imd pathways work synergistically to limit infectivity and that cellular immunity plays a more critical role in overcoming P. aeruginosa infection than previously reported. This work reveals novel pathways by which Drosophila can survive infection with a virulent pathogen that may be rare in wild populations, however, due to their cost. The fruit fly is commonly used as a model organism to understand the mechanistic nature of the immune response to bacterial pathogens. The fly is also commonly used to understand what immunity costs hosts in terms of other traits such as life span and reproductive success. Here, we examine these two questions together in flies selected for improved defense against the bacterium Pseudomonas aeruginosa. We show that selected flies develop from egg to adult more rapidly than unselected flies. It appears that the selected flies invest more heavily in a wing of the immune system that involves engulfment and walling off of invading bacteria. This investment can also explain the shift in developmental rate, as these two biological pathways are controlled by shared sets of genes. These latter two findings are counter to the conventional wisdom and reveal a costly, but effective, means for the fly to circumvent the virulence of Pseudomonas aeruginosa. This bacterium is normally deadly, as it has specific mechanisms to evade the host immune response. Our work is significant for demonstrating a pathway for flies to survive bacterial infection with Pseudomonas aeruginosa and for offering a reason why such a defense is not normally present in wild populations.

Background: The native Australian fly Drosophila serrata belongs to the highly speciose montium subgroup of the melanogaster species group. It has recently emerged as an excellent model system with which to address a number of important questions, including the evolution of traits under sexual selection and traits involved in climatic adaptation along latitudinal gradients. Understanding the molecular genetic basis of such traits has been limited by a lack of genomic resources for this species. Here, we present the first expressed sequence tag (EST) collection for D. serrata that will enable the identification of genes underlying sexually-selected phenotypes and physiological responses to environmental change and may help resolve controversial phylogenetic relationships within the montium subgroup. Results: A normalized cDNA library was constructed from whole fly bodies at several developmental stages, including larvae and adults. Assembly of 11,616 clones sequenced from the 3' end allowed us to identify 6,607 unique contigs, of which at least 90% encoded peptides. Partial transcripts were discovered from a variety of genes of evolutionary interest by BLASTing contigs against the 12 Drosophila genomes currently sequenced. By incorporating into the cDNA library multiple individuals from populations spanning a large portion of the geographical range of D. serrata, we were able to identify 11,057 putative single nucleotide polymorphisms (SNPs), with 278 different contigs having at least one "double hit" SNP that is highly likely to be a real polymorphism. At least 394 EST-associated microsatellite markers, representing 355 different contigs, were also found, providing an additional set of genetic markers. The assembled EST library is available online at http://www.chenowethlab.org/serrata/index.cgi. Conclusion: We have provided the first gene collection and largest set of polymorphic genetic markers, to date, for the fly D. serrata. The EST collection will provide much needed genomic resources for this model species and facilitate comparative evolutionary studies within the montium subgroup of the D. melanogaster lineage.

Wolbachia pipientisis an endosymbiotic bacterium present in diverse insect species. Although it is well studied for its dramatic effects on host reproductive biology, little is known about its effects on other aspects of host biology, despite its presence in a wide array of host tissues. This study examined the effects of threeWolbachiastrains on two differentDrosophilaspecies, using a laboratory performance assay for insect locomotion in response to olfactory cues. The results demonstrate thatWolbachiainfection can have significant effects on host responsiveness that vary with respect to theWolbachiastrain-host species combination. ThewRi strain, native toDrosophila simulans, increases the basal activity level of the host insect as well as its responsiveness to food cues. In contrast, thewMel strain and the virulentwMelPop strain, native toDrosophila melanogaster, cause slight decreases in responsiveness to food cues but do not alter basal activity levels in the host. Surprisingly, the virulentwMelPop strain has very little impact on host responsiveness inD. simulans. This novel strain-host relationship was artificially created previously by transinfection. These findings have implications for understanding the evolution and spread ofWolbachiainfections in wild populations and forWolbachia-based vector-borne disease control strategies currently being developed.

Background: The endosymbiontWolbachia pipientisinfects a broad range of arthropod and filarial nematode hosts. These diverse associations form an attractive model for understanding host:symbiont coevolution.Wolbachia's ubiquity and ability to dramatically alter host reproductive biology also form the foundation of research strategies aimed at controlling insect pests and vector-borne disease. TheWolbachiastrains that infect nematodes are phylogenetically distinct, strictly vertically transmitted, and required by their hosts for growth and reproduction. Insects in contrast form more fluid associations withWolbachia. In these taxa, host populations are most often polymorphic for infection, horizontal transmission occurs between distantly related hosts, and direct fitness effects on hosts are mild. Despite extensive interest in theWolbachiasystem for many years, relatively little is known about the molecular mechanisms that mediate its varied interactions with different hosts. We have compared the genomes of theWolbachiathat infectDrosophila melanogaster,wMel and the nematodeBrugia malayi,wBm to that of an outgroupAnaplasma marginaleto identify genes that have experienced diversifying selection in theWolbachialineages. The goal of the study was to identify likely molecular mechanisms of the symbiosis and to understand the nature of the diverse association across different hosts.Results: The prevalence of selection was far greater inwMel thanwBm. Genes contributing to DNA metabolism, cofactor biosynthesis, and secretion were positively selected in both lineages. InwMel there was a greater emphasis on DNA repair, cell division, protein stability, and cell envelope synthesis.Conclusion: Secretion pathways and outer surface protein encoding genes are highly affected by selection in keeping with host:parasite theory. If evidence of selection on various cofactor molecules reflects possible provisioning, then both insect as well as nematodeWolbachiamay be providing substances to hosts. Selection on cell envelope synthesis, DNA replication and repair machinery, heat shock, and two component switching suggest strategies insectWolbachiamay employ to cope with diverse host and intra-host environments.