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Travassos, M.A., A. Niangaly, J.A. Bailey, A. Outtara, D. Coulibaly, K.E. Lyke, M.B. Laurens, J. Pablo, A. Jasinskas, R. Nakajima, A.A. Berry, M. Adams, C.G. Jacob, A. Pike, S. Takala-Harrison, L. Liang, B. Kouriba, A.K. Kone, J.A. Rowe, J. Moulds, D.A. Diallo, O.K. Doumbo, M.A. Thera, P.L. Felgner and C.V. Plowe. 2018. "Children with cerebral malaria or severe malarial anaemia lack immunity to distinct variant surface antigen subsets." Scientific Reports 8:6281.
Variant surface antigens (VSAs) play a critical role in severe malaria pathogenesis. Defining gaps, or “lacunae”, in immunity to these Plasmodium falciparum antigens in children with severe malaria would improve our understanding of vulnerability to severe malaria and how protective immunity develops. Using a protein microarray with 179 antigen variants from three VSA families as well as more than 300 variants of three other blood stage P. falciparum antigens, reactivity was measured in sera from Malian children with cerebral malaria or severe malarial anaemia and age-matched controls. Sera from children with severe malaria recognized fewer extracellular PfEMP1 fragments and were less reactive to specific fragments compared to controls. Following recovery from severe malaria, convalescent sera had increased reactivity to certain non-CD36 binding PfEMP1s, but not other malaria antigens. Sera from children with severe malarial anaemia reacted to fewer VSAs than did sera from children with cerebral malaria, and both of these groups had lacunae in their seroreactivity profiles in common with children who had both cerebral malaria and severe malarial anaemia. This microarray-based approach may identify a subset of VSAs that could inform the development of a vaccine to prevent severe disease or a diagnostic test to predict at-risk children.

Pan , X., A. Pike, D. Joshi, G. Bian, M. J. McFadden, P. Lu, X. Liang, F. Zhang, A. S. Raikhel, and Z. Xi. 2017. "The bacterium Wolbachia exploits host innate immunity to establish a symbiotic relationship with the dengue vector mosquito Aedes aegypti." ISME Journal.
A host’s immune system plays a central role in shaping the composition of the microbiota and, in return, resident microbes influence immune responses. Symbiotic associations of the maternally transmitted bacterium Wolbachia occur with a wide range of arthropods. It is, however, absent from the dengue and Zika vector mosquito Aedes aegypti in nature. When Wolbachia is artificially forced to form symbiosis with this new mosquito host, it boosts the basal immune response and enhances the mosquito’s resistance to pathogens, including dengue, Zika virus and malaria parasites. The mechanisms involved in establishing a symbiotic relationship between Wolbachia and A. aegypti, and the long-term outcomes of this interaction, are not well understood. Here, we have demonstrated that both the immune deficiency (IMD) and Toll pathways are activated by the Wolbachia strain wAlbB upon its introduction into A. aegypti. Silencing the Toll and IMD pathways via RNA interference reduces the wAlbB load. Notably, wAlbB induces peptidoglycan recognition protein (PGRP)-LE expression in the carcass of A. aegypti, and its silencing results in a reduction of symbiont load. Using transgenic mosquitoes with stage-specific induction of the IMD and Toll pathways, we have shown that elevated wAlbB infection in these mosquitoes is maintained via maternal transmission. These results indicate that host innate immunity is utilized to establish and promote host-microbial symbiosis. Our results will facilitate a long-term projection of the stability of the Wolbachia–A. aegypti mosquito system that is being developed to control dengue and Zika virus transmission to humans.

Pike, A., Y. Dong, N. B. Dizaji, A. Gacita, E. F. Mongodin and G. Dimopoulos. 2017. "Changes in the microbiota cause genetically modified Anopheles to spread in a population." Science 357(6358):1296-1399.
The mosquito’s innate immune system controls both Plasmodium and bacterial infections. We investigated the competitiveness of mosquitoes genetically modified to alter expression of their own anti-Plasmodium immune genes in a mixed-cage population with wild-type mosquitoes. We observed that genetically modified mosquitoes with increased immune activity in the midgut tissue did not have an observed fitness disadvantage and showed reduced microbial loads in both the midgut and reproductive organs. These changes result in a mating preference of genetically modified males for wild-type females, whereas wild-type males prefer genetically modified females. These changes foster the spread of the genetic modification in a mosquito cage population.

Anglero-Rodriguez Y.I., B.J. Blumberg, Y. Dong, S.L. Sandiford, A. Pike, A.M. Clayton, and G. Dimopoulos. 2016. “A natural Anopheles-associated Penicillium chrysogenum enhances mosquito susceptibility to Plasmodium infection.” Scientific Reports 6:34084.
Whereas studies have extensively examined the ability of bacteria to influence Plasmodium infection in the mosquito, the tripartite interactions between non-entomopathogenic fungi, mosquitoes, and Plasmodium parasites remain largely uncharacterized. Here we report the isolation of a common mosquito-associated ascomycete fungus, Penicillium chrysogenum, from the midgut of field-caught Anopheles mosquitoes. Although the presence of Pe. chrysogenum in the Anopheles gambiae midgut does not affect mosquito survival, it renders the mosquito significantly more susceptible to Plasmodium infection through a secreted heat-stable factor. We further provide evidence that the mechanism of the fungus-mediated modulation of mosquito susceptibility to Plasmodium involves an upregulation of the insect's ornithine decarboxylase gene, which sequesters arginine for polyamine biosynthesis. Arginine plays an important role in the mosquito's anti-Plasmodium defense as a substrate of nitric oxide production, and its availability therefore has a direct impact on the mosquito's susceptibility to the parasite. While this type of immunomodulatory mechanism has already been demonstrated in other host-pathogen interaction systems, this is the first report of a mosquito-associated fungus that can suppress the mosquito's innate immune system in a way that would favor Plasmodium infection and possibly malaria transmission.

Mesquita, R.D., A. Pike, et al. 2015. “The genome of Rhodnius prolixus, an Insect Vector of Chagas Disease, Reveals Unique Adaptations to Hematophagy and Parasite Infection.” PNAS 112 (48) 14936-14941
Rhodnius prolixus not only has served as a model organism for the study of insect physiology, but also is a major vector of Chagas disease, an illness that affects approximately seven million people worldwide. We sequenced the genome of R. prolixus, generated assembled sequences covering 95% of the genome (∼ 702 Mb), including 15,456 putative protein-coding genes, and completed comprehensive genomic analyses of this obligate blood-feeding insect. Although immune-deficiency (IMD)-mediated immune responses were observed, R. prolixus putatively lacks key components of the IMD pathway, suggesting a reorganization of the canonical immune signaling network. Although both Toll and IMD effectors controlled intestinal microbiota, neither affected Trypanosoma cruzi, the causal agent of Chagas disease, implying the existence of evasion or tolerance mechanisms. R. prolixus has experienced an extensive loss of selenoprotein genes, with its repertoire reduced to only two proteins, one of which is a selenocysteine-based glutathione peroxidase, the first found in insects. The genome contained actively transcribed, horizontally transferred genes from Wolbachia sp., which showed evidence of codon use evolution toward the insect use pattern. Comparative protein analyses revealed many lineage-specific expansions and putative gene absences in R. prolixus, including tandem expansions of genes related to chemoreception, feeding, and digestion that possibly contributed to the evolution of a blood-feeding lifestyle. The genome assembly and these associated analyses provide critical information on the physiology and evolution of this important vector species and should be instrumental for the development of innovative disease control methods.

Sandiford, S.L., Y. Dong, A. Pike, B.J. Blumberg, A.C. Bahia, and G. Dimopoulos. 2015. "Cytoplasmic Actin is an Extracellular Insect Immune Factor which is Secreted upon Immune Challenge and Mediates Phagocytosis and Direct Killing of Bacteria, and is a Plasmodium Agonist." PLoS Pathogens 11(2): e1004631
Actin is a highly versatile, abundant, and conserved protein, with functions in a variety of intracellular processes. Here, we describe a novel role for insect cytoplasmic actin as an extracellular pathogen recognition factor that mediates antibacterial defense. Insect actins are secreted from cells upon immune challenge through an exosome-independent pathway. Anopheles gambiae actin interacts with the extracellular MD2-like immune factor AgMDL1, and binds to the surfaces of bacteria, mediating their phagocytosis and direct killing. Globular and filamentous actins display distinct functions as extracellular immune factors, and mosquito actin is a Plasmodium infection antagonist.

Pike, A., A. Vadlamani, S.L. Sandiford, A. Gacita and G. Dimopoulos. 2014. "Characterization of the Rel2-Regulated Transcriptome and Proteome of Anopheles stephensi Identifies new anti-Plasmodium Factors." Insect Biochemistry and Molecular Biology 52:82-93.
Mosquitoes possess an innate immune system that is capable of limiting infection by a variety of pathogens, including the Plasmodium spp. parasites responsible for human malaria. The Anopheles immune deficiency (IMD) innate immune signaling pathway confers resistance to Plasmodium falciparum. While some previously identified Anopheles anti-Plasmodium effectors are regulated through signaling by Rel2, the transcription factor of the IMD pathway, many components of this defense system remain uncharacterized. To begin to better understand the regulation of immune effector proteins by the IMD pathway, we used oligonucleotide microarrays and iTRAQ to analyze differences in mRNA and protein expression, respectively, between transgenic Anopheles stephensi mosquitoes exhibiting blood meal-inducible overexpression of an active recombinant Rel2 and their wild-type conspecifics. Numerous genes were differentially regulated at both the mRNA and protein levels following induction of Rel2. While multiple immune genes were up-regulated, a majority of the differentially expressed genes have no known immune function in mosquitoes. Selected up-regulated genes from multiple functional categories were tested for both anti-Plasmodium and anti-bacterial action using RNA interference (RNAi). Based on our experimental findings, we conclude that increased expression of the IMD immune pathway-controlled transcription factor Rel2 affects the expression of numerous genes with diverse functions, suggesting a broader physiological impact of immune activation and possible functional versatility of Rel2. Our study has also identified multiple novel genes implicated in anti-Plasmodium defense.

Hamm, C.A., C.A. Handley, A. Pike, M.L. Forister, J.A. Fordyce, and C.C. Nice. 2014. "Wolbachia infection and Lepidoptera of conservation concern." Journal of Insect Science 14.6.

Conservation of at-risk species requires multi-faceted and carefully-considered management approaches to be successful. For arthropods, the presence of endosymbiotic bacteria, such as Wolbachia (Rickettsiales: Rickettsiaceae), may complicate management plans and exacerbate the challenges faced by conservation managers. Wolbachia poses a substantial and underappreciated threat to the conservation of arthropods because infection may induce a number of phenotypic effects, most of which are considered deleterious to the host population. In this study the prevalence of Wolbachia infection in lepidopteran species of conservation concern was examined. Using standard molecular techniques, 22 species of Lepidoptera were screened, of which 19 were infected with Wolbachia. This rate is comparable to that observed in insects as a whole. However, this is likely an underestimate because geographic sampling was not extensive and may not have included infected segments of the species' ranges. Wolbachia infections may be particularly problematic for conservation management plans that incorporate captive propagation or translocation. Inadvertent introduction of Wolbachia into uninfected populations or introduction of a new strain may put these populations at greater risk for extinction. Further sampling to investigate the geographic extent of Wolbachia infections within species of conservation concern and experiments designed to determine the nature of the infection phenotype(s) are necessary to manage the potential threat of infection.

Pike, A., C.M. Cirimotich and G. Dimopoulos. 2013. “Impact of Transgenic Immune Deployment on Mosquito Fitness.” In W. Takken and C.J. Koenraadt [eds.], Ecology of Parasite-Vector Interactions. Wageningen Academic Publishers, Wageningen. 2013.
Mosquitoes are the vectors of pathogens causing numerous human diseases, including dengue and malaria. Due to increases in drug resistance among pathogens or the lack of effective treatments for these diseases and increasing insecticide resistance among mosquito populations, new methods of control are urgently needed to limit the morbidity and mortality caused by vector-borne diseases. Mosquitoes possess an innate immune system capable of limiting infection with human pathogens, and the creation and deployment of transgenic mosquitoes with an enhanced immune system has been suggested as a novel means to reduce mosquito vector competence. However, activation of the immune system is often associated with a cost to the host, which could limit the ability of the transgenic insects to replace their wild-type conspecifics. Here, we discuss recent research into the effects of increased immune deployment and insect transgenesis on the fitness of the mosquitoes.

Dong, Y., C. M. Cirimotich, A. Pike, R. Chandra and G. Dimopoulos. 2012. Anopheles NF-κB-Regulated Splicing Factors Direct Pathogen-Specific Repertoires of the Hypervariable Pattern Recognition Receptor AgDscam. Cell Host and Microbe 12(4):521-30.
Insects rely on innate immune responses controlled by the immune deficiency (IMD), Toll, and other immune signaling pathways to combat infection by a broad spectrum of pathogens. These pathways signal to downstream NF-κB family transcription factors that control specific antipathogen action via direct transcriptional control of immune effectors, hematopoiesis, and melanization. Here we show that in the Anopheles malaria vector, IMD and Toll pathways mediate species-specific defenses against Plasmodium and bacteria through the transcriptional regulation of splicing factors Caper and IRSF1 that, in turn, determine the production of pathogen-specific splice variant repertoires of the hypervariable pattern recognition receptor AgDscam. This mechanism represents an additional level of immune response regulation that may provide a previously unrecognized level of plasticity to the insect immune pathway-regulated antipathogen defenses.

Hughes, G.*, A. Pike*, P. Xue, and J. Rasgon. 2012. Invasion of Wolbachia into Anopheles and other insect germlines in an ex vivo organ culture system. PLoS One 7: e36277.
The common bacterial endosymbiont Wolbachia manipulates its host's reproduction to promote its own maternal transmission, and can interfere with pathogen development in many insects making it an attractive agent for the control of arthropod-borne disease. However, many important species, including Anopheles mosquitoes, are uninfected. Wolbachia can be artificially transferred between insects in the laboratory but this can be a laborious and sometimes fruitless process. We used a simple ex vivo culturing technique to assess the suitability of Wolbachia-host germline associations. Wolbachia infects the dissected germline tissue of multiple insect species when the host tissue and bacteria are cultured together. Ovary and testis infection occurs in a density-dependent manner. Wolbachia strains are more capable of invading the germline of their native or closely related rather than divergent hosts. The ability of Wolbachia to associate with the germline of novel hosts is crucial for the development of stably-transinfected insect lines. Rapid assessment of the suitability of a strain-host combination prior to transinfection may dictate use of a particular Wolbachia strain. Furthermore, the cultured germline tissues of two major Anopheline vectors of Plasmodium parasites are susceptible to Wolbachia infection. This finding further enhances the prospect of using Wolbachia for the biological control of malaria.

Guo, X., Y. Xu, G. Bian, A. Pike, Y Xie, and Z. Xi. 2010. Response of the mosquito protein interaction network to dengue infection. BMC Genomics 11:380.

BACKGROUND:

Two fifths of the world's population is at risk from dengue. The absence of effective drugs and vaccines leaves vector control as the primary intervention tool. Understanding dengue virus (DENV) host interactions is essential for the development of novel control strategies. The availability of genome sequences for both human and mosquito host greatly facilitates genome-wide studies of DENV-host interactions.

RESULTS:

We developed the first draft of the mosquito protein interaction network using a computational approach. The weighted network includes 4,214 Aedes aegypti proteins with 10,209 interactions, among which 3,500 proteins are connected into an interconnected scale-free network. We demonstrated the application of this network for the further annotation of mosquito proteins and dissection of pathway crosstalk. Using three datasets based on physical interaction assays, genome-wide RNA interference (RNAi) screens and microarray assays, we identified 714 putative DENV-associated mosquito proteins. An integrated analysis of these proteins in the network highlighted four regions consisting of highly interconnected proteins with closely related functions in each of replication/transcription/translation (RTT), immunity, transport and metabolism. Putative DENV-associated proteins were further selected for validation by RNAi-mediated gene silencing, and dengue viral titer in mosquito midguts was significantly reduced for five out of ten (50.0%) randomly selected genes.

CONCLUSIONS:

Our results indicate the presence of common host requirements for DENV in mosquitoes and humans. We discuss the significance of our findings for pharmacological intervention and genetic modification of mosquitoes for blocking dengue transmission.

Bosch, R. and A. Pike. 2009. “Map-Colored Mosaics” Bridges Banff II: Mathematical Connections in art, music, and science 139-146.
We describe how to construct map-colored mosaics. When viewed from afar, they resemble familiar images. When viewed from up close, they look like properly colored maps.