Michael A. Riehle

Activation of Akt Signaling Reduces the Prevalence and Intensity of Malaria Parasite Infection and Lifespan in Anopheles stephensi Mosquitoes

Malaria (Plasmodium spp.) kills nearly one million people annually and this number will likely increase as drug and insecticide resistance reduces the effectiveness of current control strategies. The most important human malaria parasite, Plasmodium falciparum, undergoes a complex developmental cycle in the mosquito that takes approximately two weeks and begins with the invasion of the mosquito midgut. Here, we demonstrate that increased Akt signaling in the mosquito midgut disrupts parasite development and concurrently reduces the duration that mosquitoes are infective to humans. Specifically, we found that increased Akt signaling in the midgut of heterozygous Anopheles stephensi reduced the number of infected mosquitoes by 60–99%. Of those mosquitoes that were infected, we observed a 75–99% reduction in parasite load. In homozygous mosquitoes with increased Akt signaling parasite infection was completely blocked. The increase in midgut-specific Akt signaling also led to an 18–20% reduction in the average mosquito lifespan. Thus, activation of Akt signaling reduced the number of infected mosquitoes, the number of malaria parasites per infected mosquito, and the duration of mosquito infectivity.

Molecular characterization of insulin-like peptide genes and their expression in the African malaria mosquito, Anopheles gambiae.

Of the seven genes encoding insulin‐like peptides (ILPs) in the mosquito, Anopheles gambiae, four are arrayed proximally as duplicate pairs on chromosome three. Amino acid substitutions encoded in the duplicate genes occur in the C peptide and not the B and A peptides. Except for one duplicated gene, sequence‐specific transcripts for all other AgamILPs were obtained from female mosquitoes. Transcript expression of each AgamILP was determined by RT‐PCR in the head, thorax, and abdomen of all life stages and both sexes of this mosquito. Two AgamILPs were ubiquitously expressed, suggesting a growth factor function, whereas the other AgamILPs were expressed primarily in heads, as confirmed by the immunostaining of ILPs in the neurosecretory cells of female brains, thus indicating a hormonal function.

Towards genetic manipulation of wild mosquito populations to combat malaria: advances and challenges

SUMMARY Malaria kills millions of people every year, yet there has been little progress in controlling this disease. For transmission to occur, the malaria parasite has to complete a complex developmental cycle in the mosquito. The mosquito is therefore a potential weak link in malaria transmission, and generating mosquito populations that are refractory to the parasite is a potential means of controlling the disease. There has been considerable progress over the last decade towards developing the tools for creating a refractory mosquito. Accomplishments include germline transformation of several important mosquito vectors, the completed genomes of the mosquito Anopheles gambiae and the malaria parasite Plasmodium falciparum, and the identification of promoters and effector genes that confer resistance in the mosquito. These tools have provided researchers with the ability to engineer a refractory mosquito vector, but there are fundamental gaps in our knowledge of how to transfer this technology safely and effectively into field populations. This review considers strategies for interfering with Plasmodium development in the mosquito, together with issues related to the transfer of laboratory-acquired knowledge to the field, such as minimization of transgene fitness load to the mosquito, driving genes through populations, avoiding the selection of resistant strains, and how to produce and release populations of males only.

Insulin-Like Peptides: Structure, Signaling, and Function

Publisher Summary This chapter discusses the insulin-like peptides: structure, signaling, and function. Insulin-like peptides (ILPs) are paragons for the conservation of hormone structure and function among invertebrates and higher animals. They are encoded by multiple, distinct genes within each species and, upon secretion, serve as hormones, neurotransmitters, and growth factors during post-embryonic life stages. These diverse messages are transduced in target cells through an insulin receptor and a signaling network of activated proteins that directly affect biochemical pathways and gene expression. Of the diverse invertebrate groups, our accumulated knowledge of ILP endocrinology is the deepest and broadest for insects. This knowledge has revealed that essentially all of the proteins that make up the mechanisms for ILP processing, secretion, and signaling are remarkably similar to those of vertebrates, in effect conserving the pleiotropic effects of ILPs between insects and vertebrates.

Genome analysis of a major urban malaria vector mosquito, Anopheles stephensi

BackgroundAnopheles stephensi is the key vector of malaria throughout the Indian subcontinent and Middle East and an emerging model for molecular and genetic studies of mosquito-parasite interactions. The type form of the species is responsible for the majority of urban malaria transmission across its range.ResultsHere, we report the genome sequence and annotation of the Indian strain of the type form of An. stephensi. The 221 Mb genome assembly represents more than 92% of the entire genome and was produced using a combination of 454, Illumina, and PacBio sequencing. Physical mapping assigned 62% of the genome onto chromosomes, enabling chromosome-based analysis. Comparisons between An. stephensi and An. gambiae reveal that the rate of gene order reshuffling on the X chromosome was three times higher than that on the autosomes. An. stephensi has more heterochromatin in pericentric regions but less repetitive DNA in chromosome arms than An. gambiae. We also identify a number of Y-chromosome contigs and BACs. Interspersed repeats constitute 7.1% of the assembled genome while LTR retrotransposons alone comprise more than 49% of the Y contigs. RNA-seq analyses provide new insights into mosquito innate immunity, development, and sexual dimorphism.ConclusionsThe genome analysis described in this manuscript provides a resource and platform for fundamental and translational research into a major urban malaria vector. Chromosome-based investigations provide unique perspectives on Anopheles chromosome evolution. RNA-seq analysis and studies of immunity genes offer new insights into mosquito biology and mosquito-parasite interactions.

Insulin-like peptides in the mosquito Anopheles stephensi: Identification and expression in response to diet and infection with Plasmodium falciparum

Insulin-like peptides (ILPs) regulate a multitude of biological processes, including metabolism and immunity to infection, and share similar structural motifs across widely divergent taxa. Insulin/insulin-like growth factor signaling (IIS) pathway elements are similarly conserved. We have shown that IIS regulates reproduction, innate immunity, and lifespan in female Anopheles stephensi, a major mosquito vector of human malaria. To further explore IIS regulation of these processes, we identified genes encoding five ILPs in this species and characterized their expression in tissues. Antisera to ILP homologs in Anopheles gambiae were used to identify cellular sources in An. stephensi females by immunocytochemistry. We analyzed tissue-specific ILP transcript expression in young and older females, in response to different feeding regimens, and in response to infection with Plasmodiumfalciparum with quantitative reverse transcriptase-PCR assays. While some ILP transcript changes were evident in older females and in response to blood feeding, significant changes were particularly notable in response to hormonal concentrations of ingested human insulin and to P. falciparum infection. These changes suggest that ILP secretion and action may be similarly responsive in Plasmodium-infected females and potentially alter metabolism and innate immunity.

The impact of larval and adult dietary restriction on lifespan, reproduction and growth in the mosquito Aedes aegypti

Dietary restriction extends lifespan in many organisms, but little is known about how it affects hematophagous arthropods. We demonstrated that diet restriction during either larval or adult stages extends Aedes aegypti lifespan. A. aegypti females fed either single or no blood meals survived 30-40% longer than those given weekly blood meals. However, mosquitoes given weekly blood meals produced far more eggs. To minimize reproductions impact on lifespan, adult mosquitoes were fed artificial blood meals containing <10% of the protein in normal human blood, minimizing egg production. A. aegypti fed artificial blood meals containing 25mg/ml of BSA had significantly shorter lifespans than those fed either 10 or 5mg/ml. To assess the impact of larval dietary restriction on adult lifespan, we maintained larval A. aegypti on 2X, 1X (normal diet), 0.5X or 0.25X diets. Adult mosquitoes fed 0.5X and 0.25X larval diets survived significantly longer than those fed the 2X larval diet regardless of adult diet. In summary, dietary restriction during both larval and adult stages extends lifespan. This diet-mediated lifespan extension has important consequences for understanding how dietary restriction regulates lifespan and disease transmission.

Ingested Human Insulin Inhibits the Mosquito NF-κB-Dependent Immune Response to Plasmodium falciparum

ABSTRACT We showed previously that ingested human insulin activates the insulin/IGF-1 signaling pathway in Anopheles stephensi and increases the susceptibility of these mosquitoes to Plasmodium falciparum. In other organisms, insulin can alter immune responsiveness through regulation of NF-κB transcription factors, critical elements for innate immunity that are also central to mosquito immunity. We show here that insulin signaling decreased expression of NF-κB-regulated immune genes in mosquito cells stimulated with either bacterial or malarial soluble products. Further, human insulin suppressed mosquito immunity through sustained phosphatidylinositol 3-kinase activation, since inhibition of this pathway led to decreased parasite development in the mosquito. Together, these data demonstrate that activation of the insulin/IGF-1 signaling pathway by ingested human insulin can alter NF-κB-dependent immunity, and ultimately the susceptibility, of mosquitoes to P. falciparum.

Sustained Activation of Akt Elicits Mitochondrial Dysfunction to Block Plasmodium falciparum Infection in the Mosquito Host

The overexpression of activated, myristoylated Akt in the midgut of female transgenic Anopheles stephensi results in resistance to infection with the human malaria parasite Plasmodium falciparum but also decreased lifespan. In the present study, the understanding of mitochondria-dependent midgut homeostasis has been expanded to explain this apparent paradox in an insect of major medical importance. Given that Akt signaling is essential for cell growth and survival, we hypothesized that sustained Akt activation in the mosquito midgut would alter the balance of critical pathways that control mitochondrial dynamics to enhance parasite killing at some cost to survivorship. Toxic reactive oxygen and nitrogen species (RNOS) rise to high levels in the midgut after blood feeding, due to a combination of high NO production and a decline in FOXO-dependent antioxidants. Despite an apparent increase in mitochondrial biogenesis in young females (3 d), energy deficiencies were apparent as decreased oxidative phosphorylation and increased [AMP]/[ATP] ratios. In addition, mitochondrial mass was lower and accompanied by the presence of stalled autophagosomes in the posterior midgut, a critical site for blood digestion and stem cell-mediated epithelial maintenance and repair, and by functional degradation of the epithelial barrier. By 18 d, the age at which An. stephensi would transmit P. falciparum to human hosts, mitochondrial dysfunction coupled to Akt-mediated repression of autophagy/mitophagy was more evident and midgut epithelial structure was markedly compromised. Inhibition of RNOS by co-feeding of the nitric-oxide synthase inhibitor L-NAME at infection abrogated Akt-dependent killing of P. falciparum that begins within 18 h of infection in 3–5 d old mosquitoes. Hence, Akt-induced changes in mitochondrial dynamics perturb midgut homeostasis to enhance parasite resistance and decrease mosquito infective lifespan. Further, quality control of mitochondrial function in the midgut is necessary for the maintenance of midgut health as reflected in energy homeostasis and tissue repair and renewal.

Expression of a mutated phospholipase A2 in transgenic Aedes fluviatilis mosquitoes impacts Plasmodium gallinaceum development

The genetic manipulation of mosquito vectors is an alternative strategy in the fight against malaria. It was previously shown that bee venom phospholipase A2 (PLA2) inhibits ookinete invasion of the mosquito midgut although mosquito fitness was reduced. To maintain the PLA2 blocking ability without compromising mosquito biology, we mutated the protein‐coding sequence to inactivate the enzyme while maintaining the proteins structure. DNA encoding the mutated PLA2 (mPLA2) was placed downstream of a mosquito midgut‐specific promoter (Anopheles gambiae peritrophin protein 1 promoter, AgPer1) and this construct used to transform Aedes fluviatilis mosquitoes. Four different transgenic lines were obtained and characterized and all lines significantly inhibited Plasmodium gallinaceum oocyst development (up to 68% fewer oocysts). No fitness cost was observed when this mosquito species expressed the mPLA2.