Mosquito immune responses and malaria transmission: lessons from insect model systems and implications for vertebrate innate immunity and vaccine development

The introduction of novel biochemical, genetic, molecular and cell biology tools to the study of insect immunity has generated an information explosion in recent years. Due to the biodiversity of insects, complementary model systems have been developed. The conceptual framework built based on these systems is used to discuss our current understanding of mosquito immune responses and their implications for malaria transmission. The areas of insect and vertebrate innate immunity are merging as new information confirms the remarkable extent of the evolutionary conservation, at a molecular level, in the signaling pathways mediating these responses in such distant species. Our current understanding of the molecular language that allows the vertebrate innate immune system to identify parasites, such as malaria, and direct the acquired immune system to mount a protective immune response is very limited. Insect vectors of parasitic diseases, such as mosquitoes, could represent excellent models to understand the molecular responses of epithelial cells to parasite invasion. This information could broaden our understanding of vertebrate responses to parasitic infection and could have extensive implications for anti-malarial vaccine development.     

Ecdysone and the cell cycle: Investigations in a mosquito cell line

Cell lines provide a tool for investigating basic biological processes that underlie the complex interactions among the tissues and organs of an intact organism. We compare the evolution of insect and mammalian populations as they progress from diploid cell strains to continuous cell lines, and review the history of the well-characterized Aedes albopictus mosquito cell line, C7-10. Like Kc and S3 cells from Drosophila melanogaster, C7-10 cells are sensitive to the insect steroid hormone, 20-hydroxyecdysone (20E), and express 20E-inducible proteins as well as the EcR and USP components of the ecdysteroid receptor. The decrease in growth associated with 20E treatment results in an accumulation of cells in the G1 phase of the cycle, and a concomitant decrease in levels of cyclin A. In contrast, 20E induces a G2 arrest in a well-studied imaginal disc cell line from the moth, Plodia interpunctella. We hypothesize that 20E-mediated events associated with molting and metamorphosis include effects on regulatory proteins that modulate the mitotic cell cycle and that differences between the 20E response in diverse insect cell lines reflect an interplay between classical receptor-mediated effects on gene expression and non-classical effects on signaling pathways similar to those recently described for the vertebrate steroid hormone, estrogen.     

Interaction of insect trypanosomatids with mosquitoes, sand fly and the respective insect cell lines

Interaction experiments between hematophagous insects and monoxenous trypanosomatids have become relevant, once cases of human infection involving these protozoa have been reported. Moreover, investigations related to the interaction of insects with trypanosomatids that harbour an endosymbiotic bacterium and thereby lack the paraflagellar rod structure are important to elucidate the role of this structure in the adhesion process. In this work, we compared the interaction of endosymbiont-bearing trypanosomatids and their aposymbiotic counterpart strains (without endosymbionts) with cell lines of Anopheles gambiae, Aedes albopictus and Lutzomyia longipalpis and with explanted guts of the respective insects. Endosymbiont-bearing strains interacted better with insect cells and guts when compared with aposymbiotic strains. In vitro binding assays revealed that the trypanosomatids interacted with the gut epithelial cells via flagellum and cell body. Flagella attached to the insect gut were enlarged, containing electrondense filaments between the axoneme and flagellar membrane at the point of adhesion. Interactions involving the flagellum lacking paraflagellar rod structure were mainly observed close to tight junctions, between epithelial cells. Endosymbiont-bearing trypanosomatids were able to colonise Aedes aegypti guts after protozoa feeding.     

Plasmodium-mosquito interactions: a tale of dangerous liaisons

To complete their life cycle, Plasmodium parasites must survive the environment in the insect host, cross multiple barriers including epithelial layers, and avoid destruction by the mosquito immune system. Completion of the Anopheles gambiae and Plasmodium falciparum genomes has opened the opportunity to apply high throughput methods to the analysis of gene function. The burst of information generated by these approaches and the use of molecular markers to investigate the cell biology of these interactions is broadening our understanding of this complex system. This review discusses our current understanding of the critical interactions that take place during the journey of Plasmodium through the mosquito host, with special emphasis on the responses of midgut epithelial cells to parasite invasion.     

Chikungunya virus infection of cell lines: analysis of the East, Central and South African lineage

Chikungunya virus (CHIKV) is a re-emerging mosquito borne alphavirus that has caused large scale epidemics in the countries around the Indian Ocean, as well as leading to autochthonous transmission in some European countries. The transmission of the disease has been driven by the emergence of an African lineage of CHIKV with enhanced transmission and dissemination in Aedes mosquito hosts. Two main genotypes of this lineage have been circulating, characterized by the presence of a substitution of a valine for an alanine at position 226 of the E1 protein. The outbreak, numbering in millions of cases in the infected areas, has been associated with increasing numbers of cases with non-classical presentation including encephalitis and meningitis. This study sought to compare the original Ross strain with two isolates from the recent outbreak of chikungunya fever in respect of infectivity and the induction of apoptosis in eight mammalian cell lines and two insect cell lines, in addition to generating a comprehensive virus production profile for one of the newer isolates. Results showed that in mammalian cells there were few differences in either tropism or pathogenicity as assessed by induction of apoptosis with the exception of Hela cells were the recent valine isolate showed less infectivity. The Aedes albopictus C6/36 cell line was however significantly more permissive for both of the more recent isolates than the Ross strain. The results suggest that the increased infectivity seen in insect cells derives from an evolution of the CHIKV genome not solely associated with the E1:226 substitution.     

Stable expression of insect GABA receptors in insect cell lines promoters for efficient expression of Drosophila and mosquito Rdl GABA receptors in stably transformed mosquito cell lines

We are interested in establishing stably transformed insect cell lines efficiently expressing the insect γ-aminobutyric acid (GABA) receptor subunit gene Resistance to dieldrin or Rdl. In order to facilitate this we utilized a system based on stable transformation of Aedes albopictus mosquito cell lines using the dihydrofolate reductase (dhfr) gene as a selectable marker. Here we report the production of stable mosquito cell lines carrying high copy numbers of Rdl genes from both Drosophila and Aedes aegypti mosquitoes and the subsequent high efficiency expression of functional GABA gated chloride ion channels. We also used this system to compare the activity of a range of immediate early baculovirus promoters in mosquito cell culture and demonstrate that IE1 promoter constructs work efficiently across insect species. Results are discussed in relation to the potential use of these constructs in the genetic transformation of non-Drosophilid insects.     

Genome-Wide RNAi Screen Identifies Broadly-Acting Host Factors That Inhibit Arbovirus Infection

Vector-borne viruses are an important class of emerging and re-emerging pathogens; thus, an improved understanding of the cellular factors that modulate infection in their respective vertebrate and insect hosts may aid control efforts. In particular, cell-intrinsic antiviral pathways restrict vector-borne viruses including the type I interferon response in vertebrates and the RNA interference (RNAi) pathway in insects. However, it is likely that additional cell-intrinsic mechanisms exist to limit these viruses. Since insects rely on innate immune mechanisms to inhibit virus infections, we used Drosophila as a model insect to identify cellular factors that restrict West Nile virus (WNV), a flavivirus with a broad and expanding geographical host range. Our genome-wide RNAi screen identified 50 genes that inhibited WNV infection. Further screening revealed that 17 of these genes were antiviral against additional flaviviruses, and seven of these were antiviral against other vector-borne viruses, expanding our knowledge of invertebrate cell-intrinsic immunity. Investigation of two newly identified factors that restrict diverse viruses, dXPO1 and dRUVBL1, in the Tip60 complex, demonstrated they contributed to antiviral defense at the organismal level in adult flies, in mosquito cells, and in mammalian cells. These data suggest the existence of broadly acting and functionally conserved antiviral genes and pathways that restrict virus infections in evolutionarily divergent hosts.     

Immune and stress response 'cross-talk' in the Drosophila Malpighian tubule

The success of insects is in large part due to their ability to survive environmental stress, including heat, cold, and dehydration. Insects are also exposed to infection, osmotic or oxidative stress, and to xenobiotics or toxins. The molecular mechanisms of stress sensing and response have been widely investigated in mammalian cell lines, and the area of stress research is now so vast to be beyond the scope of a single review article. However, the mechanisms by which stress inputs to the organism are sensed and integrated at the tissue and cellular level are less well understood. Increasingly, common molecular events between immune and other stress responses are observed in vivo; and much of this work stems of efforts in insect molecular science and physiology. We describe here the current knowledge in the area of immune and stress signalling and response at the level of the organism, tissue and cell, focussing on a key epithelial tissue in insects, the Malpighian tubule, and drawing together the known pathways that modulate responses to different stress insults. The tubules are critical for insect survival and are increasingly implicated in responses to multiple and distinct stress inputs. Importantly, as tubule function is central to survival, they are potentially key targets for insect control, which will be facilitated by increased understanding of the complexities of stress signalling in the organism.     

In vitro cultivation of Wolbachia in insect and mammalian cell lines

Wolbachia infecting the small brown planthopper, Laodelphax striatellus, were successfully maintained and cultivated in two insect and one mammalian cell lines. The bacteria with the planthopper ovary were introduced into the flasks with the cultures of the cell lines. The Wolbachia proliferated in mosquito (Aedes albopictus) and lepidopteran (Heliothis zea) cell lines and in the mouse cell line, L929. Proliferation of Wolbachia was confirmed by electron microscopy and quantitative polymerase chain reaction. This simple method for the cultivation of Wolbachia was applicable to other strains of Wolbachia, such as the one found in the lepidopteran eggs, and should facilitate fundamental and applied studies of this important group of microorganisms.     

Characterization of hemocytes from the yellow fever mosquito,Aedes aegypti

Mosquitoes are the most important arthropod disease vectors, transmitting a broad range of pathogens that cause diseases such as malaria, lymphatic filariasis, and yellow fever. Mosquitoes and other insects are able to mount powerful cellular and humoral immune responses against invading pathogens. To date, most studies have concentrated on the humoral response. In the current study we describe the hemocytes (blood cells) of the yellow fever mosquito, Aedes aegypti, by means of morphology, lectin binding, and enzyme activity and immunocytochemistry. Our light and electron microscopic studies suggest the presence of four distinct hemocyte types: granulocytes, oenocytoids, adipohemocytes, and thrombocytoids. We believe granulocytes and oenocytoids are true circulating hemocytes, but adipohemocytes and thrombocytoids are likely adhered to fixed tissues. Granulocytes, the most abundant cell type, have acid phosphatase and alpha-naphthyl acetate esterase activity, and bind the exogenous lectins WGA, HPA, and GNL. Phenoloxidase, an essential enzyme in the melanotic encapsulation immune response, was detected inside oenocytoids. This is, to our knowledge, the first report that has detected phenoloxidase inside mosquito hemocytes at the ultrastructural level. These results have begun to form a knowledge base for our ongoing studies on the function of Ae. aegypti hemocytes, and their involvement in controlling infections.     

Coupled small molecules target RNA interference and JAK/STAT signaling to reduce Zika virus infection in Aedes aegypti

The recent global Zika epidemics have revealed the significant threat that mosquito-borne viruses pose. There are currently no effective vaccines or prophylactics to prevent Zika virus (ZIKV) infection. Limiting exposure to infected mosquitoes is the best way to reduce disease incidence. Recent studies have focused on targeting mosquito reproduction and immune responses to reduce transmission. Previous work has evaluated the effect of insulin signaling on antiviral JAK/STAT and RNAi in vector mosquitoes. Specifically, insulin-fed mosquitoes resulted in reduced virus replication in an RNAi-independent, ERK-mediated JAK/STAT-dependent mechanism. In this work, we demonstrate that targeting insulin signaling through the repurposing of small molecule drugs results in the activation of both RNAi and JAK/STAT antiviral pathways. ZIKV-infected Aedes aegypti were fed blood containing demethylasterriquinone B1 (DMAQ-B1), a potent insulin mimetic, in combination with AKT inhibitor VIII. Activation of this coordinated response additively reduced ZIKV levels in Aedes aegypti. This effect included a quantitatively greater reduction in salivary gland ZIKV levels up to 11 d post-bloodmeal ingestion, relative to single pathway activation. Together, our study indicates the potential for field delivery of these small molecules to substantially reduce virus transmission from mosquito to human. As infections like Zika virus are becoming more burdensome and prevalent, understanding how to control this family of viruses in the insect vector is an important issue in public health.     

Virulence and Pathogenesis of Yellow Fever Virus Serially Passaged in Cell Culture

Viscerotropic virulence of the Asibi strain of yellow fever virus (YFV) for monkeys has been known to be lost after serial passage in HeLa cell monolayers. This phenomenon was investigated in several other mammalian and insect tissue cell lines. Assay in monkeys of original seed virus and of virus after 7 and 11 passages in a porcine kidney cell line (PK) indicated essentially equal infectivity and mortality. Moreover, monkeys receiving the passaged virus exhibited more rapid onset of disease and death than animals infected with original seed virus. Histological changes in animals inoculated with passaged virus were identical to those in animals receiving the seed virus. Virus from later passages in PK cells was also lethal for approximately 50% of the monkeys; however, evidence for progressive attenuation was seen in these preparations. Similar results were obtained with a mosquito (Aedes aegypti) cell line. In contrast to results obtained in PK and mosquito cells, YFV became essentially avirulent (nonlethal and less infective) for monkeys after only seven passages in HeLa cell cultures.     

Chromatin condensation and differential sensitivity of mammalian and insect cells to DNA strand breaks induced by bleomycin

Bleomycin (BLM) induces DNA damage in living cells. In this report we analyzed the role of chromatin compactness in the differential response of mosquito (ATC-15) and mammalian (CHO) cells to DNA strand breaks induced by BLM. We used cells unexposed and exposed to sodium butyrate (NaB), which induces chromatin decondensation. By nucleoid sedimentation assay and digestions of nuclei with DNAse I, untreated mosquito cells (no BLM; no NaB) were shown to have more chromatin condensation than untreated CHO cells. By alkaline unwinding ATC-15 cells treated with NaB showed more BLM-induced DNA strand breaks than NaB-untreated CHO cells. The time-course of BLM-induced DNA damage to nuclear DNA was similar for NaB-untreated mammalian and insect cells, but with mosquito cells showing less DNA strand breaks, both at physiological temperatures and at 4 °C. However, when DNA repair was inhibited by low temperatures and chromatin was decondensed by NaB treatments, differences in BLM-induced DNA damage between these cells lines were no longer observed. In both cell lines, NaB did not affect BLM action on cell growth and viability. On the other hand, the low sensitivity of ATC-15 cells to BLM was reflected in their better growth efficiency. These cells exhibited a satisfactory growth at BLM doses that produced a permanent arrest of growth in CHO cells. The data suggest that mosquito cells might have linker DNAs shorter than those of mammalian cells, which would result in the observed both greater chromatin condensation and greater resistance to DNA damage induced by BLM as compared to CHO cells.     

Secreted ferritin: Mosquito defense against iron overload?

The yellow fever mosquito, Aedes aegypti, must blood feed in order to complete her life cycle. The blood meal provides a high level of iron that is required for egg development. We are interested in developing control strategies that interfere with this process. We show that A. aegypti larval cells synthesize and secrete ferritin in response to iron exposure. Cytoplasmic ferritin is maximal at low levels of iron, consists of both the light chain (LCH) and heavy chain (HCH) subunits and reflects cytoplasmic iron levels. Secreted ferritin increases in direct linear relationship to iron dose and consists primarily of HCH subunits. Although the messages for both subunits increase with iron treatment, our data indicate that mosquito HCH synthesis could be partially controlled at the translational level as well. Importantly, we show that exposure of mosquito cells to iron at low concentrations increases cytoplasmic iron, while higher iron levels results in a decline in cytoplasmic iron levels indicating that excess iron is removed from mosquito cells. Our work indicates that HCH synthesis and ferritin secretion are key factors in the response of mosquito cells to iron exposure and could be the primary mechanisms that allow these insects to defend against an intracellular iron overload.     

Comparison of Bacillus thuringiensis subsp. israelensis CryIVA and CryIVB cloned toxins reveals synergism in vivo

When the gene for the mosquitocidal protein CryIVA was expressed in two strains of Bacillus thuringiensis (Bt) cured of their resident delta-endotoxin genes, the protein accumulated as large inclusions. The inclusions produced in the Bt subsp. kurstaki recipient strain were twice as soluble at alkaline pH as the inclusions produced in Bt subsp. israelensis. Solubilized protoxins were activated by treatment with mosquito gut extracts or trypsin for varying lengths of time and tested for in vitro cytotoxicity on cell lines of three genera of mosquito. CryIVA treated with any of the mosquito gut extracts for 6 h showed significant toxicity against Anopheles gambiae cells and slight activity on Culex quinquefasciatus cells. For CryIVB, the only significant cytotoxicity observed was against Aedes aegypti cells after treatment with Aedes gut extract. In in vivo bioassays, both CryIVA, purified from either of the Bt recipient strains, and CryIVB inclusions were similarly toxic to A. aegypti and A. gambiae larvae but CryIVA was 25-fold more toxic to C. quinquefasciatus. Synergism in vivo between the two toxins was revealed when results from assaying single toxins and mixtures were compared. Mixtures of CryIVA and CryIVB proved to be 5-fold more toxic to Culex than either toxin used singly and showed a reduced but similar synergism when tested against Aedes and Anopheles larvae. The synergism was not duplicated in vitro using cell lines from these three insects.     

New approaches to insect tissue culture

Conclusion Current methods of insect cell culture have produced a limited variety of cell types in an ever expanding list of insect cell lines. In developing midgut epithelial cell lines, we found that traditional methods in insect cell culture failed to provide healthy cells from mature tissues. Examination of mammalian cell culture literature for this particular cell type provided the insight required to successfully develop a cell-specific line (Baines et al., 1994). The potential applications for cell-specific lines from insects are numerous. This paper is a compilation of ideas that will hopefully enable other researchers to develop additional cell-specific lines.     

Induction of reaper ortholog mx in mosquito midgut cells following baculovirus infection

Many vertebrate and insect viruses possess antiapoptotic genes that are required for their infectivity. This led to the hypothesis that apoptosis is an innate immunoresponse important for limiting virus infections. The role of apoptosis may be especially important in insect antiviral defense because of the lack of adaptive immunity. However, the cellular mechanism that elicits apoptosis in response to viral infection in insects has not been determined. Using an in vivo infection system with the mosquito baculovirus CuniNPV (Culex nigripalpus nucleopolyhedrovirus), we demonstrated that michelob_x (mx), the mosquito ortholog of Drosophila proapoptotic gene reaper, is specifically induced in larval midgut cells following viral infection. Interestingly, the dynamics of mx induction corresponds with the outcome of the infection. In the permissive mosquito C. quinquefasciatus, a slow induction of mx failed to induce prompt apoptosis, and the infected cells eventually undergo necrosis with heavy loads of encapsulated viruses. In contrast, in the refractory mosquito Aedes aegypti, a rapid induction of mx within 30 min p.i. is followed by apoptosis within 2-6 h p.i., suggesting a possible role for apoptosis in limiting viral infection. When the execution of apoptosis was delayed by caspase inhibitors, viral gene expression became detectable in the A. aegypti larvae.