Infectivity of Plasmodium parasites to Aedes aegypti and Anopheles stephensi mosquitoes maintained on blood-free meals of SkitoSnack.

BACKGROUND: Aedes and Anopheles mosquitoes are responsible for tremendous global health burdens from their transmission of pathogens causing malaria, lymphatic filariasis, dengue, and yellow fever. Innovative vector control strategies will help to reduce the prevalence of these diseases. Mass rearing of mosquitoes for research and support of these strategies presently depends on meals of vertebrate blood, which is subject to acquisition, handling, and storage issues. Various blood-free replacements have been formulated for these mosquitoes, but none of these replacements are in wide use, and little is known about their potential impact on competence of the mosquitoes for Plasmodium infection. METHODS: Colonies of Aedes aegypti and Anopheles stephensi were continuously maintained on a blood-free replacement (SkitoSnack; SS) or bovine blood (BB) and monitored for engorgement and hatch rates. Infections of Ae. aegypti and An. stephensi were assessed with Plasmodium gallinaceum and P. falciparum, respectively. RESULTS: Replicate colonies of mosquitoes were maintained on BB or SS for 10 generations of Ae. aegypti and more than 63 generations of An. stephensi. The odds of engorgement by SS- relative to BB-maintained mosquitoes were higher for both Ae. aegypti (OR = 2.6, 95% CI 1.3-5.2) and An. stephensi (OR 2.7, 95% CI 1.4-5.5), while lower odds of hatching were found for eggs from the SS-maintained mosquitoes of both species (Ae. aegypti OR = 0.40, 95% CI 0.26-0.62; An. stephensi OR = 0.59, 95% CI 0.36-0.96). Oocyst counts were similar for P. gallinaceum infections of Ae. aegypti mosquitoes maintained on SS or BB (mean ratio = [mean on SS]/[mean on BB] = 1.11, 95% CI 0.85-1.49). Similar oocyst counts were also observed from the P. falciparum infections of SS- or BB-maintained An. stephensi (mean ratio = 0.76, 95% CI 0.44-1.37). The average counts of sporozoites/mosquito showed no evidence of reductions in the SS-maintained relative to BB-maintained mosquitoes of both species. CONCLUSIONS: Aedes aegypti and An. stephensi can be reliably maintained on SS over multiple generations and are as competent for Plasmodium infection as mosquitoes maintained on BB. Use of SS alleviates the need to acquire and preserve blood for mosquito husbandry and may support new initiatives in fundamental and applied research, including novel manipulations of midgut microbiota and factors important to the mosquito life cycle and pathogen susceptibility.

Aedes aegypti SGS1 is critical for Plasmodium gallinaceum infection of both the mosquito midgut and salivary glands.

BACKGROUND: The invasion of the mosquito salivary glands by Plasmodium sporozoites is a critical step that defines the success of malaria transmission and a detailed understanding of the molecules responsible for salivary gland invasion could be leveraged towards control of vector-borne pathogens. Antibodies directed against the mosquito salivary gland protein SGS1 have been shown to reduce Plasmodium gallinaceum sporozoite invasion of Aedes aegypti salivary glands, but the specific role of this protein in sporozoite invasion and in other stages of the Plasmodium life cycle remains unknown. METHODS: RNA interference and CRISPR/Cas9 were used to evaluate the role of A. aegypti SGS1 in the P. gallinaceum life cycle. RESULTS: Knockdown and knockout of SGS1 disrupted sporozoite invasion of the salivary gland. Interestingly, mosquitoes lacking SGS1 also displayed fewer oocysts. Proteomic analyses confirmed the abolishment of SGS1 in the salivary gland of SGS1 knockout mosquitoes and revealed that the C-terminus of the protein is absent in the salivary gland of control mosquitoes. In silico analyses indicated that SGS1 contains two potential internal cleavage sites and thus might generate three proteins. CONCLUSION: SGS1 facilitates, but is not essential for, invasion of A. aegypti salivary glands by P. gallinaceum and has a dual role as a facilitator of parasite development in the mosquito midgut. SGS1 could, therefore, be part of a strategy to decrease malaria transmission by the mosquito vector, for example in a transgenic mosquito that blocks its interaction with the parasite.

Angiotensin II-derived constrained peptides with antiplasmodial activity and suppressed vasoconstriction.

Angiotensin II (Ang II) is a natural mammalian hormone that has been described to exhibit antiplasmodial activity therefore constituting a promising alternative for the treatment of malaria. Despite its promise, the development of Ang II as an antimalarial is limited by its potent induction of vasoconstriction and its rapid degradation within minutes. Here, we used peptide design to perform targeted chemical modifications to Ang II to generate conformationally restricted (disulfide-crosslinked) peptide derivatives with suppressed vasoconstrictor activity and increased stability. Designed constrained peptides were synthesized chemically and then tested for antiplasmodial activity. Two lead constrained peptides were identified (i.e., peptides 1 and 2), each composed of 10 amino acid residues. These peptides exhibited very promising activity in both our Plasmodium gallinaceum (>80%) and Plasmodium falciparum (>40%) models, an activity that was equivalent to that of Ang II, and led to complete suppression of vasoconstriction. In addition, peptide 5 exhibited selective activity towards the pre-erythrocytic stage (98% of activity against P. gallinaceum), thus suggesting that it may be possible to design peptides that target specific stages of the malaria life cycle. The Ang II derived stable scaffolds presented here may provide the basis for development of a new generation of peptide-based drugs for the treatment of malaria.

Vector competence of Aedes albopictus (Skuse) and Aedes aegypti (Linnaeus) for Plasmodium gallinaceum infection and transmission.

Avian malaria caused by Plasmodium gallinaceum is an important mosquito-borne disease. Eradication of this disease remains problematic since its competent vectors are diverse and widely distributed across the globe. Several mosquito species were implicated as competent vectors for this parasite. However, studies on vector competence for P. gallinaceum remain limited. In this study, vector competence in the two most predominant mosquito vectors in tropical countries, Aedes albopictus and Ae. aegypti, was compared. In order to determine their infection rates, Ae. albopictus (>F10), Ae. aegypti (>F10), and Ae. aegypti ( F10) and Ae. aegypti (> F10) and 40-60% by infected Ae. aegypti (F10) and Ae. aegypti (>F10) were highly competent vectors for P. gallinaceum infections. These mosquitoes play a crucial role in the transmission cycle of this parasite in nature.

Diet-Induced Nutritional Stress and Pathogen Interference in Wolbachia-Infected Aedes aegypti.

The pathogen interference phenotype greatly restricts infection with dengue virus (DENV) and other pathogens in Wolbachia-infected Aedes aegypti, and is a vital component of Wolbachia-based mosquito control. Critically, the phenotype's causal mechanism is complex and poorly understood, with recent evidence suggesting that the cause may be species specific. To better understand this important phenotype, we investigated the role of diet-induced nutritional stress on interference against DENV and the avian malarial parasite Plasmodium gallinaceum in Wolbachia-infected Ae. aegypti, and on physiological processes linked to the phenotype. Wolbachia-infected mosquitoes were fed one of four different concentrations of sucrose, and then challenged with either P. gallinaceum or DENV. Interference against P. gallinaceum was significantly weakened by the change in diet however there was no effect on DENV interference. Immune gene expression and H2O2 levels have previously been linked to pathogen interference. These traits were assayed for mosquitoes on each diet using RT-qPCR and the Amplex Red Hydrogen Peroxide/Peroxidase Assay Kit, and it was observed that the change in diet did not significantly affect immune expression, but low carbohydrate levels led to a loss of ROS induction in Wolbachia-infected mosquitoes. Our data suggest that host nutrition may not influence DENV interference for Wolbachia-infected mosquitoes, but Plasmodium interference may be linked to both nutrition and oxidative stress. This pathogen-specific response to nutritional change highlights the complex nature of interactions between Wolbachia and pathogens in mosquitoes.

Endogenously-expressed NH2-terminus of circumsporozoite protein interferes with sporozoite invasion of mosquito salivary glands.

BACKGROUND: The circumsporozoite protein is the most abundant polypeptide expressed by sporozoites, the malaria parasite stage capable of infecting humans. Sporozoite invasion of mosquito salivary glands prior to transmission is likely mediated by a receptor/ligand-like interaction of the parasites with the target tissues, and the amino (NH2)-terminal portion of CSP is involved in this interaction but not the TSR region on the carboxyl (C)-terminus. Peptides based on the NH2-terminal domain could compete with the parasites for the salivary gland receptors and thus inhibit penetration. METHODS: Peptides based on the NH2-terminus and TSR domains of the CSP from avian or human malaria parasites, Plasmodium gallinaceum and Plasmodium falciparum, respectively, were expressed endogenously in mosquito haemolymph using a transient (Sindbis virus-mediated) or stable (piggyBac-mediated transgenesis) system. RESULTS: Transient endogenous expression of partial NH2-terminus peptide from P. falciparum CSP in P. gallinaceum-infected Aedes aegypti resulted in a reduced number of sporozoites in the salivary glands. When a transgenic approach was used to express a partial CSP NH2-terminal domain from P. gallinaceum the number of sporozoites in the salivary glands did not show a difference when compared to controls. However, a significant difference could be observed when mosquitoes with a lower infection were analysed. The same result could not be observed with mosquitoes endogenously expressing peptides based on the TSR domain from either P. gallinaceum or P. falciparum. CONCLUSION: These results support the conclusion that CSP partial NH2-terminal domain can be endogenously expressed to promote a competition for the receptor used by sporozoites to invade salivary glands, and they could be used to block this interaction and reduce parasite transmission. The same effect cannot be obtained with peptides based on the TSR domain.

Effects of the angiotensin II Ala-scan analogs in erythrocytic cycle of Plasmodium falciparum (in vitro) and Plasmodium gallinaceum (ex vivo).

The anti-plasmodium activity of angiotensin II and its analogs have been described in different plasmodium species. Here we synthesized angiotensin II Ala-scan analogs to verify peptide-parasite invasion preservation with residue replacements. The analogs were synthesized by 9-fluorenylmethoxycarbonyl (Fmoc) and tert-butyloxycarbonyl (t-Boc) solid phase methods, purified by liquid chromatography and characterized by mass spectrometry. The results obtained in Plasmodium falciparum assays indicated that all analogs presented some influence in parasite invasion, except [Ala(4)]-Ang II (18% of anti-plasmodium activity) that was not statistically different from control. Although [Ala(8)]-Ang II presented a lower biological activity (20%), it was statistically different from control. The most relevant finding was that [Ala(5)]-Ang II preserved activity (45%) relative to Ang II (47%). In the results of Plasmodium gallinaceum assays all analogs were not statistically different from control, except [Ala(6)]-Ang II, which was able to reduce the parasitemia about 49%. This approach provides insight for understanding the importance of each amino acid on the native Ang II sequence and provides a new direction for the design of potential chemotherapeutic agents without pressor activity.

Effects of artesunate treatment on Plasmodium gallinaceum transmission in the vectors Aedes aegypti and Culex quinquefasciatus.

In the absence of vaccines, chemotherapy is an effective and economical way for controlling malaria. Development of anti-malarial drugs that target pathogenic blood stage parasites and gametocytes is preferable for the treatment as it can alleviate the host's morbidity and mortality and block transmission of the Plasmodium parasite. Recently, our laboratory has developed an in vivo transmission blocking assay that involves administration of 7 consecutive daily doses of a test compound into domestic chickens (Gallus gallus domesticus) infected with the avian malaria parasite Plasmodium gallinaceum with 10% parasitaemia and 1% gametocytaemia. To compromise the cost and time for artesunate (ATN) treatment, this study aimed to investigate effects of a 5-day consecutive administration of 10 milligrams per kilogram (mg/kg) ATN on P. gallinaceum infection in chickens and transmission to two natural vectors, Aedes aegypti and Culex quinquefasciatus. Our study showed that the treatment with 10 mg/kg ATN for 7 days, but not 5 days, completely eliminated blood stage infections, prevented recrudescence and blocked gametocyte production and transmission of P. gallinaceum to its vectors, thereby confirming the potent schizontocidal and gametocytocidal activities of ATN. This regimen should be further evaluated in field trials.

Chickens treated with a nitric oxide inhibitor became more resistant to Plasmodium gallinaceum infection due to reduced anemia, thrombocytopenia and inflammation.

Malaria is a serious infectious disease caused by parasites of the Plasmodium genus that affect different vertebrate hosts. Severe malaria leads to host death and involves different pathophysiological phenomena such as anemia, thrombocytopenia and inflammation. Nitric oxide (NO) is an important effector molecule in this disease, but little is known about its role in avian malaria models. Plasmodium gallinaceum-infected chickens were treated with aminoguanidine (AG), an inhibitor of inducible nitric oxide synthase, to observe the role of NO in the pathogenesis of this avian model. AG increased the survival of chickens, but also induced higher parasitemia. Treated chickens demonstrated reduced anemia and thrombocytopenia. Moreover, erythrocytes at different stages of maturation, heterophils, monocytes and thrombocytes were infected by Plasmodium gallinaceum and animals presented a generalized leucopenia. Activated leukocytes and thrombocytes with elongated double nuclei were observed in chickens with higher parasitemia; however, eosinophils were not involved in the infection. AG reduced levels of hemozoin in the spleen and liver, indicating lower inflammation. Taken together, the results suggest that AG reduced anemia, thrombocytopenia and inflammation, explaining the greater survival rate of the treated chickens.

RNAi knock-downs support roles for the mucin-like (AeIMUC1) gene and short-chain dehydrogenase/reductase (SDR) gene in Aedes aegypti susceptibility to Plasmodium gallinaceum.

The mosquito midgut represents the first barrier encountered by the Plasmodium parasite (Haemosporida: Plasmodiidae) when it is ingested in blood from an infected vertebrate. Previous studies identified the Aedes aegypti (L.) (Diptera: Culicidae) mucin-like (AeIMUC1) and short-chain dehydrogenase/reductase (SDR) genes as midgut-expressed candidate genes influencing susceptibility to infection by Plasmodium gallinaceum (Brumpt). We used RNA inference (RNAi) by double-stranded RNA (dsRNA) injections to examine ookinete survival to the oocyst stage following individual gene knock-downs. Double-stranded RNA gene knock-downs were performed 3 days prior to P. gallinaceum infection and oocyst development was evaluated at 7 days post-infection. Mean numbers of parasites developing to the oocyst stage were significantly reduced by 52.3% in dsAeIMUC1-injected females and by 36.5% in dsSDR-injected females compared with females injected with a dsß-gal control. The prevalence of infection was significantly reduced in dsAeIMUC1- and dsSDR-injected females compared with females injected with dsß-gal; these reductions resulted in a two- and three-fold increase in the number of uninfected individuals, respectively. Overall, these results suggest that both AeIMUC1 and SDR play a role in Ae. aegypti vector competence to P. gallinaceum.

Susceptibility of Anopheles stephensi to Plasmodium gallinaceum: a trait of the mosquito, the parasite, and the environment.

BACKGROUND: Vector susceptibility to Plasmodium infection is treated primarily as a vector trait, although it is a composite trait expressing the joint occurrence of the parasite and the vector with genetic contributions of both. A comprehensive approach to assess the specific contribution of genetic and environmental variation on "vector susceptibility" is lacking. Here we developed and implemented a simple scheme to assess the specific contributions of the vector, the parasite, and the environment to "vector susceptibility." To the best of our knowledge this is the first study that employs such an approach. METHODOLOGY/PRINCIPAL FINDINGS: We conducted selection experiments on the vector (while holding the parasite "constant") and on the parasite (while holding the vector "constant") to estimate the genetic contributions of the mosquito and the parasite to the susceptibility of Anopheles stephensi to Plasmodium gallinaceum. We separately estimated the realized heritability of (i) susceptibility to parasite infection by the mosquito vector and (ii) parasite compatibility (transmissibility) with the vector while controlling the other. The heritabilities of vector and the parasite were higher for the prevalence, i.e., fraction of infected mosquitoes, than the corresponding heritabilities of parasite load, i.e., the number of oocysts per mosquito. CONCLUSIONS: The vector's genetics (heritability) comprised 67% of "vector susceptibility" measured by the prevalence of mosquitoes infected with P. gallinaceum oocysts, whereas the specific contribution of parasite genetics (heritability) to this trait was only 5%. Our parasite source might possess minimal genetic diversity, which could explain its low heritability (and the high value of the vector). Notably, the environment contributed 28%. These estimates are relevant only to the particular system under study, but this experimental design could be useful for other parasite-host systems. The prospects and limitations of the genetic manipulation of vector populations to render the vector resistant to the parasite are better considered on the basis of this framework.

Effects of Plasmodium gallinaceum on hemolymph physiology of Aedes aegypti during parasite development.

Insect disease vectors show diminished fecundity when infected with Plasmodium. This phenomenon has already been demonstrated in laboratory models such as Aedes aegypti, Anopheles gambiae and Anopheles stephensi. This study demonstrates several changes in physiological processes of A. aegypti occurring upon infection with Plasmodium gallinaceum, such as reduced ecdysteroid levels in hemolymph as well as altered expression patterns for genes involved in vitellogenesis, lipid transport and immune response. Furthermore, we could show that P. gallinaceum infected A. aegypti presented a reduction in reproductive fitness, accompanied by an activated innate immune response and increase in lipophorin expression, with the latter possibly representing a nutritional resource for Plasmodium sporozoites.

Blocking of Plasmodium transmission by cooperative action of Cecropin A and Defensin A in transgenic Aedes aegypti mosquitoes.

To overcome burden of mosquito-borne diseases, multiple control strategies are needed. Population replacement with genetically modified mosquitoes carrying antipathogen effector genes is one of the possible approaches for controlling disease transmission. However, transgenic mosquitoes with antipathogen phenotypes based on overexpression of a single type effector molecule are not efficient in interrupting pathogen transmission. Here, we show that co-overexpression of two antimicrobial peptides (AMP), Cecropin A, and Defensin A, in transgenic Aedes aegypti mosquitoes results in the cooperative antibacterial and antiPlasmodium action of these AMPs. The transgenic hybrid mosquitoes that overexpressed both Cecropin A and Defensin A under the control of the vitellogenin promoter exhibited an elevated resistance to Pseudomonas aeruginosa infection, indicating that these AMPs acted cooperatively against this pathogenic bacterium. In these mosquitoes infected with P. gallinaceum, the number of oocysts was dramatically reduced in midguts, and no sporozoites were found in their salivary glands when the mosquitoes were fed twice to reactivate transgenic AMP production. Infection experiments using the transgenic hybrid mosquitoes, followed by sequential feeding on naive chicken, and then naive wild-type mosquitoes showed that the Plasmodium transmission was completely blocked. This study suggests an approach in generating transgenic mosquitoes with antiPlasmodium refractory phenotype, which is coexpression of two or more effector molecules with cooperative action on the parasite.

The role of NF-kappaB factor REL2 in the Aedes aegypti immune response.

Mosquitoes transmit numerous diseases that continue to be an enormous burden on public health worldwide. Transgenic mosquitoes impervious to vector-borne pathogens, in concert with vector control and drug and vaccine development, comprise an arsenal of means anticipated to defeat mosquito-spread diseases in the future. Mosquito transgenesis allows tissue-specific manipulation of their major immune pathways and enhances the ability to study mosquito-pathogen interactions. Here, we report the generation of two independent transgenic strains of Aedes aegypti overexpressing the NF-?B transcriptional factor REL2, a homologue of Drosophila Relish, which is shown to be under the control of the vitellogenin promoter in the mosquito fat body after a blood meal. We show that this REL2 overexpression in the fat body results in transcriptional activation of Defensins A, C, and D, and Cecropins A and N, as well as translation and secretion of Defensin A protein into the hemolymph. We also demonstrate that induction of REL2 results in the increased resistance of the mosquito to tested Gram-negative and Gram-positive bacteria. Importantly, induction of transgenic REL2 leads to the significant decrease in susceptibility of A. aegypti to Plasmodium gallinaceum infection. Consistently, RNAi knockdown of REL2 in wild-type mosquitoes results in a delay in Defensin A and Cecropin A expression in response to infection and in increased susceptibility to both bacteria and P. gallinaceum. Moreover, our transgenic assays demonstrate that the N-terminus of the mosquito REL2, which includes the His/Gln-rich and serine-rich regions, plays a role in its transactivation properties.

A Wolbachia symbiont in Aedes aegypti limits infection with dengue, Chikungunya, and Plasmodium.

Wolbachia are maternally inherited intracellular bacterial symbionts that are estimated to infect more than 60% of all insect species. While Wolbachia is commonly found in many mosquitoes it is absent from the species that are considered to be of major importance for the transmission of human pathogens. The successful introduction of a life-shortening strain of Wolbachia into the dengue vector Aedes aegypti that halves adult lifespan has recently been reported. Here we show that this same Wolbachia infection also directly inhibits the ability of a range of pathogens to infect this mosquito species. The effect is Wolbachia strain specific and relates to Wolbachia priming of the mosquito innate immune system and potentially competition for limiting cellular resources required for pathogen replication. We suggest that this Wolbachia-mediated pathogen interference may work synergistically with the life-shortening strategy proposed previously to provide a powerful approach for the control of insect transmitted diseases.

Evaluation of the function of a type I peritrophic matrix as a physical barrier for midgut epithelium invasion by mosquito-borne pathogens in Aedes aegypti.

In addition to modulating blood meal digestion and protecting the midgut epithelial cells from mechanical and chemical damage, a biological function attributed to the mosquito type I peritrophic matrix (PM) is preventing or reducing pathogen invasion, especially from Plasmodium spp. Previously, we demonstrated that chitin is an essential component of the PM and is synthesized de novo in response to blood feeding in Aedes aegypti. Therefore, knocking down chitin synthase expression by RNA interference severely disrupts formation of the PM. Utilizing this artificial manipulation, we determined that the absence of the PM has no effect on the development of Brugia pahangi or on the dissemination of dengue virus. However, infectivity of Plasmodium gallinaceum is lower, as measured by oocyst intensity, when the PM is absent. Our findings also suggest that the PM seems to localize proteolytic enzymes along the periphery of the blood bolus during the first 24 hours after blood feeding. Finally, the absence of the PM does not affect reproductive fitness, as measured by the number and viability of eggs oviposited.

Proteomic analysis of zygote and ookinete stages of the avian malaria parasite Plasmodium gallinaceum delineates the homologous proteomes of the lethal human malaria parasite Plasmodium falciparum.

Delineation of the complement of proteins comprising the zygote and ookinete, the early developmental stages of Plasmodium within the mosquito midgut, is fundamental to understand initial molecular parasite-vector interactions. The published proteome of Plasmodium falciparum does not include analysis of the zygote/ookinete stages, nor does that of P. berghei include the zygote stage or secreted proteins. P. gallinaceum zygote, ookinete, and ookinete-secreted/released protein samples were prepared and subjected to Multidimensional protein identification technology (MudPIT). Peptides of P. gallinaceum zygote, ookinete, and ookinete-secreted proteins were identified by MS/MS, mapped to ORFs (> 50 amino acids) in the extent P. gallinaceum whole genome sequence, and then matched to homologous ORFs in P. falciparum. A total of 966 P. falciparum ORFs encoding orthologous proteins were identified; just over 40% of these predicted proteins were found to be hypothetical. A majority of putative proteins with predicted secretory signal peptides or transmembrane domains were hypothetical proteins. This analysis provides a more comprehensive view of the hitherto unknown proteome of the early mosquito midgut stages of P. falciparum. The results underpin more robust study of Plasmodium-mosquito midgut interactions, fundamental to the development of novel strategies of blocking malaria transmission.

Efficiency of salivary gland invasion by malaria sporozoites is controlled by rapid sporozoite destruction in the mosquito haemocoel.

For successful transmission to the vertebrate host, malaria sporozoites must migrate from the mosquito midgut to the salivary glands. Here, using purified sporozoites inoculated into the mosquito haemocoel, we show that salivary gland invasion is inefficient and that sporozoites have a narrow window of opportunity for salivary gland invasion. Only 19% of sporozoites invade the salivary glands, all invasion occurs within 8h at a rate of approximately 200 sporozoites per hour, and sporozoites that fail to invade within this time rapidly die and are degraded. Then, using natural release of sporozoites from oocysts, we show that haemolymph flow through the dorsal vessel facilitates proper invasion. Most mosquitoes had low steady-state numbers of circulating sporozoites, which is remarkable given the thousands of sporozoites released per oocyst, and suggests that sporozoite degradation is a rapid immune process most efficient in regions of high haemolymph flow. Only 2% of Anopheles gambiae haemocytes phagocytized Plasmodium berghei sporozoites, a rate insufficient to explain the extent of sporozoite clearance. Greater than 95% of haemocytes phagocytized Escherichia coli or latex particles, indicating that their failure to sequester large numbers of sporozoites is not due to an inability to engage in phagocytosis. These results reveal the operation of an efficient sporozoite-killing and degradation machinery within the mosquito haemocoel, which drastically limits the numbers of infective sporozoites in the mosquito salivary glands.

Laminin and a Plasmodium ookinete surface protein inhibit melanotic encapsulation of Sephadex beads in the hemocoel of mosquitoes.

In refractory mosquitoes, melanotic encapsulation of Plasmodium ookinetes and oocysts is a commonly observed immune response. However, in susceptible mosquitoes, Plasmodium oocysts develop extracellularly in the body cavity without being recognized by the immune system. Like Plasmodium gallinaceum oocysts, negatively charged carboxymethyl (CM)-Sephadex beads implanted in the hemocoel of Aedes aegypti female mosquitoes were not usually melanized, but were coated with mosquito-derived laminin. Conversely, electrically neutral G-Sephadex beads were routinely melanized. Since mosquito laminin coated both CM-Sephadex beads and P. gallinaceum oocysts, we hypothesized that laminin prevents melanization of both. To test this hypothesis, we coated cyanogen-bromide-activated G-Sephadex beads with laminin, recombinant P. gallinaceum ookinete surface protein (PgS28) or bovine serum albumin (BSA). Beads were implanted into the abdominal body cavity of female Aedes aegypti and retrieved 4 days later. Uncoated controls as well as BSA-coated G-Sephadex beads were melanized in a normal manner. However, melanization of beads coated with mouse laminin, Drosophila L2-secreted proteins or PgS28 was markedly reduced. Fluorescent antibody labeling showed that PgS28-coated beads had adsorbed mosquito laminin on their surface. Thus, mosquito laminin interacting with Plasmodium surface proteins probably masks oocysts from the mosquito's immune system, thereby facilitating their development in the body cavity.

Regulation of lipid metabolism genes, lipid carrier protein lipophorin, and its receptor during immune challenge in the mosquito Aedes aegypti.

In the mosquito Aedes aegypti, the expression of two fat body genes involved in lipid metabolism, a lipid carrier protein lipophorin (Lp) and its lipophorin receptor (LpRfb), was significantly increased after infections with Gram (+) bacteria and fungi, but not with Gram (-) bacteria. The expression of these genes was enhanced after the infection with Plasmodium gallinaceum. RNA interference (RNAi) knockdown of Lp strongly restricted the development of Plasmodium oocysts, reducing their number by 90%. In Vg-DeltaREL1-A transgenic mosquitoes, with gain-of-function phenotype of Toll/REL1 immune pathway activated after blood feeding, both the Lp and LpRfb genes were overexpressed independently of septic injury. The same phenotype was observed in the mosquitoes with RNAi knockdown of Cactus, an IkappaB inhibitor in the Toll/REL1 pathway. These results showed that, in the mosquito fat body, both Lp and LpRfb gene expression were regulated by the Toll/REL1 pathway during immune induction by pathogen and parasite infections. Indeed, the proximal region of the LpRfb promoter contained closely linked binding motifs for GATA and NF-kappaB transcription factors. Transfection and in vivo RNAi knockdown experiments showed that the bindings of both GATA and NF-kappaB transcription factors to the corresponding motif were required for the induction of the LpRfb gene. These findings suggest that lipid metabolism is involved in the mosquito systemic immune responses to pathogens and parasites.