Mosquito pericardial cells upregulate Cecropin expression after an immune challenge.

Most mosquito-transmitted pathogens grow or replicate in the midgut before invading the salivary glands. Pathogens are exposed to several immunological factors along the way. Recently, it was shown that hemocytes gather near the periostial region of the heart to efficiently phagocytose pathogens circulating in the hemolymph. Nerveless, not all pathogens can be phagocyted by hemocytes and eliminated by lysis. Interestingly, some studies have shown that pericardial cells (PCs) surrounding periostial regions, may produce humoral factors, such as lysozymes. Our current work provides evidence that Anopheles albimanus PCs are a major producer of Cecropin 1 (Cec1). Furthermore, our findings reveal that after an immunological challenge, PCs upregulate Cec1 expression. We conclude that PCs are positioned in a strategic location that could allow releasing humoral components, such as cecropin, to lyse pathogens on the heart or circulating in the hemolymph, implying that PCs could play a significant role in the systemic immune response.

Plasmodium exposure alters midgut epithelial cell dynamics during the immune memory in Anopheles albimanus.

Immunological priming in insects is defined as a previous contact with non-virulent pathogens, which induces protection after a second virulent infection. The mechanism of this process is not well understood. We have observed midgut DNA synthesis (endoreplication) in Plasmodium berghei exposure mosquitoes (primed) and after the immune challenge, which could be an essential component of the priming response in the mosquito. Endoreplication requires cell cycle components re-direction to make multiple DNA copies. Therefore, it is fundamental to understand the role of cell cycle components in priming. Here, we analyzed the expression of the cyclins A, B, E, and AurkA, and the endoreplication components NOTCH and HNT in the mosquito Anopheles albimanus; after priming with non-infective Plasmodium berghei and challenged with an infective P. berghei. The overexpression of cell cycle elements occurred seven days after priming with a quick reduction 24 h after the challenge. Hnt and NOTCH overexpression occurred 24 h after priming. Antimicrobial peptide cecropin is quickly overexpressed after 24 h in primed mosquitoes, then is downregulated at day seven and overexpressed again after parasite challenge. We also found that DNA synthesis occurs in cells with different nuclear sizes, suggesting a change in midgut epithelial dynamics after Plasmodium exposure. Inhibition of DNA synthesis via cisplatin revealed that DNA synthesis is required for priming to limit Plasmodium infection. Our results indicate the importance of cell cycle components on DNA synthesis and Notch pathway during priming response in An. albimanus mosquitoes.

Metal ions in insect reproduction: a crosstalk between reproductive physiology and immunity.

Most insects exhibit high reproductive capacity, which demands large amounts of energy, including macronutrients and micronutrients. Interestingly, many proteins involved in oogenesis depend on metals ions, in particular iron (Fe), zinc (Zn), and copper (Cu). Mechanisms by which metal ions influence reproduction have been described in Drosophila melanogaster, but remain poorly understood in hematophagous insects where blood meals include significant ingestion of metal ions. Moreover, there is evidence that some proteins involved in reproduction and immunity could have dual function in both processes. This review highlights the importance of metal ions in the reproduction of non-hematophagous and hematophagous insects. In addition, we discuss how insects optimize physiological processes using proteins involved in crosstalk between reproductive physiology and immunity, which is a double-edge sword in allocating their functions to protect the insect and ensure reproduction.

Heat Shock Causes Lower Plasmodium Infection Rates in Anopheles albimanus.

The immune response of Anopheles mosquitoes to Plasmodium invasion has been extensively studied and shown to be mediated mainly by the nitric oxide synthase (NOS), dual oxidase (DUOX), phenoloxidase (PO), and antimicrobial peptides activity. Here, we studied the correlation between a heat shock insult, transcription of immune response genes, and subsequent susceptibility to Plasmodium berghei infection in Anopheles albimanus. We found that transcript levels of many immune genes were drastically affected by the thermal stress, either positively or negatively. Furthermore, the transcription of genes associated with modifications of nucleic acid methylation was affected, suggesting an increment in both DNA and RNA methylation. The heat shock increased PO and NOS activity in the hemolymph, as well as the transcription of several immune genes. As consequence, we observed that heat shock increased the resistance of mosquitoes to Plasmodium invasion. The data provided here could help the understanding of infection transmission under the ever more common heat waves.

Mosquito metallomics reveal copper and iron as critical factors for Plasmodium infection.

Iron and copper chelation restricts Plasmodium growth in vitro and in mammalian hosts. The parasite alters metal homeostasis in red blood cells to its favor, for example metabolizing hemoglobin to hemozoin. Metal interactions with the mosquito have not, however, been studied. Here, we describe the metallomes of Anopheles albimanus and Aedes aegypti throughout their life cycle and following a blood meal. Consistent with previous reports, we found evidence of maternal iron deposition in embryos of Ae. aegypti, but less so in An. albimanus. Sodium, potassium, iron, and copper are present at higher concentrations during larval developmental stages. Two An. albimanus phenotypes that differ in their susceptibility to Plasmodium berghei infection were studied. The susceptible white stripe (ws) phenotype was named after a dorsal white stripe apparent during larval stages 3, 4, and pupae. During larval stage 3, ws larvae accumulate more iron and copper than the resistant brown stripe (bs) phenotype counterparts. A similar increase in copper and iron accumulation was also observed in the susceptible ws, but not in the resistant bs phenotype following P. berghei infection. Feeding ws mosquitoes with extracellular iron and copper chelators before and after receiving Plasmodium-infected blood protected from infection and simultaneously affected follicular development in the case of iron chelation. Unexpectedly, the application of the iron chelator to the bs strain reverted resistance to infection. Besides a drop in iron, iron-chelated bs mosquitoes experienced a concomitant loss of copper. Thus, the effect of metal chelation on P. berghei infectivity was strain-specific.

Transcriptome analysis uncover differential regulation in cell cycle, immunity, and metabolism in Anopheles albimanus during immune priming with Plasmodium berghei.

In invertebrates, "immunological priming" is considered as the ability to acquire a protective (adaptive) immune response against a pathogen due to previous exposure to the same organism. To date, the mechanism by which this type of adaptive immune response originates in insects is not well understood. In the Anopheles albimanus - Plasmodium berghei model, a DNA synthesis that probably indicates an endoreplication process during priming induction has been evidenced. This work aimed to know the transcriptomic profile in the midguts of An. albimanus after priming induction. Our analysis indicates the participation of regulatory elements of the cell cycle in the immunological priming and points out the importance of the cell cycle regulation in the mosquito midgut.

Lysozyme c-1 gene is overexpressed in Anopheles albimanus pericardial cells after an immune challenge.

Different evidences suggest that pericardial cells play an important role during the immune response against pathogens that invade the mosquito hemocoel. Previously, we identified two lysozyme genes in Anopheles albimanus heart transcriptome. The present study showed that one of these genes (IDVB: AALB004517) has high percentage of identity to mosquito lysozyme genes related to immunity, suggesting its possible participation during the mosquito immune response. This An. albimanus gen, constitutively expressed lysozyme c-1 mRNA (albLys c-1) in mosquito heart; however, it was overexpressed in bacteria-injected mosquitoes. In heart extract samples, we identified a protein of approximately 14 kDa (likely lysozyme c-1), which lysed M. luteus. In addition, mRNA-FISH assay in heart samples, showed specific fluorescent hybridization signal in pericardial cells from M. luteus-injected mosquitos. We conclude that for the first time an inducible immune factor (lysozyme c-1) is identified in Anopheles albimanus mosquito pericardial cells, which could be a key component in the response against pathogens that interact with the mosquito heart.

DNA synthesis increases during the first hours post-emergence in Anopheles albimanus mosquito midgut.

In hematophagous insects, the midgut is a fundamental barrier against infections and limits the development and transmission of pathogens. However, in mosquitoes, cell differentiation, proliferation, and cell cycle process in the midgut have not been characterized. Here we provide evidence of how cell cycle progression occurs in the newly emerged Anopheles albimanus mosquito midgut and describing cyclins expression as mediators of the cell cycle. The cell cycle at different post-emergence times was evaluated in disaggregated cells from midgut tissue using flow cytometry. Also, cyclins A, B, and E were identified by bioinformatics tools. These cyclins were used to analyze cell cycle progression. Flow cytometry data and the expression-pattern of the cyclins by qRT-PCR supported a polyploidy process, besides mitosis marker was marginally detected and only in newly emerged mosquitoes. Our results suggest that DNA increment in midguts occurs by polyploidy during the first hours post-emergence.