Manipulating insulin signaling to enhance mosquito reproduction

<p>Abstract</p> <p>Backgrond</p> <p>In the mosquito <it>Aedes aegypti </it>the insulin/insulin growth factor I signaling (IIS) cascade is a key regulator of many physiological processes, including reproduction. Two important reproductive events, steroidogenesis in the ovary and yolk synthesis in the fat body, are regulated by the IIS cascade in mosquitoes. The signaling molecule phosphatase and tensin homolog (PTEN) is a key inhibitor of the IIS cascade that helps modulate the activity of the IIS cascade. In <it>Ae. aegypti</it>, six unique splice variants of AaegPTEN were previously identified, but the role of these splice variants, particularly AaegPTEN3 and 6, were unknown.</p> <p>Results</p> <p>Knockdown of AaegPTEN or its specific splice variant AaegPTEN6 (the splice variant thought to regulate reproduction in the ovary and fat body) using RNAi led to a 15–63% increase in egg production with no adverse effects on egg viability during the first reproductive cycle. Knockdown of AaegPTEN3, expressed predominantly in the head, had no effect on reproduction. We also characterized the protein expression patterns of these two splice variants during development and in various tissues during a reproductive cycle.</p> <p>Conclusion</p> <p>Previous studies in a range of organisms, including <it>Drosophila melanogaster </it>and <it>Caenorhabditis elegans</it>, have demonstrated that disruption of the IIS cascade leads to decreased reproduction or sterility. In this study we demonstrate that knockdown of the IIS inhibitor PTEN can actually increase reproduction in the mosquito, at least during the first reproductive cycle.</p

Expression of the receptor tyrosine kinase ephb2 on dendritic cells is modulated by toll-like receptor ligation but is not required for t cell activation

The Eph receptor tyrosine kinases interact with their ephrin ligands on adjacent cells to facilitate contact-dependent cell communication. Ephrin B ligands are expressed on T cells and have been suggested to act as co-stimulatory molecules during T cell activation. There are no detailed reports of the expression and modulation of EphB receptors on dendritic cells, the main antigen presenting cells that interact with T cells. Here we show that mouse splenic dendritic cells (DC) and bone-marrow derived DCs (BMDC) express EphB2, a member of the EphB family. EphB2 expression is modulated by ligation of TLR4 and TLR9 and also by interaction with ephrin B ligands. Co-localization of EphB2 with MHC-II is also consistent with a potential role in T cell activation. However, BMDCs derived from EphB2 deficient mice were able to present antigen in the context of MHC-II and produce T cell activating cytokines to the same extent as intact DCs. Collectively our data suggest that EphB2 may contribute to DC responses, but that EphB2 is not required for T cell activation. This result may have arisen because DCs express other members of the EphB receptor family, EphB3, EphB4 and EphB6, all of which can interact with ephrin B ligands, or because EphB2 may be playing a role in another aspect of DC biology such as migration

Increased Insulin Signaling in Fat Body of Aedes Aegypti Mosquitoes Enhance Lifespace

The mosquito Aedes aegypti is a well known vector of many viral diseases such as yellow fever, dengue and dengue hemorrhagic fever (DHF). In particular, dengue and DHF, though normally considered tropical diseases, are steadily increasing in prevalence throughout the world with cases being reported as far north as the Texas-Mexico border and in Florida. There is currently no vaccine for these diseases and vector control has proved challenging due to the urban preferences of A. aegypti. The insulin/insulin-growth factor signaling (IIS) pathway may provide an endogenous solution to vector control. In mosquitoes, the IIS plays an important role in the regulation of many physiological processes, including longevity and reproduction. Studies in several species, including Drosophila melanogaster, Caenorhabolitis elegans and Anopheles stephensi, have shown that decreasing insulin signaling leads to an increase in longevity [Hwangbo et al., 2004; Kimura et al., 1997]. Inversely, similar studies have shown that increasing insulin signaling results in a shortened lifespan and often an increase in reproduction [Corby-Harris et al., 2010]. Here we aimed to increase insulin signaling in the fat body of Aedes aegypti mosquitoes by creating a transgenic line that expresses an active form of Akt, a key component of the IIS, specifically in the fat body tissue. We observed the effects on longevity and reproduction in both a heterozygous and a homozygous line. However, contrary to the expected results, we observed an increase in the lifespan of heterozygous females positive for the transgene. We also observed no significant difference in the reproductive potential of heterozygous positive versus heterozygous negative females, although expression of vitellogenin was increased. This led us to consider the possibility that under these conditions insulin signaling may have been down-regulated or shut off completely, instead of being up-regulated as was expected. Further investigations need to be made into the possible causes of these results

The Innate Immune Playbook for Restricting West Nile Virus Infection

West Nile virus (WNV) is an emerging mosquito-borne flavivirus that causes annual epidemics of encephalitic disease throughout the world. Despite the ongoing risk to public health, no approved vaccines or therapies exist for use in humans to prevent or combat WNV infection. The innate immune response is critical for controlling WNV replication, limiting virus-induced pathology, and programming protective humoral and cell-mediated immunity to WNV infection. The RIG-I like receptors, Toll-like receptors, and Nod-like receptors detect and respond to WNV by inducing a potent antiviral defense program, characterized by production of type I IFN, IL-1β and expression of antiviral effector genes. Recent research efforts have focused on uncovering the mechanisms of innate immune sensing, antiviral effector genes that inhibit WNV, and countermeasures employed by WNV to antagonize innate immune cellular defenses. In this review, we highlight the major research findings pertaining to innate immune regulation of WNV infection

BMDCs stimulated with EphrinB2 upregulate <i>EphB2</i> mRNA.

<p>Naïve BMDCs were plated onto plate-bound Ephrin-B1-Fc fusion protein (A, B, C) or plate bound Ephrin-B2-Fc fusion protein (D, E, F). The transcription of <i>EphB2</i> with and without the addition of recombinant interferon-γ (IFN-γ) was monitored by qPCR over 24 hours of culture (A, D). The efficiency of the Eph receptor ligation was monitored by assessing tyrosine phosphorylation on DCs by western blot (B, E) and the phosphorylation quantified using densitometry (C, F). All graphs represent the median value of pooled data from 3 independent DC preparations ±SD.</p

EphB2 co-localizes with MHC-II on BMDCs.

<p>Two examples are shown: (A) Representative BMDCs stimulated with 1μg/ml lipopolysaccharide (LPS) and 20ng/ml recombinant mouse interferon (IFN)-γ for 22 hours; (B) shows unstimulated cells. EphB2 is shown in red (Northernlights557), MHC-II is shown in green (FITC) and DAPI is shown to demarcate the nuclei of the cells. Magnification 100x; Scale bar, 20μm.</p

Stimulation of BMDCs from EphB2+/+ and EphB2-/- mice with TLR receptor agonists results in similar levels of secreted cytokine.

<p>BMDCs from EphB2+/+ and EphB2-/- mice were incubated with TLR ligands LPS and CpG1668 with and without the addition of recombinant interferon (IFN)-γ for 20 hours. Interleukin (IL)-12p40 (A), IL-12p70 (B), IL-10 (C) and tumor necrosis factor (TNF)-α (D) were measured in the culture supernatant using Luminex. The graphs are representative of 3 individual experiments and bars represent the mean ±SD of 2 replicate wells plated.</p

EphB2 expression on BMDCs can be modulated by ligation with Toll-like receptor ligands.

<p>(A) BMDC were incubated with a Toll Like receptor (TLR)-4 agonist (lipopolysaccharide (LPS) 1μg/ml) and (B) a TLR-9 agonist (CpG1668 1μM) with or without recombinant mouse interferon (IFN)-γ at a concentration of 20ng/ml. <i>EphB2</i> mRNA was quantified by qPCR at different time points post-stimulation. The modulation of transcription for known co-stimulatory molecules CD80 and CD86 as well as the cytokine interleukin (IL)-12p40 in response to TLR stimulation in the same experiments are shown for comparison. (C) The change in EphB2 protein expression at 22 hours post-incubation with LPS and IFN-γ is shown and the mean fluorescence quantified for different conditions. All graphs represent the median value of pooled data across 3 independent BMDC preparations ±SD and data analyzed using One-way ANOVA– Kruskal Wallis test and Dunn’s multiple comparisons post-test. *P<0.05. MFI = Mean Fluorescence Intensity.</p