Salivary gland-specific <i>P. berghei</i> reporter lines enable rapid evaluation of tissue-specific sporozoite loads in mosquitoes

Malaria is a life-threatening human infectious disease transmitted by mosquitoes. Levels of the salivary gland sporozoites (sgs), the only mosquito stage infectious to a mammalian host, represent an important cumulative index of &lt;i&gt;Plasmodium&lt;/i&gt; development within a mosquito. However, current techniques of sgs quantification are laborious and imprecise. Here, transgenic &lt;i&gt;P. berghei&lt;/i&gt; reporter lines that produce the green fluorescent protein fused to luciferase (GFP-LUC) specifically in sgs were generated, verified and characterised. Fluorescence microscopy confirmed the sgs stage specificity of expression of the reporter gene. The luciferase activity of the reporter lines was then exploited to establish a simple and fast biochemical assay to evaluate sgs loads in whole mosquitoes. Using this assay we successfully identified differences in sgs loads in mosquitoes silenced for genes that display opposing effects on &lt;i&gt;P. berghei&lt;/i&gt; ookinete/oocyst development. It offers a new powerful tool to study infectivity of &lt;i&gt;P. berghei&lt;/i&gt; to the mosquito, including analysis of vector-parasite interactions and evaluation of transmission-blocking vaccines

Transgenic Expression of the Anti-parasitic Factor TEP1 in the Malaria Mosquito Anopheles gambiae

Mosquitoes genetically engineered to be resistant to Plasmodium parasites represent a promising novel approach in the fight against malaria. The insect immune system itself is a source of anti-parasitic genes potentially exploitable for transgenic designs. The Anopheles gambiae thioester containing protein 1 (TEP1) is a potent anti-parasitic protein. TEP1 is secreted and circulates in the mosquito hemolymph, where its activated cleaved form binds and eliminates malaria parasites. Here we investigated whether TEP1 can be used to create malaria resistant mosquitoes. Using a GFP reporter transgene, we determined that the fat body is the main site of TEP1 expression. We generated transgenic mosquitoes that express TEP1r, a potent refractory allele of TEP1, in the fat body and examined the activity of the transgenic protein in wild-type or TEP1 mutant genetic backgrounds. Transgenic TEP1r rescued loss-of-function mutations, but did not increase parasite resistance in the presence of a wild-type susceptible allele. Consistent with previous reports, TEP1 protein expressed from the transgene in the fat body was taken up by hemocytes upon a challenge with injected bacteria. Furthermore, although maturation of transgenic TEP1 into the cleaved form was impaired in one of the TEP1 mutant lines, it was still sufficient to reduce parasite numbers and induce parasite melanization. We also report here the first use of Transcription Activator Like Effectors (TALEs) in Anopheles gambiae to stimulate expression of endogenous TEP1. We found that artificial elevation of TEP1 expression remains moderate in vivo and that enhancement of endogenous TEP1 expression did not result in increased resistance to Plasmodium. Taken together, our results reveal the difficulty of artificially influencing TEP1-mediated Plasmodium resistance, and contribute to further our understanding of the molecular mechanisms underlying mosquito resistance to Plasmodium parasites

Targeted Mutagenesis in the Malaria Mosquito Using TALE Nucleases

Anopheles gambiae, the main mosquito vector of human malaria, is a challenging organism to manipulate genetically. As a consequence, reverse genetics studies in this disease vector have been largely limited to RNA interference experiments. Here, we report the targeted disruption of the immunity gene TEP1 using transgenic expression of Transcription-Activator Like Effector Nucleases (TALENs), and the isolation of several TEP1 mutant A. gambiae lines. These mutations inhibited protein production and rendered TEP1 mutants hypersusceptible to Plasmodium berghei. The TALEN technology opens up new avenues for genetic analysis in this disease vector and may offer novel biotechnology-based approaches for malaria control

Salivary gland-specific P. berghei reporter lines enable rapid evaluation of tissue-specific sporozoite loads in mosquitoes

Malaria is a life-threatening human infectious disease transmitted by mosquitoes. Levels of the salivary gland sporozoites (sgs), the only mosquito stage infectious to a mammalian host, represent an important cumulative index of Plasmodium development within a mosquito. However, current techniques of sgs quantification are laborious and imprecise. Here, transgenic P. berghei reporter lines that produce the green fluorescent protein fused to luciferase (GFP-LUC) specifically in sgs were generated, verified and characterised. Fluorescence microscopy confirmed the sgs stage specificity of expression of the reporter gene. The luciferase activity of the reporter lines was then exploited to establish a simple and fast biochemical assay to evaluate sgs loads in whole mosquitoes. Using this assay we successfully identified differences in sgs loads in mosquitoes silenced for genes that display opposing effects on P. berghei ookinete/oocyst development. It offers a new powerful tool to study infectivity of P. berghei to the mosquito, including analysis of vector-parasite interactions and evaluation of transmission-blocking vaccines

Transgenic Expression of the Anti-parasitic Factor TEP1 in the Malaria Mosquito Anopheles gambiae

Mosquitoes genetically engineered to be resistant to Plasmodium parasites represent a promising novel approach in the fight against malaria. The insect immune system itself is a source of anti-parasitic genes potentially exploitable for transgenic designs. The Anopheles gambiae thioester containing protein 1 (TEP1) is a potent anti-parasitic protein. TEP1 is secreted and circulates in the mosquito hemolymph, where its activated cleaved form binds and eliminates malaria parasites. Here we investigated whether TEP1 can be used to create malaria resistant mosquitoes. Using a GFP reporter transgene, we determined that the fat body is the main site of TEP1 expression. We generated transgenic mosquitoes that express TEP1r, a potent refractory allele of TEP1, in the fat body and examined the activity of the transgenic protein in wild-type or TEP1 mutant genetic backgrounds. Transgenic TEP1r rescued loss-of-function mutations, but did not increase parasite resistance in the presence of a wild-type susceptible allele. Consistent with previous reports, TEP1 protein expressed from the transgene in the fat body was taken up by hemocytes upon a challenge with injected bacteria. Furthermore, although maturation of transgenic TEP1 into the cleaved form was impaired in one of the TEP1 mutant lines, it was still sufficient to reduce parasite numbers and induce parasite melanization. We also report here the first use of Transcription Activator Like Effectors (TALEs) in Anopheles gambiae to stimulate expression of endogenous TEP1. We found that artificial elevation of TEP1 expression remains moderate in vivo and that enhancement of endogenous TEP1 expression did not result in increased resistance to Plasmodium. Taken together, our results reveal the difficulty of artificially influencing TEP1-mediated Plasmodium resistance, and contribute to further our understanding of the molecular mechanisms underlying mosquito resistance to Plasmodium parasites

Characterization of functional polymorphisms and glucocorticoid-responsive elements in the promoter of TDO2, a candidate gene for ethanol-induced behavioural disorders

Aims: In response to acute ethanol consumption, tryptophan 2,3-dioxygenase (TDO) induces the kynurenine pathway (KP) through a glucocorticoid-mediated mechanism, which could lead to a dramatic accumulation of neurotoxic metabolites in association with serotonin depletion. As a result, interindividual variability in ethanol-induced behavioural disorders, such as black-outs and violent impulsive behaviours (BOVIBs) following binge drinking, could be partly explained by genetic polymorphisms affecting the KP. The aim of this study was to identify polymorphisms on the promoter of the TDO2 gene that could affect expression and/or activity of TDO through glucocorticoid inductio.Methods: Polymorphisms were screened using a PCR-sequencing strategy applied to 31 alcohol-dependent patients and 49 unrelated healthy volunteers, and functionally analysed with bioinformatic prediction tools and gene reporter assays in HepG2 and A549 cell lines. Results: We identified 12 polymorphisms in the human TDO2 promoter region, 2 of them corresponding to previously unknown single-nucleotide polymorphisms (SNPs) and 3 of them located in putative glucocorticoid-responsive elements (GREs). Gene reporter assays using HepG2 and A549 cell lines confirmed the presence of several functional GREs in the promoter region of TDO2 and revealed that some of the identified polymorphisms affect the promoter activity under glucocorticoid receptor over-expression and dexamethasone exposure conditions. Conclusions: Correlational studies in larger samples could help to determine whether these polymorphisms are responsible for variations of expression and/or activity of TDO, inparticular under conditions where release of glucocorticoids is increased, such as acute ethanol intake. If confirmed, such results would be of major interest in explaining part of the interindividual variability observed in behavioural responses to acute ethanol consumption.11 page(s

Tools for Anopheles gambiae Transgenesis

Transgenesis is an essential tool to investigate gene function and to introduce desired characters in laboratory organisms. Setting-up transgenesis in non-model organisms is challenging due to the diversity of biological life traits and due to knowledge gaps in genomic information. Some procedures will be broadly applicable to many organisms, and others have to be specifically developed for the target species. Transgenesis in disease vector mosquitoes has existed since the 2000s but has remained limited by the delicate biology of these insects. Here, we report a compilation of the transgenesis tools that we have designed for the malaria vector Anopheles gambiae, including new docking strains, convenient transgenesis plasmids, a puromycin resistance selection marker, mosquitoes expressing cre recombinase, and various reporter lines defining the activity of cloned promoters. This toolbox contributed to rendering transgenesis routine in this species and is now enabling the development of increasingly refined genetic manipulations such as targeted mutagenesis. Some of the reagents and procedures reported here are easily transferable to other nonmodel species, including other disease vector or agricultural pest insects

Activation of presynaptic oxytocin receptors enhances glutamate release in the ventral hippocampus of prenatally restraint stressed rats

Oxytocin receptors are known to modulate synaptic transmission and network activity in the hippocampus, but their precise function has been only partially elucidated. Here, we have found that activation of presynaptic oxytocin receptor with the potent agonist, carbetocin, enhanced depolarization-evoked glutamate release in the ventral hippocampus with no effect on GABA release. This evidence paved the way for examining the effect of carbetocin treatment in "prenatally restraint stressed" (PRS) rats, i.e., the offspring of dams exposed to repeated episodes of restraint stress during pregnancy. Adult PRS rats exhibit an anxious/depressive-like phenotype associated with an abnormal glucocorticoid feedback regulation of the hypothalamus-pituitary-adrenal (HPA) axis, and, remarkably, with a reduced depolarization-evoked glutamate release in the ventral hippocampus. Chronic systemic treatment with carbetocin (1mg/kg, i.p., once a day for 2-3 weeks) in PRS rats corrected the defect in glutamate release, anxiety- and depressive-like behavior, and abnormalities in social behavior, in the HPA response to stress, and in the expression of stress-related genes in the hippocampus and amygdala. Of note, carbetocin treatment had no effect on these behavioral and neuroendocrine parameters in prenatally unstressed (control) rats, with the exception of a reduced expression of the oxytocin receptor gene in the amygdala. These findings disclose a novel function of oxytocin receptors in the hippocampus, and encourage the use of oxytocin receptor agonists in the treatment of stress-related psychiatric disorders in adult life

Relative luciferase activity in <i>TEP1</i> and <i>Lp</i> knockdown mosquitoes infected with <i>glyc::GFP-LUC</i>.

<p>Mosquitoes were injected with dsRNA prior to infection with <i>glyc::GFP-LUC</i>. Surviving mosquitoes 18–21 dpi were freeze dried, ground and the luciferase activity was measured. The values are normalised to the values in control treatment (<i>dsLacZ</i>). Shown are the results of 3 independent experiments for each treatment expressed as means of duplicate or triplicate measurements; error bars represent the standard error of the mean. The dotted line marks the value 1, corresponding to the respective controls. Sample sizes in experiment 1: <i>dsLacZ</i> (n = 21), <i>dsTEP1</i> (n<b> = </b>17); experiment 2: <i>dsLacZ</i> (n = 19), <i>dsTEP1</i> (n<b> = </b>17); experiment 3: <i>dsLacZ</i> (n = 26), <i>dsTEP1</i> (n<b> = </b>11); experiment 4: <i>dsLacZ</i> (n = 21), <i>dsLp</i> (n<i> = </i>20); experiment 5: <i>dsLacZ</i> (n = 26), <i>dsLp</i> (n = 8); experiment 6: <i>dsLacZ</i> (n = 44), <i>dsLp</i> (n = 39).</p