Using capillary electrophoresis to identify Anopheline species in routine sampling sites.

In the Anopheles genus, various mosquito species are able to transmit the Plasmodium parasites responsible for malaria, while others are non-vectors. In an effort to better understand the biology of Anopheles species and to quantify transmission risk in an area, the identification of mosquito species collected in the field is an essential but problematic task. Morphological identification requires expertise and cannot be checked after processing samples in a destructive treatment, while sequencing of numerous samples is costly. Here, we introduce a method of Species identification via Simple Observation Coupled with Capillary Electrophoresis Technology (SOCCET). This molecular technique of species identification is based on precise determination of ITS2 length combined with a simple visual observation, the colour of mosquito hindleg tip. DNA extracted from field-collected Anopheles mosquitoes was amplified with universal Anopheles ITS2 primers and analysed with a capillary electrophoresis device, which precisely determines the size of the fragments. We defined windows of amplicon sizes combined with fifth hind tarsus colour, which allows discrimination of the major Anopheles species found in our collections. We validated our parameters via Sanger sequencing of ITS2 amplicons. Using the SOCCET method, we characterised the composition of Anopheles populations in five locations of French Guiana, where we detected a total of nine species. Anopheles braziliensis and Anopheles darlingi were detected in four locations each and represented 13 and 67% of our samples, respectively. The SOCCET method can be particularly useful when working with routine sampling sites with a moderate species diversity, that is, when the number of local species is too high to define species-specific primers but low enough to avoid individual ITS2 sequencing. This tool will be of interest to evaluate local malaria transmission risk and this approach may be further implemented for other mosquito genera.

The Effect of Secondary Metabolites Produced by Serratia marcescens on Aedes aegypti and Its Microbiota.

Serratia marcescens is a bacterial species widely found in the environment, which very efficiently colonizes mosquitoes. In this study, we isolated a red-pigmented S. marcescens strain from our mosquito colony (called S. marcescens VA). This red pigmentation is caused by the production of prodigiosin, a molecule with antibacterial properties. To investigate the role of prodigiosin on mosquito-S. marcescens interactions, we produced two white mutants of S. marcescens VA by random mutagenesis. Whole genome sequencing and chemical analyses suggest that one mutant has a nonsense mutation in the gene encoding prodigiosin synthase, while the other one is deficient in the production of several types of secondary metabolites including prodigiosin and serratamolide. We used our mutants to investigate how S. marcescens secondary metabolites affect the mosquito and its microbiota. Our in vitro tests indicated that S. marcescens VA inhibits the growth of several mosquito microbiota isolates using a combination of prodigiosin and other secondary metabolites, corroborating published data. This strain requires secondary metabolites other than prodigiosin for its proteolytic and hemolytic activities. In the mosquito, we observed that S. marcescens VA is highly virulent to larvae in a prodigiosin-dependent manner, while its virulence on adults is lower and largely depends on other metabolites.

[Mosquito microbiota and its influence on disease vectorial transmission]. / Le microbiote de moustique et son influence sur la transmission vectorielle.

Mosquitoes (Diptera: Culicidae) are found worldwide. Around 100 among 3500 mosquito species are known to be vectors of parasites and viruses, responsible for infectious diseases including malaria and dengue. Mosquitoes host diverse microbial communities that influence disease transmission, either by direct interference or via affecting host immunity and physiology. These microbial communities are present within diverse tissues, including the digestive tract, and vary depending on the sex of the mosquito, its developmental stage, and ecological factors. This review summarizes the current knowledge about the mosquito microbiota, defined as a community of commensal, symbiotic or pathogenic microbes harboured by a host. We first describe the current knowledge on the diversity of the microbiota, that includes bacteria, fungi, parasites and viruses and on its modes of acquisition throughout the mosquito life cycle. We then focus on microbial interactions within the mosquito gut, which notably affect vector competence, and on host-microbe interactions affecting mosquito fitness. Finally, we discuss current or potential methods based on the use of microbes or microbial products to interfere with pathogen transmission or to reduce mosquito lifespan and reproduction.

Molecular and serological investigation of Trypanosoma cruzi infection in dogs in French Guiana.

Clinical cases of Chagas disease, an infection caused by the parasite Trypanosoma cruzi, have been recently described in humans and dogs in French Guiana, a French overseas department located in South America. Elsewhere in endemic countries for this disease, cases of asymptomatic infections have been described. We performed a prevalence survey of the infection in dogs in Cayenne and Kourou, the main cities of French Guiana. In 2014 and 2016, blood samples were taken from 153 dogs from Cayenne and Kourou. All dogs were apparently healthy at the time of sampling. Sex and age of the dogs were recorded as well as the location where they lived. Serum samples from dogs were screened using a rapid immunochromatographic test (Chagas Stat-Pak®Assay, Chembio, USA) detecting anti-T. cruzi antibodies. Simultaneously, a real-time PCR targeting T. cruzi kDNA was performed on the blood samples of the dog. Six dogs (3.9%) were positive only in serology and one (0.6%) only in qPCR. Two dogs were positive for both tests. The prevalence of infection (positivity for one of the two tests) was 5.8% (9/153). There was no significant difference (χ2 test) between Cayenne (5/100) and Kourou (4/53), between males (3/60) and females (6/93), or between 2014 (2/55) and 2016 (7/98). Canine surveillance is a useful tool for the public health risk assessment of Chagas disease. Positive dogs, even when asymptomatic, should be treated as they can serve as a reservoir for T. cruzi.

Reduced Notch signalling leads to postnatal skeletal muscle hypertrophy in Pofut1cax/cax mice.

Postnatal skeletal muscle growth results from the activation of satellite cells and/or an increase in protein synthesis. The Notch signalling pathway maintains satellite cells in a quiescent state, and once activated, sustains their proliferation and commitment towards differentiation. In mammals, POFUT1-mediated O-fucosylation regulates the interactions between NOTCH receptors and ligands of the DELTA/JAGGED family, thus initiating the activation of canonical Notch signalling. Here, we analysed the consequences of downregulated expression of the Pofut1 gene on postnatal muscle growth in mutant Pofut1(cax/cax) (cax, compact axial skeleton) mice and differentiation of their satellite cell-derived myoblasts (SCDMs). Pofut1(cax/cax) mice exhibited muscle hypertrophy, no hyperplasia and a decrease in satellite cell numbers compared with wild-type C3H mice. In agreement with these observations, Pofut1(cax/cax) SCDMs differentiated earlier concomitant with reduced Pax7 expression and decrease in PAX7(+)/MYOD(-) progenitor cells. In vitro binding assays showed a reduced interaction of DELTA-LIKE 1 ligand (DLL1) with NOTCH receptors expressed at the cell surface of SCDMs, leading to a decreased Notch signalling as seen by the quantification of cleaved NICD and Notch target genes. These results demonstrated that POFUT1-mediated O-fucosylation of NOTCH receptors regulates myogenic cell differentiation and affects postnatal muscle growth in mice.

Ubiquitous Gasp1 overexpression in mice leads mainly to a hypermuscular phenotype.

BACKGROUND: Myostatin, a member of the TGFß superfamily, is well known as a potent and specific negative regulator of muscle growth. Targeting the myostatin signalling pathway may offer promising therapeutic strategies for the treatment of muscle-wasting disorders. In the last decade, various myostatin-binding proteins have been identified to be able to inhibit myostatin activity. One of these is GASP1 (Growth and Differentiation Factor-Associated Serum Protein-1), a protein containing a follistatin domain as well as multiple domains associated with protease inhibitors. Despite in vitro data, remarkably little is known about in vivo functions of Gasp1. To further address the role of GASP1 during mouse development and in adulthood, we generated a gain-of-function transgenic mouse model that overexpresses Gasp1 under transcriptional control of the human cytomegalovirus immediate-early promoter/enhancer. RESULTS: Overexpression of Gasp1 led to an increase in muscle mass observed not before day 15 of postnatal life. The surGasp1 transgenic mice did not display any other gross abnormality. Histological and morphometric analysis of surGasp1 rectus femoris muscles revealed an increase in myofiber size without a corresponding increase in myofiber number. Fiber-type distribution was unaltered. Interestingly, we do not detect a change in total fat mass and lean mass. These results differ from those for myostatin knockout mice, transgenic mice overexpressing the myostatin propeptide or follistatin which exhibit both muscle hypertrophy and hyperplasia, and show minimal fat deposition. CONCLUSIONS: Altogether, our data give new insight into the in vivo functions of Gasp1. As an extracellular regulatory factor in the myostatin signalling pathway, additional studies on GASP1 and its homolog GASP2 are required to elucidate the crosstalk between the different intrinsic inhibitors of the myostatin.