An improved method for generating axenic entomopathogenic nematodes.

BACKGROUND: Steinernema carpocapsae are parasitic nematodes that invade and kill insects. The nematodes are mutualistically associated with the bacteria Xenorhabdus nematophila and together form an excellent model to study pathogen infection processes and host anti-nematode/antibacterial immune responses. To determine the contribution of S. carpocapsae and their associated X. nematophila to the successful infection of insects as well as to investigate the interaction of each mutualistic partner with the insect immune system, it is important to develop and establish robust methods for generating nematodes devoid of their bacteria. FINDINGS: To produce S. carpocapsae nematodes without their associated X. nematophila bacteria, we have modified a previous method, which involves the use of a X. nematophila rpoS mutant strain that fails to colonize the intestine of the worms. We confirmed the absence of bacteria in the nematodes using a molecular diagnostic and two rounds of an axenicity assay involving appropriate antibiotics and nematode surface sterilization. We used axenic and symbiotic S. carpocapsae to infect Drosophila melanogaster larvae and found that both types of nematodes were able to cause insect death at similar rates. CONCLUSION: Generation of entomopathogenic nematodes lacking their mutualistic bacteria provides an excellent tool to dissect the molecular and genetic basis of nematode parasitism and to identify the insect host immune factors that participate in the immune response against nematode infections

Challenging the Wigglesworthia, Sodalis, Wolbachia symbiosis dogma in tsetse flies : Spiroplasma is present in both laboratory and natural populations

Profiling of wild and laboratory tsetse populations using 16S rRNA gene amplicon sequencing allowed us to examine whether the “Wigglesworthia-Sodalis-Wolbachia dogma” operates across species and populations. The most abundant taxa, in wild and laboratory populations, were Wigglesworthia (the primary endosymbiont), Sodalis and Wolbachia as previously characterized. The species richness of the microbiota was greater in wild than laboratory populations. Spiroplasma was identified as a new symbiont exclusively in Glossina fuscipes fuscipes and G. tachinoides, members of the palpalis sub-group, and the infection prevalence in several laboratory and natural populations was surveyed. Multi locus sequencing typing (MLST) analysis identified two strains of tsetse-associated Spiroplasma, present in G. f. fuscipes and G. tachinoides. Spiroplasma density in G. f. fuscipes larva guts was significantly higher than in guts from teneral and 15-day old male and female adults. In gonads of teneral and 15-day old insects, Spiroplasma density was higher in testes than ovaries, and was significantly higher density in live versus prematurely deceased females indicating a potentially mutualistic association. Higher Spiroplasma density in testes than in ovaries was also detected by fluorescent in situ hybridization in G. f. fuscipe

Evolution and Function of Thioester-Containing Proteins and the Complement System in the Innate Immune Response

The innate immune response is evolutionary conserved among organisms. The complement system forms an important and efficient immune defense mechanism. It consists of plasma proteins that participate in microbial detection, which ultimately results in the production of various molecules with antimicrobial activity. Thioester-containing proteins (TEPs) are a superfamily of secreted effector proteins. In vertebrates, certain TEPs act in the innate immune response by promoting recruitment of immune cells, phagocytosis, and direct lysis of microbial invaders. Insects are excellent models for dissecting the molecular basis of innate immune recognition and response to a wide range of microbial infections. Impressive progress in recent years has generated crucial information on the role of TEPs in the antibacterial and antiparasite response of the tractable model insect Drosophila melanogaster and the mosquito malaria vector Anopheles gambiae. This knowledge is critical for better understanding the evolution of TEPs and their involvement in the regulation of the host innate immune system

Supplementary Material for: Thioester-Containing Protein-4 Regulates the Drosophila Immune Signaling and Function against the Pathogen Photorhabdus

<br>Despite important progress in identifying the molecules that participate in the immune response of <i>Drosophila melanogaster</i> to microbial infections, the involvement of thioester-containing proteins (TEPs) in the antibacterial immunity of the fly is not fully clarified. Previous studies mostly focused on identifying the function of TEP2, TEP3 and TEP6 molecules in the <i>D. melanogaster</i> immune system. Here, we investigated the role of TEP4 in the regulation and function of <i>D. melanogaster</i> host defense against 2 virulent pathogens from the genus <i>Photorhabdus</i>, i.e. the insect pathogenic bacterium <i>Photorhabdus luminescens</i> and the emerging human pathogen <i>P. asymbiotica</i>. We demonstrate that <i>Tep4</i> is strongly upregulated in adult flies following the injection of <i>Photorhabdus</i> bacteria. We also show that <i>Tep4</i> loss-of-function mutants are resistant to <i>P. luminescens </i>but not to <i>P. asymbiotica</i> infection<i>.</i> In addition, we find that inactivation of <i>Tep4 </i>results in the upregulation of the Toll and Imd immune pathways, and the downregulation of the Jak/Stat and Jnk pathways upon <i>Photorhabdus</i> infection. We document that loss of <i>Tep4</i> promotes melanization and phenoloxidase activity in the mutant flies infected with <i>Photorhabdus</i>. Together, these findings generate novel insights into the immune role of TEP4 as a regulator and effector of the <i>D. melanogaster</i> antibacterial immune response