A PCR-based method for estimating parasitism rates in the olive fly parasitoids Psyttalia concolor and P. lounsburyi (Hymenoptera: Braconidae)

International audienceSeveral parasitoids of the genus Psyttalia have been repeatedly introduced as biological control agents against the principal pest of olive, the fly Bactrocera oleae. However, few of the parasitoids released have become established and proved effective against B. oleae. It may however still be possible to find effective biological control agents adapted to local environmental conditions among the highly diverse Psyttalia species and populations infesting B. oleae worldwide. For this purpose, we have developed a rapid, sensitive molecular method based on the polymerase chain reaction (PCR) for estimating and comparing the parasitism success of Psyttalia parasitoids through the detection of eggs and larvae within the host. This method was tested and shown to be appropriate for two Psyttalia species (Psyttalia concolor and Psyttalia lounsburyi). The possible detection of DNA was also demonstrated for several populations of these species and for other Psyttalia species, namely Psyttalia humilis and Psyttalia ponerophaga. For P. concolor and P. lounsburyi, a strong correlation was observed between the parasitism rates estimated by PCR, host larva dissection and counts of emerging parasitoids. No significant difference was found between the rates of parasitism estimated by host larva dissection and PCR, whereas the rates of parasitism estimated by PCR were significantly higher than those estimated from emergence, suggesting occurrence of mortality during the parasitoid development. This PCR method is thus highly reliable and provides an objective criterion for estimating the efficacy of biological control agent candidates from diverse taxa and populations of Psyttalia. ⇑ Corresponding author

Adverse effects of Bacillus thuringiensis bioinsecticide on non-target Drosophila species 1

Biopesticides based on Bacillus thuringiensis kurstaki (Btk) and israelensis (Bti) spores and toxins are widely used to control insect pests, increasing environmental risks to non-target biodiversity. Here, we tested for potential effects of larval ingestion of Bt commercial formulations on Drosophila species. Doses equivalent to those recommended for field application (⩽10^6 CFU/g of fly medium) had no effect whereas Btk doses 10 to 100-fold higher (10^7-10^8 CFU/g) altered the development (decreased emergence due to larval mortality and increased development time), and moderately influenced adult fitness-related traits. At the highest Btk and Bti dose (10^9 CFU/g), all larvae died before pupation. The impact of Btk formulations resulted from the spores/cleaved toxins synergy, but also additives. While recommended doses had no effect on non-target Drosophila species, the accumulation of Bt bioinsecticides in the environment could have adverse side-effects on the populations of these species and therefore their associated communities

Expression, immunolocalization and processing of fertilins ADAM-1 and ADAM-2 in the boar (sus domesticus) spermatozoa during epididymal maturation

Fertilin alpha (ADAM-1) and beta (ADAM-2) are integral membrane proteins of the ADAM family that form a fertilin complex involved in key steps of the sperm-oocyte membrane interaction. In the present work, we analyzed the presence of ADAM-1 and ADAM-2 mRNAs, the spermatozoa proteins' processing and their sub-cellular localization in epididymal samples from adult boars. ADAM-1 and ADAM-2 mRNAs were highly produced in the testis, but also in the vas efferens and the epididymis. On immunoblots of sperm extracts, ADAM-1 subunit appeared as a main reactive band of ~50-55 kDa corresponding to occurrence of different isoforms throughout the epididymal duct, especially in the corpus region where isoforms ranged from acidic to basic pI. In contrast, ADAM-2 was detected as several bands of ~90 kDa, ~75 kDa, ~50-55 kDa and ~40 kDa. The intensity of high molecular mass bands decreased progressively in the distal corpus where lower bands were also transiently observed, and only the ~40 kDa was observed in the cauda. The presence of bands of different molecular weights likely results from a proteolytic processing occurring mainly in the testis for ADAM-1, and also throughout the caput epididymis for ADAM-2. Immunolocalization showed that fertilin migrates from the acrosomal region to the acrosomal ridge during the sperm transit from the distal corpus to the proximal cauda. This migration is accompanied by an important change in the extractability of a part of ADAM-1 from the sperm membrane. This suggests that the fertilin surface migration may be triggered by the biochemical changes induced by the epididymal post-translational processing of both ADAM1 and ADAM-2. Different patterns of fertilin immunolocalization then define several populations of spermatozoa in the cauda epididymis. Characterization of such fertilin complex maturation patterns is an important step to develop fertility markers based on epididymal maturation of surface membrane proteins in domestic mammals

Drosophila Cellular Immunity Against Parasitoid Wasps: A Complex and Time-Dependent Process

Host-parasitoid interactions are among the most studied interactions between invertebrates because of their fundamental interest – the evolution of original traits in parasitoids – and applied, parasitoids being widely used in biological control. Immunity, and in particular cellular immunity, is central in these interactions, the host encapsulation response being specific for large foreign bodies such as parasitoid eggs. Although already well studied in this species, recent data on Drosophila melanogaster have unquestionably improved knowledge of invertebrate cellular immunity. At the same time, the venomics of parasitoids has expanded, notably those of Drosophila. Here, we summarize and discuss these advances, with a focus on an emerging “time-dependent” view of interactions outcome at the intra- and interspecific level. We also present issues still in debate and prospects for study. Data on the Drosophila-parasitoid model paves the way to new concepts in insect immunity as well as parasitoid wasp strategies to overcome it

The adult boar testicular and epididymal transcriptomes

<p>Abstract</p> <p>Background</p> <p>Mammalians gamete production takes place in the testis but when they exit this organ, although spermatozoa have acquired a specialized and distinct morphology, they are immotile and infertile. It is only after their travel in the epididymis that sperm gain their motility and fertility. Epididymis is a crescent shaped organ adjacent to the testis that can be divided in three gross morphological regions, head (caput), body (corpus) and tail (cauda). It contains a long and unique convoluted tubule connected to the testis via the efferent ducts and finished by joining the <it>vas deferens </it>in its caudal part.</p> <p>Results</p> <p>In this study, the testis, the efferent ducts (<it>vas efferens</it>, VE), nine distinct successive epididymal segments and the deferent duct (<it>vas deferens</it>, VD) of four adult boars of known fertility were isolated and their mRNA extracted. The gene expression of each of these samples was analyzed using a pig generic 9 K nylon microarray (AGENAE program; GEO accession number: GPL3729) spotted with 8931 clones derived from normalized cDNA banks from different pig tissues including testis and epididymis. Differentially expressed transcripts were obtained with moderated t-tests and F-tests and two data clustering algorithms based either on partitioning around medoid (top down PAM) or hierarchical clustering (bottom up HCL) were combined for class discovery and gene expression analysis. Tissue clustering defined seven transcriptomic units: testis, <it>vas efferens </it>and five epididymal transcriptomic units. Meanwhile transcripts formed only four clusters related to the tissues. We have then used a specific statistical method to sort out genes specifically over-expressed (markers) in testis, VE or in each of the five transcriptomic units of the epididymis (including VD). The specific regional expression of some of these genes was further validated by PCR and Q-PCR. We also searched for specific pathways and functions using available gene ontology information.</p> <p>Conclusion</p> <p>This study described for the first time the complete transcriptomes of the testis, the epididymis, the <it>vas efferens </it>and the <it>vas deferens </it>on the same species. It described new genes or genes not yet reported over-expressed in these boar tissues, as well as new control mechanisms. It emphasizes and fulfilled the gap between studies done in rodents and human, and provides tools that will be useful for further studies on the biochemical processes responsible for the formation and maintain of the epididymal regionalization and the development of a fertile spermatozoa.</p

Rapid and dfferential evolution of the venom composition of a parasitoid wasp depending on the host strain

Abstract: Parasitoid wasps rely primarily on venom to suppress the immune response and regulatethe physiology of their host. Intraspecific variability of venom protein composition has beendocumented in some species, but its evolutionary potential is poorly understood. We performed anexperimental evolution initiated with the crosses of two lines of Leptopilina boulardi of differentvenom composition to generate variability and create new combinations of venom factors. Theoffspring were maintained for 10 generations on two strains of Drosophila melanogaster differing inresistance/susceptibility to the parental parasitoid lines. The venom composition of individuals wascharacterized by a semi-automatic analysis of 1D SDS-PAGE electrophoresis protein profiles whoseaccuracy was checked by Western blot analysis of well-characterized venom proteins. Results madeevident a rapid and differential evolution of the venom composition on both hosts and showed thatthe proteins beneficial on one host can be costly on the other. Overall, we demonstrated the capacityof rapid evolution of the venom composition in parasitoid wasps, important regulators of arthropodpopulations, suggesting a potential for adaptation to new hosts. Our approach also proved relevantin identifying, among the diversity of venom proteins, those possibly involved in parasitism successand whose role deserves to be deepened

Variability of venom components in immune suppressive parasitoid wasps: From a phylogenetic to a population approach

International audienceEndoparasitoid wasps develop at the expense of other insects, leading to their death. Eggs deposited inside the host body induce an immune response, which results in the formation of a melanized cellular capsule around the egg. To evade or counteract this response, endoparasitoids have evolved different strategies, the most often reported being injection into the host of immunosuppressive factors, notably venom proteins, along with the egg. The analysis of venom components has been performed independently in species of different taxa, but the present picture is far from complete. Intriguingly, the question of the level of venom variability inside species has been neglected, although it may partly determine the potential for parasitoid adaptation. Here, we present a short review of our present knowledge of venom components in endoparasitoids, as well as of the only well-known example of intraspecific variability in a venom immune suppressive protein being responsible for variation in parasitoid virulence. We then present data evidencing inter-individual variation of venom protein profiles, using a gel electrophoresis approach, both in laboratory strains and field populations of a figitid and a braconid species. Whether occurrence of such variability may permit a selection of parasitoid venom components driven by the host remains to be tested, notably in the context of the production and use of biological control auxiliaries

Variation in the Venom of Parasitic Wasps, Drift, or Selection? Insights From a Multivariate QST Analysis

Differentiation of traits among populations can evolve by drift when gene flow is low relative to drift or selection when there are different local optima in each population (heterogeneous selection), whereas homogeneous selection tends to prevent evolution of such a differentiation. Analyses of geographical variations in venom composition have been done in several taxa such as wasps, spiders, scorpions, cone snails and snakes, but surprisingly never in parasitoid wasps, although their venom should constrain their ability to succeed on locally available hosts. Such a study is now facilitated by the development of an accurate method (quantitative digital analysis) that allows analyzing the quantitative variation of large sets of proteins from several individuals. This method was used here to analyse the venom-based differentiation of four samples of Leptopilina boulardi and five samples of L. heterotoma from populations along a 300 km long south-north gradient in the Rhône-Saône valley (South-East of France). A major result is that the composition of the venom allows to differentiate the populations studied even when separated by few kilometers. We further analyzed these differentiations on the populations (reared under similar conditions to exclude environmental variance) with a QST analysis which compared the variance of a quantitative trait (Q) among the subpopulations (S) to the total variance (T). We also used random forest clustering analyses to detect the venom components the most likely to be adapted locally. The signature of the natural selection was strong for L. heterotoma and L. boulardi. For the latter, the comparison with the differentiation observed at some neutral markers revealed that differentiation was partly due to some local adaptation. The combination of methods used here appears to be a powerful framework for population proteomics and for the study of eco-evolutionary feedbacks between proteomic level and population and ecosystem levels. This is of interest not only for studying field evolution at an intermediate level between the genome and phenotypes, or for understanding the role of evolution in chemical ecology, but also for more applied issues in biological control

Efficient generation of CD34+ progenitorâ derived dendritic cells from Gâ CSFâ mobilized peripheral mononuclear cells does not require hematopoietic stem cell enrichment

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/141647/1/jlb0957.pd

Microarray analysis of peripheral blood lymphocytes from ALS patients and the SAFE detection of the KEGG ALS pathway

<p>Abstract</p> <p>Background</p> <p>Sporadic amyotrophic lateral sclerosis (sALS) is a motor neuron disease with poorly understood etiology. Results of gene expression profiling studies of whole blood from ALS patients have not been validated and are difficult to relate to ALS pathogenesis because gene expression profiles depend on the relative abundance of the different cell types present in whole blood. We conducted microarray analyses using Agilent Human Whole Genome 4 × 44k Arrays on a more homogeneous cell population, namely purified peripheral blood lymphocytes (PBLs), from ALS patients and healthy controls to identify molecular signatures possibly relevant to ALS pathogenesis.</p> <p>Methods</p> <p>Differentially expressed genes were determined by LIMMA (Linear Models for MicroArray) and SAM (Significance Analysis of Microarrays) analyses. The SAFE (Significance Analysis of Function and Expression) procedure was used to identify molecular pathway perturbations. Proteasome inhibition assays were conducted on cultured peripheral blood mononuclear cells (PBMCs) from ALS patients to confirm alteration of the Ubiquitin/Proteasome System (UPS).</p> <p>Results</p> <p>For the first time, using SAFE in a global gene ontology analysis (gene set size 5-100), we show significant perturbation of the KEGG (Kyoto Encyclopedia of Genes and Genomes) ALS pathway of motor neuron degeneration in PBLs from ALS patients. This was the only KEGG disease pathway significantly upregulated among 25, and contributing genes, including <it>SOD1</it>, represented 54% of the encoded proteins or protein complexes of the KEGG ALS pathway. Further SAFE analysis, including gene set sizes >100, showed that only neurodegenerative diseases (4 out of 34 disease pathways) including ALS were significantly upregulated. Changes in <it>UBR2 </it>expression correlated inversely with time since onset of disease and directly with ALSFRS-R, implying that <it>UBR2 </it>was increased early in the course of ALS. Cultured PBMCs from ALS patients accumulated more ubiquitinated proteins than PBMCs from healthy controls in a serum-dependent manner confirming changes in this pathway.</p> <p>Conclusions</p> <p>Our study indicates that PBLs from sALS patients are strong responders to systemic signals or local signals acquired by cell trafficking, representing changes in gene expression similar to those present in brain and spinal cord of sALS patients. PBLs may provide a useful means to study ALS pathogenesis.</p