Statistical analysis of the individual variability of 1D protein profiles as a tool in ecology: an application to parasitoid venom.

Understanding the forces that shape eco-evolutionary patterns often requires linking phenotypes to genotypes, allowing characterization of these patterns at the molecular level. DNA-based markers are less informative in this aim compared to markers associated with gene expression and, more specifically, with protein quantities. The characterization of eco-evolutionary patterns also usually requires the analysis of large sample sizes to accurately estimate interindividual variability. However, the methods used to characterize and compare protein samples are generally expensive and time-consuming, which constrains the size of the produced data sets to few individuals. We present here a method that estimates the interindividual variability of protein quantities based on a global, semi-automatic analysis of 1D electrophoretic profiles, opening the way to rapid analysis and comparison of hundreds of individuals. The main original features of the method are the in silico normalization of sample protein quantities using pictures of electrophoresis gels at different staining levels, as well as a new method of analysis of electrophoretic profiles based on a median profile. We demonstrate that this method can accurately discriminate between species and between geographically distant or close populations, based on interindividual variation in venom protein profiles from three endoparasitoid wasps of two different genera (Psyttalia concolor, Psyttalia lounsburyi and Leptopilina boulardi). Finally, we discuss the experimental designs that would benefit from the use of this method.

Variation of Leptopilina boulardi success in Drosophila hosts: what is inside the black box?

Interactions between Drosophila hosts and parasitoid wasps are among the few examples in which occurrence of intraspecific variation of parasite success has been studied in natural populations. Such variations can originate from three categories of factors: environmental, host and parasitoid factors. Under controlled laboratory conditions, it is possible to focus on the two last categories, and, using specific reference lines, to analyze their respective importance. Parasitoid and host contributions to variations in parasite success have largely been studied in terms of evolutionary and mechanistic aspects in two Drosophila parasitoids, Asobara tabida and, in more details, in Leptopilina boulardi. This chapter focuses on the physiological and molecular aspects of L. boulardi interactions with two Drosophila host species, while most of the evolutionary hypotheses and models are presented in Chapter 11 of Dupas et al.

The nucleotide sequence and genome organization of the whitefly transmitted sweetpotato mild mottle virus: a close relationship with members of the family Potyviridae.

Primers corresponding to conserved regions in the RNA-dependent RNA polymerase and the RACE procedure led to the cloning of the complete sweetpotato mild mottle virus (SPMMV) RNA genome. The assembled SPMMV genomic sequence was 10,818 nucleotides in length with a polyadenylated tract at the 3' terminus. The structure and organization of the SPMMV genome appear to be similar to those of potyviruses and rymoviruses. A 5' untranslated region, rich in A and U residues, is present between nucleotides 1 and 139. A putative initiation codon, at nucleotides 140-142, marks the beginning of a large open reading frame (ORF) which ends in UAA at positions 10,508-10,510. A 308-nucleotide untranslated region is present between the termination codon of the ORF and the beginning of the 3' polyadenylated region. Almost all known potyvirus motifs are present in the polyprotein of SPMMV. However, motifs in the putative helper-component and coat protein of SPMMV are incomplete or missing, which may account for its vector relations. Despite similarities with rymoviruses, potyviruses and, to a lesser extent, bymoviruses, comparative sequence analyses demonstrated that SPMMV belongs to a distinct genus of the family Potyviridae.

Differentiation Among Potyviruses Infecting Sweet Potato Based on Genus-and Virus-Specific Reverse Transcription Polymerase Chain Reaction.

Knowledge of virus diseases affecting sweet potato has been complicated due to the frequent occurrence of mixed infections and difficulties in isolating and purifying sweet potato viruses. A combined assay of reverse transcription and polymerase chain reaction (PCR) utilizing degenerate genus-specific primers POT1 and POT2 was applied to 18 sweet potato clones from China. The primers were designed to amplify the variable 5' terminal region of the potyvirus coat protein gene. Molecular analysis of the amplified fragments identified the Chinese strains of sweet potato feathery mottle virus (SPFMV-CH), sweet potato latent virus (SPLV-CH), and sweet potato virus G (SPVG-CH). Among the detected potyviruses, a distantly related strain of SPFMV-CH, tentatively named SPFMV-CH2, was identified in sweet potatoes from China. On the basis of sequence identity, SPFMV-CH2 was closely related to the common (-C) strain of that virus. Identification of a closely related strain of SPVG-CH in one sweet potato clone from China further illustrated the usefulness of broad-spectrum PCR for detecting uncharacterized viruses. The acquisition of sequence information permitted the design of virus-specific primers for detecting and differentiating SPFMV, SPLV, and SPVG.

Detection of Apple Stem Grooving Virus in Dormant Apple Trees with Crude Extracts as Templates for One-Step RT-PCR.

Partial nucleotide sequences of amplification products obtained from four European apple stem grooving virus (ASGV) isolates using degenerate primers showed 80 to 85% similarity with the published ASGV sequence of a Japanese strain but 98 to 100% identities among themselves. Based on these sequences, two ASGV-specific primers (ASGV4F-ASGV4R) were designed to amplify a 574-bp fragment located in the putative viral RNA polymerase. With these primers, six European and five American ASGV isolates, maintained in herbaceous hosts (Chenopodium quinoa, Nicotiana glutinosa, and N. occidentalis) or in apple trees, were readily detected by reverse transcription-polymerase chain reaction (RT-PCR). Using these specific ASGV primers, dsRNA preparations have been shown to constitute good templates for reliable amplification of ASGV sequences from leaves and bark tissues of apple trees, both in a two-step RT-PCR protocol and in the one-step Titan One-Tube RT-PCR. System. Furthermore, the one-step RT-PCR system allowed a specific amplification of ASGV sequences directly from clarified crude extracts of leaves and bark tissues of apple trees during both active growth and the dormant season.

Evidence for the assignment of two strains of SPLV to the genus Potyvirus based on coat protein and 3' non-coding region sequence data.

The use of potyvirus-specific primers and subsequent application of the RACE procedure allowed the cloning of the 3' terminal 1088 nucleotides of the genomic RNA of the Taiwan isolate of sweetpotato latent virus (SPLV-T) and the 3' genomic 1085 nucleotides of a SPLV-like virus from China (SPLV-CH). The sequence of an internal part of the presumptive nuclear inclusion b gene was also determined for both isolates. Detailed sequence analyses revealed the presence of consensus motifs which indicated that SPLV-CH and SPLV-T should be regarded as members of the genus Potyvirus. Multiple sequence alignments and phylogenetic analyses were also performed and unambiguously assessed these isolates as strains of a distinct Potyvirus. SPLV was not related to other potyviruses infecting sweetpotato nor to any other sequenced virus. From the presence of the DAG box, SPLV-CH is expected to be a typical aphid transmitted Potyvirus whereas a conceivable explanation is proposed for the non-aphid transmission of SPLV-T.

Molecular evidence that the whitefly-transmitted sweetpotato mild mottle virus belongs to a distinct genus of the Potyviridae.

Complementary DNA representing 2 108 nucleotides at the 3' end of the genomic RNA of the whitefly-transmitted sweetpotato mild mottle virus (SPMMV) was cloned after PCR. Sequence analysis revealed an open reading frame of 1 797 nucleotides which codes for a protein of 599 amino acids, followed by a 3' non-coding region of 311 nucleotides. Alignment of the deduced amino acid sequence with corresponding sequences of other members of the Potyviridae demonstrated that part of the presumptive RNA-dependent RNA polymerase and the coat protein coding regions of SPMMV are found at the 3' end of its genome, in that order. Alignment of the amino acid sequence of the core of SPMMV coat protein with those of selected members of the Potyviridae showed limited identity, thus demonstrating--with phylogenetic analysis--that SPMMV belongs to a distinct genus of the family Potyviridae.

Determination of the taxonomic position and characterization of yam mosaic virus isolates based on sequence data of the 5'-terminal part of the coat protein cistron.

The sequences of the N-terminal part of the coat protein cistron from six isolates of yam mosaic virus (YMV-TOG, YMV-COT, YMV-12, YMV-CAR, YMV-BU1 and YMV-BU2) were determined. The analysis of the deduced amino acid sequences revealed the presence of consensus motifs characteristic of the potyvirus genus supporting the classification of YMV as a potyvirus member. The alignment of the N-terminal part of the coat protein of YMV-TOG, YMV-COT, YMV-12 and YMV-CAR showed that they were identical in size (152 aa) while YMV-BU1 and YMV-BU2 were shorter (140 aa) due to a deletion of 12 aa. These amino acid sequences exhibit an overall sequence identity ranging from 70.4% to 97.4% while the identity level with the other potyviruses sequenced in the considered region is below 50%, confirming that YMV is a distinct member of the potyvirus genus. The detailed analysis of the amino acid sequence alignment and of the identity levels observed between the N-terminal part of the coat protein of the six YMV isolates lead us to suggest that they have to be considered as distantly related strains of YMV rather than closely related but distinct viruses.

The complete nucleotide sequences of the coat protein cistron and the 3' non-coding region of a newly-identified potyvirus infecting sweetpotato, as compared to those of sweetpotato feathery mottle virus.

Complementary DNA representing 728 nucleotides of the 3' end of the genomic RNA of sweetpotato virus G (SPV-G) a newly-identified potyvirus infecting sweetpotato, was cloned and sequenced. This sequence was combined with that previously determined for the 5' terminal part of the coat protein cistron of the virus. The whole sequence contained a single open reading frame (ORF) of 1065 nucleotide, with the capacity to encode a coat protein of 355 amino acids, significantly larger than that of other potyviruses. The ORF was followed by an untranslated region of 222 nucleotides and a poly (A) tail. The coat protein of SPV-G was only distantly related to that of known potyviruses, with the exception of sweetpotato feathery mottle virus (SPFMV). Indeed, sequence identity in the C-terminal three quarters of the coat protein (more than 80%) and in the 3' untranslated region (more than 70%) indicate that SPV-G should be considered as closely related to, though distinct from SPFMV. This subset relationship is similar to that previously reported for members of the bean yellow mosaic virus subgroup or the bean common mosaic virus subgroup.

Identification of a sweet potato feathery mottle virus isolate from China (SPFMV-CH) by the polymerase chain reaction with degenerate primers.

Four degenerate oligonucleotide primers derived from conserved regions of the genome of potyviruses have been designed. A combined assay of reverse transcription and the polymerase chain reaction utilizing these primers on total RNA extracted from Ipomoea purpurea infected with a sweet potato feathery mottle virus isolate from China (SPFMV-CH), amplified a 1.35 kb and a 830 bp fragment. These amplified fragments were cloned into the Bluescript plasmid and sequenced. The comparison of the sequence of the N-terminal part of the coat protein of SPFMV-CH with those published for two other strains of SPFMV, showed a strong relationship between SPMV-CH and -RC.