Miguel López-Ferber

Defective or effective? Mutualistic interactions between virus genotypes

Defective viruses lack genes essential for survival but they can co–infect with complete virus genotypes and use gene products from the complete genotype for their replication and transmission. As such, they are detrimental to the fitness of complete genotypes. Here, we describe a mutualistic interaction between genotypes of an insect baculovirus (nucleopolyhedrovirus of Spodoptera frugiperda (Lepidoptera)) that increases the pathogenicity of the viral population. Mixtures of a complete genotype able to be transmitted orally and a deletion mutant unable to be transmitted orally resulted in a phenotype of increased pathogenicity. Because the infectiousness of mixed genotype infections was greater than that of single genotype infections, we predict that the transmissibility of mixed genotype occlusion bodies will be greater than that of any of their single genotype components. Such interactions will be subject to frequency–dependent selection and will influence the impact of these viruses on insect population dynamics and their efficacy as biological insecticides.

SPODOBASE : an EST database for the lepidopteran crop pest Spodoptera

BackgroundThe Lepidoptera Spodoptera frugiperda is a pest which causes widespread economic damage on a variety of crop plants. It is also well known through its famous Sf9 cell line which is used for numerous heterologous protein productions. Species of the Spodoptera genus are used as model for pesticide resistance and to study virus host interactions. A genomic approach is now a critical step for further new developments in biology and pathology of these insects, and the results of ESTs sequencing efforts need to be structured into databases providing an integrated set of tools and informations.DescriptionThe ESTs from five independent cDNA libraries, prepared from three different S. frugiperda tissues (hemocytes, midgut and fat body) and from the Sf9 cell line, are deposited in the database. These tissues were chosen because of their importance in biological processes such as immune response, development and plant/insect interaction. So far, the SPODOBASE contains 29,325 ESTs, which are cleaned and clustered into non-redundant sets (2294 clusters and 6103 singletons). The SPODOBASE is constructed in such a way that other ESTs from S. frugiperda or other species may be added. User can retrieve information using text searches, pre-formatted queries, query assistant or blast searches. Annotation is provided against NCBI, UNIPROT or Bombyx mori ESTs databases, and with GO-Slim vocabulary.ConclusionThe SPODOBASE database provides integrated access to expressed sequence tags (EST) from the lepidopteran insect Spodoptera frugiperda. It is a publicly available structured database with insect pest sequences which will allow identification of a number of genes and comprehensive cloning of gene families of interest for scientific community. SPODOBASE is available from URL: http://bioweb.ensam.inra.fr/spodobase

Genetic Structure of a Spodoptera frugiperda Nucleopolyhedrovirus Population: High Prevalence of Deletion Genotypes

ABSTRACT A Nicaraguan field isolate (SfNIC) of Spodoptera frugiperda nucleopolyhedrovirus was purified by plaque assay on Sf9 cells. Nine distinct genotypes, A to I, were identified by their restriction endonuclease profiles. Variant SfNIC-B was selected as the standard because its restriction profile corresponded to that of the wild-type isolate. Physical maps were generated for each of the variants. The differences between variants and the SfNIC-B standard were confined to the region between map units 9 and 32.5. This region included PstI-G, PstI-F, PstI-L, PstI-K and EcoRI-L fragments. Eight genotypes presented a deletion in their genome compared with SfNIC-B. Occlusion body-derived virions of SfNIC-C, -D and -G accounted for 41% of plaque-purified clones. These variants were not infectious per os but retained infectivity by injection into S. frugiperda larvae. Median 50% lethal concentration values for the other cloned genotypes were significantly higher than that of the wild type. The variants also differed in their speed of kill. Noninfectious variants SfNIC-C and -D lacked the pif and pif-2 genes. Infectivity was restored to these variants by plasmid rescue with a plasmid comprising both pif and pif-2. Transcription of an SfNIC-G gene was detected by reverse transcription-PCR in insects, but no fatal disease developed. Transcription was not detected in SfNIC-C or -D-inoculated larvae. We conclude that the SfNIC population presents high levels of genetic diversity, localized to a 17-kb region containing pif and pif-2, and that interactions among complete and deleted genotypic variants will likely influence the capacity of this virus to control insect pests.

Two key mutations in the host-range specificity domain of the p143 gene of Autographa californica nucleopolyhedrovirus are required to kill Bombyx mori larvae.

Autographa californica nucleopolyhedrovirus (AcMNPV) does not replicate in Bombyx mori cells (Bm5, BmN). We have shown previously that when a short DNA sequence within AcMNPV ORF95, which encodes the viral helicase P143, is replaced with the colinear region of B. mori nucleopolyhedrovirus (BmNPV), AcMNPV gains the ability to replicate in Bm5 cells. To determine the mutational events in the p143 gene required to allow AcMNPV replication in B. mori cells, AcMNPV recombinants produced in Sf9 cells were screened in vivo in B. mori larvae, which are more permissive to baculovirus infection than B. mori cell lines. Eight combinations of mutations were tested and characterization of viral DNA extracted from dead larvae showed that amino acid changes at position 564 and 577 are required to kill B. mori larvae.

Cydia pomonella granulovirus Genotypes Overcome Virus Resistance in the Codling Moth and Improve Virus Efficiency by Selection against Resistant Hosts

ABSTRACT Cydia pomonella granulovirus (CpGV) has been used for 15 years as a bioinsecticide in codling moth (Cydia pomonella) control. In 2004, some insect populations with low susceptibility to the virus were detected for the first time in southeast France. RGV, a laboratory colony of codling moths resistant to the CpGV-M isolate used in the field, was established with collection of resistant insects in the field followed by an introgression of the resistant trait into a susceptible colony (Sv). The resistance level (based on the 50% lethal concentrations [LC50s]) of the RGV colony to the CpGV-M isolate, the active ingredient in all commercial virus formulations in Europe, appeared to be over 60,000-fold compared to the Sv colony. The efficiency of CpGV isolates from various other regions was tested on RGV. Among them, two isolates (I12 and NPP-R1) presented an increased pathogenicity on RGV. I12 had already been identified as effective against a resistant C. pomonella colony in Germany and was observed to partially overcome the resistance in the RGV colony. The recently identified isolate NPP-R1 showed an even higher pathogenicity on RGV than other isolates, with an LC50 of 166 occlusion bodies (OBs)/μl, compared to 1.36 × 106 OBs/μl for CpGV-M. Genetic characterization showed that NPP-R1 is a mixture of at least two genotypes, one of which is similar to CpGV-M. The 2016-r4 isolate obtained from four successive passages of NPP-R1 in RGV larvae had a sharply reduced proportion of the CpGV-M-like genotype and an increased pathogenicity against insects from the RGV colony.

Functional Importance of Deletion Mutant Genotypes in an Insect Nucleopolyhedrovirus Population

ABSTRACT A Nicaraguan isolate of a nucleopolyhedrovirus (SfNIC) that attacks the fall armyworm, Spodoptera frugiperda, survives as a mixture of nine genotypes (SfNIC A to I) that all present genomic deletions, except variant B (complete genotype). Sequencing of cloned restriction fragments revealed that genotypic variants lack between 5 and 16 of the open reading frames present in a contiguous sequence of 18 kb of the SfNIC genome. The absence of oral infectivity of SfNIC-C and -D variants is related to the deletion of the pif and/or pif-2 gene, while that of SfNIC-G remains unexplained. The presence of open reading frame 10, homolog of Se030, also appeared to influence pathogenicity in certain variants. Previous studies demonstrated a significant positive interaction between genotypes B and C. We compared the median lethal concentration of single genotypes (A, B, C, D, and F) and co-occluded genotype mixtures (B+A, B+D, B+F, A+C, and F+C in a 3:1 ratio). Mixtures B+A and B+D showed increased pathogenicity, although only B+D restored the activity of the mixture to that of the natural population. Mixtures of two deletion variants (A+C and F+C) did not show interactions in pathogenicity. We conclude that minority genotypes have an important influence on the overall pathogenicity of the population. These results clearly demonstrate the value of retaining genotypic diversity in virus-based bioinsecticides.

Dynamics of deletion genotypes in an experimental insect virus population

Defective viruses, that are deficient in certain essential genes, are maintained in the population by transcomplementation, exploiting the gene products of complete genotypes in co-infected cells. This process becomes prevalent only when cells are frequently infected by several virus particles, and only then will the fitness of defective viruses be subjected to frequency-dependent selection. Deletion variants that are not infectious per os are present in a multicapsid nucleopolyhedrovirus (SfMNPV, Baculoviridae) that infects the fall armyworm, Spodoptera frugiperda. These variants enhance the pathogenicity and, therefore, the likelihood of transmission of the virus when co-infecting cells with complete genotypes, resulting in occlusion bodies (OBs) that may contain both genotypes co-occluded. Mixtures of complete (B) and defective (C) variants in ratios of 90% B+10% C, 50% B+50% C and 10% B+90% C were used to inoculate by injection S. frugiperda larvae. Viral OBs extracted from diseased insects were subjected to four or five successive rounds of per os infection. Following successive passages, genotype frequencies in all three experimental populations converged to a single equilibrium frequency comprising ∼20% of deletion genotype C and ∼80% of complete genotype B. This mirrors the relative proportions of deletion (22%) and complete (78%) genotypes observed in the wild-type SfMNPV population. The pathogenicity of experimental populations at the final passage was not significantly different from that of the wild-type isolate. In contrast, OBs of all genotype mixtures were significantly more pathogenic than OBs of genotype B alone. A population genetics model, in which virus populations were assigned linear frequency-dependent transmissibility values, was in remarkably close agreement to empirical data. Clearly, non-infectious deletion variants can profoundly affect the likelihood of transmission and the genetic structure and stability of virus populations.

Estrogenic and anti-estrogenic activity of 23 commercial textile dyes.

The presence of dyes in wastewater effluent of textile industry is well documented. In contrast, the endocrine disrupting effects of these dyes and wastewater effluent have been poorly investigated. Herein, we studied twenty-three commercial dyes, usually used in the textile industry, and extracts of blue jean textile wastewater samples were evaluated for their agonistic and antagonistic estrogen activity. Total estrogenic and anti-estrogenic activities were measured using the Yeast Estrogen Screen bioassay (YES) that evaluates estrogen receptor binding-dependent transcriptional and translational activities. The estrogenic potencies of the dyes and wastewater samples were evaluated by dose-response curves and compared to the dose-response curve of 17β-estradiol (E2), the reference compound. The dose-dependent anti-estrogenic activities of the dyes and wastewater samples were normalized to the known antagonistic effect of 4-hydroxytamoxifen (4-OHT) on the induction of the lac Z reporter gene by E2. About half azo textile dyes have anti-estrogenic activity with the most active being Blue HFRL. Most azo dyes however have no or weak estrogenic activity. E2/dye or E2/waste water ER competitive binding assays show activity of Blue HFRL, benzopurpurine 4B, Everzol Navy Blue FBN, direct red 89 BNL 200% and waste water samples indicating a mechanism of action common to E2. Our results indicate that several textile dyes are potential endocrine disrupting agents. The presence of some of these dyes in textile industry wastewater may thus impact the aquatic ecosystem.

Rapid Visual Tests: Fast and Reliable Detection of Ochratoxin A

This paper reviews the early detection strategies that have been employed for the rapid monitoring of ochratoxin A (OTA) contamination of food. OTA, a mycotoxin mainly produced by some Aspergillus and Penicillium species, is found in cereals, coffee, wine, pork and grapes. To minimize the entry of this mycotoxin into the food chain, rapid diagnostic tools are required. To this end, the potential use of lateral flow devices has also been developed. In this study, we analyze the robustness of test strips using published methods for colorimetric detection. Different test formats are discussed, and challenges in the development of lateral flow devices for on-site determination of OTA, with requirements such as robustness, speed, and cost-effectiveness, are discussed.

Resistance to bio-insecticides or how to enhance their sustainability: a review

After more than 70 years of chemical pesticide use, modern agriculture is increasingly using biological control products. Resistances to conventional insecticides are wide spread, while those to bio-insecticides have raised less attention, and resistance management is frequently neglected. However, a good knowledge of the limitations of a new technique often provides greater sustainability. In this review, we compile cases of resistance to widely used bio-insecticides and describe the associated resistance mechanisms. This overview shows that all widely used bio-insecticides ultimately select resistant individuals. For example, at least 27 species of insects have been described as resistant to Bacillus thuringiensis toxins. The resistance mechanisms are at least as diverse as those that are involved in resistance to chemical insecticides, some of them being common to bio-insecticides and chemical insecticides. This analysis highlights the specific properties of bio-insecticides that the scientific community should use to provide a better sustainability of these products.