BUSCADOR RECOLECTA

Helicoverpa armigera single nucleopolyhedrovirus (HearSNPV) is a virulent pathogen of lepidopterans in the genera Heliothis and Helicoverpa, whereas Helicoverpa armigera multiple nu-cleopolyhedrovirus (HearSNPV) is a different virus species with a broader host range. This study aimed to examine the consequences of coocclusion of HearSNPV and HearMNPV on the patho-genicity, stability and host range of mixed-virus occlusion bodies (OBs). HearSNPV OBs were approximately 6-fold more pathogenic than HearMNPV OBs, showed faster killing by approximately 13 h, and were approximately 45% more productive in terms of OB production per larva. For coocclusion, H. armigera larvae were first inoculated with HearMNPV OBs and subsequently inoculated with HearSNPV OBs at intervals of 0-72 h after the initial inoculation. When the interval between inoculations was 12-24 h, OBs collected from virus-killed insects were found to comprise 41¿57% of HearSNPV genomes, but the prevalence of HearSNPV genomes was greatly reduced (3- 4%) at later time points. Quantitative PCR (qPCR) analysis revealed the presence of HearSNPV genomes in a small fraction of multinucleocapsid ODVs representing 0.47¿0.88% of the genomes quan-tified in ODV samples, indicating that both viruses had replicated in coinfected host cells. End-point dilution assays on ODVs from cooccluded mixed-virus OBs confirmed the presence of both viruses in 41.9¿55.6% of wells that were predicted to have been infected by a single ODV. A control exper-iment indicated that this result was unlikely to be due to the adhesion of HearSNPV ODVs to HearMNPV ODVs or accidental contamination during ODV band extraction. Therefore, the dispar-ity between the qPCR and end-point dilution estimates of the prevalence of mixed-virus ODVs likely reflected virus-specific differences in replication efficiency in cell culture and the higher in-fectivity of pseudotyped ODVs that were produced in coinfected parental cells. Bioassays on H. armigera, Spodoptera frugiperda and Mamestra brassicae larvae revealed that mixed-virus OBs were capable of infecting heterologous hosts, but relative potency values largely reflected the proportion of HearMNPV present in each mixed-virus preparation. The cooccluded mixtures were unstable in serial passage; HearSNPV rapidly dominated during passage in H. armigera whereas HearMNPV rapidly dominated during passage in the heterologous hosts. We conclude that mixed-virus coocclusion technology may be useful for producing precise mixtures of viruses with host range properties suitable for the control of complexes of lepidopteran pests in particular crops, although this requires validation by field testing., This research was funded by the Ministerio de Economía y Competitividad, Spain, project numbers AGL2014-57752-C2-1-R, AGL2017-83498-C2-1-R, PID2020-117062RB-C21 and Gobierno de Navarra grant numbers IIQ14065.RI1 and IIM14200.RI1. The APC was funded by Ministerio de Economía y Competitividad, Spain, project number PID2020-117062RB-C21. M.A. received a predoctoral scholarship from CSIC, Spain.

Nucleopolyhedroviruses (NPV, Baculoviridae) that infect lepidopteran pests have an established record as safe and effective biological insecticides. Here, we describe a new approach for the development of NPV-based insecticides. This technology takes advantage of the unique way in which these viruses are transmitted as collective infectious units, and the genotypic diversity present in natural virus populations. A ten-step procedure is described involving genotypic variant selection, mixing, coinfection and intraspecific coocclusion of variants within viral occlusion bodies. Using two examples, we demonstrate how this approach can be used to produce highly pathogenic virus preparations for pest control. As restricted host range limits the uptake of NPV-based insecticides, this technology has recently been adapted to produce custom-designed interspecific mixtures of viruses that can be applied to control complexes of lepidopteran pests on particular crops, as long as a shared host species is available for virus production. This approach to the development of NPV-based insecticides has the potential to be applied across a broad range of NPV-pest pathosystems., This review was funded by the Ministerio de Economía y Competitividad, Spain, project number AGL2017-83498-C2-1-R and previous projects AGL2014-57752-C2-1-R, AGL2011-30352-CO2-01, AGL2008-05456-C03-01, AGL2005-07909-CO3-01, and AGL2002-04320-C02-01.

Phylogenetic analyses suggest that Mamestra brassicae multiple nucleopolyhedrovirus (MbMNPV) and Helicoverpa armigera multiple nucleopolyhedrovirus (HearMNPV) may be strains of the same virus species. Most of the studies comparing their biological activities have been performed in their homologous hosts. A comparison of host range and stability in alternative hosts was performed. The host range of these viruses was compared using high concentrations of inoculum to inoculate second instars of six species of Lepidoptera. One semi-permissive host (Spodoptera littoralis) and one permissive host (S. exigua) were then selected and used to perform six serial passages involving a concentration corresponding to the ~25% lethal concentration for both viruses. Restriction endonuclease analysis showed fragment length polymorphisms in every hostvirus system studied. In S. littoralis, serial passage of MbMNPV resulted in decreased pathogenicity and an increase in speed-of-kill, whereas no significant changes were detected for HearMNPV with respect to the initial inoculum. In contrast, both viruses showed a similar trend in S. exigua. These results highlight the low genetic diversity and a high phenotypic stability of HearMNPV with respect to the original inoculum after six successive passages in both insect hosts. This study concludes that host-baculovirus interactions during serial passage are complex and the process of adaptation to a novel semi-permissive host is far from predictable., This research was funded by project AGL2014-57752-C2-1-R (Ministerio Español de Economía, Industria y Competitividad). I.M.B. received a studentship from Ministerio Español de Economía, Industria y Competitividad. I.B. received a Torres Quevedo postdoctoral fellowship from Ministerio de Ciencia, Innovación y Universidades.

Quantitative laboratory bioassay methods are required to evaluate the toxicity of novel insecticidal compounds for pest control and to determine the presence of resistance traits. We used a radioactive tracer based on P-32-ATP to estimate the volume of a droplet ingested by two dipteran pests: Ceratitis capitata (Tephritidae) and Drosophila suzukii (Drosophilidae). Using blue food dye it was possible to distinguish between individuals that ingested the solution from those that did not. The average volume ingested by C. capitata adults was 1.968 mu l. Females ingested a similar to 20% greater volume of solution than males. Adults of D. suzukii ingested an average of 0.879 mu l and females ingested similar to 30% greater volume than males. The droplet feeding method was validated using the naturally-derived insecticide spinosad as the active ingredient (a.i.). For C. capitata, the concentration-mortality response did not differ between the sexes or among three different batches of insects. Lethal dose values were calculated based on mean ingested volumes. For C. capitata LD50 values were 1.462 and 1.502 ng a.i./insect for males and females, respectively, equivalent to 0.274 and 0.271 ng a.i./mg for males and females, respectively, when sex-specific variation in body weight was considered. Using the same process for D. suzukii, the LD50 value was estimated at 2.927 ng a.i./insect, or 1.994 ng a.i./mg based on a mean body weight of 1.67 mg for both sexes together. We conclude that this technique could be readily employed for determination of the resistance status and dose-mortality responses of insecticidal compounds in many species of pestiferous Diptera., The study was funded by the Ministerio de Economía y Competitividad (MEC), Spain, projects AGL2014-57752-C2-1-R and AGL2017-83498-C2-1-R awarded to PC and TW. DDdL received a student scholarship from MEC project AGL2014-57752-C2-1-R. MV and IMM were funded by Gobierno de Navarra, Spain, project no. BTMOL-PI028. JC received a predoctoral scholarship from the Universidad Publica de Navarra, Spain.

Virus transmission and the prevalence of infection depend on multiple factors, including
the interaction with other viral pathogens infecting the same host. In this study,
active replication of an iflavirus, Spodoptera exigua iflavirus 1 (order Picornavirales)
was observed in the offspring of insects that survived following inoculation with a
pathogenic baculovirus, Spodoptera exigua multiple nucleopolyhedrovirus. Tracking
the origin of the iflavirus suggested the association of this virus with the occlusion
bodies of the baculovirus. Here we investigated the effect of this association on the
stability and infectivity of both viruses. A reduction in baculovirus pathogenicity,
without affecting its infectivity and productivity, was observed when associated with
the iflavirus. In contrast, viral association increased the infectivity of the iflavirus and
its resistance to ultraviolet radiation and high temperature, two of the main factors
affecting virus stability in the field. In addition, electron microscopy analysis revealed
the presence of particles resembling iflavirus virions inside the occlusion bodies of the
baculovirus, suggesting the possible co-occlusion of both viruses. Results reported here
are indicative of facultative phoresis of a virus and suggest that virus virus interactions
may be more common than currently recognized, and may be influential in the ecology
of baculovirus and host populations and in consequence in the use of baculoviruses as
biological insecticides., This study received financial support from the Spanish Ministry for Science and Technology (AGL2011-30352-C02 and AGL2014-57752-C2).

Genetic variation in baculoviruses is recognized as a key factor, not only due to the influence of such variation on pathogen transmission and virulence traits, but also because genetic variants can form the basis for novel biological insecticides. In this study, we examined the genetic variability of Chrysodeixis includens nucleopolyhedrovirus (ChinNPV) present in field isolates obtained from virus-killed larvae. Different ChinNPV strains were identified by restriction endonuclease analysis, from which genetic variants were isolated by plaque assay. Biological characterization studies were based on pathogenicity, median time to death (MTD), and viral occlusion body (OB) production (OBs/larva). Nine different isolates were obtained from eleven virus-killed larvae collected from fields of soybean in Mexico. An equimolar mixture of these isolates, named ChinNPV-Mex1, showed good insecticidal properties and yielded 23 genetic variants by plaque assay, one of which (ChinNPV-R) caused the highest mortality in second instars of C. includens. Five of these variants were selected: ChinNPV-F, ChinNPV-J, ChinNPV-K, ChinNPV-R, and ChinNPV-V. No differences in median time to death were found between them, while ChinNPV-F, ChinNPV-K, ChinNPV-R and ChinNPV-V were more productive than ChinNPV-J and the original mixture of field isolates ChinNPV-Mex1. These results demonstrate the high variability present in natural populations of this virus and support the use of these new genetic variants as promising active substances for baculovirus-based bioinsecticides., This research was funded by the Ministerio de Economía y Competitividad; grant number AGL2014-57752-C2-1-R. E.A. received an UPNA studentship and I.B. received a Torres-Quevedo grant.

Chrysodeixis chalcites, an important pest of banana crops on the Canary Islands, is usually
controlled by chemical insecticides. The present study aimed to evaluate the efficacy of the
most prevalent isolate of the Chrysodeixis chalcites nucleopolyhedrovirus (ChchNPV, Baculoviridae)
as a biological insecticide. Overall the prevalence of ChchNPV infection in C. chalcites
populations was 2.3% (103 infected larvae out of 4,438 sampled), but varied from
0±4.8% on Tenerife and was usually low (0±2%) on the other islands. On Tenerife, infected
larvae were present at 11 out of 17 plantations sampled. The prevalence of infection in larvae
on bananas grown under greenhouse structures was significantly higher (3%) than in
open-field sites (1.4%). The ChchNPV-TF1 isolate was the most abundant and widespread
of four genetic variants of the virus. Application of 1.0x109 viral occlusion bodies (OBs)/l of
ChchNPV-TF1 significantly reduced C. chalcites foliar damage in young banana plants as
did commonly used pesticides, both in greenhouse and open-field sites. The insecticidal efficacy
of ChchNPV-TF1 was similar to that of indoxacarb and a Bacillus thuringiensis (Bt)-
based insecticide in one year of trials and similar to Bt in the following year of trails in greenhouse
and field crops. However, larvae collected at different time intervals following virus
treatments and reared in the laboratory experienced 2±7 fold more mortality than insects
from conventional insecticide treatments. This suggests that the acquisition of lethal dose
occurred over an extended period (up to 7 days) compared to a brief peak in larvae on plants
treated with conventional insecticides. These results should prove useful for the registration
of a ChchNPV-based insecticide for integrated management of this pest in banana crops on
the Canary Islands., This study received financial support from the National Institute of the Agricultural Research from the Spanish Ministry of Economy, Industry and Competitiveness project numbers RTA2010-00016-C02 and RTA2013-00114-C02 and from the Spanish Ministry for Science and Technology AGL2014-57752-C02-1R). E. G. F. received a student scholarship from National Institute of the Agricultural Research.