BUSCADOR RECOLECTA

Los grandes costes asociados a la producción masiva de baculovirus hacen necesaria la búsqueda de productos sinérgicos que reduzcan la cantidad de materia activa. En la presente tesis, se expresó y produjo un factor sinérgico con gran potencial para formulaciones basadas en baculovirus utilizando un sistema de expresión de baculovirus. El principal logro del trabajo es que la producción in vivo de sobrenadante con enhancinas utilizando sistemas de expresión basados en baculovirus puede utilizarse para mejorar la eficacia de los insecticidas biológicos contra plagas de lepidópteros, reduciendo la materia activa de los bioinsecticidas y haciéndolos competitivos comercialmente en comparación con los productos químicos., The increased costs associated with baculovirus mass-production urge the search for synergistic products that reduce the amount of active matter. In the present thesis, a synergistic factor with great potential for baculovirus-based formulations was expressed and produced using a baculovirus expression system. The main achievement of the present thesis is that the in vivo production of solubilized enhancins using baculovirus-based expression systems can be used to improve the efficacy of biological insecticides against lepidopteran pests, reducing the active matter of bioinsecticides and making them commercially competitive with chemicals., Beca predoctoral de la Universidad Pública de Navarra y posteriormente obtuvo contratos como ayudante de proyecto a cargo del proyecto AGL2017-83498-C2-1-R., Programa de Doctorado en Biotecnología (RD 99/2011), Bioteknologiako Doktoretza Programa (ED 99/2011)

Outbreaks of Anticarsia gemmatalis (Hübner, 1818) (Lepidoptera: Erebidae), a major pest of soybean, can be controlled below economic thresholds with methods that do not involve the application of synthetic insecticides. Formulations based on natural isolates of the Anticarsia gemmatalis multiple nucleopolyhedrovirus (AgMNPV) (Baculoviridae: Alphabaculovirus) played a significant role in integrated pest management programs in the early 2000s, but a new generation of chemical insecticides and transgenic soybean have displaced AgMNPV-based products over the past decade. However, the marked genotypic variability present among and within alphabaculovirus isolates suggests that highly insecticidal genotypic variants can be isolated and used to reduce virus production costs or overcome isolate-dependent host resistance. This study aimed to select novel variants of AgMNPV with suitable insecticidal traits that could complement the existing AgMNPV active ingredients. Three distinct AgMNPV isolates were compared using their restriction endonuclease profile and in terms of their occlusion body (OB) pathogenicity. One isolate was selected (AgABB51) from which eighteen genotypic variants were plaque purified and characterized in terms of their insecticidal properties. The five most pathogenic variants varied in OB pathogenicity, although none of them was faster-killing or had higher OB production characteristics than the wild-type isolate. We conclude that the AgABB51 wild-type isolates appear to be genotypically structured for fast speed of kill and high OB production, both of which would favor horizontal transmission. Interactions among the component variants are likely to influence this insecticidal phenotype., This research was funded by the Ministerio de Ciencia e Innovacion, grant number AGL2017-83498-C2-1-R. A.P.-J. received a predoctoral grant PRE2018-086829 from the Agencia Estatal de Investigación (Ministerio de Ciencia e Innovación) of the Spanish Government.

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.

The mode of action underlying the insecticidal activity of the Bacillus thuringiensis (Bt) binary pesticidal protein Vpa1/Vpa2 is uncertain. In this study, three recombinant baculoviruses were constructed using Bac-to-Bac technology to express Vpa2Ac1 and two novel Vpa2-like genes, Vpa2-like1 and Vpa2-like2, under the baculovirus p10 promoter in transfected Sf9 cells. Pairwise amino acid analyses revealed a higher percentage of identity and a lower number of gaps between Vpa2Ac1 and Vpa2-like2 than to Vpa2-like1. Moreover, Vpa2-like1 lacked the conserved Ser-Thr-Ser motif, involved in NAD binding, and the (F/Y)xx(Q/E)xE consensus sequence, characteristic of the ARTT toxin family involved in actin polymerization. Vpa2Ac1, Vpa2-like1 and Vpa2-like2 transcripts and proteins were detected in Sf9 culture cells, but the signals of Vpa2Ac1 and Vpa2-like2 were weak and decreased over time. Sf9 cells infected by a recombinant bacmid expressing Vpa2-like1 showed typical circular morphology and produced viral occlusion bodies (OBs) at the same level as the control virus. However, expression of Vpa2Ac1 and Vpa2-like2 induced cell polarization, similar to that produced by the microfilament-destabilizing agent cytochalasin D and OBs were not produced. The presence of filament disrupting agents, such as nicotinamide and nocodazole, during transfection prevented cell polarization and OB production was observed. We conclude that Vpa2Ac1 and Vpa2-like2 proteins likely possess ADP-ribosyltransferase activity that modulated actin polarization, whereas Vpa2-like1 is not a typical Vpa2 protein. Vpa2-like2 has now been designated Vpa2Ca1 (accession number AAO86513) by the Bacillus thuringiensis delta-endotoxin nomenclature committee., This research was funded by the Spanish Ministry project AGL2017-83498-C2-1-R.

Baculoviruses (BV) are highly effective against lepidopteran pests of economic importance such as Spodoptera exigua. The combined use of entomopathogens and macrobiological control agents requires the study of their relationships. Laboratory bioassays were developed to evaluate the interactions between the multiple nucleopolyhedroviruses of S. exigua (SeMNPV) and Autographa californica (AcMNPV), and the predator Chrysoperla carnea. The microscopic examination of predator’s excreta (larval drops and meconia) after the ingestion of BV-infected S. exigua revealed the presence of viral occlusion bodies (OBs). The reinfection of S. exigua larvae with BVs-contaminated excreta by using OBs water suspensions or by direct application both yielded high mortality values but different speed-of-kill results. Meconia killed before in suspensions due to their higher viral load and larval excretion drops did so in direct application due to their liquid nature and their easiness of consumption. The prey-mediated ingestion of SeMNPV and AcMNPV triggered slight effects in C. carnea, which were probably derived from the food nutritional quality. Chrysoperla carnea larvae did not discriminate between healthy and BV-infected S. exigua, while a preference was shown for S. exigua (healthy or infected) vs. Macrosiphum euphorbiae. Our findings present C. carnea, and particularly its larvae, as a promissory candidate for BV dispersion in the field., This research was funded by Spanish Ministry of Science, Iinnovation and Universitites, grant number AGL2017-83498-C2-1-R and AGL2017-83498-C2-R awarded to Primitivo Caballero and Pilar Medina, respectively. O. G. Gutiérrez-Cárdenas acknowledges the Government of Mexico and the National Council of Science and Technology (CONACYT) for the Ph.D. research grant (no. of scholarship 717840-2018).

The joint use of baculoviruses and synthetic insecticides for integrated pest management requires the study of the additive, synergistic or antagonistic effects among them on pest mortality. Droplet bioassays were conducted with Autographa californica multiple nucleopolyhedrovirus (AcMNPV), Spodoptera littoralis nucleopolyhedrovirus (SpliNPV) and seven insecticides (azadirachtin, Bacillus thuringiensis, cyantraniliprole, emamectin, metaflumizone, methoxyfenozide and spinetoram) on Spodoptera exigua and Spodoptera littoralis. The lethal concentrations LC50 and LC95 were calculated through probit regressions. Then, the sequential feeding of insecticides and nucleopolyhedroviruses was studied. Larvae were provided with the LC50 of one insecticide, followed by the LC50 of one nucleopolyhedrovirus 24 h later. The inverse order was also conducted. The insecticide LC50 and LC95 were higher for S. littoralis than for S. exigua. AcMNPV showed greater toxicity on S. exigua than SpliNPV on S. littoralis. Emamectin showed synergy with AcMNPV when the chemical was applied first, and metaflumizone and AcMNPV were synergistic regardless of the order of application, both from the first day of evaluation. SpliNPV was synergistic with azadirachtin and emamectin when it was applied first, but synergy was reached after 12–13 days. Excellent control is possible with the LC50 of azadirachtin, emamectin and metaflumizone in combination with nucleopolyhedroviruses, and merits further study as a means of controlling lepidopteran pests., This research was funded by Ministerio de Ciencia e Innovación, projects AGL2017-83498-C2-1-R and AGL2017-83498-C2-2-R awarded to P.C. and P.M., respectively, and postdoctoral fellowship FJCI-2016-28443 awarded to B.D.; Universidad Politécnica de Madrid, project PINV18XFWLGK24S2US6D awarded to B.D.; and Universidad Pública de Navarra, studentship awarded to E.A.

Incluye material complementario, Enhancins are metalloproteinases that facilitate baculovirus infection in the insect midgut.
They are more prevalent in granuloviruses (GVs), constituting up to 5% of the proteins of viral
occlusion bodies (OBs). In nucleopolyhedroviruses (NPVs), in contrast, they are present in the
envelope of the occlusion-derived virions (ODV). In the present study, we constructed a recombinant
Autographa californica NPV (AcMNPV) that expressed the Trichoplusia ni GV (TnGV) enhancin 3
(En3), with the aim of increasing the presence of enhancin in the OBs or ODVs. En3 was successfully
produced but did not localize to the OBs or the ODVs and accumulated in the soluble fraction of
infected cells. As a result, increased OB pathogenicity was observed when OBs were administered
in mixtures with the soluble fraction of infected cells. The mixture of OBs and the soluble fraction
of Sf9 cells infected with BacPhEn3 recombinant virus was ~3- and ~4.7-fold more pathogenic than
BacPh control OBs in the second and fourth instars of Spodoptera exigua, respectively. In contrast,
when purified, recombinant BacPhEn3 OBs were as pathogenic as control BacPh OBs. The expression
of En3 in the soluble fraction of insect cells may find applications in the development of virus-based
insecticides with increased efficacy., This study received financial support from the Spanish Ministry project AGL2017-83498-C2-1-R. Bioinsectis SL funded the activities of ABF.

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.

The mechanisms generating variability in viruses are diverse. Variability allows baculoviruses
to evolve with their host and with changes in their environment. We examined the role of
one genetic variant of Chrysodeixis includens nucleopolyhedrovirus (ChinNPV) and its contribution
to the variability of the virus under laboratory conditions. A mixture of natural isolates (ChinNPVMex1)
contained two genetic variants that dominated over other variants in individual larvae that
consumed high (ChinNPV-K) and low (ChinNPV-E) concentrations of inoculum. Studies on the
ChinNPV-K variant indicated that it was capable of generating novel variation in a concentrationdependent
manner. In cell culture, cells inoculated with high concentrations of ChinNPV-K produced
OBs with the ChinNPV-K REN profile, whereas a high diversity of ChinNPV variants was recovered
following plaque purification of low concentrations of ChinNPV-K virion inoculum. Interestingly,
the ChinNPV-K variant could not be recovered from plaques derived from low concentration inocula
originating from budded virions or occlusion-derived virions of ChinNPV-K. Genome sequencing
revealed marked differences between ChinNPV-K and ChinNPV-E, with high variation in the
ChinNPV-K genome, mostly due to single nucleotide polymorphisms. We conclude that ChinNPV-K
is an unstable genetic variant that is responsible for generating much of the detected variability in
the natural ChinNPV isolates used in this study., This research was funded by the Ministerio de Economía y Competitividad; grant number
AGL2017-83498-C2-1-R. E.A. received an UPNA studentship. I.B. received a Torres-Quevedo grant
and was funded by Bioinsectis SL during the preparation of this manuscript.