Open Access
The role of Chrysoperla carnea (Steph.) (neuroptera: Chrysopidae) as a potential dispersive agent of noctuid baculoviruses
(MDPI, 2020) Gutiérrez Cárdenas, Oscar Giovanni; Adán, Ángeles; Beperet Arive, Inés; Medina, Pilar; Caballero Murillo, Primitivo; Garzón, Agustín; Agronomía, Biotecnología y Alimentación; Agronomia, Bioteknologia eta Elikadura
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.
Open Access
Baculovirus expression and functional analysis of Vpa2 proteins from Bacillus thuringiensis
(MDPI, 2020) Simón de Goñi, Oihane; Palma Dovis, Leopoldo; Fernández González, Ana Beatriz; Williams, Trevor; Caballero Murillo, Primitivo; Institute for Multidisciplinary Research in Applied Biology - IMAB
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.
Open Access
Gender-mediated differences in vertical transmission of a nucleopolyhedrovirus
(Public Library of Science, 2013) Virto Garayoa, Cristina; Zárate Chaves, Carlos Andrés; López Ferber, Miguel; Murillo Pérez, Rosa; Caballero Murillo, Primitivo; Williams, Trevor; Nekazaritza Ekoizpena; Producción Agraria; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua
With the development of sensitive molecular techniques for detection of low levels of asymptomatic pathogens, it becoming clear that vertical transmission is a common feature of some insect pathogenic viruses, and likely to be essential to virus survival when opportunities for horizontal transmission are unfavorable. Vertical transmission of Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV) is common in natural populations of S. exigua. To assess whether gender affected transgenerational virus transmission, four mating group treatments were performed using healthy and sublethally infected insects: i) healthy males (H=)6healthy females (HR); ii) infected males (I=)6healthy females (HR); iii) healthy males (H=)6infected females (IR) and iv) infected males (I=)6infected females (IR). Experimental adults and their offspring were analyzed by qPCR to determine the prevalence of infection. Both males and females were able to transmit the infection to the next generation, although female-mediated transmission resulted in a higher prevalence of infected offspring. Malemediated venereal transmission was half as efficient as maternally-mediated transmission. Egg surface decontamination studies indicated that the main route of transmission is likely transovarial rather than transovum. Both male and female offspring were infected by their parents in similar proportions. Incorporating vertically-transmitted genotypes into virusbased insecticides could provide moderate levels of transgenerational pest control, thereby extending the periods between bioinsecticide applications.
Open Access
Stability of a Spodoptera frugiperda nucleopolyhedrovirus deletion recombinant during serial passage in insects
(American Society for Microbiology, 2009) Simón de Goñi, Oihane; Williams, Trevor; Possee, Robert D.; López Ferber, Miguel; Caballero Murillo, Primitivo; Nekazaritza Ekoizpena; Producción Agraria; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua
The stabilities of the Spodoptera frugiperda multiple nucleopolyhedrovirus (SfMNPV) complete genome bacmid (Sfbac) and a deletion recombinant (Sf29null) in which the Sf29 gene was replaced by a kanamycin resistance cassette were determined during sequential rounds of per os infection in insect larvae. The Sf29 gene is a viral factor that determines the number of virions in occlusion bodies (OBs). The Sf29null bacmid virus was able to recover the Sf29 gene during passage. After the third passage (P3) of Sf29null bacmid OBs, the population was observed to reach an equilibrium involving a mixture of those with a kanamycin resistance cassette and those with the Sf29 gene. The biological activity of Sf29null bacmid OBs at P3 was similar to that of Sfbac OBs. The recovered gene in the Sf29null virus was 98 to 100% homologous to the Sf29 genes of different SfMNPV genotypes. Reverse transcription-PCR analysis of uninoculated S. frugiperda larvae confirmed the expression of the SfMNPV ie-0 and Sf29 genes, indicating that the insect colony harbors a covert SfMNPV infection. Additionally, the nonessential bacterial artificial chromosome vector was spontaneously deleted from both viral genomes upon passage in insects.
Open Access
Bacillus thuringiensis Cyt proteins as enablers of activity of Cry and Tpp toxins against Aedes albopictus
(2023) Lai, Liliana; Villanueva, Maite; Muruzabal Galarza, Ane; Fernández González, Ana Beatriz; Unzue Pozas, Argiñe; Toledo Arana, Alejandro; Caballero Murillo, Primitivo; Caballero Sánchez, Carlos; Institute for Multidisciplinary Research in Applied Biology - IMAB
Aedes albopictus is a species of mosquito, originally from Southeast Asia, that belongs to the Culicidae family and the Dipteran insect order. The distribution of this vector has rapidly changed over the past decade, making most of the temperate territories in the world vulnerable to important human vector-borne diseases such as dengue, yellow fever, zika or chikungunya. Bacillus thuringiensis var. israeliensis (Bti)-based insecticides represent a realistic alternative to the most common synthetic insecticides for the control of mosquito larvae. However, several studies have revealed emerging resistances to the major Bti Crystal proteins such as Cry4Aa, Cry4Ba and Cry11Aa, making the finding of new toxins necessary to diminish the exposure to the same toxicity factors overtime. Here, we characterized the individual activity of Cyt1Aa, Cry4Aa, Cry4Ba and Cry11Aa against A. albopictus and found a new protein, Cyt1A-like, that increases the activity of Cry11Aa more than 20-fold. Additionally, we demonstrated that Cyt1A-like facilitates the activity three new Bti toxins: Cry53-like, Cry56A-like and Tpp36-like. All in all, these results provide alternatives to the currently available Bti products for the control of mosquito populations and position Cyt proteins as enablers of activity for otherwise non-active crystal proteins.
Open Access
Analysis of a naturally-occurring deletion mutant of Spodoptera frugiperda multiple nucleopolyhedrovirus reveals sf58 as a new per os infectivity factor of lepidopteran-infecting baculoviruses
(Elsevier, 2012-10-21) Simón de Goñi, Oihane; Palma Dovis, Leopoldo; Williams, Trevor; López Ferber, Miguel; Caballero Murillo, Primitivo; Producción Agraria; Nekazaritza Ekoizpena; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua; Gobierno de Navarra / Nafarroako Gobernua
The Nicaraguan population of Spodoptera frugiperda multiple nucleopolyhedrovirus, SfMNPV-NIC, is structured as a mixture of nine genotypes (A–I). Occlusion bodies (OBs) of SfMNPV-C, -D and -G pure genotypes are incapable of oral transmission; a phenotype which in SfMNPV-C and -D is due to the absence of pif1 and pif2 genes. The complete sequence of the SfMNPV-G genome was determined to identify possible factors involved in this phenotype. Deletions of 4860 bp (22,366–27,225) and 60 bp (119,759–119,818) were observed in SfMNPV-G genome compared with that of the predominant complete genotype SfMNPV-B (132,954 bp). However no genes homologous to previously described per os infectivity factors were located within the deleted sequences. Significant differences were detected in the nucleotide sequence in sf58 gene (unknown function) that produced changes in the amino acid sequence and the predicted secondary structure of the corresponding protein. This gene is conserved only in lepidopteran baculoviruses (alpha- and betabaculoviruses). To determine the role of sf58 in peroral infectivity a deletion mutant was constructed using bacmid technology. OBs of the deletion mutant (Sf58null) were not orally infectious for S. frugiperda larvae, whereas Sf58null rescue virus OBs recovered oral infectivity. Sf58null DNA and occlusion derived virions (ODVs) were as infective as SfMNPV bacmid DNA and ODVs in intrahemocelically infected larvae or cell culture, indicating that defects in ODV or OB morphogenesis were not involved in the loss of peroral infectivity. Addition of optical brightener or the presence of the orally infectious SfMNPV-B OBs in mixtures with SfMNPV-G OBs did not recover Sf58null OB infectivity. According to these results sf58 is a new per os infectivity factor present only in lepidopteran baculoviruses.
Open Access
Functional importance of deletion mutant genotypes in an insect nucleopolyhedrovirus population
(American Society for Microbiology, 2005) Simón de Goñi, Oihane; Williams, Trevor; López Ferber, Miguel; Caballero Murillo, Primitivo; Producción Agraria; Nekazaritza Ekoizpena
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.
Open Access
Population genetic structure determine the virulence and transmissibility of Spodoptera frugiperda multiple necleopolyhedrovirus
(Elsevier, 2007-12-28) Simón de Goñi, Oihane; Williams, Trevor; López Ferber, Miguel; Taulemesse, Jean-Marie; Caballero Murillo, Primitivo; Producción Agraria; Nekazaritza Ekoizpena; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua
A Nicaraguan isolate of Spodoptera frugiperda multiple nucleopolyhedrovirus (SfNIC) survives as a complex mixture of genotypes (named A to I). The speed of kill, time-mortality distribution, and occlusion body (OB) production of single genotypes (A, B and F) and co-occluded mixtures of genotypes, in a 75% + 25% ratio, were compared to determine the contribution of each genotype to the transmissibility of the viral population. Pure genotypes differed markedly in their speed of kill in second instar S. frugiperda. The speed of kill of SfNIC was attenuated compared to that of the dominant genotype B, indicating that interactions involving two or more genotypes likely determine host killing traits in the virus population. Genotypes A, F and defective genotype C, had no significant effects on the distribution of insect deaths over time when present as minority components in mixtures comprising 75% of genotype B. Similarly, the mortality pattern over time of insects infected by genotype F, the fastest-killing genotype tested, was not affected by the presence of genotypes A or C. Semi-quantitative PCR studies indicated that the genetic composition did not differ significantly between SfNIC-infected insects that died soon (67 h) or late (139 h) after inoculation, suggesting that stability in genotypic composition is important for virus survival. Median OB production per insect was correlated with mean time to death so that attenuated speed of kill of SfNIC resulted in high OB yields. We conclude that (i) minority genotypes play a functional role in determining the timing of mortality of infected hosts and (ii) the genotypic structure of the virus population is stably maintained to maximize the likelihood of survival.
Open Access
Insecticidal traits of variants in a genotypically diverse natural isolate of anticarsia gemmatalis multiple nucleopolyhedrovirus (AgMNPV)
(MDPI, 2023) Parras-Jurado, Ana; Muñoz Labiano, Delia; Beperet Arive, Inés; Williams, Trevor; Caballero Murillo, Primitivo; Institute for Multidisciplinary Research in Applied Biology - IMAB
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.
Open Access
Domain shuffling between Vip3Aa and Vip3Ca: chimera stability and insecticidal activity against European, American, African, and Asian pests
(MDPI, 2020) Gomis Cebolla, Joaquín; Santos, Rafael Ferreira dos; Wang, Yueqin; Caballero Sánchez, Javier; Caballero Murillo, Primitivo; He, Kanglai; Jurat Fuentes, Juan Luis; Ferré, Juan; Institute for Multidisciplinary Research in Applied Biology - IMAB
The bacterium Bacillus thuringiensis produces insecticidal Vip3 proteins during the vegetative growth phase with activity against several lepidopteran pests. To date, three different Vip3 protein families have been identified based on sequence identity: Vip3A, Vip3B, and Vip3C. In this study, we report the construction of chimeras by exchanging domains between Vip3Aa and Vip3Ca, two proteins with marked specificity differences against lepidopteran pests. We found that some domain combinations made proteins insoluble or prone to degradation by trypsin as most abundant insect gut protease. The soluble and trypsin-stable chimeras, along with the parental proteins Vip3Aa and Vip3Ca, were tested against lepidopteran pests from different continents: Spodoptera exigua, Spodoptera littoralis, Spodoptera frugiperda, Helicoverpa armigera, Mamestra brassicae, Anticarsia gemmatalis, and Ostrinia furnacalis. The exchange of the Nt domain (188 N-terminal amino acids) had little effect on the stability and toxicity (equal or slightly lower) of the resulting chimeric protein against all insects except for S. frugiperda, for which the chimera with the Nt domain from Vip3Aa and the rest of the protein from Vip3Ca showed a significant increase in toxicity compared to the parental Vip3Ca. Chimeras with the C-terminal domain from Vip3Aa (from amino acid 510 of Vip3Aa to the Ct) with the central domain of Vip3Ca (amino acids 189–509 based on the Vip3Aa sequence) made proteins that could not be solubilized. Finally, the chimera including the Ct domain of Vip3Ca and the Nt and central domain from Vip3Aa was unstable. Importantly, an insect species tolerant to Vip3Aa but susceptible to Vip3Ca, such as Ostrinia furnacalis, was also susceptible to chimeras maintaining the Ct domain from Vip3Ca, in agreement with the hypothesis that the Ct region of the protein is the one conferring specificity to Vip3 proteins.