New Paralogs of the Heliothis virescens ABCC2 Transporter as Potential Receptors for Bt Cry1A Proteins.

The ATP-binding cassette (ABC) transporters are a superfamily of membrane proteins. These active transporters are involved in the export of different substances such as xenobiotics. ABC transporters from subfamily C (ABCC) have also been described as functional receptors for different insecticidal proteins from Bacillus thuringiensis (Bt) in several lepidopteran species. Numerous studies have characterized the relationship between the ABCC2 transporter and Bt Cry1 proteins. Although other ABCC transporters sharing structural and functional similarities have been described, little is known of their role in the mode of action of Bt proteins. For Heliothis virescens, only the ABCC2 transporter and its interaction with Cry1A proteins have been studied to date. Here, we have searched for paralogs to the ABCC2 gene in H. virescens, and identified two new ABC transporter genes: HvABCC3 and HvABCC4. Furthermore, we have characterized their gene expression in the midgut and their protein topology, and compared them with that of ABCC2. Finally, we discuss their possible interaction with Bt proteins by performing protein docking analysis.

Mutations associated with pyrethroid resistance in Varroa mite, a parasite of honey bees, are widespread across the United States.

BACKGROUND: Managed honey bees are key pollinators of many crops and play an essential role in the United States food production. For more than ten years, beekeepers in the United States have been reporting high rates of colony losses. One of the drivers of these losses is the parasitic mite Varroa destructor. Maintaining healthy honey bee colonies in the United States is dependent on a successful control of this mite. The pyrethroid tau-fluvalinate (Apistan®) was among the first synthetic varroacides registered in the United States. With over 20 years of use, mites resistant to Apistan® have emerged, and so it is unsurprising that treatment failures have been reported. Resistance to tau-fluvalinate in US mite populations is associated with point mutations at position 925 of the voltage-gated sodium channel. RESULTS: Here, we have generated a distribution map of pyrethroid resistance alleles in Varroa samples collected from US apiaries in 2016 and 2017, using a high throughput allelic discrimination assay based on TaqMan®. Our results evidence that knockdown resistance (kdr)-type mutations are widely distributed in Varroa populations across the country showing high variability among apiaries. We used these data to predict the phenotype of the mites in the case of treatments with pyrethroids. CONCLUSION: We highlight the relevance of monitoring the resistance in mite populations to achieve an efficient control of this pest. We also put forward the benefits of implementing this methodology to provide data for designing pest management programs aiming to control Varroa. © 2021 Society of Chemical Industry.

Reduced Membrane-Bound Alkaline Phosphatase Does Not Affect Binding of Vip3Aa in a Heliothis virescens Resistant Colony.

The Vip3Aa insecticidal protein from Bacillus thuringiensis (Bt) is produced by specific transgenic corn and cotton varieties for efficient control of target lepidopteran pests. The main threat to this technology is the evolution of resistance in targeted insect pests and understanding the mechanistic basis of resistance is crucial to deploy the most appropriate strategies for resistance management. In this work, we tested whether alteration of membrane receptors in the insect midgut might explain the >2000-fold Vip3Aa resistance phenotype in a laboratory-selected colony of Heliothis virescens (Vip-Sel). Binding of 125I-labeled Vip3Aa to brush border membrane vesicles (BBMV) from 3rd instar larvae from Vip-Sel was not significantly different from binding in the reference susceptible colony. Interestingly, BBMV from Vip-Sel larvae showed dramatically reduced levels of membrane-bound alkaline phosphatase (mALP) activity, which was further confirmed by a strong downregulation of the membrane-bound alkaline phosphatase 1 (HvmALP1) gene. However, the involvement of HvmALP1 as a receptor for the Vip3Aa protein was not supported by results from ligand blotting and viability assays with insect cells expressing HvmALP1.

Identification of new viral variants specific to the honey bee mite Varroa destructor.

Large-scale colony losses among managed Western honey bees have become a serious threat to the beekeeping industry in the last decade. Multiple factors contribute to these losses, but the impact of Varroa destructor parasitism is by far the most important, along with the contribution of some pathogenic viruses vectored by the mite. So far, more than 20 viruses have been identified infecting the honey bee, most of them RNA viruses. They may be maintained either as covert infections or causing severe symptomatic infections, compromising the viability of the colony. In silico analysis of available transcriptomic data obtained from mites collected in the USA and Europe, as well as additional investigation with new samples collected locally, allowed the description of three RNA viruses, two of them variants of the previously described VDV-2 and VDV-3 and the other a new species reported here for the first time. Our results showed that these viruses were widespread among samples and that they were present in the mites as well as in the bees but with differences in the relative abundance and prevalence. However, we have obtained strong evidence showing that these three viruses were able to replicate in the mite, but not in the bee, suggesting that they are selectively infecting the mite. This opens the door to future applications that may help controlling the mite through biological control approaches.

Novel RNA viruses producing simultaneous covert infections in Ceratitis capitata. Correlations between viral titers and host fitness, and implications for SIT programs.

The Mediterranean fruit fly (medfly), Ceratitis capitata is a highly polyphagous pest, which infests multiple species of fruits and vegetables worldwide. In addition to the traditional control with chemical insecticides, sterile insect technique (SIT) has been implemented in integrated programs worldwide, and has become an essential measure for the control of this pest. A key issue for SIT is to release sterile males that are sufficiently competitive with males from the wild population. Using sequence information available in public databases, three novel picornaviruses infecting medflies were discovered and named as C. capitata iflavirus 1 and 2 (CcaIV1 and CcaIV2), and C. capitata noravirus (CcaNV). Additional analyses have revealed the presence of CcaIV2 and CcaNV covertly infecting most of the medfly strains used in the different SIT programs around the world, as well as in field captures in the east of Spain. High viral titers of CcaNV were associated with a reduction in the lifespan of males released to the field for the control of this pest, suggesting the possibility that CcaNV may impair the fitness of sterile flies produced by SIT programs.

In search of pathogens: transcriptome-based identification of viral sequences from the pine processionary moth (Thaumetopoea pityocampa).

Thaumetopoea pityocampa (pine processionary moth) is one of the most important pine pests in the forests of Mediterranean countries, Central Europe, the Middle East and North Africa. Apart from causing significant damage to pinewoods, T. pityocampa occurrence is also an issue for public and animal health, as it is responsible for dermatological reactions in humans and animals by contact with its irritating hairs. High throughput sequencing technologies have allowed the fast and cost-effective generation of genetic information of interest to understand different biological aspects of non-model organisms as well as the identification of potential pathogens. Using these technologies, we have obtained and characterized the transcriptome of T. pityocampa larvae collected in 12 different geographical locations in Turkey. cDNA libraries for Illumina sequencing were prepared from four larval tissues, head, gut, fat body and integument. By pooling the sequences from Illumina platform with those previously published using the Roche 454-FLX and Sanger methods we generated the largest reference transcriptome of T. pityocampa. In addition, this study has also allowed identification of possible viral pathogens with potential application in future biocontrol strategies.

Simultaneous occurrence of covert infections with small RNA viruses in the lepidopteran Spodoptera exigua.

Viral covert infections in invertebrates have been traditionally attributed to sublethal infections that were not able to establish an acute infection. Recent studies are revealing that, although true for some viruses, other viruses may follow the strategy of establishing covert or persistent infections without producing the death of the host. Recently, and due to the revolution in the sequencing technologies, a large number of viruses causing covert infections in all type of hosts have been identified. The beet armyworm, Spodoptera exigua (Lepidoptera: Noctuidae) is a worldwide pest that causes significant losses to agricultural and ornamental plant industries. In a previous project we used NGS to obtain a comprehensive transcriptome of the larval stage, revealing the presence of an important number of unigenes belonging to novel RNA viruses, most of them from the order Picornavirales. In order to characterize S. exigua viral complex, in this work we have completed the genomic sequences of two picorna-like viruses, and compared them to a SeIV1, a member of Iflaviridae previously described by our group. We performed additional studies to determine virus morphology, horizontal transmission, tissue and life stage distribution and abundance in the hosts. We discuss the role of virus persistent infections on insect populations.

Genome sequence of SeIV-1, a novel virus from the Iflaviridae family infective to Spodoptera exigua.

Analysis of the transcriptome of Spodoptera exigua larvae revealed the presence of several ESTs with homology to virus of the order Picornavirales and with the highest similarity to Infectious flacherie virus (Iflaviridae) that infects Bombyx mori larvae. Iflaviridae is a recently defined family of insect-infecting viruses that consist of positive single strand RNA genomes translated into a single polyprotein of around 3000 amino acids long. Using the sequence information derived from the obtained ESTs, we have completed the genomic sequence of this virus. The novel S. exigua iflavirus (SeIV-1) has a genome of 10.3 kb and codes for a 3222 aa polyprotein. Expression analysis has revealed the presence of the virus in all tissues tested and insect stages, being more abundant in the midgut of the larvae. High infectivity of this virus against S. exigua has been demonstrated after observing the presence of this virus in different colonies that were reared in the same chamber with the virus-infected colony, despite no evidence of pathological effects. Further study of viral covert infections of SeIV-1 could lead to a better understanding of its pathological effect as well as any possible interaction with other microbial pathogens used for the control of this pest.