The distribution and characteristic of two transposable elements in the genome of Cydia pomonella granulovirus and codling moth.

Baculoviruses are capable to acquire insect host transposable elements (TEs) in their genomes and are hypothesized as possible vectors of insect transposons between Lepidopteran species. Here, we investigated the host origin of two TEs, namely the Tc1/mariner-like element TCp3.2 and a 0.7 kbp insertion sequence (IS07), found in the genome of different isolates of Cydia pomonella granulovirus (CpGV), a member of the Betabaculovirus genus. The sequences of both TEs were searched for in the full genome sequence database of codling moth (CM, Cydia pomonella L.). A total of eleven TCp3.2 TE copies and 76 copies of the IS07 fragments were identified in the CM genome. These TEs were distributed over the 22 autosomes and the Z chromosome (chr1) of CM, except chr6, chr12, chr16, chr23, chr27 and the W chromosome (chr29). TCp3.2 copies with two transposase genes in opposite direction, representing a novel feature, were identified on chr10 and chr18. The TCp3.2 transposase was characterized by DD41D motif of classic Tc1/mariner transposons, consisting of DNA-binding domain, catalytic domain and nuclear localization signal (NLS). Transcription analyses of uninfected and CpGV-infected CM larvae suggested a doubling of the TCp3.2 transposase transcription rate in virus infected larvae. Furthermore, IS07 insertion into the CpGV genome apparently added new transcription initiation sites to the viral genome. The global analysis of the distribution of two TEs in the genome of CM addressed the influx of mobile TEs from CM to CpGV, a genetic process that contributes to the population diversity of baculoviruses.

First Evidence of CpGV Resistance of Codling Moth in the USA.

Codling moth (Cydia pomonella L.) is a very important pest in apple, pear, and walnut orchards worldwide, including the USA. Cydia pomonella granulovirus (CpGV) is used to control codling moth in organic and conventional production. Due to increasing codling moth infestations from organic apple orchards in Washington State, USA, five codling moth colonies (WA1-WA5) were screened for their susceptibility relative to the isolate GV-0001, the main active ingredient of Cyd-X®, using a discriminating concentration of 6 × 104 OB/mL. Compared to a susceptible laboratory colony, the observed results indicated that GV-0001 lacked efficacy against codling moth colony WA3. It was confirmed that WA3 was the first case of codling moth resistance to CpGV in the USA. Further testing of WA3 was performed on a range of CpGV isolates and a lack of efficacy was observed against additional isolates. However, three newly developed CpGV preparations can efficiently infect larvae from the resistant colony WA3. Our results suggest that there is an urgent need to monitor the situation in the USA, aiming to prevent the emergence or spread of additional codling moth populations with CpGV resistance. Strategies to sustain the efficacy of codling moth control using novel CpGV formulations need to be developed.

Correction: Fan et al. First Evidence of CpGV Resistance of Codling Moth in the USA. Insects 2022, 13, 533.

In the original publication [...].

Monitoring Insect Transposable Elements in Large Double-Stranded DNA Viruses Reveals Host-to-Virus and Virus-to-Virus Transposition.

The mechanisms by which transposable elements (TEs) can be horizontally transferred between animals are unknown, but viruses are possible candidate vectors. Here, we surveyed the presence of host-derived TEs in viral genomes in 35 deep sequencing data sets produced from 11 host-virus systems, encompassing nine arthropod host species (five lepidopterans, two dipterans, and two crustaceans) and six different double-stranded (ds) DNA viruses (four baculoviruses and two iridoviruses). We found evidence of viral-borne TEs in 14 data sets, with frequencies of viral genomes carrying a TE ranging from 0.01% to 26.33% for baculoviruses and from 0.45% to 7.36% for iridoviruses. The analysis of viral populations separated by a single replication cycle revealed that viral-borne TEs originating from an initial host species can be retrieved after viral replication in another host species, sometimes at higher frequencies. Furthermore, we detected a strong increase in the number of integrations in a viral population for a TE absent from the hosts' genomes, indicating that this TE has undergone intense transposition within the viral population. Finally, we provide evidence that many TEs found integrated in viral genomes (15/41) have been horizontally transferred in insects. Altogether, our results indicate that multiple large dsDNA viruses have the capacity to shuttle TEs in insects and they underline the potential of viruses to act as vectors of horizontal transfer of TEs. Furthermore, the finding that TEs can transpose between viral genomes of a viral species sets viruses as possible new niches in which TEs can persist and evolve.

Gene expression patterns of Cydia pomonella granulovirus in codling moth larvae revealed by RNAseq analysis.

The Cydia pomonella granulovirus (CpGV) has been used as a biological control agent of codling moth (Cydia pomonella), a severe global pest on pome fruit. Despite the economic importance, our knowledge of its molecular biology is still limited and a detailed picture of its gene expression is still missing. Here, we sequenced the transcriptome of codling moth larvae infected with the Mexican isolate CpGV-M and analyzed the expression of viral genes at 12, 48, and 96 h post infection (hpi). The results showed that two genes (p6.9 and pp31/39K) related to DNA binding of virus production, were highly expressed at 48 and 96 hpi. From 48 to 96 hpi, the expression of genes associated with virus replication and dissemination decreased, whereas the expression of genes related to infectious virion production and per os infectivity increased. This study provides a comprehensive view of CpGV gene expression patterns in host larvae.

Population structure of Cydia pomonella granulovirus isolates revealed by quantitative analysis of genetic variation.

Genetic diversity of viruses is driven by genomic mutations and selection through its host, resulting in differences in virulence as well as host responses. For baculoviruses, which are naturally occurring pathogens of insects and which are frequently sprayed on hundred thousands to millions of hectares as biocontrol agents of insect pests, the phenomenon of virus-host co-evolution is of particular scientific interest and economic importance because high virulence of baculovirus products is essential and emergence of host resistance needs to be avoided as much as possible. In the present study, the population structure of twenty isolates of the Cydia pomonella granulovirus (CpGV), including twelve isolates from different geographic origins and eight commercial formulations, were studied on the basis of next-generation sequencing data and by analyzing the distribution of single nucleotide polymorphisms (SNPs). An entirely consensus sequence-free quantitative SNP analysis was applied for the identification of 753 variant SNP sites being specific for single as well as groups of CpGV isolates. Based on the quantitative SNP analysis, homogenous, heterogenous as well as mixed isolates were identified and their proportions of genotypes were deciphered, revealing a high genetic diversity of CpGV isolates from around the world. Based on hierarchical clustering on principal components (HCPC), six distinct isolate/group clusters were identified, representing the proposed main phylogenetic lineages of CpGV but comprising full genome information from virus mixtures. The relative location of different isolates in HCPC reflected the proportion of variable compositions of different genotypes. The established methods provide novel analysis tools to decipher the molecular complexity of genotype mixtures in baculovirus isolates, thus depicting the population structure of baculovirus isolates in a more adequate form than consensus based analyses.

Bacsnp: Using Single Nucleotide Polymorphism (SNP) Specificities and Frequencies to Identify Genotype Composition in Baculoviruses.

Natural isolates of baculoviruses (as well as other dsDNA viruses) generally consist of homogenous or heterogenous populations of genotypes. The number and positions of single nucleotide polymorphisms (SNPs) from sequencing data are often used as suitable markers to study their genotypic composition. Identifying and assigning the specificities and frequencies of SNPs from high-throughput genome sequencing data can be very challenging, especially when comparing between several sequenced isolates or samples. In this study, the new tool "bacsnp", written in R programming langue, was developed as a downstream process, enabling the detection of SNP specificities across several virus isolates. The basis of this analysis is the use of a common, closely related reference to which the sequencing reads of an isolate are mapped. Thereby, the specificities of SNPs are linked and their frequencies can be used to analyze the genetic composition across the sequenced isolate. Here, the downstream process and analysis of detected SNP positions is demonstrated on the example of three baculovirus isolates showing the fast and reliable detection of a mixed sequenced sample.

Novel Diversity and Virulence Patterns Found in New Isolates of Cydia pomonella Granulovirus from China.

Cydia pomonella granulovirus (CpGV) is successfully used worldwide as a biocontrol agent of the codling moth (CM) (Cydia pomonella). The occurrence of CM populations with different modes of resistance against commercial CpGV preparations in Europe, as well as the invasiveness of CM in China, threatening major apple production areas there, requires the development of new control options. Utilizing the naturally occurring genetic diversity of CpGV can improve such control strategies. Here, we report the identification of seven new CpGV isolates that were collected from infected CM larvae in northwest China. Resistance testing using a discriminating CpGV concentration and the determination of the median lethal concentration (LC50) were performed to characterize their levels of virulence against susceptible and resistant CM larvae. The isolates were further screened for the presence of the 2 × 12-bp-repeat insertion in CpGV gene pe38 (open reading frame 24 [ORF24]), which was shown to be the target of type I resistance. It was found that three isolates, CpGV-JQ, -KS1, and -ZY2, could break type I resistance, although delayed mortality was observed in the infection process. All isolates followed the pe38 model of breaking type I resistance, except for CpGV-WW, which harbored the genetic factor but failed to overcome type I resistance. However, CpGV-WW was able to overcome type II and type III resistance. The bioassay results and sequencing data of pe38 support previous findings that pe38 is the major target for type I resistance. The new isolates show some distinct virulence characteristics when infection of different CM strains is considered.IMPORTANCE CpGV is a highly virulent pathogen of the codling moth (CM). It is registered and widely applied as a biocontrol agent in nearly all apple-growing countries worldwide. The emergence of CpGV resistance and the increasing lack of chemical control options require improvements to current control strategies. Natural CpGV isolates, as well as resistance-breaking isolates selected in resistant CM strains, have provided resources for improved resistance-breaking CpGV products. Here, we report novel CpGV isolates collected in China, which have new resistance-breaking capacities and may be an important asset for future application in the biological control of codling moths.

Single nucleotide polymorphism (SNP) frequencies and distribution reveal complex genetic composition of seven novel natural isolates of Cydia pomonella granulovirus.

The co-evolution between baculoviruses and their insect hosts results in selection of virus populations. To explore this phenomenon at the molecular level, seven natural isolates of Cydia pomonella granulovirus (CpGV) collected from orchards in northwest China were studied using Illumina next generation sequencing (NGS). A total of 540 genome positions with single nucleotide polymorphisms (SNPs) were detected in comparison with known CpGV isolates. New members of previously defined phylogenetic genome groups A, D and E of CpGV, as well as two novel phylogenetic lines, termed genome group F and G, were identified. Combining SNP frequency distribution with the prevalence of genome group-specific SNPs, revealed that six isolates of CpGV were mixtures of different ratios of at least two genotypes, whereas only one isolate, CpGV-WW, was genetically highly homogeneous. This study significantly extends our current understanding of the genetic diversity of CpGV and opens new lines of application of this virus.

Partial Loss of Inheritable Type I Resistance of Codling Moth to Cydia pomonella qranulovirus.

Current knowledge of the field resistance of codling moth (CM, Cydia pomonella, L) against Cydia pomonella granulovirus (CpGV) is based mainly on the interaction between the Mexican isolate CpGV-M and CpRR1, a genetically homogeneous CM inbreed line carrying type I resistance. The resistance level of laboratory-reared CpRR1 to CpGV-M was recently found to have decreased considerably, compared to the initially high resistance. To understand the background of this phenomenon, CpRR1 larvae were exposed over several generations to CpGV-M for re-selection of the original resistance level. After five and seven generations of selection, new CpRR1_F5 and CpRR1_F7 lines were established. The resistance ratio of these selected lines was determined by full range bioassays. The CpRR1_F5 strain regained a higher level of resistance against CpGV up to 104-fold based on LC50 values compared to susceptible larvae (CpS), which indicated that the absence of virus selection had resulted in a reduction of resistance under laboratory rearing conditions. In addition, some fitness costs of fecundity were observed in CpRR1_F5. Single-pair crossings between CpRR1_F5 or CpRR1_F7 with susceptible CpS moths revealed a dominant but not fully sex-linked inheritance, which suggests a partial loss of previous resistance traits in CpRR1.