Engineering Phage Host-Range and Suppressing Bacterial Resistance through Phage Tail Fiber Mutagenesis.

The rapid emergence of antibiotic-resistant infections is prompting increased interest in phage-based antimicrobials. However, acquisition of resistance by bacteria is a major issue in the successful development of phage therapies. Through natural evolution and structural modeling, we identified host-range-determining regions (HRDRs) in the T3 phage tail fiber protein and developed a high-throughput strategy to genetically engineer these regions through site-directed mutagenesis. Inspired by antibody specificity engineering, this approach generates deep functional diversity while minimizing disruptions to the overall tail fiber structure, resulting in synthetic "phagebodies." We showed that mutating HRDRs yields phagebodies with altered host-ranges, and select phagebodies enable long-term suppression of bacterial growth in vitro, by preventing resistance appearance, and are functional in vivo using a murine model. We anticipate that this approach may facilitate the creation of next-generation antimicrobials that slow resistance development and could be extended to other viral scaffolds for a broad range of applications.

Genomics and evolutionary analysis of Chlorella variabilis-infecting viruses demarcate criteria for defining species of giant viruses.

Chloroviruses exhibit a close relationship with their hosts with the phenotypic aspect of their ability to form lytic plaques having primarily guided the taxonomy. However, with the isolation of viruses that are only able to complete their replication cycle in one strain of Chlorella variabilis, systematic challenges emerged. In this study, we described the genomic features of 53 new chlorovirus isolates and used them to elucidate part of the evolutionary history and taxonomy of this clade. Our analysis revealed new chloroviruses with the largest genomes to date (>400 kbp) and indicated that four genomic features are statistically different in the viruses that only infect the Syngen 2-3 strain of C. variabilis (OSy viruses). We found large regions of dissimilarity in the genomes of viruses PBCV-1 and OSy-NE5 when compared with the other genomes. These regions contained genes related to the interaction with the host cell machinery and viral capsid proteins, which provided insights into the evolution of the replicative and structural modules in these giant viruses. Phylogenetic analysis using hallmark genes of Nucleocytoviricota revealed that OSy-viruses evolved from the NC64A-viruses, possibly emerging as a result of the strict relationship with their hosts. Merging phylogenetics and nucleotide identity analyses, we propose strategies to demarcate viral species, resulting in seven new species of chloroviruses. Collectively, our results show how genomic data can be used as lines of evidence to demarcate viral species. Using the chloroviruses as a case study, we expect that similar initiatives will emerge using the basis exhibited here.IMPORTANCEChloroviruses are a group of giant viruses with long dsDNA genomes that infect different species of Chlorella-like green algae. They are host-specific, and some isolates can only replicate within a single strain of Chlorella variabilis. The genomics of these viruses is still poorly explored, and the characterization of new isolates provides important data on their genetic diversity and evolution. In this work, we describe 53 new chlorovirus genomes, including many isolated from alkaline lakes for the first time. Through comparative genomics and molecular phylogeny, we provide evidence of genomic gigantism in chloroviruses and show that a subset of viruses became highly specific for their hosts at a particular point in evolutionary history. We propose criteria to demarcate species of chloroviruses, paving the way for an update in the taxonomy of other groups of viruses. This study is a new and important piece in the complex puzzle of giant algal viruses.

The adenovirus DNA-binding protein DBP.

Adenoviruses are a group of double-stranded DNA viruses that can mainly cause respiratory, gastrointestinal, and eye infections in humans. In addition, adenoviruses are employed as vector vaccines for combatting viral infections, including SARS-CoV-2, and serve as excellent gene therapy vectors. These viruses have the ability to modulate the host cell machinery to their advantage and trigger significant restructuring of the nuclei of infected cells through the activity of viral proteins. One of those, the adenovirus DNA-binding protein (DBP), is a multifunctional non-structural protein that is integral to the reorganization processes. DBP is encoded in the E2A transcriptional unit and is highly abundant in infected cells. Its activity is unequivocally linked to the formation, structure, and integrity of virus-induced replication compartments, molecular hubs for the regulation of viral processes, and control of the infected cell. DBP also plays key roles in viral DNA replication, transcription, viral gene expression, and even host range specificity. Notably, post-translational modifications of DBP, such as SUMOylation and extensive phosphorylation, regulate its biological functions. DBP was first investigated in the 1970s, pioneering research on viral DNA-binding proteins. In this literature review, we provide an overview of DBP and specifically summarize key findings related to its complex structure, diverse functions, and significant role in the context of viral replication. Finally, we address novel insights and perspectives for future research.

Exploiting breakdown in nonhost effector-target interactions to boost host disease resistance.

Plants are resistant to most microbial species due to nonhost resistance (NHR), providing broad-spectrum and durable immunity. However, the molecular components contributing to NHR are poorly characterised. We address the question of whether failure of pathogen effectors to manipulate nonhost plants plays a critical role in NHR. RxLR (Arg-any amino acid-Leu-Arg) effectors from two oomycete pathogens, Phytophthora infestans and Hyaloperonospora arabidopsidis, enhanced pathogen infection when expressed in host plants (Nicotiana benthamiana and Arabidopsis, respectively) but the same effectors performed poorly in distantly related nonhost pathosystems. Putative target proteins in the host plant potato were identified for 64 P. infestans RxLR effectors using yeast 2-hybrid (Y2H) screens. Candidate orthologues of these target proteins in the distantly related non-host plant Arabidopsis were identified and screened using matrix Y2H for interaction with RxLR effectors from both P. infestans and H. arabidopsidis. Few P. infestans effector-target protein interactions were conserved from potato to candidate Arabidopsis target orthologues (cAtOrths). However, there was an enrichment of H. arabidopsidis RxLR effectors interacting with cAtOrths. We expressed the cAtOrth AtPUB33, which unlike its potato orthologue did not interact with P. infestans effector PiSFI3, in potato and Nicotiana benthamiana. Expression of AtPUB33 significantly reduced P. infestans colonization in both host plants. Our results provide evidence that failure of pathogen effectors to interact with and/or correctly manipulate target proteins in distantly related non-host plants contributes to NHR. Moreover, exploiting this breakdown in effector-nonhost target interaction, transferring effector target orthologues from non-host to host plants is a strategy to reduce disease.

Host Plant Effects on Sexual Selection Dynamics in Phytophagous Insects.

Natural selection is notoriously dynamic in nature, and so, too, is sexual selection. The interactions between phytophagous insects and their host plants have provided valuable insights into the many ways in which ecological factors can influence sexual selection. In this review, we highlight recent discoveries and provide guidance for future work in this area. Importantly, host plants can affect both the agents of sexual selection (e.g., mate choice and male-male competition) and the traits under selection (e.g., ornaments and weapons). Furthermore, in our rapidly changing world, insects now routinely encounter new potential host plants. The process of adaptation to a new host may be hindered or accelerated by sexual selection, and the unexplored evolutionary trajectories that emerge from these dynamics are relevant to pest management and insect conservation strategies. Examining the effects of host plants on sexual selection has the potential to advance our fundamental understanding of sexual conflict, host range evolution, and speciation, with relevance across taxa.

The dimensionality of infection networks among viruses infecting microbial eukaryotes and bacteria.

Diverse viruses and their hosts are interconnected through complex networks of infection, which are thought to influence ecological and evolutionary processes, but the principles underlying infection network structure are not well understood. Here we focus on network dimensionality and how it varies across 37 networks of viruses infecting eukaryotic phytoplankton and bacteria. We find that dimensionality is often strikingly low, with most networks being one- or two-dimensional, although dimensionality increases with network richness, suggesting that the true dimensionality of natural systems is higher. Low-dimensional networks generally exhibit a mixture of host partitioning among viruses and nestededness of host ranges. Networks of bacteria-infecting and eukaryote-infecting viruses possess comparable distributions of dimensionality and prevalence of nestedness, indicating that fundamentals of network structure are similar among domains of life and different viral lineages. The relative simplicity of many infection networks suggests that coevolutionary dynamics are often driven by a modest number of underlying mechanisms.

The virtue of training: extending phage host spectra against vancomycin-resistant Enterococcus faecium strains using the Appelmans method.

Phage therapy has (re)emerged as a serious possibility for combating multidrug-resistant bacterial infections, including those caused by vancomycin-resistant Enterococcus faecium strains. These opportunistic pathogens belong to a specific clonal complex 17, against which relatively few phages have been screened. We isolated a collection of 21 virulent phages growing on these vancomycin-resistant isolates. Each of these phages harbored a typical narrow plaquing host range, lysing at most 5 strains and covering together 10 strains of our panel of 14 clinical isolates. To enlarge the host spectrum of our phages, the Appelmans protocol was used. We mixed four out of our most complementary phages in a cocktail that we iteratively grew on eight naive strains from our panel, of which six were initially refractory to at least three of the combined phages. Fifteen successive passages permitted to significantly improve the lytic activity of the cocktail, from which phages with extended host ranges within the E. faecium species could be isolated. A single evolved phage able to kill up to 10 of the 14 initial E. faecium strains was obtained, and it barely infected nearby species. All evolved phages had acquired point mutations or a recombination event in the tail fiber genetic region, suggesting these genes might have driven phage evolution by contributing to their extended host spectra.

Virus-host coevolutionary analyses of an Alphabaculovirus with a wide host range.

Baculoviruses are highly host specific, and their host range is usually restricted to a single or a few closely related insect species, except for few virus species, e.g. Alphabaculovirus aucalifonicae and Alphabaculovirus mabrassicae. In this study, two new alphabaculovirus isolates were isolated from the larvae of Mamestra brassicae and Mythimna separata, which were named as Mamestra brassicae multiple nucleopolyhedrovirus isolate QD (MbMNPV-QD) and Mythimna separata multiple nucleopolyhedrovirus isolate Hb (MyseMNPV-Hb), respectively. The Kimura two-parameter values based on the concatenated 38 core genes of baculovirus revealed that MbMNPV (isolates QD/CHb1/K1/CTa), MyseMNPV-Hb, Helicoverpa armigera multiple nucleopolyhedrovirus (HearMNPV) and Mamestra configurata nucleopolyhedrovirus B (MacoNPV-B) were different isolates of a same virus species. A phylogenetic tree of baculoviruses and nudiviruses constructed from their 20 homologous gene sequences, and that of their isolated hosts constructed from 13 protein-coding genes of the insect mitochondrial genomes, were used to analyse the coevolution of baculoviruses with their isolated hosts. The results showed that M. brassicae was the most likely ancestral host of these virus isolates, included MbMNPV isolates, MyseMNPV-Hb, HearMNPV, and MacoNPV-B. Therefore, we concluded that these virus isolates belong to the existing virus species - Alphabaculovirus mabrassicae with M. brassicae as their ancestral host.

Evolutionary analysis and biological characterization of a novel alphabaculovirus isolated from Mythimna separata.

Baculoviruses are insect-specific pathogens. Novel baculovirus isolates provide new options for the biological control of pests. Therefore, research into the biological characteristics of newly isolated baculoviruses, including accurate classification and nomenclature, is important. In this study, a baculovirus was isolated from Mythimna separata and its complete genome sequence was determined by next-generation sequencing. The double-stranded DNA genome was 153 882 bp in length, encoding 163 open reading frames. The virus was identified as a variant of Mamestra brassicae multiple nucleopolyhedrovirus (MbMNPV) and designated Mamestra brassicae multiple nucleopolyhedrovirus CHN1 (MbMNPV-CHN1) according to ultrastructural analysis, genome comparison and phylogenetic analysis. Phylogenetic inference placed MbMNPV-CHN1 in a clade containing isolates of MacoNPV-A, MacoNPV-B and MbMNPV, which we have designated the Mb-McNPV group. The genomes of isolates in the Mb-McNPV group exhibited a high degree of collinearity with relatively minor differences in the content of annotated open reading frames. The development of codon usage bias in the Mb-McNPV group was affected mainly by natural selection. MbMNPV-CHN1 shows high infectivity against seven species of Lepidoptera. The yield of MbMNPV-CHN1 in the fourth- and fifth-instar M. separata larvae was 6.25×109-1.23×1010 OBs/cadaver. Our data provide insights into the classification, host range and virulence differences among baculoviruses of the Mb-McNPV group, as well as a promising potential new baculoviral insecticide.

Energy input, habitat heterogeneity and host specificity drive avian haemosporidian diversity at continental scales.

The correct identification of variables affecting parasite diversity and assemblage composition at different spatial scales is crucial for understanding how pathogen distribution responds to anthropogenic disturbance and climate change. Here, we used a database of avian haemosporidian parasites to test how the taxonomic and phylogenetic diversity and phylogenetic structure of the genera Plasmodium, Haemoproteus and Leucocytozoon from three zoogeographic regions are related to surrogate variables of Earth's energy input, habitat heterogeneity (climatic diversity, landscape heterogeneity, host richness and human disturbance) and ecological interactions (resource use), which was measured by a novel assemblage-level metric related to parasite niche overlap (degree of generalism). We found that different components of energy input explained variation in richness for each genus. We found that human disturbance influences the phylogenetic structure of Haemoproteus while the degree of generalism explained richness and phylogenetic structure of Plasmodium and Leucocytozoon genera. Furthermore, landscape attributes related to human disturbance (human footprint) can filter Haemoproteus assemblages by their phylogenetic relatedness. Finally, assembly processes related to resource use within parasite assemblages modify species richness and phylogenetic structure of Plasmodium and Leucocytozoon assemblages. Overall, our study highlighted the genus-specific patterns with the different components of Earth's energy budget, human disturbances and degree of generalism.

Making the leap from technique to treatment - genetic engineering is paving the way for more efficient phage therapy.

Bacteriophages (phages) are viruses specific to bacteria that target them with great efficiency and specificity. Phages were first studied for their antibacterial potential in the early twentieth century; however, their use was largely eclipsed by the popularity of antibiotics. Given the surge of antimicrobial-resistant strains worldwide, there has been a renaissance in harnessing phages as therapeutics once more. One of the key advantages of phages is their amenability to modification, allowing the generation of numerous derivatives optimised for specific functions depending on the modification. These enhanced derivatives could display higher infectivity, expanded host range or greater affinity to human tissues, where some bacterial species exert their pathogenesis. Despite this, there has been a noticeable discrepancy between the generation of derivatives in vitro and their clinical application in vivo. In most instances, phage therapy is only used on a compassionate-use basis, where all other treatment options have been exhausted. A lack of clinical trials and numerous regulatory hurdles hamper the progress of phage therapy and in turn, the engineered variants, in becoming widely used in the clinic. In this review, we outline the various types of modifications enacted upon phages and how these modifications contribute to their enhanced bactericidal function compared with wild-type phages. We also discuss the nascent progress of genetically modified phages in clinical trials along with the current issues these are confronted with, to validate it as a therapy in the clinic.

Directed evolution of bacteriophages: thwarted by prolific prophage.

Various directed evolution methods exist that seek to procure bacteriophages with expanded host ranges, typically targeting phage-resistant or non-permissive bacterial hosts. The general premise of these methods involves propagating phage(s) on multiple bacterial hosts, pooling the lysate, and repeating this process until phage(s) can form plaques on the target host(s). In theory, this produces a lysate containing input phages and their evolved phage progeny. However, in practice, this lysate can also include prophages originating from bacterial hosts. Here, we describe our experience implementing one directed evolution method, the Appelmans protocol, to study phage evolution in the Pseudomonas aeruginosa phage-host system, where we observed rapid host-range expansion of the phage cocktail. Further experimentation and sequencing revealed that the observed host-range expansion was due to a Casadabanvirus prophage originating from a lysogenic host that was only included in the first three rounds of the experiment. This prophage could infect five of eight bacterial hosts initially used, allowing it to persist and proliferate until the termination of the experiment. This prophage was represented in half of the sequenced phage samples isolated from the Appelmans experiment, but despite being subjected to directed evolution conditions, it does not appear to have evolved. This work highlights the impact of prophages in directed evolution experiments and the importance of genetically verifying output phages, particularly for those attempting to procure phages intended for phage therapy applications. This study also notes the usefulness of intraspecies antagonism assays between bacterial host strains to establish a baseline for inhibitory activity and determine the presence of prophage.IMPORTANCEDirected evolution is a common strategy for evolving phages to expand the host range, often targeting pathogenic strains of bacteria. In this study, we investigated phage host-range expansion using directed evolution in the Pseudomonas aeruginosa system. We show that prophages are active players in directed evolution and can contribute to observation of host-range expansion. Since prophages are prevalent in bacterial hosts, particularly pathogenic strains of bacteria, and all directed evolution approaches involve iteratively propagating phage on one or more bacterial hosts, the presence of prophage in phage preparations is a factor that needs to be considered in experimental design and interpretation of results. These results highlight the importance of screening for prophages either genetically or through intraspecies antagonism assays during selection of bacterial strains and will contribute to improving the experimental design of future directed evolution studies.

Plant virus community structuring is shaped by habitat heterogeneity and traits for host plant resource utilisation.

Host plants provide resources critical to viruses and the spatial structuring of plant communities affects the niches available for colonisation and disease emergence. However, large gaps remain in the understanding of mechanisms that govern plant-virus disease ecology across heterogeneous plant assemblages. We combine high-throughput sequencing, network, and metacommunity approaches to test whether habitat heterogeneity in plant community composition corresponded with virus resource utilisation traits of transmission mode and host range. A majority of viruses exhibited habitat specificity, with communities connected by key generalist viruses and potential host reservoirs. There was an association between habitat heterogeneity and virus community structuring, and between virus community structuring and resource utilisation traits of host range and transmission. The relationship between virus species distributions and virus trait responses to habitat heterogeneity was scale-dependent, being stronger at finer (site) than larger (habitat) spatial scales. Results indicate that habitat heterogeneity has a part in plant virus community assembly, and virus community structuring corresponds to virus trait responses that vary with the scale of observation. Distinctions in virus communities caused by plant resource compartmentalisation can be used to track trait responses of viruses to hosts important in forecasting disease emergence.

Methylated chalcones are required for rhizobial nod gene induction in the Medicago truncatula rhizosphere.

Legume nodulation requires the detection of flavonoids in the rhizosphere by rhizobia to activate their production of Nod factor countersignals. Here we investigated the flavonoids involved in nodulation of Medicago truncatula. We biochemically characterized five flavonoid-O-methyltransferases (OMTs) and a lux-based nod gene reporter was used to investigate the response of Sinorhizobium medicae NodD1 to various flavonoids. We found that chalcone-OMT 1 (ChOMT1) and ChOMT3, but not OMT2, 4, and 5, were able to produce 4,4'-dihydroxy-2'-methoxychalcone (DHMC). The bioreporter responded most strongly to DHMC, while isoflavones important for nodulation of soybean (Glycine max) showed no activity. Mutant analysis revealed that loss of ChOMT1 strongly reduced DHMC levels. Furthermore, chomt1 and omt2 showed strongly reduced bioreporter luminescence in their rhizospheres. In addition, loss of both ChOMT1 and ChOMT3 reduced nodulation, and this phenotype was strengthened by the further loss of OMT2. We conclude that: the loss of ChOMT1 greatly reduces root DHMC levels; ChOMT1 or OMT2 are important for nod gene activation in the rhizosphere; and ChOMT1/3 and OMT2 promote nodulation. Our findings suggest a degree of exclusivity in the flavonoids used for nodulation in M. truncatula compared to soybean, supporting a role for flavonoids in rhizobial host range.

The Threat of the Fall Armyworm to Asian Rice Production Is Amplified by the Brown Planthopper.

The recent invasion of the fall armyworm (FAW) into Asia not only has had a major impact on maize yield but is feared to also pose a risk to rice production. We hypothesized that the brown planthopper (BPH) may aggravate this risk based on a recently discovered mutualism between the planthopper and the rice striped stem borer. Here we show that BPH may indeed facilitate a shift of FAW to rice. FAW females were found to strongly prefer to oviposit on BPH-infested rice plants, which emitted significantly elevated levels of five volatile compounds. A synthetic mixture of these compounds had a potent stimulatory effect on ovipositing females. Although FAW caterpillars exhibited relatively poor growth on both uninfested and BPH-infested rice, a considerable portion completed their development on young plants. Moreover, FAW were found to readily pupate and survive in exceedingly moist soils typical for rice cultivation, further highlighting FAW's potential to switch to rice. We conclude that BPH, by changing the bouquet of volatiles emitted by rice plants, may greatly facilitate this switch. These findings, together with a current increase of nonflooded upland rice in Asia, warrant careful monitoring and specific control measures against FAW to safeguard Asian rice production.

High parasite diversity maintained after an alga-virus coevolutionary arms race.

Arms race dynamics are a common outcome of host-parasite coevolution. While they can theoretically be maintained indefinitely, realistic arms races are expected to be finite. Once an arms race has ended, for example due to the evolution of a generalist-resistant host, the system may transition into coevolutionary dynamics that favour long-term diversity. In microbial experiments, host-parasite arms races often transition into a stable coexistence of generalist-resistant hosts, (semi-)susceptible hosts, and parasites. While long-term host diversity is implicit in these cases, parasite diversity is usually overlooked. In this study, we examined parasite diversity after the end of an experimental arms race between a unicellular alga (Chlorella variabilis) and its lytic virus (PBCV-1). First, we isolated virus genotypes from multiple time points from two replicate microcosms. A time-shift experiment confirmed that the virus isolates had escalating host ranges, i.e., that arms races had occurred. We then examined the phenotypic and genetic diversity of virus isolates from the post-arms race phase. Post-arms race virus isolates had diverse host ranges, survival probabilities, and growth rates; they also clustered into distinct genetic groups. Importantly, host range diversity was maintained throughout the post-arms race phase, and the frequency of host range phenotypes fluctuated over time. We hypothesize that this dynamic polymorphism was maintained by a combination of fluctuating selection and demographic stochasticity. Together with previous work in prokaryotic systems, our results link experimental observations of arms races to natural observations of long-term host and parasite diversity.

Characterization of a novel phage against multidrug-resistant Klebsiella pneumoniae.

Multidrug-resistant Klebsiella pneumoniae (MDR-KP) poses a significant challenge in global healthcare, underscoring the urgency for innovative therapeutic approaches. Phage therapy emerges as a promising strategy amidst rising antibiotic resistance, emphasizing the crucial need to identify and characterize effective phage resources for clinical use. In this study, we introduce a novel lytic phage, RCIP0100, distinguished by its classification into the Chaoyangvirus genus and Fjlabviridae family based on International Committee on Taxonomy of Viruses (ICTV) criteria due to low genetic similarity to known phage families. Our findings demonstrate that RCIP0100 exhibits broad lytic activity against 15 out of 27 tested MDR-KP strains, including diverse profiles such as carbapenem-resistant K. pneumoniae (CR-KP). This positions phage RCIP0100 as a promising candidate for phage therapy. Strains resistant to RCIP0100 also showed increased susceptibility to various antibiotics, implying the potential for synergistic use of RCIP0100 and antibiotics as a strategic countermeasure against MDR-KP.

Parasitic plants show striking convergence in host preference across angiosperm lineages.

BACKGROUND AND AIMS: The host specificity of a parasite underpins its ecology, distribution, invasive potential and adaptability, yet for most parasitic plants host ranges are poorly understood. We examine host-parasite relationships across lineages to infer how host specificity may have influenced the evolution of parasitism in plants. METHODS: Host preference data for all plant holoparasite species were manually collected from literature and herbarium specimens, then analysed to investigate and visualise host diversity and specificity. KEY RESULTS: We reveal a disproportionality in host preference across host lineages: the Asteraceae contains 10% of angiosperm diversity but is infected by 31% of parasite species; meanwhile Monocots comprise 23% but are infected by just 3.2%. Furthermore, we observe striking convergence in host preference: Asteraceae, Euphorbiaceae and Fabaceae are infected by six, five and four independent parasite lineages, respectively. We also demonstrate considerable variation in the degree of host specificity among closely related parasite species; a result that does not reflect the expectation of holoparasites - especially endoparasites - as host specialists. CONCLUSIONS: The marked pattern of convergence in preference across disparate lineages points to a common pathway in the evolution of parasitism of eudicots in preference to monocots, which may have in turn have been driven by a divergence in host root and vascular architecture. The unexpected variation in host specificity among closely related species suggests that even apparent generalists may comprise cryptic host-specific taxa. This highlights the value of host preference as an additional consideration in parasitic plant taxonomy. Together, our data point to a complex interplay between ecological and physiological factors driving the evolution of host-parasite interactions. Moreover, they emphasize how little is known about the ecology of most holoparasitic plants, a group of organisms that are especially vulnerable at a time of unprecedented biodiversity loss and extinction.

Probing of plant transcriptomes reveals the hidden genetic diversity of the family Secoviridae.

Secoviruses are single-stranded RNA viruses that infect plants. In the present study, we identified 61 putative novel secoviral genomes in various plant species by mining publicly available plant transcriptome data. These viral sequences represent the genomes of 13 monopartite and 48 bipartite secovirids. The genome sequences of 52 secovirids were coding-complete, and nine were partial. Except for small open reading frames (ORFs) determined in waikaviral genomes and RNA2 of torradoviruses, all of the recovered genomes/genome segments contained a large ORF encoding a polyprotein. Based on genome organization and phylogeny, all but three of the novel secoviruses were assigned to different genera. The genome organization of two identified waika-like viruses resembled that of the recently identified waika-like virus Triticum aestivum secovirus. Phylogenetic analysis revealed a pattern of host-virus co-evolution in a few waika- and waika-like viruses and increased phylogenetic diversity of nepoviruses. The study provides a basis for further investigation of the biological properties of these novel secoviruses.

Electrophysiological Responses of Trissolcus japonicus, T. basalis, and T. oenone (Hymenoptera: Scelionidae) to Volatile Compounds Associated with New Zealand Stink Bugs (Hemiptera: Pentatomidae).

Parasitoid biological control agents rely heavily on olfaction to locate their hosts. Chemical cues associated with hosts and non-hosts are known to influence the expression of host preferences and host-specificity. A better understanding of how and why parasitoids attack some species and not others, based on volatile organic compounds associated with potential hosts, can provide key information on the parasitoid's host preferences, which could be applied to pre-release risk assessments for classical biological control agents. Electrophysiological techniques such as electroantennography (EAG) and GC-EAD (gas chromatography coupled with electroantennographic detection) are widely used to identify bioactive semiochemicals. But the application of these techniques to understanding how chemical ecological cues mediate parasitoid host specificity has not been as thoroughly explored. We conducted GC-EAD and EAG studies to identify olfactory-active compounds associated with adult females of nine stink bug species from Aotearoa/New Zealand on the antennae of three closely related parasitoid species: Trissolcus japonicus Ashmead, a pre-emptively (= proactively) approved biocontrol agent against brown marmorated stink bug; T. basalis (Wollaston), a biocontrol agent introduced against Nezara viridula L. in 1949; and T. oenone Johnson, a native Australasian pentatomid parasitoid. Eight compounds associated with stink bugs elicited antennal responses from all three parasitoids, and we were able to identify seven of these. (E)-2-hexenal, (E)-4-oxo-2-hexenal, (E)-2-octenal and (E)-2-decenal generally elicited stronger responses in the three parasitoids, while n-tridecane, n-dodecane, and (E)-2-decenyl acetate elicited weaker responses. We discuss how and why the results from electrophysiological experiments can be applied to non-target risk assessments within biological control programmes.