Immune system modulation & virus transmission during parasitism identified by multi-species transcriptomics of a declining insect biocontrol system.

BACKGROUND: The Argentine stem weevil (ASW, Listronotus bonariensis) is a significant pasture pest in Aotearoa New Zealand, primarily controlled by the parasitoid biocontrol agent Microctonus hyperodae. Despite providing effective control of ASW soon after release, M. hyperodae parasitism rates have since declined significantly, with ASW hypothesised to have evolved resistance to its biocontrol agent. While the parasitism arsenal of M. hyperodae has previously been investigated, revealing many venom components and an exogenous novel DNA virus Microctonus hyperodae filamentous virus (MhFV), the effects of said arsenal on gene expression in ASW during parasitism have not been examined. In this study, we performed a multi-species transcriptomic analysis to investigate the biology of ASW parasitism by M. hyperodae, as well as the decline in efficacy of this biocontrol system. RESULTS: The transcriptomic response of ASW to parasitism by M. hyperodae involves modulation of the weevil's innate immune system, flight muscle components, and lipid and glucose metabolism. The multispecies approach also revealed continued expression of venom components in parasitised ASW, as well as the transmission of MhFV to weevils during parasitism and some interrupted parasitism attempts. Transcriptomics did not detect a clear indication of parasitoid avoidance or other mechanisms to explain biocontrol decline. CONCLUSIONS: This study has expanded our understanding of interactions between M. hyperodae and ASW in a biocontrol system of critical importance to Aotearoa-New Zealand's agricultural economy. Transmission of MhFV to ASW during successful and interrupted parasitism attempts may link to a premature mortality phenomenon in ASW, hypothesised to be a result of a toxin-antitoxin system. Further research into MhFV and its potential role in ASW premature mortality is required to explore whether manipulation of this viral infection has the potential to increase biocontrol efficacy in future.

Chromosome-level genome assemblies of two parasitoid biocontrol wasps reveal the parthenogenesis mechanism and an associated novel virus.

BACKGROUND: Biocontrol is a key technology for the control of pest species. Microctonus parasitoid wasps (Hymenoptera: Braconidae) have been released in Aotearoa New Zealand as biocontrol agents, targeting three different pest weevil species. Despite their value as biocontrol agents, no genome assemblies are currently available for these Microctonus wasps, limiting investigations into key biological differences between the different species and strains. METHODS AND FINDINGS: Here we present high-quality genomes for Microctonus hyperodae and Microctonus aethiopoides, assembled with short read sequencing and Hi-C scaffolding. These assemblies have total lengths of 106.7 Mb for M. hyperodae and 129.2 Mb for M. aethiopoides, with scaffold N50 values of 9 Mb and 23 Mb respectively. With these assemblies we investigated differences in reproductive mechanisms, and association with viruses between Microctonus wasps. Meiosis-specific genes are conserved in asexual Microctonus, with in-situ hybridisation validating expression of one of these genes in the ovaries of asexual Microctonus aethiopoides. This implies asexual reproduction in these Microctonus wasps involves meiosis, with the potential for sexual reproduction maintained. Investigation of viral gene content revealed candidate genes that may be involved in virus-like particle production in M. aethiopoides, as well as a novel virus infecting M. hyperodae, for which a complete genome was assembled. CONCLUSION AND SIGNIFICANCE: These are the first published genomes for Microctonus wasps which have been deployed as biocontrol agents, in Aotearoa New Zealand. These assemblies will be valuable resources for continued investigation and monitoring of these biocontrol systems. Understanding the biology underpinning Microctonus biocontrol is crucial if we are to maintain its efficacy, or in the case of M. hyperodae to understand what may have influenced the significant decline of biocontrol efficacy. The potential for sexual reproduction in asexual Microctonus is significant given that empirical modelling suggests this asexual reproduction is likely to have contributed to biocontrol decline. Furthermore the identification of a novel virus in M. hyperodae highlights a previously unknown aspect of this biocontrol system, which may contribute to premature mortality of the host pest. These findings have potential to be exploited in future in attempt to increase the effectiveness of M. hyperodae biocontrol.

Plant-Mediated Behavioural Avoidance of a Weevil Towards Its Biological Control Agent.

New Zealand pastures largely comprising Lolium ryegrass species (Poales: Poaceae) are worth $19.6B and are subject to major pest impacts. A very severe pest is the Argentine stem weevil Listronotus bonariensis (Kuschel) (Coleoptera: Curculionidae). This has been previously suppressed by the importation biological control agent, Microctonus hyperodae Loan (Hymenoptera: Braconidae). However, this suppression has recently declined and is subject to investigation. It has been hypothesised that grass type influences the parasitism avoidance behaviour by the weevil and thus parasitism rates. This study explored the hypothesis using three common pasture grasses: a diploid Lolium perenne x Lolium multiflorum hybrid ryegrass (cv. Manawa), a tetraploid Italian ryegrass L. multiflorum Lam. (cv. Tama), and a diploid perennial ryegrass L. perenne L. (cv. Samson). The described laboratory-based microcosm methodology determined the extent of weevil avoidance behaviour on each of these three grasses when subjected to the parasitoid. Such reaction was gauged by the extent of reduced weevil on-plant presence and feeding compared to the control populations. In the absence of the parasitoid, the hybrid cv. Manawa ryegrass is as highly favoured by the weevil as the tetraploid cv. Tama. On diploid cv. Samson, feeding is considerably less. In the presence of the parasitoid, weevils on the tetraploid cv. Tama plants showed little avoidance activity in response to the parasitoid and it can be argued that the benefits of staying on this plant outweighed the possibility of parasitism. Conversely and surprisingly, in the parasitoid's presence, weevils on diploid cv. Manawa showed very strong avoidance behaviour leading to levels of exposure similar to those found on the less-preferred diploid cv. Samson. These findings reflect how weevil parasitism rates have declined in most Lolium grasses, particularly diploids, since the 1990s, but not in the tetraploid L. multiflorum. This contribution supports the hypothesis that the decline in weevil parasitism rates has been the result of rapid evolution arising from parasitoid-induced selection pressure and the countervailing effect of the nutritional quality of the host plants.

A network perspective for sustainable agroecosystems.

Nature-based management aims to improve sustainable agroecosystem production, but its efficacy has been variable. We argue that nature-based agroecosystem management could be significantly improved by explicitly considering and manipulating the underlying networks of species interactions. A network perspective can link species interactions to ecosystem functioning and stability, identify influential species and interactions, and suggest optimal management approaches. Recent advances in predicting the network roles of species from their functional traits could allow direct manipulation of network architecture through additions or removals of species with targeted traits. Combined with improved understanding of the structure and dynamics of networks across spatial and temporal scales and interaction types, including social-ecological, applying these tools to nature-based management can contribute to sustainable agroecosystems.

Towards Post-Pandemic Sustainable and Ethical Food Systems.

The current global COVID-19 pandemic has led to a deep and multidimensional crisis across all sectors of society. As countries contemplate their mobility and social-distancing policy restrictions, we have a unique opportunity to re-imagine the deliberative frameworks and value priorities in our food systems. Pre-pandemic food systems at global, national, regional and local scales already needed revision to chart a common vision for sustainable and ethical food futures. Re-orientation is also needed by the relevant sciences, traditionally siloed in their disciplines and without adequate attention paid to how the food system problem is variously framed by diverse stakeholders according to their values. From the transdisciplinary perspective of food ethics, we argue that a post-pandemic scheme focused on bottom-up, regional, cross-sectoral and non-partisan deliberation may provide the re-orientation and benchmarks needed for not only more sustainable, but also more ethical food futures.

Genetic Diversity in Invasive Populations of Argentine Stem Weevil Associated with Adaptation to Biocontrol.

Modified, agricultural landscapes are susceptible to damage by insect pests. Biological control of pests is typically successful once a control agent has established, but this depends on the agent's capacity to co-evolve with the host. Theoretical studies have shown that different levels of genetic variation between the host and the control agent will lead to rapid evolution of resistance in the host. Although this has been reported in one instance, the underlying genetics have not been studied. To address this, we measured the genetic variation in New Zealand populations of the pasture pest, Argentine stem weevil (Listronotus bonariensis), which is controlled with declining effectiveness by a parasitoid wasp, Microctonus hyperodae. We constructed a draft reference genome of the weevil, collected samples from a geographical survey of 10 sites around New Zealand, and genotyped them using a modified genotyping-by-sequencing approach. New Zealand populations of Argentine stem weevil have high levels of heterozygosity and low population structure, consistent with a large effective population size and frequent gene flow. This implies that Argentine stem weevils were able to evolve more rapidly than their biocontrol agent, which reproduces asexually. These findings show that monitoring genetic diversity in biocontrol agents and their targets is critical for long-term success of biological control.

Severe Insect Pest Impacts on New Zealand Pasture: The Plight of an Ecological Outlier.

New Zealand's intensive pastures, comprised almost entirely introduced Lolium L. and Trifolium L. species, are arguably the most productive grazing-lands in the world. However, these areas are vulnerable to destructive invasive pest species. Of these, three of the most damaging pests are weevils (Coleoptera: Curculionidae) that have relatively recently been controlled by three different introduced parasitoids, all belonging to the genus Microctonus Wesmael (Hymenoptera: Braconidae). Arguably that these introduced parasitoids have been highly effective is probably because they, like many of the exotic pest species, have benefited from enemy release. Parasitism has been so intense that, very unusually, one of the weevils has now evolved resistance to its parthenogenetic parasitoid. This review argues that New Zealand's high exotic pasture pest burden is attributable to a lack of pasture plant and natural enemy diversity that presents little biotic resistance to invasive species. There is a native natural enemy fauna in New Zealand that has evolved over millions of years of geographical isolation. However, these species remain in their indigenous ecosystems and, therefore, play a minimal role in creating biotic resistance in the country's exotic ecosystems. For clear ecological reasons relating to the nature of New Zealand pastures, importation biological control can work extremely well. Conversely, conservation biological control is less likely to be effective than elsewhere.

Asymmetry in reproduction strategies drives evolution of resistance in biological control systems.

The success of biological control may depend on the control agent co-evolving with its target pest species, precluding the emergence of resistance that often undermines chemical control. However, recent evidence of a decline in attack rates of a sexual pest weevil by its asexual parasitoid suggests that evolutionary arms races may not prevent the emergence of resistance if the host and parasitoid do not have reproductive strategies that generate equal amounts of genetic variation. To understand how these asymmetries in reproductive strategies may drive the emergence of resistance, we combined life history data from two pest weevils and their parasitoids (one sexual and one asexual) in the New Zealand pastoral ecosystem, with a population dynamic model that allows the coevolution of hosts and parasitoids. We found that the ratio of the genetic variance of hosts to parasitoids was a key determinant of the emergence of resistance. Host resistance eventually occurred unless the parasitoids had considerably greater additive genetic variance than their host. The higher reproductive rate of asexual parasitoids did little to offset the cost of reduced additive genetic variance. The model predictions were congruent with long-term parasitism rates observed in the field for both of the pests considered (one with a sexual and one with an asexual parasitoid). We then explored the consequences of introducing two parasitoids with different reproductive strategies that attack the same sexual host. The model showed that the sexually reproducing parasitoid always out-competed the asexually reproducing one. Our study shows that any asymmetry in reproductive strategies is extremely important for predicting the long-term success of biological control agents. Fortunately, introduction of sexually reproducing individuals after an initial introduction of asexual strains may overcome the problems of host resistance. We conclude that evolution must be considered when evaluating the long-term outcomes of importation biological control.

Identification of volatiles released by diapausing brown marmorated stink bug, Halyomorpha halys (Hemiptera: Pentatomidae).

The brown marmorated stink bug, Halyomorpha halys, is an agricultural and urban pest that has become widely established as an invasive species of major concern in the USA and across Europe. This species forms large aggregations when entering diapause, and it is often these aggregations that are found by officials conducting inspections of internationally shipped freight. Identifying the presence of diapausing aggregations of H. halys using their emissions of volatile organic compounds (VOCs) may be a potential means for detecting and intercepting them during international freight inspections. Headspace samples were collected from aggregations of diapausing H. halys using volatile collection traps (VCTs) and solid phase microextraction. The only compound detected in all samples was tridecane, with small amounts of (E)-2-decenal found in most samples. We also monitored the release of defensive odors, following mechanical agitation of diapausing and diapause-disrupted adult H. halys. Diapausing groups were significantly more likely to release defensive odors than diapause-disrupted groups. The predominant compounds consistently found from both groups were tridecane, (E)-2-decenal, and 4-oxo-(E)-2-hexenal, with a small abundance of dodecane. Our findings show that diapausing H. halys do release defensive compounds, and suggest that volatile sampling may be feasible to detect H. halys in freight.

Intensified agriculture favors evolved resistance to biological control.

Increased regulation of chemical pesticides and rapid evolution of pesticide resistance have increased calls for sustainable pest management. Biological control offers sustainable pest suppression, partly because evolution of resistance to predators and parasitoids is prevented by several factors (e.g., spatial or temporal refuges from attacks, reciprocal evolution by control agents, and contrasting selection pressures from other enemy species). However, evolution of resistance may become more probable as agricultural intensification reduces the availability of refuges and diversity of enemy species, or if control agents have genetic barriers to evolution. Here we use 21 y of field data from 196 sites across New Zealand to show that parasitism of a key pasture pest (Listronotus bonariensis; Argentine stem weevil) by an introduced parasitoid (Microctonus hyperodae) was initially nationally successful but then declined by 44% (leading to pasture damage of c. 160 million New Zealand dollars per annum). This decline was not attributable to parasitoid numbers released, elevation, or local climatic variables at sample locations. Rather, in all locations the decline began 7 y (14 host generations) following parasitoid introduction, despite releases being staggered across locations in different years. Finally, we demonstrate experimentally that declining parasitism rates occurred in ryegrass Lolium perenne, which is grown nationwide in high-intensity was significantly less than in adjacent plots of a less-common pasture grass (Lolium multiflorum), indicating that resistance to parasitism is host plant-dependent. We conclude that low plant and enemy biodiversity in intensive large-scale agriculture may facilitate the evolution of host resistance by pests and threaten the long-term viability of biological control.

Invertebrate Biosecurity Challenges in High-Productivity Grassland: The New Zealand Example.

To protect productive grasslands from pests and diseases, effective pre- and at-border planning and interventions are necessary. Biosecurity failure inevitably requires expensive and difficult eradication, or long-term and often quite ineffective management strategies. This is compared to the early intervention more likely for sectors where there is public and political interest in plants of immediate economic and/or social value, and where associated pests are typically located above-ground on host plantings of relatively limited distribution. Here, biosecurity surveillance and responses can be readily designed. In contrast, pastures comprising plants of low inherent unit value create little, if any, esthetic interest. Yet, given the vast extent of pasture in New Zealand and the value of the associated industries, these plants are of immense economic importance. Compounding this is the invasibility of New Zealand's pastoral ecosystems through a lack of biotic resistance to incursion and invasion. Further, given the sheer area of pasture, intervention options are limited because of costs per unit area and the potential for pollution if pesticides are used. Biosecurity risk for pastoral products differs from, say, that of fruit where at least part of an invasive pathway can be recognized and risks assessed. The ability to do this via pastoral sector pathways is much reduced, since risk organisms more frequently arrive via hitchhiker pathways which are diffuse and varied. Added to this pasture pests within grassland ecosystems are typically cryptic, often with subterranean larval stages. Such characteristics make detection and response particularly difficult. The consequences of this threaten to add to the already-increasing stressors of production intensification and climate change. This review explores the unique challenges faced by pasture biosecurity and what may be done to confront existing difficulties. While there is no silver bullet, and limited opportunity pre- and at-border for improving pasture biosecurity, advancement may include increased and informed vigilance by farmers, pheromone traps and resistant plants to slow invasion. Increasingly, there is also the potential for more use of improved population dispersal models and surveillance strategies including unmanned aerial vehicles, as well as emerging techniques to determine invasive pest genomes and their geographical origins.

Apparent Acquired Resistance by a Weevil to Its Parasitoid Is Influenced by Host Plant.

Field parasitism rates of the Argentine stem weevil Listronotus bonariensis (Kuschel; Coleoptera: Curculionidae) by Microctonus hyperodae Loan (Hymenoptera: Braconidae) are known to vary according to different host Lolium species that also differ in ploidy. To further investigate this, a laboratory study was conducted to examine parasitism rates on tetraploid Italian Lolium multiflorum, diploid Lolium perenne and diploid hybrid L. perenne ×L. multiflorum; none of which were infected by Epichloë endophyte. At the same time, the opportunity was taken to compare the results of this study with observations made during extensive laboratory-based research and parasitoid-rearing in the 1990s using the same host plant species. This made it possible to determine whether there has been any change in weevil susceptibility to the parasitoid over a 20 year period when in the presence of the tetraploid Italian, diploid perennial and hybrid host grasses that were commonly in use in the 1990's. The incidence of parasitism in cages, in the presence of these three grasses mirrored what has recently been observed in the field. When caged, weevil parasitism rates in the presence of a tetraploid Italian ryegrass host were significantly higher (75%) than rates that occurred in the presence of either the diploid perennial (46%) or the diploid hybrid (52%) grass, which were not significantly different from each other. This is very different to laboratory parasitism rates in the 1990s when in the presence of both of the latter grasses high rates of parasitism (c. 75%) were recorded. These high rates are typical of those still found in weevils in the presence of both field and caged tetraploid Italian grasses. In contrast, the abrupt decline in weevil parasitism rates points to the possibility of evolved resistance by the weevil to the parasitoid in the diploid and hybrid grasses, but not so in the tetraploid. The orientation of plants in the laboratory cages had no significant effect on parasitism rates under any treatment conditions suggesting that plant architecture may not be contributing to the underlying mechanism resulting in different rates of parasitism. The evolutionary implications of what appears to be plant-mediated resistance of L. bonariensis to parasitism by M. hyperodae are discussed.

Is the Invasive Species Listronotus bonariensis (Kuschel) (Coleoptera: Curculionidae) (Argentine Stem Weevil) a Threat to New Zealand Natural Grassland Ecosystems?

Listronotus bonariensis (Argentine stem weevil) is a stem-boring weevil that has become a major pasture pest in New Zealand, and cool climate turf grass in Australia. This species is also frequently found in native tussock grassland in New Zealand. Laboratory and field trials were established to determine the risk posed to both seedlings and established plants of three native grass species compared to what happens with a common host of this species, hybrid ryegrass (L. perenne X L. multiflorum). Adult weevil feeding damage scores were higher on Poa colensoi and Festuca novae-zelandiae than Chionochloa rigida. Oviposition was lower on P. colensoi than hybrid ryegrass, and no eggs were laid on F. novae-zelandiae. In field trials using the same four species established as spaced plants L. bonariensis laid more eggs per tiller in ryegrass in a low altitude pasture site than in ryegrass in a higher altitude site. No eggs were found on the three native grass species at the tussock sites, and only low numbers were found on other grasses at the low altitude pasture site. Despite this, numbers of adult weevils were extracted from the plants in the field trials. These may have comprised survivors of the original weevils added to the plants, together with new generation weevils that had emerged during the experiment. Irrespective, higher numbers were recovered from the tussock site plants than from those from the pasture site. It was concluded that L. bonariensis is likely to have little overall impact, but a greater impact on native grass seedling survival than on established plants.

Postdispersal removal and germination of seed dispersed by Cercopithecus nictitans in a West African Montane Forest.

Factors that determine the effectiveness of primates as seed dispersers include (i) the microsite into which they deposit seed, (ii) secondary removal of seed by other taxa and (iii) the effect of gut passage and/or spitting on subsequent seed germination. This contribution evaluated these factors in the little studied putty-nosed monkey, Cercopithecus nictitans, in a Nigerian montane forest. Field experiments showed that C. nictitans has greatly increased in its importance as a disperser of medium-sized seed (>5 mm) because other large primates have been hunted to near extinction. C. nictitans disperses seed across habitats by spitting and defaecation. Rates of secondary seed removal were high for all seed species irrespective of the presence or absence of C. nictitans faecal matter, size or microsite variables. Gut passage enhanced germination relative to hand-cleaned seed, while spitting had either no effect or decreased the germination rate.

Potential of solid phase microextraction and gas chromatography for quarantine-required detection of wood packaging in shipping containers.

Solid phase microextraction (SPME) coupled with gas chromatography (GC) was used to detect terpene hydrocarbons inside shipping containers entering New Zealand. The utility of this system for the rapid detection of undeclared wood packaging for quarantine purposes was demonstrated. A portable dynamic air-sampling device was built to house a SPME fibre and allow the air from shipping containers to be sampled. The effects of sample flow rate and sampling time were investigated and sampling conditions of 100 mL/min for 30 s were chosen to keep sampling within the linear range. A CV of less than 15% (n = 12) was obtained for all the compounds analysed under these conditions. To obtain an estimate for the limit of detection (LOD) for the terpene hydrocarbons of interest, small quantities of lime oil were placed in an empty shipping container and the air inside was analysed. LOD (S/N = 3) was estimated to be in the order of 50-100 ng/L of air using GC with flame ionisation detection (GC-FID). Finally, the device was tested in fully laden containers and was shown to be effective for trapping terpene hydrocarbons indicative of wood packaging.