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.

Species-wide genomics of kakapo provides tools to accelerate recovery.

The kakapo is a critically endangered, intensively managed, long-lived nocturnal parrot endemic to Aotearoa New Zealand. We generated and analysed whole-genome sequence data for nearly all individuals living in early 2018 (169 individuals) to generate a high-quality species-wide genetic variant callset. We leverage extensive long-term metadata to quantify genome-wide diversity of the species over time and present new approaches using probabilistic programming, combined with a phenotype dataset spanning five decades, to disentangle phenotypic variance into environmental and genetic effects while quantifying uncertainty in small populations. We find associations for growth, disease susceptibility, clutch size and egg fertility within genic regions previously shown to influence these traits in other species. Finally, we generate breeding values to predict phenotype and illustrate that active management over the past 45 years has maintained both genome-wide diversity and diversity in breeding values and, hence, evolutionary potential. We provide new pathways for informing future conservation management decisions for kakapo, including prioritizing individuals for translocation and monitoring individuals with poor growth or high disease risk. Overall, by explicitly addressing the challenge of the small sample size, we provide a template for the inclusion of genomic data that will be transformational for species recovery efforts around the globe.

Germline progenitors and oocyte production in the honeybee queen ovary.

Understanding the reproduction of honeybee queens is crucial to support populations of this economically important insect. Here we examine the structure of the honeybee ovary to determine the nature of the germline progenitors in the ovary. Using a panel of marker genes that mark somatic or germline tissue in other insects we determine which cells in the honeybee ovary are somatic and which germline. We examine patterns of cell division and demonstrate that, unlike Drosophila, there is no evidence of single germline stem cells that provide the germline in honeybees. Germline progenitors are clustered in groups of 8 cells, joined by a polyfusome, and collections of these, in each ovariole, appear to maintain the germline during reproduction. We also show that these 8-cell clusters can divide and that their division occurs such that the numbers of germline progenitors are relatively constant over the reproductive life of queen honeybees. This information helps us to understand the diversity of structures in insect reproduction, and provide information to better support honeybee reproduction.

The promise and challenges of characterizing genome-wide structural variants: A case study in a critically endangered parrot.

There is growing interest in the role of structural variants (SVs) as drivers of local adaptation and speciation. From a biodiversity genomics perspective, the characterization of genome-wide SVs provides an exciting opportunity to complement single nucleotide polymorphisms (SNPs). However, little is known about the impacts of SV discovery and genotyping strategies on the characterization of genome-wide SV diversity within and among populations. Here, we explore a near whole-species resequence data set, and long-read sequence data for a subset of highly represented individuals in the critically endangered kakapo (Strigops habroptilus). We demonstrate that even when using a highly contiguous reference genome, different discovery and genotyping strategies can significantly impact the type, size and location of SVs characterized genome-wide. Further, we found that the mean number of SVs in each of two kakapo lineages differed both within and across generations. These combined results suggest that genome-wide characterization of SVs remains challenging at the population-scale. We are optimistic that increased accessibility to long-read sequencing and advancements in bioinformatic approaches including multireference approaches like genome graphs will alleviate at least some of the challenges associated with resolving SV characteristics below the species level. In the meantime, we address caveats, highlight considerations, and provide recommendations for the characterization of genome-wide SVs in biodiversity genomic research.

Hidden impacts of conservation management on fertility of the critically endangered kakapo.

Background: Animal conservation often requires intensive management actions to improve reproductive output, yet any adverse effects of these may not be immediately apparent, particularly in threatened species with small populations and long lifespans. Hand-rearing is an example of a conservation management strategy which, while boosting populations, can cause long-term demographic and behavioural problems. It is used in the recovery of the critically endangered kakapo (Strigops habroptilus), a flightless parrot endemic to New Zealand, to improve the slow population growth that is due to infrequent breeding, low fertility and low hatching success. Methods: We applied Bayesian mixed models to examine whether hand-rearing and other factors were associated with clutch fertility in kakapo. We used projection predictive variable selection to compare the relative contributions to fertility from the parents' rearing environment, their age and previous copulation experience, the parental kinship, and the number of mates and copulations for each clutch. We also explored how the incidence of repeated copulations and multiple mates varied with kakapo density. Results: The rearing status of the clutch father and the number of mates and copulations of the clutch mother were the dominant factors in predicting fertility. Clutches were less likely to be fertile if the father was hand-reared compared to wild-reared, but there was no similar effect for mothers. Clutches produced by females copulating with different males were more likely to be fertile than those from repeated copulations with one male, which in turn had a higher probability of fertility than those from a single copulation. The likelihood of multiple copulations and mates increased with female:male adult sex ratio, perhaps as a result of mate guarding by females. Parental kinship, copulation experience and age all had negligible associations with clutch fertility. Conclusions: These results provide a rare assessment of factors affecting fertility in a wild threatened bird species, with implications for conservation management. The increased fertility due to multiple mates and copulations, combined with the evidence for mate guarding and previous results of kakapo sperm morphology, suggests that an evolutionary mechanism exists to optimise fertility through sperm competition in kakapo. The high frequency of clutches produced from single copulations in the contemporary population may therefore represent an unnatural state, perhaps due to too few females. This suggests that opportunity for sperm competition should be maximised by increasing population densities, optimising sex ratios, and using artificial insemination. The lower fertility of hand-reared males may result from behavioural defects due to lack of exposure to conspecifics at critical development stages, as seen in other taxa. This potential negative impact of hand-rearing must be balanced against the short-term benefits it provides.

Honeybee queen mandibular pheromone induces a starvation response in Drosophila melanogaster.

Eusocial insect societies are defined by the reproductive division of labour, a social structure that is generally enforced by the reproductive dominant(s) or 'queen(s)'. Reproductive dominance is maintained through behavioural dominance or production of queen pheromones, or a mixture of both. Queen mandibular pheromone (QMP) is a queen pheromone produced by queen honeybees (Apis mellifera) which represses reproduction in worker honeybees. How QMP acts to repress worker reproduction, the mechanisms by which this repression is induced, and how it has evolved this activity, remain poorly understood. Surprisingly, QMP is capable of repressing reproduction in non-target arthropods. Here we show that in Drosophila melanogaster QMP treatment mimics the starvation response, disrupting reproduction. QMP exposure induces an increase in food consumption and activation of checkpoints in the ovary that reduce fecundity and depresses insulin signalling. The magnitude of these effects is indistinguishable between QMP-treated and starved individuals. As QMP triggers a starvation response in an insect diverged from honeybees, we propose that QMP originally evolved by co-opting nutrition signalling pathways to regulate reproduction.

The venom composition and parthenogenesis mechanism of the parasitoid wasp Microctonus hyperodae, a declining biocontrol agent.

A biocontrol system in New Zealand using the endoparasitoid Microctonus hyperodae is failing, despite once being one of the most successful examples of classical biocontrol worldwide. Though it is of significant economic importance as a control agent, little is known about the genetics of M. hyperodae. In this study, RNA-seq was used to characterise two key traits of M. hyperodae in this system, the venom, critical for the initial success of biocontrol, and the asexual reproduction mode, which influenced biocontrol decline. Expanded characterisation of M. hyperodae venom revealed candidates involved in manipulating the host environment to source nutrition for the parasitoid egg, preventing a host immune response against the egg, as well as two components that may stimulate the host's innate immune system. Notably lacking from the venom-specific expression list was calreticulin, as it also had high expression in the ovaries. In-situ hybridisation revealed this ovarian expression was localised to the follicle cells, which may result in the deposition of calreticulin into the egg exochorion. Investigating the asexual reproduction of M. hyperodae revealed core meiosis-specific genes had conserved expression patterns with the highest expression in the ovaries, suggesting M. hyperodae parthenogenesis involves meiosis and that the potential for sexual reproduction may have been retained. Upregulation of genes involved in endoreduplication provides a potential mechanism for the restoration of diploidy in eggs after meiosis.

Two new tardigrade genera from New Zealand's Southern Alp glaciers display morphological stasis and parallel evolution.

Tardigrada is an invertebrate phylum that often constitutes a dominant micrometazoan group on glaciers worldwide. We investigated tardigrades residing in surface ice above the equilibrium line altitude (ELA) on three temperate glaciers of New Zealand's Southern Alps. Morphological, morphometric and multilocus DNA analyses (CO1, 18S rRNA, 28S rRNA, ITS-2) revealed two new genera comprising four species, of which two are formally described here: Kopakaius gen. nov. nicolae sp. nov. and Kararehius gen. nov. gregorii sp. nov. The former is represented by three genetically distinct phyletic lineages akin to species. According to CO1, Kopakaius gen. nov. nicolae sp. nov. inhabits Whataroa Glacier only while the remaining two Kopakaius species occur on Fox and Franz Joseph Glaciers, suggesting low dispersal capabilities. Although morphological characteristics of the new genera could indicate affinity with the subfamily Itaquasconinae, phylogenetic analysis placed them confidently in the subfamily Diphasconinae. Kopakaius gen. nov. lack placoids in the pharynx similar with some Itaquasconinae, whereas dark pigmentation and claw shape aligns them with the glacier-obligate genus, Cryobiotus (subfamily Hypsibiinae), which is an example of parallel evolution. The second genus, Kararehius gen nov. could be classified as Adropion-like (subfamily Itaquasconinae), but differs greatly by genetics (placed in the subfamily Diphasconinae) as well as morphology (e.g., lack of septulum), exemplify deep stasis in Hypsibiidae. Our results suggest that glacier fragmentation during the Pleistocene triggered tardigrade speciation, making it a suitable model for studies on allopatric divergence in glacier meiofauna.

A single fungal strain was the unexpected cause of a mass aspergillosis outbreak in the world's largest and only flightless parrot.

Kakapo are a critically endangered species of parrots restricted to a few islands off the coast of New Zealand. Kakapo are very closely monitored, especially during nesting seasons. In 2019, during a highly successful nesting season, an outbreak of aspergillosis affected 21 individuals and led to the deaths of 9, leaving a population of only 211 kakapo. In monitoring this outbreak, cultures of aspergillus were grown, and genome sequenced. These sequences demonstrate that, very unusually for an aspergillus outbreak, a single strain of aspergillus caused the outbreak. This strain was found on two islands, but only one had an outbreak of aspergillosis; indicating that the strain was necessary, but not sufficient, to cause disease. Our analysis provides an understanding of the 2019 outbreak and provides potential ways to manage such events in the future.

Evolution: Hidden homologies may underpin the diversity of arthropods.

Arthropods are remarkable for the diversity of their exoskeletons. A new study shows that these structures, from crustacean carapaces to insect wings, may be homologous and derived from hidden developmental structures preserved through arthropod evolution.

Noggin proteins are multifunctional extracellular regulators of cell signaling.

Noggin is an extracellular cysteine knot protein that plays a crucial role in vertebrate dorsoventral patterning. Noggin binds and inhibits the activity of bone morphogenetic proteins via a conserved N-terminal clip domain. Noncanonical orthologs of Noggin that lack a clip domain ("Noggin-like" proteins) are encoded in many arthropod genomes and are thought to have evolved into receptor tyrosine kinase ligands that promote Torso/receptor tyrosine kinase signaling rather than inhibiting bone morphogenic protein signaling. Here, we examined the molecular function of noggin/noggin-like genes (ApNL1 and ApNL2) from the arthropod pea aphid using the dorso-ventral patterning of Xenopus and the terminal patterning system of Drosophila to identify whether these proteins function as bone morphogenic protein or receptor tyrosine kinase signaling regulators. Our findings reveal that ApNL1 from the pea aphid can regulate both bone morphogenic protein and receptor tyrosine kinase signaling pathways, and unexpectedly, that the clip domain is not essential for its antagonism of bone morphogenic protein signaling. Our findings indicate that ancestral noggin/noggin-like genes were multifunctional regulators of signaling that have specialized to regulate multiple cell signaling pathways during the evolution of animals.

Phenotypic Plasticity: What Has DNA Methylation Got to Do with It?

How does one genome give rise to multiple, often markedly different, phenotypes in response to an environmental cue? This phenomenon, known as phenotypic plasticity, is common amongst plants and animals, but arguably the most striking examples are seen in insects. Well-known insect examples include seasonal morphs of butterfly wing patterns, sexual and asexual reproduction in aphids, and queen and worker castes of eusocial insects. Ultimately, we need to understand how phenotypic plasticity works at a mechanistic level; how do environmental signals alter gene expression, and how are changes in gene expression translated into novel morphology, physiology and behaviour? Understanding how plasticity works is of major interest in evolutionary-developmental biology and may have implications for understanding how insects respond to global change. It has been proposed that epigenetic mechanisms, specifically DNA methylation, are the key link between environmental cues and changes in gene expression. Here, we review the available evidence on the function of DNA methylation of insects, the possible role(s) for DNA methylation in phenotypic plasticity and also highlight key outstanding questions in this field as well as new experimental approaches to address these questions.

The Nasonia pair-rule gene regulatory network retains its function over 300 million years of evolution.

Insect segmentation is a well-studied and tractable system with which to investigate the genetic regulation of development. Though insects segment their germband using a variety of methods, modelling work implies that a single gene regulatory network can underpin the two main types of insect segmentation. This means limited genetic changes are required to explain significant differences in segmentation mode between different insects. This idea needs to be tested in a wider variety of species, and the nature of the gene regulatory network (GRN) underlying this model has not been tested. Some insects, e.g. Nasonia vitripennis and Apis mellifera segment progressively, a pattern not examined in previous studies of this segmentation model, producing stripes at different times progressively through the embryo, but not from a segment addition zone. Here, we aim to understand the GRNs patterning Nasonia using a simulation-based approach. We found that an existing model of Drosophila segmentation ( Clark, 2017) can be used to recapitulate the progressive segmentation of Nasonia, if provided with altered inputs in the form of expression of the timer genes Nv-caudal and Nv-odd paired. We predict limited topological changes to the pair-rule network and show, by RNAi knockdown, that Nv-odd paired is required for morphological segmentation. Together this implies that very limited changes to the Drosophila network are required to simulate Nasonia segmentation, despite significant differences in segmentation modes, implying that Nasonia use a very similar version of an ancestral GRN used by Drosophila, which must therefore have been conserved for at least 300 million years.

Genomic signatures of parallel alpine adaptation in recently evolved flightless insects.

Natural selection along elevational gradients has potential to drive predictable adaptations across distinct lineages, but the extent of such repeated evolution remains poorly studied for many widespread alpine taxa. We present parallel genomic analyses of two recently evolved flightless alpine insect lineages to test for molecular signatures of repeated alpine adaptation. Specifically, we compare low-elevation vs. alpine stonefly ecotypes from parallel stream populations in which flightless upland ecotypes have been independently derived. We map 67,922 polymorphic genetic markers, generated across 176 Zelandoperla fenestrata specimens from two independent alpine stream populations in New Zealand's Rock and Pillar Range, to a newly developed plecopteran reference genome. Genome-wide scans revealed 31 regions with outlier single nucleotide polymorphisms (SNPs) differentiating lowland vs. alpine ecotypes in Lug Creek, and 37 regions with outliers differentiating ecotypes in Six Mile Creek. Of these regions, 13% (8/60) yielded outlier SNPs across both within-stream ecotype comparisons, implying comparable genomic shifts contribute to this repeated alpine adaptation. Candidate genes closely linked to repeated outlier regions include several with documented roles in insect wing-development (e.g., dishevelled), suggesting that they may contribute to repeated alpine wing reduction. Additional candidate genes have been shown to influence insect fecundity (e.g., ovo) and lifespan (e.g., Mrp4), implying that they might contribute to life history differentiation between upland and lowland ecotypes. Additional outlier genes have potential roles in the evolution of reproductive isolation among ecotypes (hedgehog and Desaturase 1). These results demonstrate how replicated outlier tests across independent lineages can potentially contribute to the discovery of genes underpinning repeated adaptation.

Evolution and genomic organization of the insect sHSP gene cluster and coordinate regulation in phenotypic plasticity.

BACKGROUND: Conserved syntenic gene complexes are rare in Arthropods and likely only retained due to functional constraint. Numerous sHSPs have been identified in the genomes of insects, some of which are located clustered in close proximity. Previous phylogenetic analyses of these clustered sHSP have been limited to a small number of holometabolous insect species and have not determined the pattern of evolution of the clustered sHSP genes (sHSP-C) in insect or Arthropod lineages. RESULTS: Using eight genomes from representative insect orders and three non-insect arthropod genomes we have identified that a syntenic cluster of sHSPs (sHSP-C) is a hallmark of most Arthropod genomes. Using 11 genomes from Hymenopteran species our phylogenetic analyses have refined the evolution of the sHSP-C in Hymenoptera and found that the sHSP-C is order-specific with evidence of birth-and-death evolution in the hymenopteran lineage. Finally we have shown that the honeybee sHSP-C is co-ordinately expressed and is marked by genomic features, including H3K27me3 histone marks consistent with coordinate regulation, during honeybee ovary activation. CONCLUSIONS: The syntenic sHSP-C is present in most insect genomes, and its conserved coordinate expression and regulation implies that it is an integral genomic component of environmental response in arthropods.

Five animal phyla in glacier ice reveal unprecedented biodiversity in New Zealand's Southern Alps.

Glacier ice is an extreme environment in which most animals cannot survive. Here we report the colonization of high elevation, climate-threatened glaciers along New Zealand's southwestern coast by species of Arthropoda, Nematoda, Platyhelminthes, Rotifera and Tardigrada. Based on DNA barcoding and haplotype-inferred evidence for deep genetic variability, at least 12 undescribed species are reported, some of which have persisted in this niche habitat throughout the Pleistocene. These findings identify not only an atypical biodiversity hotspot but also highlight the adaptive plasticity of microinvertebrate Animalia.

Population genomics of the critically endangered kakapo.

The kakapo is a flightless parrot endemic to New Zealand. Once common in the archipelago, only 201 individuals remain today, most of them descending from an isolated island population. We report the first genome-wide analyses of the species, including a high-quality genome assembly for kakapo, one of the first chromosome-level reference genomes sequenced by the Vertebrate Genomes Project (VGP). We also sequenced and analyzed 35 modern genomes from the sole surviving island population and 14 genomes from the extinct mainland population. While theory suggests that such a small population is likely to have accumulated deleterious mutations through genetic drift, our analyses on the impact of the long-term small population size in kakapo indicate that present-day island kakapo have a reduced number of harmful mutations compared to mainland individuals. We hypothesize that this reduced mutational load is due to the island population having been subjected to a combination of genetic drift and purging of deleterious mutations, through increased inbreeding and purifying selection, since its isolation from the mainland ∼10,000 years ago. Our results provide evidence that small populations can survive even when isolated for hundreds of generations. This work provides key insights into kakapo breeding and recovery and more generally into the application of genetic tools in conservation efforts for endangered species.

Including Digital Sequence Data in the Nagoya Protocol Can Promote Data Sharing.

The Nagoya Protocol (NP), a legal framework under the Convention on Biological Diversity (CBD), formalises fair and equitable sharing of benefits arising from biological diversity. It encompasses biological samples and associated indigenous knowledge, with equitable return of benefits to those providing samples. Recent proposals that the use of digital sequence information (DSI) derived from samples should also require benefit-sharing under the NP have raised concerns that this might hamper research progress. Here, we propose that formalised benefit-sharing for biological data use can increase willingness to participate in research and share data, by ensuring equitable collaboration between sample providers and researchers, and preventing exploitative practices. Three case studies demonstrate how equitable benefit-sharing agreements might build long-term collaborations, furthering research for global benefits.