Point mutations including a novel Pro-197-Phe mutation confer cross-resistance to acetolactate synthase (ALS) inhibiting herbicides in Lactuca serriola in Australia.

BACKGROUND: Control of prickly lettuce has become increasingly difficult for lentil growers in southern Australia because of widespread resistance to common herbicides, a lack of alternative herbicide options and the prolific production of highly mobile seed. This study aimed to quantify acetolactate synthase (ALS)-inhibiting herbicide resistance in the Mid North (MN) and Yorke Peninsula (YP) of South Australia, characterize the resistance mutations present and investigate population structure and gene flow in this species. RESULTS: Resistance was identified in all populations tested, with average survival of 92% to chlorsulfuron and 95% to imazamox + imazapyr. Five different amino acid substitutions were identified at proline 197 of the ALS gene. There was no significant difference in the median lethal dose (LD50 ) between plants with these five different substitutions when treated with metsulfuron-methyl; however, the imidazolinone resistance level was higher in plants with a phenylalanine substitution and lower in plants with a serine. Population structure based on 701 single nucleotide polymorphisms and 271 individuals provided evidence for both independent evolution of the same mutation in different populations, as well as frequent short- to medium-distance dispersal accompanied by occasional long-distance dispersal events. The overall inbreeding coefficient (FIS ) was calculated at 0.5174, indicating an intermediate level of outcrossing despite the cross-pollination experiment showing only low outcrossing. In the structure analyses, most individuals from YP were assigned to a single cluster, whereas most individuals from MN were assigned 50% to each of two clusters, indicating some genetic differences between these two regions, but also evidence for dispersal between them. CONCLUSIONS: Use of imidazolinone herbicides has selected for mutations conferring higher levels of resistance, such as the Pro-197-Phe mutation, and resulted in further spread of resistance in this species. © 2023 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Population structure of ALS-inhibiting herbicide-resistant Sonchus oleraceus in South Australia.

BACKGROUND: Annual sowthistle is a weed that is difficult to control in lentil crops in southern Australia due to a lack of herbicide options, widespread herbicide resistance and prolific production of highly mobile seed. This study investigates herbicide resistance in annual sowthistle in the Mid-North (MN) and Yorke Peninsula (YP) regions of South Australia, identifies and characterizes the mechanisms of acetolactate-synthase (ALS)-inhibitor resistance in this amphidiploid species, and combines this with analyses of population structure and gene flow. RESULTS: ALS-inhibitor-resistant annual sowthistle is widespread across the YP and MN of South Australia and is associated with a variety of Proline-197 mutations of the ALS gene, including leucine, alanine, arginine, serine, threonine and histidine. These mutations were found in different combinations on either of the two copies of the ALS gene. An additional 200 tissue samples were collected from across a single field on the YP and the ALS gene was sequenced for all these individuals. Different ALS-inhibitor resistance profiles were evident between mutation combinations and within mutation combinations, possibly mediated by differing subgenome assortment of the mutations, or altered gene experession of the two ALS homeologs. Population genetics analysis showed evidence of long-distance dispersal, resulting in highly mobile resistance genes, and multiple instances of resistance mutation evolution. CONCLUSIONS: Continuing selection of Sonchus oleraceus populations with ALS-inhibiting herbicides has resulted in the accumulation of additional mutations within the ALS gene. New practices to control herbicide-resistant S. oleraceus should be examined, and control should focus on reducing seed set and dispersal to prevent the spread of emerging cases of resistance. © 2023 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Taxonomic Delineation of the Old World Species Stomphastis thraustica (Lepidoptera: Gracillariidae) Feeding on Jatropha gossypiifolia (Euphorbiaceae) that Was Collected in the New World and Imported as a Biocontrol Agent to Australia.

We provide the identification and species delineation of this biocontrol agent as Stomphastis thraustica (Meyrick in Trans Ent Soc Lond 80(1):107-120, 1908) belonging to the family Gracillariidae. We clarify the distribution pattern of S. thraustica, its host plant preferences, and present taxonomic and molecular diagnoses based on original morphological and genetic data as well as data retrieved from historic literature and genetic databases. Following our own collecting efforts in three continents Africa, South America, and Australia as well as our study of historic museum collection material, we present many new distribution records of S. thraustica for countries and territories in the world including the new discovery of this species in the Neotropical region and we report its introduction in Australia as a biocontrol agent. Using mitogenomic and COI gene data, we clarified that the closest relative of S. thraustica is Stomphastis sp. that occurs in Madagascar and Australia and feeds on the same host plant as S. thraustica - Jatropha gossypiifolia L. (Euphorbiaceae). The molecular sequence divergence in the mitochondrial DNA barcode fragment between these two closely related species S. thraustica and Stomphastis sp. is over 5.7% supporting that they are different species.

Corrigendum to ``Examination of population genetics of the Coconut Rhinoceros Beetle (Oryctes rhinoceros) and the incidence of its biocontrol agent (Oryctes rhinoceros nudivirus) in the South Pacific Islands Current'' [Research in Insect Science 1 (2021) 100015].

[This corrects the article DOI: 10.1016/j.cris.2021.100015.].

A high-quality de novo genome assembly based on nanopore sequencing of a wild-caught coconut rhinoceros beetle (Oryctes rhinoceros).

BACKGROUND: An optimal starting point for relating genome function to organismal biology is a high-quality nuclear genome assembly, and long-read sequencing is revolutionizing the production of this genomic resource in insects. Despite this, nuclear genome assemblies have been under-represented for agricultural insect pests, particularly from the order Coleoptera. Here we present a de novo genome assembly and structural annotation for the coconut rhinoceros beetle, Oryctes rhinoceros (Coleoptera: Scarabaeidae), based on Oxford Nanopore Technologies (ONT) long-read data generated from a wild-caught female, as well as the assembly process that also led to the recovery of the complete circular genome assemblies of the beetle's mitochondrial genome and that of the biocontrol agent, Oryctes rhinoceros nudivirus (OrNV). As an invasive pest of palm trees, O. rhinoceros is undergoing an expansion in its range across the Pacific Islands, requiring new approaches to management that may include strategies facilitated by genome assembly and annotation. RESULTS: High-quality DNA isolated from an adult female was used to create four ONT libraries that were sequenced using four MinION flow cells, producing a total of 27.2 Gb of high-quality long-read sequences. We employed an iterative assembly process and polishing with one lane of high-accuracy Illumina reads, obtaining a final size of the assembly of 377.36 Mb that had high contiguity (fragment N50 length = 12 Mb) and accuracy, as evidenced by the exceptionally high completeness of the benchmarked set of conserved single-copy orthologous genes (BUSCO completeness = 99.1%). These quality metrics place our assembly ahead of the published Coleopteran genomes, including that of an insect model, the red flour beetle (Tribolium castaneum). The structural annotation of the nuclear genome assembly contained a highly-accurate set of 16,371 protein-coding genes, with only 2.8% missing BUSCOs, and the expected number of non-coding RNAs. The number and structure of paralogous genes in a gene family like Sigma GST is lower than in another scarab beetle (Onthophagus taurus), but higher than in the red flour beetle (Tribolium castaneum), which suggests expansion of this GST class in Scarabaeidae. The quality of our gene models was also confirmed with the correct placement of O. rhinoceros among other members of the rhinoceros beetles (subfamily Dynastinae) in a phylogeny based on the sequences of 95 protein-coding genes in 373 beetle species from all major lineages of Coleoptera. Finally, we provide a list of 30 candidate dsRNA targets whose orthologs have been experimentally validated as highly effective targets for RNAi-based control of several beetles. CONCLUSIONS: The genomic resources produced in this study form a foundation for further functional genetic research and management programs that may inform the control and surveillance of O. rhinoceros populations, and we demonstrate the efficacy of de novo genome assembly using long-read ONT data from a single field-caught insect.

Comparisons of chemosensory gene repertoires in human and non-human feeding Anopheles mosquitoes link olfactory genes to anthropophily.

We investigate the genetic basis of anthropophily (human host use) in a non-model mosquito species group, the Anopheles farauti complex from the southwest Pacific. This complex has experienced multiple transitions from anthropophily to zoophily, contrasting with well-studied systems (the global species Aedes aegypti and the African Anopheles gambiae complex) that have evolved to be specialist anthropophiles. By performing tests of selection and assessing evolutionary patterns for >200 olfactory genes from nine genomes, we identify several candidate genes associated with differences in anthropophily in this complex. Based on evolutionary patterns (phylogenetic relationships, fixed amino acid differences, and structural differences) as well as results from selection analyses, we identify numerous genes that are likely to play an important role in mosquitoes' ability to detect humans as hosts. Our findings contribute to the understanding of the evolution of insect olfactory gene families and mosquito host preference as well as having potential applied outcomes.

The complete mitochondrial genome sequence of Oryctes rhinoceros (Coleoptera: Scarabaeidae) based on long-read nanopore sequencing.

BACKGROUND: The coconut rhinoceros beetle (CRB, Oryctes rhinoceros) is a severe and invasive pest of coconut and other palms throughout Asia and the Pacific. The biocontrol agent, Oryctes rhinoceros nudivirus (OrNV), has successfully suppressed O. rhinoceros populations for decades but new CRB invasions started appearing after 2007. A single-SNP variant within the mitochondrial cox1 gene is used to distinguish the recently-invading CRB-G lineage from other haplotypes, but the lack of mitogenome sequence for this species hinders further development of a molecular toolset for biosecurity and management programmes against CRB. Here we report the complete circular sequence and annotation for CRB mitogenome, generated to support such efforts. METHODS: Sequencing data were generated using long-read Nanopore technology from genomic DNA isolated from a CRB-G female. The mitogenome was assembled with Flye v.2.5, using the short-read Illumina sequences to remove homopolymers with Pilon, and annotated with MITOS. Independently-generated transcriptome data were used to assess the O. rhinoceros mitogenome annotation and transcription. The aligned sequences of 13 protein-coding genes (PCGs) (with degenerate third codon position) from O. rhinoceros, 13 other Scarabaeidae taxa and two outgroup taxa were used for the phylogenetic reconstruction with the Maximum likelihood (ML) approach in IQ-TREE and Bayesian (BI) approach in MrBayes. RESULTS: The complete circular mitogenome of O. rhinoceros is 20,898 bp in length, with a gene content canonical for insects (13 PCGs, two rRNA genes, and 22 tRNA genes), as well as one structural variation (rearrangement of trnQ and trnI) and a long control region (6,204 bp). Transcription was detected across all 37 genes, and interestingly, within three domains in the control region. ML and BI phylogenies had the same topology, correctly grouping O. rhinoceros with one other Dynastinae taxon, and recovering the previously reported relationship among lineages in the Scarabaeidae. In silico PCR-RFLP analysis recovered the correct fragment set that is diagnostic for the CRB-G haplogroup. These results validate the high-quality of the O. rhinoceros mitogenome sequence and annotation.

Examination of population genetics of the Coconut Rhinoceros Beetle (Oryctes rhinoceros) and the incidence of its biocontrol agent (Oryctes rhinoceros nudivirus) in the South Pacific Islands.

Recently, incursions of the Coconut rhinoceros beetle (CRB), Oryctes rhinoceros, have been detected in south Pacific countries that were previously free of the pest. It has been suggested that this range expansion is related to an O. rhinoceros haplotype that is reported to show reduced susceptibility to the well-established classical biocontrol agent, Oryctes rhinoceros nudivirus (OrNV). We investigated O. rhinoceros population genetics and the OrNV status of specimens collected in Fiji, New Caledonia, Papua New Guinea (PNG), Samoa, Solomon Islands, Tonga, Vanuatu and the Philippines. Based on the sequence of the mitochondrial CoxI gene, we found three major mitochondrial haplotype groups (CRB-G, CRB-PNG and CRB-S) across the region. Haplotype diversity varied between and within countries and a high incidence of OrNV infection was detected in all haplotypes wherever they occurred. The O. rhinoceros population in some countries was monotypic and all individuals tested belonged to a single haplotype group. However, in Samoa we detected CRB-S and CRB-PNG and in Solomon Islands we detected all three haplotype groups. Genotyping-by-Sequencing (GBS) showed genetic differentiation in the O. rhinoceros nuclear genome across populations on different islands and provided evidence for gene flow, resulting in a well-mixed population, despite the presence of different CoxI haplotypes in Solomon Islands. Evidence of admixture was also detected on both islands of Samoa. The current CoxI based method is not a reliable diagnostic marker for phenotypic traits, especially in countries such as Solomon Islands where the mitochondrial haplotypes have come back into sympatry and are mixed. To identify possible mechanisms of resistance to OrNV, further molecular analyses O. rhinoceros in response to virus infection is required. To improve biological control of O. rhinoceros, such analyses will need to be combined with an improved understanding of the population genetics of the pest and the evolutionary history of OrNV in the region.

Migration dynamics of an important rice pest: The brown planthopper (Nilaparvata lugens) across Asia-Insights from population genomics.

INTRODUCTION: Brown planthoppers (Nilaparvata lugens) are the most serious insect pests of rice, one of the world's most important staple crops. They reproduce year-round in the tropical parts of their distribution, but cannot overwinter in the temperate areas where they occur, and invade seasonally from elsewhere. Decades of research have not revealed their source unambiguously. METHODS AND RESULTS: We sequenced the genomes of brown planthopper populations from across temperate and tropical parts of their distribution and show that the Indochinese peninsula is the major source of migration into temperate China. The Philippines, once considered a key source, is not significant, with little evidence for their migration into China. We find support for immigration from the west of China contributing to these regional dynamics. DISCUSSION: The lack of connectivity between the Philippine population and the mainland Chinese populations explains the different evolution of Imidacloprid resistance in these populations. This study highlights the promise of whole-genome sequence data to understand migration when gene flow is high-a situation that has been difficult to resolve using traditional genetic markers.

Multiple invasions of a generalist herbivore-Secondary contact between two divergent lineages of Nezara viridula Linnaeus in Australia.

The presence of distinct evolutionary lineages within herbivorous pest insect taxa requires close attention. Scientific understanding, biosecurity planning and practice, and pest management decision-making each suffer when such situations remain poorly understood. The pest bug Nezara viridula Linnaeus has been recorded from numerous host plants and has two globally distributed mitochondrial (mtDNA) lineages. These mtDNA lineages co-occur in few locations globally, and the consequences of their divergence and recent secondary contact have not been assessed. We present evidence that both mtDNA lineages of N. viridula are present in Australia and their haplotype groups have a mostly separate distribution from one another. The north-western population has only Asian mtDNA haplotypes, and the population with an eastern distribution is characterized mostly by European mtDNA haplotypes. Haplotypes of both lineages were detected together at only one site in the north of eastern Australia, and microsatellite data indicate that this secondary contact has resulted in mating across the lineages. Admixture and the movement of mtDNA haplotypes outside of this limited area of overlap has not, however, been extensive. Some degree of mating incompatibility or differences in the climatic requirements and tolerances of the two lineages, and perhaps a combination of these influences, might limit introgression and the movement of individuals, but this needs to be tested. This work provides the foundation for further ecological investigation of the lineages of N. viridula, particularly the consequences of admixture on the ecology of this widespread pest. We propose that for now, the Asian and European lineages of N. viridula would best be investigated as subspecies, so that "pure" and admixed populations of this bug can each be considered directly with respect to management and research priorities.

The recognition concept and genetic approaches to interpreting species.

1. Introduction. 2. The Specific-mate Recognition System (SMRS). 3. The Genetic Change As sociated with Speciation.

Gene flow across host-associated populations of the rice stem borer Chilo suppressalis Walker (Lepidoptera: Crambidae): implications for Bt resistance management in rice.

BACKGROUND: The rice stem borer, Chilo suppressalis, is a serious pest of rice, but also damages an aquatic vegetable, water oats (Zizania latifolia Turcz.). The time at which mating occurs is different between populations of rice stem borer associated with rice and those associated with water-oats, which suggests that undetected cryptic species may be associated with these plant hosts. If true, this would have significant management implications. This study is the first empirical test of this idea, using population genetic tools from two sampling cohorts. We genotyped 320 rice stem borer individuals from 2014, collected from rice and water-oats across five locations (where they exist in sympatry), using seven microsatellite loci. RESULTS: We found no genetic structuring associated with host plant species. On water oats, some rice stem borers were found that had a similar mating time to the rice population, so in 2016, a second cohort of samples was screened by their timing of mating to get 'pure rice feeders' and 'pure water oats feeders'. These samples were genotyped with microsatellites, mitochondrial DNA (mtDNA) (COI and COII), and a nuclear gene (EF1-α). Our mtDNA data suggest a relatively low amount of population subdivision associated with plant host, but the microsatellite data revealed no such genetic structure, and we were only able to identify one haplotype of EF1-α. CONCLUSIONS: Our results indicate gene flow between rice and water oats populations of rice stem borer, indicating that water oats will likely provide a refuge for resistance management of Bt rice. © 2019 Society of Chemical Industry.

Origins, Divergence, and Contrasting Invasion History of the Sweet Potato Weevil Pests Cylas formicarius (Coleoptera: Brentidae) and Euscepes batatae (Coleoptera: Curculionidae) in the Asia-Pacific.

Cylas formicarius F. and Euscepes batatae Waterhouse are the most damaging sweet potato insect pests globally. Both weevils are thought to have invaded the Pacific alongside the movement of sweet potato (Ipomoea batatas (L.) Lam. Convolvulaceae), with C. formicarius having originated in India and E. batatae in Central or South America. Here we compare the genetic relationships between populations of the pests, primarily in the Asia-Pacific, to understand better their contemporary population structure and their historical movement relative to that of sweet potato. Cylas formicarius has divergent mitochondrial lineages that indicate a more complex biogeographic and invasive history than is presently assumed for this insect, suggesting it was widespread across the Asia-Pacific before the arrival of sweet potato. Cylas formicarius must have originally fed on Ipomoea species other than I. batatas but the identity of these species is presently unknown. Cylas formicarius was formerly designated as three species or subspecies and the genetic data presented here suggests that these designations should be reinvestigated. Euscepes batatae has very low genetic diversity which is consistent with its historical association with sweet potato and a recent introduction to the Asia-Pacific from the Americas. The distribution of E. batatae may be narrower than that of C. formicarius in the Asia-Pacific because it has relied relatively more on human-assisted movement. Consequently, E. batatae may become more widespread in the future. Investigating the invasion history of both species will help to understand the probability and nature of future invasions.

Herbicide resistance evolution can be tamed by diversity in irrigated Australian cotton: a multi-species, multi-herbicide modelling approach.

BACKGROUND: Because herbicide resistance evolves in very large populations over periods of many years, modelling is an important tool for investigating the dynamics of the problem. The Diversity model tracks the simultaneous evolution of resistance to multiple herbicides, using multiple genetic pathways, in several weed species at once. Tracking multiple species and simultaneous resistances is an important development in resistance modelling. We used the Diversity model to test weed management strategies for new cropping cotton varieties with multiple herbicide tolerances ('triple-stacked' varieties), in an Australian context. RESULTS: The diversity required for long-term control of three key weeds in Australian cotton goes beyond using three herbicides, especially where there is already a substantial background of existing resistance to one or more of these herbicides. Assuming some glyphosate resistance is already present, simulations showed that glyphosate-resistant summer grass populations reach 20 000 seeds m-2 within 12 years using the triple-stack herbicides (glyphosate, glufosinate and dicamba) and a minimum of other tactics. Adding three pre-emergent modes of action plus cultivation to the system effectively controls glyphosate-resistant grasses for over 30 years. In conditions where resistance genes are as frequent as 1 in 100, however, highly fecund weeds such as Conyza bonariensis are hard to control beyond 15 years even with very highly diverse management. CONCLUSIONS: Stacked herbicide tolerances in new crop varieties offers potential for increased herbicide diversity, but existing glyphosate-resistant weed populations need substantial extra management beyond what a glyphosate/glufosinate/dicamba resistance stack provides. More diverse systems can provide robust management over 30 years in the absence of very high levels of background resistance to other herbicides. © 2018 Society of Chemical Industry.

Complete chloroplast genome of glyphosate resistant Sonchus oleraceus L. from Australia, with notes on the small single copy (SSC) region orientation.

Sonchus oleraceus, common sowthistle, is an asteraceous weed in Australian agricultural systems and has recently developed resistance to glyphosate. We present the complete chloroplast sequence of S. oleracueus reconstructed from Illumina whole genome shotgun sequencing. This is the first complete chloroplast genome available for the genus Sonchus. The complete chloroplast sequence is 151,808 bp long. A Bayesian phylogeny of the chloroplast coding regions of the tribe Cichorieae (Asteraceae) is presented. The S. oleraceus chloroplast genome is deposited at GenBank under accession number MG878405.

The complete mitochondrial genome of the five-lined cardinalfish Cheilodipterus quinquelineatus (Apogonidae).

Cheilopdipterus quinquelineatus (Apogonidae) is an interesting species for investigating processes driving population divergence in marine system due to its wide distribution and life-history traits. However, to date, there is a limited genetic resource available for this species, or the family Apogonidae as exemplified by the availability of only two mitogenomes. In this study, we assembled the whole mitochondrial genome of this species yielding a 16,537 bp circular assembly composed of the typical vertebrate mitochondrial features. Phylogenetic inference of the supraordinal group of C. quiquelineatus showed monophyly of the major families.

The complete mitochondrial genome of Bathygobius cocosensis (Perciformes, Gobiidae).

In this study, we sequenced the full mitochondrial genome of Bathygobius cocosensis, an abundant intertidal fish species, which may provide insights into the evolutionary genetics of chaotic genetic patchiness and range expansion in marine systems. The mitochondrial genome is 16,692 bp, and contains 13 protein-coding genes along with 22 tRNA and 2 rRNA genes and a D-loop region, arranged similarly to other Gobiidae species. A Bayesian phylogeny of Gobiidae species indicates close relationships to the genus Glossogobius. The B. cocosensis mitochondrial genome is now available through GenBank (Accession = MG704838).

The complete chloroplast genome of the invasive fern Lygodium microphyllum (Cav.) R. Br.

The Old World climbing fern, Lygodium microphyllum, is a rapidly spreading environmental weed in Florida, United States. We reconstructed the complete chloroplast genome of L. microphyllum from Illumina whole-genome shotgun sequencing, and investigate the phylogenetic placement of this species within the Leptosporangiate ferns. The chloroplast genome is 158,891 bp and contains 87 protein-coding genes, four rRNA genes, and 27 tRNA genes. Thirty-three genes contained internal stop codons, a common feature in Leptosporangiate fern genomes. The L. microphyllum genome has been deposited in GenBank under accession number MG761729.

Gene expression in response to glyphosate treatment in fleabane (Conyza bonariensis) - glyphosate death response and candidate resistance genes.

BACKGROUND: This study takes a whole-transcriptome approach to assess gene expression changes in response to glyphosate treatment in glyphosate-resistant fleabane. We assessed gene expression changes in both susceptible and resistant lines so that the glyphosate death response could be quantified, and constitutively expressed candidate resistance genes identified. There are three copies of the glyphosate target site (5-enolpyruvylshikimate-3-phosphate; EPSPS) gene in Conyza and because Conyza bonariensis is allohexaploid, there is a baseline nine copies of the gene in any individual. RESULTS: Many genes were differentially expressed in response to glyphosate treatment. Known resistance mutations are present in EPSPS2 but they are present in a glyphosate-susceptible line as well as resistant lines and therefore not sufficient to confer resistance. EPSPS1 is expressed four times more than EPSPS2, further reducing the overall contribution of these mutations. CONCLUSION: We demonstrate that glyphosate resistance in C. bonariensis is not the result of EPSPS mutations or overexpression, but due to a non-target-site mechanism. A large number of genes are affected by glyphosate treatment. We present a list of candidate non-target-site-resistance (NTSR) genes in fleabane for future studies into these mechanisms. © 2017 Society of Chemical Industry.