Short Communication |
Corresponding author: Paul K. Abram ( paul.abram@canada.ca ) Academic editor: Richard Shaw
© 2023 Paul K. Abram, Tyler D. Nelson, Valerie Marshall, Tara D. Gariepy, Tim Haye, Jinping Zhang, Tracy Hueppelsheuser, Susanna Acheampong, Chandra E. Moffat.
This is an open access article distributed under the terms of the CC0 Public Domain Dedication.
Citation:
Abram PK, Nelson TD, Marshall V, Gariepy TD, Haye T, Zhang J, Hueppelsheuser T, Acheampong S, Moffat CE (2023) Genetic relationships among laboratory lines of the egg parasitoid Trissolcus japonicus from native and adventive populations. NeoBiota 82: 145-161. https://doi.org/10.3897/neobiota.82.97881
|
Candidate biological control agents of invasive insect pests are increasingly being found in new geographic regions as a result of unintentional introductions. However, testing the degree of genetic differentiation among adventive and native-range populations of these agents is rarely done. We used reduced-representation sequencing of genomic DNA to investigate the relationships among laboratory lines of Trissolcus japonicus (Ashmead) (Hymenoptera, Scelionidae), an egg parasitoid and biological control agent of the brown marmorated stink bug, Halyomorpha halys (Stål) (Hemiptera, Pentatomidae). We compared sequences from multiple adventive populations in North America (Canada, USA) and Europe (Switzerland) with populations sourced from part of its native range in China. We found considerably more genetic variation among lines sourced from adventive populations than among those within native populations. In the Pacific Northwest of North America (British Columbia, Canada and Washington State, USA), we found preliminary evidence of three distinct genetic clusters, two of which were highly dissimilar from all other lines we genotyped. In contrast, we found that other adventive lines with close geographic proximity (two from Ontario, Canada, three from Switzerland) had limited genetic variation. These findings provide a basis for testing biological differences among lines that will inform their use as biological control agents, and provide evidence to support a hypothesis of several independent introductions of T. japonicus in western North America from different source areas.
classical biological control, ddRAD, Halyomorpha halys, Scelionidae, unintentional biological control
There are now numerous documented instances where natural enemies of invasive insects have been discovered following the establishment of their host or prey species, presumably as a result of unintentional introductions (
In addition to determining invasion histories, molecular techniques could determine relationships among laboratory cultures of adventive natural enemy populations that have been sourced from different regions. When applied to these ‘living genetic resources’, genetic analyses could identify distinct populations that may differ in biological attributes (e.g., host range, life history, climate tolerance) that affect establishment success and suitability as biological control agents, and could inform future introductions (e.g., to increase genetic diversity of unintentionally introduced populations) or redistributions within new geographic areas that aim to improve biological control outcomes (
Trissolcus japonicus (Ashmead) (Hymenoptera, Scelionidae) is an egg parasitoid of the brown marmorated stink bug Halyomorpha halys (Stål) (Hemiptera, Pentatomidae) whose presumed native range includes China, southeastern Russia, South Korea, Japan, and Taiwan (
Between 2017 and 2020, we established 19 laboratory lines of T. japonicus in a containment facility certified by the Canadian Food Inspection Agency at Agriculture and Agri-Food Canada’s Agassiz Research and Development Centre (Agassiz, British Columbia, Canada). We originally collected progenitors of these lines from wild populations in Switzerland, China, the USA, and Canada between 2009 and 2021 (Fig.
Collection locations for 19 laboratory lines of Trissolcus japonicus. Inset maps depict sampling regions a the Pacific Northwest of North America b Switzerland, and c Beijing and Hebei provinces, China. Symbol shape depicts geographic area of collection: diamond = Switzerland, triangle = Pacific Northwest of North America, circle = China, square = Ontario, Canada. Square symbols for the two locations in London, Ontario, Canada have been jittered for visibility.
We extracted genomic DNA using DNeasy Blood and Tissue DNA kits (QIAGEN, Hilden, Germany) by following the manufacturer’s protocol, but we added a bovine ribonuclease A treatment (RNaseA, 4 uL at 100 mg/mL, QIAGEN) to digest RNA. We eluted DNA into 2× 50 uL of 56 °C Buffer AE to increase DNA concentration and yield, then we stored DNA at -20 °C until ddRAD library preparation. PstI-MspI library preparation and sequencing were performed by sequencing facility staff as outlined in
Obtaining high-concentration, high-quality DNA from small-bodied organisms is an inherent challenge when preparing DNA libraries (e.g.,
The number of individual pools of 10 female wasps from each Trissolcus japonicus laboratory line included in each of the three analyses of population genetic structure. See Suppl. material
Laboratory line | Number of pools included in dataset | ||
---|---|---|---|
full | geographic | Pacific Northwest | |
British Columbia, Canada (Langley) | 7 | – | 7 |
British Columbia, Canada (Chilliwack) | 5 | – | 5 |
British Columbia, Canada (Agassiz) | 7 | 7 | 7 |
British Columbia, Canada (Kelowna) | 7 | 7 | 7 |
Washington, USA (Vancouver) | 5 | 4 | 4 |
Washington, USA (Walla Walla) | 5 | – | 3 |
Switzerland (Basel-Stadt) | 4 | – | – |
Switzerland (Zurich) | 5 | – | – |
Switzerland (Ticino) | 5 | 5 | – |
Ontario, Canada (London, 2019–2020) | 4 | 4 | – |
Ontario, Canada (London, 2021) | 3 | – | – |
China (Beijing line 1) | 5 | – | – |
China (Beijing line 2) | 5 | – | – |
China (Beijing line 3) | 5 | 5 | – |
China (Beijing USDA line) | 4 | – | – |
China (Hebei line 1) | 5 | – | – |
China (Hebei line 2) | 4 | – | – |
China (Hebei line 3) | 5 | – | – |
China (Heilongjang) | 5 | 4 | – |
We used Stacks 2 version 2.55 (
We assessed population genetic structuring for three datasets: one containing all 19 laboratory lines (‘full dataset’), one with seven lines chosen for proportional representation of potential geographic clusters (‘geographic dataset’), and one with all six lines from the Pacific Northwest of North America (‘Pacific Northwest dataset’) (Table
DNA sequences are available as fastq files in the National Center for Biotechnology Information Sequence Read Archive (NCBI SRA) as BioProject PRJNA933214.
In total, we sequenced 109 pools of T. japonicus (1090 individuals) from the 19 laboratory lines, resulting in 109,473,577 raw Illumina reads. In a preliminary PCA, two pools had unexpected behaviour. They may have been contaminated during sample preparation or DNA extraction: neither clustered with any other pool, and both were placed intermediate to other pools extracted from the same line and pools from other lines. We removed both before all subsequent analyses. After our final filtering of raw reads, the full dataset contained 1,889 SNPs across 95 pools with a mean SNP read depth of 64.9×, 11,360,339 filtered reads, and 3.82% total missing data. We used VCFtools to re-filter this dataset before running structure, using the same filtering parameters but sub-selecting near-equal sample sizes for pools from 18 lines in accordance with
Principal component and structure analyses of SNP data from the full datasets comprising 18–19 Trissolcus japonicus laboratory lines. We present structure results with greatest LnP(K) and ΔK statistical support. Symbol shape depicts geographic area of collection: diamond = Switzerland, triangle = Pacific Northwest of North America, circle = China, square = Ontario, Canada (blue square = London, Ontario, Canada 2021 line). Colours behind laboratory line names correspond with geographic genetic clusters (Fig.
In the geographic dataset, we retained 36 pools across the 7 laboratory lines after filtering raw reads. The dataset contained 2,896 SNPs with a mean SNP read depth of 72.9×, 7,498,995 filtered reads, and 2.03% total missing data. We used VCFtools to sub-select four pools per line before running structure, ensuring equal sample size. Our final geographic structure dataset had a mean SNP read depth of 67.4×, 5,386,771 filtered reads, and 2.45% total missing data across 28 pools. We found greatest statistical support for K=11 (LnP(K) method) and K=10 (ΔK method) in this dataset (Fig.
Structure analysis of SNP data from the geographic structure dataset comprising seven Trissolcus japonicus laboratory lines. We present structure results with greatest ΔK statistical support. Symbol shape depicts geographic area of collection: diamond = Switzerland, triangle = Pacific Northwest of North America, circle = China, square = Ontario, Canada.
In the Pacific Northwest dataset, we retained 33 pools across the 6 laboratory lines after filtering raw reads. This dataset contained 1,976 SNPs with a mean SNP read depth of 78.8×, 5,069,119 filtered reads, and 2.15% total missing data. We used VCFtools to sub-select four pools per line before running structure, ensuring equal sample size; however we could only select three high quality pools from the Walla Walla laboratory line. Our final Pacific Northwest structure dataset had a mean SNP read depth of 71.3×, 3,186,750 filtered reads, and 2.83% total missing data across 23 pools. We found greatest statistical support for K=12 (LnP(K) method) and K=3 (ΔK method) in this dataset (Fig.
Principal component and structure analyses of SNP data from six Trissolcus japonicus laboratory lines collected across the Pacific Northwest of North America. We present the structure results with greatest ΔK and LnP(K) statistical support. We present both modes of K=12 across its 10 replicate runs.
Overall, we found T. japonicus lines had the greatest genetic similarity when collected in close geographic proximity (Figs
Among adventive T. japonicus populations in Canada, the lines from London, Ontario and Kelowna, British Columbia were the most similar to populations from China, the parasitoid’s native range, suggesting that these two populations may have originated from an area in proximity to our sampled Chinese populations. The two lines from London, Ontario were more closely related to the Beijing and Hebei cluster than was the Heilongjiang line in the full dataset (PCA and structure plots, Fig.
We had expected that westernmost lines from Canada and the USA (Langley, Chilliwack, Agassiz, and Vancouver, WA) would be most closely related, as would those from the interior of BC and WA (Kelowna, BC and Walla Walla, WA). Instead, both lines from Washington State are members of the same cluster despite being separated by more than 350 kilometres, providing good evidence that T. japonicus in Walla Walla and Vancouver are either 1) descendants of a single introduction event in Washington or 2) two separate introduction events from the same region (Fig.
Our study demonstrates that there is relatively strong population genetic structuring between T. japonicus laboratory lines collected at relatively small geographic scales, such as the Pacific Northwest of North America. One caveat of these analyses is the relatively low level of biological replication in certain genetic clusters. Nonetheless, clusters with more replicates of independently collected lines (n≥3: Switzerland; Beijing/Hebei, China; and western British Columbia) did tend to have high genetic similarity relative to the much larger between-cluster variation. Because the geographic limits of these clusters are not yet known, it may be difficult to increase biological replication of the adventive populations. Several regions of the native and adventive ranges of T. japonicus are missing from the analyses (e.g., Japan, Italy, Eastern and Central USA), so more work is required to comprehensively describe the worldwide population genetic structure of this species. Secondly, the analyses compared inbred laboratory lines, possibly leading to greater perceived genetic differences between lines than the ‘true’ wild relationships due to high genetic similarity of each individual in a pool. However, the lines show little evidence of genetic drift towards a common ‘lab genotype’, and lines that have been in culture for many generations are still genetically similar to more recently established lines from the same genetic cluster, strongly suggesting that these living genetic resources are maintaining their individual integrity and are a close representation of the wild genotypic relationships. To build on this study and clarify the genetic relationships among these laboratory lines, we recommend further research comparing behavioural and life history attributes of each line to inform their use for biological control of H. halys. In addition, we suggest that for investigating patterns of invasion history for adventive or invasive species of parasitoids, data from RRS or other genome-wide methods be used, as inferences from single-gene sequencing can over-estimate genetic relatedness among disjunct populations.
We thank Josh Milnes, Betsy Beers, and Kim Hoelmer for laboratory lines of Trissolcus japonicus collected in 2016–2017 from Washington State and the strain collected in 2009 from Beijing, respectively. For ddRAD sequencing, we thank Sophie Dang and staff at the Molecular Biology Service Unit at the University of Alberta. We also thank Warren Wong, Jade Sherwood, Peggy Clarke, Chris Hou, Caitlyn MacDonald, Emily Grove, Laura Keery, Allison Briun, and Jason Thiessen for insect collection and rearing support. We again thank Warren Wong and Jason Thiessen for photos of Trissolcus japonicus. Finally, we thank one anonymous reviewer for their thoughtful comments on the manuscript. Funding to PKA and CEM is from Agriculture and Agri-Food Canada, ABASE #2955 and APMS #4609. Funding for TH and SA is from the Canadian Agricultural Partnership, a federal provincial territorial initiative.
Supplementary information
Data type: table and figures (word document)