Short Communication |
Corresponding author: Judith M. Stahl ( judith.stahl@csiro.au ) Academic editor: Miles Zhang
© 2024 Judith M. Stahl, Fabrizio Lisi, Thaliana Samarin, Xingeng Wang, Elizabeth H. Beers, Kent M. Daane.
This is an open access article distributed under the terms of the CC0 Public Domain Dedication.
Citation:
Stahl JM, Lisi F, Samarin T, Wang X, Beers EH, Daane KM (2024) Compatibility of released and adventive populations of Ganaspis kimorum Buffington, 2024, (Cynipoidea, Figitidae), parasitoid of the spotted-wing drosophila Drosophila suzukii (Matsumura, 1931). Journal of Hymenoptera Research 97: 1403-1415. https://doi.org/10.3897/jhr.97.137087
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Taxonomic and host associations have been closely studied within the Ganaspis brasiliensis (Ihering, 1905) (Hymenoptera, Figitidae) complex as parasitoids of the spotted-wing drosophila, Drosophila suzukii (Matsumura, 1931) (Diptera, Drosophilidae). Initially, five genetic groups (G1–G5) were identified that suggested the existence of cryptic species that vary in their host ranges and geographic distributions. What was referred to as the “G1” strain was recently described as G. kimorum Buffington, 2024, and approved for release as a classical biological control agent in the United States and parts of Europe. Concurrently, an adventive population of G. kimorum was found in British Columbia, Canada and is likely spreading through parts of the Pacific Northwest such as Washington State, USA. Here, we compare the reproductive compatibility and molecular similarity of laboratory-bred G. kimorum (collected in Tokyo, Japan) used for release in the USA and Europe with the adventive population found in Washington State, USA. Cross-breeding experiments between the Tokyo and the adventive population showed successful mating and the production of female offspring, indicating that they are reproductively compatible. For both populations, the mitochondrial COI barcode region was sequenced and further confirmed the conspecificity of the Tokyo and adventive Washington populations with published G. kimorum. These findings will help to better understand and document the effects of releases of G. kimorum and the reproductive success of adventive and released populations.
Biological control, cryptic species, reproductive compatibility
Some insect species are widely distributed geographically and, as a result, isolated populations can exhibit biological, ecological, and genetic differences due to selective environmental pressure and local adaptation (
In this context, understanding the reproductive status among populations of biological control agents can be crucial to predict the success of multiple introductions and subsequent admixture impact on pest control efforts (
As the most host specific Asian D. suzukii parasitoid, G. kimorum was selected for release as a biological control agent in parts of Europe (
Two colonies of G. kimorum were maintained at the University of California Kearney Agricultural Research and Extension Center in California. The first colony was established from material originally collected in Tokyo, Japan, in 2015 (
The two G. kimorum populations were cross-bred in 2022–2023 with the following treatments: ‘rel’ female × ‘adv’ male, and ‘adv’ female × ‘rel’ male. To establish what proportion of G. kimorum females produces female offspring under the experimental conditions and also possible cytoplasmic incompatibility, the controls ‘rel’ female × ‘rel’ male, and ‘adv’ female × ‘adv’ male were added. If cytoplasmic incompatibility occurs, cross-breedings between infected males with uninfected females can result in reproductive incompatibility. Unmated individuals of G. kimorum were collected the day they emerged and placed with a mate for 1 h inside a plastic vial (70 mm high, 20.5 mm diameter) with a sponge lid that was coated in a thin layer of honey. They were observed for the first 10 min, and once every 10 min after that within the hour. The occurrence of mating behavior was recorded when mating was observed. After 1 h, the couple was placed into a rearing container, as previously described, that contained blueberries with 0–24 h old D. suzukii eggs and larvae. After 72 h, the parasitoids were removed and placed into 1.5 ml Eppendorf tubes filled with 90% ethanol to be stored at -80 °C. Emerged offspring was sexed and counted. Since G. kimorum is haplodiploid, meaning that males emerge from unfertilized eggs and females from fertilized eggs, the presence of daughters indicates successful mating. To confirm the viability of emerged daughters, replicates with both female and male offspring were used for a secondary experiment. Offspring were collected and stored in vials with honey until females and males had had at least 24 h of time to mate before one female and one male from each vial were placed onto new blueberries with D. suzukii immatures, as described previously. Only a maximum of three daughters per replicate was used to establish F1 viability. Afterwards, all F1 offspring were similarly stored in 90% ethanol.
The proportion of female G. kimorum producing daughters was compared between treatments and controls with a generalized linear model (GLM) with a binomial error distribution. Only cross-breedings that produced offspring were included. The relationship between observed mating and the occurrence of female offspring was investigated with a Pearson’s product-moment correlation. All statistics were conducted in R version 3.6.2 (
A total of 127 adult parasitoids were used for DNA barcoding. DNA was extracted from the whole body of each specimen using the FastDNA® kit and the FastPrep® Instrument (MP Biomedicals, Santa Ana, California, USA), according to the manufacturer’s instructions, with minor modifications. Extracted DNA was quantified using a Nanodrop 2000c spectrophotometer (Thermo Fischer Scientific, USA), diluted to approximately 50 ng/µL and stored at -20 °C.
The barcode region of the mitochondrial coding gene Cytochrome Oxidase subunit 1 (COI) was amplified with the universal primer pairs LCO/HCO (LCO: 5’- GGTCAACAAATCATAAAGATATTGG – 3’ and HCO: 5’ – TAAACTTCAGGGTGACCAAAAAATCA - 3’, 440–688 bp) (
The COI target region was amplified by setting the following profile of the T100 Thermal Cycler (Bio-Rad): 94 °C for 10 min, 35 cycles of 94 °C for 1 min, 50 °C for 1 min and 72 °C for 1.5 min, followed by final extension at 72 °C for 1.5 min. PCR products were sent to the University of California, Berkeley DNA sequencing facility for direct sequencing of both strands using the ABI Big Dye V3.1 terminator sequencing reaction kit (Perkin-Elmer/ABI, Weiterstadt, Germany) on an ABI 3707xl DNA Analyzer (Perkin-Elmer) with POP 7 and a 50 cm array. A Basic Local Alignment Search Tool (BLAST) comparison of amplified COI sequences was performed using the NCBI database.
Sequences were trimmed using FinchTV (Geospiza, Inc., Seattle, Washington, USA) software by removing terminal ambiguous regions and a multiple sequence alignment was performed using the MUSCLE algorithm (
A neighbor-joining tree (
All four cross-breedings yielded G. kimorum offspring (Table
Females producing daughters after cross-breedings. ‘rel’ = released population, ‘adv’ = adventive population, ‘WA’ = Washington State, USA.
Cross-breeding | Origin of female | Origin of male | Parental females (n) | Total progeny (n) | Females producing offspring (n) | Females producing daughters (n) | Females producing daughters (%) |
---|---|---|---|---|---|---|---|
‘rel’ × ‘adv’ | Tokyo | WA | 68 | 143 | 25 | 18 | 72.0 |
‘rel’ × ‘adv’ | WA | Tokyo | 28 | 29 | 9 | 7 | 77.8 |
‘rel’ × ‘rel’ | Tokyo | Tokyo | 36 | 125 | 17 | 11 | 64.7 |
‘adv’ × ‘adv’ | WA | WA | 22 | 29 | 8 | 5 | 62.5 |
Sixty-three percent of the females producing offspring were observed to have mated with their male during the first hour. There was no correlation between observed mating and the occurrence of female offspring (Pearson’s correlation, t = -0.41, DF = 57, p = 0.684); some females that had not been observed mating produced daughters and vice versa.
The majority of the F1 generation did not produce offspring. The one daughter from the ‘rel’ female × ‘adv’ male cross-breeding that did produce offspring, also produced female offspring. Similarly, the two cross-breedings between ‘adv’ females x ‘rel’ males F1 generation producing offspring included daughters, indicating viable original cross-breedingss.
The molecular characterization based on the mitochondrial COI gene confirmed that generated high quality reads from specimens showed 99–100% sequence similarity with G. brasiliensis isolate USNMENTO (accession number: MT559420.1), G. xanthopoda isolate suz21 (accession number: LC122451.1) and isolate suzukii type 3 (accession number: AB678736.1), and sequences were submitted to GenBank (accession number: PP980973 and PQ493166–PQ493291). Moreover, both the parental ‘rel’ and ‘adv’ G. kimorum used in the cross-breeding experiment matched with the same accession numbers showing a high sequence similarity (99–100%).
The phylogenetic tree showed two main clusters with an extended group referring to the G. kimorum samples (Fig.
Neighbor-joining tree; Neighbor-joining tree based on COI sequences for G. kimorum specimens used in the cross-breeding experiment (arrows) and on sequences retrieved from NCBI. The accession number, collection locality and host are also reported for each sequence. Ganaspis xanthopoda AB624299 was used as outgroup. Bootstrap values are indicated on the branches (values < 50% are not shown).
Our results confirm that the G. kimorum currently being released in the framework of the classical biological control program against D. suzukii is reproductively compatible with the adventive population of G. kimorum reported in North America. This was confirmed by cross-breeding of the adventive and the imported G. kimorum populations producing female progeny providing evidence for reproductive compatibility, and with the clustering of the COI barcoding region.
Taxonomic status and reproductive compatibility of introduced parasitoids and previously released, adventive or native parasitoids can be crucial for the outcome of biological control programs (
Given the wide distribution of G. kimorum in East Asia (
This study has practical implications for the classical biological control program of D. suzukii. Since the laboratory colony descends from only a small subset of the adventive population, our results cannot eliminate the possibility of additional species within the G. brasiliensis species group being present in the adventive range, which might have less or no compatibility with the released material. However, aligning with
Our results settle potential concerns about mating incompatibility and pave the way for re-distribution in the USA of the adventive G. kimorum population and imported G. kimorum from Tokyo. Overall, this study contributes to a better understanding of the reproductive status of two G. kimorum populations being used for classical biological control and improves predictions of the outcome of field releases for classical biological control against D. suzukii in North America. Still there is the possibility of viability or phenotypic effects resulting from the admixture of the two populations that has yet to be determined.
We thank Jeanne Gourlaouen, Emily Henry, Gadiel Leon, Michael Lopez, and Thomas Bultez for their technical assistance and fly and parasitoid colony maintenance. The authors are grateful to Paul Abram for his helpful comments on an earlier version of this manuscript. This project received funding from USDA NIFA SCRI 2020-51181-32140, USDA-NIFA OREI 2022-51300-37890, USDA APHIS Farm Bill Fund (60-8010-4-001), and USDA ARS Areawide Pest Management Program (administered by Stephen Young, National Program leader). Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the USDA. USDA is an equal opportunity provider and employer.