Research Article |
Corresponding author: Elijah J. Talamas ( billy.jenkins@GMAIL.COM ) Academic editor: Petr Janšta
© 2020 Fatemeh Ganjisaffar, Elijah J. Talamas, Marie Claude Bon, Thomas M. Perring.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Ganjisaffar F, Talamas EJ, Bon MC, Perring TM (2020) First report and integrated analysis of two native Trissolcus species utilizing Bagrada hilaris eggs in California. Journal of Hymenoptera Research 80: 49-70. https://doi.org/10.3897/jhr.80.57024
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Surveys with sentinel eggs of Bagrada hilaris (Hemiptera: Pentatomidae) in southern California retrieved two parasitoids that were not previously known to be associated with this stink bug, Trissolcus hullensis and T. utahensis (Hymenoptera: Scelionidae). Molecular and morphological analysis of these specimens is used to modify the concept of T. utahensis and assess the factors that contribute to intraspecific variation. We provide an updated couplet to separate T. utahensis from a morphologically similar species, T. cosmopeplae.
Bagrada bug, egg parasitoids, painted bug, Scelionidae, sentinel eggs
Bagrada hilaris (Burmeister) (Hemiptera: Pentatomidae) is native to Africa, Asia, and the Middle East (
Chemical applications have been the main approach used against this pest (
Our analysis includes T. utahensis reared from sentinel eggs of Podisus maculiventris (Say) (Hemiptera: Pentatomidae) in British Columbia, Canada, to assess the limits of this species and determine its intraspecific variation using molecular and morphological data. Previous identification of the Canadian specimens brought attention to problems with the couplet that separates T. utahensis and Trissolcus cosmopeplae (Gahan) in the key to Nearctic Trissolcus of
The Agricultural Operations of the University of California, Riverside, was the main site for the surveys. Fields that had been planted for various research were used for our sentinel egg deployments. A mixed vegetable field available from October 2017 to March 2018, an alfalfa field (Medicago sativa L.) available from October 2017 to January 2019, and a squash field (Cucurbita moschata L., variety black futsu) available from January 2018 to September 2019 were used. The mixed vegetable field was selected particularly because of its two rows of broccoli (Brassica oleracea L., variety Italica), a favorable host for B. hilaris. The alfalfa field hosted several stink bug species during spring and summer, serving as a potential source of stink bug egg parasitoids. The squash field was selected because B. hilaris adults were found on shortpod mustard weeds, Hirschfeldia incana (L.) Lagr.-Foss., within the field. Surveys also were conducted at other locations on the Agricultural Operations property where mustard weeds were found. In addition, various locations in the urban area with brassicaceous weeds surrounding the University of California, Riverside, were surveyed (Fig.
Survey locations are displayed in red dots. The black rectangle shows the Agricultural Operations of the University of California, Riverside, where most of our surveys were conducted. Five Trissolcus hullensis were recovered from an alfalfa field (33.96508°N, 117.34084°W), one Trissolcus utahensis was recovered from a squash field with mustard weeds (33.96611°N, 117.34230°W), and eleven T. utahensis were recovered from roadside mustard weeds (33.99105°N, 117.33360°W).
Bagrada hilaris eggs (≤ 24 hours old) were used for the surveys. Adult mating pairs of greenhouse-grown B. hilaris (
Genomic DNA was non-destructively isolated from the entire specimen using the Qiagen DNeasy Blood and Tissue kit (Hilden, Germany) as described in
Collection data on samples of Trissolcus utahensis, T. hullensis and T. colemani obtained in this study, and corresponding CO1 information.
Sample ID | Collection Unit identifier | Species (Hymenoptera: Scelionidae) | Host (Hemiptera: Pentatomidae) | Location | Date collected; name of the collector | Primers for mt-CO1 PCR and sequencing | DNA depository | CO1 Genbank Accession number |
---|---|---|---|---|---|---|---|---|
TSP274 | FSCA 00000302 | Trissolcus utahensis | Podisus maculiventris | Penticton, BC, Canada | July 2017; W. Wong & Paul Abram | LCO1490/HCO2198 | EBCL | MT804738 |
TSP276 | FSCA 00033040 | Trissolcus utahensis | Podisus maculiventris | Penticton, BC, Canada | June 2017; W. Wong & Paul Abram | LCO1490/HCO2198 | EBCL | MT804739 |
TSP277 | FSCA 00033041 | Trissolcus utahensis | Podisus maculiventris | Penticton, BC, Canada | June 2017; W. Wong & Paul Abram | LCO1490/HCO2198 | EBCL | MT804740 |
TSP278 | FSCA 00033042 | Trissolcus utahensis | Podisus maculiventris | Penticton, BC, Canada | August 2017; W. Wong & Paul Abram | LCO1490/HCO2198 | EBCL | MT804741 |
TSP279 | FSCA 00033043 | Trissolcus utahensis | Podisus maculiventris | Penticton, BC, Canada | August 2017; W. Wong & Paul Abram | LCO1490/HCO2198 | EBCL | MT804742 |
TSP280 | FSCA 00033044 | Trissolcus utahensis | Podisus maculiventris | Penticton, BC, Canada | June 2017; W. Wong & Paul Abram | LCO1490/HCO2198 | EBCL | MT804743 |
TSP281 | FSCA 00033045 | Trissolcus utahensis | Podisus maculiventris | Penticton, BC, Canada | June 2017; W. Wong & Paul Abram | LCO1490/HCO2198 | EBCL | MT804744 |
TSP288 | FSCA 00033239 | Trissolcus utahensis | Bagrada hilaris | Riverside California, USA | October 2018; Fatemeh Ganjisaffar | LCO1490/HCO2198 | EBCL | MT804745 |
PL141 | FSCA 00091859 | Trissolcus utahensis | Bagrada hilaris | Riverside California, USA | October 2018; Fatemeh Ganjisaffar | LCO1490/HCO2198 | FSCA | MT804746 |
PL142 | FSCA 00091872 | Trissolcus utahensis | Bagrada hilaris | Riverside California, USA | October 2018; Fatemeh Ganjisaffar | LCO1490/HCO2198 | FSCA | MT804747 |
PL259 | FSCA 00094712 | Trissolcus utahensis | Podisus maculiventris | Penticton, BC, Canada | July 2017; W. Wong & Paul Abram | LCO1490/HCO2198 | FSCA | MT804748 |
PL260 | FSCA 00094711 | Trissolcus utahensis | Podisus maculiventris | Penticton, BC, Canada | May 2017; W. Wong & Paul Abram | LCO1490/HCO2198 | FSCA | MT804749 |
PL261 | FSCA 00094713 | Trissolcus utahensis | Podisus maculiventris | Penticton, BC, Canada | August 2017; W. Wong & Paul Abram | LCO1490/HCO2198 | FSCA | MT804750 |
PL262 | FSCA 00094714 | Trissolcus utahensis | Podisus maculiventris | Penticton, BC, Canada | August 2017; W. Wong & Paul Abram | LCO1490/HCO2198 | FSCA | MT804751 |
PL263 | FSCA 00094715 | Trissolcus utahensis | Podisus maculiventris | Penticton, BC, Canada | August 2017; W. Wong & Paul Abram | LCO1490/HCO2198 | FSCA | MT804752 |
TSP291 | FSCA 00090585 | Trissolcus hullensis | Halyomorpha halys | St. Helena, CA, USA | August 2017; Kent Daane | LCO1490/HCO2198 | EBCL | MT804753 |
PL143 | FSCA 00091873 | Trissolcus hullensis | Bagrada hilaris | Riverside, CA, USA | March 2019; Fatemeh Ganjisaffar | LCO1490/HCO2198 | FSCA | MT804754 |
PL144 | FSCA 00091874 | Trissolcus hullensis | Bagrada hilaris | Riverside, CA, USA | March 2019; Fatemeh Ganjisaffar | LCO1490/HCO2198 | FSCA | MT804755 |
TSP398 | FSCA 00094753 | Trissolcus colemani | Piezodorus lituratus | Assas, France | June 2011; Marie Roche | LCO-1490puc/C1-N-2353 | EBCL | MT804756 |
TSP400 | FSCA 00094754 | Trissolcus colemani | Eurydema ventralis | Montferrier le Lez, France | May 2019; Marie Roche | LCO-1490puc/C1-N-2353 | EBCL | MT804757 |
TSP401 | FSCA 00094755 | Trissolcus colemani | Eurydema ventralis | Montferrier le Lez, France | May 2019; Marie Roche | LCO-1490puc/C1-N-2353 | EBCL | MT804758 |
TSP403 | FSCA 00094756 | Trissolcus colemani | Dolycoris baccarum (Hemiptera: Pentatomidae) | Montferrier le Lez, France | July 2010; Marie Roche | LCO-1490puc/C1-N-2353 | EBCL | MT804759 |
TSP409 | FSCA 00094757 | Trissolcus colemani | Graphosoma italicum | Montferrier le lez, France | July 2019; Marie Roche | LCO-1490puc/C1-N-2353 | EBCL | MT804760 |
TSP410 | FSCA 00094758 | Trissolcus colemani | Graphosoma italicum | Montferrier le lez, France | July 2019; Marie Roche | LCO-1490puc/C1-N-2353 | EBCL | MT804761 |
Voucher specimens which have been reexamined following the molecular analysis are presently archived at FSCA (Table
Terminology follows that of
aem anteroventral extension of the metapleuron (Figs
gc genal carina (Fig.
msct metascutellum (Figs
mshs mesoscutal humeral sulcus (Figs
mtnm metanotum (Figs
mtpm metapostnotum (Figs
not notaulus (Figs
oc occipital carina (Figs
ppm propodeum (Figs
Our survey period of March 6–9, 2018, in the alfalfa field, yielded a sentinel card with 6 parasitized eggs from which 5 specimens of T. hullensis and 1 specimen of T. basalis wasps emerged. A month later during our April 6–9, 2018, survey in the roadside mustard weeds (33.99105N, 117.33360W), two sentinel cards were parasitized; one of them had 5, and the other one had 7 parasitized eggs, which yielded 11 T. utahensis. One T. utahensis wasp was recovered from a sentinel card that was deployed in the squash field with mustard weeds on October 12–16, 2018.
The phylogenetic analysis based on the COI barcode data revealed a relatively well-resolved and supported topology identifying six terminal taxa (Fig.
Genetic distances (p-distances %, 1,000 bootstrap replications) for the CO1 barcode at the levels of intra-species, inter-species and clades of T. utahensis. Data are expressed as mean ± S.E.
T. colemani (East Asia) | T. semistriatus | T. hyalinipennis | T. colemani (Europe, Middle East) | T. hullensis | T. utahensis Clade 1 | T. utahensis Clade 2 | T. utahensis Clade 3 | T. utahensis Clade 4 | |
---|---|---|---|---|---|---|---|---|---|
T. colemani (East Asia) | 4.2 ± 0.6 | ||||||||
T. semistriatus | 10.6 ± 1.18 | 0.7 ± 0.2 | |||||||
T hyalinipennis | 13.3 ± 1.2 | 12.1 ± 1.3 | 0.4 ± 0.2 | ||||||
T. colemani (Europe, Middle East) | 12.7 ± 1.31 | 14.1 ± 1.4 | 14.3 ± 0.1 | 0.4 ± 0.1 | |||||
T. hullensis | 12.5 ± 1.3 | 12.4 ± 1.3 | 13.1 ± 1.3 | 12.3 ± 1.4 | 0.2 ± 0.1 | ||||
T. utahensis Clade 1 | 11.4 ± 1.2 | 10.8 ± 1.3 | 14.2 ± 1.4 | 14.1 ± 1.4 | 13.1 ± 1.4 | 0.7 ± 0.2 | |||
T. utahensis Clade 2 | 11.6 ± 1.2 | 10.8 ± 1.3 | 14.1 ± 1.4 | 14.3 ± 1.4 | 12.8 ± 1.3 | 2.8 ± 0.6 | 0 | ||
T. utahensis Clade 3 | 12.6 ± 1.3 | 11.8 ± 1.3 | 16 ± 1.5 | 14.5 ± 1.4 | 12.2 ± 1.3 | 7.78 ± 1.1 | 7.12 ± 1.1 | 0 | |
T. utahensis Clade 4 | 11.9 ± 1.2 | 12.9 ± 1.3 | 12.6 ± 1.3 | 14.7 ± 1.5 | 11.9 ± 1.3 | 9.97 ± 1.1 | 8.65 ± 1.1 | 9.61 ± 1.2 | 0.4 ± 0.1 |
The Bayesian 50% majority rule consensus tree inferred from the 56 CO1 sequences of the six Trissolcus species including T. hullensis and T. utahensis. Only posterior probabilities >90% are indicated on the nodes. The tree is rooted with the outgroup Trissolcus thyantae (GenBank MN615574). The scale bar corresponds to 0.1 estimated substitutions per site.
TCS COI haplotype network for the four clades of T. utahensis by fixing connection limits at 50 steps. Each haplotype is represented by a colored circle. Lines represent one mutational step between haplotypes, and dark circles represent unsampled haplotypes inferred from the data. Interrupted lines were used when haplotypes were separated by a long branch of more than 7 mutation steps.
The identification of Trissolcus hullensis is straightforward using the characters presented in
5 females, FSCA 00091873–00091874, 00091886–00091888 (deposited in FSCA) USA: CA: UC Riverside Agricultural Operations, 33.96508N, 117.34084W, alfalfa field, ex. Bagrada hilaris sentinel eggs deployed 6–9.III.2018, parasitoids emerged 23.III.2018, Coll. Ganjisaffar.
The concept of Trissolcus utahensis was most recently treated in a revisionary context by
14 | Anteroventral extension of the metapleuron long, extending to base of mesocoxa (Fig. |
T. cosmopeplae (Gahan) |
– | Anteroventral extension of the metapleuron short, not approaching base of mesocoxa (Figs |
T. utahensis (Ashmead) |
Figs
The shape of the gena varies between and within the four clades of T. utahensis. In clades 1 and 3, the specimens have a rather narrow gena, and in clades 2 and 4 the gena is moderately to distinctly bulging in lateral view. Figs
Specimens with a bulging gena tend not to have the genal carina indicated, whereas specimens with a narrow gena often have it clearly expressed, but this is not an exact correlation. The specimen in Figs
Microsculpture on the poster gena is less developed in specimens reared from B. hilaris eggs (Fig.
Trissolcus utahensis 20 FSCA 00033239 (ex. B. hilaris), head, mesosoma, metasoma, dorsal view 21 FSCA 00033041 (ex. P. maculiventris), head, mesosoma, metasoma, dorsolateral view 22 FSCA 00033040 (ex. P. maculiventris), head, mesosoma, metasoma, dorsal view. Scale bars in millimeters.
Specimens of T. utahensis reared from both B. hilaris and P. maculiventris have the notaulus indicated by short, shallow grooves present at the posterior margin of the mesoscutum (Figs
The form of the mesoscutal humeral sulcus was used by
The length of this structure, reaching to the mesocoxa in T. cosmopeplae (Fig.
Most species in Trissolcus have a black metasoma. The most notable exception is a Palearctic species, T. rufiventris Mayr, in which T2–T7 vary from bright yellow to dark brown. The specimens reared from B. hilaris eggs have T2–T7 notably lighter in color than the head and mesosoma (Figs
The most obvious color variation in T. utahensis is in the legs. Specimens that parasitized B. hilaris eggs have legs that are pale brown to orange distal to the coxae (clades 1, 2; Figs
11 females, FSCA 00091859, 00091872, 00094741–00094749, USA: CA: 33.99105N, 117.33360W, roadside mustard weeds, ex. Bagrada hilaris sentinel eggs deployed 6–9.IV.2018, parasitoids emerged 22–23.IV.2018, Coll. F. Ganjisaffar; 1 female, FSCA 00033239, USA: CA: UC Riverside Agricultural Operations, 33.96611N, 117.34230W, squash field with mustard weeds, ex: Bagrada hilaris sentinel eggs deployed 12–16-X-2018; parasitoid emerged 27–28-X-2018, Coll. F. Ganjisaffar; 9 females, 2 males, CANADA, BC, Penticton, reared from Podisus maculiventris, JUN–AUG-2017, Coll. W. Wong & P. Abram. Egg mass #181: FSCA 00094713–00094715, 00033042–00033043; Egg mass #144: FSCA 00000302; Egg mass #160: FSCA 00094712; Egg mass #92: 00033044–00033045; Egg mass #102; FSCA 00033040–00033041. 1 female, FSCA 00094711, CANADA, BC, Kelowna, reared from Podisus maculiventris, 23.V.2017, Coll. W. Wong & P. Abram. Egg mass #171.
The reports of this study indicate that at least four species of Trissolcus (T. basalis, T. hyalinipennis, T. hullensis, and T. utahensis) are actively parasitizing B. hilaris eggs in southern California (
Trissolcus utahensis exhibits a striking degree of variation in COI among specimens from only a few localities. An expansion of this analysis with additional samples is certain to expand our understanding of genetic diversity in this species. It is worth noting that specimens FSCA 00094713–00094715 emerged from the same egg mass and were retrieved in different clades (1 and 3), indicating that interbreeding opportunities exist between these maternally definable populations. Future work should include nuclear genes to provide a broader view of population dynamics and genetic diversity in this species.
The systematics of Trissolcus is undergoing perpetual improvement as it continues to receive attention for the species that attack the eggs of economically important stink bugs. This study demonstrates how a seemingly routine activity of rearing and identifying specimens can require a multifaceted research endeavor to reach a satisfactory answer while also providing new lines of inquiry. For example, our molecular analysis retrieved T. colemani in two clades, one comprised of specimens from East Asia and one from Europe and the Middle East. This study also demonstrates how ongoing parasitoid surveys continue to be productive by providing fresh specimens with host association data. These specimens and data are instrumental for an integrated approach to systematics in which morphological, molecular, and behavioral data are combined to provide robust and holistic species concepts.
We thank Shayla Hampel and Colt Bellman for their assistance with bagrada bug colony maintenance and Tim Lewis for his assistance in mapping survey locations and placing and retrieving sentinel egg cards. Brian Hogg and Charlie Pickett developed the sentinel card design used in this study. We also thank Matthew Moore, Cheryl Roberts and Lynn Combee (FDCAS-DPI) for their assistance with generating COI barcodes; and Marie Roche (EBCL) for rearing and identifying specimens of T. colemani in France. This project was supported in part by the Florida Department of Agriculture and Consumer Services-Division of Plant Industry, USDA-APHIS Farm Bill: Biological Control of Bagrada Bug, and California Department of Food and Agriculture Specialty Crops Grant Program # SCB16053.