Research Article |
Corresponding author: Tetsuya Adachi-Hagimori ( tadachi@cc.miyazaki-u.ac.jp ) Academic editor: Andreas Köhler
© 2020 Serguei V. Triapitsyn, Tetsuya Adachi-Hagimori, Paul F. Rugman-Jones, Natsuko Kado, Nobuo Sawamura, Yutaka Narai.
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:
Triapitsyn SV, Adachi-Hagimori T, Rugman-Jones PF, Kado N, Sawamura N, Narai Y (2020) Egg parasitoids of Arboridia apicalis (Nawa, 1913) (Hemiptera, Cicadellidae), a leafhopper pest of grapevines in Japan, with description of a new species of Anagrus Haliday, 1833 (Hymenoptera, Mymaridae). ZooKeys 945: 129-152. https://doi.org/10.3897/zookeys.945.51865
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Several species of egg parasitoids (Hymenoptera: Mymaridae and Trichogrammatidae) of the leafhopper pest of grapevines in Japan, Arboridia (Arboridia) apicalis (Nawa) (Hemiptera, Cicadellidae), were reared and identified for the first time. Using a combination of genetic and morphological evidence, Anagrus (Anagrus) arboridiae Triapitsyn & Adachi-Hagimori, sp. nov. (Mymaridae) is described and illustrated from Honshu Island (Shimane Prefecture) and Kyushu Island (Miyazaki Prefecture). It is shown to be different from Anagrus (Anagrus) japonicus Sahad and A. flaviapex Chiappini & Lin, to which it is most similar; the latter species was originally described from China and is newly recorded here from Okinawa Island, Japan. Mitochondrial and nuclear ribosomal DNA sequence data provide clear evidence for the separation of A. arboridiae from A. flaviapex, A. japonicus, and some other members of the Anagrus (Anagrus) atomus (L.) species group. Two other species of Anagrus Haliday, A. (Anagrus) avalae Soyka and A. atomus, are also identified in Japan from eggs of the leafhoppers Edwardsiana ishidae (Matsumura) and Eurhadina ? betularia Anufriev, respectively. An updated key to females of the Japanese species of Anagrus is given. Oligosita pallida Kryger (a new record for Japan), Oligosita sp., and an Aphelinoidea (Aphelinoidea) sp. (Trichogrammatidae) were the other, although much less abundant, apparent egg parasitoids of A. apicalis in Shimane Prefecture, mainly in non-organic vineyards.
Aphelinoidea sp., egg parasitoid, grapevine pest, identification key, natural enemy, Oligosita spp., taxonomy
The leafhoppers Arboridia (Arboridia) apicalis (Nawa) (Fig.
Arboridia (Arboridia) apicalis and its damage to cultivated grapevines in Japan a adult (Takayama, Gifu Prefecture, Honshu Island) b heavy damage to grape leaves in a covered vineyard (Shimane Prefecture, Honshu Island, also c–f) c numerous adults on the underside of a grape leaf d nymphs on the underside of a grape leaf e light damage to a grape leaf by a few nymphs f heavy damage to a grape leaf.
Egg parasitoids of Arboridia spp. have been unknown in Japan, yet elsewhere several species of Mymaridae and Trichogrammatidae (Hymenoptera: Chalcidoidea) were reported from eggs of other species of this genus (
As the first step towards the establishment of a biological control-based integrated pest management (IPM) of these grape leafhoppers in Japan, we identified egg parasitoids of A. apicalis collected mainly from organic vineyards.
Both adults and nymphs of A. apicalis were collected in Japan by sweeping from the grapevines at several vineyards in Shimane Prefecture, Honshu Island, as well as from the organic vineyard in Aya, Miyazaki Prefecture, Kyushu Island (26°06'38"N, 129°41'16"E, 52 m). Arboridia suzukii was not present in these sites. Parasitized eggs, which turn dark orange as the parasitoid larva develops and then pupates, were documented by dissections in Petri dishes of the heavily leafhopper-infested grape leaves from the organic Oku-Izumo vineyard (Black Olympia cultivar) in Unnan, Shimane Prefecture. In early October 2019, leaves and vines infested with A. apicalis were collected from one organic and two non-organic vineyards in Shimane Prefecture and also in the above-mentioned organic vineyard in Miyazaki Prefecture; these were placed in tightly taped carton boxes. To collect adult parasitoids and host leafhoppers (both adults and nymphs), two clear glass vials were inserted through the holes into each of the boxes, one on the top and the other on the side near the top of the box, the latter to maximize exposure to daylight coming through the window. The vials were also exposed to a constant light source in the laboratory, and the emerging individuals were collected in 80% ethanol at least once almost daily (except for some weekends) for at least 30 days. Both host leafhoppers and parasitoids were sorted to morphospecies and identified by the first author at the second author’s laboratory at the University of Miyazaki Kibana Campus, Miyazaki City. The parasitoids were stored at -20 °C until they were shipped to the first author’s laboratory at the Entomology Research Museum, University of California at Riverside, California, USA (
Voucher specimens of the grape leafhoppers resulting from this study are deposited in the insect collections of the Entomological Laboratory, Faculty of Agriculture, Kyushu University, Fukuoka, Japan (ELKU), Illinois Natural History Survey, University of Illinois at Urbana-Champaign, Champaign, Illinois, USA (INHS) and the
Morphological identifications of the Anagrus sp., made by the first author, were based mainly on females because males of many species of Anagrus are often similar and difficult to determine beyond a species group.
For the taxonomic description of the new species, the morphological terms of
F funicle segment of the female antenna or flagellomere of the male antenna;
mps multi-porous plate sensillum or sensilla on the antennal flagellar segments (= longitudinal sensillum or sensilla, or sensory ridge(s)).
Specimens from ethanol were dried using a critical point drier, then point-mounted and labeled. Selected specimens were dissected and slide-mounted in Canada balsam. Slide mounts were examined under a Zeiss Axioskop 2 plus compound microscope (Carl Zeiss Microscopy, LLC, Thornwood, New York, USA) and photographed using the Auto-Montage system (Syncroscopy, Princeton, New Jersey, USA). Photographs were retouched where necessary using Adobe Photoshop (Adobe Systems, Inc., San Jose, California, USA).
Specimens of the parasitoids examined are deposited in the collections with the following acronyms:
CNC Canadian National Collection of Insects, Arachnids and Nematodes, Ottawa, Ontario, Canada;
DNA was extracted from two individual female wasps of the new species described herein using the “HotSHOT” method of
In addition to the specimens of the newly described species, the same methods were used to extract, amplify, and sequence the DNA from two individual females of Anagrus (Anagrus) japonicus Sahad, which represent the voucher specimens of the study by Adachi-Hagimori et al. (unpublished) (P. F. Rugman-Jones’ molecular vouchers PR19-501 [UCRC_ENT 005517343] and PR19-502 [UCRC_ENT 005517344]), and also from one female of A. (Anagrus) flaviapex Chiappini & Lin (data given below under “Comments”). Both species belong to the atomus species group of the nominate subgenus of Anagrus; all three specimens were recently collected in Okinawa Island, Ryukyu Islands, Japan.
All sequences generated in this study were deposited in GenBank (
The COI sequences generated from the five specimens in this study (GenBank accessions MT396446–MT396450) were combined with 45 others retrieved from GenBank, each of which was a unique haplotype identified in three earlier studies of members of the Anagrus ‘atomus’ species complex, which is altogether comprised of six nominal species (
Since phylogenetic inference from ITS2 is typically problematic due to large interspecific differences that make alignment of this region difficult and somewhat ambiguous, ITS2 sequences were examined “by eye” to corroborate the differentiation of our specimens based on COI. Furthermore, BLAST searches of the NCBI database were performed to assess their similarity to other members of the ‘atomus’ species complex.
Mymaridae
Holotype
♀ (Fig.
Paratypes. Japan: Honshu Island, Shimane Prefecture, Unnan, Oku-Izumo vineyard, 35°17'20"N, 132°55'46"E, 155 m (organic Black Olympia table grapes heavily infested with A. apicalis in a covered vineyard), collected 4.x.2019, N. Kado, N. Sawamura, T. Adachi-Hagimori, S. V. Triapitsyn, emerged from grape leaves: 5.x.2019, S. V. Triapitsyn [4 ♀♀, 6 ♂♂,
Other (non-type) material examined. Japan: Honshu Island, Shimane Prefecture, Unnan, Oku-Izumo vineyard, 35°17'20"N, 132°55'46"E, 155 m (organic Black Olympia table grapes heavily infested with A. apicalis in a covered vineyard), collected 4.x.2019, N. Kado, N. Sawamura, T. Adachi-Hagimori, S. V. Triapitsyn: sweeping on vines [1 ♀]; emerged from grape leaves: 5.x.2019, S. V. Triapitsyn [10 ♀♀, 10 ♂♂]; 6.x.2019, S. V. Triapitsyn [27 ♀♀, 22 ♂♂]; 7.x.2019, N. Kado [35 ♀♀, 29 ♂♂]; 8.x.2019, N. Kado [27 ♀♀, 18 ♂♂]; 9.x.2019, N. Kado [24 ♀♀, 12 ♂♂]; 10.x.2019, N. Kado [6 ♀♀, 6 ♂♂]; 11.x.2019, N. Kado [12 ♀♀, 7 ♂♂]; 13.x.2019, N. Kado [7 ♀♀]; 15.x.2019, N. Kado [1 ♀♀, 3 ♂♂]. Kyushu Island, Miyazaki Prefecture, Higashimorokata, Aya, Kitamata, 32°00'32"N, 131°14'26"E, 86 m (Katsuki Wines LLC organic vineyard, Chardonnay wine grapes lightly infested with A. apicalis in a covered vineyard), leaves collected 30.x.2019, T. Adachi-Hagimori, S. V. Triapitsyn: emerged 31.x.2019, S. V. Triapitsyn [1 ♂]; emerged 2.xi.2019, T. Adachi-Hagimori [1 ♂]; emerged 4.xi.2019, T. Adachi-Hagimori [2 ♀♀]; emerged 2.xi.2019, T. Adachi-Hagimori [1 ♂]; emerged 5.xi.2019, S. V. Triapitsyn [1 ♀]. All in 80% ethanol in a freezer [
The new species is a member of the atomus species group of Anagrus (Anagrus) as defined by
Morphologically, A. arboridiae is most similar to A. flaviapex, to which its female specimens with a more or less distinct bare area on the fore wing disc key in
Females of A. arboridiae are also similar to those A. japonicus that sometimes have an mps on F3, but the former always have distinct light brown and brown patches on the mesoscutum and the basal gastral terga respectively, which the latter species lacks.
An updated key to females of the Japanese species of Anagrus is provided below, as the latest key by
Female (holotype and paratypes). Body length of dry-mounted, critical point-dried paratypes 400–500 µm, and of the slide-mounted paratypes 415–520 µm. Body (Fig.
Measurements (µm) of the holotype (as length or length: width). Body: 545; mesosoma 197; gaster 251; ovipositor 212. Antenna: scape 75; pedicel 38; F1 13; F2 27; F3 39; F4 43; F5 42; F6 48; clava 100. Fore wing 463: 74; longest marginal seta 185. Hind wing 424: 20; longest marginal seta 148.
Male (paratypes). Body length of dry-mounted, critical point-dried paratypes 330–460 µm, and of the slide-mounted paratypes 400–520 µm. Body color mainly as in female except most of mesoscutum brown and most of gaster dark brown (light brown basally) (Fig.
This new species is named after the host leafhopper genus.
Palaearctic region: Japan (Honshu and Kyushu Islands).
Cicadellidae: Arboridia (Arboridia) apicalis (Nawa). Only A. apicalis was present on the grapevines both in Aya, Miyazaki Prefecture and in Unnan, Shimane Prefecture, with any other leafhoppers being absent, so this host association of A. arboridiae is obvious.
In the dissected parasitized eggs of A. apicalis in the organic vineyard in Unnan, Shimane Prefecture, A. arboridiae was observed to develop as a solitary endoparasitoid. Other aspects of its biology are unknown and thus would require further investigations.
The following species of Anagrus are newly recorded for the fauna of Japan.
Anagrus (Anagrus) flaviapex: Japan, Ryukyu Islands, Okinawa Prefecture, Okinawa Island, Itoman, Makabe, Okinawa Prefectural Agricultural Research Center (26°06'37.9"N, 129°41'16.1"E, 52 m), okra (organic experimental plot), 15–18.x.2019, S.V. Triapitsyn, T. Adachi-Hagimori, T. Uesato, Malaise trap [1 ♀,
The following dry-mounted specimens of Anagrus were found in T. Tachikawa’s collection during the first author’s visit of
Anagrus (Anagrus) atomus (L.): Japan, Shikoku Island, Ehime Prefecture, Kihoku, 24.iii.1966, emerged 28.iii.1966 from eggs of Eurhadina ? betularia Anufriev (a tentative identification of the host per the original label in Japanese) [4 ♀♀,
Anagrus (Anagrus) avalae Soyka: Japan, Honshu Island, Aomori Prefecture, Hirosaki City, vi.1964, R. Tsugawa, from eggs of Edwardsiana ishidae (Matsumura) [13 ♀♀, 4 ♂♂,
1 | Ocelli on a stemmaticum | 3 |
– | Ocelli not on a stemmaticum (Anagrus (Anagrella) Bakkendorf) | 2 |
2 | F2 approximately 1.5× F1 length | Anagrus (Anagrella) brevis Chiappini & Lin |
– | F2 at least 4.0× F1 length | Anagrus (Anagrella) hirashimai Sahad |
3 | Frenum with triangular paramedial plates widely separated from each other; metafemur short, less than 2× trochanter length, trochantellus incision almost half-way between coxa-trochanter and femur-tibia articulations (Anagrus (Paranagrus) Perkins) | 4 |
– | Frenum with triangular paramedial plates very close to each other; metafemur long, more than 2× trochanter length, trochantellus incision ca. one-third way between coxa-trochanter and femur-tibia articulations (Anagrus (Anagrus) Haliday [sensu sricto]) | 5 |
4 | Ovipositor projecting beyond apex of gaster by approximately 1/3 of its total length; ovipositor length: protibia length ratio at least 3.5 | Anagrus (Paranagrus) perforator (Perkins) |
– | Ovipositor not projecting or at most slightly projecting beyond apex of gaster; ovipositor length: protibia length ratio at most 2.5 | Anagrus (Paranagrus) optabilis (Perkins) |
5 | Clava with 3 mps (atomus species group) | 6 |
– | Clava with 5 mps (incarnatus species group) | 10 |
6 | Fore wing length: width ratio more than 10 | Anagrus (Anagrus) frequens Perkins |
– | Fore wing length: width ratio less than 10 | 7 |
7 | Body light brown or brown (at most frenum, propodeum and apex of gaster yellow) | 8 |
– | Body pale yellow, yellow, or greyish yellow (at most parts of mesoscutum and 2 basal gastral terga light brown or brown) | 9 |
8 | F3 without mps; gaster uniformly colored | Anagrus (Anagrus) atomus (L.) |
– | F3 with 1 mps; 2 apical gastral terga contrastingly yellow | Anagrus (Anagrus) flaviapex Chiappini & Lin |
9 | Body pale yellow except mesoscutum mostly light brown and 2 basal gastral terga contrastingly brown | Anagrus (Anagrus) arboridiae Triapitsyn & Adachi-Hagimori, sp. nov. |
– | Body yellow or greyish yellow, with 2 basal gastral terga concolorous with the rest of metasoma | Anagrus (Anagrus) japonicus Sahad |
10 | Midlobe of mesoscutum with adnotaular setae | 11 |
– | Midlobe of mesoscutum without adnotaular setae | 12 |
11 | Body light yellow except mesoscutum partially a little darker; each external plate of ovipositor (second valvifer) with 2 setae | Anagrus (Anagrus) avalae Soyka |
– | Body brown; each external plate of ovipositor with 3 setae | Anagrus (Anagrus) subfuscus Foerster |
12 | Fore wing approximately 6.3× as long as wide | Anagrus (Anagrus) takeyanus Gordh |
– | Fore wing at least 7.0× as long as wide | 13 |
13 | F2 the longest funicular segment | Anagrus (Anagrus) incarnatus Haliday |
_ | F2 at least slightly shorter than following funicular segments | Anagrus (Anagrus) rugmanjonesi Triapitsyn & Adachi-Hagimori |
Oligosita pallida Kryger, 1919: 318–319. 2 syntype ♀♀ [Zoological Museum, Natural History Museum of Denmark, University of Copenhagen, Copenhagen, Denmark (ZMUC)] (not examined). Type locality: Gentofte, Denmark.
Oligosita pallida
Kryger:
Oligosita sp.: Triapitsyn, 1998: 83 (reared from eggs of an Arboridia sp. in Turkmenistan).
Japan, Honshu Island, Shimane Prefecture: Izumo, Taisha (emerged from leaves of non-organic table grape infested with A. apicalis in a covered vineyard): 35°21'11"N, 132°40'59"E, 8 m, Shine Muscat grapes: collected 25.ix.2019, N. Kado, N. Sawamura, emerged 2–6.x.2019, S. V. Triapitsyn, N. Kado [2 ♂♂,
France, Gironde, Sainte Colombe, M. van Helden, in vineyards: 44°52'N, 00°02'W [3 ♀♀,
Palaearctic region: China (
Cicadellidae: Arboridia (Arboridia) apicalis (Nawa), A. (Arboridia) hussaini (Ghauri) (
Females of this distinctive species can be recognized by the uniformly pale color of the body (Fig.
Other examined female specimens of an Oligosita sp. in
Japan, Honshu Island, Shimane Prefecture, Izumo, Taisha, 35°21'21"N, 132°41'22"E, 3 m, 5.x.2019, N. Kado, N. Sawamura, T. Adachi-Hagimori, S. V. Triapitsyn (sweeping upon leaves of non-organic Delaware table grapes infested with A. apicalis in a covered Watanabe vineyard) [1 ♀,
Palaearctic region: Japan (Honshu Island).
Cicadellidae: Arboridia (Arboridia) apicalis (Nawa). This tentative host association will need to be confirmed by experimental work using sentinel eggs of this leafhopper.
Because taxonomy of the genus Oligosita Walker is in flux and no working keys are available, we could not positively identify this specimen to the species. Its body color is more or less light brown except dorsum of mesosoma is mostly yellow (Fig.
Japan, Honshu Island, Shimane Prefecture, Unnan, Oku-Izumo vineyard, 35°17'20"N, 132°55'46"E, 155 m, leaves of organic Black Olympia table grapes heavily infested with A. apicalis in a covered vineyard collected 4.x.2019, N. Kado, N. Sawamura, T. Adachi-Hagimori, S. V. Triapitsyn, emerged 5.x.2019, S. V. Triapitsyn [1 ♂,
Nearctic region: USA (Nebraska); Palaearctic region: Japan (Honshu Island).
Cicadellidae: Arboridia (Arboridia) apicalis (Nawa). This tentative host association will need to be confirmed by experimental work using sentinel eggs of this leafhopper.
This specimen is similar to Aphelinoidea (Aphelinoidea) waterhousei (Blood & Kryger) which belongs to the semifuscipennis species group of the nominate subgenus of the genus Aphelinoidea Girault, formerly placed in the synonymized subgenus Aphelinoidea (Diaclava Blood & Kryger) (
Sequences of the COI gene provided strong evidence that A. arboridiae is distinct from A. flaviapex and also from members of the A. atomus species complex for which comparative sequence data are available. The two A. arboridiae specimens (both from Unnan, Shimane Prefecture, Honshu Island) shared a single haplotype which, based on uncorrected p-distance, differed from the nearest taxon, A. japonicus, by 5%, and from A. flaviapex by 7.3% (Fig.
Relationship of Anagrus arboridiae sp. nov. with other members of the A. atomus species group for which reliable DNA sequences are available, based on a 587 bp fragment of COI. Optimal NJ tree with the sum of branch length = 0.34117946. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches and the tree is drawn to scale, with branch lengths indicating uncorrected p-distance. Analyses conducted in MEGA 6.06.
Genetic divergence between Anagrus arboridiae sp. nov., A. japonicus, A. flaviapex, and other members of the A. atomus species group including those in the A. atomus species complex*, based on DNA sequences of the mitochondrial COI gene. Diagonal element shows intraspecific variation (only when more than one sequence was considered). Average pairwise uncorrected p-distances calculated using MEGA 6.06.
A. arboridiae | A. japonicus | A. flaviapex | A. atomus* | A. incarnatus § | |
---|---|---|---|---|---|
A. arboridiae | 0.000 | ||||
A. japonicus | 0.050 | 0.005 | |||
A. flaviapex | 0.073 | 0.074 | – | ||
A. atomus* | 0.073 | 0.071 | 0.064 | 0.034 | |
A. incarnatus§ | 0.073 | 0.078 | 0.082 | 0.080 | – |
The results of this study are well within the expected composition of the genera of the parasitoids, as outside of Japan members of both Anagrus and Oligosita are known to parasitize eggs of other species of Arboridia; however, at species level the parasitoids turned out to be mostly different. Besides the above-mentioned A. turpanicus (parasitizing eggs of the invasive A. kakogawana in Xinjiang, China), which unlike A. arboridiae belongs to the incarnatus species group of the nominate subgenus of Anagrus (
Of note is the fact that in Japan, most of the obtained Oligosita spp. from Shimane Prefecture emerged from table grape leaves collected in non-organic vineyards where no A. arboridiae were present, whereas in the organic vineyard in the same prefecture A. arboridiae was abundant while only one specimen of O. pallida was collected. That perhaps could be due to the longer developmental times of Oligosita spp. versus Anagrus spp. and the fact that the former spin a cocoon at pupal stage and the latter do not (
These results could be of importance for other agricultural crops as well because A. apicalis is also known to feed on very common, aforementioned fruit trees in Japan. Furthermore, knowledge of the egg parasitoids of Arboridia spp. in Japan would be important for the potential classical biological control programs against A. kakogawana in other countries of Asia and Europe within the Palaearctic region, where it has established recently as an invasive pest of grapes (
We thank Dmitry A. Dmitriev (