Introduction

The brown-winged green bug, Plautia stali Scott (Hemiptera: Pentatomidae), is a serious pest of various fruit trees, such as apple, pears, citrus, persimmon, and peach, in Japan because feeding by P. stali leads to deterioration of fruit quality (e.g., Ide et al. 1997; Murai and Ogasawara 2003; Sugiura et al. 2007; The Japanese Society of Applied Entomology and Zoology 2006). In spring, overwintering adults of P. stali lay their eggs on conifers, such as Japanese cedar and Japanese cypress, and then adults of the first generation move to orchards to feed on fruits. Because the growing area of conifers covers at least 21 % of Japan (Forestry Agency, Japan 2011), it is extremely difficult to suppress P. stali populations on conifers using chemical insecticides. At present, chemical insecticides are often applied to orchards for their control. Thus, the pest management system largely relies on chemical insecticides, but it has a risk of causing the resurgence of other fruit-tree pests, such as coccoids and spider mites (Morishita 2005; Tsutsumi 2004). Under such a situation, integrated pest management by different control measures including chemical insecticides are needed for this stink bug pest. Natural enemies of the pest are among the different control measures, and egg parasitoids are the most promising control agents as natural enemies of P. stali (Goto and Adachi 2004; Ohno 1987a, b).

As an egg parasitoid of P. stali, Watanabe (1954) first described Asolcus plautiae Watanabe (Hymenoptera: Platygastridae, but Scelionidae at that time of his description) from Zentsuji, Kagawa, Japan. His description was based on nine females and one male bred from field-collected eggs of P. stali and ten females and one male probably reared from field-collected eggs of P. splendens Distant. Tachikawa et al. (1977) used the scientific name Trissolcus plautiae (Watanabe) instead of A. plautiae because Masner and Muesebeck (1968) transferred A. plautiae to the genus Trissolcus. Recording four species as egg parasitoids of P. stali reared in Fukuoka, Japan, Tachikawa et al. (1977) stated that T. plautiae seems to be the most effective in eliminating Plautia eggs among the four species. Yamada and Miyahara (1979) demonstrated that T. plautiae is the most dominant egg parasitoid of P. stali, thus corroborating the statement of Tachikawa et al. (1977). Since then, the view that T. plautiae has considerable ability to be an effective control agent against P. stali has been supported by several workers, such as Goto and Adachi (2004), Ohno (1987a, b), and Toyama and Mishiro (2010).

Four years after Hirashima and Yamagishi (1981), Tachikawa et al. (1977) synonymized T. plautiae with T. japonicus (Ashmead), described as Dissolcus japonicus by Ashmead (1904) on the basis of a single female collected from Hakone, Kanagawa, Japan, without host record. One of the authors, YH, considered further confirmation was required for the taxonomic treatment proposed by Hirashima and Yamagishi (1981) because they did not examine the type specimens of T. plautiae that had been preserved in Hokkaido University in Sapporo, Japan. Preceding the comparison of morphological characteristics between types of T. plautiae and T. japonicus, YH sent some specimens of Trissolcus that were reared from eggs of P. stali to one of the authors, NJ, in 1983 and requested him to compare the specimens with the type of T. japonicus that had been deposited in the US National Museum. In 1984, NJ found one substantial difference among them: the specimens from YH have a pair of sublateral setae on the T1, whereas the type of T. japonicus does not have the setae. Because the type of T. plautiae was reared from an egg of P. stali, specimens from YH might be T. plautiae. Therefore, this finding strongly suggested that T. plautiae and T. japonicus are different from each other.

As a result of unpublished information that it is possible that there are two species involved, two scientific names, i.e., T. plautiae (Watanabe) and T. japonicus (Ashmead), have been used for an egg parasitoid of P. stali in Japan since Hirashima and Yamagishi (1981): T. plautiae by Arakawa and Namura (2002), Goto and Adachi (2004), Ohno (1987a, b, 1999), and Toyama and Mishiro (2010) and T. japonicus by Buhl (1996), Johnson (1992), Ryu and Hirashima (1984), and Talamas et al. (2013).

As exemplified by Masner (1958), the genus Trissolcus, which was referred to as Asolcus by him, contains many species characterized by a high degree of variability in adult morphology. Thus, we see the possibility that cryptic species could be frequently found in this genus. The first case of finding cryptic species in the genus is that of T. crypticus Clarke reported by Clarke (1993) as a cryptic species of T. basalis (Wollaston). Cryptic species of parasitoids may differ in their biological traits that affect their efficiency as control agents, such as Torymus species (Hymenoptera: Torymidae) attacking Dryocosmus kuriphilus Yasumatsu (Hymenoptera: Cynipidae), a serious pest of chestnut production (Moriya et al. 2003; Yara et al. 2010). We therefore consider precise identification is essential to promote IPM programs. In this context, we attempted to resolve the above-mentioned confusion about the taxonomy of the two species, T. plautiae and T. japonicus. In this article, we present the results of morphological, biological and genetic studies of these two species to confirm their taxonomic status.

Materials and methods

Insect collecting and culture

A host culture was established not only for rearing Trissolcus wasps, but also for obtaining hosts for host deployment to collect these wasps in the field and for laboratory mating experiments to determine reproductive isolation of T. plautiae from T. japonicus. The culture was initiated using P. stali adults, which were collected from Fukuoka, Japan, in 2011–2012. The adults were kept in plastic cages containing filter paper, water and peanuts under 23 °C and 14L–10D conditions.

In the host deployment in the field, egg clusters of P. stali, which were laid on filter paper, were attached to leaves of Morus alba Linnaeus (Moraceae) by paper clips during the period from May to September 2012 in Hakozaki, Fukuoka. Each egg cluster was deployed for 2–4 days. On the occasion of the host deployment, we collected egg masses of P. stali and Halyomorpha halys (Stål) (Hemiptera: Pentatomidae) that were laid under natural conditions to clarify natural host ranges of Trissolcus wasps. After being brought back into the laboratory, the deployed and field-collected eggs were kept under 23 °C and 14L–10D conditions until adult wasp emergence. Adults of parasitoid wasps were preserved in 99 % ethanol for morphological observation and DNA analysis.

Morphological examination and terminology

For microscopic study, the ethanol-stored specimens were dried by the method described in Matsuo and Yukawa (2009). Specimens were observed under a binocular microscope (LEICA S8APO). Several specimens were gold-coated for microphotography with a JEOL JSM-5600LV scanning electronic microscope. Male genitalia were mounted on slides in Canada balsam using ethanol and clove oil. Drawings were made with the aid of a drawing tube. For morphological comparison, we paid special attention to characteristics mentioned by Clarke (1993) and those of the pleural and genital morphologies that had not been described in the earlier studies (Ashmead 1904; Hirashima and Yamagishi 1981; Ryu and Hirashima 1984; Watanabe 1954). Morphological terminology of adults follows usage in Mikó et al. (2007) for the head and mesosoma, Johnson (1984) for the metasoma, and Polaszek and Kimani (1990) for the male genitalia.

Specimens examined and depositories

The holotypes of T. plautiae and T. japonicus are deposited in the Hokkaido University Museum, Hokkaido, Japan (HUM), and the US National Museum of Natural History, Washington, DC, USA (USNM), respectively. For morphological comparison, we examined these holotypes. We also examined Trissolcus specimens reared from eggs of P. stali deposited in the collection of the Entomological Laboratory, Faculty of Agriculture, Kyushu University, Japan (ELKU). We further examined specimens used in Arakawa and Namura (2002), Ohno (1981, 1987a, b, 1999), and Toyama and Mishiro (2010) that have been kept in the ELKU.

Mating experiment and statistical analysis

For mating experiments, adults of T. plautiae and T. japonicus were kept in glass tubes (8 mm in width and 50 mm in height) with honey under the same conditions as those of P. stali. Laboratory experiments to determine reproductive isolation of T. plautiae from T. japonicus were conducted using 0-day-old females and 0–4-day-old males under 23 °C and 14L–10D conditions. An unmated female and male were introduced to the same glass tube (8 mm in width and 50 mm in height) for mating. Four mating combinations were tested: T. japonicus (♂) × T. plautiae (♀), T. plautiae (♂) × T. japonicus (♀), T. japonicus (♂) × T. japonicus (♀) and T. plautiae (♂) × T. plautiae (♀). The number of replicates conducted for each mating test was varied from 75 to 84. A pair of wasps was isolated 24–36 h after copulation. To each isolated female, 6–28 eggs of P. stali (0–2 days old) were presented for 24–36 h to assess mating success. The egg masses were then removed and kept in the tube until offspring emerged. Females that produced female offspring were regarded as successfully mated. We compared the percentage of mating success among the four combinations. The significance of results in the mating test was assessed with a chi-square test.

DNA analysis

Specimens stored in 99 % ethanol were used for DNA analysis (Table 1). For every individual specimen, total DNA was extracted from the whole body with the DNeasy Blood and Tissue Kit following the manufacturer’s instructions. The primers used in the analysis were: forward, CI-J-1751 5′-GGA TCA CCT GAT ATA GCA TTC CC-3′ and reverse, CI-N-2191 5′-CCC GGT AAA ATT AAA ATA TAA ACT TC-3′ (Simon et al. 1994). A region of the cytochrome oxidase subunit I (COI) gene of mitochondrial DNA was amplified by following procedures: denaturation step at 94 °C for 60 s, annealing for 90 s at 48 °C and extension at 72 °C for 90 s, with 35 cycles being performed. The sequencing reaction was performed using the BigDye Terminator Cycle Sequencing Kit and an ABI 3100 sequencer. The sequence data were analyzed with the neighbor-joining (NJ) method using MEGA 4 (Tamura et al. 2007). In constructing an NJ tree, Telenomus chrysopae Ashmead (Hymenoptera: Platygastridae) that had been reared from an egg of an unidentified chrysopid (Neuroptera) in Fukuoka, Japan, was employed as an outgroup taxon. Evolutionary distances were computed by Kimura’s two-parameter distances (Kimura 1980). The resulting trees were subjected to bootstrap analysis (Efron 1982; Felsenstein 1985) with 1,000 replications using MEGA 4.

Table 1 Specimens of two species of Trissolcus and an outgroup taxon which were used for DNA analysis
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Results

Morphological examination

We reconfirmed NJ’s observation that T. plautiae and T. japonicus were distinguished by the presence or absence of the sublateral setae on the T1 based on morphological comparison between their type and other specimens. Trissolcus plautiae has a pair of sublateral setae on the T1, whereas T. japonicus does not (Figs. 1–4). This characteristic was available in both sexes in discriminating these two species. Additional morphological characters are given in the descriptions below. Specimens used in Arakawa and Namura (2002), Ohno (1981, 1987a, b, 1999), and Toyama and Mishiro (2010) had a pair of sublateral setae on the T1. This finding showed that they were identical to T. plautiae. Thus, the ecological traits revealed by the aforementioned papers should be regarded as those of T. plautiae. The result of morphological examination of specimens deposited in the ELKU (Table 2) demonstrated that both T. plautiae and T. japonicus parasitize P. stali eggs and have sympatric distributions.

Figs. 1–4
figure 1

Trissolcus plautiae and T. japonicus. 1: Female of T. plautiae, metasoma dorsal view. 2: Female of T. plautiae, side of T1, an arrow points toward the sublateral setae. 3: Female of T. japonicus, metasoma dorsal view. 4: Female of T. japonicus, side of T1

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Figs. 5–10
figure 2

T. plautiae. 5: Female head, dorsal view. 6: Female head, frontal view. 7: Female antenna. 8: Male antenna. 9: Female mesosoma, dorsal view. 10: Female mesosoma, lateral view

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Figs. 11–16
figure 3

T. japonicus. 11: Female head, dorsal view. 12: Female head, frontal view. 13: Female antenna. 14: Male antenna. 15: Female mesosoma, dorsal view. 16: Female mesosoma, lateral view

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Figs. 17–18
figure 4

T. plautiae and T. japonicus. 1: Male genitalia of T. plautiae. 2: Male genitalia of T. japonicus. Scale bar: 0.1 mm

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Table 2 Number of individuals of two Trissolcus species reared from field-collected eggs of P. stali in Japan. These specimens have been deposited in the collection of the ELKU
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Mating experiments

Most females used in intraspecific mating combinations successfully reproduced female offspring, whereas females of interspecific mating combinations completely failed to reproduce female offspring (Fig. 19). Mating success was significantly different between T. japonicus (♂) × T. plautiae (♀) and T. japonicus (♂) × T. japonicus (♀) (p < 0.01); T. japonicus (♂) × T. plautiae (♀) and T. plautiae (♂) × T. plautiae (♀) (p < 0.01); T. plautiae (♂) × T. japonicus (♀) and T. japonicus (♂) × T. japonicus (♀) (p < 0.01); T. plautiae (♂) × T. japonicus (♀) and T. plautiae (♂) × T. plautiae (♀) (p < 0.01).

Fig. 19
figure 5

Percentage of mating success of inter- and intraspecific mating in T. plautiae and T. japonicus. The number of replicates is shown above each bar

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DNA analysis

The length of the amplified COI gene fragment was 424 bp. DNA sequencing data supported the results of morphological examination and mating experiments with high bootstrap values (Fig. 20). The maximum and minimum sequence divergences between T. plautiae and T. japonicus were 13.9 and 13.4 %, respectively.

Fig. 20
figure 6

Neighbor-joining tree for T. plautiae and T. japonicus based on 424 bp of the mtDNA COI gene. Bootstrap values are indicated for nodes gaining more than 90 % support in 1,000 replications. Telenomus chrysopae was used as an outgroup species. DDBJ accession numbers are shown in Table 1

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Taxonomy

We resurrect T. plautiae from T. japonicus based on the results of morphological observation, mating experiments, and DNA analysis. Host records are restricted to field-collected hosts.

Trissolcus plautiae (Watanabe, 1954) stat. rev.

Figs. 1, 2, 5–10, and 17

  • Asolcus plautiae Watanabe, 1954, Trans. Shikoku Entomol. Soc. 2: 18 (original description).

  • Trissolcus plautiae (Watanabe): Masner and Muesebeck, 1968, Bull. U.S. Nat. Mus. 270: 72 (generic transfer).

  • Trissolcus plautiae (Watanabe): Hirashima and Yamagishi, 1981, J. Fac. Agr. Kyushu Univ. 25: 153 (junior synonym of Trissolcus japonicus (Ashmead)).

Specimens examined. Holotype: female, emerged on 4 vii 1952 from an egg of P. stali collected by T. Kobayashi from Zentsuji, Kagawa, Japan (HUM). Paratypes: five females and one male, same data as holotype; nine females and one male, emerged on 20 vi 1951 from eggs of a Plautia species (probably P. splendens Distant) on Catalpa ovata G. Don (Bignoniaceae) collected by T. Kobayashi from Zentsuji, Kagawa, Japan (HUM). Other specimens: one female and one male, emerged on 11-16 viii 2011 from eggs of P. stali on Pinus thunbergii Parlatore (Pinaceae) collected by K. Matsuo on 4 viii 2011 from Hakozaki, Fukuoka, Fukuoka, Japan (ELKU); four females and three males, emerged in x 2012 from eggs of P. stali deployed on M. alba and collected by K. Matsuo in ix 2012 from Hakozaki, Fukuoka, Fukuoka, Japan (ELKU); four females, emerged on 25 v 2012 from eggs of H. halys on Distylium racemosum Siebold et Zuccarini (Hamamelidaceae) collected by K. Matsuo on 17 v 2012 from Hakozaki, Fukuoka, Fukuoka, Japan (ELKU). For other specimens examined, see Table 2.

Redescription. Female. Body length 1.2-1.7 mm. Head, mesosoma, and metasoma black. Antenna black except for A1, which is brownish at base, sometimes A1 and A2 brown. Coxae of all legs black; all femora infuscate; all tibiae brownish yellow.

Head 1.1–1.2 times as wide as mesosoma; hyperoccipital carina complete but not very sharp (Fig. 5); lateral ocellus connected to orbital furrow by a short sulcus; orbital furrow smooth, expanded ventrally; eye with very short setae; frons shiny with coriaceous microsculpture, below preocellar pit with extensive smooth area; a longitudinal groove present below preocellar pit in larger individuals; frontal depression transversely striated (Fig. 6). Antenna 11 segmented; A3 distinctly longer than A2 (Fig. 7).

Mesosoma with distinct notauli (Fig. 9); mesoscutum coarsely sculptured, smoother posteriorly; scutellum shiny and smooth, sometimes with superficial reticulation; setal base on scutellum smooth; netrion well developed (Fig. 10); mesopleural carina connects to pleural pit, not reaching ventral margin of mesopleuron; postacetabular patch coriaceous, with several setae; mesepisternum below postacetabular patch sometimes with numerous (15–20) short setae; posterior mesepimeral area smooth; anterior extension of metapleuron toward mid coxa long and acute.

T1 with a pair of sublateral setae (Figs. 1, 2); T2 smooth, sparsely setose laterally; T3 and beyond punctulate anteriorly.

Male. Differs from female as follows: Body length 1.2–1.4 mm. Antenna with 12 segments (Fig. 8). Genitalia (Fig. 17); basal ring 0.3–0.4 times as long as aedeago-volsellar shaft; aedeago-volsellar parallel sided; end of laminae volsellars strongly incised; digitus with four digital teeth; aedeagal lobe weakly emarginated or sometimes entirely truncate.

Distribution. Japan.

Host information. This species parasitizes eggs of P. stali and H. halys.

Trissolcus japonicus (Ashmead, 1904)

Figs. 3, 4, 11–16, and 18

  • Dissolcus japonicus Ashmead, 1904, J. New York Entomol. Soc. 12: 73 (original description).

  • Trissolcus japonicus (Ashmead): Masner and Muesebeck, 1968, Bull. U.S. Nat. Mus. 270: 72 (generic transfer).

  • Trissolcus japonicus (Ashmead): Hirashima and Yamagishi, 1981, J. Fac. Agr. Kyushu Univ. 25: 153 (senior synonym of Trissolcus plautiae (Watanabe)).

  • Trissolcus halyomorphae Yang: Talamas et al., 2013, J. Hym. Res. 33: 116 (junior synonym of Trissolcus japonicus (Ashmead)).

Specimens examined. Holotype: female, collected by A. Koebele from Hakone, Kanagawa, Japan (USNM, type no. 7127). Other specimens: four females and three males, emerged in x 2012 from eggs of P. stali deployed on M. alba and collected by K. Matsuo in ix 2012 from Hakozaki, Fukuoka, Fukuoka, Japan (ELKU); four females, emerged on 25 v 2012 from eggs of H. halys on D. racemosum collected by K. Matsuo on 17 v 2012 from Hakozaki, Fukuoka, Fukuoka, Japan (ELKU). For other specimens examined, see Table 2.

Morphology. Female and male. Morphological characteristics are largely identical to those of T. plautiae, differing in the following: upper frons almost entirely sculptured, smooth field limited to area just below the preocellar pit; microsculpture of mesoscutum more extensive, reaching transscutal articulation between notauli; mesoscutellum with coriaceous microsculpture anteriorly; T1 without sublateral setae.

Distribution. Japan and P.R. China (Yang et al. 2009).

Host information. This species parasitizes eggs of P. stali and H. halys.

Discussion

Taxonomic remarks

Talamas et al. (2013) have recently synonymized T. halyomorphae Yang with T. japonicus. The characters noted in their paper are consistent with our observations. Some of the features noted by Yang et al. (2009), such as the presence of a longitudinal groove below the median ocellus, may be influenced by host size in that the host from which those specimens were reared, H. halys, is larger than P. stali. The effect of host size on character expression in these parasitoids remains to be carefully examined. Similarly, in the absence of controlled rearing conditions, the morphological differences in sculpture that we report here would likely be treated as insignificant individual variation. The fact that the differences are observed under identical temperature and photoperiod and from specimens reared from the same host species suggests that they accurately reflect that T. plautiae and T. japonicus are different species.

Host range

Arakawa and Namura (2002) showed that T. plautiae can utilize eggs of H. halys under laboratory conditions. We newly confirmed that H. halys is one of the natural hosts of T. plautiae in Japan. Yang et al. (2009) recorded H. halys as a natural host of T. japonicus (= T. halyomorphae) from P.R. China. We have also reared T. japonicus from field-collected eggs of H. halys in Fukuoka and showed that the Japanese population of T. japonicus parasitizes P. stali and H. halys as natural hosts.

Ryu and Hirashima (1984) recorded Elasmucha putoni Scott (Hemiptera: Acanthosomatidae) as one of the hosts of T. plautiae or T. japonicus. However, they never provided information on voucher specimens with such collecting data and their depositories. Therefore, we were unable to identify which species, T. plautiae or T. japonicus, parasitizes eggs of E. putoni. Elasmucha putoni females guard their eggs (Honbo and Nakamura 1985; Kudo 1990), but P. stali females do not. Thus, egg parasitoids of E. putoni may be required to develop parasitic strategies to overcome host parental care. In this context, a series of oviposition behaviors of T. plautiae and T. japonicus (antennation, ovipositing, and marking) takes much time and would make it impossible to parasitize E. putoni eggs. We believe that the host E. putoni recorded by Ryu and Hirashima (1984) needs to be reconfirmed.