Two new records of the genus Trioxys (Hymenoptera, Braconidae, Aphidiinae) parasitic on bamboo aphids from South Korea

Abstract

Background

The genus Trioxys Haliday, 1833 consists of more than 80 species worldwide with three species being recorded in South Korea. In this study, we report the first observation of the two additional species, T.liui Chou & Chou, 1993 from Takecallisarundinariae (Essig, 1917) on Phyllostachysbambusoides Siebold & Zucc., 1843 and T.remaudierei Starý & Rakhshani, 2017 from T.taiwana (Takahashi, 1926) on Sasaborealis (Hack.) Makino & Shibata, 1901.

New information

Trioxysliui and T.remaudierei are described and reported with phototographs of the diagnostic morphological characters and the mitochondrial cytochrome c oxidase subunit I (COI) data (barcode region) and Bayesian tree of the phylogenetic analysis amongst the closely-related taxa are provided.

Introduction

The genus Trioxys Haliday, 1833 (Hymenoptera, Braconidae, Aphidiinae) consists of more than 80 known species around the world (Rakhshani et al. 2012, Yu et al. 2016, Rakhshani et al. 2017, Čkrkić et al. 2021, Davidian 2021, Petrović et al. 2021), with three species being recorded in South Korea (National Institute of Biological Resources 2022). As a key character for classification to the two morphologically similar genera, Trioxys Haliday, 1833 and Binodoxys Mackauer, 1960, a pair of accessory prongs are evident on the abdominal sternite. The former has only a spiracular (primary) tubercle, while the latter has not only a spiracular tubercle, but also has a secondary tubercle (Mackauer 1961, Starý 1981).

The plant subfamily Bambusoideae Bambusoideae Luerss., (1893) (Cyperales, Poaceae), commonly known as Bamboo, consists of 120 genera with more than 1600 species in the world (Soreng et al. 2017) and five genera with 13 species being recorded in South Korea (National Institute of Biological Resources 2022). Bamboo, an evergreen perennial flowering plant, is represented by three main species in Korea: Phyllostachysbambusoides Siebold & Zucc., 1843, P.nigravar.henonis (Mitford) Stapf ex Rendle, 1904 and Pseudosasajaponica (Siebold & Zucc. ex Steud.) Makino ex Nakai, 1920, these being the most widely distributed in the region (Kelchner 2013, Zhao et al. 2019).

The genus Takecallis Mastumura, 1917 (Hemiptera, Aphididae, Calaphidinae) consists of eight valid species in the world (Remaudière and Remaudière 1997, Lee and Lee 2018, Wieczorek and Sawka-Gądek 2023) with five species recorded in South Korea (National Institute of Biological Resources 2022). Takecallis species was regarded as a pest in various bamboos, Arundinaria spp., Bambusa spp., Dendrocalamus spp., Phyllostachys spp., Pleioblastus spp., Pseudosasa spp., Sasa spp. and Yushania sp. (Quednau 2003, Qiao and Zhang 2004). In this study, we provide the dignostic characters for two species, Trioxysliui and T.remaudierei, from the Takecallis species in bamboo spp. and analyse their phylogenetic amongst closely-related congeneric species, using the COI barcode region.

Materials and methods

Field and Taxonomic works

Samples were collected by searching for Takecallis mummies (T.taiwana (Takahashi, 1926) and T.arundinariae (Essig, 1917)) on various bamboo species (Phyllostachysbambusoides and Sasaborealis). Leaves containing mummified aphids were then collected and placed in a clean insect breeding dish (SPL Life Sciences, Korea). To ensure a sufficient number of samples, these dishes were kept in the laboratory at room temperature. The emergence of parasitoid wasps was monitored daily and they were collected using an insect aspirator. Subsequently, the collected wasps were preserved in 80% ethyl alcohol at -19℃.

Morphological identification of Trioxys species was referred from Chou and Chou (1993), Davidian (2007), Yu et al. (2016), Das and Chakrabarti (2017), Rakhshani et al. (2017), Rakhshani et al. (2020) and that of Takecallis species from Lee and Lee (2018) and Wieczorek and Sawka-Gądek (2023). Terminology of morphological characters for the Aphidiinae follows Wharton et al. (1997) (for the venation of wing) and Takada (1968). We first performed morphological sorting of similar phenotypes and labelling of this sample using a stereomicroscope (OLYMPUS SZX16, Leica M205C), after which DNA extraction was performed.

After conducting both morphological and molecular identification, measurements of unrecorded species were carried out. A LEICA DMC2900 digital camera and a LEICA M205 C microscope (Leica Geosystems AG) were utilised for photography and characterisation. Multiple pictures were taken at various heights using multifocusing technology. LAS V4.11 (Leica Geosystems AG) and HeliconFocus 7 (Helicon Soft) software were used for the stacking process. After the stacking procedure, illustrations were generated using Adobe Photoshop CS6. LAS V.4.11 (Leica Geosystems AG) was utilised to determine the shape of the specimens (Berkovitch et al. 2009).

Molecular analysis

Total genomic DNA extraction was performed using a LaboPass Tissue Kit (COSMOgenetech, Korea) following the manufacturer’s protocol. To preserve a morphologically complete specimen, the DNA extraction method was slightly modified from the “freezing method” used by Yaakop et al. (2013). In the original protocol, the sample was incubated for 30 minutes at 56°C with 200 μl of TL buffer + 20 μl of proteinase K. In the slightly modified DNA extraction methods, a 2 hour incubation period at the same temperature was used. Genomic DNA was extracted individually from each sample.

The target site for molecular identification was the front partial region of mitochondrial COI, a 658-bp fragment, amplified using primers, LCO1490 (forward) 5’-GGTCAACAAATCATAAAGATATTGG-3’ and HCO2198 (reverse) 5'-TAAACTTCAGGGTGACCAAAAAATCA-3’ (Folmer et al. 1994), with AccuPower PCR PreMix (Bioneer Corp., Daejeon, Korea). Polymerase chain reaction (PCR) amplification was conducted with 20 ml of a reaction mixture consisting of 3 ml of DNA extract, 2 ml of primer and 15 ml of ddH₂O. It was carried out as follows: denaturation for 5 min at 95℃; 4 cycles of 20 s at 95℃, 30 s at 55℃ (decreasing incrementally by 2℃ per cycle) and 40 s at 72℃, 31 cycles of 20 s at 95℃, 30 s at 48℃ and 40 s at 72℃; and final extension at 72℃ for 5 min. PCR products were visualised by electrophoresis on agar gel and, if a band existed, we commissioned Macrogen (Daejeon, Korea) for purification and sequencing analysis.

Using MEGA version 7.0 (Kumar et al. 2016), sequences were aligned by ClustalW default settings and their frame-shifts checked to avoid pseudogenes. Alignments were translated to amino acids using MEGA version 7.0. We calculated sequence divergences using the ‘p-distance’ model commonly with 1,000 bootstrapping replications and complete deletion in data gaps.

A phylogenetic tree was constructed with the Bayesian method using BEAST2 (Bouckaert et al. 2014). To produce dated phylogenies, we used an optimised relaxed clock model (Drummond et al. 2006) in BEAUti and other options were set at default. MCMC analysis was performed and checked using Tracer, DensiTree. After that, we constructed the consensus tree using TreeAnnotator with posterior probability limit 1.0 setting.

Molecular identification was based on Ratnasingham and Herbert (2007). The front partial region 658 bp of the COI fragment was sequenced from T.liui and T.remaudierei and deposited in GenBank. Altogether, 14 sequences of six species, containing the outgroup, were retrieved from GenBank and BOLD (http://www.boldsystems.org) and were used to compare them with T.liui and T.remaudierei (Table 1).

Table 1.

Analysis sample list from GenBank (1-11, 16-18) with our own sample (12-15).

No	Species	NCBI accession number	BOLD ID
1	Trioxyscomplanatus	KJ848479.1	-
2	T.liui	MT324249.1	-
3	T.liui	MT324250.1	-
4	T.pallidus	KM973397.1	GBAHB12494-15
5	T.pallidus	KM973342.1	GBAHB1349-15
6	T.pallidus	KM973234.1	GBAHB1457-15
7	T.sunnysidensis	KR789189.1	JSJUN008-11
8	T.sunnysidensis	MG438589.1	BARSE352-16
9	T.ulmi	MT873046.1	-
10	T.ulmi	MT873047.1	-
11	T.ulmi	MT873048.1	-
12	T.liui	PP373116.1	-
13	T.liui	PP373117.1	-
14	T.remaudierei	PP373118.1	-
15	T.remaudierei	PP373119.1	-
16	Aphidiusuzbekistanicus	ON759206.1	-
17	Aphidiusuzbekistanicus	ON827042.1	-
18	Aphidiusuzbekistanicus	ON827045.1	-

Taxon treatments

Trioxys liui

Chou & Chou, 1993

E24207D1-7A28-5DD0-A101-6E2156743BD0

PP373116

PP373117

• Trioxysliui Chou & Chou, 1993 - Chou and Chou (1993): 375-378.

• Trioxys sp. Starý and Schlinger (1967), 1967: 127.

• Trioxysbambusa Liu, 1975 - Liu (1975): 69 (nomen nudum).

Materials

1. Type status: Other material. Occurrence: individualCount: 6; sex: female; lifeStage: adult; occurrenceStatus: present; preparations: in 80% ethanol and dry-specimen; occurrenceID: 1A829D92-1D9D-5A03-BB52-E23958988DE4; Taxon: scientificName: Trioxysliui Chou & Chou, 1993; kingdom: Animalia; phylum: Arthropoda; class: Insecta; order: Hymenoptera; family: Braconidae; genus: Trioxys; specificEpithet: liui; taxonRank: species; scientificNameAuthorship: Chou & Chou, 1993; Location: higherGeography: East Asia; country: South Korea; countryCode: KR; stateProvince: Jeollabuk-do; municipality: Gunsan-si; locality: 290-2, Singwan-dong; Identification: identifiedBy: Sangjin Kim, JuHyeong Sohn, Hyojoong Kim; Event: eventDate: 08-11-2022; year: 2022; month: 11; day: 8; Record Level: institutionCode: KSNU; basisOfRecord: preserved specimen

Description

Female. Length of body about 1.99 mm (Figure Fig. 1A). Length of forewing 1.67 mm (Fig. 1K).

Figure 1.

Trioxysliui: A Habitus; B Antenna; C F1 and F2; D Head; E Mesoscutum; F Propodeum; G Ovipositor; H Prong; I Dorsal view of petiole; J Lateral view of petiole; K Forewing.

Head. Eyes oval, sparsely setose. Tentorial index 0.20 (Fig. 1D). Clypeus oval with 6 setae. Malar space 0.09 times as long as longitudinal eye diameter. Antenna 11-segmented (Fig. 1B). F1 equal with F2 (Fig. 1C). F1 and F2 4.1 and 3.4 times as long as their width at the middle, respectively. F1 and F2 are with two and three longitudinal placodes, respectively. Maxillary palp with four palpomeres, labial palp with two palpomeres. Ratio of eye to temple in dorsal view 0.7. Face width/height ratio 1.3 (Fig. 1D).

Mesosoma. Mesoscutum with notaulices on anterior part, dorsal surface smooth, scarcely setose (Fig. 1E). Head width/mesoscutum width ratio 1.4. Propodeum areolated, areola length/width ratio subequal (1.01×) (Fig. 1F). Pterostigma 3.6 times as long as width. Ratio of pterostigma length to R1 vein (= metacarpus) length 1.4. r and RS veins extended (Fig. 1D).

Metasoma. Petiole 1.5 times as long as wide at spiracles (Fig. 1I and J). Ovipositor sheath stout, concave on ventral margin. Ratio of ovipositor sheath width/length 2.0 at base (Fig. 1G). Anal prongs (= accessory prongs) almost straight, slightly curved upwards at apex. Dorsal side of prongs four setae, with one claw-like apical bristle and two setae at apex (Figure 2H).

Colour. Antenna brown; scape, pedicel and F1 yellowish-brown. Head and face dark brown, clypeus with mouth-parts yellowish-brown. Mesosoma and metasoma brown; Petiole yellowish-brown. Legs yellowish-brown with dark apices.

Parasite of: Takecallistaiwana on Sasaborealis

Trioxys remaudierei

Starý & Rakhshani, 2017

BF6C6285-558C-5605-ABB4-58A92A4784D8

PP373118

PP373119

• Trioxysremaudierei Starý & Rakhshani, 2017 - Rakhshani et al. (2017): 1237-1248.

Materials

1. Type status: Other material. Occurrence: individualCount: 7; sex: female; lifeStage: adult; occurrenceStatus: present; preparations: in 80% ethanol, dry-specimen; occurrenceID: 98714FFC-467A-5148-97A4-D142E794F19F; Taxon: scientificName: Trioxy remaudierei Starý & Rakhshani, 2017; kingdom: Animalia; phylum: Arthropoda; class: Insecta; order: Hymenoptera; family: Braconidae; genus: Trioxys; specificEpithet: remaudierei; scientificNameAuthorship: Starý & Rakhshani, 2017; Location: higherGeography: East Asia; country: South Korea; countryCode: KR; stateProvince: Jeollabuk-do; municipality: Gimje-si; locality: Cheongha-myeon; verbatimLocality: 119, Jangsan-ri; Identification: identifiedBy: Sangjin Kim, JuHyeong Sohn, Hyojoong Kim; Event: eventDate: 07-11-2022; year: 2022; month: 11; day: 7; Record Level: language: en; institutionCode: KSNU

Description

Female. Length of body about 2.3 mm (Fig. 2A). Length of forewing 1.9 mm (Fig. 2K).

Figure 2.

Trioxysremaudierei: A Habitus; B Antenna; C F1 and F2; D Head; E Mesoscutum; F Propodeum; G Ovipositor; H Prong; I Dorsal view of petiole; J Lateral view of petiole; K Forewing.

Head. Eyes oval, sparsely setose. Tentorial index 0.14 (Fig. 2D). Clypeus oval with seven setae. Malar space 0.08 times as long as longitudinal eye diameter. Antenna 11-segmented (Fig. 2B). F1 slightly shorter than F2 (F2 1.07 times as long as F1) (Fig. 2C). F1 and F2 5.0 and 4.1 times as long as their width at the middle, respectively. F1 without longitudinal placodes and F2 with 3-4 longitudinal placodes. Maxillary palp with four palpomeres, labial palp with two palpomeres. Ratio of eye to temple in dorsal view 0.6. Face width/height ratio 1.2 (Fig. 2D).

Mesosoma. Mesoscutum with notaulices on anterior part, dorsal surface smooth, two rows of scarcely setose start at end of notaulus (Fig. 2E). Head width/mesoscutum width ratio 1.3. Propodeum areolated, areola length/width ratio 1.2 (Fig. 2F). Pterostigma 3.5 times as long as width. Ratio of pterostigma length to R1 vein (= metacarpus) length 2.5 (Fig. 2K). r and RS veins extended.

Metasoma. Petiole 2.0 times as long as wide at spiracles (Fig. 2I and J). Ovipositor sheath elongate, gently concave on ventral margin. Ratio of ovipositor sheath width/length 2.8 at base (Fig. 2G). Anal prongs (= accessory prongs) long and straight upwards, bifurcated on apical one-third to two-thirds (apical one-third are four, apcial half is two, apical two-third is one), each having one claw-like apical bristle and one seta at apex with two or three setae at dorsal side (Fig. 2H).

Colour. Antenna brown; scape, pedicel, F1 and F2 yellowish-brown, sometimes dorsal part of F2 brown; Head, face and clypeus with mouth-parts dark brown. Dorsal side of mesoscutum and metasoma dark brown, except light brown propodeum and yellowish-brown petiole. Legs yellowish-brown with dark apices.

Parasite of: T.arundinariae on Phyllostachysbambusoides.

Notes

In the original description, the antenna of this species was 13-segmented. However, it was a mistake in the email from the author. This species is really 11-segmented, but two segments are artificially repeated in the line drawing.

Analysis

A Bayesian tree was constructed with twelve sequences from seven species, including an outgroup. Three clades were identified: Clade A represented Trioxyssunnysidensis, Clade B included T.remaudierei, both identified as monophyletic and the remaining species formed Clade C (Fig. 3). Within Clade C, T.liui was observed as the sister group to T.pallidus, T.companatus and T.ulmi. In comparison to a previous study by Čkrkić et al. (2021), T.liui exhibited a consistent pattern, but in this study, it was positioned between T.ulmi and the T.pallidus-complanatus group. Trioxysremaudierei was situated between Clade A and Clade C (Fig. 3).

Figure 3.

Phylogenetic tree of six Trioxys spp. estimated by the Bayesian method using their COI DNA barcode data. Aphidiusuzbekistanicus was used as an outgroup. Bootstrap support values more than 50% are indicated above branches. Scale-bar means the expected rate of a nucleotide substitution.

Intraspecific and interspecific distances ranged from 0.000 to 0.011 (averaging 0.003) and 0.057 to 0.128 (averaging 0.108), respectively (Table 2). In Clade C, interspecific genetic distances ranged from 0.102 to 0.120 (averagint 0.113) between T.liui and the other species (T.pallidus, T.complanatus and T.ulmi). Genetic distances between T.remaudierei and T.sunnysidensis were 0.105 and genetic distances within Clade C were 0.111 (Table 2).

Table 2.

Calculated genetic distances, based on COI sequences between Trioxys spp. used in the analysis.

	T.liui (n = 4)	T.remaudierei (n = 2)	T.complanatus	T.pallidus (n = 3)	T.sunnysidensis (n = 2)	T.ulmi (n = 3)
Trioxysliui	(0.000)	-	-	-	-	-
T.remaudierei	0.111	(0.000)	-	-	-	-
T.complanatus	0.120	0.120	(0.000)	-	-	-
T.pallidus	0.118	0.113	0.057	(0.011)	-	-
T.sunnysidensis	0.110	0.105	0.121	0.128	(0.005)	-
T.ulmi	0.102	0.101	0.115	0.106	0.100	(0.000)

Discussion

Most of the Trioxys species typically exhibit morphology characterised by two prongs (Mackauer 1960, Mackauer 1961). However, T.remaudierei stands out as it starts with a single prong that graudally bifucates into two towards the apex (Fig. 2G and H). This morphology is not unique to T.remaudierei alone, other species like T.tenuicaudus Starý, 1978, T.udalovi Davidian, 2005, T.betulae Marshall, 1896 and T.artistigma Telenga, 1953 also exhibit this mophology (Davidian 2005, Davidian 2007). The genus Trioxys uses anal prongs (= accessory prongs) for grasping and restraining host movement (Eidmann 1924, Schlinger and Hall 1961, Shaw and Huddleston 1991) and may have evolved into two strands to maximise it. Therefore, such prong morphology was considered a potential indicator of evolutionary ancestral traits even although it seemed to be not a clear correlation between taxa due to the limited number of samples in this study.

In case of T.liui, it is parasitic on Cranaphisformosanus (Takahashi) (Liu, 1975) and parasitic on Phyllaphoidesbambusicola Takahashi, 1921 on Phyllostachysmakinoi Hayata, 1915 in China (Chou and Chou 1993). Moreover, Takecallis sp. has been recorded on Phyllostachysaurea Carrière ex. Rivière & C. Rivière, 1878, T.taiwana and T.arundinariae on Phyllostachys sp. and captured on Indocalamustessellatus (Munro) Keng f., 1957 in Spain (Rakhshani et al. 2020). Similarly, T.remaudierei parasite T.taiwana on Phyllostachys sp. in France and T.arundinariae on Phyllostachys sp. in Spain (Rakhshani et al. 2017). This study newly records of T.liui is parasitic on T.taiwana on Sasaborealis, and T.remaudierei is parasitic on T.arundinariae on P.bambusoides in South Korea.

Both species have already been recorded in Europe and are considered invasive or subsequent adaptation into western Europe (Rakhshani et al. 2020). Aphidiinae species exhibit strong host specificity (Starý 1978) and both species show a strong host specificity for Tacekallis species. Futhermore, since Takecallis species have been recorded only on bamboo species (Lee and Lee 2018), the distribution of T.liui and T.remaudierei parasitising on it seems to be limited to areas where bamboo is located. In the natural range of Tacekallis species, all known species of this genus are restricted to East Asia (China, India, Japan, Korea, Manchurian subregion, Taiwan), T.arundicolens (Clarke, 1903), T.arundinariae and T.taiwana, T.nigroantennatus Wieczorek, 2023 are now widely distributed and introduced to other countries, including Africa (Algeria), USA (California), England, Netherlands, Hungary, Madeira, Australia, New Zealand, Argentina, Spain and Poland (Blackman and Eastop 2023, Wieczorek and Sawka-Gądek 2023). Consequently, parasitoids of Takecallis species are likely to be invasive species outside East Asia, with invasive countries potentially hosting distributions of these species.

Author contributions

Sangjin Kim wrote the text; Sangjin Kim, JuHyeong Sohn and Hyjooing Kim collected material, carried out the DNA experiment and provided photographs; all authors revised the manuscript.