Research Article |
Corresponding author: Shunpei Fujie ( shunpei.fujie@gmail.com ) Academic editor: Jose Fernandez-Triana
© 2021 Shunpei Fujie, So Shimizu, Koichi Tone, Kazunori Matsuo, Kaoru Maeto.
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:
Fujie S, Shimizu S, Tone K, Matsuo K, Maeto K (2021) Stars in subtropical Japan: a new gregarious Meteorus species (Hymenoptera, Braconidae, Euphorinae) constructs enigmatic star-shaped pendulous communal cocoons. Journal of Hymenoptera Research 86: 19-45. https://doi.org/10.3897/jhr.86.71225
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A new gregarious braconid parasitoid wasp of Euphorinae, Meteorus stellatus Fujie, Shimizu & Maeto sp. nov., is described from the Ryukyu Islands in Japan, based on an integrative taxonomic framework. The phylogenetic position of the new species within the Meteorini was analyzed based on DNA fragments of the mitochondrial cytochrome c oxidase 1 (CO1) and the nuclear 28S rDNA genes. The new species was recovered as a member of the versicolor complex of the versicolor + rubens subclade within the pulchricornis clade. The new species is a gregarious parasitoid of two Macroglossum species (Lepidoptera: Sphingidae) and constructs single or several unique star-shaped cocoon masses separately suspended by very long threads. The evolution of gregariousness and spherical cocoon masses is discussed.
Endoparasitoid, female-biased sex ratio, integrative taxonomy, Lepidoptera, Macroglossum, phylogeny, species delimitation, Sphingidae
The pupae of parasitoid wasps cannot actively escape various risks, such as predation, parasitism, pathogenesis, and environmental stresses. Therefore, cocoons and mummies play important roles in protecting soft and exarate pupae from such risks (
Members of Braconidae, one of the most diverse hymenopteran families, form various types of cocoons and mummies to adapt to various natural enemies and environmental threats. Many gregarious braconids produce communal cocoon masses, while discrete cocoons for each individual are also constructed (
The cosmopolitan braconid genus Meteorus Haliday consists of more than 300 valid species (
Meteorus species are solitary or gregarious koinobiont endoparasitoids of Lepidoptera or Coleoptera larvae (
Types | Characteristics | Species | References |
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A | loosely clumped within a host pupal chamber | Palearctic species | |
M. acerbiavorus |
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M. heliophilus |
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M. rubens |
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B | individually suspended from host plant by a thread | Neotropical species | |
M. oviedoi |
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M. papiliovorus |
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C | sparsely arranged and suspended by a common cable | Palearctic species | |
M. kurokoi |
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Neotropical species | |||
M. restionis |
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D | loosely clumped and suspended by a common cable | Neotropical species | |
M. cecavorum |
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M. juliae |
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E | congregated and directly attached to host plant without a cable | Neotropical species | |
M. congregatus |
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F | communal and suspended by a common cable | Afrotropical species | |
M. komensis |
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Neotropical species | |||
M. townsendi |
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undescribed species |
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Oriental species | |||
M. stellatus sp. nov. | Present study |
Although the cocoon structure of Meteorus is quite mysterious, larval behavior associated with cocoon formation has received little attention, with only a few reports examining it (
The field collection of host moth larvae to observe the cocoon formation behavior of emerged larvae of wasps was conducted at Okinawa Municipal Museum, Okinawa City, Okinawa-hontô, Okinawa Prefecture, Japan. Some materials were also collected within Okinawa-hontô (Okinawa Prefecture) and Amami-ôshima (Kagoshima Prefecture), Japan. All materials were from the middle part of the Ryukyu Islands, the subtropical Oriental region in Japan.
Morphological observation was conducted with a stereoscopic microscope (SMZ800N, Nikon, Tokyo, Japan). Specimens and cocoons were photographed using a Digital Microscope (VHX-1000, Keyence, Osaka, Japan) with a 10–130× lens. Multi-focus photographs were stacked in the software associated with the Keyence System. Multi-focus photographs of cocoon masses were taken using a single lens reflex camera (α7II, Sony, Tokyo, Japan) with a micro-lens (A FE 50 mm F2.8 Macro SEL50M28, Sony). The RAW format photographs were developed using Adobe Lightroom CC v.2.2.1 (Adobe Systems Inc., San Jose, CA, USA), and stacked using Zerene Stacker v.1.04 (Zerene Systems LLC., Richland, WA, USA). The holotype of M. komensis, deposited in the Natural History Museum, London, UK was also examined by the second author using a stereoscopic microscope (SMZ1500, Nikon). Multi-focus photographs were taken using an α7II camera with micro-lenses (LAOWA 25 mm F2.8 2.5–5× ULTRA MACRO, Anhui Changgeng Optics Technology Co., Ltd, Hefei, China). The captured RAW format photographs were developed and stacked as per the aforementioned photo technique used for the cocoon masses. The figures were edited in Microsoft PowerPoint 2019.
The description style mostly follows that of
The abbreviations for repositories are listed below:
NARO Institute for Agro-Environmental Sciences, NARO (= NIAES: National Institute for Agro-Environmental Sciences), Tsukuba, Japan;
NSMT National Museum of Nature and Science, Tsukuba, Japan;
OMM Okinawa Municipal Museum, Okinawa, Japan;
RUM Ryukyu University Museum, Okinawa, Japan;
To investigate the secondary sex ratio of wasps, the number of males and females of all enclosed wasps that emerged from each host larva was counted for 11 host larvae (Suppl. material
The cocoon formation behavior of wasp larvae was observed at a laboratory of OMM, in June 2019 by the third author. It was recorded with video cameras (Sony Handycam, HDR-CX470 and HDR-XR150, Sony). The suspended larvae were blown with air currents created by breathing, as there was no wind, which would enhance the merging of each individually suspended larva in the laboratory as it would in natural conditions. A single silk thread spun by an individual larva is called a "thread", and intertwined threads are called a "cable" as in
To delimit a species, fragments of a mitochondrial protein encoding gene, cytochrome c oxidase 1 (CO1), were selected, because its evolutionary rate is more or less rapid and it is one of the most common genes used for population to species level phylogenetic analysis (this is well-known as the DNA barcoding gene). To infer the phylogenetic relationships among species of Meteorini (Meteorus and Zele), CO1 and a nuclear noncoding gene, 28S rRNA (28S), were selected. 28S is a gene that has evolved more slowly than CO1 and is usually used for species-groups or higher-level phylogeny; therefore, the combined CO1 and 28S analysis can provide a higher resolution of species phylogeny.
A total of 44 species of Meteorus including five morphospecies were sampled as ingroups. Five species of Zele were also sampled as ingroups because Zele is deeply nested within the Meteorus tree (
The newly collected samples from Okinawa were stored in 99.9% ethanol for DNA extraction. DNA was extracted from a right mid or/and hind leg. The protocols followed from PCR to sequencing were according to the work of
Target | Primer name | Sequence (5’ to 3’) | References |
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CO1 | CO1 lco hym | CAA ATC ATA AAG ATA TTG G |
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CO1 hco extB* | CCT ATT GAW ARA ACA TAR TGA AAA TG |
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28S | 28SD1F | ACC CGC TGA ATT TAA GCA TAT | Harry et al. (1997) |
28SD5R* | CCC ACA GCG CCA GTT CTG CTT ACC |
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Partial fragments of CO1 were used for species delineation. A total of 189 sequences were used for analysis (Suppl. material
To delimit the species, both distance- and topology-based methods were employed as below. Using both methods, three types of datasets were analyzed: (1) Meteorus + Zele + outgroups, (2) Meteorus + Zele, and (3) Meteorus. Prior to the analysis, identical haplotypes were removed from the datasets on the web server of ALTER (
The barcoding gap based analysis, Automatic Barcode Gap Discovery (ABGD) (
The General Mixed Yule Coalescent (GMYC) analysis was employed. GMYC analysis requires an ultrametric tree (UTree) as an input. To construct the UTree, the model and parameters was selected on a web server of the smart model selection (SMS) (
The phylogeny of Meteorini species was inferred with both the Bayesian Inference (BI) and maximum likelihood (ML) approaches using a concatenated CO1 and 28S fragments.
Although the MSA of CO1 was already performed in the species delimitation, MSA for 28S was conducted in the MAFFT online service (
In order to exclude the taxon sampling bias, a single sequence for each species was selected based on the conservative results of the species delimitation analysis by ABGD: sequences of 61 Meteorus species, six Zele species, and three outgroup species were finally selected (Table
Nomenclature systems for Meteorini species. The following abbreviations are used: pulchri. = pulchricornis.
Species | Present study |
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Maeto (1990) |
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clade / subclade / complex | Clade | group / subgroup | |
Meteorus ictericus A, B | ictericus / – / – | I | ictericus / – |
M. ruficeps | ictericus / – / – | I | ictericus / – |
M. aff. ruficeps | ictericus / – / – | I | ictericus / – |
Meteorus sp. | ictericus / – / – | – | – |
M. artocercus | pulchri. / colon / – | IIA | – |
M. cinctellus | pulchri. / colon / – | IIA | pulchri. / colon |
M. colon | pulchri. / colon / – | IIA | pulchri. / colon |
M. stenomastax | pulchri. / colon / – | IIA | – |
M. tenellus A–C | pulchri. / colon / – | IIA | – |
M. pendulus A–C | pulchri. / pendulus / – | IIB | pulchri. / gyrator |
M. abscissus | pulchri. / pulchri. / – | IIB | – |
M. limbatus | pulchri. / pulchri. / – | IIB | pulchri. / gyrator |
M. pulchricornis | pulchri. / pulchri. / – | IIB | pulchri. / pulchri. |
Meteorus sp. | pulchri. / pulchri. / – | – | – |
M. acerbiavorus | pulchri. / rubens-versicolor / rubens | IIB | – |
M. rubens A–C | pulchri. / rubens-versicolor / rubens | IIB | rubens / – |
M. aff. versicolor A, B | pulchri. / rubens-versicolor / versicolor | – | pulchri. / versicolor |
M. arizonensis | pulchri. / rubens-versicolor / versicolor | – | – |
M. obsoletus | pulchri. / rubens-versicolor / versicolor | IIB | pulchri. / versicolor |
M. tarius | pulchri. / rubens-versicolor / versicolor | – | – |
M. stellatus sp. nov. | pulchri. / rubens-versicolor / versicolor | – | – |
M. versicolor | pulchri. / rubens-versicolor / versicolor | IIB | pulchri. / versicolor |
M. micropterus | micropterus / – / – | IIC | micropterus / – |
M. abdominator A, B | Unresolved | III | – |
M. affinis A–D | Unresolved | III | – |
M. cespitator A, B | Unresolved | III | hirsutipes / – |
M. cis A, B | Unresolved | III | hirsutipes / – |
M. consimilis | Unresolved | III | – |
M. densipilosus | Unresolved | III | – |
M. eklundi | Unresolved | III | – |
M. filator A, B | Unresolved | III | – |
M. gigas | Unresolved | – | – |
M. hirsutipes | Unresolved | III | hirsutipes / – |
M. jaculator | Unresolved | III | – |
M. kyushuensis | Unresolved | III | hirsutipes / – |
M. longicaudis | Unresolved | III | – |
M. obfuscatus | Unresolved | III | – |
M. aff. obfuscatus | Unresolved | III | – |
M. oculatus | Unresolved | III | – |
M. sibyllae | Unresolved | III | – |
Meteorus sp. | Unresolved | – | – |
Meteorus sp. | Unresolved | – | – |
M. sulcatus | Unresolved | III | corax / – |
M. tabidus | Unresolved | III | – |
M. vexator | Unresolved | III | – |
Zela albiditarsus | Zele | IV | Zele / – |
Z. caligatus | Zele | IV | Zele / – |
Z. chlorophthalmus | Zele | IV | Zele / – |
Z. deceptor | Zele | IV | Zele / – |
Z. niveitarsis | Zele | – | Zele / – |
Zela sp. | Zele | – | – |
Each codon position within the CO1 fragment was treated as a different data block, but not for noncoding 28S. The best-fit substitution model was determined using PartitionFinder v.2.1.1 (
The BI analyses were conducted using MrBayes v.3.2.2 (
Although the molecular species delimitation was conducted using (1) whole datasets (i.e., Meteorus plus Zele plus outgroups), (2) Meteorus plus Zele datasets, and (3) Meteorus datasets, the results were congruent among all datasets in both the ABGD and GMYC methods. The number of recognized species was higher in the GMYC than in the ABGD method (Fig.
Species delimitation of Meteorus plus Zele plus closely related outgroups based on ABGD and GMYC methods, shown using a Bayesian consensus ultrametric tree generated using BEAST. Although the species delimitation was conducted using (1) Meteorus plus Zele plus outgroups, (2) Meteorus plus Zele, and (3) Meteorus sequences or topologies, all results were congruent; therefore, all results are shown as a summarized unit for each method.
Baded on morphological data, M. stellatus sp. nov. ran to the versicolor subgroup of the pulchricornis group based on
The specific name is a masculine Latin word, “stellatus”, meaning “starry”, which is derived from the unique shape of the cocoon masses.
41♀♀ 40♂♂ (all from Japan). Holotype ♀ (
Paratypes: 1♀2♂♂ (OMM), same as holotype; 2♀♀2♂♂ (OMM), Okinawa-kodomonokuni, Goya, Okinawa City /Shimabukuro, Kitanakagusuku Vil, Okinawa-hontô Is., collected as cocoon masses on 13.V.2019 and emerged on 19.V.2019, Koichi Tone et. al leg.; 2♀♀2♂♂ (
323♀♀228♂♂ adults; 29 cocoon masses (see Suppl. material
Japan (Ryukyus: Okinawa-hontô Island and Amami-ôshima Island).
Meteorus stellatus sp. nov. is most similar to M. komensis (Fig.
Meteorus stellatus sp. nov., ♀ holotype (exceptionally L is a paratype) A habitus B head, frontal view C head, dorsal view D mesopleuron and scutellum, dorsal view E mesosoma, lateral view F head, dorso-lateral view G forewing H basal antennal segments I apical antennal segments J propodeum and T1, dorsal view K T2 and following tergites, dorsal view L T1, ventral view.
In the key to species of Meteorus from the West Palaearctic region (
The results of a GenBank BLAST search showed that the CO1 sequences of M. stellatus sp. nov. were closest to those of M. arizonensis Muesebeck and M. tarius Huddleston. However, M. stellatus sp. nov. can be distinguished from M. arizonensis by its smaller body (the body lengths of M. stellatus sp. nov. and M. arizonensis are 2.9–3.9 mm and 4.6–5.5 mm, respectively), the longer malar space (the malar space length 1.0–1.4× the basal mandibular width in M. stellatus sp. nov. whereas 0.6–0.7× in M. arizonensis), the shorter ovipositor sheaths (the ovipositor sheath length 1.1–1.2× length of the first tergite in M. stellatus sp. nov. and 1.6–1.9× in M. arizonensis); the species can be distinguished from M. tarius by its broader face (the face width 1.5–1.7× its height in M. stellatus sp. nov. whereas approximately 1.0× in M. tarius) and the position of the forewing vein m-cu (slightly antefurcal to interstitial in M. stellatus sp. nov., but far antefurcal in M. tarius).
Female (holotype; Fig.
Head
(Fig.
Mesosoma
(Fig.
Wings
(Fig.
Legs. Tarsal claws with a distinct submedial lobe. Hind leg: outer surface of coxa punctate; femur 4.7× longer than wide, and distinctly and densely punctate.
Metasoma
(Fig.
Color
(Fig.
Variation. Body length 2.9–3.9 mm. Width of head 1.6–1.8× median height. Length of eye 1.5–1.7× length of temple in dorsal view. Face with width 1.5–1.7× height. OOL / OD = 1.2–1.6. POL / OD = 1.3–1.7. Frons with a longitudinal carina or a pair of obscure carinae. Length of malar space 1.0–1.4× basal mandibular width. Antennae with 26–31 segments; 4th segment 2.9–3.6× longer than wide; and penultimate one 1.7–2.0× longer than wide. Mesosoma length 1.4–1.5× height. Fore wing length 2.7–3.5 mm with length of pterostigma 2.8–3.3× maximum width, 3-SR / r = 0.8–1.4, m-cu distinctly postfurcal to interstitial, 1-CU1 / cu-a = 0.6–1.1. Hind wing with 1M / cu-a = 0.6–1.0, 1M / 1r-m = 0.5–0.7. Hind femur 4.6–4.9× longer than wide. 1st metasomal tergite 1.5–1.8× longer than apical width; longitudinally strigose with often some rugosity medially; length of ovipositor sheath 0.6–0.8× C+SC+R and 1.1–1.2× 1st tergite. 2nd tergite brownish yellow to infuscate anteromedially. Pterostigma unicolored or faintly paler basally.
Males (Fig.
Two species of Sphingidae (Lepidoptera) were identified as hosts of M. stellatus sp. nov.: Macroglossum passalus (Drury) feeding on Daphniphyllum glaucescens Blume (Daphniphyllaceae) and M. pyrrhosticta Butler feeding on Paederia foetida Linnaeus [= P. scandens (Lour.) Merr.] (Rubiaceae). All wasp larvae of M. stellatus sp. nov. emerged from mature larvae of the host sphingids.
Some hymenopteran hyper-parasitoids emerged from the cocoon masses after the emergence of M. stellatus sp. nov. adults. The following three species were identified as morphospecies at the generic level: Tetrastichus sp. (Eulophidae), Eurytoma sp. (Eurytomidae), and Aphanogmus sp. (Ceraphronidae) (Suppl. material
Despite the multiple field collection sessions at primary forest areas in the Okinawa-hontô and Amami-ôshima Islands, only one specimen of M. stellatus sp. nov. was sampled from a secondary evergreen forest in the latter island. Most other specimens of M. stellatus sp. nov. were collected from a campus of the University of the Ryukyus, urban parks, and back yards in Okinawa-hontô Island, by finding suspended cocoon masses or rearing host larvae. As the host sphingids and their host plants are abundant in or around the edges of sparse forests, M. stellatus sp. nov. likely prefers rather open forests.
The emergence of adult wasps occurred from April to June and from October to January, but not during the hottest season from July to September (Fig.
The proportion of males (secondary sex ratio) ranged from 0.20 to 0.64, showing a gradual increase with the total number of wasps per host larva (Fig.
The third author observed a case of larval emergence and subsequent cocoon formation in the laboratory. At approximately 1:30 p.m. on June 9, 2019, approximately 100 larvae of M. stellatus sp. nov. emerged from the abdomen of a matured larva of M. pyrrhosticta on the vine of P. foetida by chewing holes (Fig.
Cocoon forming behavior of Meteorus stellatus sp. nov. A emerging from a host larva (start time) B hanging down from the host plant substance (2 min) C intertwining with threads: arrows show larvae looking for other threads (39 min) D almost merging into three masses (57 min) E–J forming spherical cocoon masses (E 30 min F 65 min G 69 min H 84 min I 105 min J 139 min).
The host sphingid died on the following day after wondering. The color of the cocoons gradually darkened over a few days. After 8 days, 68 females and 23 males of M. stellatus sp. nov. emerged from these three cocoon masses. The wasps emerged simultaneously, cutting the tip of each cocoon.
The cocoon masses of M. stellatus sp. nov. (Fig.
Cocoon masses of Meteorus stellatus sp. nov. A habitus, medium-sized B habitus, exceptionally large-sized and somewhat collapsed in an artificial condition C a medium-sized cocoon mass D, E small-sized cocoon masses F independent cocoons near a cocoon mass G a part of suspending thread, consisting of individual cable.
The Meteorini phylogeny is illustrated in Fig.
Meteorus stellatus sp. nov. was recovered as an ingroup of the versicolor complex of the rubens subclade within the pulchricornis clade and sister to M. tarius.
Our observation of M. stellatus sp. nov. shows that gregarious cocoon masses were constructed by the highly elaborated cooperation of larvae. The larvae never merged immediately after emergence from their host, but initially just descended. This seems to reinforce the idea that the suspended cocoon makes the pupating wasp inaccessible to some potential enemies (
The cable of gregarious Meteorus is thought to be very resistant to breaking and highly tolerant to environmental stress (
The star-shaped cocoon masses of M. stellatus sp. nov. can reduce the risk of hyperparasitism, because the exposed area of each individual cocoon is apparently smaller than the solitary cocoon or non-star-shaped cocoon masses, as suggested by the spherical cocoons of M. komensis (
The sex ratio has been studied in gregarious species of Meteorus, while a similar pattern of female-biased sex ratio has been shown in Macrostomion sumatranum (Enderlein) (Braconidae, Rogadinae), which is also a gregarious parasitoid of matured sphingid larvae (
The pulchricornis and rubens species-groups belong to the monophyletic lineage of pulchricornis clade (Fig.
We are grateful to Yu Erh Chen, Tamami Gushiken, Kazuo Minato, Toshimasa Mitamura, Kozue Miyagi, Masashi Sugimoto, Koichi Sugino, Nakatada Wachi, Masako Yafuso, and research volunteers of Okinawa Zoo and Museum for collecting and offering the materials; to Kota Sakagami for identifying the host sphingid; to Kees van Achterberg and Jose Fernandez-Triana for variable suggestions on the manuscript; to Gavin Broad (
This research is partially supported by the Grants-in-Aid for JSPS KAKENHI (Grant numbers 19H00942) to KM and the Grant-in-Aid for JSPS Fellows (Grant Number 18J20333) to SS from the Japan Society for the Promotion of Science. The JSPS Overseas Challenge Program for Young Researchers enabled SS to carry out research at
Table S1
Data type: excel (.xslx) file
Explanation note: Table S1. Examined materials Meteorus stellatus sp. nov.
Table S2
Data type: excel (.xslx) file
Explanation note: Table S2. Gene bank accession numbers for the sampled taxa in the analyses.