Corresponding author: Shunpei Fujie (
Academic editor: Jose Fernandez-Triana
A new gregarious braconid parasitoid wasp of
Fujie S, Shimizu S, Tone K, Matsuo K, Maeto K (2021) Stars in subtropical Japan: a new gregarious
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
The cosmopolitan braconid genus
The types of cocoon masses in gregarious
Types | Characteristics | Species | References |
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A | loosely clumped within a host pupal chamber |
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B | individually suspended from host plant by a thread |
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C | sparsely arranged and suspended by a common cable |
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D | loosely clumped and suspended by a common cable |
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E | congregated and directly attached to host plant without a cable |
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F | communal and suspended by a common cable |
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undescribed species |
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Present study |
Although the cocoon structure of
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
The description style mostly follows that of
The abbreviations for repositories are listed below:
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
To delimit a species, fragments of a mitochondrial protein encoding gene, cytochrome c oxidase 1 (
A total of 44 species of
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
Primer information for PCR.
Target | Primer name | Sequence (5’ to 3’) | References |
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CAA ATC ATA AAG ATA TTG G |
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CCT ATT GAW ARA ACA TAR TGA AAA TG |
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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
To delimit the species, both distance- and topology-based methods were employed as below. Using both methods, three types of datasets were analyzed: (1)
The barcoding gap based analysis, Automatic Barcode Gap Discovery (
The General Mixed Yule Coalescent (
The phylogeny of
Although the
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
Nomenclature systems for
Species | Present study |
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Maeto (1990) |
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clade / subclade / complex | Clade | group / subgroup | |
I | |||
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I | ||
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I | ||
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IIA | – | |
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IIA |
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IIA |
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IIA | – | |
IIA | – | ||
IIB |
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IIB | – | |
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IIB |
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IIB |
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– | – | ||
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IIB | – | |
IIB | |||
– |
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– | – | |
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IIB |
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– | – | |
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– | – | |
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IIB |
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IIC | ||
Unresolved | III | – | |
Unresolved | III | – | |
Unresolved | III | ||
Unresolved | III | ||
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Unresolved | III | – |
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Unresolved | III | – |
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Unresolved | III | – |
Unresolved | III | – | |
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Unresolved | – | – |
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Unresolved | III | |
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Unresolved | III | – |
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Unresolved | III | |
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Unresolved | III | – |
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Unresolved | III | – |
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Unresolved | III | – |
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Unresolved | III | – |
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Unresolved | III | – |
Unresolved | – | – | |
Unresolved | – | – | |
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Unresolved | III | |
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Unresolved | III | – |
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Unresolved | III | – |
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IV | |
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IV | |
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IV | |
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IV | |
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– | |
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– | – |
Each codon position within the
The
Although the molecular species delimitation was conducted using (1) whole datasets (i.e.,
Species delimitation of
Baded on morphological data,
The specific name is a masculine Latin word, “
41♀♀ 40♂♂ (all from Japan).
323♀♀228♂♂ adults; 29 cocoon masses (see Suppl. material
Japan (Ryukyus: Okinawa-hontô Island and Amami-ôshima Island).
In the key to species of
The results of a GenBank BLAST search showed that the
Two species of
Some hymenopteran hyper-parasitoids emerged from the cocoon masses after the emergence of
Despite the multiple field collection sessions at primary forest areas in the Okinawa-hontô and Amami-ôshima Islands, only one specimen of
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.
Seasonal changes in the adult emergence of
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.
Sex ratios of emerged adults of
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
Cocoon forming behavior of
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
The cocoon masses of
Cocoon masses of
The
Maximum likelihood tree of
Our observation of
The cable of gregarious
The star-shaped cocoon masses of
The sex ratio has been studied in gregarious species of
The
Phylogenetic relationships among the species of
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
excel (.xslx) file
Table S1. Examined materials
Table S2
excel (.xslx) file
Table S2. Gene bank accession numbers for the sampled taxa in the analyses.