A new species of Oomyzus Rondani (Hymenoptera, Eulophidae) reared from the pupae of Coccinella septempunctata (Coleoptera, Coccinellidae) in China

Abstract Oomyzus spiraculus Song, Fei & Cao sp. nov. (Hymenoptera, Eulophidae) is described and illustrated as a gregarious larval-pupal endoparasitoid of Coccinella septempunctata L. (Coleoptera, Coccinellidae). Differentiation between O. spiraculus and its similar species is discussed and a key to differentiate the female and male of these species is provided. DNA barcodes of O. spiraculus and O. scaposus are analyzed and compared.


introduction
The seven-spotted lady beetle, Coccinella septempunctata L. (Coleoptera, Coccinellidae), is widely recorded from the Palearctic and has a large distribution in China. It plays a significant role as an effective predator by suppressing populations of homopteran pests (e.g. aphids, whiteflies, and scales), which cause severe damage to agricultural crops (Pervez 2002). It is attacked by multiple parasitoids from Hymenoptera and Diptera (Honet et al. 2019).
Because of the ecological and economic significance of C. septempunctata, the interactions between C. septempunctata and its parasitoids have been traced and studied for many years (Li 1984;Semyanov 1986;Schaefer and Semyanov 1992;Triltsch 1996;Ceryngier et al. 2012). Among these parasitoids, Oomyzus scaposus (Thomson) is a common parasitoid wasp of coccinellids (Song et al. 2017). During a recent project related to interactions between coccinellids and their parasitoids, O. scaposus and another Oomyzus species were reared from the pupae of C. septempunctata. Here, this other Oomyzus species is described as new to science and compared to other known Oomyzus species.
Oomyzus is one of the smaller genera in Tetrastichinae (Hymenoptera, Eulophidae), with 26 described species prior to this study. Graham (1991) revised the European species of Oomyzus and included keys to the females of 12 species and males of 11 species. Although some species of Oomyzus (e.g. O. gallerucae (Fonscolombe) and O. sokolowskii (Kurdjumov)) have been recorded as cosmopolitan and some others have a large distribution across different continents owing to biocontrol introductions (e.g. O. brevistigma (Gahan)) (Noyes 2019), most species are rarely found or recorded from more than one zoogeographical region. This is possible because Oomyzus is difficult to characterize and shares some features with some other genera of Tetrastichinae, such as Baryscapus Förster, Tetrastichus Haliday, and Quadrastichus Girault. Species of Oomyzus are mainly larval or pupal parasitoids of Coleoptera, sometimes of Lepidoptera, Neuroptera, and Diptera, and sometimes as egg parasitoids of their hosts (Graham 1991). Some species, such as O. incertus (Ratzeburg), O. brevistigma, and O. sokolowskii, have been successfully used as biological control agents against some important agricultural pests of leaf beetles (LaSalle 1994). Graham (1991) preliminarily discussed assignments of species groups for some European species of Oomyzus based on morphological studies. However, to confirm these species groups further evidence is required. Although DNA barcode fragments of O. scaposus and O. spiraculus were generated in this study, the assignment to species group is not included because of the absence of data for the other species. Therefore, only COI fragments of these two species were analyzed and compared, even though they do not seem very close using morphological data. Despite the lack of molecular data, the differences and similarities of this new species between some possibly close species are discussed based on morphology.

Parasitoid wasp collection and rearing
The pupae of C. septempunctata were collected during field surveys in Nanjing, China, 2018. The host pupae were placed in plastic cups covered with mesh cloth and moved to the Laboratory of Biological Control in Nanjing Agricultural University and maintained in an insectary (25 ± 1 °C, 60 ± 5% RH, photoperiod L16: D 8 h) to rear adults of lady beetles or parasitoid wasps. Emerged wasps were then used to establish colonies using healthy larvae of C. septempunctata. Sample individuals from reared colonies were preserved in 95% ethanol after emergence for further use.

Taxonomy
Specimens used for morphological studies were critical-point dried with a Leica EM CPD300 automated critical point dryer. Then some specimens were mounted on cards and some others were dissected into head, mesosoma, metasoma, and gaster for scanning electron microscopy (SEM). Specimens were examined using a Nikon SMZ 1500 stereomicroscope fitted with a 10 mm ocular grid having 100 divisions. Habitus pictures were taken with a Nikon D7000 digital camera connected to the stereomicroscope. Dissected parts used for SEM were sputter-coated with gold using a Leica EM SCD050 super cool sputter coater. Micrographs were taken using an FEI Quanta 450 environmental scanning electron microscope. Photographs of appendages (fore wings, antennae, and legs) were taken with a Canon 550D digital camera connected to a Leica DM-2500 compound microscope. All color pictures were stacked using Helicon Focus software. The images were processed and combined into plates using Adobe Photoshop CC 2015.
The terminology follows Gibson (1997). Abbreviations are as follows: F1-F3, funiculars 1-3; MLM, midlobe of mesoscutum; Gtn, gastral tergite number; POL, the shortest distance between the posterior ocelli; OOL, the shortest distance between an eye and posterior ocellus. The type specimens were deposited in the Institute of Zoology, Chinese Academy of Sciences (IZCAS) and Nanjing Agricultural University (NAU).

Molecular analysis
A total of 16 specimens of Oomyzus (ten O. spiraculus and six O. scaposus) were used for extractions of whole genomic DNA by using the DNeasy Blood & Tissue Kit (Qiagen) following manufacturer's instructions. The primer pair LCO1490 (5′-GGTCA ACAAA TCATA AAGAT ATTGG-3′) and HCO2198 (5′-TAAAC TTCAG GGTGA CCAAA AAATCA-3′) (Folmer et al. 1994) were used to amplify the fragments of mitochondrial cytochrome c oxidase I (COI). All PCR procedures were performed using MyCycler Thermal Cycler (Bio-Rad, California, USA). The PCR reactions were carried out with Ex-Taq polymerase (Takara, Japan) under the following conditions: initial denaturation for 3 min at 94 °C, 35 cycles at 94 °C for 30 s, 52 °C for 40 s, and 72 °C at 30 s, followed by extension at 72 °C for 10 min. Sequencing was performed in both directions. Sequences of both directions were assembled and edited in Sequencher version 4.5 (Gene Codes Corporation, Ann Arbor, MI, USA) and aligned in BioEdit version 7.0.9.0 (Hall 1999). The COI matrix was translated into the amino acids in MEGA7.0 (Kumar et al. 2016) to check for stop codons. The Neighbor-Joining (NJ) phylogenetic tree based on the Kimura 2-parameter (K2P) distances was constructed by using MEGA7.0 with 1000 bootstrap replicates to generate support value for nodes.
Voucher specimens are deposited in the Nanjing Agricultural University. The obtained DNA sequences in this study have been deposited in Genbank (accession numbers MT259797-MT259812).

Results
Two species of Oomyzus were reared from coccinellid pupae collected during field surveys in Jiangsu Province. One species was identified as O. scaposus and the other as a new species which is described and illustrated here.
A COI matrix containing 16 individuals of Oomyzus (ten O. spiraculus and six O. scaposus) with a length of 581 base pairs was generated after alignment and trimming, without insertion or deletion. Graphical representation of K2P distances between these 16 individuals based on COI is presented as an NJ tree in Figure 4.  Graham), which can be differentiated from Baryscapus by the combination of other diagnostic characters listed above. See also discussions in Graham (1991) and LaSalle (1994).

Key to Oomyzus species similar to O. spiraculus
In this key both sexes are included and if 'female' or 'male' is not specified, then the features apply to both.
Head slightly broader than mesoscutum, and very easily collapsing when dried. OOL 1.58× POL (0.70: 0.44) (Fig. 3g). Ocelli arranged in an obtuse triangle (Fig. 3g), almost in a line if head is collapsed. Ocellar triangle slightly raised. Head in anterior view 1.23× as broad as high (2.7: 2.2), with vertex convex (Fig. 3h). Frons with a short and narrow V-shaped frontofacial suture connecting to ocellar area; upper face with a thin and raised carina between depressed scrobes; head easily collapsed along frontofacial sutures and outer margins of scrobes (Fig. 3h). Face with longitudinal reticulation, scrobes with indistinct reticulation. Toruli inserted slightly above lower margin of eyes. Eyes with short and sparse white hairs, diameter larger than malar space. Malar space 0.8-0.9× as long as mouth opening, and malar sulcus more or less straight. Anterior margin of clypeus weakly bilobed (Fig. 3h).
Pronotum strongly sloping and almost invisible in dorsal view; pronotum distinctly reticulate, neck and collar not delimited, without posterior carina (Fig. 3a, b). Mesoscutum with engraved and longitudinal reticulation, notaular grooves deep and curved; MLM with 3 adnotaular setae, without median line or with a trace of median line only posteriorly, with posterior margin straight (Fig. 3b). Axillae strongly shifted forward, with engraved reticulation in anterior 2/3 and with strongly raised striations, like carinae, in posterior 1/3 (Fig. 3b). Scutellum convex in profile, slightly broader than long (1.3: 1.2), with engraved, longitudinal reticulation; scutellum with distinct submedian lines that are slightly nearer to sublateral lines than to each other, enclosed space between submedian lines 2.1-2.2× as long as broad; with two pairs of setae on scutellum, subequal in length, anterior pair situated slightly before middle and posterior pair situated near posterior margin; scutellum with depressed frenum, delimited by groove and scattered, irregular carinae (Fig. 3b, d). Dorsellum about 2.6× as broad as long, with coarser engraved reticulation than scutellum; slightly incised in middle of posterior margin; lateral panel of metanotum smooth, with a carina medially (Fig. 3b, c). Propodeum incised medially along anterior and posterior margins, thus shortest medially, medially slightly longer than dorsellum; propodeum with median carina, broadening caudad and then extending laterally; propodeum with paraspiracular carinae, median area, delimited by paraspiracular carinae and median carina, with slightly raised reticulation and with irregular oblique carinae posteriorly (Fig. 3b, c); spiracles with entire rim exposed; callus reticulate, with 4 setae. Lateral panel of pronotum, prepectus, mesepimeron and mesepisternum reticulate, except a small area between mesepimeron and mesepisternum that is smooth; acropleuron smooth; metapleuron reticulate (Fig. 3d).
Petiole short and hidden (Figs 1a, 3e). Gaster (Fig. 3e) 1.2-1.6× as long as broad and easily collapsed, especially Gt 2-4 easily shrink or expand after death, and thus the relative length of mesosoma and metasoma is variable; gastral tergites each with weak raised reticulation; Gt 7 with 4 cercal setae, the longest seta slightly longer than the other three setae that are subequal in length.
Etymology. From the Latin word spiraculus (spiracle), referring to the propodeum with paraspiracular carinae. Host and offspring information. This species was reared as a gregarious endoparasitoid from pupae of C. septempunctata. In the laboratory, each instar of the host larva could be parasitized and would pupate successfully when provided with adequate number of aphids. The parasitoid offspring emerged from the host pupa, after a development time of 14-18 days. The brood size ranged from 4-23 after a single bout of parasitization, and the male number ranged from 0-3, mostly 2 (n =28). Another two common ladybird species, Harmonia axyridis Pallas and Propylaea japonica (Thunberg), were also included in the study and the result showed that the Japanese lady beetle, P. japonica, was a potential host.

Discussion
Coccinella septempunctata is a dominant predator attacking aphids in agroecological ecosystems and has great potential as a biological control agent in the development of a green agricultural economy. Prior to this study, quite a few Chalcidoidea parasitoids of C. septempunctata have been reported, including species belonging to Encyrtidae, Eulophidae, and Pteromalidae. Homalotylus spp. (Hymenoptera: Encyrtidae) seem to be especially well associated with C. septempunctata (Noyes 2019). In China, Oomyzus scaposus and Homalotylus flaminius (Dalman) are the most common parasitoids of C. septempunctata (Jing and Huang 2002;Song et al. 2017). This study demonstrates that O. spiraculus is a new parasitoid species of C. septempunctata, and it provides an ideal model system together with O. scaposus for further studies of interactions with C. septempunctata, as well as competition among themselves. In addition, the description of this new species will facilitate the discussions of phylogenetic relationships between close species of Oomyzus and the divergence and speciation of parasitoids in the same niche.