A new species of Diglyphus Walker (Hymenoptera, Eulophidae) from China, with morphological characterizations and molecular analysis.

Diglyphus Walker, 1844 (Hymenoptera: Eulophidae) is an economically important genus including species acting as biocontrol agents against agromyzid leafminer pests. A new species of Diglyphus, Diglyphusdifasciatus Liu, Hansson & Wan, sp. nov., was discovered during the identification of agromyzid leafminers and their associated parasitoid wasps collected from 2016 to 2022 in China, based on morphological characteristics and molecular analyses of COI, ITS2 and 28S genes. Diglyphusdifasciatus is similar to D.bimaculatus Zhu, LaSalle & Huang, distinguished by two interconnected infuscate vertical bands on the fore wing and the color of the scape. Molecular data support D.difasciatus and D.bimaculatus as two different species. The mean genetic distances between D.difasciatus and D.bimaculatus were 11.33%, 8.62%, and 0.18%, based on the COI, ITS2, and 28S genes, respectively.

A One-Step Multiplex PCR Method to Rapidly Distinguish Two Strains of Diglyphus wani (Hymenoptera: Eulophidae) Against Agromyzid Leafminers.

Hymenopteran parasitoids generally show a haplo-diploid sex determination system. Haploid males are produced from unfertilized eggs, whereas diploid females develop from fertilized eggs (arrhenotokous). In some cases, diploid females develop from unfertilized eggs (thelytokous). Diglyphus wani (Hymenoptera: Eulophidae) is a biological control agent for agromyzid leafminers and have arrhenotokous and thelytokous strains. However, the morphological characteristics of two strains of D. wani are so similar that it is difficult to accurately distinguish them based on morphology. Here, a rapid molecular identification method was developed based on the mitochondrial gene cytochrome c oxidase I (COI) and one-step multiplex PCR. Two primer combinations, PC1 (Ar-F1/Th-F1/WR2) and PC2 (Ar-F1/Th-F4/WR2), were designed and repeatedly screened to distinguish two strains simultaneously, of which two special forward primers Th-F1/Th-F4 were used for the thelytokous strain and one special forward primer Ar-F1 was used for the arrhenotokous strain. In addition, a common reverse primer, WR2, was used for both strains. The PC1 and PC2 PCR assays were effective in distinguishing the two strains at different developmental stages and field colonies. This method provides a reliable, highly sensitive, and cost-effective tool for the rapid identification of the two strains of D. wani.

Morphological and molecular identification of arrhenotokous strain of Diglyphuswani (Hymenoptera, Eulophidae) found in China as a control agent against agromyzid leafminers.

Diglyphus species are ecologically and economically important on agromyzid leafminers. In 2018, a thelytokous species, Diglyphuswani Liu, Zhu & Yefremova, was firstly reported and described. Subsequently, the arrhenotokous D.wani were discovered in Yunnan and Guizhou Provinces of China. We compared the morphological characteristics of thelytokous and arrhenotokous strains. However, the females of two strains had a strongly similar morphology and showed subtle differences in fore- and hind-wings. The difference was that forewing of arrhenotokous female was with denser setae overall, showing that costal cell with 2 ~ 4 rows of setae on dorsal surface and the setae of basal cell with 15 ~ 21 hairs and forewing of thelytokous female was with two rows of setae on dorsal surface and basal cell with 10 ~ 15 hairs generally. The setation beneath the marginal vein of the hind-wing of arrhenotokous female is denser than the same area of thelytokous female. To explore the genetic divergence between thelytokous and arrhenotokous strains of D.wani, the mitochondrial and nuclear gene were applied and sequenced. The polygenic analyses revealed that two strains can be distinguished by COI, ITS1 and ITS2. The mean sequence divergence between the two strains was 0.052, 0.010 and 0.007, respectively. Nevertheless, the 28S gene was unfeasible due to its containing a sharing haplotype between different strains. The two strains of D.wani are dominant parasitoids against agromyzid leafminers and such effective discernible foundation provides future in-depth studies on biological characteristics, along with insight into field application of two strains of D.wani.