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.

The thelytokous strain of the parasitoid Neochrysocharis formosa outperforms the arrhenotokous strain in reproductive capacity and biological control of agromyzid leafminers.

BACKGROUND: Both arrhenotoky (sexual reproduction of females and asexual reproduction of males) and thelytoky (asexual reproduction of females) occur within the order Hymenoptera. The existence of both thelytokous and arrhenotokous strains within one species provides an opportunity to compare the biocontrol efficiency between two reproductive modes. The parasitoid Neochrysocharis formosa (Westwood) (Hymenoptera: Eulophidae) has thelytokous and arrhenotokous strains with sympatric distributions. This parasitoid is used to control invasive leafminers through feeding, stinging, and parasitization. To compare the biocontrol efficiency of the two strains, we analyzed life tables and host-killing parameters of these two strains reared on the leafminer Liriomyza sativae Blanchard using the age-stage, two-sex life table and the CONSUME-MSChart software. RESULTS: Our results showed that the intrinsic rate of increase (r), finite rate of increase (λ), and net reproduction rate (R0 ) of the thelytokous strain were significantly higher than those of the arrhenotokous strain. The thelytokous females also performed better than the arrhenotokous females for the net host-feeding rate, net host-stinging rate, and net host-killing rate, but not the finite parasitism rate. Conclusively, the finite host-killing rate of the thelytokous strain (0.8720 ± 0.0516) was significantly higher than that of the arrhenotokous strain (0.5914 ± 0.0832). CONCLUSION: We concluded that thelytokous N. formosa is a better candidate as a biocontrol agent than arrhenotokous N. formosa to control leafminers. Our results shed light on how to choose a better biocontrol agent for integrated pest management (IPM) based on biological control, especially for co-occurring thelytokous and arrhenotokous parasitoids. © 2022 Society of Chemical Industry.

Apomixis for no bacteria-induced thelytoky in Diglyphus wani (Hymenoptera: Eulophidae).

In Hymenoptera species, the reproductive mode is usually arrhenotoky, where haploid males arise from unfertilized eggs and diploid females from fertilized eggs. In addition, a few species reproduce by thelytoky, where diploid females arise from unfertilized eggs. Diploid females can be derived through various cytological mechanisms in thelytokous Hymenoptera species. Hitherto, these mechanisms were revealed mainly in endosymbiont-induced thelytokous Hymenoptera species. In contrast, thelytokous Hymenoptera species in which a reproductive manipulator has not been verified or several common endosymbionts have been excluded were paid less attention in their cytological mechanisms, for instance, Diglyphus wani (Hymenoptera: Eulophidae). Here, we investigated the cytological mechanism of D. wani using cytological methods and genetic markers. Our observations indicated that the diploid karyotypes of two strains of D. wani consist of four pairs of relatively large metacentric chromosomes and one pair of short submetacentric chromosomes (2n = 10). The arrhenotokous strains could complete normal meiosis, whereas the thelytokous strain lacked meiosis and did not expulse any polar bodies. This reproductive type of lacking meiosis is classified as apomictic thelytoky. Moreover, a total of 636 microsatellite sequences were obtained from thelytokous D. wani, dominated by dinucleotide repeats. Genetic markers results showed all three generations of offspring from thelytokous strain maintained the same genotype as their parents. Our results revealed that D. wani is the first eulophid parasitoid wasp in Hymenoptera whose thelytoky was not induced by bacteria to form an apomictic thelytoky. These findings provide a baseline for future inner molecular genetic studies of ameiotic thelytoky.

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.

Multiple Data Demonstrate That Bacteria Regulating Reproduction Could Be Not the Cause for the Thelytoky of Diglyphus wani (Hymenoptera: Eulophidae).

In Hymenoptera parasitoids, the reproductive mode is arrhenotoky, while a few species reproduce by thelytoky. The thelytoky of Hymenoptera parasitoids is generally genetically determined by the parasitoids themselves or induced by bacteria, including Wolbachia, Cardinium, and Rickettsia. Diglyphus wani (Hymenoptera: Eulophidae), a recently reported thelytokous species is a main parasitoid attacking agromyzid leafminers. To assess whether endosymbionts induce thelytoky in D. wani, we performed universal PCR detection and sequenced the V3-V4 region of 16S ribosomal RNA gene. In addition, bacteria were removed through high-temperature and antibiotic treatments, and the localized bacteria were detected using FISH. Based on general PCR detection, Wolbachia, Cardinium, Rickettsia, Arsenophonus, Spiroplasma, and Microsporidia were absent in laboratory and field individuals of thelytokous D. wani. Furthermore, 16S rRNA gene sequencing revealed that the dominant endosymbionts in thelytokous D. wani were not reproductive manipulators. High-temperature and antibiotic treatment for five consecutive generations cannot reverse the thelytokous pattern of D. wani, and no male offspring were produced. Moreover, no bacterial spots were found in the ovaries of D. wani. Thus, it is considered that the thelytoky of D. wani does not result in the presence of endosymbionts. This species is thus the second reported eulophid parasitoid whose thelytoky appears not to be associated with endosymbionts.

Life history and biocontrol potential of the first female-producing parthenogenetic species of Diglyphus (Hymenoptera: Eulophidae) against agromyzid leafminers.

Diglyphus species are dominant biocontrol agents for suppressing outbreaks of agromyzid leafminers in fields. In July 2015, we collected a new thelytokous species of Diglyphus in Qinghai, China. The wasp is here named as D. wani Liu, Zhu & Yefremova sp. nov., based on morphological and molecular analyses. The life history and biocontrol potential of the wasp were studied in the laboratory and by providing Liriomyza sativae larvae. The intrinsic rate of increase, finite rate of increase, and mean generation time were 0.2373 d-1, 1.2678 d-1, and 15.9 d, respectively. The wasps showed three types of host-killing behaviour, namely parasitism, host feeding and host stinging, resulting in 47.6, 94.4 and 3.4 host larvae killed per wasp over a lifetime, respectively. The finite total host-killing rate was 1.0071 and the wasp showed strong synovigeny. The wasp may be a crucial biocontrol agent against agromyzid leafminers.