First report of Ovomermis sinensis (Nematoda: Mermithidae) parasitizing fall armyworm Spodoptera frugiperda (Lepidoptera: Noctuidae) in China

From May 8 to July 24, 2019, the collection of FAW was carried out in the corn fields of Qiongzhong County, Ledong County, Yazhou District, Danzhou City, and Qionghai City, Hainan Province. Large numbers of larvae were collected by a chessboard sampling method in maize fields in the above areas. The instar and number of FAW larvae obtained were recorded and all insects were brought back to the laboratory for feeding and observation. Digital images were obtained using Olympus BX63. For the molecular description, the nematodes were removed from the parasitized FAW.

Genomic DNA was extracted from the nematodes using a Universal Genomic DNA Kit (CWBIO, China) according to the manufacturer’s protocol. Amplifications of 18 S and partial 28 S ribosomal DNA (D3 region) were performed according to Kobylinski et al. (2012) and Wang et al. (2007). As for 18 S, the following primers were used: 18S-F: 5’-CAAGGACGAAAGTTAGAGGTTC-3’ (forward) and 18S-R: 5’-GGAAACCTTGTTACGACTTTTA-3’ (reverse) (Kobylinski et al., 2012). A pair of primers of 28 S, 28S-5F: 5’-ACCCGTCTTGAAACACGGA-3’ (forward) and 28S-9R: 5’-TCGGAAGGAACCAGCTACTA-3’ (reverse) adopted from Wang et al. (2007) were used in D3 region study. Each polymerase chain reaction (PCR) was made in a total volume of 25 μl containing 12.5 μl × Es Taq MasterMix (CWBIO, China) 0.5 μl 10 μM of each primer, 2 μl template DNA and 9.5 μl ddH₂O. PCR products were electrophoresed in 1.5% agarose gel, purified and sequenced in both directions with ABI 3730 (Suzhou Genewiz Biotechnology Co., Ltd., Tianjin, China). Sequences were aligned using Clustalw with the default settings in MEGA X software package (Kumar et al., 2018). Sequences were visually proofread, edited, and assembled into contigs in Bioedit v7.1.7 (Hall, 2012). The resulting sequences were submitted to GenBank. Sequences of mermithid from Genbank were searched and involved into the phylogenetic analysis. Pairwise distances and neighbor-joining (NJ) (Saitou and Nei, 1987) phylogenetic analysis were done using MEGA X software package (Kumar et al., 2018) under a Kimura 2-parameter (Kimura, 1980) model. Bootstrap analysis was computed with 500 replicates.

According to morphological characteristics, these nematodes obtained from Hainan province belong to the family Mermithidae. This nematode has a stylet on the anterior portion of post-parasitic juvenile (Fig. 1A) and post-parasitic tail appendage (0.62 mm) on posterior end of post-parasitic juvenile (Fig. 1B). The mermithids are white and 228.5 mm in length (Fig. 2). S. frugiperda parasitized by the nematodes is shown in Figure 3.

Figure 1:

Figure 2:

Figure 3:

In the molecular analyses, the two individuals analyzed showed no polymorphism in the 18 S rDNA gene fragment detected (accession number MN367956 and MN367957). The D3 fragment of 28 S rDNA gene was uploaded with accession numbers MN367954 and MN367955. Based on the NJ trees of 18 S and D3 region in 28 S (Figs. 4 and 5), respectively, we confirmed that the nematodes isolated from S. frugiperda in Hainan province are O. sinensis and form an OTU with very small genetic distance.

Figure 4:

Figure 5:

The 18 S rDNA sequences of HN-1 and HN-2 nematode specimens from Hainan Province were analyzed by Blast, and the similarity with O. sinensis (KU177046) was 99.35%. There are five base differences between them and the difference sites as shown in Figure S1. The corresponding genetic distance between the two tested samples (HN-1 and HN-2) and the known species O. sinensis from GenBank was 0.0040 and 0.0080 based on 18 S rDNA gene, 0.0836 and 0.0825 based on D3 region in 28 S rDNA gene, respectively. This genetic distance is significantly smaller than that between any two species, which is enough to suggest that the nematodes found in Hainan province belong to the species O. sinensis.

Figure S1: