Due to intensive pesticide application, FAW has developed resistance to a variety of chemical pesticides in several countries (Yu et al., 2003; Zhao et al., 2019), i.e. substantially high levels of resistance to omethoate was observed in FAW captured in China (Zhao et al., 2019). Based on the adverse effects of some chemicals on human health, the environment and living organisms, researchers are focusing on potential biological control agents (Harrison et al., 2019). FAW are attacked by various natural biocontrol agents such as parasitoids (López et al., 2018), bacteria (del Valle Loto et al., 2019), fungi (Shylesha et al., 2018), nematodes (Ruiz-Nájera et al., 2013; Viteri et al., 2018), and virus (Souza et al., 2019). There is a dearth of information on natural enemies of FAW, in China, particularly because the insect only invaded the region within the past year. It is high time to develop biological control methods for FAW in China. Although assessing the virulence of commercial biological control agents is a very important way to control FAW from the perspective of environmental protection, there is also a great need to understand, promote, and maximize the effectiveness of indigenous populations of natural enemies. Nematodes are the most abundant metazoan on earth. Nematodes in several families are able to kill insects, known to be entomoparasitic nematodes. Of them, Mermithidae, Steinernematidae, and Heterorhabditidae have been more studied and some are considered as biological control agents for FAW (Huot et al., 2019; Ruiz-Nájera et al., 2013; Tarla et al., 2015; Viteri et al., 2018).
In general, mermithids nematode parasites can infect various hosts, such as spiders, mosquitoes, grasshoppers, or cockroaches (Košulič and Mašová, 2019; Kobylinski et al., 2012; Tarla et al., 2015). In addition, mermithids are also fatal to the insect host (Nikdel et al., 2011). Mermithid parasites have a great degree of species-specificity, so they are more promising to control target pests (Sáringer-Kenyeres et al., 2017). For example, in China the mermithid
Recently, we found FAW naturally parasitized by a mermithid nematode in a field located in Hainan Province, China. This is the first report of
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.
The prevalence of parasitic nematodes in parts of Hainan province is shown in Table 1. According to the survey data, the parasitic rate of the nematodes in FAW is about 2%. During the cultivation process, FAW was found and the survey distribution of nematode infection rate is shown in Table 1.
Location, date, coordinates, and number of larvae, parasitic nematodes of fall armyworm (FAW)
Date | Location | Coordinates | Total FAW (No.) | Parasitized FAW (No.) | Cornfield size (m2) | Plant height (cm) | Plant status | Planting gap (cm) | Spray insecticide situation |
---|---|---|---|---|---|---|---|---|---|
May 08, 2019 | 1 Wuna road, Qiongzhong Li and Miao autonomous county, Hainan Province, China | N 19°2´20˝ |
43 | 1 | 600 | 35 | The late seedling | 50 | No spray insecticide |
June 13, 2019 | Qionghai X356, Hainan Province, China | N 19°15´51˝ |
52 | 1 | 2300 | 120 | Jointing and booting stage | 40 | No spray insecticide |
June 11, 2019 | Foluo town, Ledong Li autonomous county, Hainan Province, China | N 18°61´97˝ |
105 | 2 | 990 | 150 | Mature stage | 45 | No spray insecticide |
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).
Microscopic photo of
Post-parasitic juvenile
Post-parasitic juvenile
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
Neighbor-joining tree of the Mermithidae family. The tree was based on 18 S rDNA data and Kimura 2-parameter model; numbers on branches represent bootstrap support (>70%) based on 500 replicates; scale represents K2P genetic distance.
Neighbor-joining tree of the Mermithidae family. The tree was based on 28 S rDNA data and Kimura 2-parameter model; numbers on branches represent bootstrap support (>70%) based on 500 replicates; scale represents K2P genetic distance.
The 18 S rDNA sequences of HN-1 and HN-2 nematode specimens from Hainan Province were analyzed by Blast, and the similarity with
Alignment of 18 S sequences for comparative purposes of
To our knowledge, this is the first report that