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

Abstract Spodoptera frugiperda invaded China in the end of 2018 and has caused severe damage to maize and other crops. Several S. frugiperda naturally parasitized by nematodes were observed in Hainan Province, China. The morphological characteristics based on the results of scanning electron microscopy indicated that the nematode belongs to the family Mermithidae. Additionally, coding sequences for the 18 S and 28 S rDNA were amplified from the nematode genome, and phylogenetic analysis revealed that the nematode belongs to Ovomermis sinensis, a known entomoparasitic nematode. Our finding is the first record that S. frugiperda was naturally parasitized by O. sinensis. The results of this study are of great significance for potential biological control of S. frugiperda by indigenous natural beneficial organisms, i.e. O. sinensis within an integrated pest management system.

Spodoptera frugiperda (J. E. Smith), fall armyworm (FAW) is a pest native to tropical and subtropical regions of the America and widely distributed throughout the American continents (Todd and Poole, 1998). The pest, which is indigenous, is highly polyphagous, causing economic damage in various crops such as maize, beans, cotton, and sorghum (Day et al., 2017). Due to its voracity, high dispersal ability, wide host range, and high fecundity, it has already invaded many countries in Africa and Asia and is causing substantial yield losses . FAW causes up to $16 billion in crop losses across Africa annually (Harrison et al., 2019). FAW invaded Yunnan province, China in late 2018. Since then FAW has dramatically spread in China Li et al., 2019), and was recorded in 1 million hectares from 26 provinces in China by December 2019.
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), First report of Ovomermis sinensis parasitizing Spodoptera frugiperda: Sun et al.
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. Nema todes 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).
Recently, we found FAW naturally parasitized by a mermithid nematode in a field located in Hainan Province, China. This is the first report of O. sinensis (Nematoda: Mermithidae) parasitizing FAW S. frugiperda (Lepidoptera: Noctuidae) in the world. Our discovery can provide urgent and useful information on policy making for the control of FAW in China and Asia.

Field survey
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.

Results
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.

Diagnostic characters
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.

Molecular analyses
In the molecular analyses, the two individuals ana lyzed 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.
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.

Discussion
To our knowledge, this is the first report that S. frugiperda was parasitized by O. sinensis in a natural context, an indigenous mermithid nematode in China. In a survey conducted in Mexico, mermithid nematode species from another genus, Hexamermis sp., was found to be parasitic on FAW, causing a mortality rate of 8.42% (Ruiz-Nájera et al., 2013). The present study confirms new associations of mermithid nematodes O. sinensis as natural enemies of S. frugiperda.
O. sinensis had been efficiently used to control pest insects from family Noctuidae, i.e. Spodoptera litura and Helicoverpa armigera (Li et al., 2003) as well as M. separata (Walker) (Chen et al., 1991;Zhang et al., 1992). S. frugiperda, also from Noctuidae, was found naturally parasitized by O. sinensis in the preset study. All these findings confirmed the great potential of O. sinensis for control against the invasive pest insect S. frugiperda and other pest insects from Noctuidae. An intriguing finding in this study is that we can exploit the great potential to use O. sinensis to control FAW. Information on the occurrence and rates of parasitism of indigenous natural enemies is of paramount importance in designing a biological control program for S. frugiperda, either through conservation of native natural enemies or the introduction of new species for augmentative release. Major field releases of mermithids have been conducted for Anopheles population control (Kobylinski et al., 2012). Therefore, further study is needed to understand the biological and ecological relationship between O. sinensis nematodes and their hosts, and to explore the approach to artificially rear this nematode for potential field application in future.