Nematoda: Rhabditidae), a Potentially Entomopathogenic Nematode from the Marshlands of Wisconsin, USA
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JOURNAL OF NEMATOLOGY Article | DOI: 10.21307/jofnem-2018-004 Issue 1 | Vol. 50 Incidence of Oscheius onirici (Nematoda: Rhabditidae), a potentially entomopathogenic nematode from the marshlands of Wisconsin, USA Weimin YE,1* Shane Foye,2 Ann E. MacGuidwin,3 and Shawn Steffan4 Abstract 1Nematode Assay Section, In a search for an entomopathogenic nematode to control cranberry Agronomic Division, North insect pests, three Oscheius populations (Rhabditidae) were Carolina Department of recovered through the Galleria-bait method from one sample taken Agriculture & Consumer Services, in a wild cranberry marsh in Jackson County, Wisconsin, USA. 4300 Reedy Creek Road, Raleigh, Morphological studies with light microscopy and scanning electron N C 276 07. microscopy, as well as molecular analyses of the near-full-length 2Department of Entomology, small subunit rDNA gene, D2/D3 expansion segments of the large University of Wisconsin-Madison, subunit rDNA gene, internal transcribed spacer, and mitochondrial 1630 Linden Drive, Madison, cytochrome oxidase subunit 1 (CoxI) genes revealed this as Oscheius WI 53706. onirici, a species recently described from a karst cave soil of central 3Department of Plant Pathology, Italy. The species belongs to the dolichura-group and is characterized University of Wisconsin-Madison, by its DNA sequences; hermaphroditic reproduction; and males not 1630 Linden Drive, Madison, WI found. A Bacillus-like bacterium appears to be associated with this 53706. nematode based on our microscopic and SEM observations; however 4United States Department of its identity and persistent association with the nematode has not Agriculture, Agricultural Research been confirmed. Nonetheless, this nematode is capable of infecting Service, Madison, WI 53706. and killing the sparganothis fruitworm Sparganothis sulfureana *E-mail: [email protected]. Clemens (Lepidoptera: Tortricidae), the brown-banded cockroach Supella longipalpa Fabricius (Blattodea: Ectobiidae), and the cranberry Weimin YE, and Shane Foye are fruitworm Acrobasis vaccinii Riley (Lepidoptera: Pyralidae), under co-first author. laboratory conditions, and each in less than 72 hr. The mealworm This article was edited by Raquel Tenebrio molitor Linnaeus (Coleoptera: Tenebrionidae) and the greater Campos-Herrera. wax moth Galleria mellonella Linnaeus (Lepidoptera: Pyralidae), are Received for Publication November also susceptible, but take 3.5 and 5.2 days to die, respectively. This 19, 2017. species is a new potential bio-control agent on insects. Key words Cranberry, characterization, DNA sequencing, entomopathogenic nematode, Dolichura-group, molecular phylogeny, morphology, rDNA. Oscheius Andrassy, 1976 belongs to the family Rhab- the Insectivora group have crochet-like needle-shaped ditidae. These bacteria-feeding nematodes live in close spicules, leptoderan bursa and common rectum versus association with insects (Stock et al., 2005; Ye et al., normal spicules, peloderan bursa and expansile rectum 2010; Campos-Herrera et al., 2015; Torrini et al., 2015). in Dolichura group (Sudhaus and Hooper, 1994). So far, Oscheius was divided into two groups: Insectivora and 43 species were described in Oscheius with 29 in Insec- Dolichura (Sudhaus and Hooper, 1994). Species of tivora and 14 in Dolichura (Tabassum et al., 2016). 9 © The Society of Nematologists 2018. Incidence of Oscheius onirici (Nematoda: Rhabditidae), a potentially entomopathogenic nematode Nematodes can be considered as entomopath- have shown that nematodes in the genus Oscheius ogenic if they fulfill the following criteria for ento- can sometimes be entomopathogenic. mopathogenicity: bearing a pathogenic bacterium A survey of the EPN fauna was initiated in Wis- within a dauer (also known as infective) juvenile nem- consin during the summer of 2015, then repeated in atode, releasing the bacterium within the host, active 2016. This survey was conducted using a previously host-seeking and -penetration by dauer juveniles, rap- employed Galleria-bait method (Orozco et al., 2014). id insect death, nematode and bacterial reproduction, A total of three cadavers were recovered (n = 540 wax reassociation of the pathogenic bacteria with new worms used during each of the seasons), all from generations of dauer juveniles; and emergence of IJs 2015. The EPN that emerged from them were main- from the cadaver so that the cycle can be repeated tained in culture. They appeared as hermaphroditic (Dillman et al., 2012). Only seven species in Insectivora without males. DNA sequencing was performed to group including O. amsactae (Ali et al., 2011), O. car- identify this population and it is identical to two her- olinensis (Ye et al., 2010), O. chongminensis (Zhang maphroditic populations of PS2068 from Oregon, et al., 2008), O. microvilli (Zhou et al., 2017), O. niazii USA and JU179 from France (Felix et al., 2001) and (Tabassum and Shahina, 2010), O. rugaoensis (Zhang it has minor differences from a recently described et al., 2012), O. siddiqii (Tabassum and Shahina, 2010) species O. onirici from a karst cave soil of central It- and one species in Dolichura group namely O. onirici aly. This paper presented the characterization of this (Torrini et al., 2015) were recognized as entomopatho- nematode through combined approaches by mor- genic. Phylogenetic analysis based on ribosomal DNA phological and molecular analysis to determine its sequences revealed these two groups are monophy- identity and tested its entomopathogenicity. letic (Ye et al., 2010, Torrini et al., 2015). Heterorhab- ditidoides was proposed as a new genus (Zhang Materials and methods et al., 2008), but it was considered as a junior syno- nym of Oscheius (Ye et al., 2010) and it is in general Isolation of EPN from cranberry marshes not accepted as a valid genus (Liu et al., 2012; Campos-Herrera et al., 2015; Torrini et al., 2015; Counties with commercial cranberry operations were Tabassum et al., 2016; Valizadeh et al., 2017). Another chosen for sampling efforts. Sites were further refined species, Heterorhabditidoides rugaoensis, was also by selecting locations that also had accessible wild transferred to Oscheius (Darsouei et al., 2014; Tabassum cranberry populations. Based on these criteria, two et al., 2016). sites were chosen in Jackson County and one was Nematodes of the genera Steinernema and Het- chosen in Warren County in Wisconsin. erorhabditis, are often entomopathogenic, and as Once sites were established, a modified method such, they have been used as bio-control agents to described in Orozco et al. (2014) was used to sam- suppress pest populations (Kaya and Gaugler, 1993). ple them. Briefly, a four square-meter grid was estab- These nematodes have been found on every conti- lished around the first wild cranberry plant encoun- nent except for Antarctica, and are lethal to a wide tered at each site. The grid was randomly sampled variety of insect pests (Poinar and Thomas, 1984). three times by taking an approximately 200 cm3 vol- For bio-control programs, nematodes represent par- ume of substrate from the surface of the soil to a ticularly promising agents because they can be read- depth of about 15 cm. This sample was placed in a ily applied to crops using standard spray equipment. plastic bag. The process was repeated twice, within In the cranberry marshlands of Washington, for ex- each site, on each sampling date. Sites were sampled ample, entomopathogenic nematodes (EPNs) have during the first week of May, June, July, and August been used to control the black vine weevil Otiorhy- 2015. nchus sulcatus Fabricius (Coleoptera: Curculionidae) Although sites were located in different geograph- (Booth et al., 2002). In the bogs of Massachusetts, ic locations, there were some similar and noteworthy populations of the strawberry root weevil have been conditions present in each sampling grid. The pres- suppressed via applications of Heterorhabditis bac- ence of Sphagnum moss (Sphagnales: Sphagnace- teriophora Poinar, 1976 (Simser and Roberts 1994). ae) was unmistakable in all sampling grids, with cran- Typically, commercially available EPN are found in the berry plants growing out of hummocks formed by genera Heterorhabditis or Steinernema; however, re- this plant. Peat could be found approximately 30 cent discoveries of nematodes in North Carolina, USA centimeters below the moss. Plants like leatherleaf, (Ye et al., 2010), Switzerland (Campos-Herrera et al., Chamaedaphne sp. (Ericales: Ericaceae), sedges 2015, Jaffuel et al., 2016) and Italy (Torrini et al., 2015) (Poales: Cyperaceae), and bog birch, Betula pumila 10 JOURNAL OF NEMATOLOGY (Fagales: Betulaceae) were typically located at the cut open and fitted with 20-μ m nylon mesh to hold sampling sites. These plants are typical of the peat- the specimens through the entire preparation proce- land habitats that support cranberry plants (Rydin and dure (Bozzola and Russell, 1999). The capped cap- Jeglend, 2006). sules were then transferred to a jar containing 3% Upon returning to the laboratory, EPN were ex- glutaraldehyde in 0.1 M sodium cacodylate buffer, pH tracted with the insect baiting technique as described 7.2, at 4°C. Samples were washed in three changes in Orozco et al. (2014). Each soil sample was baited by of the same buffer; post-fixed in 2% osmium tetrox- placing six fifth instar wax worms Galleria mellonella ide in 0.1 M sodium cacodylate buffer; pH 7.2, for 16 hr Linnaeus (Lepidoptera: Pyralidae) into the plastic bags at 4°C in the dark in a refrigerator; washed in three containing