Steinernema ethiopiense sp. n. (Rhabditida: Steinernematidae), a new entomopathogenic nematode from Ethiopia

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Three isolates (Dero-1, Dero-8 and Mosisa-1) of a new entomopathogenic nematode, S. ethiopiense sp. n., were isolated by baiting soil samples from the Mendi area, Western Wollega, Ethiopia, with last instar wax moth larvae Galleria mellonella. Infective juveniles of S. ethiopiense sp. n. have a body length of 898 (768-1010) μm, a maximum of eight identical ridges (i.e., nine lines) in the lateral field, excretory pore located at mid-pharynx, hyaline layer occupying approximately half of the tail and c′ = 3.2. First generation males lack a caudal mucron, whereas second generation males possess a short spine-like mucron. The spicules are slightly arcuate, golden-brown in colour and have an ellipsoid or oblong manubrium. First generation females lack a postanal swelling and have a minute protuberance on the tail tip whereas second generation females have a postanal swelling and protruding vulva. Based on the morphology, morphometrics and DNA analysis, the new species belongs to the glaseri group. The closest relative species is the afro-tropical S. karii recorded from Kenya. The BLAST analysis of the ITS region of the rDNA revealed a similarity of 93% with S. karii, supporting the validity of S. ethiopiense sp. n. as a new species. In the phylogenetic trees the new species groups together only with S. karii (bootstrap value of 100%), but is also separated from S. karii by a bootstrap value of 100% or 70%.

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References
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Figures
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    Steinernema ethiopiense sp. n. A-C: Female. A: First generation tail; B: Second generation tail; C: First generation vulva. D-J: Male. D: Head and pharynx; E: First generation tail with genital papillae (ventral); F: First generation tail with genital papillae (slightly rolled); G: Second generation tail with genital papillae and mucron (slightly rolled); H: First generation spicule and variability of proximal end; I: First generation gubernaculum and variability of proximal end; J: Gubernaculum (ventral). K, L: Infective juvenile. K: Lateral field with eight ridges; L: Tail with hyaline region and phasmids. (Scale bar: A = 145 μm; B, H = 50 μm; C = 240 μm; D = 105 μm; E = 100 μm; F = 70 μm; G = 55 μm; I = 45 μm; J = 25 μm; K = 40 μm; L = 65 μm.)

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    Steinernema ethiopiense sp. n. DIC microscopy. A: First generation female vulva; B: First generation female tail showing minute protuberance; C: First generation male tail with spicules and gubernaculum; D: First generation male tail with two protuberances on gubernaculum; E: Second generation male tail with a short mucron (arrow) and irregular proximal end of gubernaculum; F: First generation male tail with single protuberance on gubernaculum; G: Infective juvenile with rounded head and pharynx (left) and bacterial vesicle (right; arrow); H: Infective juvenile tail. (Scale bar: A = 145 μm; B = 57 μm; C = 80 μm; D-F = 27 μm; G = 47 μm; H = 70 μm.)

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    Steinernema ethiopiense sp. n. SEM microscopy. A: Lateral field of infective juvenile with eight ridges (1-8) at mid-body; B: Lateral field of infective juvenile showing gradual disappearance of ridges on tail, four ridges reducing to two at phasmid level; C: First generation female tail; D: First generation female head with two circles of papillae and pair of amphidial apertures; E: First generation male tail with genital papillae and spicules; F: First generation male head with two circles of papillae and pair of amphidial apertures.

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    Phylogenetic relationships (strict consensus tree, Maximum Parsimony analysis) of Dero-1, Dero-8 and Mosisa-1 with 53 Steinernema spp. based on ITS-rDNA sequences. Caenorhabditis elegans (EU131007) was used as outgroup. Bootstrap values >60 are indicated.

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    Phylogenetic relationships (strict consensus tree, Minimum Evolution analysis) of Dero-1, Dero-8 and Mosisa-1 with 53 Steinernema spp. based on ITS-rDNA sequences. Caenorhabditis elegans (EU131007) was used as outgroup. Bootstrap values >60 are indicated.

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    Phylogenetic relationships (strict consensus tree, Maximum Parsimony analysis) of Dero-1, Dero-8 and Mosisa-1 with 49 Steinernema spp. based on D2D3 sequences of the 28S rRNA gene. Cervidellus alutus (AF331911) were used as outgroups. Bootstrap values >60 are indicated.

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    Phylogenetic relationships (strict consensus tree, Minimum Evolution analysis) of Dero-1, Dero-8 and Mosisa-1 with 49 Steinernema spp. based on D2D3 sequences of the 28S rRNA gene. Cervidellus alutus (AF331911) was used as outgroup. Bootstrap values >60 are indicated.

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