Nematology, 2008, Vol. 10(4), 561-574

A new entomopathogenic nematode, Steinernema colombiensen. sp. (Nematoda: Steinernematidae), from Colombia

Juan Carlos LÓPEZ-NÚÑEZ 1, Kathryn PLICHTA 2, Carmenza E. GÓNGORA-BOTERO 1

and S. Patricia STOCK 2,∗1 Disciplina de Entomología, Centro Nacional de Investigaciones de Café, Cenicafé, Cenicafé Planalto,

Kilómetro 4, Vía Antigua a Manizales, Chinchiná, Caldas, Colombia2 Department of Entomology, The University of Arizona, Forbes 410, 1140 E, South Campus Drive,

Tucson, AZ 85721-0036, USA

Received: 26 October 2007; revised: 17 December 2007Accepted for publication: 19 December 2007

Summary – A new entomopathogenic nematode, Steinernema colombiense n. sp., is described from Colombia. Morphological,molecular (28S and ITS rDNA sequence data) and cross-hybridisation studies were used for diagnostics and identification purposes. Inaddition, 28S and ITS rDNA sequence data were used to assess evolutionary relationships of the new species with other Steinernemaspp. Morphological diagnostic features for S. colombiense n. sp. include morphometric features of the third-stage infective juvenile,including body length of 636 (549-732) µm, narrow body diam. (31 (22-36) µm), position of the excretory pore (35 (31-40) µm), taillength (41 (32-53) µm), D% = 29 (25-33) and E% = 205 (138-284). In addition, males of first and second generations are characterisedby the morphology of the spicules and gubernaculum, the number and arrangement of the genital papillae and the excretory poreposition (at 67 (56-76) and 54 (46-63) µm, for first and second generations, respectively). In addition to these traits, 28S and ITS rDNAsequences analyses both showed this species to be a distinct and unique entity.

Keywords – description, molecular, morphology, morphometrics, new species, phylogeny, South America, taxonomy.

Public awareness of the problems developed from theuse of chemical pesticides (i.e., adverse impact on humanhealth, wildlife, environmental pollution, pesticide resis-tance and pest resurgence) has dramatically increased overthe past year in most Latin American countries, includ-ing Colombia. Environmentalists, scientists and labourgroups from various countries not only demand laws toregulate chemical pesticide use but also to disseminateand sponsor the search and consideration of natural en-emies and entomopathogens to control a wide array ofinvertebrate pests of agricultural forestry and urban sig-nificance.

Entomopathogenic nematodes (EPN) (Steinernemati-dae and Heterorhabditidae) are one of the beneficial or-ganisms currently considered in biological control pro-grammes in Colombia (López et al., 2007). Empha-sis has been place on the discovery of native speciesand strains, as well as on the introduction of exoticspecies, particularly, currently available EPN formula-tions.

∗ Corresponding author, e-mail: spstock@ag.arizona.edu

EPN have been considered for control of several nox-ious agricultural pests, including the cassava bug (Cyr-tomenus bergi Froeschner), oil palm borer (Sagalassa val-ida Walker), coffee berry borer (Hypothenemus hampei(Ferrari)) and white grub complex (Phyllophaga and Ano-mala spp.) (see Caicedo & Belloti, 1996; Saenz, 1998;López, 2002, 2003; Caicedo et al., 2004; Lara et al., 2004;Mello & Gail, 2004; Sáenz et al., 2005; López et al.,2007).

In one of the earliest surveys in this country, a steiner-nematid nematode was isolated from a coffee plantation(Maracay Experimental Station, National Federation ofColombian Coffee Growers) on the western slopes ofthe Central Andean range (López, 2002). This unchar-acterised Steinernema sp. was designated as isolate SNI-0198. Morphological and molecular characterisation indi-cated that this nematode was a new, undescribed, species.We herein provide a formal description of this species us-ing a combination of morphological and molecular meth-ods.

© Koninklijke Brill NV, Leiden, 2008 561Also available online - www.brill.nl/nemy

J.C. López-Núñez et al.

Materials and methods

ISOLATION AND NEMATODE PROPAGATION

Isolate SNI-0198 was recovered from soil samplesin a coffee plantation at Maracay Experimental Station(National Federation of Colombian Coffee Growers) onthe western slopes of the Central Andean mountain range.The insect-baiting method described by Bedding andAkhurst (1975) was used for isolation of this nematode.Baits (ten, last instar Galleria mellonella (L.) larvae)were placed in 250 ml plastic containers (five containersper sample) with moistened soil obtained from eachsample. Containers were covered with a lid, turned upsidedown and kept at room temperature (20 ± 3◦C). Galleriamellonella larvae were checked every 2-3 days anddead larvae were replaced by fresh ones. After 7 days,dead insects were removed from the baiting containers,thoroughly rinsed in distilled water and placed in modifiedWhite traps (Kaya & Stock, 1997) until emergence ofthird-stage infective juveniles.

MORPHOLOGICAL OBSERVATIONS

Specimens for morphological studies were obtainedfrom last instar G. mellonella larvae exposed to ca 100third-stage infective juveniles (IJ) per larva on moistenedfilter paper in Petri dishes and incubated at 25◦C in thedark. First and second generation adults and third-stageinfective juveniles were randomly collected from infectedcadavers following procedures described by Kaya andStock (1997). Twenty-five randomly selected specimensof each nematode stage were examined either live or heat-relaxed and relaxed in Ringer’s solution (60◦C). Killedspecimens were fixed in triethanolamine formalin (TAF)at 50-60◦C (Courtney et al., 1965), slowly dehydratedand processed to anhydrous glycerin (Seinhorst, 1959).Nematodes were mounted on glass slides with glass fibreused as cover slip supports to avoid flattening of thespecimens. Quantitative measurements of each specimenwere made using an Olympus BX81 microscope equippedwith differential interference contrast optics and OlympusMicrosuite software (Soft Imaging System, Lakewood,CO, USA). Illustrations were prepared from digitisedcamera lucida images.

Morphological characters measured were based onthe recommendations of Hominick et al. (1997). Thefollowing abbreviations have been used in the text ortables: D = (head to excretory pore/pharynx length) ×100; E = (head to excretory pore/tail length) × 100;

GS = gubernaculum length/spicule length; H = lengthof hyaline portion of tail; H% = H as % of tail length;SW = spicule length/anal body diam. Spicule length wasmeasured along the curved median line.

SCANNING ELECTRON MICROSCOPY (SEM)

Adults were dissected from G. mellonella larvae inRinger’s solution (pH 7.3). They were rinsed three timesfor 5 min in Ringer’s solution. Three-day-old IJ wererinsed for 3×15 min in 0.05% NaCl. All nematodes wererelaxed and killed by heating in a water bath (60◦C) for2-3 min and were then fixed in 8% glutaraldehyde/25%EM grade (diluted in Ringer’s solution) for 2 h at roomtemperature. Fixed nematodes were rinsed three timesin distilled water, post-fixed in OsO4 for 1 h, rinsed indistilled water and dehydrated at 15 min intervals through30, 50, 70, 90, 95 and 100% ethanol. They were thencritical point-dried in liquid CO2, mounted on SEM stubs,coated with gold and scanned using a SES DS-130 at 15kV accelerating voltage.

MOLECULAR CHARACTERISATION AND

PHYLOGENETIC ANALYSIS

Total genomic DNA isolation, PCR amplification (reac-tion, cycling conditions and primers) and sequence analy-sis followed protocols described by Stock et al. (2001) andNguyen et al. (2001). LSU and ITS rDNA sequences andphylogenetic relationships with other Steinernema specieswere compared using an existing library (P. Stock’s lab-oratory, University of Arizona) of more that 50 Steiner-nema spp. and available sequences deposited in GenBank.Phylogenetic analyses (maximum parsimony analysis) ofsequence data were made using PAUP* v 4.0b (Swof-ford, 2001) following criteria described by Stock et al.(2001) and Nadler et al. (2006). Caenorhabditis eleganswas considered in both phylogenetic analyses as the out-group taxon according to criteria described by Nadleret al. (2006). Ribosomal DNA sequences for the newSteinernema spp. were deposited in GenBank with acces-sion numbers EU345420 and EU345421 for 28S and ITSrDNA sequences, respectively.

CROSS-HYBRIDISATION

Reproductive isolation of the new species was testedusing the modified hanging-blood assay as describedby Kaya and Stock (1997). Steinernema carpocapsae(Weiser, 1955), S. monticolum Stock, Choo & Kaya,

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1997 and S. siamkayai Stock, Somsook & Kaya, 1998,depicted as close relatives to the new species from thephylogenetic analyses, were used for these experiments.Controls consisted of crosses with male and single femalenematodes of the same species and single female. Therewere ten replicates per cross and tests were repeatedtwice.

Steinernema colombiense* n. sp.(Figs 1-3)

MEASUREMENTS

See Tables 1 and 2.

DESCRIPTION

First generation male

Body slender, ventrally curved posteriorly, J-shapedwhen heat-relaxed. First generation male larger (aver-age = 1542 µm) than second generation male (average =870 µm). Cuticle smooth under light microscopy. Lat-eral field and phasmids inconspicuous. Head truncate toslightly rounded, continuous with body. Six lips, amal-gamated, but tips distinct, bearing one labial papillaeach. Four cephalic papillae present. Amphidial aper-tures small, located posterior to lateral labial papillae.Stoma reduced (cheilo-, gymno- and stegostom vestigial),short and wide, with inconspicuous sclerotised walls. Pha-rynx muscular; procorpus cylindrical; metacorpus slightlyswollen, non-valvate; indistinct isthmus followed by pyri-form basal bulb containing reduced valve. Pharynx set offfrom intestine. Nerve-ring usually surrounding isthmus oranterior part of basal bulb. Excretory pore opening cir-cular, located anterior to nerve ring at anterior one-thirdof metacorpus. Testis single, reflexed, consisting of ger-minal growth zone leading to seminal vesicle. Vas defe-rens with inconspicuous walls. Spicules paired, symmet-rical, curved, ochre-brown in colour. Manubrium rhom-boidal, with a rounded, ‘knob-like’, protrusion. Calomus(shaft) distinct. Lamina with rostrum or retinaculum andtwo internal ribs. Velum present, narrow. Blade terminusblunt. Gubernaculum arcuate, ca 75% of spicule length.Manubrium of gubernaculum curved ventrally and bi-furcate. Tail conoid and non-mucronate. Gubernaculummore slender and longer than that of first generation male.

* Specific epithet named after the country in which the specieswas found.

Twenty-five genital papillae (12 pairs and one single)arranged as follows: five subventral precloacal pairs, sin-gle midventral precloacal papilla (located between pre-cloacal pairs 4 and 5); one pair of cloacal papillae (lo-cated laterally on cloacal opening); two pairs adcloacal(one pair subventral, one pair subdorsal); four pairs post-cloacal (one pair subventral, two pairs ventral, one pairsubdorsal).

Second generation male

General morphology similar to that of first generationmales, but smaller in size. Tail with or without mucro.Spicules with manubrium morphology slightly differentto that of first generation male. Manubrium wider thanlong, with two small ribs. Calomus indistinct, continuouswith shaft. Lamina with a small rostrum, velum present.

First generation female

Cuticle, lip region, stoma and pharyngeal region as inmale. Body C-shaped when heat-relaxed. First generationfemales larger (average = 4608 µm) than second gen-eration females (average = 1470 µm). Excretory porelocated about mid-procorpus level. Ovaries opposed, re-flexed dorsally; oviduct well developed; glandular sper-matheca and uterus in ventral position. Vagina short, withmuscular walls. Vulva located near mid-body, with pro-truding asymmetric lips (posterior lip larger than ante-rior). Tail blunt, conoid, lacking mucro. Anal lips usuallyprotruding, asymmetric with anterior lip smaller than pos-terior.

Second generation female

Body open C-shaped when heat-relaxed. Similar to firstgeneration female, but smaller. Vulva with protruding,symmetrical lips. Tail conoid, with post-anal swelling.

Third-stage infective juvenile

Body of heat-relaxed specimens almost straight, slen-der, gradually tapering posteriorly. Cuticle with fine trans-verse striae. Lateral field distinct with six longitudinalridges (i.e., seven lines/incisures) in mid-body region.Head region continuous with body, slightly truncate. Sixdistinct labial papillae and four cephalic papillae present.Amphidial apertures pore-like. Lip region smooth, con-tinuous; stoma closed. Pharynx long, narrow, with slightlyexpanded procorpus, narrower isthmus and pyriform basalbulb with valve. Cardia present. Nerve-ring located at isth-mus level. Excretory pore located ca mid-corpus level.Hemizonid not observed. Anterior portion of intestinewith bacterial receptacle. Intestine filled with numerous

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Fig. 1. Steinernema colombiense n. sp. A: Anterior end of first generation female, lateral view; B: Vulva of first generation female,lateral view; C: Vulva of second generation female, lateral view; D: Tail of first generation female, lateral view; E: Tail of secondgeneration female, lateral view; F: First generation male, in toto, lateral view; G: First generation male spicule, lateral view; H:Second generation male spicule, lateral view; I: First generation male gubernaculum, dorsal view; J: Anterior end of third-stageinfective juvenile, lateral view; K: Tail of third-stage infective juvenile, lateral view. (Scale bars: A, J = 23 µm; B-D = 35 µm; E, G-I,K = 15.5 µm; F = 170 µm.)

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Fig. 2. Light and scanning electron microscope photographs of male specimens of Steinernema colombiense n. sp. A: First generationmale, in toto; B: Anterior end, showing excretory pore location (arrow); C, D: Scanning electron microscopy images of tail showingdistribution of several genital papillae (pr = precloacal, c = cloacal, ad = adcloacal, po = postcloacal, v = length from anteriorend to vulva opening); E: First generation male spicule, lateral view showing ‘knob-like’ manubrium (arrow); F: First generation malegubernaculum, ventral view showing bifurcate head and cuneus (arrow); G: Second generation male spicule, lateral view. (Scales arebased on scale bar in A: A = 100 µm; B, D, E = 16 µm; C, F, G = 12 µm.)

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Table 1. Morphometrics of Steinernema colombiense n. sp. All measurements are in µm and in the form: mean ± s.d. (range).

Male Female Infectivejuvenile

1st 2nd 1st 2ndgeneration generation generation generation

Holotype Paratypes Paratypes Paratypes Paratypes Paratypes

n – 19 20 19 20 20L 1678 1542 ± 226 870 ± 61 4608 ± 102 1470 ± 211 636 ± 15

(1209-2040) (762-980) (2796-6752) (996-2123) (549-732)a – – – – – 21 ± 3

(18-28)b – – – – – 5.4 ± 0.6

(4.4-6.4)V – – – 52 ± 2 59 ± 2 –

(49-55) (55-64)Max. body diam. 95 102 ± 20 56 ± 6 140 ± 15 75 ± 18 31 ± 4

(58-121) (46-68) (115-163) (51-116) (22-36)Pharynx length 158 156 ± 15 122 ± 7 185 ± 17 147 ± 12 118 ± 6

(123-199) (105-140) (155-215) (111-179) (106-128)Head to excretory pore 70 67 ± 5 54 ± 4 76 ± 7 67 ± 11 35 ± 4

(56-76) (46-63) (62-89) (52-115) (31-40)Head to nerve ring 134 121 ± 14 92 ± 6 137 ± 14 120 ± 12 83 ± 5

(96-155) (80-105) (112-173) (102-155) (73-92)Tail length 40 31 ± 6 25 ± 4 48 ± 10 45 ± 5 41 ± 5

(23-41) (21-35) (33-74) (28-60) (32-53)Anal body diam. 38 37 ± 6 32 ± 5 64 ± 11 36 ± 7 11.5 ± 2

(26-49) (23-43) (47-81) (38-62) (9-14)Spicule length 70 71 ± 4 54 ± 5 – – –

(64-77) (43-62)Gubernaculum length 61 50 ± 8 35 ± 5 – – –

(47-63) (23-43)SW 1.8 2.0 ± 0.3 1.7 ± 0.4 – – –

(1.5-2.7) (1.0-2.4)GS 0.9 1.5 ± 0.3 1.6 ± 0.2 – – –

(1.1-1.8) (1.3-2.3)D% 44 40 ± 4 44 ± 4 – – 29 ± 2

(30-50) (39-49) (25-33)E% 57 52 ± 9 218 ± 3 – – 205 ± 31

(35-69) (166-263) (138-284)H – – – – – 14 ± 2

(11-19)H% – – – – – 35 ± 7

(27-50)

Fig. 3. Light and scanning electron microscope photographs of female and third-stage infective juvenile specimens of Steinernemacolombiense n. sp. A: Anterior end of female, lateral view showing position of excretory pore (arrow); B: Vulva, lateral view of firstgeneration female; C: Posterior end of first generation female, lateral view; D: Vulva, lateral view of second generation female; E:Posterior end of second generation female, lateral view; F-H: Third-stage infective juvenile. F: Anterior end, lateral view, showingposition of excretory pore (arrow); G: Posterior end, lateral view, showing anus position (arrow); H: Bacterial receptacle; I: Lateralfield pattern at mid-body level. (Scales are based on scale bar in A: A, G, H = 24 µm; B, E, F = 32 µm; C = 38.5 µm; D = 9 µm;I = 4.5 µm.)

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Table 2. Comparison of morphometric traits of infective juveniles of Steinernema colombiense n. sp. with other morphologically similarSteinernema spp. All measurements in µm and in the form: mean (range).

Species L Max. body diam. Excretory pore Tail D% E%

S. colombiense n. sp. 636 31 35 41 29 205(549-732) (22-36) (31-40) (32-53) (25-33) (138-284)

S. scapterisci1 572 24 39 54 31 73(517-609) (18-30) (36-38) (48-60) (27-40) (60-80)

S. carpocapsae2 558 25 38 53 26 60(438-650) (25-30) (30-60) (46-61) (23-28) (54-66)

S. riobrave3 622 28 56 54 49 105(561-701) (26-30) (51-64) (46-59) (45-55) (93-111)

S. siamkayai4 446 21 35 36 37 96(398-495) (18-24) (29-38) (31-41) (31-43) (95-112)

1 After Nguyen and Smart (1992).2 After Poinar (1990).3 After Cabanillas et al. (1994).4 After Stock et al. (1998).

fat globules, narrow lumen. Rectum long, straight; anusdistinct. Genital primordium evident. Tail conoid withpointed terminus. Hyaline portion occupying ca 25-50%of tail length.

TYPE HOST AND LOCALITY

The natural host is unknown as S. colombiense n. sp.(isolate SNI-0198) was collected by baiting from soilsamples from a coffee plantation (Coffea arabica var.Castillo) in Maracay Experimental Station, QuimbayaCounty, Quindío department, Colombia. Elevation of thisregion is 1402 m a.s.l. and specific geographic coordinateswhere EPN-positive samples were obtained are: 04◦36′N,75◦44′W. Soil in this region is acidic (pH 4.9) with loworganic matter (5.9%) and N content (0.29%).

TYPE MATERIAL

Holotype first generation male, five first generation pa-ratype males, five first generation paratype females andfive paratype third-stage infective juveniles deposited inthe USDA Nematode Collection, Beltsville, MD, USA,and five first generation paratype males, five first genera-tion paratype females and five paratype third-stage infec-tive juveniles deposited in the University of CaliforniaDavis Nematode Collection, Davis, CA, USA.

DIAGNOSIS AND RELATIONSHIPS

Steinernema colombiense n. sp. is characterised by themorphometrics of the third-stage infective juvenile whichhas a small body length of 636 (549-732) µm, narrowbody diam. (31 (22-36) µm), the position of the excretorypore (35 (31-40) µm) and a short tail (41 (32-53) µm).Additional diagnostic traits include D% = 29 (25.5-33), E% = 205 (138-284) and H% = 35 (27-50). Firstgeneration males can be distinguished by the morphologyof the spicules and gubernaculum, the values of ratiosSW = 2.0 (1.5-2.7) and GS = 1.5 (1.1-1.8) and thenumber and arrangement of the genital papillae. Firstgeneration females can be recognised by the presence ofprotruding and asymmetric vulval lips.

Phylogenetic analysis of LSU sequences data placed S.colombiense n. sp. in a clade that includes four species:S. carpocapsae, S. monticolum, S. siamkayai and S.scapterisci Nguyen & Smart, 1990, all of which arecharacterised by having the IJ with a short body length(average = 572 µm). However, the new species can bedifferentiated from these taxa by several morphometricdifferences of the IJ and first generation male. Forexample, third-stage infective juveniles of the new specieshave a larger body size (average = 636 µm) and highervalue of E% (average = 205) than S. carpocapsae, S.scapterisci and S. monticolum.

First and second generation males of S. colombiensen. sp. also differ from those of S. carpocapsae, S. scap-terisci and S. monticolum by the number and distribu-

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Fig. 4. Evidence of large subunit (LSU) ribosomal DNA lineage independence for Steinernema colombiense n. sp. based on maximumparsimony analysis. Clades are in roman numbers. Number in bold indicates bootstrap value. Numbers after species names correspondto GenBank accessions.

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Fig. 5. Evidence of internal transcribed spacer region (ITS) of ribosomal DNA lineage independence for Steinernema colombiense n.sp. based on maximum parsimony analysis. Numbers at each node indicate bootstrap values. Numbers after species names correspondto GenBank accessions.

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–10

.S.b

icor

nutu

m30

2725

2525

2828

2825

9–

11.S

.sic

huan

ense

4239

3634

3837

3630

2831

–12

.S.a

ffine

4237

3636

3837

3634

2629

6–

13.S

.int

erm

ediu

m41

3635

3537

3837

3428

319

5–

14.S

.bed

ding

i42

3936

3638

4239

4031

3512

1011

–15

.S.n

eocu

rtil

lae

3732

3131

3237

3438

2526

3332

3134

–16

.S.c

uban

um35

3328

2829

3535

3022

2429

3026

2928

–17

.S.g

lase

ri33

3126

2627

3535

2820

2229

3026

2926

2–

18.S

.are

nari

um34

3227

2728

3635

2820

2227

2824

2826

64

–19

.S.g

uang

dong

ense

3533

2828

2534

3327

1921

2928

2829

268

66

–20

.S.l

ongi

caud

um32

3025

2525

3332

2518

2028

2828

2926

75

72

–21

.S.d

iapr

epes

i36

3429

2926

3335

2919

2129

3028

2924

97

88

7–

22.S

.kho

isan

ae39

3732

3229

3532

3124

2530

2927

3027

97

77

78

–23

.S.a

ciar

i34

3226

2627

3133

2818

2028

2827

2825

97

1010

86

12–

24.S

.kar

ii33

3126

2627

3534

2820

2228

2927

2621

1311

1312

117

116

–25

.S.h

ebei

ense

3026

2322

2529

2422

1818

3126

2627

2222

2018

1415

1917

1720

–26

.S.s

angi

3029

2120

2429

2923

1719

3129

2827

2114

1214

98

1113

1011

9–

27.S

.fel

tiae

3026

2323

2331

2825

1717

3429

2930

2019

1717

1312

1616

1518

65

–28

.S.k

raus

sei

3129

2424

2531

3123

1919

3231

3130

2416

1618

1210

1717

1516

94

6–

29.S

.ore

gone

nse

3127

2424

2533

3027

1919

3429

2930

2118

1616

1011

1513

1417

66

46

–30

.S.m

onti

colu

m37

3330

2931

3532

3427

2639

3636

3826

2725

2622

2123

2322

2512

916

1816

–31

.S.r

arum

3535

2929

3133

3533

2928

3737

3537

3426

2322

2223

2320

2224

2623

2523

2223

–32

.C.e

lega

ns13

513

714

113

714

413

414

413

313

614

115

415

916

015

514

113

913

714

013

513

413

513

813

613

310

911

013

413

513

413

511

7–

572 Nematology

A new Steinernema from Colombia

tion of genital papillae and by the overall morphology ofthe spicules and gubernaculum (see Table 2). In the newspecies the spicules are characterised by having a rhom-boidal manubrium, with a rounded, ‘knob-like’, protru-sion. The gubernaculum is also unique in its morphologywith the manubrium curved ventrally and bifurcate. In ad-dition to these qualitative differences, males of the newspecies can be differentiated by the SW value (average =2.0 and 1.7 for first and second generation males, respec-tively; see Table 2).

Both first and second generation females of S. colombi-ense n. sp. are characterised by having protruding vulvallips. This feature is shared with females of S. carpocap-sae. Females of S. monticolum lack protruding vulval lipsand S. siamkayai and S. scapterisci have only slightly pro-truding vulval lips. Both first and second generation fe-males of the new species lack a tail mucro, whereas S. car-pocapsae, S. scapterisci and S. monticolum may or maynot have a mucro.

MOLECULAR CHARACTERISATION AND

PHYLOGENETIC ANALYSIS

Maximum parsimony (MP) analysis of the 28S rDNAsequences set yielded 399 parsimony informative charac-ters and produced 15 equally parsimonious trees (Fig. 4)with a tree length of 1096 steps (CI = 0.64). MP analysisplaced S. colombiense n. sp. as a relative of Clade I. Thisclade comprises Steinernema spp. known to have infectivejuveniles with small body size (average < 600 µm). As-sociation of the new species with this clade (Fig. 5) wasstrongly supported by bootstrap resampling (100%). Pair-wise distances (Table 3) also shows that S. colombiensen. sp. differs from closely related species by 16-20 basepairs (numbers in bold font).

Analysis of ITS rDNA sequences yielded two equallyparsimonious trees with a tree length of 3358 steps (CI =0.45) (Fig. 5). A total of 615 parsimony informative traitswas depicted for this analysis. Steinernema colombiensen. sp. was placed as a member of a clade encompassingspecies with small IJ, including S. tami Luc, Nguyen, Reid& Spiridonov, 2000, S. carpocapsae, S. scapterisci and S.siamkayai. The new species was considered to be a sistertaxon of S. carpocapsae from which it differs by threebase pairs (numbers in bold font). Affiliation of these twospecies was strongly supported by bootstrap resampling(100%). These observations and those from the 28S rDNAanalysis provide further evidence for the distinctness ofthis species.

CROSS-HYBRIDISATION TESTS

Cross-hybridisation assays between males and femalesof S. colombiense n. sp., with S. carpocapsae, S.siamkayai and S. scapterisci yielded no progeny. Inthe controls, offspring were produced in all self-crossedspecies. No progeny were observed in the single femalecontrol plates.

Acknowledgements

We are thankful to Y. Flores-Lara for assistance withthe scanning electron microscopy images. The authorsalso express their gratitude to Cenicafé research leadersG. Cadena-Gómez, A.E. Bustillo-P., D. Rodríguez and C.Quintero for their support on the EPN project. This studywas funded in part by a Colombia Ministry of the Envi-ronment grant (Project ENT1801) and The National Fed-eration of Colombian Coffee Growers, Coffee ResearchCentre (Cenicafé).

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