Morphological and morphometric studies (Table 1, Figs. 1-3)
Table 1. Morphometrics of Steinernema feltiae isolate Lican Ray, Chile. Measurements are in µm, except the indexes, in form: mean ± standard deviation (range).
Character
|
Males generation
|
Females generation
|
Third juvenile
stage
|
1°
|
2°
|
1°
|
2°
|
|
|
|
|
|
|
n
|
25
|
25
|
25
|
25
|
25
|
|
|
|
|
|
|
L
|
1,428±86
|
866±75
|
5,318±907
|
3,269±286
|
807±21
|
|
(1,309-1,578)
|
(731-1,004)
|
(3,856-7,327)
|
(2,890-3,930)
|
(779-841)
|
|
|
|
|
|
|
MBW
|
95±7
|
53±5
|
189±27
|
154±11
|
31±4
|
|
(85-116)
|
(44-64)
|
(156-243)
|
(131-173)
|
(27-41)
|
|
|
|
|
|
|
|
|
|
|
|
|
ES
|
143±10
|
115±9
|
167±17
|
166±15
|
119±7
|
|
(122-177)
|
(99-138)
|
(141-200)
|
(136-200)
|
(104-130)
|
|
|
|
|
|
|
EP
|
101±9
|
72±7
|
90±17
|
85±14
|
55±3
|
|
(80-118)
|
(61-86)
|
(46-118)
|
(67-117)
|
(50-60)
|
|
|
|
|
|
|
NR
|
113±11
|
85±7
|
117±16
|
114±13
|
87±5
|
|
(90-144)
|
(71-101)
|
(93-149)
|
(86-140)
|
(80-98)
|
|
|
|
|
|
|
TL
|
39±3
|
35±3
|
55±6
|
48±7
|
74±3
|
|
(35-47)
|
(31-40)
|
(42-64)
|
(37-62)
|
(68-78)
|
|
|
|
|
|
|
ML
|
5±1
|
11±2
|
|
|
|
|
(4-8)
|
(8-14)
|
|
|
|
|
|
|
|
|
|
ABD
|
40±4
|
32±3
|
65±15
|
47±6
|
16±1
|
|
(35-48)
|
(25-39)
|
(42-91)
|
(37-60)
|
(14-20)
|
|
|
|
|
|
|
SpL
|
68±5
|
57±6
|
|
|
|
|
(58-78)
|
(42-65)
|
|
|
|
|
|
|
|
|
|
GuL
|
46±3
|
36±4
|
|
|
|
|
(40-55)
|
(29-45)
|
|
|
|
|
|
|
|
|
|
SW
|
1.7±0.2
|
1.8±0.3
|
|
|
|
|
(1.3-2.1)
|
(1.3-2.4)
|
|
|
|
|
|
|
|
|
|
GS
|
0.7±0.05
|
0.6±0.1
|
|
|
|
|
(0.6-0.8)
|
(0.5-0.7)
|
|
|
|
|
|
|
|
|
|
V
|
|
|
50±2
|
53±3
|
|
|
|
|
(46-55)
|
(49-58)
|
|
|
|
|
|
|
|
a
|
|
|
|
|
26±3
|
|
|
|
|
|
(19-31)
|
|
|
|
|
|
|
b
|
|
|
|
|
7±0.4
|
|
|
|
|
|
(6-8)
|
|
|
|
|
|
|
c
|
|
|
|
|
11±0,4
|
|
|
|
|
|
(10-12)
|
|
|
|
|
|
|
D%
|
71±7
|
62±6
|
54±10
|
51±7
|
46± 4
|
|
(56-84)
|
(52-79)
|
(33-67)
|
(39-62)
|
(40-55)
|
|
|
|
|
|
|
E%
|
261±32
|
207±27
|
165±36
|
180±44
|
75± 5
|
|
(202-326)
|
(162-252)
|
(76-232)
|
(118-263)
|
(67-83)
|
|
|
|
|
|
|
H
|
|
|
|
|
26±4
|
|
|
|
|
|
(17-33)
|
|
|
|
|
|
|
H%
|
|
|
|
|
36±6
|
|
|
|
|
|
(23-45)
|
|
|
|
|
|
|
L: total body length; MBW: maximum body width; ES: esophagus length; EP: anterior end to excretory pore; NR: anterior end to nerve ring; TL: tail length; ML: mucron length; ABD: anal body diameter; SpL: spicule length; GuL: gubernaculum length; H: hyaline portion; V: position of vulva (%); D%=(EP/ES)x100; E%=(EP/TL)x100; SW=SpL/ABD; GS=GuL/SpL; H%=(H/TL)x100; a=L/MBW; b=L/ES; c=L/TL.
Male, first generation
Body C- or J-shaped posteriorly when heat-killed (Fig. 1A). Cuticle with fine, annular striation under SEM but smooth under light microscope; lateral fields and phasmids inconspicuous. Anterior end slightly rounded, continuous with the body. Six prominent lips, each lip bearing a labial papilla. Four cephalic papillae, also notorious (Fig. 3B, D). Small amphidial opening, behind to lateral lip papillae. Deirids conspicuous, located in the first third, after than excretory pore. Stoma short and wide, inconspicuous sclerotized walls. Esophagous muscular with cylindrical procorpus, metacorpus slightly swollen, isthmus fairly notorious, ending in a pyriform basal bulb. Nerve ring surrounding the isthmus or the anterior end of basal bulb. Excretory pore anterior to the nerve ring, around first third of the isthmus (Fig. 1C). Simple testis, reflexed. Vas deferens with inconspicuous walls. Spicules paired, symmetrical, curved, ocher brown color (Fig. 1G); head (manubrium) oblong, shaft (calomus) notorious, velum present. Gubernaculum curved, approximately 2/3 of spicule's length; boat-shaped in lateral view, anterior end curved ventrally (Fig. 1G); in ventral view, corpus with two projections. Tail conoid, tail terminus with a mucron (Fig. 1E). One single, midventral, precloacal papilla, and 11 pairs of papillae. Six pairs are precloacal, subventral, one pair lateral precloacal, one pair adanal, two pairs subterminal subventral, and one pair post cloacal, lateral (Fig. 1F).
Male, second generation
Similar to the first-generation male, but more slender and smaller in body length and other morphometric characters. Deirids not observed. Mucron on tail terminus present and longer than that in the first generation.
Females, first generation
Body robust, habitus C-shaped (Fig. 2A). Cuticle, lips, stoma and esophageal region as in males. Excretory pore at mid of metacorpus (Fig. 2B). Reproductive system didelphic-amphidelphic, ovary reflexed dorsally. Vulva a transverse slit at midbody region, protuberant, with a double epiptygma (Fig. 2D). Vagina short, leading into paired uteri. Tail conoid with ventral postanal swelling (Fig. 2E). Mucron absent.
Females, second generation
Similar to first generation females (Fig. 2C), but smaller in size. Vulva located slightly back compared to the first generation females, symmetric and protuberant lips with a double epiptygma. Relation excretory pore/tail length bigger than in the first generation. Tail conoid, with a slight postanal swelling (Fig. 2F).
Third juvenile stage
Body slender, habitus straight. Cuticle with fine transverse striae. Head continuous with body contour, slightly truncate (Fig. 3A), not annulated. Labial papillae not observed, amphidial opening like a pore at the level of four distinct cephalic papillae (Fig. 3B). Oral aperture and anus closed. Lateral fields with 8 notorious ridges at midbody region (Fig. 3C). Long esophagous, narrow, procorpus slightly expanded, narrowing in isthmus and base bulb pyriform (Fig. 3A). Excretory pore at mid-esophagous level, isthmus surrounded by nerve ring. Deirids not observed. Cardia present. Small bacterium receptacle in the anterior part of intestine. Tail conoid, tapering gradually (Fig. 3D, E), hyaline portion equivalent to 36% of tail length (Fig. 3D).
Molecular characterization
For the ITS region a fragment of 859 bp was obtained for the Chilean S. feltiae isolate. This sequence was tested in BLAST with data deposited in GenBank, showing approximately 97-99% similarity with sequences of the same species. The majority-rule consensus tree of the Bayesian inference showed a well-supported group (100% bootstrap) that comprised the LR isolate (MK504438) and known sequences of S. feltiae from different countries, including one from Chillán, Chile (MK504439) sequenced in the present work as reference (Fig. 4). For 28S, a fragment of 894 bp was obtained (MK509752) showing 99% similarity with published sequences of S. feltiae and other species from the feltiae group. The phylogenetic relationships revealed a clade (100% bootstrap) that included sequences of S. feltiae from different geographical origins including Chillán, Chile (MK509780) and from Steinernema jollieti, Steinernema puntauvense, Steinernema litorale, Steinernema ichnusae, Steinernema weiseri and Steinernema silvaticum (Fig. 5). Based on the BLAST search and phylogenetic analysis of 16S rRNA, the symbiotic bacterium of S. feltiae LR is Xenorhabdus bovienii (BLAST similarities 99%). The Bayesian inference showed that the sequence obtained (MK504451) formed a well-supported group with sequences of the same species deposited in the GenBank (Fig. 6). ML analysis produced trees with the same topology for all the genes considered.
Ecological characteristics
Observations of the cycle of LR isolate
The life cycle of the LR isolate was similar to those described for other Steinernema species. The IJs were able to kill G. mellonella larvae between the first and second day after inoculation at 20 °C. Male and females of the first generation were present on the third or fourth day. On the fifth day, the first IJs were observed in the insect cadaver. A second adult generation occurred between the seventh and eighth day. IJs emerged massively from the insect body on the tenth day. The color of insect larvae turned brown when they died.
Effect of temperature
The effect of IJs on G. mellonella mortality at different temperatures is shown in Table 2. On the second day, most larvae incubated at 20 °C or higher were immobile and starting to change color. While at 5 °C, no mortality was recorded, at 10 °C, a few dead larvae (10%) were observed on day five after inoculation (DAI). Mortality increased dramatically with higher temperatures, reaching 100% at 15, 20 and 25 ºC on third DAI. The last two temperatures seemed to be optimal for reaching the highest mortality in the shortest period, with a 90-97.5% mortality on the second DAI. The highest temperature (30 °C) had an effect on mortality, reaching the maximum value on the fifth DAI.
Table 2. Percent mortality of Galleria mellonella larvae at different temperatures during the five days after inoculation with infective juveniles of Steinernema feltiae LR.
Temperature (ºC)
|
Days after inoculation
|
|
1
|
2
|
3
|
4
|
5
|
5
|
0 ax
|
0 a
|
0 a
|
0 a
|
0 a
|
10
|
0 a
|
0 a
|
0 a
|
0 a
|
10 a
|
15
|
0 a
|
0 a
|
100 c
|
100 b
|
100 b
|
20
|
0 a
|
97.5 c
|
100 c
|
100 b
|
100 b
|
25
|
0 a
|
90 c
|
100 c
|
100 b
|
100 b
|
30
|
12.5 b
|
60 b
|
90 b
|
95 b
|
100 b
|
X: Means (n = 20) in columns followed by the same letter do not differ significantly according to Tukey´s multiple range test (P < 0.05).
The number of invader IJs per G. mellonella larva at different temperatures is presented in Table 3. At the lowest evaluated temperatures (5 and 10 °C), nematode presence was not detected by dissection. IJ penetration was higher at 15 and 20 °C (approximately 25% of IJs inoculated) and significantly decreased as temperature increased. Females with eggs were observed on the second day after larval death at 15, 20 and 25 °C. The time for emergence of IJs (Table 3) from insect cadaver was optimal for medium temperatures (15 and 20 °C), coming out of the insect around the tenth DAI. At lowest (5 and 10 °C) and highest (30 °C) temperatures, neither IJs emergence nor offspring were observed. The greatest IJs production occurred at 20 °C. The maximum IJs recovery occurred during the fifth DAI, and the emergence lasted for 30 days (data not shown). The optimal temperature for host invasion and reproduction of S. feltiae LR was 20 °C, so this temperature was used for the following assays.
Table 3. Number of invader infective juveniles (IJs) per Galleria mellonella larva, time to emerge from the cadaver and offspring production (IJs/larva) after inoculation at different temperatures.
Temperature (ºC)
|
Invader IJs
|
Emergence days
|
Offspring
|
5
|
nd
|
nd
|
nd
|
10
|
nd
|
nd
|
nd
|
15
|
26.6±11.4 cx
|
17.3±1.4 c
|
72884.9±26417 b
|
20
|
26.4±11.2 c
|
10.6±1.4 b
|
102807.3±23256 c
|
25
|
12.4±7.2 b
|
9.1±0.7 a
|
52107.8±15452 a
|
30
|
3.1±2.6 a
|
nd
|
nd
|
nd: nematodes no detected.
X: Means (n = 20) in columns followed by the same letter do not significantly differ according to Tukey´s multiple range test (P < 0.05).
Lethal concentration
Insect mortality percentages obtained during four DAI are presented in Table 4, showing that mortality increased as the inoculum and DAI increased. On the third day, 100% mortality was reached for all nematode doses; however, on the second day, over 80 IJs were enough to achieve maximum control. The CL50 and CL90 at 48 h were 7.2 and 40.4 IJs/larva, respectively.
Table 4. Percentage of mortality of Galleria mellonella larvae during the four days after inoculation with different infective juvenile (IJs) doses at 20 ºC.
Doses (IJs/larva)
|
Days after inoculation
|
1
|
2
|
3
|
4
|
10
|
0 ax
|
70 a
|
100 a
|
100 a
|
20
|
0 a
|
70 a
|
100 a
|
100 a
|
40
|
0 a
|
80 ab
|
100 a
|
100 a
|
80
|
0 a
|
100 b
|
100 a
|
100 a
|
120
|
0 a
|
100 b
|
100 a
|
100 a
|
240
|
20 b
|
100 b
|
100 a
|
100 a
|
X: Means (n = 20) in columns followed by the same letter do not significantly differ according to Tukey´s multiple range test (P < 0.05).
The number of nematodes invading the host increased with dose (Table 5); however, efficacy of penetration, ranging from 24% to 49.6%, did not show statistical differences. Independent of IJs inoculum dose, no significant differences were observed respect to the time needed for new IJs to start to emerge from the insect cadaver, varying between 11 and 13.5 days. Differences were observed in the offspring emerging from the host, where doses of 120 IJs/larva were the most prolific. This nematode dose was used to investigate effects of soil water content.
Table 5. Number of invader infective juveniles (IJs) per Galleria mellonella larva, percentage of penetration efficacy, emergence time and offspring (number of IJs) at 20º C and different doses of Steinernema feltiae LR.
Doses (IJs/larva)
|
Invader IJs
|
Penetration efficacy
|
Emergence time (days)
|
Offspring
|
10
|
2.4±1.7 ax
|
24±16.7 a
|
13.5±5.7 a
|
64919.3±41294.8 a
|
20
|
6.6±1.8 b
|
33±9.1 a
|
12±1.7 a
|
79130.2±16801.7 ab
|
40
|
10±5.2 bc
|
25±13.3 a
|
11.2±0.5 a
|
95787.8±18570.1 abc
|
80
|
20.8±3.9 d
|
26.4±4.8 a
|
11.4±0.6 a
|
90316.2±8542.4 abc
|
120
|
40.8±17.3 de
|
34±14.3 a
|
11±0.7 a
|
106152±13569.3 c
|
240
|
118.8±46.8 f
|
49.6±19.6 a
|
11.2±1.5 a
|
86240±67172 abc
|
X: Means (n = 5) in columns followed by the same letter do not significantly differ according to Tukey´s multiple range test (P < 0.05).
Effect of soil water content
The effect of soil water content on the mortality of G. mellonella over five days is shown in Table 6. On the second and third days, differences were observed between water content; however, on the fourth day, mortality was statistically similar for all treatments, showing a delay in achieving full mortality. Water content did not affect the number of IJ invaders or penetration efficacy (Table 7). The time of emergence of the IJs and offspring per larva showed no differences between treatments.
Table 6. Assessment of mortality (%) of Galleria mellonella larvae during 5 days after inoculation with Steinernema feltiae LR infective juveniles (IJs) in soil with different water contents.
Water content
|
Days after inoculation
|
1
|
2
|
3
|
4
|
5
|
Permanent wilting point
|
0 ax
|
73 a
|
93 a
|
98 a
|
100 a
|
Field capacity
|
2.5 a
|
93 b
|
100 b
|
100 a
|
100 a
|
Saturation
|
2.5 a
|
100 b
|
100 b
|
100 a
|
100 a
|
X: Means (n = 20) in columns followed by the same letter do not differ according to Tukey´s multiple range test (p < 0.05).
Table 7. Incidence of soil water content in number of invader infective juveniles (IJs) per insect larva, percentage of penetration, emergence days and offspring (number of IJs) of Steinernema feltiae LR.
Water content
|
Invader IJs
|
Penetration efficacy
|
Time of emergence
|
Offspring
|
Permanent wilting point
|
24.9±11.5 ax
|
20.8±11.5 a
|
10.1±1.6 a
|
75180.1±37210.5 a
|
Field capacity
|
36.8±21.4 a
|
30.8±21.4 a
|
10.5±0.7 a
|
81518.3±24150.1 a
|
Saturation
|
38.4±18.2 a
|
38.4±18.2 a
|
10.1±0.2 a
|
70975.5±23629.5 a
|
X: Means (n = 20) in columns followed by the same letter do not differ according to Tukey´s multiple range test (P < 0.05).