In the present investigation, four soil samples out of 360 were found positive for the H. indica. The pH of the soil was 5.4-8.8 and the texture of the soil was found to be alluvial, clay loam and sandy loam. Most of the sampling was done in the winter season mainly in December-March. For the morphological characterizations, permanent slides were prepared and were deposited in the Nematology Laboratory, Department of Zoology, Ch. Charan Singh University, India.
Morphological Characterization
The morphological and morphometrical data of all the generations showed much resemblance with the originally described species of H. indica which was first isolated from sugarcane top borer, Scirpophaga excerptalis (Pyralidae: Lepidoptera) by Poinar, Karunakar and David, 1992 in Coimbatore, Tamil Nadu, India. The present specimens showed close proximity to already described species with minor deviations both in morphology and morphometry. The posterior part of hermaphrodite and amphimictic females of the present specimens showed the mucron (Online Resource 1) whereas the originally described species lack this. The originally described species has prominent anal swelling in the first generation females, however, in the present specimens, anal swellings were observed in both first and second generation females (Online Resource 1). The hermaphroditic females were found to be larger than males in the present specimen and they bear truncate cephalic extremities. Lips six in number, protruding outward with prominent labial papilla. Amphidial apertures are pore-like, located immediately posterior to lips. Pharynx rhabditioid type without distinct metacarpus and isthmus is present. Vulval region distinct, smooth and located centrally. Tail length greater than anal body diameter. Mucron present and prominent. The amphimictic females of present isolate found to be smaller in size than hermaphrodites and become C-shaped when heat killed. Vulva narrow and mated female bear copulation plug. Anus swelling present and anal pore at the anterior third of anal swelling. Tail short and pointed. The infective juveniles characterized by straight and cylindrical body upon heat killing. They remain ensheathed within second stage cuticle. Large dorsal tooth on labial region and with open stoma walls. The pharynx with large isthmus than adult generations. Hyaline present and distinct in some isolates. Bacteria were present in the gut and also occur in anterior intestinal lumen. They appear luminescent in dark because of bioluminescent bacteria. Photomicrographs of all generations and comparative morphometry have been shown in Online Resource 1, 2.
Principal Component Analysis
The results of the PCA showed that there are variations in the morphometry between the different developmental stages (hermaphroditic females, males and IJ) of four isolates of H. indica in the present investigation and the originally described H. indica species and with the other species of Indica clade (Spiridonov &Subbotin, 2016). The analyzed parameters of morphology demonstrated a clear difference between the isolated nematodes of H. indica i.e., CH22, CH23, CH25 and CH26 and the original population of H. indica. Besides this, the isolates showed differences from the Indica clade species (Fig. 1).
The PCA based on the morphometry of IJs showed an accumulated variability of 68.1%. The contribution of PC1 and PC2 was found out to be 38.3% and 29.8%, respectively (Fig. 1a). Four parameters, length (r=0.183), tail (r=0.45), anterior to excretory pore (r=0.054) and D% (r=0.139) were found to be positively correlated across nematode/ species. Seven characters out of eleven were found to be positively correlated across isolates and the remaining characters were negatively correlated (Fig 1a). The highest coefficient of correlation was observed in pharynx length (r=0.521) in PC2 which is consistent with the previous findings (Bhat et al., 2020). An accumulated variability of 57.4% was observed in the male-based PCA (PC1: 27.9% and PC2: 29.5%). Eight morphometric characters out of thirteen were found to be positively correlated across isolates/ species and the remaining were found to be negatively correlated (Fig. 1b). Specifically, spicule length (r = 0.361) and E% (r = 0.359) exhibited the highest correlation coefficient within the PC1 (Fig. 1b). In the case of PC2, all characters except E% (r = – 0.121) and SW% (r = – 0.16) were negatively correlated. The PCA based on the morphometry of a Hermaphroditic female showed maximum variability of 76.9% with PC1 at 28.8% and PC2 at 48.7% (Fig, 1c). Twelve characters out of thirteen were found to be positively correlated across isolates/ species except V% (r=-0.3) which is negatively correlated (Fig. 1b). In the case of PC1 and PC2, the highest coefficient of correlation was observed in Body length (r=0.395) and D% (r=0.409), respectively.
Molecular analysis
The ITS sequence of the present strain (CH22, CH23, CH26) differ by 2 base pair (bp) (positions 331 and 632) from the original ITS sequence. The ITS rDNA sequences of present strains of Heterorhabditis are separated from those of other described Heterorhabditis species by 15-268 bp. (Online Resource 3).As far as D2 and D3 expansion fragments of 28S rDNA are concerned, present strains (CH22, CH23, CH25) differ from original D2D3 rRNA sequences by 6 bp. The D2D3 rRNA sequences of present strains of Heterorhabditis are separated from those of other described Heterorhabditis species by 22-82 bp (Online Resource 4).
Phylogenetic analysis
The morphological and molecular data of the present strains was also supported by the ITS and D2-D3 rDNA phylogenetic analysis. ITS and D2-D3 region are well conserved regions in the nematodes and are used to ascertain the relationship between closely and distantly related species. Phylogenetic analyses based on ITS rDNA showed a clear monophyly of the group formed by the isolates viz., CH22, CH23, CH26 and already described H. indica Poinar, Karunakar and David (1992) (Fig. 2a). Sequences of H. indica formed a monophyletic group with Heterorhabditis noenieputensis Malan, Knoetze and Tiedt (2012) and this pair formed a sister pair with the species of Indica-group. Similar results were obtained in the D2-D3 sequences too. Here also, the present strain (CH22, CH23, CH25) formed a monophyletic clade with the already described isolates of H.indica from different geographical areas and formed a sister clade with Heterorhabditis noenieputensis Malan, Knoetze and Tiedt (2014). The above pair formed a sister pair with the species of Indica- group (Fig. 2b).
Pathogenicity tests
The pathogenicity tests against G. mellonella showed that the CH22 and CH23 isolates of H. indica were highly virulent. On the contrary, the CH25 and CH26 strains were the least pathogenic against the G. mellonella (Fig. 3, Table 1). There was a significant effect of time on the lethal concentration LC50 of EPN isolate against pests (p<0.05; one-way ANOVA, Table 1).
There was effect of concentration (p< 0.05, 2- way repeated measures ANOVA, Table 2) and time (p< 0.0001, 2- way repeated measures ANOVA, Table 2) but not of interaction between the concentration and time (p> 0.05, 2- way repeated measures ANOVA, Table 2) of H. indica isolate CH22 against G. mellonella and S. litura. Specifically, mortality % of G. mellonella larvae was found to be significantly higher in the 100 and 200 IJs/ larva concentration in comparison to 25 IJs/ larva at 36 hrs of post infection period (PIP) (p< 0.0001, Tukey’s multiple comparison post-hoc test). Specifically, mortality % of Spodoptera litura larvae was found to be significantly higher in the 100 and 200 IJs/ larva concentration in comparison to 25 IJs/ larva at 36 hrs of PIP (p< 0.0001, Tukey’s multiple comparison test, Fig 3). The effect of the concentration and exposure time along with the interaction between the concentrations and time (p < 0.0001, p< 0.05, 2- way repeated measures ANOVA, Fig 3, Table 2) of H. indica isolate CH23 against G. mellonella were found statistically significant. At 24 hrs of PIP, there was significantly higher mortality of larvae in the 200IJs/ larva dose as compared to 25 IJs/ larva and 50 IJs/larva (p< 0.0001, Tukey’s multiple comparison post-hoc test). The effect of the concentration and exposure time were found statistically significant of H. indica isolate CH23 against Spodoptera litura larvae (p< 0.0001, 2- way RM ANOVA, Table 2). After 36 hrs of PIP, % mortality was found to be higher in 200 IJs/ larva than 25 IJs/ larva (p< 0.0001, Tukey’s multiple comparison post-hoc tests, Fig 3, Table 2).
In CH25 isolate of H. indica against G. mellonella and S. litura, there was effect of the concentration and exposure time along with the interaction between the concentration and time (p < 0.0001, p< 0.05, 2- way repeated measures ANOVA, Table 2). The mortality was found to be significantly higher in the 200 IJs/larva in comparison to the 25 and 50 IJs/ larva at 24 hrs of PIP (p< 0.0001, Tukey’s multiple comparison post-hoc tests). The mortality was found to be significantly higher in the 100 and 200 IJs/larva in comparison to the 25 IJs/ larva at 24 hrs of PIP against S. litura (p< 0.0001, Tukey’s multiple comparison post-hoc tests). At 60 hrs of PIP, it was observed that the % mortality was significantly lower in the 25 IJs/larva as compared to 100 and 200 IJs/ larva (p< 0.0001, Tukey’s multiple comparison post-hoc tests).In CH26 isolate of H. indica against G. mellonella and S. litura, there was effect of the concentration and exposure time along with the interaction between the concentration and time (p< 0.0001, p < 0.05, 2- way RM ANOVA, Table 2). The % mortality was found to be higher in the 200 IJs/larva than the 25 IJs/ larva dose at 24 hrs (p< 0.0001, Tukey’s multiple comparison post-hoc tests). At 72 hrs of PIP, it was found that there were no significant differences in the % mortalities between 25 and 50 IJs/ larva and 100 and 200 IJs/ larva (p> 0.05, Tukey’s multiple comparison post-hoc tests). In S. litura, 72 hrs of PIP, it was found that the % mortality was highest in the 200 IJs/ larva in comparison to other doses i.e., 25 IJs/ larva, 50 IJs/ larva and 100 IJs/ larva (p< 0.0001, Tukey’s multiple comparison post-hoc tests).
Relationship between the concentration and % mortality
Two isolates (CH23, highly pathogenic and CH26, least pathogenic) of H. indica were processed for further analysis to find out the possible relationship between the dose and the % mortality at an interval of 12 hrs PIP. There was a significant positive correlation between the concentration (dose) and % mortality at 24 hrs in CH23 against G. mellonella (r= 0.9834; p=0.0166, Pearson’s correlation) and S. litura (r= 0.9799; p=0.0201, Pearson’s correlation, Fig. 4a). Similar results were obtained from linear regression analysis which showed positive relation between the dose and % mortality at 24 hrs against G. mellonella and S. litura (p< 0.0001; Pearson’s correlation, Table 3; Fig. 4a). There was a significant positive correlation found between the concentration (dose) and % mortality at 24 hrs in CH26 against G. mellonella (r= 0.9834; p=0.0166, Pearson’s correlation, Fig 4b). There were positive relationship found between the dose and % mortality at 36 (r= 0.9834; p=0.0166, Pearson’s correlation) and 72 hrs against S. litura (r= 0.9984; p=0.0016, Pearson’s correlation). Results obtained from the linear regression analysis also showed positive relationship between dose and % mortality at 24 hrs against G. mellonella larvae and at 36 and 72 hrs against S. litura (p< 0.0001; Pearson’s correlation, Table 3; Fig. 4b).