# Variability study of entomopathogenic nematode populations (Heterorhabditidae) from Argentina/ Estudo da variabilidade entomopatogenicos nematoides populacoes (Heterorhabditidae) da Argentina.

1. IntroductionEntomopathogenic nematodes (Heterorhabditidae), are generalized consumers of insects in soil food webs that occur widely in natural and agricultural ecosystems. Their associations with symbiotic pathogenic bacteria make them highly virulent, constituting an alternative for the control of insect pests. The third juvenile stage of entomopathogenic nematodes is referred to as the "infective juvenile" or "dauer" stage and is the only free-living stage. They are capable of surviving in the soil without nourishment for prolonged periods up it locates, attacks, and infects an insect host. Once inside the host hemocoel, the IJ releases symbiotic bacteria and kills the host by a combination of toxins and septicemia. Soon after, the IJs begin to feed on the bacteria and develop to reproductive stages. Infective juveniles of the genus Heterorhabditis present a first generation of adult hermaphrodites and generally, a second generation with males and true females. Finally, IJs leave the cadavers in response to declining nutrients. Thus, a single IJ has the potential to colonise a new habitat (Boff et al., 2000).

Two species of heterorhabditid nematodes were cited for Argentina: Heterorhabditis argentinensis Stock, 1993 in Santa Fe and La Pampa provinces, and Heterorhabditis bacteriophora Poinar, 1976, reported in Buenos Aires, Cordoba, La Pampa, Neuquen, Rio Negro, Mendoza and Santa Fe provinces (Stock, 1993, 1995; Doucet and Bertolotti, 1996; Giayetto and Cichon, 2006; Del Valle et al., 2013). These strains were characterized mainly by morphological and morphometric features. In this way, Heterorhabditis argentinensis was reported and characterized at first, by the large size of the adult males and first-generation females, the longer tail, and the peloderan bursa with 9 pairs of genital papillae in the arrangement 1, 2, 1, 2, 3 (Stock, 1993). However, Adams et al. (1998) considered H. argentinensis and H. bacteriophora species as sister taxa and possibly even conspecific by PCR amplification of the ITS-1 region. Even, Nguyen (2016) mentioned the arrangement of the last three genital papillae of the bursa, as highly variable in strains of Argentina.

The nematode stage and main traits that should be used for Heterorhabditis species distinctions is a matter of discussion. Several authors considered morphological and morphometric features of males and IJs as the most suitable for the distinction among heterorhabditid populations (Adams et al., 1998; Dolinski et al., 2008). Body (L) and tail (T) length in IJs, plus body (L) and reflection of testis in males were used to identify Heterorhabditis species (Hominick et al., 1997). However, Phan et al. (2003) suggest length in IJs, and spicule and gubernaculum length and shape in males as ones that should be considered.

Stock and Mracek (2000), suggested that geographical origin and habitat can influence morphometric data. Intraspecific morphometric variations were observed among strains of the entomopathogenic nematode Steinernema feltiae from UK, Pakistan, Rioja and Catalonia (Spain). Also, differences were observed for IJs and males from different infected hosts (Campos Herrera et al., 2006).

Considering the number of Heterorhabditis populations isolated from different regions from Argentina, the lack of variability studies for this region and the discussion about the position of H. argentinensis, the aims of this work was to analyze morphometric variability between Argentinian strains of Heterorhabditis spp. and to evaluate the taxonomic position of H. argentinensis.

2. Material and Methods

2.1. Morphometric study of Heterorhabditis spp. populations

Five populations of Heterorhabditis spp. isolated from Argentina were considered. Measurements of morphometric features were taken from the literature: Heterorhabditis bacteriophora RIV from Rio Cuarto city, Cordoba province (33[degrees]08'00"S; 64[degrees]21'00"W) OLI strain, from Oliva, Cordoba province (32[degrees]02'00"S; 63[degrees]34'00" W), (Doucet and Bertolotti, 1996; Doucet et al., 1996), RN strain from Rio Negro province (38[degrees]56'00"S 68[degrees]01'00"W) (Doucet and Bertolotti, 1996), VELI strain from Villa Elisa, Buenos Aires province (34[degrees]51'12" S, 58[degrees]04'45"W) (Salas et al., 2013), and H. argentinensis isolated from Rafaela, Santa Fe province (31[degrees]16'00"S 61[degrees]29'00"W) (Stock, 1993). Data of the climatic conditions for regions where nematodes were isolated, are shown in Table 1.

Principal component analyses (PCA) were performed on the morphometric variables representing data of hermaphroditic females, amphimictic females, males and infective juveniles of the Heterorhabditis species, to examine the general grouping of all individuals with Infostat statistical program (version 2014). Six variables were considered for hermaphroditic females: L, MBW, NR, EP, ES, TL, and V; eight for amphimictic females: L, MBW, NR, EP, ES, TL, ABW and V; nine for males: L, MBW, NR, EP, ES, TRL, TL, SpL and GuL; and five for infective juveniles: L, MBW, EP, ES and TL (as shown in Tables 2-5).

2.2. Molecular-genetic analyses

The genetic variability among Argentinian populations was analyzed. Heterorhabditis argentinensis was molecularly characterized by DNA sequences of the ITS1 region (Adams et al., 1998), so this nucleotide sequence was considered as limit length, of the segments of analyzed species. A set of 37 homologous sequences recovered from GenBank were aligned with other ITS1 rDNA for bioinformatic analyses. Longer sequences were shortened in order to have a common informative genome segment.

Among five isolates compared in the morphometric study, only H. bacteriophora (VELI strain) and H. argentinensis were included in the phylogenetic analysis because there are no records of sequences for ITS-1 region in the Genbank for the rest. The evolutionary history was inferred by means of the neighbor-joining method (Saitou and Nei, 1987). The evolutionary distances were computed by means of the maximum-composite-likelihood method (Tamura et al., 2004) and the rate of variation among loci modeled with a gamma distribution (shape parameter = 2.25). These evolutionary analyses were carried out by the MEGA5 software (Tamura et al., 2011).

3. Results

3.1. PCA analysis

Morphometric variation was greatest between H. argentinensis and all other H. bacteriophora isolates. Heterorhabditis argentinensis was separated from the rest of H. bacteriophora populations for juveniles, males, amphimictic and hermaphroditic females (see Figure 1 A-D).

An accumulated variability of 91% was reached in hermaphroditic females by the PC1 (76%) and PC2 (15%). Except for NR and V, all variables showed positive correlation between them and were responsible of the great variability of the PC1. Heterorhabditis argentinensis hermaphroditic females differed from the other H. bacteriophora populations due to larger dimensions. Heterorhabditis bacteriophora OLI and RIV strains were more similar than the other H. bacteriophora strains. The variable V was responsible to the major variability of the PC2 (see Figure 1A).

As well as hermaphrodites, all morphometric characters for amphimictic females, except V and NR, explained the PC1 variability (72.8% of the total) and had a positive and high correlation. The variable V was correlated with PC2 and separated one of isolates of H. bacteriophora (OLI). RN and RIV strains ofH. bacteriophora were most similar. Heterorhabditis argentinensis was separated from the rest of populations (see Figure 1B).

Males showed the greatest morphometric differences, being the five strains individually separated by PCA analysis. Results of the first two principal components for males explained 68.9%. The PC1 separated H. argentinensis from the other H. bacteriophora populations by MBW, TL, L and EP variables. L, MBW, EP, ES, TL and GuL were responsible of the major variability in the PC1, while TRL and SpL defined PC2. Heterorhabditis bacteriophora VELI strain was separated from the others by a high value of TRL (see Figure 1C).

The infective juvenile was the stage with lower variability between strains and the best to discriminate Argentinian populations. The principal components 1 and 2 accounted for 48% (PC1) and 28% (PC2) respectively of the total variation of juveniles (76.3%). Three groups were evident, one formed by H. bacteriophora OLI, RIV and RN strains, one for H. bacteriophora VELI strain, and one for H. argentinensis (see Figure 1D). The variable L and MBW were highly related to H. argentinensis, while ES and TL separated H. bacteriophora (OLI, RIV, RN strains) and EP H. bacteriophora VELI strain.

3.2. Phylogenetic analysis

In our phylogenetic tree, Heterorhabditis spp. formed three separate clades. The bioinformatic study placed H. argentinensis as a member of the clade B (100% support of the most ancestral node), related to six populations of H. bacteriophora (among them the argentine VELI strain), five populations of H. georgiana, one isolate of H. zealandica, and two isolates of Heterorhabditis sp. (see Figure 2; Table 6). Within the above set of sequences, the genetic similarities between H. argentinensis and the other members of the clade B varied between 98.3 and 100%; and this isolate proved to be included in a subgroup together with five isolates of H. bacteriophora, where the similarities were between 99.4% and 99.7%.

In clade B,H. bacteriophora HP88, N-KMD7, and 190-C strains had identical nucleotide sequences. Similarly, the strains of H. georgiana N-SPCM3, N-GPS29, N-KMD82 and H. zealandica NZH3 contained no differences (Table 6). However, the Argentinian H. bacteriophora VELI strain, differed from the above three H. bacteriophora populations in one nucleotide at position 176 (a T vs. C transition), whereas H. argentinensis did so with respect to an insertion of a C at position 348. The latter species also exhibited variation at three positions with respect to H. bacteriophora N-KMD6 (at nucleotides 97, 176, and 231) and to H. bacteriophora 51-C (at nucleotides 176, 295, and 310). Greater differences were observed between VELI strain and H. georgiana populations (between 4 and 5 different nucleotides; Table 6).

The clade A (100% support of the most ancestral node) comprised all species isolated from the Southern Asia and Indian-Ocean region: five isolates of H. indica, one isolate of H. gerrardi, and two Heterorhabditis sp.

The remaining group, clade C (100% support of the most ancestral node), comprised five species: H. marelatus, H. safricana, H. atacamensis, H. downesi, and H. megidis. Also, this clade was composed of five subgroups (51-100% support; see Figure 2). Length of expected amplicon for the species analyzed in this phylogenetic study (without considering primers used for each case) varied between 829 and 830 bp in clade A, 861-862 bp in clade B, and 836- 851 bp in clade C. These results are in accordance with the similarity levels data obtained within clades by phylogenetic inference, indicating a major homogeneity between H. bacteriophora populations.

4. Discussion

Two species of heterorhabditid nematodes were reported for Argentina: six isolates ofH. argentinensis in Santa Fe and La Pampa provinces (Stock, 1993, 1995), and more than 20 populations of Heterorhabditis bacteriophora reported in the central-pampean region, Cuyo and Patagonia (Doucet and Bertolotti, 1996; Giayetto and Cichon, 2006; Del Valle et al., 2013).

Studies on the morphometric variability are useful to provide valuable information about geographical and ecological requirements for EPN. Morphometric differences can be observed in nematode strains isolated from different sites and hosts (Campos Herrera et al., 2006).

In our study, morphometric variations were observed according to Nguyen (2016) which considered some morphological features as highly variables for Argentinian strains. The greatest differences were registered between H. argentinensis and all other H. bacteriophora populations, unlike between H. bacteriophora strains from different regions.

Results of PCA analysis for morphometric characters separated H. argentinensis from the rest of H. bacteriophora strains from Argentina, considering the four nematode stages. The infective juvenile was a stage with high weight in the separation between Heterorhabditis populations. Three groups were separated considering PC1 and PC2 for this stage; one formed by H. bacteriophora OLI, RIV and RN strains, one for H. bacteriophora VELI strain and one for H. argentinensis. The variable L was negatively correlated with TL for juveniles, showing that longer specimens had shorter tails. Measurements of males and infective juveniles were suggested for several authors as the most suitable for the distinction among heterorhabditid populations (Adams et al., 1998; Dolinski et al., 2008) although in our study males were not as good as juveniles.

Morphometric characteristics of H. bacteriophora strain VELI, were slightly larger respect to other Argentinian isolates of the same species, which was observed always separated from the rest of populations by PCA analysis. However, these variations were not as considerable as in H. argentinensis.

Even though morphometric study separated H. argentinensis from the rest of H. bacteriophora isolates, the phylogenetic analysis placed in the same clade H. argentinensis and six populations of H. bacteriophora. The genetic similarity between H. bacteriophora populations and H. argentinensis was over 99%. Heterorhabditis bacteriophora VELI strain and H. argentinensis had the same number of nucleotide-sequence variations with respect to the rest of the H. bacteriophora isolates analyzed in our study. The Argentinian isolations exhibited two differences in nucleotide sequence: a T vs. C at position 176 of ITS1-rDNA (constituting a new variation in coding sequence) in H. bacteriophora (VELI strain) and a C inserted at position 348 in Heterorhabditis argentinensis. These nucleotide variations were not present in the rest of H. bacteriophora member of the clade B. Results obtained in our study are according to Adams et al. (1998) and Phan et al. (2003) who analyzing DNA sequences of the ITS1 of the Heterorhabditis genus, showed the type strain H. bacteriophora (HB1) differing from H. argentinensis in a single transversion (G vs. C) at position 620 (Adams et al., 1998; Phan et al., 2003) and in a single insertion of a C at position 348 as we mentioned (Phan et al., 2003). Therefore, by the results obtained in our study, H. argentinensis would be a sister taxa of H. bacteriophora, as was considered by Adams et al. (1998).

Morphometry of the parasites can be influenced by nutritional conditions inside the host, and environmental factors (Phan et al., 2003; Canto-Silva, et al., 2005). Stock and Mracek (2000), mentioned that the geographical origin and habitat can influence on morphometric data, so even values obtained from a same host can change based on abiotic factors and rearing conditions. Boff et al. (2000) observed that a larger body size of the host and a lower dose of infection increased the size of IJs of Heterorhabditis megidis. These variations could be produced because measurements often are taken from the progeny of a few soil-baited insect hosts, so is unlikely that they represent the range of variation present in the population (Adams et al., 1998).

In Argentina, Heterorhabditis spp. populations were isolated from different locations and hosts. Heterorhabditis argentinensis was found parasitizing the alfalfa weevil Graphognathus sp. (Coleoptera: Curculionidae) (Stock, 1993), from Santa Fe province, H. bacteriophora RIV strain from Heliothis sp. (Lepidoptera: Noctuidae) from Cordoba province, and both were later maintained in the laboratory in Galleria mellonella larvae (Lepidoptera) for the identification; meanwhile Heterorhabditis bacteriophora OLI strain, from Cordoba province, RN strain from Rio Negro province and VELI strain from Buenos Aires province were isolated directly from field, in Galleria mellonella baits, leaving the natural hosts unknown (Aguera de Doucet and Doucet, 1986; Stock, 1995; Doucet and Bertolotti, 1996; Giayetto and Cichon, 2006). Geographically, Argentina is divided in different regions determined by a homogeneous relief and climate. Rainfalls decrease from east to west, from Mesopotamia to the mountains by the rainfall regime of the Atlantic. From there we can find humid climates, with over 800 mm per year, less than 400 mm dry climates and arid and semiarid in the transition zone between both (Bianchi and Cravero, 2010). Buenos Aires and Santa Fe provinces, where H. bacteriophora VELI and H. argentinensis strains were isolated, present more rainfalls and humidity conditions, respect the other sites. These requirements could have influenced on the larger dimensions observed in H. bacteriophora VELI and H. argentinensis populations, as the conditions of the host at the time to be isolated from the field.

In the same way, the average annual temperature declines in the plains of central and northeastern of Argentina with increasing latitude. The "Rio Negro" river where the upper Valley of Rio Negro province is located, and where H. bacteriophora RN strain was isolated, born at the eastern end of the province of Neuquen, and flows to the Rio Negro territory in southeast direction to reach the Atlantic Ocean, being in its last leg the natural boundary between the provinces of Rio Negro and

Buenos Aires. The presence of mountains and plateaus in the west and south of the 'Rio Negro " river, deflects the isotherms, which take a parallel course to the mountain, producing a decreasing of the temperatures to the south of the country. According to this parameter, in Argentina can be distinguished subtropical climates, with annual average temperatures above 18 [degrees]C, temperate, ranging between 18 [degrees]C and 12 [degrees]C, and cold climates, less than 12 [degrees]C (Bianchi and Cravero, 2010). While all Argentinian strains were isolated from temperate climates, the average annual temperature for Santa Fe province (18 [degrees]C) is at the limit between a mild and subtropical climate, which also could have influenced the largest morphometry reached by H. argentinensis.

Considering the results of this study, morphometric variations are present between Argentinian populations of Heterorhabditis spp. isolated from different regions. In this way, Heterorhabditis argentinensis would be conspecific to H. bacteriophora, constituting a strain with a great morphometric variation where the host and climatic conditions could have influenced on the measurements.

This work constituted the first comparative and phylogenetic study of heterorhabditid populations from Argentina.

http://dx.doi.org/10.1590/1519-6984.20015

Acknowledgements

This study was partially supported by Consejo Nacional de Investigaciones Cientificas y Tecnicas (CONICET), PIP 2010/0170, Universidad Nacional de La Plata, (UNLP), and Agencia Nacional de Promocion Cientifica y Tecnologica, Argentina, PICT 2011/1439. We thank to Dra. Graciela Minardi, and Carlos A. Galliari (CEPAVE), for statistical assistance and Dr. Larry Duncan (UF/IFAS) a native English speaker, and referent in entomopathogenic nematodes for his suggestions and editing of the final version of the manuscript.

References

ADAMS, B.J., BURNELL, A.M. and POWERS, T.O., 1998. A phylogenetic analysis of Heterorhabditis (Nemata: Rhabditidae) based on internal transcribed spacer 1 DNA sequence data. Journal of Nematology, vol. 30, no. 1, pp. 22-39. PMid:19274196.

AGUERA DE DOUCET, M.M. and DOUCET, M.E., 1986. Nuevos datos para el conocimiento de Heterorhabditis bacteriophora Poinar, 1975. Revista de Investigaciones Agropecuarias INTA, vol. XXI, pp. 1-10.

BIANCHI, A. and CRAVERO, S., 2010. Atlas climatico digital de la Republica Argentina. Buenos Aires: Editorial INTA, Instituto Nacional de Tecnologia Agropecuaria. 55 p.

BOFF, M.I.C., WIEGERS, G.L. and SMITS, P.H., 2000. Influences of host size and host species on the infectivity and development of Heterorhabditis megidis (strain NLH E87.3). BioControl, vol. 45, no. 4, pp. 469-482. http://dx.doi.org/10.1023/A:1026560208285.

CAMPOS-HERRERA, R., ESCUER, M., ROBERTSON, L. and GUTIERREZ, C., 2006. Morphological and Ecological Characterization of Steinernema feltiae (Rhabditida: Steinernematidae) Rioja Strain Isolated from Bibio hortulanus (Diptera: Bibionidae) in Spain. Journal of Nematology, vol. 38, no. 1, pp. 68-75. PMid:19259432.

CANTO-SILVA, C.R., ROMANOWSKI, H.P and REDAELLI, L. R., 2005. Effect of temperature on the development and viability of Gryon gallardoi (Brethes) (Hymenoptera: Scelionidae) parasitizing Spartocera dentiventris (Berg) (Hemiptera: Coreidae) eggs. Brazilian Journal of Biology = Revista Brasileira de Biologia, vol. 65, no. 3, pp. 415-421. http://dx.doi.org/10.1590/ S1519-69842005000300006. PMid:16341419.

DEL VALLE, E., LAX, P., RONDAN DUENAS, J. and DOUCET, M., 2013. Effects of insect cadavers infected by Heterorhabditis bacteriophora and Steinernema diaprepesi on Meloidogyne incognita parasitism in pepper and summer squash plants. Ciencia eInvestigacionAgraria, vol. 40, no. 1, pp. 109-118. http://dx.doi. org/10.4067/S0718-16202013000100009.

DOLINSKI, C., KAMITANI, F., MACHADO, I. and WINTER, C., 2008. Molecular and morphological characterization of heterorhabditid entomopathogenic nematodes from the tropical rainforest in Brazil. Memorias do Instituto Oswaldo Cruz, vol. 103, no. 2, pp. 150-159. http://dx.doi.org/10.1590/S007402762008000200005. PMid:18425267.

DOUCET, M.M.A. and BERTOLOTTI, M.A., 1996. Una nueva poblacion de Heterorhabditis bacteriophora Poinar, 1975 para Argentina. Caracterizacion y accion sobre el huesped. Nematologia Mediterranea, vol. 24, pp. 169-174.

DOUCET, M.M.A., BERTOLOTTI, M.A., and CAGNOLO, S.R., 1996. On a new isolate of Heterorhabditis bacteriophora Poinar, 1975 (Nematoda: Heterorhabditidae) from Argentina: life cycle and description of infective juveniles, female, males and hermaphrodites of 2nd and 3rd generations. Fundamental and Applied Nematology, vol. 19, pp. 415-420.

FELSENSTEIN, J., 1985. Confidence limits on phylogenies: an approach using the bootstrap. E-volution, International Journal of Organic Evolution, vol. 39, no. 4, pp. 783-791. http://dx.doi. org/10.2307/2408678.

GIAYETTO, A.L. and CICHON, L.I., 2006. Distribucion, gama de huespedes y especificidad de cinco poblaciones de Heterorhabditis bacteriophora (Nematoda: Heterorhabditidae) del Alto Valle de Rio Negro y Neuquen, Argentina. RIA, vol. 35, no. 2, pp. 163-183.

HOMINICK, W.M., BRISCOE, B.R., DEL PINO, F.G, HENG, J., HUNT, D.J., KOZODOY, E., MRACEK, Z., NGUYEN, K. B., REID, A.P., SPIRIDONOV, S., STOCK, P., STURHAN, D., WATURU, C. and YOSHIDA, M., 1997. Biosystematics of entomopathogenic nematodes: current status, protocols and definitions. Journal of Helminthology, vol. 71, no. 4, pp. 271-298. http://dx.doi.org/10.1017/S0022149X00016096. PMid:9443947.

NGUYEN, K.B., 2016 [viewed 4 April, 2016]. Morphology of bursa of Heterorhabditis spp. [online]. Gainesville: Institute of Food and Agricultural Sciences, University of Florida. Available from: http://entnemdept.ufl.edu/nguyen/ morph/het-bursa.htm

PHAN, K.L., SUBBOTIN, S.A., NGUYEN, N.C. and MOENS, M., 2003. Heterorhabditis baujardi sp. n. (Rhabditida: Heterorhabditidae) from Vietnam and morphometric data for H. indica populations. Nematology, vol. 5, no. 3, pp. 367-382. http://dx.doi.org/10.1163/156854103769224368.

SAITOU, N. and NEI, M., 1987. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Molecular Biology andEvolution, vol. 4, no. 4, pp. 406-425. PMid:3447015.

SALAS, A., ELICECHE, D., BELAICH, M. and ACHINELLY, M., 2013. Biologia, patogenicidad y multiplicacion de un aislamiento nativo del nematodo entomopatogeno Heterorhabditis bacteriophora proveniente de huertas de cultivo organico en Argentina. Nematropica, vol. 43, pp. 323.

STOCK, S.P. and MRACEK, Z., 2000. Morphological variation between allopatric populations of Steinernema krausei (Steiner, 1923) (Rhabditida: Steinernematidae). Nematology, vol. 2, no. 2, pp. 143-152. http://dx.doi.org/10.1163/156854100509033.

STOCK, S.P., 1993. A new species of the genus Heterorhabditis Poinar, 1976 (Nematoda: Heterorhabditidae) parasitizing Graphognathus sp. Larva (Coleoptera: Curculionidae) from Argentina. Research and Reviews in Parasitology, vol. 53, pp. 103-107.

STOCK, S.P., 1995. Natural populations of entomopathogenic nematodes in the Pampean region of Argentina. Nematropica, vol. 25, pp. 143-148.

TAMURA, K., NEI, M. and KUMAR, S., 2004. Prospects for inferring very large phylogenies by using the neighbor-joining method. Proceedings of the National Academy of Sciences of the United States of America, vol. 101, no. 30, pp. 11030-11035. http://dx.doi.org/10.1073/pnas.0404206101. PMid:15258291.

TAMURA, K., PETERSON, D., PETERSON, N., STECHER, G., NEI, M. and KUMAR, S., 2011. MEGA5: Molecular Evolutionary Genetics Analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Molecular Biology and Evolution, vol. 28, no. 10, pp. 2731-2739. http://dx.doi. org/10.1093/molbev/msr121. PMid:21546353.

M. F Achinelly (a), *, D. P. Eliceche (a), M. N. Belaich (b), P. D. Ghiringhelli (b)

(a) Consejo Nacional de Investigaciones Cientificas y Tecnicas--CONICET, Centro de Estudios Parasitologicos y de Vectores--CEPAVE, Facultad de Ciencias Naturales y Museo, Universidade Nacional de La Plata--UNLP, Calle 60 y 121, s/n, La Plata, Buenos Aires, Argentina

(b) Laboratorio de Ingenieria Genetica y Biologia Celular y Molecular, Consejo Nacional de Investigaciones Cientificas y Tecnicas--CONICET, Universidad Nacional de Quilmes--UNQ, Roque Saenz Pena, 352, Bernal, Buenos Aires, Argentina

* e-mail: fachinelly@cepave.edu.ar

Received: November 24, 2015--Accepted: April 14, 2016--Distributed: August 31, 2017 (With 2 figures)

Caption: Figure 1. Principal component analysis of Heterorhabditis spp., based on mean values of morphometric characters for all nematode stages. (A) = Hermaphroditic females, (B) = Amphimictic females, (C) = Males, (D) = Infective juveniles. (H. bacteriophora strains: 1. VELI, 2. OLI, 3. RIV, 4. RN; 5: H. argentinensis).

Caption: Figure 2. Evolutionary relationships among Heterorhabditis taxa. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1,000 replicates) is shown next to the branches, Felsenstein (1985). Only node consistencies above 40% are shown. The tree is drawn to scale, with branch lengths in the same proportions as the evolutionary distances used to infer the phylogenetic tree.

Table 1. Climatic conditions of origin regions of entomopathogenic nematodes strains considered in this study (H. b: H. bacteriophora). H.b. VELI H.b. OLI Origin Villa Elisa Oliva Coordinates 34[degrees]51'12" S, 32[degrees]02'00"S; 58[degrees]04'45" W 63[degrees]34'00"W Geography Plain Plain Clime temperate and temperate and humid semi arid Mean annual max. 11 10 temp. ([degrees]C) Mean annual min. 21 24 temp. ([degrees]C) Mean annual temp. 15.8 16.9 ([degrees]C) Total annual 1007 711 rainfall (mm) Humidity (%) 77 67 H.b. RIV H.b. RN Origin Rio Cuarto Rio Negro valley Coordinates 33[degrees]08'00"S; 38[degrees]56'00"S; 64[degrees]21'00"W 68[degrees]01'00"W Geography Plain Plain Clime temperate and temperate and semi arid dry Mean annual max. 10 8 temp. ([degrees]C) Mean annual min. 22 21 temp. ([degrees]C) Mean annual temp. 16.8 14.1 ([degrees]C) Total annual 846 213 rainfall (mm) Humidity (%) 68 56 H. argentinensis Origin Rafaela Coordinates 31[degrees]16'00"S 61[degrees]29'00"W Geography Plain Clime temperate and humid Mean annual max. 12 temp. ([degrees]C) Mean annual min. 25 temp. ([degrees]C) Mean annual temp. 18 ([degrees]C) Total annual 959 rainfall (mm) Humidity (%) 76

Table 2. Morphometrics (mean and range) of hermaproditic females of Heterorhabditis isolates provided by the bibliography. All measurements are in pm (H. b: H. bacteriophora; NA: not available). Characters Hermaprhroditic females H.b. VELI H.b. OLI L 2,835.46 [+ or -] 482.72 4,800 [+ or -] 550 (2,160-3,840) (3,900-5,800) MBW 117.64 [+ or -] 14.32 215.7 [+ or -] 27.64 (97.4-138.65) (177.5-255) VBW 123.60 [+ or -] 17.54 NA (109.04-153.12) STL 8.91 [+ or -] 2.10 11.13 [+ or -] 1.9 (6.25-11.6) (7.5-15) STW 8.16 [+ or -] 1.34 11.13 [+ or -] 1.89 (5.87-9.28) (7.5-12.5) NR 88.95 [+ or -] 1.34 77.88 [+ or -] 7.71 (69.6-118.7) (67.5-92.5) EP 157.65 [+ or -] 19.89 194.38 [+ or -] 15.53 (132.24-200) (175-225) ES 152.58 [+ or -] 16.41 183.7 [+ or -] 14.06 (132.22-181.25) (162.5-207) TL 47.87 [+ or -] 9.46 81 [+ or -] 7.09 (37.5-51.04) (70-95) ABW 52.41 [+ or -] 17.80 48.5 [+ or -] 6.24 (37.12-63.45) (35.5-59) V 42.15 [+ or -] 12.79 41.83 [+ or -] 2.1 (39.86-47) (36.1-45.1) Characters Hermaprhroditic females H.b. RIV H.b. RN L 5,010 [+ or -] 410 3,460 [+ or -] 780 (4,200-5,600) (2,400-4,800) MBW 202.6 [+ or -] 16.99 185.75 [+ or -] 24.03 (175-242) (145-222.5) VBW NA NA STL NA NA STW NA NA NR 138.86 [+ or -] 8.37 136.41 [+ or -] 17.26 (125-152) (107.5-167.5) EP 189.93 [+ or -] 13.65 175.58 [+ or -] 23.27 (163-225) (142.5-212.5) ES 199.03 [+ or -] 8.25 189.41 [+ or -] 23.83 (180-220) (155-225) TL 72.8 [+ or -] 7.09 61.16 [+ or -] 8.97 (50-87) (47.5-87.5) ABW 53.3 [+ or -] 3.66 35.16 [+ or -] 9.3 (45-62) (22.5-55) V 40.53 [+ or -] 1.54 45.8 [+ or -] 2.96 (35-45) (39.53-50.9) Characters Hermaprhroditic females H. argentinensis L 6,500 (5,000-7,500) MBW 360 (250-275) VBW NA STL 13 (10.0-16.0) STW 10 (6.0-12) NR 160 (132-196) EP 294 (250-340) ES 274 (235-300) TL 118 (100-140) ABW 86 (70-120) V 44.5 (40-50)

Table 3. Morphometrics (mean and range) of amphimictic females of Heterorhabditis isolates provided by the bibliography. All measurements are in [micro]m (H. b: H. bacteriophora; NA: not available). Characters Amphimictic females H.b. VELI H.b. OLI L 1,646.34 [+ or -] 282.42 2,220 [+ or -] 210 (1,251-2,286) (1,800-2,600) MBW 97.08 [+ or -] 11.89 114.13 [+ or -] 9.81 (78.8-113.68) (97.5-133) STL 7.76 [+ or -] 1.36 7.88 [+ or -] 1.47 (6.96-11.6) (5.0-10.0) STW 7.22 [+ or -] 0.69 8.25 [+ or -] 1.43 (6.96-9.28) (5.0-10.0) NR 73.69 [+ or -] 13.56 65 [+ or -] 7.43 (58-104.4) (55-87.5) EP 117.18 [+ or -] 20.88 165 [+ or -] 7.4 (83.52-173.32) (152.5-175) ES 123.13 [+ or -] 10.04 137.25 [+ or -] 6.48 (104.4-141.52) (125-148) TL 41.58 [+ or -] 0.39 71.5 [+ or -] 4.32 (20.88-53.36) (62.5-80.0) ABW 48.36 [+ or -] 12.30 NA (27.84-69.6) V 45.61 [+ or -] 2.69 49.88 [+ or -] 5.2 (39.2-49.68) (40.7-69.7) Characters Amphimictic females H.b. RIV H.b. RN L 2,140 [+ or -] 170 1,830 [+ or -] 120 (1,800-2,400) (1,650-2,150) MBW 123.46 [+ or -] 13.39 112.9 [+ or -] 8.2 (100-162) (95-127.5) STL NA NA STW NA NA NR 93.23 [+ or -] 5.2 95.75 [+ or -] 6.47 (83-102) (82.5-107.5) EP 149.76 [+ or -] 8.33 131.25 [+ or -] 8.34 (122-162) (117.5-150) ES 133.63 [+ or -] 6.77 135.83 [+ or -] 5.62 (108-145) (125-150) TL 54.46 [+ or -] 4.83 67.41 [+ or -] 4.61 (40-65) (60-77.5) ABW 27.7 [+ or -] 3.79 26.25 [+ or -] 2.6 (23-40) (22.5-32.5) V 46.73 [+ or -] 2.37 46.51 [+ or -] 1.52 (41-50) (42.7-48.9) Characters Amphimictic females H. argentinensis L 3,000 (2,000-3,500) MBW 130 (90-180) STL 9.5 (7-12) STW 8 (5-10) NR 114 (88-140) EP 203 (105-240) ES 180 (162-200) TL 93 (75-108) ABW 45 (33-55) V 45 (42-48)

Table 4. Morphometrics (mean and range) of males of Heterorhabditis isolates provided by the bibliography. All measurements are in [micro]m (H. b: H. bacteriophora; NA: not available). Characters Males H.b. VELI H.b. OLI L 822.46 [+ or -] 83.16 910 [+ or -] 60 (711-972) (800-1005) MBW 46.89 [+ or -] 6.61 48.05 [+ or -] 3.86 (42.3-62.6) (40-57) STL 5.22 [+ or -] 1.71 7.15 [+ or -] 0.93 (2.35-4.28) (6-9) STW 4.264 [+ or -] 1.15 5.25 [+ or -] 0.55 (2.35-6.9) (4-6) NR 66.39 [+ or -] 10.21 51.9 [+ or -] 4.45 (39.9-81.2) (45-62) EP 110.17 [+ or -] 22.31 125.9 [+ or -] 7.6 (95.12-134.5) (113-140) ES 116.07 [+ or -] 12.40 101.4 [+ or -] 4.69 (104.4-141.52) (92.5-107.5) TRL 200.78 [+ or -] 77.98 96 (54-210) (103.4-229.68) TL 27.95 [+ or -] 8.47 28.9 [+ or -] 2.27 (22.0-41.76) (24-33) ABW 36.91 [+ or -] 10.97 NA (27-8-44.08) SpL 45.00 [+ or -] 4.76 46.8 [+ or -] 2.86 (34.8-48.7) (40-51) SpW 4.64 [+ or -] 0.66 NA (3.48-5.8) GuL 22.5 [+ or -] 6.49 22.9 [+ or -] 2.22 (18.5-21.1) (19-27) GuW 5.21 [+ or -] 1.37 NA (3.94-6.96) GS (GuL/SpL) 0.53 [+ or -] 0.13 NA (0.48-0.55) SW (SpL/ABW) 10.28 [+ or -] 2.09 NA (7.29-14.0) E (EP/TL) 4.32 [+ or -] 3.11 NA (2.96-5.65) D (EP/ES) 1.11 [+ or -] 0.18 NA (0.89-1.17) L/TL 35.62 [+ or -] 19.58 NA (21.36-44.18) L/MBW 19.31 [+ or -] 3.64 NA (13.5-22.97) MBW/TL 1.57 [+ or -] 1.09 NA (1.1-2.7) Characters H.b. RIV H.b. RN L 845 [+ or -] 57 938 [+ or -] 44 (700-940) (850-1,003) MBW 44.5 [+ or -] 2.7 43.56 [+ or -] 1.63 (37-47) (40-47) STL 6 [+ or -] 0.6 NA (5-7) STW 5.05 [+ or -] 0.51 NA (4-6) NR 77.25 [+ or -] 3.43 77.46 [+ or -] 3.78 (70-85) (70-85) EP 129.15 [+ or -] 4.57 88.36 [+ or -] 4.52 (120-137) (78-98) ES 102.3 [+ or -] 3.34 101.9 [+ or -] 3.06 (95-110) (94-107) TRL 88.7 [+ or -] 11.5 97.23 (80-114) (75-115) TL 24.35 [+ or -] 1.56 30.33 [+ or -] 2.03 (20-27) (26-35) ABW NA 20.2 [+ or -] 1.42 (18-25) SpL 43.2 [+ or -] 1.96 48.03 [+ or -] 2.1 (39-47) (45-53) SpW NA NA GuL 20.6 [+ or -] 1.35 19.2 [+ or -] 2.32 (18-24) (14-23) GuW NA NA GS (GuL/SpL) NA NA SW (SpL/ABW) NA NA E (EP/TL) NA NA D (EP/ES) NA NA L/TL NA NA L/MBW NA NA MBW/TL NA NA Characters H. argentinensis L 1,500 (1,000-2,000) MBW 56 (42-70) STL 5 (3.5-6.0) STW 4 (2.5-5.0) NR 72 (64-82) EP 157 (145-170) ES 113 103-120 TRL 133 (100-194) TL 37 (28-49) ABW 24 (21-30) SpL 46 (42-49) SpW NA GuL 23 (20-26) GuW NA GS (GuL/SpL) NA SW (SpL/ABW) NA E (EP/TL) NA D (EP/ES) NA L/TL NA L/MBW NA MBW/TL NA

Table 5. Morphometrics (mean and range) of infective juveniles of Heterorhabditis isolates provided by the bibliography. All measurements are in [micro]m (H. b: H. bacteriophora ; NA: not available). Characters Infective juveniles H.b. VELI H.b. OLI L 616.75 [+ or -] 60.95 540 [+ or -] 0.03 (505.04-675.12) (490-610) MBW 25.04 [+ or -] 1.6 23 [+ or -] 1.03 (23.2-27.84) (22-45) EP 100.11 [+ or -] 12.34 93.95 [+ or -] 2.96 (78.8-109.04) (87-101) ES 101.93 [+ or -] 14.46 112 [+ or -] 4.68 (74.24-113.68) (103-119) TL 38.54 [+ or -] 14.3 89.6 [+ or -] 10.22 (32.16-51.4) (72-105) ABW 18.63 [+ or -] 3.25 NA (15.08-27.84) Ratio a (L/MBW) 23.76 [+ or -] 3.03 23.44 [+ or -] 1.01 (22.2-26.2) (20.4-25) Ratio b (L/ES) 6.19 [+ or -]1.2 4.77 [+ or -] 0.24 (5.3-8.8) (4.3-5.2) Ratio c (L/TL) 9.87 [+ or -] 3.63 6.07 [+ or -] 0.79 (6.2-14.4) (4.9-7.6) Ratio d (EP/ES) 0.99 [+ or -] 0.14 0.84 [+ or -] 0.02 (0.62-1.07) (0.8-0.88) Ratio e (EP/TL) 1.54 [+ or -] 0.39 1.0 [+ or -] 0.14 (1.9-2.1) (0.8-1.3) Characters Infective juveniles H.b. RIV H.b. RN H. argentinensis L 590 [+ or -] 29.57 603 [+ or -] 19.67 (540-640) (560-640) MBW 25.45 [+ or -] 2.35 23 [+ or -] 0.78 (22-29) (22-25) EP 100.4 [+ or -] 3.74 96.76 [+ or -] 4.93 (96-110) (90-112) ES 121.4 [+ or -] 4.92 116.3 [+ or -] 4.16 (110-130) (108-122) TL 93.4 [+ or -] 4 101.5 [+ or -] 6.72 (85-100) (88-113) ABW 15.9 [+ or -] 0.75 15.25 [+ or -] 1.2 (14-17) (13-17) Ratio a (L/MBW) 23.6 [+ or -] 1.5 26.24 [+ or -] 1.16 (20-26) (23.75-28.63) Ratio b (L/ES) 4.92 [+ or -] 0.3 5.18 [+ or -] 0.17 (4.5-5.8) (4.9-5.6) Ratio c (L/TL) 6.39 [+ or -] 0.19 5.95 [+ or -] 0.37 (6.1-6.8) (5.22-6.66) Ratio d (EP/ES) 0.82 [+ or -] 0.05 0.83 [+ or -] 0.03 (0.75-0.9) (0.77-0.94) Ratio e (EP/TL) 1.07 [+ or -] 0.06 0.95 [+ or -] 0.07 (0.98-1.2) (0.82-1.1) Characters Infective juveniles L 657 (610-710) MBW 31 (24-38) EP 95 (82-116) ES 107 (68-122) TL 84 (70-105) ABW NA Ratio a (L/MBW) NA Ratio b (L/ES) NA Ratio c (L/TL) NA Ratio d (EP/ES) NA Ratio e (EP/TL) NA

Table 6. Nucleotide differences between populations of Heterorhabditis spp. within the Clade B. Alignment HQ225898_H. HQ225855_H. EF043440_H. HQ225879_H. relative georgiana_ georgiana_ zealandica_ georgiana_ position N-SPCM3 N-GPS29 NZH3 N-KMD82 5 T T T T 97 T T T T 176 T T T T 204 G G G G 214 T T T T 225 A A A A 229 A A A A 230 C C C C 231 A A A A 233 T T T T 295 C C C C 310 C C C C 348 Alignment HQ225885_H. HQ225863_H. AY321477_H. relative georgiana_ georgiana_ bacteriophora_ position N-OH25 N-KMD1 HP88 5 G G G 97 T T T 176 T T T 204 G G C 214 T T T 225 A G A 229 A A A 230 C C C 231 A A A 233 T T A 295 C C G 310 C C C 348 Alignment HQ225877_H. FJ217349_H. AF029706_H. relative bacteriophora_ bacteriophora_ argentinensis position N-KMD7 190-C 5 G G G 97 T T T 176 T T T 204 C C C 214 T T T 225 A A A 229 A A A 230 C C C 231 A A A 233 A A A 295 G G G 310 C C C 348 C Alignment KJ575524_H. HQ225875_H. EU796074_H. relative bacteriophora_ bacteriophora_ bacteriophora_ position VELI N-KMD6 51-C 5 G G G 97 T C T 176 C T T 204 C C C 214 T T T 225 A A A 229 A A A 230 C C C 231 A G A 233 A A A 295 G G C 310 C C A 348 Alignment HQ896630_ JX465738_ relative Heterorhabditis Heterorhabditis position sp_QZL-2011 sp_M6 5 G G 97 T T 176 T T 204 C C 214 C C 225 A A 229 T T 230 A A 231 A A 233 C C 295 C C 310 C C 348

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Title Annotation: | Original Article |
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Author: | Achinelly, M.F.; Eliceche, D.P.; Belaich, M.N.; Ghiringhelli, P.D. |

Publication: | Brazilian Journal of Biology |

Date: | Jul 1, 2017 |

Words: | 6158 |

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