Survey of Entomopathogenic Nematodes (Rhabditida: Heterorhabditidae, Steinernematidae) in Rio Grande do Sul State, Brazil

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Nematologia Brasileira 189

Survey of Entomopathogenic Nematodes (Rhabditida: Heterorhabditidae,

Steinernematidae) in Rio Grande do Sul State, Brazil

Carla R.C. Barbosa-Negrisoli1_{, Mauro S. Garcia}1_{, Claudia Dolinski}2_{*, Aldomario S.}

Negrisoli Jr.1_{, Daniel Bernardi}1_{& Fioravante J. dos Santos}3

1_{Laboratório de Biologia de Insetos e Controle Biológico, Universidade Federal de Pelotas, P.O. Box 354, 96010-900,}

Pelotas (RS) Brazil.

2_{Laboratório de Fitopatologia e Entomologia, Universidade Estadual do Norte Fluminense Darcy Ribeiro, 28013-602,}

Campos de Goytacazes (RJ) Brazil.

3_{Laboratório de Solos, Universidade Federal de Pelotas.}

*Corresponding author: claudia.dolinski@censanet.com.br Recebido para publicação em 18 / 04 / 2010. Aceito em 17 / 11 / 2010.

Editado por Luiz Carlos C.B. Ferraz

Summary - Barbosa-Negrisoli, C.R.C., M.S. Garcia, C. Dolinski, A.S. Negrisoli Jr., D. Bernardi & F.J. Santos.

2010. Survey of entomopathogenic nematodes (Rhabditida: Heterorhabditidae, Steinernematidae) in Rio Grande do Sul State, Brazil.

Entomopathogenic nematodes (EPNs) have been found in different ecosystems. It is generally accepted that natural habitats have the highest probability of the occurrence of native species suitable for mass release against local pests, because they are adapted to the climate and other population regulators. The introduction of exotic EPNs may have a negative impact on the native non-target organisms. Therefore, it is important to determine the occurrence of native EPNs in a particular area, and this study aimed to search for EPN species in Rio Grande do Sul State, Brazil. From the 121 soil samples collected, 15.7 % contained EPNs (9.9 % steinernematids and 5.8 % heterorhabditids). Steinernema feltiae, S. rarum and S. riobrave were isolated for the first time in the country. Steinernema rarum and Heterorhabditis bacteriophora were the most common EPNs found (73.6 %). The data showed highest prevalence of EPNs in the north-northeast regions, in sandy soils, at temperatures up to 25 ºC, from 700 to 1,100 m of altitude. H. bacteriophora and S. rarum were recorded at sea level. Although the number of samples collected was not sufficient to be representative of all the species that occur in this region of Brazil, the current data are expected to be important for use in local pest control programs.

Key words: Heterorhabditis, Steinernema, occurrence, abiotic factors.

Resumo - Barbosa-Negrisoli, C.R.C., M.S. Garcia, C. Dolinski, A.S. Negrisoli Jr., D. Bernardi & F.J. Santos.

2010. Isolamento de nematóides entomopatogênicos (Rhabditida: Heterorhabditidae, Steinernematidae) no Estado do Rio Grande do Sul, Brasil.

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Introduction

Entomopathogenic nematodes (EPNs) of the genera Steinernema and Heterorhabditis (Rhabditida: Steinernematidae, Heterorhabditidae) are cosmopolitan, being present in soils and sediments in several ecosystems, limited by water availability. They move through the pores and water film that cover soil particles traveling short distances, depending on environmental conditions, in their search for a host to feed and reproduce on (Treonis & Wall, 2005). EPNs have an important role in controlling various soil insect species (Kaya, 1990), and they are frequently detected in most terrestrial habitats either in natural, agricultural or other disturbed soils (Hominick, 2002).

Various searches of EPNs have been carried out in natural habitats throughout the world, as previously summarized by Hominick (2002). In Brazil, EPNs were found in the states of Minas Gerais (Molina-Acevedo et al., 2005), Rondônia (Dolinski et al., 2008) and Piauí (Barbosa-Negrisoli et al., 2007), among others.

As a result of these studies, new species of EPNs have being described worldwide and investigation dealing with their population dynamics are crucial to understand their persistence, distribution and effect on local insect populations (Hominick & Reid, 1990). In addition, climate parameters such as precipitation, relative humidity, air and soil temperature influence persistence of EPNs in the soil (Mrácek et al., 2005). Natural habitats present a higher probability of occurrence of native species, serving as an important source in relation to biodiversity and use in biocontrol (Stock & Gress, 2006). These species can be more suitable to mass release against local pests due to their adaptation to the local climate and other population regulators (Bedding, 1990). In addition, many countries consider that the introduction of exotic EPNs may result in negative impact on the native non-target organisms (Bathon, 1996; Dolinski & Moino Jr., 2006). The present study aimed to determine the

occurrence and prevalence of the genera Heterorhabditis and Steinernema in different areas of Rio Grande do Sul State (RS), Brazil, in relation to abiotic factors such as habitat, altitude, and soil (type, texture, and temperature).

Material and Methods

Soil sampling was done in the regions of Central Depression (CD), Central Plateau (CP), Coastal Plain (CTP), Medium Plain (MP) and South Plain (SP), comprising agronomic crops, pastures and native forests cultivated in RS, covering an area of

approximately 150.000 km2_.

Soil samples were collected by hoeing and shoveling to a depth of 15 to 20 cm, and each sample consisted of ten 100 g-sub-samples. The sub-samples were mixed in buckets, placed in plastic bags and transported in Styrofoam boxes to keep them at 20 º to 25 ºC (Kaya & Stock, 1997). Each bag was labeled with: date, place, associated habitat, altitude, geographical position (latitude and longitude) and soil temperature taken at 20cm depth. The sampling route was planned to comprise the different RS regions, and the number of samples for each region was taken at random.

In the laboratory, soil samples were wetted with distilled water when necessary, up to field capacity. According to the modified methodology of Bedding & Akhurst (1975), each sample was divided into two plastic containers (14 x 14 x 7 cm), to which ten last instar larvae of Galleria mellonella (Lepidoptera: Pyralidae) were added. Containers were closed and inverted so that the insects were in full contact with soil. The containers were kept at room temperature (25 ± 3 ºC) and after seven days, the dead larvae with infection symptoms by EPNs were superficially disinfested with sodium hypochlorite at 0.1%, placed in a dry chamber (9 cm Petri dish with filter paper) and incubated in a germination chamber at 25 ºC, RH 70 ± 10 % and 12 h photophase. After three altitude. H. bacteriophora e S. rarum foram registrados ao nível do mar. Embora o número de amostras coletadas não fosse suficiente para descrever todas as espécies de NEPs ocorrentes no Estado, os dados obtidos deverão ser úteis em programas futuros de biocontrole de pragas locais.

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days, the larvae were transferred to modified White traps (White, 1927) for harvesting of infective juveniles (IJs). Parasitism was confirmed through the Koch’s postulates, and the nematodes were multiplied for five generations in G. mellonella larvae. The isolates were identified using the morphometrics of IJs and males (Nguyen & Smart, 1995) and molecular technique (Dolinski et al., 2008).

The sufficiency of samples was determined by collector curve or logarithmic regression analysis, from the cumulative curve of additional species adapted from Santana & Souto (2006). In order to determine the relationship of the abiotic factors with the incidence of EPNs, the percentage (P) of isolated EPNs for each observed variable [regions, habitats, altitudes and soils (type, granulometry and temperature)] was calculated through the relationship between the number of positive EPN samples and total number of samples. Also, the total percentage isolation (TP) was obtained through the relationship between the total positive samples with nematodes and the total collected samples, as follows: P = (number of positive samples in each variable / number of samples taken in that variable) x 100, and TP = 100 (total number of positive samples / total number of samples) x 100.

Results and Discussion

From the 121 soil samples collected, 19 (15.7 %) were EPN positive (Figure 1, Table 1). EPNs have been isolated all over the world and the available data of their prevalence, i.e., percentage of samples that are positive, can vary drastically, as pointed out and discussed by Hominick (2002). This variation is largely due to the fact that they are aggregated, rather than at random, in distribution, thus requiring a great sampling effort (= size of sample area, number of samples, size of samples) and the use of a combination of extraction techniques to provide that the losses may be kept to a minimum. The prevalence can also vary with time, as the existence of local dominant and comparatively rare EPN species is already well established; many samples are necessary to recover all species occurring in a determined region, if this is accepted as possible. Habitat type and other factors can also affect the prevalence. In Brazil, previous attempts to correlate the occurrence of EPNs with abiotic factors are not available.

Steinernema rarum (36.8 %) and Heterorhabditis bacteriophora (31.6%) were the most common species followed by Steinernema sp. (10.4 %), H. amazonensis (5.2 %), S. feltiae (5.2 %), S. glaseri (5.2 %), and S. riobrave (5.2 %). Two Steinernema isolates could not be

Table 1 - Occurrence of entomopathogenic nematodes in different regions of Rio Grande do Sul State, Brazil.

1_{Total percentage.} Regions Central Depression 8 2 25.00 1 0 0 0 0 0 1 Central Plateau 4 1 25.00 0 0 1 0 0 0 0 Coastal Plain 43 2 6.97 1 0 1 0 0 0 0 Medium Plain 38 13 34.20 4 1 5 1 1 0 1 South Plain 3 1 33.33 0 0 0 0 0 1 0 Others 25 0 0.00 0 0 0 0 0 0 0 Total 121 19 - 6 1 7 1 1 1 2 TP1 _15.7 _31.6 _5.2 _36.8 _5.2 _5.2 _5.2 _10.4 T otal n umber of samples T otal n umber of positi ve samples

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identified as they did not multiply in G. mellonella. This study revealed for the first time in Brazil the occurrence of S. feltiae, S. rarum and S. riobrave. S. rarum was first isolated in the Province of Córdoba, Argentina (Doucet, 1986). H. bacteriophora is considered cosmopolitan (Hominick, 2002) and has been found in other regions of Brazil, such as in the states of Pernambuco (Poinar, 1990) and São Paulo (Vasconcelos et al. 2004). S. glaseri was first recorded in Brazil in the state of São Paulo, as a parasite of eggs of Migdolus fryanus, a sugarcane soil pest (Pizano et al., 1985). H. amazonensis, so far only found in Brazil, it was originally described from the state of Amazonas (Andaló et al., 2006).

The EPNs were found in all parts of RS, but most frequently in the north-northeast region (MP – Medium Plateau and Above) (Table 1). Despite the fact that Brazil is considered a tropical country, RS has a temperate climate, favoring the occurrence of

steinernematids (Salas-Luévano, 2001), as also reported in Ireland, where they form 98 % of the insect-parasitic nematofauna (Griffin et al., 1991). Heterorhabditids prevail most in tropical regions (Roman & Beavers, 1982; Hominick & Briscoe, 1990; Hara et al. 1991). In Brazil, several authors mentioned a higher number of heterorhabditids adapted to mild and hot environments, for example in the states of Amazonas (Andaló et al., 2006), Minas Gerais (Molina-Acevedo et al., 2005), Rio de Janeiro (C. Dolinski, UENF, personal observation), Rondônia (Dolinski et al., 2008), and São Paulo (Fowler, 1988).

Entomopathogenic nematodes were present in 7.69 % to 18.18 % of samples from vegetables, forest, native pastures, fruit trees and corn crop, and between 21.42 % and 25.00 % of samples within forest, soybean and tobacco (Table 2). Mrácek et al. (1999) observed higher frequency of EPNs (> 50%) in areas with fruit trees, forests and pastures. In agricultural Figure 1 - Geographic location of the sites from where entomopathogenic nematodes were isolated in Rio Grande do Sul State,

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habitats, the occurrence was lower than 50 % in corn crop and pasture. Our results differed from the ones obtained by Mrácek et al. (2005), regarding to the Czech Republic, who observed a higher prevalence in fruit trees in relation to native pasture. The same authors observed low prevalence (10 – 32 %) in managed apple orchard, cereals and managed pasture; intermediate prevalence (36.4 – 50 %) in corn crop, vineyard, and acacia and cowpea plant; high prevalence (57 – 80 %) in non-managed apple orchard and native pasture. In the same country, a higher frequency was observed of S. feltiae (35.9 %), prevailing in pasture and forests (Mrácek et al., 1999); this was also seen in the UK and Holland, where the species occupied pasture and refuge areas (Hominick et al., 1995).

Regarding the soil type, a high incidence of EPNs was observed in Vertisols (udert) and Alisols (udult) (100 %), followed by Nitosols (kandiudult) (33.33 %) and Latosols (undox) (32.14 %) (Table 3). As for

granulometry, heterorhabditids (85.7 %) and steinernematids (58.4 %) were predominant in sandy soils (68.4 %) (Table 4). Due to the large diversity, the soil type has not been a factor related to the prevalence of EPNs. However, in agreement with the results of this study, in the Brazilian state of Minas Gerais, Molina-Acevedo et al. (2005) observed higher EPN prevalence (66.6 %) in soil with higher sand proportion (yellow-red Latosol) than in soil with high clay content (purple Latosol, dark-red Latosol and yellow-red Podzolic). Similarly, higher prevalence was observed in light soils in the Czech Republic (Mrácek et al., 2005), in Hawaii (Hara et al., 1991) and Ireland (Blackshaw, 1988).

The isolated EPNs were grouped in three soil temperature ranges (Table 5). In the samples in which the temperature was below 20ºC (11.76 %) and above 25ºC (11.11 %) there was a higher prevalence of EPNs. S. feltiae was the only species isolated in the Table 2 - Occurrence of entomopathogenic nematodes as sampled in different crops of Rio Grande do Sul State, Brazil.

1_{Orange, apple and vine.}

2_{Splash, potato, pea, watermelon, strawberry, radish and cucumber.} 3_{Acacia, eucalyptus and pine.}

4_{Total percentage.}

T

otal n

umber of

samples

Number of positive samples % of positive samples H. bacteriophora H. amazonensis S. rarum S. feltiae S. glaseri S. riobrave Steinernema

sp

Crop

Rice 1 0 0.0 0 0 0 0 0 0 0

Oat 1 0 0.0 0 0 0 0 0 0 0

Sugarcane 1 0 0.0 0 0 0 0 0 0 0

Area under Fallow 15 0 0.0 0 0 0 0 0 0 0

Beans 3 0 0.0 0 0 0 0 0 0 0

Fruit trees 1 ₆ ₁ _16.66 ₀ ₁ ₀ ₀ ₀ ₀ ₀

Tobacco 4 1 25.00 0 0 1 0 0 0 0

Forest trees (generic) 8 1 12.50 1 0 0 0 0 0 0 Horticultural plants 2 ₁₃ ₁ _7.69 ₁ ₀ ₀ ₀ ₀ ₀ ₀

Wheat 3 2 66.66 1 0 1 0 0 0 0

Native pasture 18 3 16.66 0 0 2 0 0 1 0

Forest trees (identified) 3 ₁₄ ₃ _21.42 ₁ ₀ ₁ ₀ ₀ ₀ ₁

Soybean 12 3 25.00 1 0 1 0 0 0 1

Corn 22 4 18.18 1 0 1 1 1 0 0

Total 121 19 - 6 1 7 1 1 1 2

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-Table 3 - Occurrence of entomopathogenic nematodes in Rio Grande do Sul State, Brazil according to the different types of soil. 1_{Total percentage.} T otal n umber of samples

Number of positive samples % of positive samples (P) H. bacteriophora H. amazonensis S. rarum S. feltiae S. glaseri S. riobrave Steinernema

sp Soil type Argisoil 21 2 9.50 1 0 1 0 0 0 Latosoil 28 9 32.14 3 1 3 1 0 0 1 Luvisoil 9 1 11.11 0 0 0 0 0 1 0 Neosoil 35 4 11.42 1 0 3 0 0 0 0 Nitosoil 3 1 33.33 0 0 0 0 1 0 0 Vertisoil 1 1 100 1 0 0 0 0 0 0 Alisoil 1 1 100 0 0 0 0 0 0 1 Others 24 0 0 0 0 0 0 0 0 0 Total 121 19 - 6 1 7 1 1 1 2 TP1 _15.7

Table 4 - Soil granulometry and correspondent geographic location of the positive samples for entomopathogenic nematodes

(isolates) found in Rio Grande do Sul State, Brazil.

Entomopathogenic nematodes Isolate Soil granulometry Latitude Longitude

Sand(%) Silt (%) Clay (%)

H. bacteriophora RS33 55.3 23.8 20.9 -31º48’9,7’’ -52º25’5,8" RS56 62.9 21.7 12.8 -28º19’45" -51º05’17,0" RS57 63.7 25.0 11.4 -28º15’06,8" -51º29’46,4" RS58 63.1 26.2 11.7 -28º15’06,8" -51º29’46,4" RS88 37.3 22.8 39.8 -28º56’41,5" -53º38’54,4" RS107 67.0 19.5 13.5 -32º06’49,7" -53º08’02,6" H. amazonensis RS72 84.2 9.7 6.0 -30º15’39" -54º58’13,1" S. rarum RS47 60.0 18.9 21.1 -27º28’28,5" -52º54’13’7" RS55 35.5 17.1 47.4 -28º19’43,1" -51º05’17,8" RS70 51.0 29.0 20.0 -30º44’30,4" -55º06’11,6" RS89 68.6 19.2 12.5 -31º23’59,6" -52º40’48" RS90 59.0 17.6 23.4 -31º20’20,1" -52º52’32" RS102 59.6 27.4 12.9 -28º50’14" -52º51’36,9" RS106 96.0 0.4 4.4 -30º10’48" -50º12’35,9" S. feltiae RS76 57.4 31.2 11.3 -29º38’56,9" -54º40’40,1" S. glaseri RS38 8.3 21.2 70.5 -28º01’40" -52º14’00,6" S. riobrave RS59 6.9 22.5 70.6 -28º14’59,9" -51º30’16,1" Steinernema sp. RS69 17.5 56.3 26.2 -30º53’55,4" -54º51’31,7" RS92 18.2 50.8 31.0 -31º00’15,3" -52º58’49,1"

samples where the soil temperature was above 25 ºC. In general, the prevalence of Heterorhabditis sp. is negatively correlated to high temperatures and

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Table 5 - Occurrence of entomopathogenic nematodes in Rio Grande do Sul State, Brazil, in relation to the soil temperatures.

T

otal n

umber of

samples

sp Soil temperature (ºC) < 20 68 8 11.76 3 1 2 0 1 1 0 20 – 25 44 10 2.27 3 0 5 0 0 0 2 > 25 9 1 11.11 0 0 0 1 0 0 0 Total 121 19 - 6 1 7 1 1 1 2 TP1 _15.7

Table 6 - Occurrence of entomopathogenic nematodes found in Rio Grande do Sul State, Brazil, according to different altitudes.

T

otal n

umber of

samples

sp Altitude (m.s.l.) 1 – 100 42 2 4.76 1 0 1 0 0 0 0 100 – 200 29 5 17.24 1 1 1 1 0 0 1 200 – 300 7 2 28.57 0 0 1 0 0 0 1 300 – 400 9 2 22.22 1 0 1 0 0 0 0 400 – 500 5 0 0 0 0 0 0 0 0 0 500 – 600 0 0 0 0 0 0 0 0 0 0 600 – 700 14 3 21.42 0 0 2 0 1 0 0 700 – 800 2 0 0 0 0 0 0 0 0 0 800 – 900 6 5 83.33 3 0 1 0 0 1 0 900 – 1,000 0 0 0 0 0 0 0 0 0 0 1,000 – 1,100 3 0 0 0 0 0 0 0 0 0 Total 121 19 - 6 1 7 1 1 1 2 TP1 _15.7

in the population dynamics of EPNs, and whether they interfere or not in incidence (Hominick & Briscoe, 1990; Puza & Mrácek, 2005).

With regard to altitude, EPNs were more prevalent between 800 and 900 m (83.33 %) followed by 200 to 300 m (28.57 %), and at sea level from 1 to 100 m (4.76 %) only H. bacteriophora and S. rarum were found (Table 6). No influence of altitude on the occurrence

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steinernematids. In our study, S. riobrave occurred at 827 m high, but Stock & Gress (2006) recorded it at 1,400 m; in opposition, S. feltiae was only present in samples collected at low altitude, as also reported by Roman & Beavers (1982) in Puerto Rico.

The number of samples collected in the present study was not sufficient to represent all the EPNs that may occur in RS, as the collector curve (Figure 2) was

not asymptotic, based on Colwell et al. (2004).

Therefore, additional investigation dealing with the prevalence, abundance and other ecological aspects related to EPNs occurring in the Rio Grande do Sul State are strongly suggested.

Acknowledgements

The authors thank Dr. Márcio Voss from Embrapa Trigo, Passo Fundo (RS) Brazil for his help with soil sampling. C.R.C. Barbosa-Negrisoli received a scholarship from Conselho Nacional de Pesquisa e Desenvolvimento Tecnológico (CNPq).

Literature Cited

ANDALÓ, V., K.B. NGUYEN & A. MOINO Jr. 2006.

Heterorhabditis amazonensis n. sp. (Rhabditida:

Heterorhabditidae) from Amazonas, Brazil. Nematology, 8(6): 853-867.

BARBOSA-NEGRISOLI, C. R. C., NEGRISOLI JUNIOR, A. S., MOINO JUNIOR, A., & SILVA, P. H. S. 2007. Ocorrência e identificação de Nematóide Entomopatogênico do Gênero Heterorhabditis em solo cultivado com arroz e feijão-caupi no estado do Piauí. Embrapa Meio-Norte, Teresina (PI). Comunicado Técnico 194, 4 p.

BATHON, H. 1996. Impact of entomopathogenic nematodes on non-target hosts. Biocontrol Science Technology, 6: 421-434.

BEDDING, R. A. 1990. Logistics and strategies for introducing entomopathogenic nematodes technology in developing contries. In: Gaugler, R. & H. K. Kaya (ed) Entomopathogenic nematodes in Biological Control. CRC Press, Boca Raton, p. 77-98.

BEDDING, R. A. & R. J. AKHURST. 1975. A simple technique for the detection of insect parasitic rhabditid nematodes in soil. Nematologica, 21: 109-110. BLACKSHAW, R. P. 1988. Survey of insect-parasitic

nematodes in Northern Ireland. Annals of Applied Biology, 113: 561-565.

CAMPOS-HERRERA, R., M. ESCUER, S. LABRADOR, L. ROBERTSON, L. BARRIOS & C. GUTIÉRREZ. 2007. Distribution of the entomopathogenic nematodes from La Rioja (Northern Spain). Journal of Invertebrate Pathology, 95: 125–139.

COLWELL, R. K., C. X. MAO & J. CHANG. 2004. Interpolando, extrapolando y comparando las curvas de acumulación de especies basadas en su incidencia. Ecology, 85: 2717-2727.

DOLINSKI, C. & A. MOINO Jr. 2006. Utilização de nematóides entomopatogênicos nativos ou exóticos: o perigo das introduções. Nematologia Brasileira, 30 (2): 139-149.

DOLINSKI, C. M., F. L. KAMITANI, I. R. MACHADO & C. E. WINTER. 2008. Molecular and morphological characterization of heterorhabditid entomopathogenic nematodes from the tropical rainforest in Brazil. Memórias do Instituto Oswaldo Cruz, 103 (2): 150-159.

DOUCET, M. M. A. 1986. A new species of Neoaplectana Steiner, 1929 (Nematoda: Steinernematidae) from Córdoba, Argentina. Revue Nématologie, 9: 317. FOWLER, H.G. 1988. Occurrence and infectivity of

entomogenous nematodes in mole crickets in Brazil. International Rice Research Newsletter, 13: 34. Figure 2 - Sample sufficiency for entomopathogenic nematode isolated in Rio Grande do Sul State, Brazil.

y = 1.9194Ln(x) - 4.3879 R2= 0.7488 0 1 2 3 4 5 1 7 13 19 25 31 37 43 49 55 61 67 73 79 85 91 97 103 109 115 121 number of samples

Observed Log. (Observed)

(9)

Nematologia Brasileira 197 GRIFFIN, C.T., J. F. MOORE & M. J. DOWNES. 1991.

Occurrence of insect-parasitic nematodes in the Republic of Ireland. Nematologica, 37: 92-100.

HARA, A.H., R. GAUGLER, H.K. KAYA & L.M. LEBECK. 1991. Natural populations of entomopathogenic nematodes (Rhabditida: Heterorhabditidae, Steinernematidae) from the Hawaiian Islands. Environmental Entomology, 20(1): 211-216.

HOMINICK, W. M. 2002. Biogeography. In: GAUGLER, R. (ed) Entomopathogenic Nematology. CABI, Wallingford, p. 115-137.

HOMINICK, W.M. & A.P. REID. 1990. Perspectives on Entomopathogenic Nematology. In: GAUGLER, R & H. K. KAYA (ed). Entomopathogenic nematodes in Biological Control. CRC Press, Boca Raton, p. 327-349. HOMINICK, W.M. & B. R. BRISCOE. 1990. Occurrence of entomopathogenic nematodes (Rhabditida: Steinernematidae and Heterorhabditidae) in British soil. Parasitology, 100: 295-302.

HOMINICK, W.M., A.P. REID & B.R. BRISCOE. 1995. Prevalence and habitat specificity of steinernematid and heterorhabditid nematodes isolated during soil surveys of the UK and the Netherlands. Journal Helminthology, 69: 27-32.

KAYA, H.K. 1990. Soil Ecology. In: GAUGLER, R. & H.K. KAYA (ed) Entomopathogenic Nematodes in Biological Control. CRC Press, Boca Raton, p. 93-111. KAYA, H.K. & S.P. STOCK. 1997. Techniques in insect

nematology. In: LACEY, L.A. (ed). Manual of Techniques in Insect Pathology. Academic Press, San Diego, p. 281-322.

MRÁCEK, Z., S. BECVÁR & P. KINDLMANN. 1999. Survey of entomopathogenic nematodes from the families Steinernematidae and Heterorhabditidae (Nematoda: Rhabditida) in the Czech Republic. Folia Parasitologica, 46: 145-148.

MRÁCEK, Z.A., S.A. BEBVÁL, P.B.C. KINDLMANN, J. JERSÁKOVÁ. 2005. Habitat preference for entomopathogenic nematodes, their insect hosts and new faunistic records for the Czech Republic. Biological Control, 34: 27–37.

MOLINA-ACEVEDO, J.P., A. MOINO JR, R.S. CAVALCANTI, C. DOLINSKI & F.A. CARVALHO. 2005. Amostragem e avaliação de técnicas para isolamento de nematóides entomopatogênicos nativos obtidos em Lavras, Minas Gerais. Nematologia Brasileira, 29 (1): 17-23.

NGUYEN, K.B. & G.C. SMART Jr., 1995. Morphometrics of infective juveniles of Steinernema spp. and

Heterorhabditis bacteriophora (Nemata: Rhabditida).

Journal of Nematology, 27: 206-212.

PIZANO, M.A., M.M. AGUILLERA, A.R. MONTEIRO & L.C.C.B. FERRAZ. 1985. Incidência de Neoaplectana

glaseri parasitando ovo de Mygdolus fryanus. Nematologia

Brasileira, 9: 9-10.

POINAR Jr., G.O. 1990. Biology and taxonomy of Steinernematidae and Heterorhabditidae. In: GAUGLER, R. & H. K. KAYA (ed). Entomopathogenic Nematodes in Biological Control. CRC Press, Boca Raton, p. 23-58.

PRASAD, S.G., P.K. SINGH & H.R. RANGANATH. 2001. Population fluctuation of entomopathogenic nematode, Heterorhabditis sp. in South Andaman as influenced by weather parameters. Current Science, 80 (8): 923-924. PUZA, V. & Z. MRÁCEK. 2005. Seasonal dynamics of

entomopathogenic nematodes of the genera Steinernema and Heterorhabditis as a response to abiotic factors and abundance of insect hosts. Journal of Invertebrate Pathology, 89: 116-122.

ROMAN, J. & J.B. BEAVERS. 1982. A survey of Puerto Rican soils for entomogenous nematodes which attack

Diaprepes abbreviatus (L.). Journal of Agriculture

University of Puerto Rico, 67: 311-316.

SALAS-LUÉVANO, M.A. 2001. Existencia de nemátodos entomopatógenos (Steinerne-matidae y Heterorhabditidae) en agrosistemas del Cañon de Juchipila Zacatecas, México. Jornadas de Investigación Universidad Autónoma de Zacatecas, 5, México. SANTANA, J.A.S. & J.S. SOUTO. 2006. Diversidade e

estrutura fitossociológica da caatinga na Estação Ecológica do Seridó-RN. Rev. de Biologia e Ciências da Terra, 16: 232-242.

SHISHINIOVA, M., I. BUDUROVA & D. GRADINAROV. 1997. Contribution to fauna of entomopathogenic nematodes (Rhabditida: Steinernematidae, Heterorhabditidae) from Bulgaria. Biotechnology & Biotechnological Equipment, 11: 45–51.

STOCK, S.P. & J.C. GRESS. 2006. Diversity and phylogenetic relationships of entomopathogenic nematodes (Steinernematidae and Heterorhabditidae) from the Sky Islands of southern Arizona. Journal of Invertebrate Pathology, 92: 66-72.

TREONIS, A.M. & D.H. WALL. 2005. Soil nematodes and desiccation survival in the extreme arid environment of the Antarctic Dry Valleys. Integrative and Comparative Biology, 45: 741–750.

VASCONCELOS, V.O., J. FURLONG, G.M. FREITAS, C. DOLINSKI, M.M. AGUILLERA, R.C.D. RODRIGUES & M. PRATA. 2004. Steinernema glaseri Santa Rosa strain and Heterorhabditis bacteriophora CCA strain as biological control agents of Boophilus microplus (Acari: Ixodidae). Parasitology Research, 94: 201-206. WHITE, G.F. 1927. A method for obtaining infective