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Diversity and distribution of entomopathogenic nematodes (Steinernematidae, Heterorhabditidae) in South Africa
Journal of Invertebrate Pathology 102 (2009) 120–128
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Diversity and distribution of entomopathogenic nematodes (Steinernematidae, Heterorhabditidae) in South Africa Justin Hatting a,*, S. Patricia Stock b, Selçuk Hazir c a
South African Agricultural Research Council, Small Grain Institute, Private Bag X29, Bethlehem 9701, South Africa Department of Entomology, University of Arizona, Tucson, AZ 85721-0036, USA c Adnan Menderes University, Faculty of Arts and Science, Department of Biology, 09010 Aydin, Turkey b
a r t i c l e
i n f o
Article history: Received 23 March 2009 Accepted 9 July 2009 Available online 15 July 2009 Keywords: South Africa Insect-pathogenic nematodes Geographic regions Natural habitats Crops Survey
a b s t r a c t A total of 1506 soil samples from different habitats in seven geographic regions of South Africa were evaluated for the presence of entomopathogenic nematodes (EPN). Nematodes were isolated from 5% of the samples. Among the steinernematids, four Steinernema sp. were recovered including Steinernema khoisanae and three new undescribed species. Although steinernematids were recovered from both humid subtropical and semiarid regions, this family accounted for 80% of EPN recovered from the semiarid climate zones characterised by sandy, acidic soils. Eight isolates of S. khoisanae were recovered from the Western Cape province. One of the new undescribed steinernematids (Steinernema sp. 1) was recovered only from the Free State and KwaZulu-Natal provinces where humid subtropical conditions prevail and soils are generally less acidic with higher clay content. A high level of adaptation, however, was noted with Steinernema sp. 2, which was recovered from a wide range of soil conditions and habitats ranging from semiarid (Western Cape province) to humid subtropical (KwaZulu-Natal province). A third undescribed steinernematid, Steinernema sp. 3, seemed better adapted to heavier soils with more than 80% of isolates recovered from fruit orchards in the Free State province. Heterorhabditis bacteriophora was the only heterorhabditid recovered during this survey. This species was particularly prevalent in four provinces ranging from humid subtropical to semiarid regions. Isolation of EPN directly from insect cadavers included Steinernema sp. 2 and one H. bacteriophora from an unidentified white grub (Scarabaeidae) cadaver (i.e., dual infection) and H. bacteriophora from the black vine weevil, Otiorhynchus sulcatus. Ó 2009 Elsevier Inc. All rights reserved.
1. Introduction Entomopathogenic nematodes (EPN) in the families Steinernematidae and Heterorhabditidae are obligate parasites of mainly soil-inhabiting insects and have great potential as biological control agents of many insect pests. They are found in a variety of soil habitats, and the various species and isolates exhibit considerable variation in terms of host range, reproduction, infectivity and conditions for survival (i.e. temperature, soil moisture, etc.) (Gaugler and Kaya, 1990). Surveys for EPN have been conducted in temperate, subtropical and tropical regions (summarized by Hominick, 2002; Adams et al., 2006). To further advance the notion of using EPN as biological control agents in South Africa (Hatting and Kaya, 2001), locally-adapted species or isolates from native habitats need to be identified and their unique characteristics documented (Stock et al., 1999).
* Corresponding author. E-mail address: [email protected] (J. Hatting). 0022-2011/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.jip.2009.07.003
In South Africa, EPN have been recovered directly from insect hosts [i.e., maize beetle, Heteronychus arator (Harington, 1953)] as well as from soil samples collected in the provinces KwaZuluNatal (Spaull, 1988, 1990, 1991), Eastern Cape (Malan et al., 2006) and the Western Cape (Grenier et al., 1996, 1997; Malan et al., 2006, 2008; Nguyen et al., 2006). However, no systematic survey has been conducted on a regional scale to assess presence and diversity of EPN occurring in both agricultural and natural (non-disturbed) habitats in this country. In contrast to human modified areas, natural habitats are more likely uncontaminated by introduced nematodes and offer a better chance for finding native species (Stock et al., 1999; Stock and Gress, 2006). South Africa, a country with a surface area of more than 1.2-million square kilometers is unique in climatic and habitat diversity, ranging from subtropical in the East to semiarid in the West. Notably, South Africa’s landscape is dominated by a high plateau in the interior, surrounded by a narrow strip of coastal lowlands. Climatic conditions are therefore largely dependent on the altitude of the area in question and its proximity to either the Indian (East) or Atlantic (West) oceans. This may contribute to an equally diverse
J. Hatting et al. / Journal of Invertebrate Pathology 102 (2009) 120–128
distribution of EPN. The goal of this study was to survey EPN diversity in five provinces, representing 10 climate zones, ranging from subtropical in the East to semiarid in the West. A combination of classical and molecular taxonomy methods were considered to identify the recovered isolates. 2. Material and methods 2.1. Collection and isolation of nematodes A total of 1506 soil samples were collected in the provinces Western Cape, Free State, Gauteng, Mpumalanga, and KwaZuluNatal of South Africa between 2003 and 2005 (Fig. 1). Four major vegetation types were represented within these provinces, viz., Fynbos and Nama karoo (Western Cape), Grassland (Free State, Gauteng and KwaZulu-Natal), and Savanna (Mpumalanga and KwaZulu-Natal) (Table 1). Habitats selected for sampling included natural undisturbed grassland, vineyards, vegetables (beans and broccoli), fruit (cherry, apple, blueberry, raspberry, guava, banana and apricot), sugarcane, indigenous red tea (i.e., Rooibos, Aspalathus linearis), and wheat. Each soil sample (approximately 400 g) was a composite of 3–10 random sub-samples, taken at least 8–10 m apart, from the surface to a depth of 20 cm. All sub-samples were mixed together and placed in a paper bag and then a polyethylene bag to prevent water loss and kept in a cooler (ca. 15 °C) during transit to the laboratory as suggested by Kaya and Stock (1997). Samples collected by collab-
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orating institutions (in provinces other than the Free State) were shipped via courier service to ARC-Small Grain Institute (SGI) for processing. EPN were recovered from the soil samples using the insect-baiting method of Bedding and Akhurst (1975). Briefly, five Galleria mellonella (L.) larvae were placed in a 600 ml plastic container (80 mm diameter) and then filled 3/4 way with moistened soil from each sample. Containers were covered with a lid, turned upside down and kept at 21 °C in complete darkness. Soil samples were checked every 2–3 days for a total of 12 days. Cadavers with signs of EPN-infection, recognized by change in color (usually red/ purple for heterorhabditids, and ochre/brown/black for steinernematids), were removed and rinsed in sterile distilled water and individually placed in modified White traps (Kaya and Stock, 1997) for emergence of the infective-stage juveniles (IJs). Emerging nematodes were pooled for each sample and used to infect fresh G. mellonella larvae to confirm Koch’s postulates of pathogenicity and to obtain nematodes for identification and establishment of cultures. For most sampling sites, a portion of the soil (ca. 100 g) was combined and submitted to the Soil Analysis Laboratory of ARCSGI, Bethlehem (member of AgriLASA – Agri Laboratory Association of Southern Africa) for pH (KCl) and soil texture (only clay percentage was measured using a Bouyoucous Hydrometer) analysis. 2.2. Nematode identification A preliminary morphological diagnosis (i.e., IJ body size and male spicule/gubernaculum morphology) of the recovered isolates
Fig. 1. Map of South Africa showing distribution of EPN-positive sampling sites. N, Steinernema sp. 1; j, Steinernema sp. 2; €, Steinernema sp. 3; , S. khoisanae; and |, H. bacteriophora.
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was performed to sort them, according to their morphological characteristics, into similar species groups (Lopez-Nuñez et al., 2008). Morphological observations followed taxonomic criteria suggested by Stock and Kaya (1996) and Hominick et al. (1997). Briefly, 20 first-generation males and 20 IJs were randomly selected from different G. mellonella cadavers. Nematodes were examined live or heat-killed in 60 °C Ringer’s solution. The heatkilled nematodes were placed in triethanolamine-formalin (TAF) fixative (Kaya and Stock, 1997) and processed to anhydrous glycerin for mounting (Seinhorst, 1959). Observations were made on live and mounted specimens using an Olympus BX51 microscope equipped with differential interference contrast optics and digital image software (Analysis Image software, Soft Imaging System Corp. CA, USA). In general, and for all the nematode stages (firstgeneration males and infective juveniles), the following characters were analyzed: total length, greatest width, distance from anterior end to excretory pore, distance from anterior end to nerve ring, distance from anterior end to base of esophagus, tail length, width at anus/cloaca, and D% and E%. According to their morphology, isolates were placed into different species groups using taxonomic criteria suggested by Stock and Kaya (1996) and Stock et al. (2001). All isolates were molecularly characterized by analysis of rDNA sequences. Two nuclear genes, the large subunit (LSU) and internal transcribed spacer region (ITS) were considered for steinernematids and heterorhabditids, respectively. DNA extraction, PCR conditions and sequencing followed methods described by Stock et al. (2001). The resulting sequences were compared to a library of more than 60 EPN species (Stock Lab Database, Univ. Arizona). Phylogenetic analyses (maximum parsimony analysis) of LSU and ITS sequence data was done using PAUP v 4.0b (Swofford, 2002) following criteria described by Stock et al. (2001) and Stock and Gress (2006).
3. Results 3.1. Isolation and identification of recovered nematode isolates From a total of 1506 samples 79 (5.2%) were EPN-positive. Of these, 44 samples contained steinernematids (55.7%) and 35 held heterorhabditids (44.3%). EPN recovery varied also among the different climate zones with 13% recovered from semiarid zones compared to 87% from subtropical zones. Heterorhabditis isolates were identified as Heterorhabditis bacteriophora (Poinar, 1976). All isolates of this species had morphological traits of this species including IJ body size (average 580 lm), male tail (number and arrangement of bursal rays) and spicules morphology. Furthermore, analysis of ITS rDNA sequences also depicted these strains as H. bacteriophora (100% sequence similarity). This species was the most wide-spread, collected in four provinces (Free State, Western Cape, KwaZulu-Natal and Mpumalanga; Fig. 1) from a wide variety of habitats/crops (Table 2). This species, however, did seem less adapted to semiarid winter rainfall conditions with only a single isolate (SGI 151) recovered from this climate zone (i.e., Clanwilliam, Western Cape province). Moreover, the pine forest habitat (cool/shaded/with potentially higher organic matter and moisture content) may have served to off-set the rather adverse soil conditions normally encountered in this area, in support of H. bacteriophora survival. Two isolates of this species were recovered directly from weevil larvae Otiorhynchus sulcatus (Coleoptera: Curculionidae) in apple orchards around Bethlehem, Free State province. Interestingly, an isolate of an unidentified steinernematid (Steinernema sp. 2, see below) and H. bacteriophora (SASRI 426a and b, respectively) was recovered from a single (unidentified) white grub (Scarabaeidae) cadaver at the Weltevreden locality near Dalton, KwaZulu-Natal.
Table 1 Number of soil samples collected per climate zone/vegetation type by collaborating institutions. Institution
Climate zonea from which EPN-positive soil samples collected
Vegetation typeb
Total # samples/ EPN+
Rooibos Ltd.
Zone 1. Humid subtropical with winter rainfall and warm (warmest month >22 °C) Zone 2. Semiarid with winter rainfall and cool (average annual temperature <18 °C) Zone 3. Semiarid with winter rainfall and warm (average annual temperature >18 °C) Zone 4. Semiarid with annual rainfall and cool (average annual temperature <18 °C) Zone 5. Semiarid with summer rainfall and cool (average annual temperature <18 °C) Zone 6. Humid subtropical with winter rainfall and cool (warmest month <22 °C) Zone 7. Humid subtropical with summer rainfall and cool (warmest month <22 °C) Zone 8. Humid subtropical with annual rainfall and warm (warmest month > 22 °C) Zone 9. Humid subtropical with annual rainfall and cool (warmest month <22 °C) Zone 10. Humid subtropical with summer rainfall and warm (warmest month >22 °C) Zone 11. Humid subtropical with annual rainfall and cool (warmest month <22 °C) Zone 8. Humid subtropical with annual rainfall and warm (warmest month >22 °C) Zone 10. Humid subtropical with summer rainfall and warm (warmest month >22 °C)
Fynbos
603/22
ARCc-Infruitec-Nietvoorbij
ARC-Small Grain Institute and ARC-Vegetable and Ornamental Plant Institute South African Sugar Research Institute
University of KwaZulu-Natal, Pietermaritzburg ARC-Institute for Tropical and Subtropical Crops Total no. samples a
Nama karoo
Fynbos Grassland
336d/39
Savanna
567/15
Grassland Savanna
1506/76
Agrometeorology Staff, 2004: Koppen Climate Zones for South Africa. Agricultural Research Council Institute for Soil, Climate and Water, Pretoria. b According to: Mucina, L., Rutherford, M.C. and Powrie, L.W., 2005. Vegetation Map of South Africa, Lesotho and Swaziland, 1:1 000 000 Scale Sheet Maps, South African National Biodiversity Institute, Pretoria. ISBN: 1-919976-22-1. c Agricultural Research Council. d No positive samples from 168 collected by ARC-Vegetable and Ornamental Plant Institute (ARC-VOPI; Gauteng province).
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Preliminary morphological observations (IJ body size and male spicule/gubernaculum morphology) assisted in placing all other isolates into different Steinernema sp. groups. Recovered steinernematid isolates represent four different species, including Steinernema khoisanae and three new undescribed taxa, hereafter referred to as Steinernema sp. 1, Steinernema sp. 2 and Steinernema sp. 3. Notably, steinernematids accounted for 80% of EPN recovered from the semiarid zones characterised by sandy, acidic soils (average pH 4.5 ± 0.5 and clay content of 4.2 ± 1.3%). Isolation of S. khoisanae was restricted to localities in the Western Cape province (i.e., winter rainfall region; Fynbos vegetation type). These isolates presented morphological similarities in their IJ body length and male spicule morphology to S. khoisanae, a species previously reported in this country (Nguyen et al., 2006). Interpretation of 28S rDNA sequence data further confirmed these isolates as S. khoisanae. However, it should be noted that sequences generated in this study for S. khoisanae strains differed only in one base pair with that of the type strain deposited in GenBank (accession no. DQ314289). Lack of access to original electropherographs for this sequence impeded further comparisons. However, we consider the existing basepair variation between the sequences generated from this study and that deposited in GenBank, may be due to editing errors of electropherographs of the latter sequence (accession no. DQ314289). Morphological examination of key diagnostic traits including male tail morphology, arrangement and number of genital papillae, size of spicules and IJ total body length, maximum width, excretory pore location, tail length and values of D% and E%, provided further evidence to confirm identity of recovered isolates. Steinernema sp. 1 was recovered from five locations around the towns of Bethlehem, Fouriesburg, Mt. Edgecombe, Umhlanga Rocks and Stanger. Notably, these localities were all situated within humid subtropical climate zones in the Free State and KwaZulu-Natal provinces. A total of 10 isolates were collected; three of which were recovered from natural habitats including grasslands and mixed weed fields, and the remaining isolates from ploughed fields, grain and sugarcane (see Table 2). Eleven of the recovered Steinernema strains were considered identical to each other based on both morphological and molecular observations. These isolates were considered to be members of a new undescribed species, Steinernema sp. 2. Most isolates (total of seven isolates) were collected in the locality of Clanwilliam, Western Cape province. This species seems well adapted to a wide range of climate conditions ranging from semiarid (Fynbos or predominantly evergreen sclerophyll plants; isolate SGI 146) to humid subtropical (Savanna; isolate SASRI 356). This species was also associated with a variety of habitats including Rooibos, lettuce, vineyards, wheat and grasslands. As mentioned above, a dual infection of a white grub (Coleoptera: Scarabaeidae) cadaver yielded one isolate of this species (SASRI 426a) and an isolate of H. bacteriophora (SASRI 426b). Steinernema sp. 3 was recovered from both semiarid and humid subtropical climates, although most isolates (>90%) were recovered from soils collected in the Bethlehem and Fouriesburg areas (Free State province) (Table 2). Dominant in these areas are the heavier Avalon and Pinedene soil forms with a predominantly fine sandy loam to sandy clay loam texture (Soil Classification Working Group, 1977). A total of eleven isolates were collected; nine from the above-mentioned orchards, one from a grassland (Bethlehem area) and one from a Rooibos field in the Western Cape province. A detailed morphological analysis, including DIC and SEM microscopy, together with cross-hybridisation tests is currently underway to formally describe all new EPN species recovered in this study.
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3.2. Phylogenetic relationships of new Steinernema sp. Maximum parsimony (MP) analysis of 28S rDNA sequences placed all three new Steinernema sp. in the glaseri-clade also known as clade V (Spiridinov et al., 2004; Nadler et al., 2006). This phylogenetic group encompasses species with large body infective juveniles (generally more than 1000 lm long) and includes 12 species: Steinernema glaseri, Steinernema khoisanae, Steinernema hermaphroditum, Steinernema scarabaei, Steinernema sangi, Steinernema cubanum, Steinernema diaprepesi, Steinernema puertoricense, Steinernema boemarei, Steinernema longicaudum, Steinernema karii and Steinernema arenarium. MP analysis yielded nine equally parsimonious trees with a tree length of 1267 steps and a consistency index (CI) of 0.57 (Fig. 2). Affiliation of each isolate into their corresponding species group was well supported by bootstrap resampling, providing further evidence for their distinctiveness as separate species. As mentioned above, analysis of ITS rDNA sequences for all Heterorhabditis isolates recovered placed them as members of the bacteriophora-group. One equally parsimonious tree was obtained with a tree length of 703 steps and a consistency index (CI) of 0.78 (Fig. 3). The bacteriophora-group currently encompasses two species Heterorhabditis georgiana and H. bacteriophora.
4. Discussion The present study represents the first systematic survey of indigenous EPN species in South Africa. Seventy-six viable EPN isolates were recovered from 1506 soil samples collected from five provinces throughout the country. These regions are quite distinct in geography and habitat diversity with typical soil forms ranging from very sandy texture (e.g., Clovelly and Fernwood forms from the West Coast of South Africa) to sandy clay loam texture (e.g., Avalon and Hutton forms from the Free State and KwaZulu-Natal, respectively) (Soil Classification Working Group, 1977). Notably, Steinernema sp. 2 and S. khoisanae were particularly tolerant of sandy, semiarid conditions with a strain of last mentioned (ROOI 334) being recovered from a locality with very low pH (3.9) and clay content (3% clay; ‘coarse sand’ soil texture). Results from this study also highlight the importance of conducting a more intensive EPN survey in other natural areas and geographic regions of South Africa. Although EPN-positive samples represented only 5% of all collected samples, the fact that five species, one Heterorhabditis and four Steinernema sp. were collected, underscores the incredible richness of EPN fauna in this country. Of all recovered species, only two, H. bacteriophora and Steinernema sp. 2 were found in association with insect hosts. The natural occurrence of these nematode species from these two important agricultural pests should be noted and their potential use as biocontrol agents further investigated. Interestingly, all recovered Steinernema sp. belong to the glaserigroup, which comprises species with IJs with large bodies (>1000 lm). Within this group four species groups were clearly depicted. Ten isolates (SASRI 186, SASRI 198, SASRI 244, SASRI 324, SGI 6, SGI 12, SGI 35, SGI 58, SGI 60 and SGI 82) clustered together representing a new unnamed sp., Steinernema sp. 1. All isolates belonging to this species, were collected in the provinces of KwaZulu-Natal and Free State. Another set of 11 isolates (ROOI 343, ROOI 352, ROOI 369, ROOI 374, SASRI 356, SASRI 426A, SGI 145B, SGI 146, SGI 148, ROOI 299 and INF 23) were considered members of another clade, that represents Steinernema sp. 2. These isolates were collected in the provinces of KwaZulu-Natal, Western Cape and Free State. Another set of 11 isolates (SGI 21, SGI 28, SGI 33, SGI 37, SGI 38, SGI 39, SGI 41, SGI 42, SGI 45, SGI 47 and SGI 152) were considered members of another clade that represents
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Isolate name
Genus
Species
Province/town
Locality
Climate zone (cf. Table 1)
Habitat/insect
Soil pH/clay content (%)
SGI SGI SGI SGI SGI SGI SGI SGI SGI SGI SGI SGI SGI SGI SGI SGI SGI SGI SGI SGI SGI SGI SGI SGI SGI SGI SGI SGI SGI SGI SGI SGI SGI SGI SGI SGI SGI SGI SGI
Steinernema Steinernema Steinernema Heterorhabditis Steinernema Heterorhabditis Heterorhabditis Steinernema Steinernema Heterorhabditis Steinernema Steinernema Steinernema Heterorhabditis Steinernema Steinernema Heterorhabditis Steinernema Heterorhabditis Steinernema Heterorhabditis Steinernema Steinernema Steinernema Heterorhabditis Heterorhabditis Heterorhabditis Steinernema Steinernema Steinernema Heterorhabditis Steinernema Heterorhabditis Heterorhabditis Heterorhabditis Heterorhabditis Heterorhabditis Heterorhabditis Heterorhabditis
new sp. 1 new sp. 1 new sp. 3 bacteriophora new sp. 3 bacteriophora bacteriophora new sp. 3 new sp. 1 bacteriophora new sp. 3 new sp. 3 new sp. 3 bacteriophora new sp. 3 new sp. 3 bacteriophora new sp. 3 bacteriophora new sp. 3 bacteriophora new sp. 1 new sp. 1 new sp. 1 bacteriophora bacteriophora bacteriophora new sp. 2 new sp. 2 new sp. 2 bacteriophora new sp. 3 bacteriophora bacteriophora bacteriophora bacteriophora bacteriophora bacteriophora bacteriophora
Free State/Bethlehem Free State/Bethlehem Free State/ Bethlehem Free State/Bethlehem Free State/Fouriesburg Free State/Fouriesburg Free State/Fouriesburg Free State/Fouriesburg Free State/Fouriesburg Free State/Fouriesburg Free State/Fouriesburg Free State/Fouriesburg Free State/Fouriesburg Free State/Fouriesburg Free State/Fouriesburg Free State/Fouriesburg Free State/Fouriesburg Free State/Fouriesburg Free State/Fouriesburg Free State/Fouriesburg Free State/Fouriesburg Free State/Bethlehem Free State/Bethlehem Free State/Bethlehem Free State/Fouriesburg Free State/Fouriesburg Free State/Fouriesburg Western Cape/Clanwilliam Western Cape/Clanwilliam Free State/Bethlehem Western Cape/Clanwilliam Western Cape/Clanwilliam Free State/Bethlehem Free State/Bethlehem Free State/Bethlehem Free State/Bethlehem Free State/Bethlehem Free State/Fouriesburg Free State/Fouriesburg
ARC-SGI ARC-SGI Utopia Utopia Denmar Denmar Denmar Denmar Denmar Denmar Denmar Denmar Denmar Denmar Denmar Denmar Denmar Denmar Denmar Denmar Denmar ARC-SGI ARC-SGI ARC-SGI Lone tree farm Lone tree farm Lone tree farm Zeekoevlei Zeekoevlei ARC-SGI Nuwedam Nuwedam OTK OTK OTK OTK OTK Lone tree farm Lone tree farm
7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 2 2 7 3 3 7 7 7 7 7 7
Ploughed field (ex wheat) Ploughed field (ex wheat) Grassland Broccoli Blueberry Blueberry Blueberry Cherry Cherry Cherry Cherry Cherry Cherry Cherry Cherry Cherry Cherry Cherry Blueberry Blueberry Blueberry Grain Grain Grain Apples (Fuji) Apples (Sundowner) Apples (Breyburn) Rooibos Fallow field (ex Rooibos) Ploughed field (ex wheat) Pine Forest Rooibos Apples (Braeburn) Apples/Otiorhynchus sulcatus Apples (Braeburn) Apples (Braeburn) Apples (Braeburn) Apples Apples
5.3/18 5.0/20 5.0/12 4.9/11 5.9/9 5.9/9 5.9/9 6.0/16 6.0/16 6.0/16 6.0/16 6.0/16 6.0/16 6.0/16 6.0/16 6.0/16 6.0/16 6.0/16 4.6/9 4.6/9 4.6/9 5.1/20 5.1/20 5.1/20 6.6/NA 6.1/10 6.4/14 4.4/4 4.1/4 4.2/4 4.2/4 4.5/4 5.3/10 5.3/10 5.3/10 5.7/9 4.6/12 6.5/NA 6.5/NA
6 12 21 22 28 29 32 33 35 36 37 38 39 40 41 42 43 45 46 47 50 58 60 82 90b 91a 96d 145b 146 148 151 152 160 161 164 165 166 170 171
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Table 2 Sample data on soils yielding EPN.
Heterorhabditis Heterorhabditis Heterorhabditis Heterorhabditis Heterorhabditis Heterorhabditis Heterorhabditis Heterorhabditis Steinernema Steinernema Steinernema Steinernema Steinernema Steinernema Steinernema Steinernema Steinernema Steinernema Steinernema Steinernema Steinernema Heterorhabditis Steinernema Steinernema Heterorhabditis Steinernema Steinernema Steinernema Steinernema Heterorhabditis Heterorhabditis Steinernema Heterorhabditis Heterorhabditis Steinernema Heterorhabditis Heterorhabditis Heterorhabditis Steinernema Heterorhabditis
bacteriophora bacteriophora bacteriophora bacteriophora bacteriophora bacteriophora bacteriophora bacteriophora khoisanae khoisanae khoisanae khoisanae khoisanae khoisanae new sp. 2 khoisanae khoisanae new sp. 2 new sp. 2 new sp. 2 new sp. 2 bacteriophora new sp. 1 new sp. 1 bacteriophora new sp. 1 new sp. 1 new sp. 2 new sp. 2 bacteriophora bacteriophora new sp. 2 bacteriophora bacteriophora unknownb bacteriophora bacteriophora bacteriophora unknownb bacteriophora
Free State/Fouriesburg Free State/Fouriesburg Free State/Fouriesburg Free State/Fouriesburg Free State/Bethlehem Free State/Bethlehem Free State/Bethlehem Free State/Bethlehem Western Cape/Clanwilliam Western Cape/Clanwilliam Western Cape/Clanwilliam Western Cape/Clanwilliam Western Cape/Clanwilliam Western Cape/Clanwilliam Western Cape/Clanwilliam Western Cape/Clanwilliam Western Cape/Clanwilliam Western Cape/Clanwilliam Western Cape/Clanwilliam Western Cape/Clanwilliam Western Cape/Clanwilliam KwaZulu-Natal/Mt Edgecombe KwaZulu-Natal/Mt Edgecombe KwaZulu-Natal/Mt Edgecombe KwaZulu-Natal/Stanger KwaZulu-Natal/Umhlanga Rocks KwaZulu-Natal/Stanger KwaZulu-Natal/Umhlanga Rocks KwaZulu-Natal/Dalton KwaZulu-Natal/Dalton Western Cape/Stellenbosch Western Cape/Stellenbosch Western Cape/Stellenbosch Western Cape/Hex River Valley Western Cape/De Doorns Western Cape/Stellenbosch Western Cape/Stellenbosch Mpumalanga/Hazyview Mpumalanga/Hazyview KwaZulu-Natal/Pietermaritzburg
Lone tree farm Lone tree farm Lone tree farm Lone tree farm Stead Stead Stead Stead Onder- Brakvlei Ysterfontein Paardekop Paardekop Paardekop Bergendal Bergendal Arbeidsend Arbeidsend De Berg De Berg Elandsfontein Elandsfontein SASRI SASRI Illovo sugar park Kearsney Umhlanga Kearsney Umhlanga Weltevreden Weltevreden Nietvoorbij Nietvoorbij Nietvoorbij Werda De Vlei Nietvoorbij Nietvoorbij Burgershall Burgershall University of KwaZulu-Natal
7 7 7 7 7 7 7 7 2 2 1 1 1 1 1 2 2 1 1 1 1 8 8 8 10 8 10 8 11 11 6 6 6 5 4 6 6 10 10 8
Apples Apples Apples Apples/Otiorhynchus sulcatus Raspberries Raspberries Raspberries Apples Ploughed field (ex Rooibos) Fallow field (ex Rooibos) Fallow field Rooibos Fallow field Grassland Apricot Rooibos Rooibos Lettuce Vineyard Grassland Fallow field Sugarcane Grassland Grassland Guava Sugarcane Mixed weeds Sugarcane Sugarcane/white gruba Sugarcane/white gruba Vineyard Vineyard Vineyard Vineyard Vineyard Beans Beans Banana Citrus Ag Fac garden plots
SGI = ARC-Small Grain Institute; ROOI = Rooibos Ltd.; SASRI = South African Sugar Research Institute; INF = ARC-Infuitec-Nietvoorbij; ITSC = ARC-Institute for Tropical and Subtropical Crops. a Dual infection of an unidentified white grub larva. b Nematodes did not multiply.
6.5/NA 6.5/NA 6.5/NA 6.5/NA NA/NA NA/NA NA/NA NA/NA 4.3/3 4.2/6 6.6/3 4.4/3 5.0/5 4.2/5 6.2/4 3.9/3 4.9/4 5.8/3 6.4/3 6.0/3 4.6/5 7.3/5 6.6/4 4.9/3 4.6/4 4.9/4 4.9/4 7.9/4 NA/NA NA/NA 5.5/4 4.1/8 5.3/3 5.6/3 5.0/7 5.7/7 5.4/7 NA/NA NA/NA 6.1/9
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SGI 172 SGI 173 SGI 174 SGI 175 SGI 178 SGI 179 SGI 180 SGI 181 ROOI 161 ROOI 229 ROOI 275 ROOI 277 ROOI 283 ROOI 293 ROOI 299 ROOI 334 ROOI 336 ROOI 343 ROOI 352 ROOI 369 ROOI 374 SASRI 75 SASRI 186 SASRI 198 SASRI 199 SASRI 244 SASRI 324 SASRI 356 SASRI 426a SASRI 426b INF 16 INF 23 INF 29 INF 61 INF 73 INF 79 INF 88 ITSC 36 ITSC 106 UNIV 17
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Fig. 2. Strict consensus (MP analysis) of 28S rDNA sequences tree showing phylogenetic relationships of Steinernema sp. Tree length = 1267. Consistency index (excluding uninformative characters = 0.57). Numbers above nodes indicate bootstrap values.
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Fig. 3. Strict consensus tree (MP analysis) of ITS rDNA sequences indicating evolutionary relationships of Heterorhabditis sp. Tree length = 703. Consistency index (excluding uninformative characters = 0.78). Numbers above nodes indicate bootstrap values.
Steinernema sp. 3. These isolates were collected in the Free State and Western Cape provinces. Eight isolates (ROOI 161, ROOI 229, ROOI 275, ROOI 277, ROOI 283, ROOI 293, ROOI 334 and ROOI 336) were collected in the Western Cape and identified as S. khoisanae, a species originally collected in three localities in this province (Malan et al., 2006; Nguyen et al., 2006). In this study, S. khoisanae was isolated from different localities around Clanwilliam in the Western Cape, indicating this species is widely distributed in this province. H. bacteriophora isolates were collected in four provinces, including the Western Cape, Free State, KwaZulu-Natal and
Mpumalanga. This species was probably recovered for the first time in KwaZulu-Natal by Spaull (1991), and later described by Grenier et al. (1996, 1997). Malan et al. (2006) also isolated H. bacteriophora in the Western Cape. However, this species had not been reported from the Free State and Mpumalanga before, expanding its geographic distribution. These native and novel strains and species provide a new alternative for their consideration in biological control and/or integrated pest management programs in South Africa. Various studies are currently underway to assess biological traits and host suitability of several of these isolates.
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Acknowledgments We acknowledge the following field collaborators responsible for collecting and sending soil samples/EPN-infected cadavers to ARC-SGI (order as per Table 1): Mr. Johan Brand (Rooibos Ltd.), Mr. Fanie van der Merwe and Dr. Pia Addison (formerly ARC-Infruitec-Nietvoorbij), Dr. Astrid Jankielsohn (ARC-SGI), Dr. Diedrich Visser (ARC-VOPI), Dr. Des Conlong (SASRI), Dr. Ray Miller (University of KZN, Pietermaritzburg), and Dr. Schalk Schoeman (ARC-ITSC). For technical assistance rendered: Me Gloria Macucwe, Me Palesa Dihemo, Me Hannetjie Jooste (formerly ARC-SGI), Me Tshima Ramakuwela, Me Lettie Opperman and Mr. Amos Mosia (ARC-SGI). We thank Me Noelien Somers and Dr. Dave Turner of the ARC-Institute for Soil, Climate and Water, Pretoria, for assistance with information regarding climate/vegetation and soil types, respectively. The project (#41404) was supported financially under Round 4 of the Innovation Fund administered by the National Research Foundation (NRF) of South Africa. We also acknowledge K. Plichta and V. Martinson (S.P. Stock laboratory) for assisting with molecular characterization of EPN. References Adams, B.J., Fodor, A., Koppenhöfer, H.S., Stackebrandt, E., Stock, S.P., Klein, M.G., 2006. Biodiversity and systematics of nematode–bacterium entomopathogens. Biol. Control 37, 32–49. Bedding, R.A., Akhurst, R.J., 1975. A simple technique for the detection of insect parasitic rhabditid nematodes in soil. Nematologica 21, 109–110. Gaugler, R., Kaya, H.K., 1990. Entomopathogenic nematodes in biological control. CRC Press, Boca Raton Florida. p. 365. Grenier, E., Bonifassi, E., Abad, P., Laumond, C., 1996. Use of species-specific satellite DNAs as diagnostic probes in the identification of Steinernematidae and Heterorhabditidae entomopathogenic nematodes. Parasitology 113, 483–489. Grenier, E., Castagnone-Sereno, P., Abad, P., 1997. Satellite DNA sequences as taxonomic markers in nematodes of agronomic interest. Parasitol. Today 13, 398–401. Harington, J.S., 1953. Observations on the biology, the parasites and the taxonomic position of the black maize beetle – Heteronychus sanctae-helenae Blanch. S. Afr. J. Sci. 50, 11–14. Hatting, J.L., Kaya, H.K., 2001. Entomopathogenic nematodes: prospects for biological control in South Africa. In: Olckers, T., Brothers, D.J. (Eds.), Proceedings of the 13th Entomological Congress, Pietermaritzburg, 2–5 July 2001. Entomological Society of Southern Africa, Hatfield, Pretoria, p. 28. Hominick, W.M., 2002. Biogeography. In: Gaugler, R. (Ed.), Entomopathogenic Nematology. CABI Publishing, Wallingford, UK, pp. 115–144. Hominick, W.M., Briscoe, B.R., Del Pino, F.G., Heng, J.A., Hunt, D.J., Kozodoy, E., Mrácek, Z., Nguyen, K.B., Reid, A.P., Spiridonov, S.E., Stock, S.P., Sturhan, D., Waturu, C., Yoshida, M., 1997. Biosystematics of entomopathogenic nematodes: current status, protocols and definitions. J. Helminthol. 71, 271–298.
Kaya, H.K., Stock, S.P., 1997. Techniques in insect nematology. In: Lacey, L.A. (Ed.), Manual of Techniques in Insect Pathology. Academic Press, San Diego, pp. 281– 322. Lopez-Nuñez, J.C., Plichta, K., Gongora-Botero, C.E., Stock, S.P., 2008. A new entomopathogenic nematode, Steinernema colombiense n. Sp. (Nematoda: Steinernematidae) from Colombia. Nematology 10, 561–574. Malan, A.P., Nguyen, K.B., Addison, M.F., 2006. Entomopathogenic nematodes (Steinernematidae and Heterorhabditidae) from the southwestern parts of South Africa. Afr. Plant Protect. 12, 65–69. Malan, A.P., Nguyen, K.B., De Waal, JY., Tiedt, L., 2008. Heterorhabditis safricana n. sp. (Rhabditida: Heterorhabditidae), a new entomopathogenic nematode from South Africa. Nematology 10, 381–396. Nadler, S.A., Bolotin, E., Stock, S.P., 2006. Phylogenetic relationships of Steinernema (Cephalobina, Steinernematidae) based on nuclear, mitochondrial, and morphological data. Syst. Parasitol. 63, 159–179. Nguyen, K.B., Malan, A.P., Gozel, U., 2006. Steinernema khoisanae n. sp. (Rhabditida: Steinernematidae), a new entomopathogenic nematode from South Africa. Nematology 8, 157–175. Poinar Jr., G.O., 1976. Description and biology of a new insect parasitic rhabditoid, Heterorhabditis bacteriophora n. gen. n. sp. (Rhabditida, Heterorhabditidae n. fam.). Nematologica 21, 463–470. Seinhorst, J.W., 1959. A rapid method for the transfer of nematodes from fixative to anhydrous glycerin. Nematologica 4, 67–69. Soil Classification Working Group, 1977. Soil Classification – A Binomial System for South Africa. Memoirs on the Agricultural Natural Resources of South Africa. ARC-Institute for Soil, Climate and Water, Pretoria. Spaull, V.W., 1988. A preliminary evaluation of entomopathogenic nematodes to control the African sugarcane stalk borer Eldana Saccharina (Lepidoptera: Pyralidae). Proc. S. Afr. Sugar Technol. Assoc. 62, 120–123. Spaull, V.W., 1990. Field tests to control the Pyralid, Eldana Saccharina, with an Entomogenous nematode, Heterorhabditis sp.. Proc. S. Afr. Sugar Technol. Assoc. 64, 103–106. Spaull, V.W., 1991. Heterorhabditis and Steinernema Species (Nematoda: Rhabditida) for the control of a sugar cane stalk borer in South Africa. Phytophylactica 23, 213–215. Spiridinov, S.E., Reid, A.P., Podrucka, K., Subbotin, S.A., Moens, M., 2004. Phylogenetic relationships within the genus Steinernema (Nematoda: Rhabditida) as inferred from analysis of sequences of the ITS-1-5. 8S-ITS2 region of rDNA and morphological features. Nematolology 6, 547– 566. Stock, S.P., Gress, J.C., 2006. Diversity and phylogenetic relationship of entomopathogenic nematodes (Steinernematidae and Heterorhabditidae) from the Sky Islands of southern Arizona. J. Invertebr. Pathol. 92, 66–72. Stock, S.P., Kaya, H.K., 1996. A multivariate analysis of morphometric characters of Heterorhabditis species (Nemata: Heterorhabditidae) and the role of morphometrics in the taxonomy of species of the genus. J. Parasitol. 82, 806– 813. Stock, P.S., Pryor, B.M., Kaya, H.K., 1999. Distribution of entomopathogenic nematodes (Steirnematidae and Heterorhabditidae in natural habitats in California, USA). Biodivers. Conserv. 8, 535–549. Stock, S.P., Campbell, J.F., Nadler, S.A., 2001. Phylogeny of Steinernema Travassos, 1927 (Cephalobina, Steinernematidae) inferred from ribosomal DNA sequences and morphological characters. J. Parasitol. 87, 877–889. Swofford, D.L., 2002. PAUP Phylogenetic Analysis Using Parsimony (and Other Methods). Sinauer Associates, Sunderland, Massachusetts.
Contents lists available at ScienceDirect
Journal of Invertebrate Pathology journal homepage: www.elsevier.com/locate/yjipa
Diversity and distribution of entomopathogenic nematodes (Steinernematidae, Heterorhabditidae) in South Africa Justin Hatting a,*, S. Patricia Stock b, Selçuk Hazir c a
South African Agricultural Research Council, Small Grain Institute, Private Bag X29, Bethlehem 9701, South Africa Department of Entomology, University of Arizona, Tucson, AZ 85721-0036, USA c Adnan Menderes University, Faculty of Arts and Science, Department of Biology, 09010 Aydin, Turkey b
a r t i c l e
i n f o
Article history: Received 23 March 2009 Accepted 9 July 2009 Available online 15 July 2009 Keywords: South Africa Insect-pathogenic nematodes Geographic regions Natural habitats Crops Survey
a b s t r a c t A total of 1506 soil samples from different habitats in seven geographic regions of South Africa were evaluated for the presence of entomopathogenic nematodes (EPN). Nematodes were isolated from 5% of the samples. Among the steinernematids, four Steinernema sp. were recovered including Steinernema khoisanae and three new undescribed species. Although steinernematids were recovered from both humid subtropical and semiarid regions, this family accounted for 80% of EPN recovered from the semiarid climate zones characterised by sandy, acidic soils. Eight isolates of S. khoisanae were recovered from the Western Cape province. One of the new undescribed steinernematids (Steinernema sp. 1) was recovered only from the Free State and KwaZulu-Natal provinces where humid subtropical conditions prevail and soils are generally less acidic with higher clay content. A high level of adaptation, however, was noted with Steinernema sp. 2, which was recovered from a wide range of soil conditions and habitats ranging from semiarid (Western Cape province) to humid subtropical (KwaZulu-Natal province). A third undescribed steinernematid, Steinernema sp. 3, seemed better adapted to heavier soils with more than 80% of isolates recovered from fruit orchards in the Free State province. Heterorhabditis bacteriophora was the only heterorhabditid recovered during this survey. This species was particularly prevalent in four provinces ranging from humid subtropical to semiarid regions. Isolation of EPN directly from insect cadavers included Steinernema sp. 2 and one H. bacteriophora from an unidentified white grub (Scarabaeidae) cadaver (i.e., dual infection) and H. bacteriophora from the black vine weevil, Otiorhynchus sulcatus. Ó 2009 Elsevier Inc. All rights reserved.
1. Introduction Entomopathogenic nematodes (EPN) in the families Steinernematidae and Heterorhabditidae are obligate parasites of mainly soil-inhabiting insects and have great potential as biological control agents of many insect pests. They are found in a variety of soil habitats, and the various species and isolates exhibit considerable variation in terms of host range, reproduction, infectivity and conditions for survival (i.e. temperature, soil moisture, etc.) (Gaugler and Kaya, 1990). Surveys for EPN have been conducted in temperate, subtropical and tropical regions (summarized by Hominick, 2002; Adams et al., 2006). To further advance the notion of using EPN as biological control agents in South Africa (Hatting and Kaya, 2001), locally-adapted species or isolates from native habitats need to be identified and their unique characteristics documented (Stock et al., 1999).
* Corresponding author. E-mail address: [email protected] (J. Hatting). 0022-2011/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.jip.2009.07.003
In South Africa, EPN have been recovered directly from insect hosts [i.e., maize beetle, Heteronychus arator (Harington, 1953)] as well as from soil samples collected in the provinces KwaZuluNatal (Spaull, 1988, 1990, 1991), Eastern Cape (Malan et al., 2006) and the Western Cape (Grenier et al., 1996, 1997; Malan et al., 2006, 2008; Nguyen et al., 2006). However, no systematic survey has been conducted on a regional scale to assess presence and diversity of EPN occurring in both agricultural and natural (non-disturbed) habitats in this country. In contrast to human modified areas, natural habitats are more likely uncontaminated by introduced nematodes and offer a better chance for finding native species (Stock et al., 1999; Stock and Gress, 2006). South Africa, a country with a surface area of more than 1.2-million square kilometers is unique in climatic and habitat diversity, ranging from subtropical in the East to semiarid in the West. Notably, South Africa’s landscape is dominated by a high plateau in the interior, surrounded by a narrow strip of coastal lowlands. Climatic conditions are therefore largely dependent on the altitude of the area in question and its proximity to either the Indian (East) or Atlantic (West) oceans. This may contribute to an equally diverse
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distribution of EPN. The goal of this study was to survey EPN diversity in five provinces, representing 10 climate zones, ranging from subtropical in the East to semiarid in the West. A combination of classical and molecular taxonomy methods were considered to identify the recovered isolates. 2. Material and methods 2.1. Collection and isolation of nematodes A total of 1506 soil samples were collected in the provinces Western Cape, Free State, Gauteng, Mpumalanga, and KwaZuluNatal of South Africa between 2003 and 2005 (Fig. 1). Four major vegetation types were represented within these provinces, viz., Fynbos and Nama karoo (Western Cape), Grassland (Free State, Gauteng and KwaZulu-Natal), and Savanna (Mpumalanga and KwaZulu-Natal) (Table 1). Habitats selected for sampling included natural undisturbed grassland, vineyards, vegetables (beans and broccoli), fruit (cherry, apple, blueberry, raspberry, guava, banana and apricot), sugarcane, indigenous red tea (i.e., Rooibos, Aspalathus linearis), and wheat. Each soil sample (approximately 400 g) was a composite of 3–10 random sub-samples, taken at least 8–10 m apart, from the surface to a depth of 20 cm. All sub-samples were mixed together and placed in a paper bag and then a polyethylene bag to prevent water loss and kept in a cooler (ca. 15 °C) during transit to the laboratory as suggested by Kaya and Stock (1997). Samples collected by collab-
121
orating institutions (in provinces other than the Free State) were shipped via courier service to ARC-Small Grain Institute (SGI) for processing. EPN were recovered from the soil samples using the insect-baiting method of Bedding and Akhurst (1975). Briefly, five Galleria mellonella (L.) larvae were placed in a 600 ml plastic container (80 mm diameter) and then filled 3/4 way with moistened soil from each sample. Containers were covered with a lid, turned upside down and kept at 21 °C in complete darkness. Soil samples were checked every 2–3 days for a total of 12 days. Cadavers with signs of EPN-infection, recognized by change in color (usually red/ purple for heterorhabditids, and ochre/brown/black for steinernematids), were removed and rinsed in sterile distilled water and individually placed in modified White traps (Kaya and Stock, 1997) for emergence of the infective-stage juveniles (IJs). Emerging nematodes were pooled for each sample and used to infect fresh G. mellonella larvae to confirm Koch’s postulates of pathogenicity and to obtain nematodes for identification and establishment of cultures. For most sampling sites, a portion of the soil (ca. 100 g) was combined and submitted to the Soil Analysis Laboratory of ARCSGI, Bethlehem (member of AgriLASA – Agri Laboratory Association of Southern Africa) for pH (KCl) and soil texture (only clay percentage was measured using a Bouyoucous Hydrometer) analysis. 2.2. Nematode identification A preliminary morphological diagnosis (i.e., IJ body size and male spicule/gubernaculum morphology) of the recovered isolates
Fig. 1. Map of South Africa showing distribution of EPN-positive sampling sites. N, Steinernema sp. 1; j, Steinernema sp. 2; €, Steinernema sp. 3; , S. khoisanae; and |, H. bacteriophora.
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was performed to sort them, according to their morphological characteristics, into similar species groups (Lopez-Nuñez et al., 2008). Morphological observations followed taxonomic criteria suggested by Stock and Kaya (1996) and Hominick et al. (1997). Briefly, 20 first-generation males and 20 IJs were randomly selected from different G. mellonella cadavers. Nematodes were examined live or heat-killed in 60 °C Ringer’s solution. The heatkilled nematodes were placed in triethanolamine-formalin (TAF) fixative (Kaya and Stock, 1997) and processed to anhydrous glycerin for mounting (Seinhorst, 1959). Observations were made on live and mounted specimens using an Olympus BX51 microscope equipped with differential interference contrast optics and digital image software (Analysis Image software, Soft Imaging System Corp. CA, USA). In general, and for all the nematode stages (firstgeneration males and infective juveniles), the following characters were analyzed: total length, greatest width, distance from anterior end to excretory pore, distance from anterior end to nerve ring, distance from anterior end to base of esophagus, tail length, width at anus/cloaca, and D% and E%. According to their morphology, isolates were placed into different species groups using taxonomic criteria suggested by Stock and Kaya (1996) and Stock et al. (2001). All isolates were molecularly characterized by analysis of rDNA sequences. Two nuclear genes, the large subunit (LSU) and internal transcribed spacer region (ITS) were considered for steinernematids and heterorhabditids, respectively. DNA extraction, PCR conditions and sequencing followed methods described by Stock et al. (2001). The resulting sequences were compared to a library of more than 60 EPN species (Stock Lab Database, Univ. Arizona). Phylogenetic analyses (maximum parsimony analysis) of LSU and ITS sequence data was done using PAUP v 4.0b (Swofford, 2002) following criteria described by Stock et al. (2001) and Stock and Gress (2006).
3. Results 3.1. Isolation and identification of recovered nematode isolates From a total of 1506 samples 79 (5.2%) were EPN-positive. Of these, 44 samples contained steinernematids (55.7%) and 35 held heterorhabditids (44.3%). EPN recovery varied also among the different climate zones with 13% recovered from semiarid zones compared to 87% from subtropical zones. Heterorhabditis isolates were identified as Heterorhabditis bacteriophora (Poinar, 1976). All isolates of this species had morphological traits of this species including IJ body size (average 580 lm), male tail (number and arrangement of bursal rays) and spicules morphology. Furthermore, analysis of ITS rDNA sequences also depicted these strains as H. bacteriophora (100% sequence similarity). This species was the most wide-spread, collected in four provinces (Free State, Western Cape, KwaZulu-Natal and Mpumalanga; Fig. 1) from a wide variety of habitats/crops (Table 2). This species, however, did seem less adapted to semiarid winter rainfall conditions with only a single isolate (SGI 151) recovered from this climate zone (i.e., Clanwilliam, Western Cape province). Moreover, the pine forest habitat (cool/shaded/with potentially higher organic matter and moisture content) may have served to off-set the rather adverse soil conditions normally encountered in this area, in support of H. bacteriophora survival. Two isolates of this species were recovered directly from weevil larvae Otiorhynchus sulcatus (Coleoptera: Curculionidae) in apple orchards around Bethlehem, Free State province. Interestingly, an isolate of an unidentified steinernematid (Steinernema sp. 2, see below) and H. bacteriophora (SASRI 426a and b, respectively) was recovered from a single (unidentified) white grub (Scarabaeidae) cadaver at the Weltevreden locality near Dalton, KwaZulu-Natal.
Table 1 Number of soil samples collected per climate zone/vegetation type by collaborating institutions. Institution
Climate zonea from which EPN-positive soil samples collected
Vegetation typeb
Total # samples/ EPN+
Rooibos Ltd.
Zone 1. Humid subtropical with winter rainfall and warm (warmest month >22 °C) Zone 2. Semiarid with winter rainfall and cool (average annual temperature <18 °C) Zone 3. Semiarid with winter rainfall and warm (average annual temperature >18 °C) Zone 4. Semiarid with annual rainfall and cool (average annual temperature <18 °C) Zone 5. Semiarid with summer rainfall and cool (average annual temperature <18 °C) Zone 6. Humid subtropical with winter rainfall and cool (warmest month <22 °C) Zone 7. Humid subtropical with summer rainfall and cool (warmest month <22 °C) Zone 8. Humid subtropical with annual rainfall and warm (warmest month > 22 °C) Zone 9. Humid subtropical with annual rainfall and cool (warmest month <22 °C) Zone 10. Humid subtropical with summer rainfall and warm (warmest month >22 °C) Zone 11. Humid subtropical with annual rainfall and cool (warmest month <22 °C) Zone 8. Humid subtropical with annual rainfall and warm (warmest month >22 °C) Zone 10. Humid subtropical with summer rainfall and warm (warmest month >22 °C)
Fynbos
603/22
ARCc-Infruitec-Nietvoorbij
ARC-Small Grain Institute and ARC-Vegetable and Ornamental Plant Institute South African Sugar Research Institute
University of KwaZulu-Natal, Pietermaritzburg ARC-Institute for Tropical and Subtropical Crops Total no. samples a
Nama karoo
Fynbos Grassland
336d/39
Savanna
567/15
Grassland Savanna
1506/76
Agrometeorology Staff, 2004: Koppen Climate Zones for South Africa. Agricultural Research Council Institute for Soil, Climate and Water, Pretoria. b According to: Mucina, L., Rutherford, M.C. and Powrie, L.W., 2005. Vegetation Map of South Africa, Lesotho and Swaziland, 1:1 000 000 Scale Sheet Maps, South African National Biodiversity Institute, Pretoria. ISBN: 1-919976-22-1. c Agricultural Research Council. d No positive samples from 168 collected by ARC-Vegetable and Ornamental Plant Institute (ARC-VOPI; Gauteng province).
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Preliminary morphological observations (IJ body size and male spicule/gubernaculum morphology) assisted in placing all other isolates into different Steinernema sp. groups. Recovered steinernematid isolates represent four different species, including Steinernema khoisanae and three new undescribed taxa, hereafter referred to as Steinernema sp. 1, Steinernema sp. 2 and Steinernema sp. 3. Notably, steinernematids accounted for 80% of EPN recovered from the semiarid zones characterised by sandy, acidic soils (average pH 4.5 ± 0.5 and clay content of 4.2 ± 1.3%). Isolation of S. khoisanae was restricted to localities in the Western Cape province (i.e., winter rainfall region; Fynbos vegetation type). These isolates presented morphological similarities in their IJ body length and male spicule morphology to S. khoisanae, a species previously reported in this country (Nguyen et al., 2006). Interpretation of 28S rDNA sequence data further confirmed these isolates as S. khoisanae. However, it should be noted that sequences generated in this study for S. khoisanae strains differed only in one base pair with that of the type strain deposited in GenBank (accession no. DQ314289). Lack of access to original electropherographs for this sequence impeded further comparisons. However, we consider the existing basepair variation between the sequences generated from this study and that deposited in GenBank, may be due to editing errors of electropherographs of the latter sequence (accession no. DQ314289). Morphological examination of key diagnostic traits including male tail morphology, arrangement and number of genital papillae, size of spicules and IJ total body length, maximum width, excretory pore location, tail length and values of D% and E%, provided further evidence to confirm identity of recovered isolates. Steinernema sp. 1 was recovered from five locations around the towns of Bethlehem, Fouriesburg, Mt. Edgecombe, Umhlanga Rocks and Stanger. Notably, these localities were all situated within humid subtropical climate zones in the Free State and KwaZulu-Natal provinces. A total of 10 isolates were collected; three of which were recovered from natural habitats including grasslands and mixed weed fields, and the remaining isolates from ploughed fields, grain and sugarcane (see Table 2). Eleven of the recovered Steinernema strains were considered identical to each other based on both morphological and molecular observations. These isolates were considered to be members of a new undescribed species, Steinernema sp. 2. Most isolates (total of seven isolates) were collected in the locality of Clanwilliam, Western Cape province. This species seems well adapted to a wide range of climate conditions ranging from semiarid (Fynbos or predominantly evergreen sclerophyll plants; isolate SGI 146) to humid subtropical (Savanna; isolate SASRI 356). This species was also associated with a variety of habitats including Rooibos, lettuce, vineyards, wheat and grasslands. As mentioned above, a dual infection of a white grub (Coleoptera: Scarabaeidae) cadaver yielded one isolate of this species (SASRI 426a) and an isolate of H. bacteriophora (SASRI 426b). Steinernema sp. 3 was recovered from both semiarid and humid subtropical climates, although most isolates (>90%) were recovered from soils collected in the Bethlehem and Fouriesburg areas (Free State province) (Table 2). Dominant in these areas are the heavier Avalon and Pinedene soil forms with a predominantly fine sandy loam to sandy clay loam texture (Soil Classification Working Group, 1977). A total of eleven isolates were collected; nine from the above-mentioned orchards, one from a grassland (Bethlehem area) and one from a Rooibos field in the Western Cape province. A detailed morphological analysis, including DIC and SEM microscopy, together with cross-hybridisation tests is currently underway to formally describe all new EPN species recovered in this study.
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3.2. Phylogenetic relationships of new Steinernema sp. Maximum parsimony (MP) analysis of 28S rDNA sequences placed all three new Steinernema sp. in the glaseri-clade also known as clade V (Spiridinov et al., 2004; Nadler et al., 2006). This phylogenetic group encompasses species with large body infective juveniles (generally more than 1000 lm long) and includes 12 species: Steinernema glaseri, Steinernema khoisanae, Steinernema hermaphroditum, Steinernema scarabaei, Steinernema sangi, Steinernema cubanum, Steinernema diaprepesi, Steinernema puertoricense, Steinernema boemarei, Steinernema longicaudum, Steinernema karii and Steinernema arenarium. MP analysis yielded nine equally parsimonious trees with a tree length of 1267 steps and a consistency index (CI) of 0.57 (Fig. 2). Affiliation of each isolate into their corresponding species group was well supported by bootstrap resampling, providing further evidence for their distinctiveness as separate species. As mentioned above, analysis of ITS rDNA sequences for all Heterorhabditis isolates recovered placed them as members of the bacteriophora-group. One equally parsimonious tree was obtained with a tree length of 703 steps and a consistency index (CI) of 0.78 (Fig. 3). The bacteriophora-group currently encompasses two species Heterorhabditis georgiana and H. bacteriophora.
4. Discussion The present study represents the first systematic survey of indigenous EPN species in South Africa. Seventy-six viable EPN isolates were recovered from 1506 soil samples collected from five provinces throughout the country. These regions are quite distinct in geography and habitat diversity with typical soil forms ranging from very sandy texture (e.g., Clovelly and Fernwood forms from the West Coast of South Africa) to sandy clay loam texture (e.g., Avalon and Hutton forms from the Free State and KwaZulu-Natal, respectively) (Soil Classification Working Group, 1977). Notably, Steinernema sp. 2 and S. khoisanae were particularly tolerant of sandy, semiarid conditions with a strain of last mentioned (ROOI 334) being recovered from a locality with very low pH (3.9) and clay content (3% clay; ‘coarse sand’ soil texture). Results from this study also highlight the importance of conducting a more intensive EPN survey in other natural areas and geographic regions of South Africa. Although EPN-positive samples represented only 5% of all collected samples, the fact that five species, one Heterorhabditis and four Steinernema sp. were collected, underscores the incredible richness of EPN fauna in this country. Of all recovered species, only two, H. bacteriophora and Steinernema sp. 2 were found in association with insect hosts. The natural occurrence of these nematode species from these two important agricultural pests should be noted and their potential use as biocontrol agents further investigated. Interestingly, all recovered Steinernema sp. belong to the glaserigroup, which comprises species with IJs with large bodies (>1000 lm). Within this group four species groups were clearly depicted. Ten isolates (SASRI 186, SASRI 198, SASRI 244, SASRI 324, SGI 6, SGI 12, SGI 35, SGI 58, SGI 60 and SGI 82) clustered together representing a new unnamed sp., Steinernema sp. 1. All isolates belonging to this species, were collected in the provinces of KwaZulu-Natal and Free State. Another set of 11 isolates (ROOI 343, ROOI 352, ROOI 369, ROOI 374, SASRI 356, SASRI 426A, SGI 145B, SGI 146, SGI 148, ROOI 299 and INF 23) were considered members of another clade, that represents Steinernema sp. 2. These isolates were collected in the provinces of KwaZulu-Natal, Western Cape and Free State. Another set of 11 isolates (SGI 21, SGI 28, SGI 33, SGI 37, SGI 38, SGI 39, SGI 41, SGI 42, SGI 45, SGI 47 and SGI 152) were considered members of another clade that represents
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Isolate name
Genus
Species
Province/town
Locality
Climate zone (cf. Table 1)
Habitat/insect
Soil pH/clay content (%)
SGI SGI SGI SGI SGI SGI SGI SGI SGI SGI SGI SGI SGI SGI SGI SGI SGI SGI SGI SGI SGI SGI SGI SGI SGI SGI SGI SGI SGI SGI SGI SGI SGI SGI SGI SGI SGI SGI SGI
Steinernema Steinernema Steinernema Heterorhabditis Steinernema Heterorhabditis Heterorhabditis Steinernema Steinernema Heterorhabditis Steinernema Steinernema Steinernema Heterorhabditis Steinernema Steinernema Heterorhabditis Steinernema Heterorhabditis Steinernema Heterorhabditis Steinernema Steinernema Steinernema Heterorhabditis Heterorhabditis Heterorhabditis Steinernema Steinernema Steinernema Heterorhabditis Steinernema Heterorhabditis Heterorhabditis Heterorhabditis Heterorhabditis Heterorhabditis Heterorhabditis Heterorhabditis
new sp. 1 new sp. 1 new sp. 3 bacteriophora new sp. 3 bacteriophora bacteriophora new sp. 3 new sp. 1 bacteriophora new sp. 3 new sp. 3 new sp. 3 bacteriophora new sp. 3 new sp. 3 bacteriophora new sp. 3 bacteriophora new sp. 3 bacteriophora new sp. 1 new sp. 1 new sp. 1 bacteriophora bacteriophora bacteriophora new sp. 2 new sp. 2 new sp. 2 bacteriophora new sp. 3 bacteriophora bacteriophora bacteriophora bacteriophora bacteriophora bacteriophora bacteriophora
Free State/Bethlehem Free State/Bethlehem Free State/ Bethlehem Free State/Bethlehem Free State/Fouriesburg Free State/Fouriesburg Free State/Fouriesburg Free State/Fouriesburg Free State/Fouriesburg Free State/Fouriesburg Free State/Fouriesburg Free State/Fouriesburg Free State/Fouriesburg Free State/Fouriesburg Free State/Fouriesburg Free State/Fouriesburg Free State/Fouriesburg Free State/Fouriesburg Free State/Fouriesburg Free State/Fouriesburg Free State/Fouriesburg Free State/Bethlehem Free State/Bethlehem Free State/Bethlehem Free State/Fouriesburg Free State/Fouriesburg Free State/Fouriesburg Western Cape/Clanwilliam Western Cape/Clanwilliam Free State/Bethlehem Western Cape/Clanwilliam Western Cape/Clanwilliam Free State/Bethlehem Free State/Bethlehem Free State/Bethlehem Free State/Bethlehem Free State/Bethlehem Free State/Fouriesburg Free State/Fouriesburg
ARC-SGI ARC-SGI Utopia Utopia Denmar Denmar Denmar Denmar Denmar Denmar Denmar Denmar Denmar Denmar Denmar Denmar Denmar Denmar Denmar Denmar Denmar ARC-SGI ARC-SGI ARC-SGI Lone tree farm Lone tree farm Lone tree farm Zeekoevlei Zeekoevlei ARC-SGI Nuwedam Nuwedam OTK OTK OTK OTK OTK Lone tree farm Lone tree farm
7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 2 2 7 3 3 7 7 7 7 7 7
Ploughed field (ex wheat) Ploughed field (ex wheat) Grassland Broccoli Blueberry Blueberry Blueberry Cherry Cherry Cherry Cherry Cherry Cherry Cherry Cherry Cherry Cherry Cherry Blueberry Blueberry Blueberry Grain Grain Grain Apples (Fuji) Apples (Sundowner) Apples (Breyburn) Rooibos Fallow field (ex Rooibos) Ploughed field (ex wheat) Pine Forest Rooibos Apples (Braeburn) Apples/Otiorhynchus sulcatus Apples (Braeburn) Apples (Braeburn) Apples (Braeburn) Apples Apples
5.3/18 5.0/20 5.0/12 4.9/11 5.9/9 5.9/9 5.9/9 6.0/16 6.0/16 6.0/16 6.0/16 6.0/16 6.0/16 6.0/16 6.0/16 6.0/16 6.0/16 6.0/16 4.6/9 4.6/9 4.6/9 5.1/20 5.1/20 5.1/20 6.6/NA 6.1/10 6.4/14 4.4/4 4.1/4 4.2/4 4.2/4 4.5/4 5.3/10 5.3/10 5.3/10 5.7/9 4.6/12 6.5/NA 6.5/NA
6 12 21 22 28 29 32 33 35 36 37 38 39 40 41 42 43 45 46 47 50 58 60 82 90b 91a 96d 145b 146 148 151 152 160 161 164 165 166 170 171
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Table 2 Sample data on soils yielding EPN.
Heterorhabditis Heterorhabditis Heterorhabditis Heterorhabditis Heterorhabditis Heterorhabditis Heterorhabditis Heterorhabditis Steinernema Steinernema Steinernema Steinernema Steinernema Steinernema Steinernema Steinernema Steinernema Steinernema Steinernema Steinernema Steinernema Heterorhabditis Steinernema Steinernema Heterorhabditis Steinernema Steinernema Steinernema Steinernema Heterorhabditis Heterorhabditis Steinernema Heterorhabditis Heterorhabditis Steinernema Heterorhabditis Heterorhabditis Heterorhabditis Steinernema Heterorhabditis
bacteriophora bacteriophora bacteriophora bacteriophora bacteriophora bacteriophora bacteriophora bacteriophora khoisanae khoisanae khoisanae khoisanae khoisanae khoisanae new sp. 2 khoisanae khoisanae new sp. 2 new sp. 2 new sp. 2 new sp. 2 bacteriophora new sp. 1 new sp. 1 bacteriophora new sp. 1 new sp. 1 new sp. 2 new sp. 2 bacteriophora bacteriophora new sp. 2 bacteriophora bacteriophora unknownb bacteriophora bacteriophora bacteriophora unknownb bacteriophora
Free State/Fouriesburg Free State/Fouriesburg Free State/Fouriesburg Free State/Fouriesburg Free State/Bethlehem Free State/Bethlehem Free State/Bethlehem Free State/Bethlehem Western Cape/Clanwilliam Western Cape/Clanwilliam Western Cape/Clanwilliam Western Cape/Clanwilliam Western Cape/Clanwilliam Western Cape/Clanwilliam Western Cape/Clanwilliam Western Cape/Clanwilliam Western Cape/Clanwilliam Western Cape/Clanwilliam Western Cape/Clanwilliam Western Cape/Clanwilliam Western Cape/Clanwilliam KwaZulu-Natal/Mt Edgecombe KwaZulu-Natal/Mt Edgecombe KwaZulu-Natal/Mt Edgecombe KwaZulu-Natal/Stanger KwaZulu-Natal/Umhlanga Rocks KwaZulu-Natal/Stanger KwaZulu-Natal/Umhlanga Rocks KwaZulu-Natal/Dalton KwaZulu-Natal/Dalton Western Cape/Stellenbosch Western Cape/Stellenbosch Western Cape/Stellenbosch Western Cape/Hex River Valley Western Cape/De Doorns Western Cape/Stellenbosch Western Cape/Stellenbosch Mpumalanga/Hazyview Mpumalanga/Hazyview KwaZulu-Natal/Pietermaritzburg
Lone tree farm Lone tree farm Lone tree farm Lone tree farm Stead Stead Stead Stead Onder- Brakvlei Ysterfontein Paardekop Paardekop Paardekop Bergendal Bergendal Arbeidsend Arbeidsend De Berg De Berg Elandsfontein Elandsfontein SASRI SASRI Illovo sugar park Kearsney Umhlanga Kearsney Umhlanga Weltevreden Weltevreden Nietvoorbij Nietvoorbij Nietvoorbij Werda De Vlei Nietvoorbij Nietvoorbij Burgershall Burgershall University of KwaZulu-Natal
7 7 7 7 7 7 7 7 2 2 1 1 1 1 1 2 2 1 1 1 1 8 8 8 10 8 10 8 11 11 6 6 6 5 4 6 6 10 10 8
Apples Apples Apples Apples/Otiorhynchus sulcatus Raspberries Raspberries Raspberries Apples Ploughed field (ex Rooibos) Fallow field (ex Rooibos) Fallow field Rooibos Fallow field Grassland Apricot Rooibos Rooibos Lettuce Vineyard Grassland Fallow field Sugarcane Grassland Grassland Guava Sugarcane Mixed weeds Sugarcane Sugarcane/white gruba Sugarcane/white gruba Vineyard Vineyard Vineyard Vineyard Vineyard Beans Beans Banana Citrus Ag Fac garden plots
SGI = ARC-Small Grain Institute; ROOI = Rooibos Ltd.; SASRI = South African Sugar Research Institute; INF = ARC-Infuitec-Nietvoorbij; ITSC = ARC-Institute for Tropical and Subtropical Crops. a Dual infection of an unidentified white grub larva. b Nematodes did not multiply.
6.5/NA 6.5/NA 6.5/NA 6.5/NA NA/NA NA/NA NA/NA NA/NA 4.3/3 4.2/6 6.6/3 4.4/3 5.0/5 4.2/5 6.2/4 3.9/3 4.9/4 5.8/3 6.4/3 6.0/3 4.6/5 7.3/5 6.6/4 4.9/3 4.6/4 4.9/4 4.9/4 7.9/4 NA/NA NA/NA 5.5/4 4.1/8 5.3/3 5.6/3 5.0/7 5.7/7 5.4/7 NA/NA NA/NA 6.1/9
J. Hatting et al. / Journal of Invertebrate Pathology 102 (2009) 120–128
SGI 172 SGI 173 SGI 174 SGI 175 SGI 178 SGI 179 SGI 180 SGI 181 ROOI 161 ROOI 229 ROOI 275 ROOI 277 ROOI 283 ROOI 293 ROOI 299 ROOI 334 ROOI 336 ROOI 343 ROOI 352 ROOI 369 ROOI 374 SASRI 75 SASRI 186 SASRI 198 SASRI 199 SASRI 244 SASRI 324 SASRI 356 SASRI 426a SASRI 426b INF 16 INF 23 INF 29 INF 61 INF 73 INF 79 INF 88 ITSC 36 ITSC 106 UNIV 17
125
126
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Fig. 2. Strict consensus (MP analysis) of 28S rDNA sequences tree showing phylogenetic relationships of Steinernema sp. Tree length = 1267. Consistency index (excluding uninformative characters = 0.57). Numbers above nodes indicate bootstrap values.
J. Hatting et al. / Journal of Invertebrate Pathology 102 (2009) 120–128
127
Fig. 3. Strict consensus tree (MP analysis) of ITS rDNA sequences indicating evolutionary relationships of Heterorhabditis sp. Tree length = 703. Consistency index (excluding uninformative characters = 0.78). Numbers above nodes indicate bootstrap values.
Steinernema sp. 3. These isolates were collected in the Free State and Western Cape provinces. Eight isolates (ROOI 161, ROOI 229, ROOI 275, ROOI 277, ROOI 283, ROOI 293, ROOI 334 and ROOI 336) were collected in the Western Cape and identified as S. khoisanae, a species originally collected in three localities in this province (Malan et al., 2006; Nguyen et al., 2006). In this study, S. khoisanae was isolated from different localities around Clanwilliam in the Western Cape, indicating this species is widely distributed in this province. H. bacteriophora isolates were collected in four provinces, including the Western Cape, Free State, KwaZulu-Natal and
Mpumalanga. This species was probably recovered for the first time in KwaZulu-Natal by Spaull (1991), and later described by Grenier et al. (1996, 1997). Malan et al. (2006) also isolated H. bacteriophora in the Western Cape. However, this species had not been reported from the Free State and Mpumalanga before, expanding its geographic distribution. These native and novel strains and species provide a new alternative for their consideration in biological control and/or integrated pest management programs in South Africa. Various studies are currently underway to assess biological traits and host suitability of several of these isolates.
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Acknowledgments We acknowledge the following field collaborators responsible for collecting and sending soil samples/EPN-infected cadavers to ARC-SGI (order as per Table 1): Mr. Johan Brand (Rooibos Ltd.), Mr. Fanie van der Merwe and Dr. Pia Addison (formerly ARC-Infruitec-Nietvoorbij), Dr. Astrid Jankielsohn (ARC-SGI), Dr. Diedrich Visser (ARC-VOPI), Dr. Des Conlong (SASRI), Dr. Ray Miller (University of KZN, Pietermaritzburg), and Dr. Schalk Schoeman (ARC-ITSC). For technical assistance rendered: Me Gloria Macucwe, Me Palesa Dihemo, Me Hannetjie Jooste (formerly ARC-SGI), Me Tshima Ramakuwela, Me Lettie Opperman and Mr. Amos Mosia (ARC-SGI). We thank Me Noelien Somers and Dr. Dave Turner of the ARC-Institute for Soil, Climate and Water, Pretoria, for assistance with information regarding climate/vegetation and soil types, respectively. The project (#41404) was supported financially under Round 4 of the Innovation Fund administered by the National Research Foundation (NRF) of South Africa. We also acknowledge K. Plichta and V. Martinson (S.P. Stock laboratory) for assisting with molecular characterization of EPN. References Adams, B.J., Fodor, A., Koppenhöfer, H.S., Stackebrandt, E., Stock, S.P., Klein, M.G., 2006. Biodiversity and systematics of nematode–bacterium entomopathogens. Biol. Control 37, 32–49. Bedding, R.A., Akhurst, R.J., 1975. A simple technique for the detection of insect parasitic rhabditid nematodes in soil. Nematologica 21, 109–110. Gaugler, R., Kaya, H.K., 1990. Entomopathogenic nematodes in biological control. CRC Press, Boca Raton Florida. p. 365. Grenier, E., Bonifassi, E., Abad, P., Laumond, C., 1996. Use of species-specific satellite DNAs as diagnostic probes in the identification of Steinernematidae and Heterorhabditidae entomopathogenic nematodes. Parasitology 113, 483–489. Grenier, E., Castagnone-Sereno, P., Abad, P., 1997. Satellite DNA sequences as taxonomic markers in nematodes of agronomic interest. Parasitol. Today 13, 398–401. Harington, J.S., 1953. Observations on the biology, the parasites and the taxonomic position of the black maize beetle – Heteronychus sanctae-helenae Blanch. S. Afr. J. Sci. 50, 11–14. Hatting, J.L., Kaya, H.K., 2001. Entomopathogenic nematodes: prospects for biological control in South Africa. In: Olckers, T., Brothers, D.J. (Eds.), Proceedings of the 13th Entomological Congress, Pietermaritzburg, 2–5 July 2001. Entomological Society of Southern Africa, Hatfield, Pretoria, p. 28. Hominick, W.M., 2002. Biogeography. In: Gaugler, R. (Ed.), Entomopathogenic Nematology. CABI Publishing, Wallingford, UK, pp. 115–144. Hominick, W.M., Briscoe, B.R., Del Pino, F.G., Heng, J.A., Hunt, D.J., Kozodoy, E., Mrácek, Z., Nguyen, K.B., Reid, A.P., Spiridonov, S.E., Stock, S.P., Sturhan, D., Waturu, C., Yoshida, M., 1997. Biosystematics of entomopathogenic nematodes: current status, protocols and definitions. J. Helminthol. 71, 271–298.
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