First record of entomopathogenic nematodes (Steinernematidae and Heterorhabditidae) in Costa Rica

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1 Journal of Invertebrate Pathology 88 (2005) First record of entomopathogenic nematodes (Steinernematidae and Heterorhabditidae) in Costa Rica Lorena Uribe-Lorío a, Marielos Mora a, S. Patricia Stock b, a Centro de Biología Celular y Molecular, Universidad de Costa Rica, Costa Rica b Division of Plant Pathology and Microbiology, Department Plant Sciences, University of Arizona, 1140 E. South Campus Dr. Tucson, AZ , USA Received 4 October 2004; accepted 18 January 2005 Available online 19 February 2005 Abstract A survey of entomopathogenic nematodes was conducted in the north PaciWc (Guanacaste Conservation Area) and southeast Caribbean (Gandoca-Manzanillo Natural Refuge) regions of Costa Rica. Out of a total of 41 soil samples, 5 were positive for entomopathogenic nematodes (20.5%), with 3 (12.3%) containing Steinernema and 2 (8.2%) Heterorhabditis isolates. Morphological and molecular studies were undertaken to characterize these isolates. The Heterorhabditis isolates were identiwed as Heterorhabditis indica and the three Steinernema isolates were identiwed as two new undescribed species. H. indica was recovered from a coastal dry forest. Steinernema n. sp. 1 was isolated from a rainforest valley, between volcanoes. Steinernema sp. n. 2 was isolated from sand dunes in the Caribbean Coast (Punta Uva) near the rainforest strip along the coast. Although limited to two geographic regions, this study suggests entomopathogenic nematodes may be diverse and perhaps widely distributed in Costa Rica. A more intensive survey, covering all geographic regions is currently undergoing Elsevier Inc. All rights reserved. Keywords: Costa Rica; Diversity; Entomopathogenic nematodes; Steinernema; Heterorhabditis 1. Introduction Entomopathogenic nematodes (EPN) of the genera Steinernema Travassos and Heterorhabditis Poinar are obligate pathogens that infect a wide range of soil insects (Kaya and Gaugler, 1993). These nematodes are mutualistically associated with bacteria in the genus Photorhabdus Boemare, Akhurst, and Mourant for Heterorhabditis or Xenorhabdus Thomas and Poinar for Steinernema (Boemare et al., 1997). The life cycle of all known entomopathogenic nematode/bacterium complexes is similar. The only stage that survives outside of the host is the non-feeding, third-stage infective juvenile (IJ). The IJ carries cells of the bacterial symbionts in its Corresponding author. Fax: address: spstock@ag.arizona.edu (S.P. Stock). intestine. When the IJ Wnds a susceptible host, it invades and penetrates into the host s hemocoel through natural openings (i.e., anus, mouth, or spiracles). The IJ then releases the symbiotic bacterium that kills the host within 48 h by septicemia. The bacterium produces antibiotics that prevent other microorganisms from colonizing the cadaver. In addition to serving as a food source for the nematode, the bacterium digests the host tissues, thereby providing suitable nutrients for nematode growth and development. In the broadest geographic sense, EPN are widespread. The only continent where they have not been found is Antarctica (GriYn et al., 1991). However, soil surveys conducted in diverent areas of the world have demonstrated variability in abundance across seasons, habitats, and geographic regions. Factors such as soil texture, moisture content, temperature, and host availability are thought to be important in determining distri /$ - see front matter 2005 Elsevier Inc. All rights reserved. doi: /j.jip

2 L. Uribe-Lorío et al. / Journal of Invertebrate Pathology 88 (2005) bution of entomopathogenic nematodes (GriYn et al., 1991; Hominick and Briscoe, 1990; Hara et al., 1991; Hominick et al., 1996; Steiner, 1996; Stock et al., 1999). In contrast to human modiwed areas, natural habitats are likely uncontaminated by introduced nematodes and over increased opportunities for Wnding native nematode species (Stock et al., 1999, 2003). In this respect, and with particular reference to entomopathogenic nematodes, the isolation of native species and/or populations provides a valuable resource not only from a biodiversity perspective but also from a more applicable standpoint (Stock et al., 1999). Indigenous EPN may result more suitable for inundative release against local insect pests because of adaptation to local climate and other population regulators (Bedding, 1990). In addition, many countries are concerned about the introduction of exotic entomopathogenic nematodes because they may have negative impact on non-target organisms (Bathon, 1996). At present, only a few studies have focused on entomopathogenic nematodes in Central America but considering only the Caribbean islands of Puerto Rico, Cuba, Guadeloupe, and the Dominican Republic (Figueroa et al., 1993; Fischer Le Saux et al., 1998; Mrábek et al., 1994; Román and Figueroa, 1994). Although, these studies have been restricted in geographical scope, at least two new Steinernema, S. cubanum and S. puertoricense, were recovered and described (Mrábek et al., 1994; Román and Figueroa, 1994). In Costa Rica, information on the diversity of entomopathogenic nematodes and their symbiotic bacteria is currently non-existent. Until now, no systematic survey has been conducted to document the presence of these nematodes in this country. Costa Rica, a country of 50,900 km 2, is the nexus of two continents and two oceans. It is located in the Central American isthmus, bordered by Panama to the south and Nicaragua to the north, with coastline on the PaciWc and Atlantic oceans. This conxuence of land and water makes the region one of nature s great and unique biodiversity bottlenecks (Carvajal-Alvarado, 1995). It has been estimated that 4% of the world s biodiversity is present in this country. Surprisingly, close to 84% of this biodiversity remains unknown to science (Janzen, 1983), and the unknown percentage exceeds 90% for groups such as insects, fungi, bacteria, viruses, and nematodes. These observations suggest that the diversity of nematodes associated to insects and other invertebrates may be vast. Therefore, and as a preliminary approach to help design a more extensive EPN diversity inventory, two of the six Costa Rican geographic regions, the Caribbean and the North PaciWc were considered in this survey. Nematodes recovered during this study were identiwed using a combination of morphological and molecular methods. Results of this survey are herein presented. 2. Materials and methods 2.1. Geography and collection of soil samples Costa Rica is divided into six geographic regions: Caribbean, Central Valley, Northern Plains, Central PaciWc, Northern PaciWc, and South PaciWc. Each of these regions has unique geographic, ecological, and climatic characteristics (Carvajal-Alvarado, 1995). In this study, two the Northern PaciWc and Caribbean regions were selected for sampling (see Fig. 1). The Area de Conservación Guanacaste (ACG) located in the North PaciWc region, comprises 110,000 ha and is located in the Northern PaciWc region. Because of its rich biological diversity it is considered one of Central America s hotspot (Myers et al., 2000). It comprises the Guanacaste Cordillera and surrounding Xatlands and coastal areas. The ACG includes a series of volcanoes, the most notable being Rincon de la Vieja, which has three craters and one lagoon. Volcanic rocks of high calcium carbonate content underlie the western region; sedimentary sandstones occur on the coastal Xank and salt Xats or salinas are found along littoral lowlands. The marine/coastal area includes various islands and islets, open ocean marine zones, beaches, and rocky coasts. The average annual precipitation is 1528 mm in Santa Rosa (ACG) with considerable variation in the Fig. 1. Map of Costa Rica showing geographic regions and sampling sites. I. Northern PaciWc; II. Central PaciWc; III. Southern PaciWc; IV. Northern Plains, V. Central Valley; and VI. Caribbean. (A) Area de Conservación Guanacaste (ACG), (B) Gandoca-Manzanillo Wildlife Refuge. 1. Playa Naranjo, 2. Santa Elena península, 3. Santa Rosa, 4. Volcano Valley, 5. Estación Pitilla, and 6. Punta Uva., Steinernematids;, Heterorhabditids.

3 228 L. Uribe-Lorío et al. / Journal of Invertebrate Pathology 88 (2005) amount of rainfall each year. The mean annual temperature is 28 C (Carvajal-Alvarado, 1995). Within ACG, Wve regions were sampled: Naranjo beach (coastal), Santa Elena península (dry forest), Santa Rosa (dry forest), volcano valleys, and Pitilla station (rain forest). Within each region, sampling sites were selected based on predominant habitats (Table 1). With the exception of the dry forest in Naranjo beach, 3 samples were taken at each site. The Gandoca-Manzanillo Wildlife Refuge (Limón Province) located in Caribbean coast, in the southeast of Costa Rica was also considered in this study. EPN were known to be present in this region (CATIE, Turrialba, unpublished data), therefore it was considered important to include this region in this study. The Caribbean region is the most humid region of Costa Rica. The rainfall in the lowlands of this region reaches 3000 mm per year and up in the highlands increases to 4500 mm. The annual average temperature oscillates between 25 and 27 C. The Gandoca-Manzanillo Wildlife Refuge is characterized by a wide variety of habitats including sandy beaches, swamps, lagoons, coral reefs, and forests. Within this region, 3 sites from Punta Cocles to Bribrí were considered for sampling. In all cases, each soil sample (approximately 1 kg) was a composite of 5 random sub-samples taken distantly located from each other in an area of 10 m 2 and at a depth of 0 20 cm. Samples were taken with a hand shovel, placed in polyethylene bags to prevent water loss and kept in coolers (ca. 15 C) during transit to the laboratory. Between samples, the shovel was thoroughly rinsed with 70% ethanol to prevent contamination of the next sampling unit Nematodes isolation and propagation EPN were recovered from soil samples using the insect bating method described by Bedding and Akhurst (1975). Insect baits (Wve last-instar Galleria mellonella (L.) larvae) were placed in 250 ml plastic containers (Wve containers/sample) with moistened soil obtained from each sample. Containers were covered with a lid, turned up side down and kept at room temperature (20 3 C) (Stock et al., 1999). Water was added to samples if they appear dry at any point during their bating. G. mellonella larvae were checked every two to three days and dead larvae were replaced by fresh ones. After seven days dead insects were and thoroughly rinsed in distilled water and placed in modiwed White traps (Kaya and Stock, 1997) until emergence of third-stage infective juveniles. Emerging nematodes were pooled for each sample and used to infect fresh G. mellonella larvae to produce nematodes for identiwcation and establishment of cultures. To maximize the recovery of nematodes from the soil samples, and after removing the dead G. mellonella (presumably infested with nematodes), a second baiting round was done by placing fresh G. mellonella into the containers with the same soil. 3. Nematode identiwcation 3.1. Taxonomic studies Morphological characterization For morphological studies, nematodes were examined live or heat-killed in 60 C Ringer s solution. The heat- Table 1 Regions, habitats, and number of samples taken in each habitat Geographic region Site Habitats Total No. of samples North PaciWc Zone (Area de Conservación Guanacaste) Playa Naranjo Sand dunes 3 Marsh 3 Dry forest 2 Dry transition forest 3 a Santa Elena Península Dry forest 3 Primary 3 Recently burned 3 Santa Rosa (Dry forest) 2 5 years regeneration years regeneration years regeneration 3 Up to 50 years regeneration 3 Valley between volcanoes Rainforest transition 3 b Estación Pitilla Caribbean humid forest 3 Caribbean Coast Gandoca-Manzanillo Sand dunes near Punta Uva 3 c a H. indica. Steinernema sp. 2. Steinernema sp. 1.

4 L. Uribe-Lorío et al. / Journal of Invertebrate Pathology 88 (2005) killed nematodes were placed in triethanolamine formalin (TAF) Wxative (Kaya and Stock, 1997) and processed to anhydrous glycerine for mounting (Seinhorst, 1959). Observations were made from live and mounted specimens using an Olympus BX81 microscope equipped with diverential interference contrast optics. Specimens measurements were made using AnalySIS Image software (Soft Imaging System, CA, USA). For morphological characterization of the isolates, 25 Wrst-generation males and 25 IJs were randomly selected from diverent G. mellonella cadavers. According to their morphological characteristics, all isolates were placed into similar species-groups using taxonomic criteria suggested by Stock and Kaya (1996) and Hominick et al. (1997). Additionally, morphological features of males and IJs of representative isolates of each species-group were examined using scanning electron microscopy. For this purpose specimens were processed following protocols described by Kaya and Stock (1997) Molecular characterization In addition to morphological studies, all isolates were molecularly characterized by analysis of the LSU (large subunit) and ITS (internal transcribed spacer region) rdna sequences for steinernematids and heterorhabditids, respectively, according to procedures described by Stock et al. (2001). The resulting sequences were compared to a library of more than 40 EPN species Bacterial symbiont isolation and identiwcation Isolation of EPN symbiotic bacteria followed procedures described by Akhurst (1980) with a small modiwcation in the sterilization process. Approximately, 100 IJs were immersed in 85% sodium hypochloride solution for 8 min until larvae sheath was completely digested. The unsheathed IJs were rinse twice in sterile Ringer solution and crushed in fresh sterile Ringer solution and then transferred to sterile nutrient broth. This suspension was streaked onto NBTA medium and incubated at 28 C for 48 h. Conventional phenotypic criteria was used for verify generic identity (Xenorhabdus and Photorhabdus) of bacterial isolates (Boemare and Akhurst, 1988). Results were compared with reference strains. All tests were conducted at 28 C. Cellular morphology was assessed by microscopic examination of 24-h-old nutrient broth cultures. Dye adsorption of bromothymol blue was tested on nutrient agar supplemented with 0.004% triphenyltetrazolium chloride and bromothymol blue (NBTA medium) for Xenorhabdus isolates. Dye adsorption of neutral red was tested on MacConkey agar for Photorhabdus isolates. A semiautomatized identiwcation system (Biolog) was also used to further identify bacterial isolates. Nucleic acids from the bacterial isolates were extracted according Boemare et al. (1992). 4. Results EPN were recovered from 5 out of the 41 samples collected from 14 habitats from two geographic regions (Table 1). Four out of the 5 positive samples contained steinernematids and 1 sample held heterorhabditids. The heterorhabditid isolates were identiwed as Heterorhabditis indica Poinar, Karunakar, and David. This species has previously been reported from tropical and subtropical regions such as Puerto Rico, SE Asia (India), and North America (Hawaii) (Hominick et al., 1996). Morphological examination and sequence analysis of 28S rdna indicated the steinernematid isolates represent two new undescribed species, hereafter refer to as Steinernema sp. 1 and Steinernema sp. 2. Heterorhabditis indica (CR# isolate) was recovered from a dry forest habitat along the coast of Naranjo beach in ACG region. Steinernema sp. 1 (Li 6 isolate) was isolated from a rainforest strip along Punta Uva coast in the Caribbean region. Steinernema sp. 2 (CR # isolate) was collected in a valley slope between the Cacao and Rincón de la Vieja volcanoes at 1200 m. This area, which comprises premontane and transitional tropical rainforest, is very unique, as many species of insects, mites, fungi, bacteria, nematodes, spiders, and protozoans encountered here, do not occur elsewhere in the ACG ( Soil types from EPN-positive samples were classiwed from sand to loam. The ph of these samples ranged from acidic (5.71) to slightly alkaline (7.24). Steinernematids were found in sandy and sandy-loam soils that ranged from acidic (5.71) to slightly alkaline (7.24). H. indica was found in a loam soil with a ph of The organic matter content of the positive samples varied from 1.1 (Guanacaste) to 5.6% (Caribbean) Nematode identiwcation Morphological examination indicated Steinernema sp. 1 (Li 6 isolate) resembles most S. feltiae (Filipjev), however there are diverences in key diagnostic traits of the third-stage infective juveniles (IJs) and males that separate these two species. Phylogenetic analysis of 28S rdna sequence data placed this species as a sister taxon of S. feltiae, within a clade that contains species with medium-size IJs (average size μm) such as S. kraussei (Steiner), S. oregonense Liu and Berry, and S. kushidai (see Stock et al., 2001 for further reference). Steinernema sp. 2 (CR9) is morphologically similar to S. riobrave Cabanillas, Poinar, and Raulston. Phylogenetic analysis of 28S rdna sequences from this species, indicates this species is

5 230 L. Uribe-Lorío et al. / Journal of Invertebrate Pathology 88 (2005) close related to a clade that contains taxa which IJs are characterized by the presence of horn-like structures in the anterior end (i.e., S. bicornutum Tallosi, Peters, and Ehlers and S. ceratophorum Jian, Reid, and Hunt) (see Stock et al., 2001 for further reference). Description of these two new species is currently undergoing Bacteria characterization Three bacterial strains corresponding to the two Steinernema spp. and Heterorhabditis indica were isolated from third-stage infective juveniles. The bacterial symbionts were identiwed as Xenorhabdus sp. and Photorhabdus sp. for the two steinernematid nematodes and for H. indica, respectively. IdentiWcation of these bacteria, to the species level is currently being conducted. 5. Discussion The present study recorded for the Wrst time the occurrence of EPN in Costa Rica. Nematodes were recovered from both the Northwest and the Caribbean regions. These two regions are quite distinct in their topology, habitat diversity, and climatic characteristics. Although, EPN were recovered only from 5 out of 41 samples (20.5 %), the recovery of two new species highlights the importance of conducting a more intensive survey considering other natural areas and geographic regions of this country. Two of the three EPN species recovered were collected from rainforest habitats with sandy or sandyloam soil types and a ph that ranged from slightly acidic to slightly alkaline. Interestingly, H. indica was recovered from a loam soil in a dry forest strip along the PaciWc coast. This species has been reported from sandy soils at coastal sites in other subtropical and tropical regions of the world (Burnell and Stock, 2000; Stack et al., 2000). However, the recovery of H. indica from a dry forest habitat adds new information on the habitat preference of this species. Until now, no EPN have been reported from volcanic soils. The isolation of Steinernema sp. 1 from a valley slope along a premontane and transitional tropical rainforest strip in a volcanic region marks the Wrst record of such Wnding. Steinernema sp. 2 was the only EPN species recovered in the Caribbean region. This nematode was isolated from a sand dune near the rainforest strip along Punta Uva coast. The soil type where this species was collected was sand. The presence of steinernematids at coastal sites has also been reported in other surveys (Constant et al., 1998; GriYn et al., 2000). According to Hominick et al. (1996) the presence of EPN along the coast may be the result of large insect populations feeding on washedup marine detritus. Based on these Wndings, we predict more new species may be discovered in future surveys adding signiwcant information to the diversity and biogeography of this group of nematodes. Knowledge of the diversity, distribution of EPN is extremely valuable not only for future ecological and biocontrol studies but also from a bioprospecting perspective. Moreover, in surveying for insect parasitic nematodes, we hope to foster tropical diversity conservation. To maintain interest in biodiversity conservation in the world, particularly in the tropics where the vast majority of the biological diversity is hosted, the preserve areas where this diversity is kept must be unraveled and seen as an economic value (Janzen, 1992; Janzen et al., 1993). Acknowledgments This study was funded in part by a WISC-AAAS grant to S. Patricia Stock and Lorena Uribe-Lorio. References Akhurst, R.J., Morphological and functional dimorphism in Xenorhabdus spp., bacteria symbiotically associated with the insect pathogenic nematodes Neoplectana and Heterorhabditis. J. Gen. Microbiol. 121, Bathon, H., Impact of entomopathogenic nematodes on non-target hosts. Biocontr. Sci. 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