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Department of Entomology, Cornell University, Ithaca, New York 14853-0901 USA
Annette Bruun Jensen
Lene Thomsen
Department of Ecology, Zoology Section, Royal Veterinary and Agricultural University, Thorvaldsensvej 40, 1871 Frederiksberg C, Denmark
Kathie T. Hodge
Department of Plant Pathology, Cornell University, Ithaca, New York 14853 USA
Jørgen Eilenberg
Department of Ecology, Zoology Section, Royal Veterinary and Agricultural University, Thorvaldsensvej 40, 1871 Frederiksberg C, Denmark
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The shape and nucleation of primary conidia are important characters in the classification of the Entomophthoraceae (Zygomycetes). The five species in the genus Eryniopsis vary in the shapes of primary conidia, although within most genera in the order Entomophthorales species have the same shapes of primary conidia. Using PCR-RFLP, we investigated two species in Eryniopsis, Ery. caroliniana with oblong-ovoid primary conidia and Ery. ptychopterae with pear-shaped primary conidia, with five species of Entomophaga, all having pear-shaped conidia. Molecular results merged with morphological data indicate that Ery. ptychopterae belongs in the genus Entomophaga while Ery. caroliniana clearly differs from Entomophaga. Ery. ptychopterae and Ery. transitans are transferred to the genus Entomophaga. Our results support the idea that morphology of primary conidia is of major importance in defining entomophthoralean genera. These results also show that such studies can be conducted with species that have not been isolated, if fungal-filled cadavers can be obtained.
Key words: Entomophthorales, genetic variation, insect pathogenic fungi, Ptychopteridae, rDNA, Tipulidae
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). This genus originally contained three species: Ery. lampyridarum, Ery. longispora and Ery. caroliniana (Table I). There was a great deal of variability among the three species, with both primary and secondary conidia from Ery. longispora being elongate, while Ery. caroliniana and Ery. lampyridarum produce two types of secondary conidia, only one of which is elongate. For these latter species, the elongate secondary conidia are produced only some of the time, either on top of thick tubes (Ery. caroliniana) or on capillary tubes (Ery. lampyridarum).
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) (Table I). It is of interest to note that these newer additions are very similar to each other in appearance but are known to attack primitive dipteran hosts of different, but closely related, families in different countries; Eryniopsis transitans is known only from Limoniidae (formerly a subfamily of crane flies, Tipulidae) in Switzerland and Eryniopsis ptychopterae is known only from Ptychopteridae (phantom crane flies, closely related to Tipulidae) in Denmark. These two species, Ery. transitans and Ery. ptychopterae, differed from other members of Eryniopsis in their pear-shaped primary conidia that resemble members of the genus Entomophaga. Because Ery. transitans and Ery. ptychopterae possessed both elongate secondary conidia like members of Eryniopsis and pear-shaped primary conidia such as Entomophaga, these species were considered a link between these two genera, suggesting a close relationship between Eryniopsis and Entomophaga (Keller & Eilenberg 1993). Curiously, one species already within Entomophaga, Ent. tipulae, known from central Europe from two crane fly families, the Limoniidae and Tipulidae, sometimes produces elongate secondary conidia (Ba
azy 1993).
Species of Entomophthorales provide few unambiguous morphological features for identification and evaluation of evolutionary affinities among groups. Molecular methods have offered the opportunity for some genuine insights in defining relationships. However, cultures of many species of Entomophthorales are not available due to the rarity of some species, the difficulty of growing many of these fungi in vitro and the cost of maintaining cultures of these often-fastidious species. Modern molecular techniques make it possible to amplify fungal DNA from tiny field-collected cadavers, eliminating the need to isolate these different fungi in axenic culture (Jensen and Eilenberg 2001).
We explored the association between members of the entomophthoralean genera Eryniopsis and Entomophaga with PCR-RFLP using entomophthoralean-specific primers. Most of the five species of Eryniopsis are not common, but two were available for use in this study. We compared these species with five species of the larger genus Entomophaga, using species from other entomophthoralean genera as outgroups.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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) were obtained from the USDA, Agricultural Research Service Collection of Entomopathogenic Fungal Cultures (ARSEF: Ithaca, NY). Cultures of Ery. caroliniana, Ery. ptychopterae, Ent. aulicae and Ent. maimaiga were obtained from ARSEF and grown in 95% Grace's insect-tissue culture medium (GIBCO-BRL, Gaithersburg, MD), plus 5% fetal bovine serum (GIBCO-BRL). To harvest fungal cells from cultures, protoplasts and hyphal bodies were centrifuged at 2000 g for 10 min. Alcohol-preserved specimens of Ery. lampyridarum and Ery. transitans also were obtained but, in both cases, it was not possible to extract detectable DNA, perhaps because the material had been stored for years.
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were followed for DNA extraction, amplification and PCR-RFLP. Both in vitro-grown fungal cells and fungus-filled cadavers were lyophilized before extracting DNA. Fungal cells then were crushed with a pestle before adding extraction buffer. To extract DNA, the methods described by Bulat et al (1998) were modified by using chloroform/octanol (24:1) instead of chloroform/phenol (1:1).
For each of the isolates used, the ITS 2 (Internal Transcribed Spacer 2) region and the first part of the LSU (nuclear large subunit ribosomal DNA) were amplified. The specific primers used were Nu-5.8S–5' (Jensen and Eilenberg 2001) and ITS 4 (White et al 1990) for ITS 2 and nu-LSU-0018-5' (Jensen and Eilenberg 2001) and nu-LSU-0805-3' (Kjøller and Rosendahl 2000) for the LSU.
PCR thermal-cycling conditions consisted of an initial denaturation lasting 5 min at 96 C, followed by 35 cycles of denaturation for 1 min at 96 C, annealing for 1 min at 65 C (ITS 2) or 52 C (LSU), and extension for 1 min at 72 C, with a final extension for 10 min at 71 C. Reactions were carried out in 25 µL volumes with 250 µM of dNTP, 0.8 µM of each primer, 3.0 mM MgCl2, 1 x DyNAzyme II buffer (10 mM Tris-HCl, 50 mM KCl and 0.1% Triton X-100), 1 unit DyNAzyme II DNA polymerase (Finnzymes, Espoo, Finland) and 1 µL of extracted DNA. The sizes of PCR-amplified fragments were monitored by electrophoresis in a 1.5% agarose gel in 1 x TBE buffer (Sambrook et al 1989) and ethidium bromide staining. To generate enough material from LSU amplifications to use with restriction enzymes, amplifications with LSU primers were repeated for all isolates in 100 µL volumes.
PCR-RFLP – LSU PCR products were cut with 9 different restriction endonucleases: HaeIII, DdeI, RsaI, aTaqI, Sau3aI, DraI, AluI, HpaII and HhaI (New England Biolabs). Reaction volumes consisted of 5 µL of the PCR product, 1 µL of the supplied 10 x buffer, and 2 units of enzymes plus distilled water to make a total volume of 10 µL. Reactions were incubated overnight at 37 C and separated on a 1.5% agarose gel, as described above. The ITS 2 PCR products were not cut with restriction enzymes because bands were dissimilar in size.
Morphological data –
We included morphological characters, discussed by Humber (1981) and used by Baazy (1993), as criteria defining entomophthoralean genera. An Olympus Provis microscope with computer facilities was used to assist morphometrics. Nuclear status was quantified as ancylistoid (nuclei small and staining weakly or not at all) versus entomophthoroid (nuclei relatively large, spherical, ovoid or oblong and staining distinctly) (Baazy 1993), and the number of nuclei was quantified as one versus >1. Sporophore branching was coded as simple or digitately branched and the presence or absence of an isthmus at the junction with conidia was recorded. Mode of primary conidial discharge was papillary eversion versus squirted. Rhizoids were coded as present or absent. The most diverse character was primary spore shape, including pear-shaped, ovoid to ellipsoid, campanulate, globose with papilla obtuse and a smooth junction or globose with papilla obtuse and conical with an abrupt junction, and oblong ovoid, subpapillate.
Data analysis – The results of the restriction analysis were coded as binary characters, with each character representing a fragment of unique length. For each of the nine enzymes used to cut the LSU, all lengths of fragments (characters) were scored as present or absent for each fungal isolate. The ITS 2 was coded as a single multistate character; each of the seven ITS "types" was scored as a separate character state. Morphological data also were coded as multistate characters, although only the shape of primary spores had more than two states. The data set comprised 83 characters (75 from restriction analysis, one from ITS 2 and seven from morphological data).
The data matrix was analyzed with PAUP* for unweighted maximum-parsimony analysis (Swofford 1998). A heuristic search with TBR branch swapping was conducted, based on starting trees obtained by 10 randomized replicates of stepwise taxon addition. The trees were rooted with Conidiobolus thromboides as an outgroup; this is the only species included with ancylistoid type nuclei and belongs to the family Ancylistaceae, while all other species have entomophthoroid nuclei and belong to the family Entomophthoraceae (Baazy 1993). To investigate the support of the data for the resulting topologies, 100 bootstrap replicates were performed using the same criteria as for the maximum parsimony analysis.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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azy (1993). Primary conidia of Ent. tipulae 2 were wider than those in this species description but fell within the ranges of primary conidial sizes and shapes of both Ery. ptychopterae and Ery. transitans.
All samples with pear-shaped conidia formed a clade. The genus Batkoa formerly was attributed to a separate subgenus within Entomophaga (Baazy 1993). The only species of Batkoa in our study, B. gigantea, was within the clade including the species of Entomophaga/Ery. ptychopterae. In three of the four most-parsimonious trees, B. gigantea fell within a clade with the species of Entomophaga; in the fourth (Fig. 2), B. gigantea was sister to the Entomophaga clade. Ent. maimaiga and Ent. aulicae, considered in the same species complex by Walsh (1996), were not closely grouped within the Entomophaga clade. The only uninucleate species tested, P. neoaphidis and P. delphacis, were distantly related to the multinucleate genera.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). However, these genera differ significantly because conidia of Eryniopsis are multinucleate while those of Erynia sl (including Pandora) are uninucleate. In agreement, our analysis demonstrated that the two evaluated uninucleate Pandora species are not closely related to Eryniopsis. Based on results from this study, Ery. ptychopterae now should be considered a member of the genus Entomophaga, while Ery. caroliniana is distinct and should remain in the genus Eryniopsis. Due to the morphological similarity of Ery. transitans to Ery. ptychopterae, we therefore transfer these species to Entomophaga, as indicated below.
The generic status of Ery. caroliniana remains uncertain. The type species for the genus Eryniopsis is Ery. lampyridarum, which was not available for inclusion in this study. The ultimate fate of the genus Eryniopsis will depend on relationships of Ery. lampyridarum with Ery. caroliniana and Ery. longispora. The primary conidia of Ery. lampyridarum are similar in shape to those of Ery. caroliniana, but secondary conidia are formed on capillaries by Ery. lampyridarum while secondary conidia of Ery. caroliniana are formed on shorter, thicker tubes. Based on the importance of primary conidia shape determined in this study, we would predict that Ery. caroliniana would be closely related to Ery. lampyridarum but this must be investigated further.
Entomophaga tipulae is reported as being isolated from Limoniidae, as is Ent. transitans, and both are from central Europe. In this study Ent. tipulae 2 was found to be more closely related to Ent. transitans or Ent. ptychopterae than to Ent. tipulae 1; key characters that could differentiate these species would be the secondary conidia and their attachment, but these characters could not be examined for the Ent. tipulae 2 specimen. More confusing still is Ent. tipulae 1, with conidia the size of Ent. tipulae but in the same clade as the lepidopteran pathogen Ent. maimaiga. It already has been suggested that the little-known Entomophaga conglomerata and Ent. tipulae could be the same because they cannot be distinguished by morphological features (Baazy 1993). These two species differ only in that Ent. conglomerata is known from mosquito larvae while Ent. tipulae is known from tipulids and limoniids. This confusion is not unusual for the Entomophthorales, which includes numerous species complexes, e.g., Entomophaga aulicae (Walsh 1996), Entomophaga grylli (Carruthers et al 1997), and Entomophthora muscae (Jensen & Eilenberg 2001), with component members very similar morphologically but differing in host range. We assume that Ent. tipulae is a complex of species within the Entomophaga clade, and we plan to further investigate relationships among Ent. tipulae, Ent. ptychopterae and Ent. transitans.
Which groups of characters define genera within the Entomophthorales has not always been agreed upon. Remaudière and Keller (1980) said that the shape of primary conidia should be the main criterion for generic classification, while Humber (1981) recommended that primary conidium shape should be secondary to nucleation, branching of sporophores and mode of discharge of primary spores. Our results support the monophyly of all species with pear-shaped conidia, thereby supporting the importance of primary conidial shape in generic grouping. Although the original five species in Eryniopsis produce at least some spore states that are elongate, whether these were primary or secondary was very important to generic grouping as revealed in this study. Our results would suggest that the morphology of secondary conidia is not as important as primary conidia in defining genera.
Our analysis reveals that Batkoa gigantea is closely related to Entomophaga. In fact, the genus Batkoa, including only five species, was once a subgenus of Entomophaga (Baazy 1993). All five species in Batkoa differ morphologically from Entomophaga because the primary conidia are globose with a sharply pointed papilla of semispherical shape while primary conidia of Entomophaga are pear-shaped with a non-apiculate papilla. In addition, the sporophores form an isthmus by which they join to the conidia while this is not seen in Entomophaga. While these two genera differ morphologically, our analysis demonstrates that Batkoa is closely related to Entomophaga. More sampling of Batkoa species and more characters might be needed to confidently infer the position of Batkoa in the Entomophthorales.
Our study brought to light the diversity of entomophthoralean species infecting Tipulidae. During our autumn sampling in Denmark, we found three entomophthoralean species infecting adult tipulids (Eryniopsis caroliniana, Entomophaga tipulae and Batkoa gigantea), often with infections from two entomophthoralean species at the same site and date. During this period, we also collected entomophthoralean infections in the closely related family Ptychopteridae. Baazy (1993) lists 10 species of Entomophthorales from Poland in five genera that are known from Tipulidae and close relatives. Such an abundance of entomophthoralean species infecting tipulids must be fostered by the moist habitats generally associated with larval and adult tipulids (Daly et al. 1978).
This study could be further elaborated by adding other species of Eryniopsis. Eryniopsis longispora was not included because it has been found only rarely and no cultures exist. Ethanol-preserved specimens of Ery. lampyridarum and Ent. transitans that had been collected four and seven years previously, respectively, were available to us, but amplification from these specimens was not successful. The in vivo specimens in this study (fungal cells in or on insect cadavers) had been collected and stored in ethanol only 1–2 months before use. Improved methods for the recovery of fungal DNA from insect cadavers in ethanol could go far toward aiding developments in entomophthoralean taxonomy and phylogeny.
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ACKNOWLEDGMENTS |
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azy for searching for Ery. longispora in the field. We thank K. Loeffler and J. Liebherr for their assistance with figures. We thank Cornell University for supporting AEH during her sabbatical at KVL. Further, the Carlsberg Foundation is thanked for financial support, enabling use of the Olympus microscope. |
FOOTNOTES |
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Accepted for publication June 27, 2002.
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LITERATURE CITED |
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azy S., 1993 Flora of Poland, Fungi (Mycota), Vol. 24. Entomophthorales. Krakow, Poland: Wydawnictwa Inst. Bot. Pan. 353 p Bulat SA, Lübeck M, Mironenko N, Jensen DF, Lübeck PS., 1998 UP-PCR analysis and ITS1 ribotyping of strains of Trichoderma and Gliocladium. Mycol. Res 102:933-943
Carruthers RI, Ramos ME, Larkin TS, Hostetter DL, Soper RS., 1997 The Entomophaga grylli (Fresenius) Batko species complex: its biology, ecology and use for biological control of pest grasshoppers. Mem. Entomol. Soc. Canada 171:329-353
Daly HV, Doyen JT, Ehrlich PR., 1978 Introduction to insect biology and diversity. New York: McGraw-Hill Book Co. 564 p
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White TJ, Bruns TD, Lee SB, Taylor JW., 1990 Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis MA, Gelfand DH, Sninsky JS, White TJ, eds. PCR protocols: a guide to methods and applications. San Diego, CA: Academic Press, p 315–321
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