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Department of Ecology, The Royal Veterinary and Agricultural University, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Denmark
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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We developed new Entomophthora-specific primers for nested-PCR of the ITS II region to be used on in vivo material and combined it with RFLP. Resting spores from Scathophaga stercoraria (3 specimens), Delia radicum (9 specimens), Botanophila fugax (1 specimen), and two syrphid host species, Platycheirus peltatus and Melanostoma mellinum (one specimen of each) were characterized genetically after analysis of RFLP-profiles of the PCR-products. The genetic characterization of the resting spore isolates was compared with twenty isolates of known primary conidial morphology (in vitro and in vivo) from the E. muscae species complex. The analysis allowed for the first time a separation of resting spore isolates into the species level, which is not possible only using morphological characters (diameter). Isolates originating from different specimens of the same host taxa appeared to be strongly clonal even they were sampled at different localities in different years. Isolates morphologically belonging to E. muscae s. str. (e.g., including E. scatophagae) could be separated genetically further into sub-groups entirely depending on the host taxa; each fungal genotype, either present at the conidial stage or at the resting spore stage, is correlated with one host species. Furthermore, E. muscae s. str. originating from D. radicum proved to be much more closely related to E. scatophagae than to E. muscae s. str. originating from M. domestica. None of the resting spore isolates could be assigned to E. schizophorae. The nested-PCR approach accompanied by RFLP proved its usefulness for identification of resting spores and for more detailed studies clarifying host-pathogen specificity and interactions. It seems that different members of the E. muscae species complex are able to complete their life cycle in only one host species and, further, that each pathogen-host system is independent.
Key words: Anthomyiidae, Diptera, Entomophthorales, fungal entomopathogens, host specificity, ITS rDNA, Muscidae, RFLP, Scathophagidae, Syrphidae, Zygomycotina
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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, Carruthers et al 1985, Eilenberg 2000, Klingen et al 2000), Muscidae (Steinkraus et al 1993, Six and Mullens 1996, Keller et al 1999), Fanniidae (Mullens et al 1987), and Psilidae (Eilenberg and Philipsen 1988). Because many of these host species are pests, E. muscae s. l. is a potential candidate for biological control of pest flies. The actual use of E. muscae s. l. for practical biological control purposes is, however, still hampered by our lack of knowledge about important aspects of the fungal life cycle.
Four separate species have been described from the E. muscae species complex. Entomophthora muscae sensu stricto was redescribed from the original host Musca domestica Linnaeus by Keller et al (1999). Entomophthora scatophagae Giard with a conidial morphology similar to E. muscae s. str. was described from Scathophaga stercoraria (Linnaeus) (Giard 1888) and emended by Steinkraus and Kramer (1988). The validity of the low-nuclear-number form, described as E. schizophorae Keller & Wilding (Keller 1987) and the high-nuclear-number form, E. syrphi Giard (Giard 1888), as separate species were recently supported by conidial morphology and molecular data (Jensen and Eilenberg 2001). A fifth, still undescribed species, E. muscae "group B" was identified by Keller (1984).
Most of the fly species acting as hosts for members of the E. muscae species complex overwinter in the temperate parts of the world as egg, larvae, or pupae. Thus overwintering as well as the timing of activity in the spring are critical points in the fungal life cycle since adult hosts are to be found. Yet, events related to infection in the spring are still very poorly understood.
Many entomophthoralean fungi produce thick-walled zygospores or azygospores, generally known as resting spores, enabling them to survive periods with unfavorable weather conditions and/or shortage of hosts (Hajek 1997). Entomophthora muscae s. l. resting spores are found in agriculturally important fly species from Anthomyiidae (Wilding and Lauckner 1974, Carruthers et al 1985, Klingen et al 2000, Thomsen and Eilenberg 2000). Both E. muscae s. str. originating from D. radicum as well as E. muscae "group B" originating from Botanophila fugax (Meigen) are able to form resting spores (Thomsen et al 2001). Resting spores of E. syrphi are described in McLeod et al (1976) from Melanostoma mellinum (Linnaeus), whereas resting spores of E. scatophagae have not been reported before. The overwintering strategy of E. schizophorae is unclear and resting spores of this species has never been observed in vivo, although it has the ability to form resting spores in vitro (Eilenberg et al 1990).
Molecular characteristics of isolates have previously been used to differentiate between species and/or isolates within Entomophthorales. Several studies have used RAPD profiles to differentiate between isolates or species (Hodge et al 1995, Hajek et al 1996, Rohel et al 1997, Nielsen et al 2001). However, as RAPD employs unspecific primed PCR, the use of this method depends on pure cultures. Entomophthoralean fungi are mostly difficult to isolate and grow in vitro and the conditions required for resting spore germination from species within the genus Entomophthora are still unknown (Thomsen et al 2001). Therefore, RAPD analysis is at present not applicable to differentiate between species of Entomophthora-resting spores.
Resting spores from field-collected grasshoppers could be assigned to one of the three generally recognized Entomophaga grylli (Fres.) Batko pathotypes within the E. grylli species complex, when pathotype-specific probes were hybridized to DNA extracts from dead infected grasshoppers (Bidochka et al 1995). This technique is useful for monitoring the distribution of already characterized species/isolates within a species complex. Though at present there is no knowledge of the genetic variation of resting spores in E. muscae s. l. and the actual number of different species/isolates within the species complex is uncertain. Consequently, the development of some species-specific probes would not explore the actual variation in the E. muscae species complex.
The use of PCR with fungus specific primers followed by digestion with different restriction endonucleases (RFLP-PCR) was successfully used to differentiate between species and/or isolates without prior cultivation for other fungi (Sanders et al 1995, Leal et al 1997). The internal transcribed spacer regions (ITS I and ITS II) of the nuclear rDNA repeat are in general more variable than the coding regions of the rDNA and have been widely used for taxonomic studies at or near the species level (Seifert et al 1995, Bridge and Arora 1998). Jensen and Eilenberg (2001), using entomophthoralean specific primers, found that the ITS II region was especially variable in the genus Entomophthora. The use of a two-step PCR with nested primers increases both the sensitivity and the specificity of the PCR significantly which can overcome problems with the relatively impure DNA extract resulting from the use of in vivo material (Albert and Fenyö 1990, Goller et al 1998).
The purposes of the present study were to develop a method to extract DNA from resting spore-bearing fly cadavers. Further, by use of a nested PCR-RFLP approach to examine the genetic variation in the E. muscae species complex in order to analyze and to compare the presence of resting spores from the different species of the E. muscae species complex in different fly hosts to isolates with a known conidial morphology. We also compared the molecular groupings with resting spore morphology.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). Protoplasts were collected by centrifugation for 10 min at 2000 x g. Conidia-forming isolates were assigned to species by the number of nuclei in the primary conidia, the size of the primary conidia, and the host identity (Keller 1987, Steinkraus and Kramer 1988, Keller et al 1999, Jensen and Eilenberg 2001). Conidia projected onto a glass slide were stained with the flourochrome DAPI (4,6-diamino-2-phenylindole) at a concentration of 5 µg/mL, and the numbers of nuclei in the primary conidia were counted using a Zeiss epirescence microscope (filter 0.5; violet, excitation wavelength 395–440 nm).
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DNA extraction and amplification –
All the material was lyophilized before DNA extraction. Resting spores were carefully crushed with a micro pestle for 1.5 mL Eppendorf tubes. Otherwise, the DNA extraction was carried out as described by Jensen and Eilenberg (2001). Amplification of the ITS II was done by nested PCR, consisting of two successive PCRs. The first PCR used DNA extracted from the sample as template and an outer pair of primers: nu-5.8S-5' (5'-TCA TCG ATG AAG AAC GTA GT-3') (Jensen and Eilenberg 2001) and ITS 4 (5'-TCC TCC GCT TCT TGA ATA GC-3') (White et al 1990). The second PCR used the first PCR product as template and a second, inner pair of primers nested within the region amplified by the first PCR: ITS II 20-5' (5'-ACA GGA GGT TTG TTT GTT TG-3') and ITS II 1500-3' (5'-CTT GCT TGA TTT GAA ATG WAG-3'). These last two primers were designed based on the sequences of ITS II from E. schizophorae, E. muscae s. str. (one isolate from M. domestica and one isolate from D. radicum), E. scatophagae, and E. syrphi (Jensen, Thomsen, and Eilenberg, unpubl) as well as the sequence of the ribosomal repeat for Entomophaga aulicae and Penicillium clavigerum with GenBank accession numbers U35394 and L14533, respectively. Both primer sets were positioned in the coding regions of the 5.8 s rDNA and LSU, respectively, and thus the whole ITS II region was amplified (Fig. 1). The PCR conditions were initial denaturation for 5 min at 96 C followed by 35 cycles with denaturation for 1 min at 96 C, annealing for 1 min at 62 C (ITS II) or 58 C (nested-ITS II), extension for 1 min at 72 C, and a final extension for 10 min at 71 C. The PCR reactions were carried out in 25 µL volume with 250 µM of each dNTP, 0.4 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), 0.5 unit DyNAzyme II DNA polymerase (Finnzymes), and 1 µL diluted DNA extraction or ITS II-PCR product as template. DNA extracts of in vivo and in vitro isolates were diluted 1:10 and 1:100 respectively, whereas the ITS II-PCR products were diluted 1:1000. The size of the PCR amplifications was estimated by electrophoresis on a 1.5% agarose gel in 1x TBE buffer, and the products visualized with ethidium bromide.
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Cluster analysis –
For each of the eight enzymes the length of the different fragments was used as a character, and then all the fragment lengths were scored as present or absent for each of the isolates, giving one data matrix. With the computer software package NTSYS-PC version 2.0 (Applied Biostatistics, Inc.) the similarities between the isolates were calculated using the Jaccard coefficient (Jaccard 1908) or the Dice coefficient (Nei and Li 1979), and dendrograms were made using the UPGMA (unweighed pair-group methods analysis). Support for internal branches was assessed with 2000 bootstrap replications using the software package NTSYS TreeCon for Windows (Van de Peer and de Wachter 1994).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Within the genus Entomophthora, length differences of the nested-ITS II amplifications were observed. The nested ITS II fragments amplified from all isolates of the E. muscae species complex, E. chromaphidis, and E. planchoniana had a length of approximately 1300 bp, while the fragment amplified for E. thripidum as expected from the ITS II was approximately 1050 bp. Two fragments with lengths of approximately 1300 and 450 bp were amplified from E. culicis (ITS II fragment approximately 2000 bp) with the nested primers. When present, the fragments amplified by the nested primers were approximately 850 bp for E. ptycopterae (ITS II 
1000 bp) and approximately 950 bp for E. maimaiga (ITS II 1100 bp) (data not shown).
RFLP – Within the E. muscae complex considerable variation of the nested-ITS II was seen using the PCR-RFLP and a total of 59 characters were scored. Polymorphism was seen with all the applied enzymes.
UPGMA analyses of the nested ITS II RFLP data using either the Jaccard or the Dice coefficient separated the E. muscae complex into three main clusters, with E. syrphi in one clade (I), E. schizophorae in a second clade (II), and E. muscae (type B and s. str. including E. scatophagae) in a third clade (III) (Fig. 2). The E. syrphi and the E. muscae clades were supported by bootstrap percentages above 72%.
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Within the E. muscae clade, isolates originating from resting spores clustered with the in vitro/in vivo isolates from the same host species. The isolates within this clade were further separated into two sub-clades: an E. muscae s. str. clade (IIIA) and an E. muscae-type B clade (IIIB) with all three isolates from Botanophila fugax.
Five, five, and four out of the eight restriction endonucleases showed polymorphism between E. muscae-type B and E. muscae s. str. from D. radicum, M. domestica, and E. scatophagae, respectively, although the bootstrap support for the type B clade was only 56%. No genetic variation was detected among the three E. muscae-type B isolates (Fig. 2).
Further, the E. muscae s. str. clade was separated into three clusters entirely depending on the host species: one S. stercoraria cluster (IIIAa), one D. radicum cluster (IIIAb), and one M. domestica cluster (IIIAc). Only very little genetic variation was detected between the D. radicum isolates, and no variation was detected between the S. stercoraria isolates (Fig. 2). The M. domestica cadaver (305*) tentatively infected by E. scatophagae (305) clustered together with the other E. scatophagae isolates from S. stercoraria (Fig. 2). None of the resting spore isolates could be assigned to E. schizophorae.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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, Thomsen and Eilenberg 2000, Thomsen et al 2001). Some host species can naturally be infected by more than one species from the E. muscae species complex (Keller et al 1999, Thomsen and Eilenberg 2000), thus impeding species determinations from naturally occurring resting spores (Thomsen and Eilenberg 2000). Although we found significant differences among resting spore isolates, we were unable to correlate these differences with host taxa or morphology. Therefore, a differentiation of resting spores from different members of the E. muscae species complex only by morphological characters is not possible.
PCR-RFLP of the ITS II has proven valuable for distinguishing among the different species from the E. muscae species complex (Jensen and Eilenberg 2001). By the aid of the nested-PCR approach implemented in this study the taxonomic determination of DNA isolated from fly cadavers bearing resting spores becomes possible for the first time. The specificity of the nested primers allowed consistent amplification only from Entomophthora species, and occasionally among species from the closely related genera Entomophaga and Eryniopsis.
Analysis of RFLP-profiles of the resting spore isolates allowed for the first time a separation of these at the species level based on the results from the in vitro isolates. None of the resting spore isolates could be assigned to E. schizophorae (Fig. 2). Thus, the mechanism for overwintering by this species remains unclear. The overall topology of the dendrogram was in concordance with a previous study on the genetic variation in the genus Entomophthora (Jensen and Eilenberg 2001). From the dendrogram, E. muscae-type B proved to be more related genetically to E. muscae s. str. than to E. schizophorae (Fig. 2). Most of the resting spore isolates were genetically identical to other isolates from the same host taxa that were found sporulating with conidia, except for the isolates originating from syrphids. We regard E. syrphi to be a complex of morphological similar species attacking different species from Syrphidae.
It seems that all species from the E. muscae complex attacking outdoor fly hosts complete a full life cycle in a single host species. Concerning E. muscae, E. scatophagae, and E. syrphi, overwintering takes place as resting spores, while E. schizophorae apparently overwinters by other means.
We found that different E. muscae isolates from the same host species appeared to be strongly clonal since they had very little variability. This lack of variability was observed despite the fact that the isolates were sampled at different localities in Denmark and Norway in different years, and some were present in the resting spore form and some in the conidial form. This agrees with the results from a study on the genetic variation of E. muscae s. str. using only in vitro isolates (Jensen et al 2001). Bidochka et al (1995) likewise found that isolates within pathotypes of E. grylli were strongly clonal. Within groups of isolates originating from the same host taxa of the widespread and host-nonspecific Zoophthora radicans, Hodge et al (1995) also found a large genetic homogeneity among isolates. In contrast, studies on the aphid pathogen P. neoaphidis demonstrated that this species is highly heterogeneous, and that groupings were more related to geographic origin of the isolates than to the host species (Rohel et al 1997, Nielsen et al 2001).
The physiological host range is relatively broad within the E. muscae species complex, as demonstrated by the reports of laboratory transmission of E. muscae s. l. across host families (Kramer and Steinkraus 1981, Steinkraus and Kramer 1987, Mullens 1989, Steenberg et al 2001) and even across insect orders (Eilenberg et al 1987). Our results show that such transmissions are unlikely to take place in the field. The ecological host range of a particular isolate seems to be limited to one species or maybe a few very closely related species. The development of epizootics is an interaction between one genotype of E. muscae and one host species.
Interestingly, our results demonstrated that E. muscae s. str. from D. radicum is much more closely related to E. scatophagae than to E. muscae s. str. from M. domestica. In an earlier study on resting spore production by different strains of E. muscae s. l. in M. domestica, E. muscae s. str. originating from M. domestica failed to produce resting spores, whereas the same morphotypes originating from D. radicum readily formed resting spores (Thomsen et al 2001). It was hypothesized that E. muscae s. str. affecting M. domestica from temperate climates had almost no interaction with isolates from fly species outdoors and is capable of overwintering without resting spores (Thomsen et al 2001). Our data support this hypothesis.
The application of the nested PCR-RFLP approach in this study to identification of resting spores from the E. muscae complex to species level proved to be very well suited for that purpose. The sensitivity and specificity of the method allows the direct use of in vivo material, irrespective of whether conidia or resting spores are formed in the cadavers. This, together with the high variability of the ITS II in the genus Entomophthora, makes the use of this approach ideal to study host-pathogen specificity and interactions in the E. muscae species complex in much more detail, including the geographical variation of the complex. Such detailed studies would improve our understanding of the spread and development of Entomophthora epizootics in insect populations at the regional level.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Accepted for publication May 13, 2002.
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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