Host Specificity of Microsporidia Pathogenic to Forest Lepidoptera

Biological Control 19, 48 –56 (2000) doi:10.1006/bcon.2000.0845, available online at http://www.idealibrary.com on

Host Specificity of Microsporidia Pathogenic to Forest Lepidoptera Leellen F. Solter,* ,1 Daniela K. Pilarska,† and Charles F. Vossbrinck‡ *Center for Economic Entomology, Illinois Natural History Survey, 607 East Peabody Drive, Champaign, Illinois 61820; †Institute of Zoology, Bulgarian Academy of Sciences, 1 Tzar Osvododite Boulevard, 1000 Sofia, Bulgaria; and ‡Department of Soil and Water, Connecticut Agricultural Experiment Station, 123 Huntington Street, New Haven, Connecticut 06504 Received January 25, 2000; accepted May 16, 2000

INTRODUCTION The host specificity of microsporidian pathogens of Lepidoptera was studied in Bulgaria where native populations of Lymantria dispar and their endemic microsporidia occur. L. dispar and sympatric lepidopteran larvae were collected from four sites in central and western Bulgaria. Three species of microsporidia, Vairimorpha sp., Nosema sp., and Endoreticulatus sp. are known to be endemic in three L. dispar populations, with one species in each population. No microsporidia were found in a fourth L. dispar population. In addition to the L. dispar microsporidia, 11 isolates of microsporidia were recovered from the 1494 individual lepidopteran hosts collected in these sites. When fed to L. dispar, 3 isolates produced infections that were atypical of infections in the natural hosts; one additional isolate produced an atypical infection in Spodoptera exigua. A Nosema sp. isolated from a noctuid host produced heavy infections in L. dispar larvae. Sequencing revealed that the noctuid microsporidium and the closely related Vairimorpha sp. and Nosema sp. microsporidia from L. dispar are distinctly different isolates. These investigations strengthen previous laboratory predictions of narrow host ranges for the Nosema and Vairimorpha microsporidia recovered from L. dispar in Europe. In addition, the Endoreticulatus sp., which was predicted from laboratory studies to be a generalist, was not found in Lepidoptera species sympatric with L. dispar. The results from our study indicate that laboratory testing may considerably underestimate the host specificity of many terrestrial microsporidia. Nevertheless, infectivity to nontarget hosts in the laboratory may set the stage for understanding the evolution of closely related microsporidia found in different host species. ©

The value of laboratory host specificity testing for predicting ecological host range and, thus, the risk of introduced organisms to nontarget species cannot be fully addressed without inclusion of field studies in the evaluation process. This presents a serious dilemma for researchers involved in classical biological control programs because introductions of exotic organisms into the field for the purposes of host specificity testing may be high risk, irretrievable, or both (Federici and Maddox, 1996). Laboratory studies have value for risk assessment of pathogens (Hajek et al., 1995, 1996; Hasan and Delfosse, 1995; Solter et al., 1997; Solter and Maddox, 1998a) but, because different organisms have different host range capabilities and biological attributes, each taxonomic group should be stringently evaluated and include the use of field studies when possible. One method for field testing the host specificity of a pathogen is to release and monitor the spread of the pathogen in the host population and to determine any effects on nontarget populations. This method presumes regulatory permission to release the pathogen, which is a circular problem in this type of study. An alternative method is to determine the host range of the pathogens of interest in the native area of the host and the pathogen. Both methods have been particularly problematic for the microsporidia because of the lack of distinguishing morphological characteristics in different but related species. Biochemical and molecular techniques are not yet well developed for identification of closely related species of microsporidia. Laboratory bioassays employed in previous studies, however, provided methods for assessing the host range of the microsporidia in sympatric populations of Lepidoptera (Solter et al., 1997; Solter and Maddox, 1998a). Long-term monitoring of Lymantria dispar (L.) populations in several sites in central and western regions of Bulgaria supplied valuable information about the presence of L. dispar microsporidia (Table 1). Three of four sites were monitored for 4 years (1995–1998) and

2000 Academic Press

Key Words: Endoreticulatus sp.; host specificity; Lepidoptera; Lymantria dispar; Nosema sp.; Vairimorpha sp.; ecological host range.

1 To whom correspondence should be addressed. Fax: (217) 3336784. E-mail: [email protected].

1049-9644/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.

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HOST SPECIFICITY OF LEPIDOPTERAN MICROSPORIDIA

TABLE 1 Collection Sites for Lymantria dispar and Nontarget Lepidoptera Site location and elevation (m)

Site

No. of years surveyed a 1984–1998

Microsporidia from L. dispar, 1984–1996

4

Endoreticulatus sp.

4 4

Nosema sp. —

Primary sites Asenovgrad Levishte Melnik Rupite

Forty Springs Reservoir 25 km SSE Plovdiv; 300 m 70 km N Sofia, Rt. 16; 800 m 22 km SSE Sandanski between Karlanovo and Sugarevo; 450 m 13 km SSW Sandanski; 200 m

11

Vairimorpha sp.

Additional Sites a Belgradchik Botev Pat Cheripish Gorni Lom Prevala Roshen Varna Veslec

1 1 1 1 1 1 1 1

— — — — — — — Nosema sp.

Note. —, No microsporidia found. a L. dispar only 1984 –1996 (Pilarska et al., 1998).

one site was monitored for 11 years during a 15-year period (1984 –1998; no collections were made during 1986 –1987 and 1990 –1991) (Pilarska et al., 1998). Three species of microsporidia in three different genera were found infecting L. dispar larvae. There was one species per site where microsporidia were found. Larvae from a fourth site were free of microsporidian disease during 4 years of monitoring. This situation suggested the possibility of screening the sympatric lepidopteran fauna for microsporidia and comparing all microsporidian species isolated to the L. dispar microsporidia found in each area, uncomplicated by multiple species infections in each L. dispar population. We hypothesized that microsporidia are species specific and that L. dispar microsporidia do not utilize other species of Lepidoptera as alternate hosts. We used feeding and transmission studies (Solter and Maddox, 1998a) to rule out microsporidia from other lepidopteran hosts that either did not infect L. dispar or produced atypical, nontransmissable infections and rDNA sequencing to determine whether infective isolates were the same as microsporidia known to be endemic in the L. dispar populations. MATERIALS AND METHODS

Collections. Four collection sites were selected in western Bulgaria based on the long-term monitoring of L. dispar populations in each of the areas (Table 1). Each site was surveyed two or three times between

May 17 and June 18, 1997, and two or three times between May 11 and June 26, 1998. L. dispar larvae were in the third to sixth stadia on host plants during this time. Along established trails of approximately 1 km in length, collections of lepidopteran larvae were made from the foliage and branches of oak trees, Quercus pubescens Willd., Q. cerris L., and Q. petraea (Matt.) Liebl., as well as adjacent Carpinus orientalis Mill. and other understory shrubs, from ground level to approximately 2 m above ground level. In the Melnik site, which did not have a trail, all oak trees and neighboring understory vegetation were examined in an area bordering a pasture of approximately 1 hectare in size. Branches and shrubs were examined visually and all observed larvae were collected. A 1-m 2 white cloth was then spread under the branches which were tapped sharply several times with a sturdy stick. All lepidopteran larvae falling onto the collecting cloths, including L. dispar larvae, were collected and placed into plastic bags with host plant foliage. L. dispar larvae were placed into separate bags. The larvae were stored at 4°C for 1 to 3 days until dissections could be completed. Examination of larvae and spore isolation. Larvae collected from each site were returned to the Bulgarian Academy of Sciences (BAS) laboratory in Sofia, separated by species, and individually examined. A cross section made at approximately the halfway point of the body length of each larva ensured that fat body, mid-

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gut, Malpighian tubules, dermal tissue, and silk glands were distinguishable, and these tissues were examined for microsporidian infection. Tissues from all individuals were examined as fresh squashes or, if necessary, were stained with Giemsa to make a definitive diagnosis (Vavra and Undeen, 1997). We were not able to identify most larvae to the genus or species level, but we identified the larvae to family level and determined whether individual specimens were the same or different species. All individual larvae that were infected with microsporidia were either identified to the genus level or vouchered in 70% ethanol for future identification. Tissues from infected larvae were Giemsastained as voucher specimens. The remaining tissues were homogenized in a glass tissue grinder to release microsporidian spores and centrifuged. The pellet of spores was resuspended in tap water in cryovials, and 5 ␮g streptomycin and 360 units fungizone were added to each vial (Undeen and Solter, 1996). The spores were stored at 4°C until they were transported to the Illinois Natural History Survey (INHS) insect pathology laboratory. Bioassays. L. dispar larvae used as hosts in laboratory studies at BAS in 1998 and at INHS in both 1997 and 1998 were supplied by the USDA/APHIS Laboratory at Otis Air Force Base, Massachusetts. At BAS, larvae were maintained on fresh oak leaves in the laboratory at room temperature and under natural light conditions. Microsporidia isolated from the dissected lepidopteran larvae, including L. dispar, were each immediately fed to five third instar L. dispar larvae. Spore suspensions at a concentration of 10 5 spores/␮l (Solter et al., 1997) were spread on the surface of small pieces of oak foliage, approximately 3 cm 2, and placed with the larvae in 100-ml plastic diet cups with paper lids. When sufficient material was available, a few additional larvae were fed spores directly from crushed host tissues smeared on the leaves. Treated larvae were fed fresh oak leaves daily and were dissected at 12–15 days postexposure to determine whether infection occurred. Microsporidia transported to the INHS laboratory were fed to third instar L. dispar larvae as well as to second and third instar Spodoptera exigua (Hu¨bner) larvae the day following arrival in the United States. The spore suspensions were spread on the surface of meridic diet (Yearian et al., 1966; Bell, 1981) and 5 larvae were allowed to feed in each 30-ml plastic diet cup for 48 h. Each isolate was fed to a minimum of 10 S. exigua larvae and 20 L. dispar larvae. More larvae were exposed when sufficient spore suspension was available. S. exigua larvae were transferred to fresh diet, 5 larvae/30-ml cup; L. dispar larvae were transferred to fresh diet in 100-ml cups, 10 larvae/cup. All larvae were reared in growth chambers at 24°C, 16/8 h light/dark at a relative humidity of approximately 60%.

S. exigua larvae were examined at 8 –10 days postexposure due to a short life cycle under the conditions in which they were held, but this duration is sufficient for development of infective spores for lepidopteran microsporidia (Maddox, 1968; Solter and Maddox, 1998b). L. dispar larvae were examined at 15–18 days postexposure. Four or 5 larvae from each treatment group were dissected, and midgut, silk glands, fat body, Malpighian tubules, dermal tissue, and gonads were individually examined for microsporidian infection under phase contrast microscopy at 400⫻ magnification. Fresh squash preparations were made of all remaining larvae and each sample was examined for presence of spores and immature forms. Giemsa stains were made in all cases in which presence of microsporidia was not obvious from fresh tissue preparations (Undeen and Vavra, 1997). Sequencing. Using the techniques of Vossbrinck et al. (1993), the small subunit ribosomal DNA (ss-rDNA) and internal transcribed spacer region were sequenced for three isolates, Vairimorpha sp. from L. dispar collected in Rupite, Nosema sp. from L. dispar collected in Levishte, and Nosema sp. from Orthosia sp. (Noctuidae) collected in Rupite. RESULTS

Isolation and identification of microsporidia from L. dispar. L. dispar larvae were collected and examined in 1997 and 1998 to confirm the presence of the microsporidian species reported in previous surveys (Table 2). The same three microsporidian species were recovered from the three host populations sampled in previous seasons, and no microsporidia were recovered from the Melnik site (Pilarska et al., 1999). No additional microsporidian species were isolated from L. dispar in these sites. In addition, several new sites were surveyed (Tables 1 and 2). Microsporidian-infected L. dispar larvae were found in one other site, Veslec, which is located near the Levishte site. The microsporidium recovered appears morphologically and biologically to be the same biotype as the Nosema sp. consistently found in Levishte. Isolation and identification of microsporidia from Lepidoptera sympatric with L. dispar. Over the two collecting seasons, 1494 insect larvae, sympatric with L. dispar and feeding on the same trees and shrubs, were collected from the four sites. These included larvae from 12 families of Lepidoptera, several species of sawflies (Hymenoptera), and a small number of coleopteran larvae (Table 3). Each larva was individually examined to determine presence of microsporidia. Eleven microsporidian isolates in three genera, Endoreticulatus, Nosema, and Vairimorpha, were recovered from noctuid and tortricid Lepidoptera. No microsporidia were recovered from the equally common geometrid species or from other, less common, species.

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HOST SPECIFICITY OF LEPIDOPTERAN MICROSPORIDIA

TABLE 2 Microsporidia Collected from Lymantria dispar in Bulgaria a Site Asenovgrad

Levishte

Melnik

Rupite

Other sites surveyed Belgradchik Botev Pat Cheripish Gorni Lom Prevala Roshen Varna b Veslec Totals 1997–1998 a b

Date collected

No. L. dispar collected

No. L. dispar infected

05/21/97 06/04/97 06/26/97 05/21/98 06/07/98 05/23/97 06/06/97 06/18/97 05/19/98 05/30/98 05/18/97 06/03/97 05/12/98 05/25/98 06/16/98 05/17/97 06/04/97 06/20/97 05/11/98 05/13/98 05/26/98

100 56 27 73 18 192 89 36 3 2 42 118 71 75 24 270 154 109 98 14 73

0 2 3 0 1 3 7 2 0 0 0 0 0 0 0 0 1 2 0 0 1

June, 1997 06/12/98 05/23/97 06/18/98 06/18/98 06/15/98 June, 1997 06/12/98

37 63 100 48 17 12 135 47 2103

0 0 0 0 0 0 0 5 27

Microsporidia collected (genus)

Endoreticulatus sp. Endoreticulatus sp. Endoreticulatus sp. Nosema sp. Nosema sp. Nosema sp.

Vairimorpha sp. Vairimorpha sp.

Vairimorpha sp.

Nosema sp.

These data included with previous years collections in Pilarska et al. (1998). Two populations.

Bioassays. Four of the 11 microsporidian isolates from larval lepidopteran hosts were infectious to L. dispar larvae in the laboratory (Table 4). Of these, 2 isolates from tortricid larvae produced atypical infections in L. dispar. These isolates appeared to have elicited an immune response or the L. dispar host was otherwise unsuitable; few mature spores were produced and those spores were atypically small, rounded, and thin walled. One of the 2 tortricid isolates infected S. exigua, but few larvae became infected and few spores were produced. Nosema-type spores from an unidentified dead host also infected L. dispar larvae fed crushed host tissues, but only primary spores (internally infectious spore forms) developed and the host larvae died 4 days postexposure. No infections developed when spores were fed at concentrations typical for 100% infection with lepidopteran Nosema spp., approximately 10 5 spores (0 infections/20 additional larvae fed). An Endoreticulatus sp. isolated from Euproctis chryssorhea (L.) was marginally infective to S. exigua, producing an unusual cellular immune response of large bubble-like membranes surrounding multiple

parasitophorous membrane-bound sporoblasts in the midgut tissues. This isolate was not infectious to L. dispar. One Nosema-type isolate from a noctuid host, Orthosia sp., collected in Rupite, produced an infection typical of those observed in natural hosts in both L. dispar and S. exigua. It infected the silk glands of L. dispar and was morphologically very similar to the Nosema sp. isolated from L. dispar in Levishte. The Orthosia isolate, the L. dispar Levishte Nosema sp., and the Rupite Vairimorpha sp. were sequenced and compared to a L. dispar Vairimorpha sp. from the Czech Republic (Table 5). The ss-rDNA sequence of the Vairimorpha sp. from the Rupite site is identical to the Czech Republic isolate. The L. dispar and the Orthosia sp. Nosema-type isolates are both closely related to the Vairimorpha isolates. The Orthosia sp. isolate differs by 1 bp from the Levishte L. dispar isolate, nucleotide No. 813 from the beginning of the approximately 1400-bp sequence reported to GenBank, and by 2 bp from the Vairimorpha spp. isolates, nucleotides Nos. 810 and 813. The Nosema isolate from Levishte differs

TABLE 3 Microsporidia Collected from Lepidoptera Sympatric with Lymantria dispar in Bulgaria Site and date collected Asenovgrad 05/21/97

06/12/97 06/26/97 05/21/98

06/07/98 Levishte 05/23/97

06/10/97

06/20/97 05/19/98

05/30/98 Melnik 05/18/97

06/03/97

05/12/98

05/25/98

06/16/98

Host family

No. species/family

Total individuals/family

No. infected

Arctiidae Geometridae Noctuidae Notodontidae Tortricidae Noctuidae Geometridae Unidentified Lepidoptera Lymantriidae Noctuidae Tortricidae Unidentified Lepidoptera

1 12 4 1 7 — — — 1 7 8 1

1 70 10 2 36 7 4 5 3 7 43 1

0 0 1a 0 3b 0 0 0 0 0 0 0

Geometridae Noctuidae Tortricidae Unidentified Lepidoptera Hymenoptera c Geometridae Notodontidae Nymphalidae Tortricidae Unidentified Lepidoptera Unidentified Lepidoptera Geometridae Tortricidae Unidentified Coleoptera larvae Hymenoptera Unidentified Lepidoptera

13 1 4 6 1 1 1 1 2 2 2 2 6 3⫹ 1 4 2

23 1 5 9 2 1 1 1 3 7 3 5 99 9 4 37 2

0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0

Geometridae Lasiocampidae Lycaenidae Noctuidae Notodontidae Tortricidae

11 2 1 15 1 12

44 10 10 24 1 62

0 0 0 0 0 5

Hymenoptera c Geometridae Lasiocampidae Noctuidae Psychidae Tortricidae Unidentified Lepidoptera Arctiidae Dilobidae Geometridae Lasiocampidae Lycaenidae Noctuidae Thyatiridae Tortricidae Unidentified Lepidoptera Hymenoptera c Dilobidae Geometridae Lasiocampidae Lymantriidae Noctuidae Tortricidae Hymenoptera c Unidentified Lepidoptera

2 1 2 3 1 5 2 1 1 16 1 1 15 1 11 5 4 1 10 1 1 7 14 1 —

5 1 4 4 1 71 5 21 1 86 11 11 18 1 211 31 18 9 16 1 6 18 123 1 3

0 0 0 0 0 1 0 0 0 0 0 0 0 0 6b 1 0 0 0 0 1e 0 2b 0 0

Microsporidia collected

Nosema sp. Vairimorpha sp.

Nosema sp. d

Vairimorpha sp. (4 larvae) Nosema sp. (4 larvae)

Nosema-type b,d sp.

Vairimorpha sp. Nosema-type d sp.

Endoreticulatus sp. Vairimorpha sp.

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HOST SPECIFICITY OF LEPIDOPTERAN MICROSPORIDIA

TABLE 3—Continued Site and date collected Rupite 05/17/97

06/04/97

06/20/97 05/11/98

05/26/98

Host family

No. species/family

Total individuals/family

No. infected

Arctiidae Geometridae Noctuidae Nymphalidae Tortricidae Unidentified Lepidoptera Hymenoptera c Geometridae Nymphalidae Tortricidae Unidentified Lepidoptera Hymenoptera c Unidentified Lepidoptera Dilobidae Geometridae Lasiocampidae Noctuidae Tortricidae Unidentified Lepidoptera Dilobidae Lymantriidae Geometridae Tortricidae

1 15 21 1 6 3 2 1 1 2 3 2 2 1 13 1 8 4 1 1 1 4 2

2 56 50 1 21 5 16 1 2 2 3 8 2 2 25 1 12 29 2 3 1 8 13 1494

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1f 0 0 0 0 0 0

Total 1997–1998

Microsporidia collected

Nosema sp.

Note. —, Pupae or dead larvae, unable to determine species. a Unknown host species. b Archips xylosteana. c Various sawfly species larvae. d Possibly Vairimorpha sp. e Euproctis chrysorrhea. f Orthosia sp.

at nucleotide No. 810 from the Vairmorpha spp. isolates. The sequences of the isolates were entered into the Genbank database, Accession No. AF141129 for the Levishte isolate from L. dispar and Accession No. AF141130 for the isolate from Orthosia sp. DISCUSSION

In an effort to determine the usefulness of laboratory studies to predict ecological host specificity, Solter and Maddox (1998a) addressed the ecological complexity of the environment in which infective spores (environmental spores) of microsporidia, produced in nontarget hosts, are encountered by conspecific nontarget individuals. They fed microsporidia isolated from other Lepidoptera to L. dispar larvae and compared horizontal transmission between infected and uninfected L. dispar larvae to horizontal transmission between individuals of the natural hosts. Of nine microsporidian species that infected L. dispar, one species was transmitted from infected L. dispar larvae to uninfected L. dispar larvae, but significantly fewer L. dispar larvae than natural host larvae became infected with this species. The findings of these experiments, as well as extensive field data showing that no North American

L. dispar populations examined have acquired native microsporidia (Campbell and Podgwaite, 1971; Podgwaite, 1981; Andreadis et al., 1983; T. G. Andreadis, personal communication), suggested that suboptimal development of the microsporidia in the nontarget hosts indicated a much higher level of host specificity than would have been predicted from interpretation of traditional bioassays (Solter et al., 1997). Solter and Maddox (1998a) also designed simple laboratory tests to better approximate ecological host specificity. Nevertheless, field data from aboriginal populations were needed to test these predictions and to assess the ecological host specificity of the microsporidia under consideration for biological control of L. dispar. Although the data collected in the field in the aboriginal area of the pathogens and L. dispar are preliminary, they support the predictions made from laboratory studies about the host specificity of the L. dispar microsporidia. No L. dispar microsporidia were found in lepidopteran larvae feeding sympatrically with L. dispar larvae. At least four species of microsporidia, probably five, were isolated from the larvae. The Vairimorpha sp. isolates recovered from Archips xylosteana L. (Tortricidae) are sufficiently similar in morphology

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TABLE 4 Susceptibility of Lymantria dispar and Spodoptera exigua to Microsporidia Collected from Sympatric Lepidopteran Hosts Collection site

Microsporidia recovered

Host (family, genus or species)

Infectivity to L. dispar (No. infected/No. tested) a

Infectivity S. exigua (No. infected/No. tested)

Asenovgrad

Vairimorpha isolate, 05/21/97 Nosema isolate c, 05/21/97 Nosema isolate, 05/19/98 Vairimorpha isolate, 05/18/97 Nosema isolate, 05/18/97 Nosema isolate c, 06/03/97 Vairimorpha isolate, 05/12/98 Nosema isolate c, 05/12/98 Endoreticulatus isolate, 05/25/98 Vairimorpha isolate, 05/25/98 Nosema isolate, 05/11/98

Noctuidae species Tortricidae species Tortricidae species Tortricidae species Tortricidae species Tortricidae species Archips xylosteana Unidentified dead larva Euproctis chrysorrhea Archips xylosteana Orthosia sp.

No (0/33) No (0/10) No (0/18) Yes b (4/25) No (0/31) Yes b (1/17) No (0/68) d Yes b,e (5/25) No (0/19) No (0/19) Yes f (39/39)

No (0/36) No (0/10) No (0/6) Yes b (2/16) No (0/24) Not tested No (0/16) No (0/6) Yes b (4/10) No (0/7) Yes f (4/8)

Levishte Melnik

Rupite a

Larvae recovered from bioassays at both BAS and INHS in 1998; infectivity data combined. Infections atypical; few or no infectious spores produced. c Possible Vairimorpha sp. d Microsporidia from three different individuals tested separately; data combined. e Atypical infection in the BAS laboratory was not duplicated in the INHS bioassays. f Infection typical of that expected in natural host; isolated from Orthosia sp. (Noctuidae). b

and pathogenesis to suggest that these isolates are conspecific. They did not infect L. dispar in laboratory tests. A Vairimorpha isolated from a different tortricid species produced atypical infections in L. dispar and S. exigua, as did one Nosema-type isolate from tortricids. It is possible that these two isolates (Vairimorphatype, 05-18-97 and Nosema-type, 06-03-97, Melnik) are conspecific, but octospores were observed only in the Vairimorpha-type isolate. The E. chrysorrhea Endoreticulatus sp. is different from the L. dispar species. Further laboratory testing confirmed its noninfectivity to L. dispar. The Nosema sp. isolate from Orthosia sp. was the only microsporidium that produced infective mature spores at the levels characteristic of infection in a natural host. A Vairimorpha sp. isolated from a noctuid host in Asenovgrad may be the same or different from the tortricid isolates. Of the isolates recovered, three produced atypical infections in L. dispar and were clearly not adapted to this host. Infections categorized as atypical produced few environmental spores, well below the spore numbers needed for horizontal transmission in previous studies (Solter and Maddox, 1998a), and three of the

isolates produced atypical development in S. exigua. The Nosema-type microsporidium that was isolated from a noctuid host, Orthosia sp., collected in the Rupite site, produced infections in L. dispar that were typical of those produced in the Orthosia sp. host. The morphology of the vegetative forms and environmental spores, as well as the life cycle and tissues infected, suggest that this species is similar to the Nosema sp. found in L. dispar populations in the Levishte site, approximately 275 km north of the Rupite site where the Orthosia sp. isolate was recovered. Nosema-type microsporidia, however, have not been found in the Rupite L. dispar population in 11 years of monitoring. Although there was only 1-bp difference between the ss-rDNA sequences of the L. dispar Nosema sp. from Levishte and the new Orthosia sp. isolate, these microsporidia, as well as the Vairimorpha sp. from Rupite, are shown to be closely related but distinctly different isolates. In addition, the rDNA sequences may be too conservative to clearly distinguish closely related microsporidian species (Baker et al., 1994). Other isolates of microsporidia recovered from sympatric lepidopteran hosts did not infect L. dispar.

TABLE 5 a

Nucleotides No. 806 – 815 of the Small Subunit Ribosomal DNA of Three Closely Related Lepidopteran Microsporidia

a

Microsporidium

Collection site

Host

Sequence

Nosema sp. Nosema sp. Vairimorpha sp. Vairimorpha sp.

Levishte, Bulgaria Rupite, Bulgaria Rupite, Bulgaria Czech Republic

Lymantria dispar Orthosia sp. Lymantria dispar Lymantria dispar

GATTTTTAATC GATTTTTAATC GATTCTTAATC GATTCTTAATC a

GenBank Accession No. AF033315.

HOST SPECIFICITY OF LEPIDOPTERAN MICROSPORIDIA

We found the same species of microsporidia to be consistently present in each L. dispar population; no new microsporidian species were found in the more than 1600 individual larvae that we examined from the four main populations. The microsporidia that we isolated from other Lepidoptera were not found in more than one host species, with the possible exception of the Vairimorpha sp. from a noctuid in Asenovgrad. The Endoreticulatus sp. from L. dispar in Asenovgrad was not recovered from other Lepidoptera, despite previous laboratory studies of an Endoreticulatus isolate from L. dispar in Portugal that demonstrated infectivity to other lepidopteran species (Solter et al., 1997). Although the host specificity data from the sympatric Lepidoptera were preliminary due to the short-term nature of the study, the long-term data set produced by monitoring microsporidian parasitism of L. dispar populations suggests that many if not most of these lepidopteran microsporidia are quite host specific. Nevertheless, the close relationship between the Orthosia sp. Nosema and the L. dispar Vairimorpha and Nosema indicate that microsporidia may adapt to new hosts, establishing the foundation for evolution of new species. Several deficiencies in these studies were unavoidable, and we address them here. The prevalences of microsporidia in both years were low for all populations of L. dispar that we surveyed. Nevertheless, previous surveys showed higher prevalences, as well as the consistent presence of each microsporidian species in the L. dispar populations (Pilarska et al., 1999). In addition, the results of the large sample of lepidopteran larvae indicate that these sympatric species probably do not serve as reservoirs for the L. dispar microsporidia. We could identify most of the sympatric lepidopteran hosts only to the family level but were able to identify to the genus level the host of the only microsporidium that was strongly infective to L. dispar. The larva was identified as Orthosia sp., possibly Orthosia gothica D. & S. (Cso´ka, 1995; (G. Cso´ka, personal communication). We believe that this situation would have been much more problematic had more species harbored microsporidia or had we been unable to identify any that produced mature infective spores in L. dispar. In a number of cases, only one or a few individual larvae of a species were collected in the L. dispar feeding sites. We attempted to address this problem, inherent in field studies of this type, by collecting for two seasons. Additional difficulties were our inability to voucher all collected larvae because of the necessity of destructive sampling, and the swelling and discoloration of larvae in ethanol that interfered with matching of species collected the following year. Thus, some species listed by family are duplicated in subsequent collections and others were collected only once.

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During the first year, we did not have uninfected L. dispar larvae in the BAS laboratory with which to conduct bioassays with freshly isolated spores. In the second year, all microsporidia were fed to second and third instar L. dispar larvae immediately upon isolation in the BAS laboratory. Bioassays with the same isolates in the INHS laboratory in 1998 produced the same results as those in the BAS laboratory when fed to L. dispar larvae. S. exigua was included as a second experimental host to assess viability of species that did not infect L. dispar and to harvest isolates that might have adequately infected a host other than L. dispar. Only the microsporidium that produced heavy, hostlike infections in L. dispar, however, produced heavy infections in S. exigua. Despite the difficulties encountered in a study of this nature, we believe that the results from these field studies strengthen the predictions that we previously made based on laboratory testing (Solter et al., 1997; Solter and Maddox, 1998a). The host ranges of the L. dispar microsporidia and those of the other lepidopteran microsporidia found in the sites that we studied are probably quite narrow. Our findings suggest that L. dispar microsporidia could play a role as an environmentally safe addition to the natural enemy complex of L. dispar in North America. ACKNOWLEDGMENTS The authors are indebted to the following persons for assistance with this project: M. Todorov, J. Donova, G. Csoka, G. Georgiev, P. Pilarski, M. Pilarski, A. Corbin, A. Benderev, I. Ilieva, V. Cerafimov, V. Golemansky, G. Markova, S. Beshkov, and T. Dudevsky. Valuable suggestions were made by J. Vavra and M. Higgs on an earlier draft of the manuscript. This project was supported in part by the Illinois Natural History Survey, the Bulgarian Academy of Sciences, Institute of Zoology, and USDA/FAS/ICD/RSED Project No. 58-31487-013.

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