First Record of a Neozygites species (Zygomycetes:Entomophthorales)Infecting Springtails (Insecta:Collembola)

T. STEENBERG,1 J. EILENBERG, AND J. BRESCIANI

Department of Ecology and Molecular Biology, Section of Zoology, Royal Veterinary and Agricultural University,Bulowsvej 13, 1870 Frederiksberg C., Denmark

Received January 11, 1996; accepted May 17, 1996

Afungal pathogen from theEntomophthorales (Zygo-mycetes) was discovered in populations of the lucerneflea Sminthurus viridis (Collembola) collected fromgrassland and leguminous crops in Denmark duringJuly to October. The morphology of the fungus wasstudied in springtails, collected live, which succumbedto fungal disease following incubation in the labora-tory. Based on these preliminary morphological stud-ies the fungus appears to be an undescribed species ofNeozygites. Field studies showed the fungus to beenzootic with a widespread geographical distributionin Denmark. At one location fungus prevalence in S.viridis reached a maximum of approximately 12% in-fected individuals. No fungus-killed cadavers wereobserved in the field. This is the first well-documentedreport of an entomophthoralean fungus infecting anapterygote insect. A single individual was infected bythe entomophthoralean fungus Conidiobolus corona-tus. Verticillium lecanii and Beauveria bassiana (Deu-teromycotina:Hyphomycetes) were also isolated fromS. viridis. r 1996 Academic Press, Inc.

KEY WORDS: Neozygites sp.; Conidiobolus coronatus;Verticillium lecanii; Beauveria bassiana; Sminthurusviridis; lucerne flea; entomopathogenic fungus; natu-ral occurrence.

INTRODUCTION

Insects from the order Collembola (springtails) usu-ally feed on decaying vegetable matter, and few areregarded as pests (Jones and Jones, 1964). However,epigeic springtails of the family Sminthuridae areknown to be occasional pests of a range of crops (Davies,1926, 1928; Smith, 1931). Plants are especially vulner-able to attack in the early stages of development, wheresminthurids have been reported to feed on seedlings of

a variety of plants. The lucerne flea Sminthurus viridisis very common in agricultural habitats, especially onlegumes, and is distributed worldwide (Wallace, 1974).In parts of Australia and in Tasmania it is presentlyconsidered a major exotic pest in pastures and legumecrops (Hopkins and Taverner, 1991; Ridshill-Smith,1991).Natural enemies of collembolans are not well known,

but include different predatory arthropods, primarilymites (Christiansen, 1964). Parasitism by pathogenicmicroorganisms has only rarely been reported fromfield populations of collembola. Purrini (1983) recordedthe occurrence of unidentified viral, bacterial, fungal,and protozoan diseases from 12 collembolan species inEurope, and found protozoan infections to predomi-nate. Bacterial and viral infections in cultures of collem-bola have also been reported (Christiansen, 1964).Among the fungi, species of the ectoparasitic Laboulbe-niaceae have been found (Paclt, 1956) as well asArthrobotrys species actively trapping collembola(Drechsler, 1944; Onofri and Tosi, 1992).Little information is available about fungal patho-

gens of collembola. A species ofHirsutella isolated froman unidentified collembolan is held in the ARS Collec-tions of Entomopathogenic Fungal Cultures (ARSEF,1992), and in the Polish host list for Zoophthoracurvispora (Zygomycetes:Entomophthorales) a collem-bolan (Podura sp.) is included (Balazy, 1993). Visseret al. (1987) described the fungal species associatedwith a collembola in a woodland habitat (Onychiurussubtenuis), listing the entomopathogenic species Beau-veria bassiana (Deuteromycotina:Hyphomycetes) alongwith different saprophytes. To our knowledge thereis no information available on the natural occurrenceof pathogens of S. viridis, including its fungal patho-gens.Recently we found S. viridis to be infected with

different species of entomopathogenic fungi, includinga zygomycete fungus of the order Entomophthorales.We report here the first well-documented finding ofsuch a fungus from an apterygote insect, including a

1 To whom correspondence should be addressed. Present affiliation:Danish Pest Infestation Laboratory, Skovbrynet 14, 2800 Lyngby,Denmark.

JOURNAL OF INVERTEBRATE PATHOLOGY 68, 97–100 (1996)ARTICLE NO. 0065

97 0022-2011/96 $18.00Copyright r 1996 by Academic Press, Inc.

All rights of reproduction in any form reserved.

preliminary morphological description, ecological dataof field prevalence, and geographical distribution inDenmark.

MATERIALS AND METHODS

Sampling Sites

The fungus was first encountered on 18 July 1994 ina single individual of S. viridis collected live in a field ofwinter wheat in Aagerup, located approximately 25 kmwest of Copenhagen. Subsequently, live sminthuridswere sampled weekly with a sweep net at this locationfor the following 4 weeks. Because sminthurid densityvaried greatly in time and place, sampling at thislocation also took place in hedgerows surrounding thefield and in a neighboring field of Phleum pratense. Inaddition, sminthurids were collected from three otherlocations: a lucerne field in Taastrup (18 km west ofCopenhagen) on 24 July, and from fields of white cloverand red clover, respectively, near Herning in westernDenmark on 6 August. Additional sampling was madein August 1995 in Aagerup and Taastrup, and inTaastrup in October 1995.

Live Incubation

Live S. viridis (a mixture of stages) were transferredindividually to 30-ml plastic cups with a layer of 2%water agar at the bottom to maintain high humidityand incubated at 20°C in constant light. A lucerneleaflet (Medicago sativa) placed in the water agar wasprovided as food. Mortality was recorded every day for10 days, and fungus-killed cadavers were removed forfurther investigation.

Identification of the Fungus

Primary and secondary spores of the fungus wereprojected under humid conditions from cadavers ontoglass slides and stained with 0.001% lactophenol-cottonblue (Keller, 1987). Nuclei in primary conidia andhyphal bodies were stained in 1% aceto-orcein usingthe methods of Keller (1987). Sporulating cadaverswere prepared for scanning electron microscopy usingthe method of Eilenberg et al. (1986). A few cadaverswere dissected to study hyphal bodies. The fungus wasidentified using the key of Keller (1987).In vitro isolation of the fungus was attempted on

solid medium (Sabouraud DextroseAgar supplementedwith 8% egg yolk and 2% milk) and with the liquidmedium of Beauvais and Latge (1988).

RESULTS

Morphological Features of Neozygites sp. (Figs. 1–7)

Fungus-infected insects collapsed soon after deathdue to their soft cuticle. Cadavers subsequently disinte-

grated quickly and putrefied. Rhizoids were not ob-served. Hyphal bodies were spherical with four nuclei.Conidiophores were unbranched. Large numbers ofprimary conidia were forcibly projected from freshlykilled cadavers. The conidia were unitunicate andpyriform with a flat but distinct papilla and containedfour nuclei stainingweakly in aceto-orcein.Mean dimen-sions of primary conidia (measurements at 4003 mag-nification of three series of each 20 conidia) were 12.2 3

6.4 µm (range of length 11–14 µm; range of width 6–8µm). Secondary conidia were more spherical than pri-mary conidia, had a more rounded base, and wereproduced from primary conidia on short, lateral conidio-phores. No capilliconidiogenesis was observed, nor werecystidia or resting spores found. This morphology fittedthe description ofNeozygites.The attempts of in vitro isolation of the fungus were

unsuccessful.

Field Prevalence of Neozygites sp.

In 1994,Neozygites sp. was found in populations of S.viridis collected from several locations in Denmarkduring July and August (Table 1). The proportion ofinfected individuals in the population reached a maxi-mum of 12.3% in a field of red clover in Jutland. Thefungus was found again in 1995 in Aagerup andTaastrup during July to October. In 1995 fungus preva-lence was very low (below 5%). In addition, Conidiobo-lus coronatus, Verticillium lecanii, and B. bassianawere found infecting a few specimens of lucerne flea.

DISCUSSION

Based on the size and morphology of primary conidiaand the number of nuclei within fungal structures, thefirst entomophthoralean fungus species to be foundinfecting Collembola was likely to belong to the genusNeozygites. However, the species described here infect-ing S. viridis is presumed to be a new species since itdoes not fit existing descriptions of Neozygites species,which comprises 9 species according to Keller (1991).So far, species of Neozygites have been described fromaphids, mites, and thrips, and appear to have relativelynarrow host ranges (Keller, 1991).TheNeozygites sp. infection in S. viridis did not reach

epizootic levels. However, it was widespread and com-mon, being found in all four locations sampled and, ontwo successive sampling seasons. The pathogenicity ofNeozygites sp. to other species of Collembola or otherinsect species still needs to be evaluated.At no time were fungus-killed cadavers observed in

the field, probably because sminthurids are tiny andinconspicuous insects, easily overlooked. Infected speci-mens were found to disintegrate quickly in the labora-tory. There is no doubt that the reason for only rarelyfinding pathogens, including fungal pathogens, in this

98 STEENBERG, EILENBERG, AND BRESCIANI

FIGS. 1–4. Micromorphology ofNeozygites sp. infecting lucerne flea Sminthurus viridis: scanning electronmicroscopy.FIG. 1. Sminthu-rus viridis killed by Neozygites sp. (scale bar 200 µm). FIG. 2. Emerging conidiophores and conidia of Neozygites sp. (scale bar 10 µm). FIG.3. Landscape of emerging conidiophores and discharged conidia on the soft cuticle of Sminthurus viridis (scale bar 10 µm).FIG. 4. Primaryconidium attached by mucus to host seta (scale bar 2 µm).

FIGS. 5–7. Micromorphology of Neozygites sp. infecting lucerne flea Sminthurus viridis: differential interference contrast. Scale bar 10µm. FIG. 5. Primary conidia of Neozygites sp. FIG. 6. Secondary conidia of Neozygites sp. FIG. 7. Formation of a secondary conidium ofNeozygites sp.

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group of insects is mainly due to not searching for them,or not conducting the investigation with live-collectedspecimens.The discovery that entomopathogenic fungi can serve

as natural mortality factors for springtails is interest-ing not only from an ecological point of view, but alsobecause they representmicrobial control agents againstsminthurids. Currently, few attempts have been madeto control sminthurids with microbial agents. Rath(1991) described field experiments with the entomo-pathogenic fungus Metarhizium anisopliae against S.viridis in field trials, in which mortalities reached up to100% at relatively low soil temperatures. Because thelucerne flea is mainly a pest in low-value crops such aspastures with high damage thresholds, this would be afurther incentive for a fungus to be used for control ofthis insect, since control need not be 100% efficient orfast acting. In addition, springtails from other familiesoccasionally cause problems in forest nurseries andglasshouse crops (Scopes and Ledieu, 1983), and arepotential target hosts for fungi in a microbial controlprogram.

ACKNOWLEDGMENTS

We express our gratitude to Dr. S. Keller (Eidg.Forschungsanstaltfur landw. Pflanzenbau, Zurich, Switzerland) and Dr. R. A. Humber(U.S. Plant, Soil and Nutrition Laboratory, Ithaca, NY) for their helpin fungus identification. D. Berthelsen, K. Dromph, C. Nielsen, andB.W. Jørgensen contributed to the field collection and preparation forSEM. This study was funded by the Danish National EnvironmentalResearch Program.

REFERENCES

ARSEF. 1992. ‘‘Collection of Entomopathogenic Fungal Cultures:Catalog of Strains, 1992.’’ U.S. Department of Agriculture, Agricul-tural Research Service, ARS-110, pp. 1–177. Washington, DC.

Balazy, S. 1993. Flora of Poland. Fungi (Mycota) XXIV.Beauvais, A., and Latge, J. P. 1988. A simple medium for growingEntomophthoralean protoplast. J. Invertebr. Pathol. 51, 175–178.

Christiansen, K. 1964. Bionomics of collembola. Annu. Rev. Entomol.9, 147–178.

Davies, W. M. 1926. Collembola injuring leaves of mangold seedlings.Bull. Entomol. Res. 17(2), 159–162.

Davies, W. M. 1928. On the economic status and bionomics ofSminthurus viridis Lubb. (Collembola). Bull. Entomol. Res. 18(3),291–296.

Drechsler, C. 1944. A species of Arthrobotrys that captures springtails.Mycologia 36, 382–399.

Eilenberg, J., Bresciani, J., and Latge, J. P. 1986. Ultrastructuralstudies of primary spore formation and discharge in the genusEntomophthora. J. Invertebr. Pathol. 48, 318–324.

Hopkins, D. C., and Taverner, P. D. 1991. Damage caused by mitesand fleas in pastures. Plant Prot. Q. 6(4), 166–167.

Jones, F. G. W., and Jones, M. 1964. ‘‘Pests of Field Crops.’’ Arnold,Sevenoaks.

Keller, S. 1987. Arthropod-pathogenic Entomophthorales of Switzer-land. I. Conidiobolus, Entomophaga and Entomophthora. Sydowia40, 122–167.

Keller, S. 1991. Arthropod-pathogenic Entomophthorales of Switzer-land. II. Erynia, Eryniopsis, Neozygites and Zoophthora. Sydowia43, 39–122.

Onofri, S., and Tosi, S. 1992. Arthrobotrys ferox new-species, aspringtail-capturing hyphomycete from continental Antarctica.Mycotaxon 44(2), 445–451.

Paclt, J. 1956. Bionomie und Okologie. In ‘‘Primar flugellosen Insek-ten,’’ pp. 91–117. Gustav Fischer, Jena.

Purrini, K. 1983. Comparison of pathogenic agents in Collembola(Insecta:Apterygota) from different forests in the Federal Republicof Germany, Austria and Spain. Pedobiologia 25, 365–371.

Rath, A. C. 1991. Pathogens for the biological control of mite andCollembolan pests. Plant Prot. Q. 6(4), 172–174.

Ridsdill-Smith, T. J. 1991. Biology and ecology of redlegged earthmite, blue oat mite and lucerne flea. Plant Prot. Q. 6(4), 159–161.

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Smith, K. M. 1931. ‘‘Agricultural Entomology.’’ Cambridge UniversityPress, Cambridge, UK.

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Wallace, M. M. H. 1974. Present and probable world distribution ofSminthurus viridis and prospects for its biological control.Pedobio-logia 14, 238–243.

TABLE 1Field Occurrence of Neozygites sp. in Lucerne Flea

(Sminthurus viridis) Collected in Different Locations in Den-mark, July andAugust, 1994

Location and crop DateNumberincubated

%Infection

Aagerup, Zealand; grass July 25 82 12.2Aagerup, Zealand; grass August 1 66 6.1Aagerup, Zealand; grass August 10 115 0.9Aagerup, Zealand; grass August 15 5 0.0Taastrup, Zealand; lucerne July 24 47 2.1Barde, Jutland; white clover August 6 105 5.7Barde, Jutland; red clover August 6 57 12.3

100 STEENBERG, EILENBERG, AND BRESCIANI