Chapter 7 Microsporidian Entomopathogens y y Leellen F. Solter,* James J. Becnel and David H. Oi * Illinois Natural History Survey, University of Illinois, Champaign, Illinois, USA, y United States Department of Agriculture, Agricultural Research Service, Gainesville, Florida, USA Chapter Outline 7.1. Introduction 221 7.4. Biological Control Programs: Case Histories 235 7.2. Classification and Phylogeny 222 7.4.1. Use of Microsporidia in Biological Control 7.2.1. Overview of Microsporidian Entomopathogens Programs 235 in a Phylogenetic Context 223 7.4.2. Aquatic Diptera 236 Amblyospora/Parathelohania Clade 223 Edhazardia aedis 237 Nosema/Vairimorpha Clade 225 Amblyospora connecticus 238 7.2.2. General Characteristics of Microsporidia 226 Life-cycle Based Management Strategies 238 Morphology 226 7.4.3. Lepidopteran Pests in Row Crop Systems 239 Genetic Characters 227 7.4.4. Grasshoppers and Paranosema locustae 240 7.3. Life History 229 7.4.5. Fire Ants in Urban Landscapes 242 7.3.1. Infection and Replication 229 Kneallhazia solenopsae 242 7.3.2. Pathology 229 Vairimorpha invictae 245 7.3.3. Transmission 230 7.4.6. Control of Forest Insect Pests 246 7.3.4. Environmental Persistence 231 Impacts of Naturally Occurring Microsporidia 7.3.5. Life Cycles 231 on Forest Pests 247 Life Cycles of Microsporidia in the Nosema/ Spruce Budworm Microsporidia 247 Vairimorpha Clade 231 Microsporidian Pathogens of Gypsy Moth 249 Life Cycle of Vavraia culicis 233 7.4.7. Microsporidia Infecting Biological Control Life Cycle of Edhazardia aedis 233 Agents 250 7.3.6. Epizootiology and Host Population Effects 234 7.5. Future Research Directions 251 7.3.7. Host Specificity 234 References 252 microsporidia in biological control programs focuses on inocu- SUMMARY lative introductions, augmentative release, and conservation Microsporidia, pathogenic protists related to the Fungi, are biology. This chapter reviews the biology, ecology, pathology, and considered to be primary pathogens of many aquatic and terres- classification of microsporidia with examples of several long-term trial insect species and have important roles in insect population research efforts to manipulate these pathogens for the suppression dynamics, managed insect disease, and biological control of insect of insect pests. pests. Hosts are infected when spores are ingested and/or by transmission via the eggs. When ingested, spores germinate in a unique fashion: a polar tube that is coiled within the spore rapidly everts and punctures the host midgut cells, injecting the 7.1. INTRODUCTION spore contents into the cell cytoplasm. Mitochondria and Golgi bodies are lacking in these obligate intracellular pathogens, and “Enigmatic”, in the words of Maddox and Sprenkel (1978) energy is evidently extracted from host cells via direct uptake of and echoed by succeeding generations of researchers ATP. Effects on the host are typically chronic; therefore, the use of (Canning et al., 1985; Bigliardi, 2001; Susko et al., 2004; Insect Pathology. DOI: 10.1016/B978-0-12-384984-7.00007-5 221 Copyright Ó 2012 Elsevier Inc. All rights reserved. 222 Insect Pathology James et al., 2006), aptly describes the group of fascinating orthopteran species (see Section 7.4.4); Vavraia (Pleisto- but elusive pathogens comprising the phylum Micro- phora) culicis introduced into Culex mosquito populations sporidia. This particular label remains a not-so-subtle in Nigeria, where establishment was not confirmed; indication of the difficulties encountered in studies of these Nosema pyrausta, a pathogen of the European corn borer, organisms and the still perplexing phylogenetic relation- Ostrinia nubilalis, introduced from Iowa as an augmenta- ships at all taxonomic levels. Nevertheless, it is important tive control in Illinois O. nubilalis populations, and per- to consider the role of the microsporidia in natural systems sisting in the Illinois populations; and the European gypsy and in biological control programs because they are nearly moth (Lymantria dispar) pathogens Nosema portugal and ubiquitous pathogens of insects and other arthropods. Most Endoreticulatus sp. (E. schubergi, ¼ Vavraia sp., ¼ pleis- of the more than 1300 described species in approximately tophora schubergi introduced into an isolated gypsy moth 186 genera are pathogens of invertebrate animals, with population in Maryland. Persistence of N. portugal was insects being type hosts of nearly half of described genera confirmed for one year (see Section 7.4.6). While probably (Becnel and Andreadis, 1999). Close inspection of most lacking utility as microbial insecticides, these typically insect taxa yields new species (Brooks, 1974). All micro- chronic pathogens clearly have a role as major components sporidia are obligate intracellular pathogens of protists and of the natural enemy complex of many insect species animals, including both warm-blooded and cold-blooded (Kohler and Wiley, 1992; Maddox, 1994; Lewis et al., vertebrates. They do not reproduce as free-living organisms 2009) and warrant continued study for their use in classical, and they are not plant pathogens. augmentative and conservation biological control Microsporidia are important primary pathogens of both programs. pest and beneficial insects. The first known micro- sporidium, Nosema bombycis, was described from the 7.2. CLASSIFICATION AND PHYLOGENY silkworm, Bombyx mori,byNa¨geli (1857), who considered the pathogen to be a yeast. Symptoms caused by Only in the past 15 years have microsporidian genes been N. bombycis infection, such as dark spots on the larval analyzed to the extent that placement of this eukaryotic integument, were first noted by Jean Louis Armand de pathogen group in the Protozoa by Balbiani (1882) was Quatrefages in the early 1800s and named “pe´brine” seriously questioned. Recent molecular studies placed the (pepper) disease. Louis Pasteur’s studies of pe´brine disease phylum Microsporidia within the kingdom Fungi (Keeling (Pasteur, 1870) provided methods of prevention and control and Doolittle, 1996; Hirt et al., 1999; Bouzat et al., 2000; and were credited with saving the silkworm industry in James et al., 2006; Hibbett et al., 2007), with evidence, France (see Chapters 2 and 12). albeit controversial, that the group is related to the ento- The silkworm was the first beneficial insect noted to be mopathogenic Zygomycetes (Keeling, 2003; Lee et al., devastated by microsporidia, but several microsporidian 2008; Corradi and Slamovits, 2010). Koestler and Ebers- species have serious impacts on other managed insects that berger (2011) point out that although Microsporidia and are reared in high-density colonies, as well as on natural Zygomycetes alone among the fungi share a three-gene populations of beneficial insects (Chapter 12). Two cluster encoding a sugar transporter, a specific transcription microsporidian species, Nosema apis and N. ceranae, are factor, and RNA helicase, clustering of these genes appears pathogens of honey bees; N. ceranae has been implicated to be ancestral in eukaryotes and is, therefore, not phylo- as a contributing factor in the decline of managed bees genetically informative. Questions about the phylogenetic (reviewed by Paxton, 2010), although effects of the disease placement of microsporidia thus remain without definitive reported in the literature are highly variable. Nosema answers. Although the predominance of evidence places bombi is a Holarctic generalist pathogen of bumble bees microsporidia within the fungal lineage (Corradi and (Bombus spp.). High prevalence levels in some Bombus Keeling, 2009), their morphology, biology, and host inter- species that are apparently in decline have led to questions actions are unique. In this chapter, the microsporidia are about the possible introduction of virulent strains and the treated as a separate group of insect pathogens. potential role these pathogens play in permanently The first modern classification for the microsporidia reducing Bombus spp. range and population numbers in was proposed by Tuzet et al. (1971) and since that time, the North America (Thorp and Shepherd, 2005; Cameron classification of the microsporidia at all taxonomic levels et al., 2011). has been controversial (reviews by Sprague, 1977a; Microsporidia are also important as regulatory factors Sprague et al., 1992; Tanada and Kaya, 1993; Franzen, in populations of insect pests. Hajek et al. (2005) listed five 2008). The classical morphological approach (based on microsporidian species that have been introduced as bio- nuclear arrangement, spore shape, structure and size, nature logical control agents globally. They include Paranosema of vesicles, etc.) and molecular-based methods have (Nosema) locustae used as a microbial insecticide for produced conflicting interpretations (Vossbrinck grasshoppers and established in Argentina in several and Debrunner-Vossbrinck, 2005; Larsson, 2005). A Chapter | 7 Microsporidian Entomopathogens 223 phylogenetic tree was created using rDNA data from 125 Amblyospora/Parathelohania species of microsporidia from vertebrate and invertebrate Clade hosts (Vossbrinck and Debrunner-Vossbrinck, 2005). Five The Amblyospora/Parathelohania clade contains more than major clades were identified, and three major groups based 122 of the approximately 150 species of microsporidia that on host habitat (freshwater, marine, and terrestrial) were have been described from mosquito hosts (Amblyospora suggested as being more consistent with evolutionary more than 100 species, Parathelohania about 22 species), relationships than