Molecular and ultrastructural characterization of Andreanna caspii n. gen., n. sp. (Microsporida: Amblyosporidae), a parasite of Ochlerotatus caspius (Diptera: Culicidae) Anastasia V. Simakova a , Charles R. Vossbrinck b , Theodore G. Andreadis b, * a Tomsk State University, Lenina Street, 32, Tomsk 634050, Russia b The Connecticut Agricultural Experiment Station, 123 Huntington Street, New Haven, CT 06511, USA article info Article history: Received 13 May 2008 Accepted 18 July 2008 Available online 25 July 2008 Keywords: Andreanna caspii Microsporidia Molecular phylogeny Ultrastructure Ochlerotatus caspius Mosquito abstract A new genus and species of microsporidia, Andreanna caspii n. gen., n. sp. is described from the mos- quito, Ochlerotatus caspius (Pallas) based on ultrastructural morphology, developmental characteristics, and comparative sequence analyses of the small subunit (SSU) ribosomal DNA (rDNA). Parasite devel- opment is confined to fat body tissue and infected larvae appear swollen with dull white masses within the thorax and abdomen. Meronts have diplokaryotic nuclei and are delineated by a simple plasmalemma contiguous with the host cell cytoplasm. Merogony occurs by synchronous binary divi- sion followed by cytokinesis. Diplokaryotic sporonts undergo meiosis and synchronous nuclear divi- sion forming sporogonial plasmodia with two, four and eight nuclei enclosed within a persistent sporophorous vesicle. Cytokinesis of sporogonial plasmodia results in the formation of eight uninucle- ate spores. The episporontal space of early sporonts is filled with a homogeneous accumulation of electron dense granular inclusions and ovoid vesicles of various dimensions, transforming into an interwoven matrix during the initial phase of sporogenesis. Spores are oval, uninucleate and measure 4.8 ± 0.3 Â 3.1 ± 0.4 lm (fixed). The spore wall is 260 lm thick with an irregular exospore consisting of two layers (150–170 lm) and a thinner endospore (90–100 lm). The anchoring disk is well devel- oped and is contiguous with a lamellar polaroplast that occupies the anterior third of the spore and possess more narrow lamellae on the posterior end. The polar filament is gradually tapered and arranged in a single row consisting of six coils ranging from 180 to 150 lm in diameter. The posterior vacuole (posterosome) is moderately sized and filled with a matrix of moderate electron density. Phy- logenetic analysis of SSU rDNA from A. caspii and 30 other species of microsporidia including 11 gen- era parasitic in mosquitoes using maximum parsimony, neighbor joining and maximum likelihood methods showed A. caspii to be a sister group to the clade containing all of the Amblyospora species, including Culicospora, Edhazardia and Intrapredatorus, as well as Culicosporella and Hyalinocysta thus providing strong support for establishment of Andreanna as a separate genus. Ó 2008 Elsevier Inc. All rights reserved. 1. Introduction Microsporidia are among the largest and most diverse group of parasitic microorganisms which infect mosquito populations in nature. At present, approximately 150 different species represent- ing 23 genera have been described, 15 of which are monotypic (only one species is known) (Andreadis, 2007). Generic classifica- tions within this group have traditionally been based on developmental cycles associated with merogony (vegetative repro- duction) and sporogony (production of spores), nuclear organiza- tion, and morphology and fine structure of developmental stages and spores (Larsson, 1986, 1988, 1999; Sprague et al., 1992; Can- ning and Vavra, 2000). More recently, small subunit ribosomal DNA (SSU rDNA) sequence data have been used to validate new genera and species and determine their phylogenetic placement (Nilsen and Chen, 2001; Andreadis and Vossbrinck, 2002; Vossbr- inck et al., 2004; Franzen et al., 2006). Unfortunately, gene se- quences have been conducted on fewer than half of the described genera infecting mosquitoes. While conducting a survey of temporary vernal pool habitats in the Tomsk region of Siberia in Russia, several larvae of Ochlerotatus caspius (Pallas) were discovered with patent infections of a novel microsporidium. In this investigation we describe the ultrastruc- tural morphology of all stages of development of the microsporidi- um in the mosquito host, and further examine SSU rDNA sequence data to determine its phylogenetic placement among other mosquito–parasitic microsporidia. Based on morphological, 0022-2011/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.jip.2008.07.004 * Corresponding author. Fax: +1 203 974 8502. E-mail address: theodore.andreadis@po.state.ct.us (T.G. Andreadis). Journal of Invertebrate Pathology 99 (2008) 302–311 Contents lists available at ScienceDirect Journal of Invertebrate Pathology journal homepage: www.elsevier.com/locate/yjipa