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Evaluating spore count and sporophorous vesicle size in Ovavesicula popilliae (Microsporidia: Ovavesiculidae) in adult Japanese beetles (Coleoptera: Scarabaeidae).

Ovavesicula popilliae (Andreadis & Hanula) (Microsporida: Ovavesiculidae) is a microsporidian pathogen of the Japanese beetle, Popillia japonica Newman (Coleoptera: Scarabaeidae), used as a biological control agent (Cappaert & Smitley 2002). This pathogen primarily infects the Malpighian tubules but often spreads systemically (Andreadis & Hanula 1987). Ovavesicula popilliae was originally described from Japanese beetles in Connecticut, USA (Andreadis & Hanula 1987) and has since been found infecting P. japonica in Michigan (Cappaert & Smitley 2002), Kentucky (Redmond & Potter 2010), and Arkansas (Petty et al. 2012). Ovavesicula popillae reportedly produces 32 ovoid spores within a persistent sporophorous vesicle with a mean size of 20.0-21.0 [micro]m x 15.0-15.5 [micro]m (Andreadis & Hanula 1987). Our objectives were to evaluate reported sizes of O. popilliae sporophorous vesicles and the number of spores contained within these vesicles.

Adult Japanese beetles were collected in Battle Creek, Michigan from the Charles Binder Park Golf Course from 12-17-VII-2012 with Jumbo Jug funnel mouth traps (1.9 L) (Trece Inc., Adair, Oklahoma) baited with compound floral and sex pheromone lures. O. popilliae infections were diagnosed by removing a small portion of Malpighian tubule from each beetle and examining them microscopically for sporophorous vesicles (Andreadis & Hanula 1987).

Beetle abdomens were macerated, and free sporophorous vesicles were examined under phase contrast microscopy at 400x magnification and measured with a calibrated ocular micrometer (n = 125). Spores within the vesicles were also counted at 1000x magnification. Due to overlapping of spores within a vesicle, vesicles were lightly crushed under their slide covers. Only vesicles that were not ruptured during this process had their spores counted (n = 100).

DNA extractions from the entire abdominal contents of an infected specimen was conducted (Sambrook & Russell 2001) and subjected to PCR using 2 [micro]L DNA template with the primers 18f and 1492r (Vossbrinck & Andreadis 2007). Amplicons were purified and sequenced in both directions (UAMS, Little Rock, Arkansas). Sequences were aligned visually and a BLAST (NCBI) search was conducted with Geneious 6.0.3 (Auckland, New Zealand).

Prevalence of O. popilliae-infected Japanese beetle adults collected from Michigan in 2012 was 25% (n = 100). Mean sporophorous vesicle size was 13.3 [micro]m (SE [+ or -] 0.1) x 9.3 [micro]m (SE [+ or -] 0.1) (Fig. 1). The size of sporophorous vesicles ranged from 9.6-16.8 [micro]m long and 7.2-13.2 [micro]m wide. Mean numbers of spores within each sporophorous vesicle was 24.7 (SE [+ or -] 0.8), ranging from 11-64 (Fig. 2), while vesicles containing 32 spores, reported as the typical number (Andreadis & Hanula 1987), occurred 27% of the time. DNA sequencing produced a 1,266 bp amplicon, the entire length of which was unambiguously aligned to the single O. popilliae SSU sequence available (GenBank, EF564602).

Our results of 24.7 spores/vesicle and vesicle size of 13.3 [micro]m x 9.3 [micro]m greatly differ from the original measurements of 32 spores/vesicle and a vesicle size of 20.0-21.0 [micro]m x 15.0-15.5 [micro]m reported by Andreadis & Hanula (1987). The variation in vesicle size and the number of spores is noteworthy for two reasons. Firstly, the vesicle size and number of spores differ from the original description, and they are used for taxonomic identification. Secondly, it is important to know the average number of spores/vesicle when planning experimental applications, such as making spore suspensions. Ovavesicula popilliae's sporophorous vesicles are highly persistent and do not readily break when disturbed. The assumption that 32 spores are present may lead to erroneous estimations of spore density.

One explanation for the difference in size of our sporophorous vesicles and those originally described may be that our O. popilliae sporophorous vesicles were collected from adult Japanese beetles. The host life stage, by some unknown mechanism, may affect vesicle size. Alternatively, the differences in sporophorous vesicle size may be evidence of the existence strains of the pathogen in addition to the one found in Michigan and the one was originally described from Connecticut.

The variability in spores may be due to failed divisions of the vegetative stages (Andreadis & Hanula 1987), an unknown effect of the adult beetle's immune system, death of spores, or some unknown factor. Number of spores/vesicle or size of vesicle may differ by host life stage or strain of O. popilliae and these variations should be recognized when working with this pathogen for practical purposes, such as counting spores in suspension or identification of taxa.

Caption: Fig. 1. Aggregated size distribution of Ovavesicula popilliae sporophorous vesicles, with the mean size (box and cross) of 13.3 [micro]m x 9.3 [micro]m (SE [+ or -] 0.1).

Caption: Fig. 2. Spore count distribution of Ovavesicula popilliae within sporophorous vesicles, with the mean spore count (bar) of 24.7 (SE [+ or -] 0.8).


ANDREADIS, T. G., AND HANULA, J. L. 1987. Ultrastructure study and description of Ovavesicula popilliae n. g., n. sp. (Microsporidia: Pleistophoridae) from the Japanese beetle, Popillia japonica (Coleoptera: Scarabaeidae). J. Protozoology 34: 15-21.

CAPPAERT, D. L., AND SMITLEY, D. R. 2002. Parasitoids and pathogens of Japanese beetle (Coleoptera: Scarabaeidae) in southern Michigan. Environ. Entomol. 31: 573-580.

PETTY, B. M., JOHNSON, D. T., AND STEINKRAUS, D. C. 2012. Survey of pathogens and parasitoids of Popillia japonica (Coleoptera: Scarabaeidae) in northwest Arkansas. Invert. Pathol. 111: 56-59.

REDMOND, C. T., AND POTTER, D. A. 2010. Incidence of turf-damaging white grubs (Coleoptera: Scarabaeidae) and associated pathogens and parasitoids on Kentucky golf courses. Environ. Entomol. 39: 1838-1847.

SAMBROOK, J., AND RUSSELL, D. W. 2001. Molecular Cloning: A Laboratory Manual 3rd edition, CSH Press, NY, 2,344 pp.

VOSSBRINCK, C. R., AND ANDREADIS, T. G. 2007. The phylogenetic position of Ovavesicula popilliae (Microsporidia) and its relationship to Antonospora and Paranosema based on small subunit rDNA analysis. J. Invert. Pathol. 96: 270-273.


Department of Entomology, 319 Agriculture Building, University of Arkansas, Fayetteville, Arkansas 72701, USA

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Article Details
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Author:Petty, B.M.; Tripodi, A.D.; Szalanski, A.L.; Johnson, D.T.; Steinkraus, D.C.
Publication:Florida Entomologist
Article Type:Report
Geographic Code:1USA
Date:Dec 1, 2013
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