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Spiroplasma leptinotarsae sp. nov., a Mollicute Uniquely Adapted to Its Host, the Colorado Potato Beetle, Leptinotarsa decemlineata (Coleoptera: Chrysomelidae)

K. J. HACKETT^1,*, R. F. WHITCOMB¹, T. B. CLARK^1,, R. B. HENEGAR¹, D. E. LYNN¹, A. G. WAGNER¹, J. G. TULLY², G. E. GASPARICH¹, D. L. ROSE², P. CARLE³, J. M. BOVɳ, M. KONAI¹, E. A. CLARK¹, J. R. ADAMS¹ and D. L. WILLIAMSON⁴

¹Insect Biocontrol Laboratory, U.S. Department of Agriculture, Beltsville, Maryland 20705
²Mycoplasma Section, Laboratory of Molecular Microbiology, National Institute of Allergy and Infectious Diseases, Frederick Cancer Research Facility, Frederick, Maryland 21702
³Laboratoire de Biologie Cellulaire et Moléculaire, Institut National de Recherche Agronomique, 33883 Villenave d'Ornon, France
⁴Department of Anatomical Sciences, State University of New York, Stony Brook, New York 11794

^* Corresponding author. Mailing address: Building 011A, Room 214, BARC-West, Beltsville, MD 20705. Phone: (301) 504-6325. Fax: (301) 504-5104.

ABSTRACT

Spiroplasma strain LD-1^T (T = type strain), which was isolated from the gut of a Colorado potato beetle (Leptinotarsa decemlineata) larva collected in Maryland, was serologically distinct from other spiroplasmas. Similar isolates were obtained from other L. decemlineata specimens collected in various parts of North America, in Poland, and in other eastern European countries and from Leptinotarsa texana specimens collected in Texas. Cells of strain LD-1^T, which in early passages were spiral, exhibited exceptionally rapid translational motility. This rapid motility and the spiral shape were lost after extended passage in culture. The organism required serum for growth. Originally isolated in coculture with insect cells in DCCM medium, strain LD-1^T adapted to several media in the absence of cocultured cells. Use of anaerobic conditions allowed primary isolation in a variety of media. The organism did not grow in serum-free media containing 2% serum fraction. Optimal growth in M1D medium occurred at 30 to 37°C (doubling time, 7.2 h). On solid M1D medium containing 2.0% Noble agar (pH 6.25) at 30°C, strain LD-1^T produced discrete colonies with numerous satellites. Strain LD-1^T hydrolyzed arginine, but did not utilize urea; there was evidence of weak fermentation of glucose. The guanine-plus-cytosine content of the DNA was determined to be 25 ± 1 mol%, and the genome size was 1,085 kb. The results of extensive studies of the ecology of this spiroplasma suggest that it is host specific for Leptinotarsa beetles. Strain LD-1 (= ATCC 43213) is designated the type strain of a new species, Spiroplasma leptinotarsae.

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