metabolites of the free-living amoeba phreatamoeba balamuthi analyzed by 13c- and 31p-nmr...

SAUER & KELLY-RESCUE OF TETRAHYMENA EXOCYTOSIS MUTANTS 183

58. Tooze, S. A. & Huttner, W. B. 1990. Cell-free protein sorting to the regulated and constitutive secretory pathways. Cell, 602337-847.

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J. Euk Microbial.. 42(2), 1995, pp. 183-191 0 1995 by the Society of Protozoologists

Metabolites of the Free-Living Amoeba Phreatamoeba balamuthi Analyzed by 13C- and 31P-NMR Spectroscopy: Occurrence of

Phosphoinositol Diphosphates JEAN-BAPTISTE MARTIN,* TILLY BAKKER-GRUNWALD** and GERARD KLEIN***.'

*Luboratoire de Rbonance Magndique en Biologie et Mkdecine, Dkparternent de Biologie Molkculaire el Structurale, CEA- Centre d'Etudes Nuclkaires de Grenoble. France,

**Abteilung Mikrobiologie, Universitat Osnabriick, Germany, and ***Labomtoire de Biologie Cellulaire (URA I130 du CNRS), Dkparternent de Biologie Molkculaire et Structurale,

CEA-Centre d%tudes Nuclkaires de Grenoble, France

ABSTRACT. Phreatarnoeba bafumuthi is a free-living heterotrophic amoeba that lacks mitochondria. Metabolites of axenically-grown cells were characterized by natural-abundance "C-NMR and )'P-NMR spectroscopy on acellular perchloric acid extracts. The amoebae were found to contain glycogen and trehalose as storage carbohydrates, together with putrescine and several amino acids, most prom- inently proline; we propose that proline and trehalose may serve in osmoregulation. Glycerophosphocholine and glycerophosphoe- thanolarnine were present with their phosphomonoester derivatives, phosphocholine and phosphoethanolarnine. Along with inorganic phosphate, inorganic pyrophosphate, nucleoside diphosphates, nucleoside triphosphates and NAD, P. balarnuthi amoebae also contained unusual phosphoinositol diphosphates in large quantities (0.5 pmoVg wet cells).

REATAMOEBA balamuthi is a free-living amoeba that I)" was isolated in 1975 by Bray from a village well in West Africa and named in 1986 [8] . P. balamuthi resembles other primitive protozoans such as Entamoeba histolytica or Giardia lamblia in that it lacks a well-developed Golgi apparatus and mitochondria. Three stages in its life cycle: amoeba, flagellate and cyst, have been described. Based on its ultrastructure, P. balamuthi has been classified as a n Archezoon, i. e. a n organism that branched off the main eukaryotic line before the primitive eukaryote captured protomitochondria [3, 4, 71. The amoebae can readily be grown axenically in complex media containing reducing agents. T h e absence of any biochemical data on this species prompted us to analyze its intracellular metabolites by I T - and "P-NMR spectroscopy. We report here that P. balamuthi amoebae contained as major carbon metabolites glycogen and trehalose together with several amino acids and putrescine.

I To whom correspondence should be addressed. Mailing address: Gtrard Klein, Laboratoire de Biologie Cellulaire, DBMSIBC, Centre d'Etudes Nuclkaires, 17, rue des Martyrs, 38054 Grenoble Cedex 9, France.

Abbreviations: Amino acids are indicated by their one-letter code (capitals). g, resonance lines from glycogen; p, putrescine; t, resonance lines from trehalose; G1 P, glucose 1-phosphate; G6P, glucose 6-phosphate, GPC, glycerophosphocholine; GPE, glycerophosphoethanolamine; InsP,PP, phosphate atoms of pentakisphosphoinositol diphosphate; InsP,PP, phosphate atoms of phosphoinositol diphosphate(s); NAD, nicotinamide adenine dinucleotide; PCho, phosphocholine; PEtn, phosphoethanolamine; Pi, inorganic phosphate; PPi, inorganic pyrophosphate; a-, @-, yATP/GTP (CTP/UTP), a-phosphate, @-phosphate, y-phosphate of purine (pyrimidine) nucleoside triphosphates; a-, @-ADPIGDP, a-phosphate, @-phosphate of purine nucleoside diphosphates; apyro, Ppyro InsP,PP/InsP,PP, a-phosphate, @-phosphate of the diphosphate groups of pentakisphosphoinositol diphosphate and phosphoinositol diphosphate(s).

Major phosphorylated compounds were various phosphomonoesters, two phosphodiesters: glycerophosphocholine and-eth- anolamine, Pi, PPi, nucleoside di- and triphosphates and NAD, along with high amounts of phosphoinositol diphosphates.

MATERIALS A N D METHODS Growth of P. balamuthi amoebae. P. balamuthi (American

Type culture collection number 30984) was grown axenically a t 36" C in TYI-S-33 (trypticase/yeast extract/iron/serum) medi- u m (ATCC culture medium 1 141). To harvest the cells, late log phase cultures were chilled on ice for 10 min and then centri- fuged a t 300 g for 10 min. Cell pellets were washed twice and resuspended in either 0.18 M NaCl containing 10 m M MOPS, pH 7.0 (31P-NMR) or in 0.15 M NaCl containing 37.5 m M KPi buffer, pH 7.2 ("C-NMR).

Perchloric acid extracts. Perchloric acid extracts of P. balamuthi amoebae were prepared as described previously [ 181. EDTA (6 mM) was added before neutralizing the perchloric acid extracts used for 3'P-NMR to avoid losses of poorly soluble salts of inositol phosphates with divalent cations [ 191. EDTA was omitted from extracts analyzed by "C-NMR. Extracts were sterilized by filtration through a 0.22 p m Millex filter (Millipore) to prevent development of bacterial growth during the long- lasting N M R experiments. '3c- and 31P-NMR spectroscopy. Proton-decoupled I3C-NMR

spectra o f perchloric acid extracts were recorded on a Bruker AM400 spectrometer equipped with a 10-mm diameter probe operating at 100.6 MHz. The N M R tube was spun at 12 rpm. Standard acquisition conditions used 60" (1 3 p s ) radio-frequency pulses at 2-s intervals and the spectral width was 22 kHz. Spectra were WALTZ- 16 proton-decoupled with two levels of decoupling: 1.5 W for 0.75 s (acquisition) and 0.5 W for 1.25 s. Free-induction decays were collected as 16K data points, zero- filled to 32K and processed with 1.5 Hz exponential line broad-

184 J. EUK. MICROBIOL., VOL. 42, NO. 2, MARCH-APRIL 1995

ening. Chemical shifts are given relative to tetramethylsilane at 0 PPm. A DEPT (Distortionless Enhancement by Polarization Transfer) sequence [ 121 was used to simplify the "C-NMR spectra and facilitate identification of metabolites. The DEPT sequence is a one dimensional pulse sequence, involving the spin echo phe- nomenon, which allows separate observation of the NMR signals for the CH, CH, and CH, groups.

P-NMR spectra were recorded in 10-mm tubes on a Bruker AMX400 WB spectrometer operating at 162 MHz. The NMR tube was spun at 12 rpm. Acquisition conditions used 60° (1 0 ps) radio-frequency pulses at 4-s (or 2 0 4 intervals, and the spectral width was 7352 Hz. For WALTZ- 16 proton-decoupled spectra, two levels of decoupling were used 2.5 W for 1.1 s (acquisition time) and 0.5 W for 2.9 s (or 18.9 s). Free-induction decays were collected as 16K data points, zero-filled to 32 K and processed with 0.5 Hz exponential line broadening. Chemical shifts are given with re- spect to 85% orthophosphoric acid at 0 ppm.

Two-dimensional I HJIP correlation spectra of perchloric acid extracts from P. balamuthi amoebae were recorded on a Bruker AMX400 spectrometer at a 400 MHz frequency for IH and 162 MHz for 31P with a HETCORR pulse sequence as described previously [ 181. Specific conditions are detailed in the legend to the figure. Such spectra give rise to a 2D-map characteristic of the examined compound and they are thus often used for identification purposes.

Quantification of cellular metabolite contents. Cellular metabolite contents were determined by a two-step method as fol- lows. Amounts of major metabolites were calculated from the intensity of the respective resonance lines. For this we used fully relaxed proton-decoupled spectra (20-s interpulse delays for P- and "C-NMR) in which the nuclear Overhauser enhancement effect (noe) was avoided by irradiating IH during the acquisition time only. A capillary of methylene diphosphonate ("P-NMR) or ethanol (IT-NMR) had been added as external calibration standard. Contents of minor metabolites were derived from proton-decoupled spectra with 2-s (IT) or 4-s ("P) interpulse delay. Data are expressed as pmoVg wet cells; if we assume that the cytoplasmic aqueous space contributes about 0.5 ml/g wet cells, a metabolite content of 1 pmoVg wet cells would roughly be equivalent to an intracellular concentration of 2 mM.

Proton-coupled or-decoupled

RESULTS W-NMR spectra of perchloric acid extracts of axenic P. baf-

amuilri amoebae. Numerous resonance lines were detected in proton-decoupled, natural abundance I3C-NMR spectra (Fig. 1) from a perchloric acid extract of axenically grown P. balamuthi ameboid cells. The identity of metabolites was established by comparison with chemical shifts published for other biological systems [ 1, 6, 14, 16, 171 and verified by use of a DEPT pulse sequence [ 121 and by direct spiking with authentic compounds when necessary. The most abundant metabolites were glycogen with its very characteristic C1 resonance at 100.6 ppm, trehalose, several amino acids: proline, lysine, alanine, leucine, glu- tamic acid, valine and the polyamine putrescine, characterized by its external C1 and C4 resonating at 40 ppm and its central C2 and C3 at 24.7 ppm. Glycogen is an a- 1,4-glycosidic polymer with a - I ,6 branch points. The resonance of carbon 4 depends on whether it belongs to a glucose unit involved in a linear stretch (C4, 77.8 ppm) or serving as a branching point (C4', 70.3 ppm) (Fig. 1). From the C4'/C4 ratio, it can be calculated that there was an a( 1 4 6 ) link every 8-9 glucose residues. Thus, the branching of the P. balamuthi glycogen is in between that of liver glycogen (every 13-14 glucose residues, [30]) and that

of Dictyostelium discoideum and E. histolytica (every 5-6 glucose residues, unpubl. observ., [16]).

31P-NMR spectra of perchloric acid extracts of axenic P. baf- amuthi amoebae. A typical 3'P-NMR spectrum of a perchloric acid extract from P. balamuthi is shown in Fig. 2. Phosphory- lated compounds identified in the spectrum were, starting from the low-field side of the spectrum, several phosphomonoesters [mainly glucose 6-phosphate (a and p anomers), phosphoethanolamine, phosphocholine, glucose 1 -phosphate], phosphoinositol diphosphates (see below for their identification), inorganic phosphate, the phosphodiesters glycerophosphoethanolamine and glycerophosphocholine, which accounted for the resonances at 0.6 and 0 ppm, respectively, pyrophosphate, the diphospho- diester ADP-ribose (see below) and nucleoside diphosphates and nucleoside triphosphates at -6, - 1 1 and -21 ppm. It was possible to discriminate the doublets of the y phosphates and the triplets of the p phosphates of purine and pyrimidine nucleoside triphosphates. No signals from UDP-sugars were detected around - 12 ppm.

Both NAD and ADP-ribose could be detected in this extract (Fig. 3). Only NAD was present in newly prepared perchloric acid extracts, but it decomposed into ADP-ribose with a tH of about 20 hours at 22" C (compare with Fig. 2). The nature of this NAD nucleosidase activity is currently unclear. We have excluded bacterial contamination (extracts were sterilized by filtration) and inherent instability of NAD at the pH used for the NMR experiments (NAD was stable in saline) as possible causes. In addition, degradation of NAD was not observed with perchloric acid extracts from other organisms (unpubl. observ.).

31P-NMR evidence for the presence of two phosphoinositol diphosphates. Without broad-band IH decoupling, the six unknown resonance lines (labeled 1-6, Fig. 2) of the phosphomonoester spectral region were split into doublets with 3JpwH coupling constants of 6-7 Hz (lines 1 4 ) and 9.6 Hz (lines 5-6) (not shown). Two clusters of 4 resonance lines were detected at -5 and at -9 ppm. The constant ratio of their intensities to those of the six previous lines suggested that they arose from phosphate groups camed by the same molecules. The a-P lines at -9 pprn had a 3JpOCH coupling constant of 9.2 Hz and a *JmP coupling constant of 16.5 Hz, that was also observed for the S-P resonance lines at - 5 ppm (Fig. 3). This unusually low *.Ipop coupling constant has only been found previously for the diphosphate group of pentakisphospho-myo-inositol diphosphate in E. histolytica [ 181 and strongly suggested the presence in our extracts of phosphoinositol diphosphates.

The same line splitting into doublets in proton-coupled spectra and the same very marked pH dependence of the chemical shifts were observed with our unknown compounds (not shown) and with InsP,PP from E. histolytica 118, Fig. 2, 31, supporting the hypothesis that phosphoinositol diphosphates are indeed present. The quadruplets at -5 and -9 ppm with the unusually low 2J,, coupling constant were indications of the presence of two diphosphate groups, which could not be camed by a single molecule, as their relative ratio was generally different from 1 (Fig. 3). P balamuthi amoebae thus contain at least two different phosphoinositol diphosphates.

Identification of pentakisphosphoinositol diphosphate. The structures of the inositol diphosphates were investigated by two- dimensional IH-,IP correlative NMR analysis [18]. The peaks that appear in the two-dimensional map originate only from phosphorus nuclei J-coupled to protons and can be used as a 2D-signature for the unambiguous identification of unknown compounds. Identifications of inositol diphosphates were fur- ther guided by previous work on E. histolytica [18] and D. discoideum [20].

Resonance lines at 3.3, 2.6 and 2.0 ppm (lines 1, 4, 5, Fig. 2)

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31 P CHEMICAL SHIFT Fig. 4. Two-dimensional 1H-31P correlation spectrum of a perchloric acid extract of P. bularnuthi amoebae. The pH of an extract obtained

from 3 g amoebae was adjusted to 8.5. The spectrum was recorded on a Bruker AMX400 spectrometer equipped with a 10 mm X-{lH} probehead at a 400 MHz frequency for 'H and 162 MHz for 31P with the following HETCORR pulse sequence [2] : RD - 9Oo(lH) - %tl - 180°(31P) - %ti - A, - 90°(1H)900(31P) - A,-acquisition ("P) with polarization transfer from 'H to IlP through J-". The time interval A, was set to 50 msec and a2 to 33 msec. The d 2 pulse lengths were I5 ps ("P) and 23 ps (IH). Acquired spectra were WALTZ-1 6 proton-decoupled and )IP acquisition time was 0.6 s with 1.7 s recycling delay. Spectral width was 3250 Hz in the F, domain (4096 points) and 1200 Hz in the F, domain (256 points). The 2D contour plot was calculated by Fourier transformation after zero-filling to a 4096 x 1024 data matrix and applying an exponential multiplication in the F, and F, dimensions. The attached IH- and "P-NMR spectra corresponding to the phosphomonoester region ( 3 . 5 / - 1 pprn) are the partial projections of the F, and F, dimensions. Resonance assignments are as described in the abbreviations listed on title page.

MARTIN ET AL-METABOLITES OF PHRE.4TAMOEBA BALAMUTHI 189

were related to one of the doublets at -5 ppm and another one at - 9 ppm. Their intensity ratio, 22: 1 : 1 : 1, was conserved in different extracts and indicated the presence of 7 phosphate groups on the molecule. The position of lines 1,4 and 5 on the 1H-31P correlation map (Fig. 4) as compared to the map of the phosphoinositol diphosphate isomer identified in E. histolytica [ 181 suggested that one of the phosphoinositol diphosphates found in P. balamuthi amoebae is indeed the pentakis-myo- inositol diphosphate (InsP,PP) isomer Ins( 1 ,2,3,4,6)P5(5)P,.

Identification of the other phosphoinositols was hampered by the fact that the intensities of the remaining lines were not consistently in a fixed ratio, an indication that they belonged to more than one compound. Their peaks in the correlation map (Fig. 4) were closely related to those of InsP,PP identified above, an indication that the basic symmetry of the molecules was conserved. The number of their phosphate groups and their positions on the inositol ring could however not be determined with certitude and they are therefore referred to as InsP,PP.

Metabolite content of axenic P. bulumuthi amoebae. As a quantitative approach is essential to understanding the relative importance of the various metabolites, we determined absolute amounts present in the perchloric acid extracts as described under Materials and Methods. As summarized in Table 1, axenic P. balamuthi amoebae contained high amounts of Pi (2 pmoVg wet cells), ATP/GTP (1.3 pmoYg wet cells), CTP/UTP (0.46 pmol/g wet cells), ADP/GDP (0.62 pmol/g wet cells), NAD (0.93 pmol/g wet cells) and glycerophosphocholine (0.98 pmoVg wet cells). Other phosphorylated compounds amounted to 0.27 pmoVg wet cells for PPi, 0.22 pmoVg wet cells for glycerophosphoethanolamine and 0.50 pmoVg wet cells for the phosphoinositol diphosphates. Cells also contained the phospholipid precursors phosphocholine (0.87 pmoVg wet cells) and phosphoethanolamine (0.1 1 pmoVg wet cells) as well as glucose 6-phosphate (0.10 pmol/g wet cells) and glucose 1 -phosphate (0.08 pmoVg wet cells). P. balamuthi amoebae also contained high concentrations of

glycogen (1 03 pmol glucose equivalentdg wet cells) and trehalose (23.5 pmol/g wet cells corresponding to 47 pmol glucose equivalentdg wet cells). Six amino acids were detectable in the 0.3-1.2 pmol/g wet cells range, except proline which was present at 3.3 pmol/g wet cells. Finally, the cells contained putrescine as major polyamine (about 0.75 pmol/g wet cells). Other polyamines were below our '3C-NMR detection limit (0.05 pmoVg wet cells).

DISCUSSION In this work, we have analyzed P. balamuthi amoebae by

natural-abundance IT-NMR and "P-NMR to explore some aspects of their carbohydrate, protein and lipid metabolism. The first important finding is the high cellular level of PPi in P. balamuthi extracts, about 15% of that of total nucleoside tn- phosphates (0.27 vs 1.80 pmol/g wet cells), a situation remi- niscent of that found in E. histolytica and other primitive ami- tochondrial protozoans [22,27-291. In these organisms, PPi has been shown to take over the function of ATP in several glycolytic reactions [27]. We propose PPi may play a similar role in P. balamuthi. Specifically, as the cells contained massive amounts of glycogen (100 pmol glucose equivalentdg wet cells), PPi together with glucose 1 -phosphate (found at about 0.1 pmoYg wet cells) might participate in glycogen cycling. In addition, PPi might be used by a PPi-dependent glucose 6-phosphatase similar to that found in E. histolytica [27] to phosphorylate glucose into glucose 6-phosphate, another phosphomonoester found in this study. There was no evidence for glucose accumulation in I T -

NMR spectra.

Table 1. Metabolite contents in P. bulurnufhi amoebae. Values were determined by a two-step method as described in Materials and Meth- ods. They represent the mean f SD of three independent determina- tions.

Cellular content Compound (pmollg wet cells)

"P-NMR Pi ATP/GTP CTP/UTP ADP/GDP PPi NAD' InsP,PP + InsP,PP GPC GPE PEtn PCho G6P G1P

"C-NMR Glycogenb Trehalose Putrescine Alanine Glutamate Leucine Lysine Pro 1 in e Valine

2.02 i- 0.57 1.33 i- 0.33 0.46 k 0.07 0.62 ? 0.35 0.27 i- 0.15 0.93 i: 0.32 0.50 i- 0.12 0.91 i- 0.36 0.22 ? 0.06 0.25 IO.08 0.71 f 0.16 0.16 i 0.06 0.08 ? 0.05

103 ? 14.7 23.5 i 5.9

0.7-0.8 1.1 ? 0.4 0.4 f 0.1 0.5 i- 0.2 1.2 i- 0.6 3.3 f 0.6 0.3 i 0.1

a Calculated from ADP-ribose + NAD. Glucose equivalents.

An interesting feature of P. balamuthi is the co-existence with glycogen of a second storage carbohydrate, trehalose, that amounts to 24 pmoVg wet cells, i.e. half the glucose equivalents contributed by glycogen. In fungi and slime molds, the accumulation of trehalose is intensive during differentiation and starvation [9, 331. In the yeast Saccharomyces cerevisiae, trehalose and glycogen are present simultaneously during the veg- etative stage [23], with glycogen triggering sporulation and trehalose playing a role in the germination process. Although a similar situation may exist in P. balamuthi, we cannot completely exclude the possibility that at least part of the trehalose detected in our NMR spectra may be correlated with the presence in the axenic cultures of a small percentage of trehalose- rich cysts.

Besides being an energy storage compound, trehalose may contribute to osmoregulation [ 1 I]. This also applies to proline, the most abundant amino acid in P. balamuthi; this compound is a prominent osmoprotectant in bacteria, algae and higher plants [lo, 1 I].

Lysine, alanine, leucine, glutamate and valine were also detected in extracts from P. balamuthi. It should be noted that growth medium TYI-S-33 is richest in these 5 amino acids [28]. Only for proline was a substantial accumulation observed, which points again towards a more specific role of this amino acid. Glutamate, glutamine and glutathione signals are close together in I3C-NMR spectra [25], but can be discriminated on close examination of the spectra. Neither glutamine nor glutathione were detectable in the I3C-NMR spectra. It remains to be established whether P. balamuthi completely lacks glutathione metabolism as does E. histolytica [ 131. It has been proposed [ 131 that eukaryotes did not acquire glutathione metabolism until they acquired mitochondria; this would be consistent with the notion [3, 4, 71 that P. balamuthi is an Archezoon.


P. balamuthi contains polyamines mainly in the form of putrescine. Other polyamines, if present, were a t a concentration at least 10-fold lower. Putrescine is widely distributed in prokaryotes, algae, plants, fungi and amoebae [ 15, 161. In another free-living amoeba, Acanthamoeba castellanii, I ,3-diaminopropane was shown to substitute for putrescine [26]. Polyamines have been recognized as regulators of cell growth and differentiation and are also involved in osmotic regulation. Their precise functions and mechanisms of action remain poorly un- derstood [24, 321.

Another result of these studies on P. balamuthi amoebae was the observation of large amounts of two phosphodiesters, glycerophosphocholine and glycerophosphoethanolamine. These are water soluble breakdown products of phosphatides; the corresponding phosphomonoesters, phosphocholine and phosphoethanolamine, were detected as well. As the levels of glycerophosphocholine and glycerophosphoethanolamine appear t o correlate with phospholipase and lysophospholipase activi- ties, these compounds may serve as endogenous lysophospholipase inhibitors to preserve membrane phospholipids [ 51.

A major finding is the occurrence in P. balamuthi amoebae of phosphoinositol diphosphates, namely InsP,PP and uniden- tified InsP,PP isomers, in high amounts (0.5 KmoVg wet cells). These compounds belong t o a new class of inositol metabolites that has been recognized recently [31]. They are also found a t high concentrations in other lower eukaryotes such as E. histolytica [ 181 and D. discoideum [3 I], but are undetectable in G. lamblia (unpubl. data). A challenging hypothesis is that they may represent a new form of high-energy phosphates [21], but additional work has to be done to understand their physiological role.

ACKNOWLEDGMENTS We thank Dr. Michel Satre for providing helpful discussions

and Dr. Eamon Rooney for critical reading of this manuscript. This work was supported by grants from the Commissariat Zi 1'Energie Atomique (Departement de Biologie Moltculaire et Structurale/Biologie Cellulaire et Rbonance Magnktique en Biologie e t MCdecine), from the Centre National de la Recherche Scientifique (Unit6 de Recherche Associk 1 130), by the Deutsche Forschungsgemeinschaft (SFB 17 1 K 2 ) and by the Fonds der Chemischen Industrie.

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Received 6-7-94, 9-20-94; accepted 10-26-94.

J. Euk. Microbrol.. 42(2), 1995. pp. 191-195 0 199s by the Society of Protozoologists

A New Method for Inoculation of Poor Germinator, Nosema sp. NIS M l l (Microsporida: Nosematidae), into an Insect Cell Culture

CHISA YASUNAGA,*,' SHIN0 INOUE,* MASAKO FUNAKOSHI,. TAKESHI KAWARABATA' and SYOJI HAYASAKA** *Institute of Biological Control, Faculty of Agriculture, Kyushu University, Fukuoka 812, Japan, and

+*National Institute of Sericulture and Entomological Science, Tsukuba. Ibaraki Prefecture 305, Japan

ABSTRACT. Spores of Nosema sp. NIS M 1 1, primed with 0.1 N KOH solution, were mixed with either physiological salt solutions or IPL-4 I medium, an insect cell culture medium, for germination. In the latter medium, only a few spores germinated, while high percentages of spore germination were obtained in physiological salt solutions, particularly in Rinaldini's solution. By using the salt solutions as inoculation media, KOH-primed NIS M11 spores were inoculated into the Spodoptera frugiperda SF2lAEII cell line. The initial infection levels were consistently higher than that obtained by using IPL-41 medium. Among the salt solutions, Rinaldini's solution, containing KCI in place of NaCI, gave the highest percentage of initial cell infection. Increased osmolarity of salt solutions did not improve the efficiency of spore germination and infection of N. sp. NIS M 1 1.

Supplementary key words. Entomopathogenic microsporidians, germination medium, inoculation medium, insect cell culture, new inoculation method, Nosema sp., poor germinator.

n recent years, growth and development of entomopathogenic I microsporidians have been investigated in insect cell cultures [8, 10, 14, 15, 20, 211. For establishment of in vitro microsporidian infection, efficient germination of microsporidian spores is the primary step, and the viability of sporoplasms extruded from spores would be a matter of secondary concern. It is widely accepted that the germination methods of microsporidian spores affect both the germination rate of spores and the initial level of infection in vitro. Many investigators [9, 10, 13, 15,2 11 have used the method in which insect cell cultures are inoculated with microsporidian spores primed with either KOH or KC1 solutions. Usually, the spores of Nosema bornbycis, a type species ofthe genus Nosema, are also treated with 0.1 N KOH solutions, then directly inoculated into insect cells suspended in the medium [8-10, 12, 201. Kawarabata et al. [12] reported that the percentage of germinated N. bornbycis spores in Spodoptera frugiperda cell culture was 97%, and 1.2% of S. frugiperda cells were infected with sporoplasms.

Nosema sp. NIS M11, a silkworm parasite found in Japan [4-61, produces spores which are difficult to germinate. When subjected to the conventional method mentioned above, the spore germination rate of this organism varies depending on the lot of spores harvested from silkworm larvae, and the percentage of germinated spores which is as low as < 1% in an insect cell culture medium. In our earlier studies [30, 3 11, we used KOH- or EDTA-priming methods for germination of N. sp. NIS M11 spores. Of the two, the former method gave extremely low germination rates of <5%, while high germination rates (5040%) were obtained in the latter. Interestingly, however, there was no significant difference in the number of host cells parasitized with sporoplasms between two cultures inoculated with differently primed spores. It is difficult to obtain higher percentages of host cells infected with sporoplasms of poor germinators.

The objective of this study was to develop a new method for germination and inoculation of KOH-primed N. sp. NIS M11 spores. We show that the new method, with KC1-containing

I To whom correspondence should be addressed.

physiological salt solutions, markedly increased the level of spore germination.

MATERIALS AND METHODS Microsporidians. Microsporidians used were Nosema sp. NIS

M 1 1 and N. bornbycis NIS 00 1, originated from the laboratory culture of the Division of Sericulture, Sericultural Experiment Station, Tsukuba, Japan. Both two microsporidians were isolated from the silkworm, Bombyx rnori. N. bombycis Y9101, isolated from the beet armyworm [32], was also used in this study. Spores were purified by Percoll density gradient centrifu- gation [ 191. Spores from the gradients were rinsed three times and stored in distilled water at 4" C. The spore concentration was determined with a hemocytometer.

Insect cell line. Spodoptera frugiperda SF2 1 AEII cell line [2, 281 was supplied by Dr. J. L. Vaughn (U.S. Department of Agriculture, Beltsville, MD). The insect cells were propagated in IPL-41 medium [7] supplemented with 10% fetal bovine serum (Whittaker M. A. Bioproducts, Walkersville, MD) and maintained at 27" C. Subculture was done every five days.

Physiological salt solutions for spore germination. The pH values, salt concentrations, and osmolarities of germinating solutions used in this study are shown in Table l . Basically, three solutions were examined for inducibility of germination: Rinal- dini's solution (RS) [ 181, Dulbecco's phosphate-buffered saline with [PBS(+)] or without [PBS(-)I Mg/Ca [3]. IPL-41 medium was used as the control.

It is generally accepted that the potassium ion is more important than the sodium ion for germination of entomopathogenic microsporidian spores [23, 291. To examine the effect of K+, NaCl in physiological salt solutions was replaced with the same concentration (molarity) of KCI [K+ RS, K+ PBS( -), and K+PBS( +), respectively].

Osmolarity of each solution was determined with an osmometer (Auto-Osmometer, OSMOSTAT OM-6020, Kyoto Daiichi Kagaku, Japan). As osmolarities of physiological salt solutions used in this study were lower than that of IPL-41 medium, K+RS and K+PBS(-) were supplemented with sucrose at the same concentration (molarity) as in IPL-41 medium [K+RS with sucrose and K+PBS( -) with sucrose, respectively].

metabolites of the free-living amoeba phreatamoeba balamuthi analyzed by 13c- and 31p-nmr...

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