Population dynamics of male-killing and non-male-killing spiroplasmas in Drosophila melanogaster

The endosymbiotic bacteria Spiroplasma spp. are vertically transmitted through female hosts and are known to cause selective death of male offspring in insects. One strain of spiroplasma, NSRO, causes male killing in Drosophila species, and a non-male-killing variant of NSRO, designated NSRO-A, has been isolated. It is not known why NSRO-A does not kill males. In an attempt to understand the mechanism of male killing, we investigated the population dynamics of NSRO and NSRO-A throughout the developmental course of the laboratory host Drosophila melanogaster by using a quantitative PCR technique. In the early development of the host insect, the titers of NSRO were significantly higher than those of NSRO-A at the first- and second-instar stages, whereas at the egg, third-instar, and pupal stages, the titers of the two spiroplasmas were almost the same. Upon adult emergence, the titers of the two spiroplasmas were similar, around 2 x 10(8) dnaA copy equivalents. However, throughout host aging, the two spiroplasmas showed strikingly different population growth patterns. The titers of NSRO increased exponentially for 3 weeks, attained a peak value of around 4 x 10(9) dnaA copy equivalents per insect, and then decreased. In contrast, the titers of NSRO-A were almost constant throughout the adult portion of the life cycle. In adult females, consequently, the titer of NSRO was significantly higher than the titer of NSRO-A except for a short period just after emergence. Although infection of adult females with NSRO resulted in almost 100% male killing, production of some male offspring was observed within 4 days after emergence when the titers of NSRO were as low as those of NSRO-A. Based on these results, we proposed a threshold density hypothesis for the expression of male killing caused by the spiroplasma. The extents of the bottleneck in the vertical transmission through host generations were estimated to be 5 x 10(-5) for NSRO and 3 x 10(-4) for NSRO-A.     

Population Dynamics of Male-Killing and Non-Male-Killing Spiroplasmas in Drosophila melanogaster

The endosymbiotic bacteria Spiroplasma spp. are vertically transmitted through female hosts and are known to cause selective death of male offspring in insects. One strain of spiroplasma, NSRO, causes male killing in Drosophila species, and a non-male-killing variant of NSRO, designated NSRO-A, has been isolated. It is not known why NSRO-A does not kill males. In an attempt to understand the mechanism of male killing, we investigated the population dynamics of NSRO and NSRO-A throughout the developmental course of the laboratory host Drosophila melanogaster by using a quantitative PCR technique. In the early development of the host insect, the titers of NSRO were significantly higher than those of NSRO-A at the first- and second-instar stages, whereas at the egg, third-instar, and pupal stages, the titers of the two spiroplasmas were almost the same. Upon adult emergence, the titers of the two spiroplasmas were similar, around 2 × 10⁸ dnaA copy equivalents. However, throughout host aging, the two spiroplasmas showed strikingly different population growth patterns. The titers of NSRO increased exponentially for 3 weeks, attained a peak value of around 4 × 10⁹ dnaA copy equivalents per insect, and then decreased. In contrast, the titers of NSRO-A were almost constant throughout the adult portion of the life cycle. In adult females, consequently, the titer of NSRO was significantly higher than the titer of NSRO-A except for a short period just after emergence. Although infection of adult females with NSRO resulted in almost 100% male killing, production of some male offspring was observed within 4 days after emergence when the titers of NSRO were as low as those of NSRO-A. Based on these results, we proposed a threshold density hypothesis for the expression of male killing caused by the spiroplasma. The extents of the bottleneck in the vertical transmission through host generations were estimated to be 5 × 10⁻⁵ for NSRO and 3 × 10⁻⁴ for NSRO-A.     

Prevalence of a non-male-killing spiroplasma in natural populations of Drosophila hydei

Male-killing phenotypes are found in a variety of insects and are often associated with maternally inherited endosymbiotic bacteria. In several species of Drosophila, male-killing endosymbionts of the genus Spiroplasma have been found at low frequencies (0.1 to 3%). In this study, spiroplasma infection without causing male-killing was shown to be prevalent (23 to 66%) in Japanese populations of Drosophila hydei. Molecular phylogenetic analyses showed that D. hydei was infected with a single strain of spiroplasma, which was closely related to male-killing spiroplasmas from other Drosophila species. Artificial-transfer experiments suggested that the spiroplasma genotype rather than the host genotype was responsible for the absence of the male-killing phenotype. Infection densities of the spiroplasma in the natural host, D. hydei, and in the artificial host, Drosophila melanogaster, were significantly lower than those of the male-killing spiroplasma NSRO, which was in accordance with the hypothesis that a threshold infection density is needed for the spiroplasma-induced male-killing expression.     

Prevalence of a Non-Male-Killing Spiroplasma in Natural Populations of Drosophila hydei

Male-killing phenotypes are found in a variety of insects and are often associated with maternally inherited endosymbiotic bacteria. In several species of Drosophila, male-killing endosymbionts of the genus Spiroplasma have been found at low frequencies (0.1 to 3%). In this study, spiroplasma infection without causing male-killing was shown to be prevalent (23 to 66%) in Japanese populations of Drosophila hydei. Molecular phylogenetic analyses showed that D. hydei was infected with a single strain of spiroplasma, which was closely related to male-killing spiroplasmas from other Drosophila species. Artificial-transfer experiments suggested that the spiroplasma genotype rather than the host genotype was responsible for the absence of the male-killing phenotype. Infection densities of the spiroplasma in the natural host, D. hydei, and in the artificial host, Drosophila melanogaster, were significantly lower than those of the male-killing spiroplasma NSRO, which was in accordance with the hypothesis that a threshold infection density is needed for the spiroplasma-induced male-killing expression.     

Asymmetrical interactions between Wolbachia and Spiroplasma endosymbionts coexisting in the same insect host

We investigated the interactions between the endosymbionts Wolbachia pipientis strain wMel and Spiroplasma sp. strain NSRO coinfecting the host insect Drosophila melanogaster. By making use of antibiotic therapy, temperature stress, and hemolymph microinjection, we established the following strains in the same host genetic background: the SW strain, infected with both Spiroplasma and Wolbachia; the S strain, infected with Spiroplasma only; and the W strain, infected with Wolbachia only. The infection dynamics of the symbionts in these strains were monitored by quantitative PCR during host development. The infection densities of Spiroplasma exhibited no significant differences between the SW and S strains throughout the developmental course. In contrast, the infection densities of Wolbachia were significantly lower in the SW strain than in the W strain at the pupal and young adult stages. These results indicated that the interactions between the coinfecting symbionts were asymmetrical, i.e., Spiroplasma organisms negatively affected the population of Wolbachia organisms, while Wolbachia organisms did not influence the population of Spiroplasma organisms. In the host body, the symbionts exhibited their own tissue tropisms: among the tissues examined, Spiroplasma was the most abundant in the ovaries, while Wolbachia showed the highest density in Malpighian tubules. Strikingly, basically no Wolbachia organisms were detected in hemolymph, the principal location of Spiroplasma. These results suggest that different host tissues act as distinct microhabitats for the symbionts and that the lytic process in host metamorphosis might be involved in the asymmetrical interactions between the coinfecting symbionts.     

High and low temperatures differently affect infection density and vertical transmission of male-killing Spiroplasma symbionts in Drosophila hosts

We investigated the vertical transmission, reproductive phenotype, and infection density of a male-killing Spiroplasma symbiont in two Drosophila species under physiological high and low temperatures through successive host generations. In both the native host Drosophila nebulosa and the nonnative host Drosophila melanogaster, the symbiont infection and the male-killing phenotype were stably maintained at 25 degrees C, rapidly lost at 18 degrees C, and gradually lost at 28 degrees C. In the nonnative host, both the high and low temperatures significantly suppressed the infection density of the spiroplasma. In the native host, by contrast, the low temperature suppressed the infection density of the spiroplasma whereas the high temperature had little effect on the infection density. These results suggested that the low temperature suppresses both the infection density and the vertical transmission of the spiroplasma whereas the high temperature suppresses the vertical transmission preferentially. The spiroplasma density was consistently higher in the native host than in the nonnative host, suggesting that the host genotype may affect the infection density of the symbiont. The temperature- and genotype-dependent instability of the symbiont infection highlights a complex genotype-by-genotype-by-environment interaction and may be relevant to the low infection frequencies of the male-killing spiroplasmas in natural Drosophila populations.     

Asymmetrical Interactions between Wolbachia and Spiroplasma Endosymbionts Coexisting in the Same Insect Host

We investigated the interactions between the endosymbionts Wolbachia pipientis strain wMel and Spiroplasma sp. strain NSRO coinfecting the host insect Drosophila melanogaster. By making use of antibiotic therapy, temperature stress, and hemolymph microinjection, we established the following strains in the same host genetic background: the SW strain, infected with both Spiroplasma and Wolbachia; the S strain, infected with Spiroplasma only; and the W strain, infected with Wolbachia only. The infection dynamics of the symbionts in these strains were monitored by quantitative PCR during host development. The infection densities of Spiroplasma exhibited no significant differences between the SW and S strains throughout the developmental course. In contrast, the infection densities of Wolbachia were significantly lower in the SW strain than in the W strain at the pupal and young adult stages. These results indicated that the interactions between the coinfecting symbionts were asymmetrical, i.e., Spiroplasma organisms negatively affected the population of Wolbachia organisms, while Wolbachia organisms did not influence the population of Spiroplasma organisms. In the host body, the symbionts exhibited their own tissue tropisms: among the tissues examined, Spiroplasma was the most abundant in the ovaries, while Wolbachia showed the highest density in Malpighian tubules. Strikingly, basically no Wolbachia organisms were detected in hemolymph, the principal location of Spiroplasma. These results suggest that different host tissues act as distinct microhabitats for the symbionts and that the lytic process in host metamorphosis might be involved in the asymmetrical interactions between the coinfecting symbionts.     

Virus-mediated change in clumping properties ofDrosophila SR spiroplasmas

Transovarially transmitted SR spiroplasmas inDrosophila cause an abnormal sex ratio (SR condition: male-specific killing) in the host fly progenies. A reaction known as clumping takes place between different SR spiroplasma strains in which spiroplasmas instantly form aggregates upon mixing of the two strains. Each strain of SR spiroplasma carries an associated virus that is lytic to certain other strains. When the virus, HIV, from the recently discovered non-male-killingDrosophila hydei spiroplasma (HIS) is injected into host flies carrying the SR spiroplasma ofD. nebulosa (NSR), the latter spiroplasmas either undergo complete lysis and disappear, or survive with decreased numbers and with an abnormal morphology, and are transmissible from generation to generation in host flies. The surviving spiroplasmas possess two viruses, the endogenous virus of thenebulosa spiroplasma, spv-1, and the newly introduced superinfecting virus, HIV. This combination leads to a change in the surface properties of the superinfected spiroplasmas that is manifested in their ability to form clumps with normalnebulosa spiroplasmas, but does not interfere with male killing. This change in spiroplasma phenotype is discussed in terms of host-phenotype modification by infecting viruses.     

Infectivity of beetle spiroplasmas for new host species

Five beetle spiroplasmas, the Colorado potato beetlespiroplasma (CPBS, strain LD-1), the Cantharis carolinusspiroplasma (CCBS, strain CC-1), the Ellychnia corrusca fireflyspiroplasma (FS, strain EC-1), the Diabrotica undecimpunctatacorn rootworm spiroplasma (CRS, strain DU-1), and the Spiroplasmafloricola fall flower spiroplasma (FFS), all associated withbeetles, were fed to beetles (Maladera matrida and Carpophilushumeralis) and mosquitoes (Aedes aegypti and Culex pipiens). CPBSand CCBS were also injected into M. matrida. Attempts to recoverspiroplasmas from regurgitates and hemolymph were conducted 1–10days after their introduction. After day 1, orally administeredspiroplasmas could not be recovered from M. matrida beetles;however, at 2–5 days, four out of five spiroplasmas wererecovered from adult C. humeralis. Injected spiroplasmas survivedin the hemolymph of M. matrida beetles for a relatively longperiod (at least 22 days). All five spiroplasmas were recoveredfrom mosquitoes 1 day post feeding, but only two (CCBS and CRS)survived for five or more days. The results show short andvariable persistence in orally challenged non-host insects, withgeneral failure to pass the gut barrier. Such evidence should beconsidered when attempting to use these microbes in biocontrolprograms.     

THE INTERACTION PHENOTYPE IN THE DROSOPHILA WILLISTONI–SPIROPLASMA SYMBIOSIS

Both the population and coevolutionary dynamics of hereditary male-lethal endosymbionts, found in a wide range of insect species, depend on host fitness and endosymbiont transmission rates. This paper reports on fitness effects and transmission rates in three lines of Drosophila willistoni infected with either male-lethal spiroplasmas or a spontaneous nonmale-lethal mutant. Overall fitness measures were reduced or unaffected by the infection; however, some infected females produced more offspring in early broods. Maternal transmission rates were high, but imperfect, and varied with a female's age, host line, and spiroplasma type. No evidence for paternal or horizontal transmission was found. If an altered temporal pattern of reproduction is not a factor in countering the loss of spiroplasma hosts through imperfect maternal transmission, persistence of this endoparasitism remains unexplained. Tolerance of the infection and ability to transmit bacteria varied with both host and spiroplasma line. Analysis of the interaction between the spontaneous nonmale-lethal mutant and its host suggests this symbiosis has undergone coevolution under laboratory culture.     

Spiroplasma taxonomy and identification of the sex ratio organisms: can they be cultivated?

The spiroplasmas that occur naturally in several species of Drosophila were the first spiroplasmas ever observed, even though their discoverers, D.F. Poulson and B. Sakaguchi, in 1961 described them as being "treponema-like spirochetes." These Drosophila spiroplasmas are transovarially, or maternally, transmitted by infected females whose progenies are composed entirely of females. A more recently discovered Drosophila spiroplasma found in flies originating in Ito, Japan, is also maternally inherited but does not result in the elimination of males from the progeny of infected females. In spite of their early discovery, their high numerical density in the hemolymph of infected females (10(6)-10(7)/microliters), and numerous attempts at in vitro cultivation, they remain prime examples of non-cultivable spiroplasmas. It is the purpose of this paper to recount some of the approaches used in attempts at their cultivation.     

Phenotype and transmission efficiency of artificial and natural male-killing Spiroplasma infections in Drosophila melanogaster

Many insect species carry inherited Spiroplasma bacteria which act as important partners and antagonists. The nature of symbioses between Spiroplasma and insects has been most extensively studied in the interaction between male-killing Spiroplasma infection and Drosophila melanogaster. For historical reasons, these studies have largely focussed on the Spiroplasma strain known as NSRO, derived from Drosophila nebulosa and transinfected into D. melanogaster. More recently, D. melanogaster naturally infected with Spiroplasma were discovered. Whilst the well studied strain NSRO is closely related to that found natively in D. melanogaster, it is unclear whether strains from D. nebulosa reflect a natural interaction when placed in D. melanogaster. In this paper, we determine if NSRO has similar or different properties from strains of Spiroplasma naturally infecting D. melanogaster in terms of transmission efficiency and the strength and timing of male-killing. Native infections were observed to have higher transmission efficiency than introduced NSRO infections during the early phases of host reproduction, but not during late reproduction. The timing and intensity of male-killing did not differ between infection classes. As a precautionary measure, it is proposed that future work seeking to reveal the nature of coevolved Spiroplasma-Drosophila interactions use the native strain.     

Spiroplasmas from coleopterous insects: New ecological dimensions

The genusSpiroplasma (helical wall-less prokaryotes) is a recently described group of microorganisms that cause disease in plants, arthropods, and experimentally, in vertebrates. Two spiroplasmas from beetles have now been discovered in a search for microorganisms suitable for biological control of economically important coleopterous insects. Colorado potato beetles (CPB) infected with spiroplasma were commonly found on potato and other solanaceous plants in Maryland. Although this spiroplasma occurred in high concentration in gut fluids and sputum, it could not be cultivated in conventional spiroplasma media. However, another spiroplasma (CN-5 and related strains) reported here to occur commonly in association with larvae and adults of the green June beetle,Cotinus nitida, could be cultivated readily in the SM-1 formulation and several other conventional spiroplasma media. The CN-5 spiroplasma was serologically distinct from representative members of all 8 major groups now recognized. Thus, it represents a ninth major spiroplasma serogroup (IX), and can be considered to be an unnamed species. The CPB spiroplasma is apparently maintained in plant surface-insect gut cycles, but details of maintenance of the CN-5 spiroplasma are incompletely understood. Isolation of CN-5 spiroplasma from soil in which host larvae had fed suggests that transmission of this agent may occur in the soil. Both CN-5 and CPB spiroplasmas exhibited unusually active translational motility in natural fluids, and CN-5 organisms exhibited such motility in culture media. Although we have no evidence that either spiroplasma is pathogenic to its usual host, the pathogenicity of spiroplasmas to many hosts, including the beetle,Melolontha melolontha, suggests possible application for biological control.     

Nutritional requirements of two flower spiroplasmas and honeybee spiroplasma.

A chemically defined medium (CC-494) was used to study the nutritional requirements of three spiroplasmas representing three distinct serogroups: flower spiroplasmas [Spiroplasma floricola and FS (SR-3)] and honeybee spiroplasma [HBS (AS-576)]. Glucose, fructose, and mannose were utilized by all three spiroplasmas. In addition, the honeybee spiroplasma could ferment trehalose, FS (SR-3) could ferment sucrose, and S. floricola could ferment trehalose, sucrose, and raffinose. The three spiroplasmas varied greatly in their requirements of amino acids for growth. S. floricola was the only strain that utilized arginine. HBS (AS-576) required at least one purine and one pyrimidine base (either free base or ribonucleoside) for growth, while both flower spiroplasmas grew with only one base in the medium. Oleic acid, cholesterol, and bovine serum albumin were essential to all three spiroplasmas. Palmitic acid, which was nonessential, promoted growth significantly.     

Temperature Ranges,Growth Optima,and Growth Rates of Spiroplasma (Spiroplasmataceae,class Mollicutes) Species

A new method was developed for determination of the doubling times of spiroplasmas. In this procedure, the time required for medium acidification of tubes in tenfold dilution series was recorded. Sixty-four spiroplasma strains, representing 24 groups and 11 subgroups, were studied. Eight strains representing putative new groups were also included in the study. Doubling times at 5, 10, 15, 20, 25, 30, 32, 37, 41, and 43°C were determined. The range of temperatures for spiroplasma growth was 5°–41°C. Twenty-three spiroplasmas had optima of 30°C, 29 had optima of 32°C, and 13 had optima of 37°C. The fastest growing spiroplasma was the MQ-4 strain (group XI), with a doubling time at optimal temperature of 0.6 h. The slowest was the Jamaican corn stunt strain B655 (subgroup I-3), with an optimal doubling time of 36.7 h. Spiroplasma strain B31 (group IV) had the widest range (5°–41°C), while the DW-1 strain and some subgroup I-3 strains had the narrowest, growing only at 25° and 30°C. Some spiroplasmas grew well at 41°C, but none grew at 43°C. The ability of spiroplasmas to withstand a wide range of temperatures may reflect the conditions to which they are exposed in nature, including the temperatures of the insect, tick, and/or plant hosts in which they are carried and the plant surfaces from which they may be acquired by arthropods.     

Relation of in vivo morphology to isolation of plant spiroplasmas

Two procedures were developed to isolate plant spiroplasmas directly onto DG-2 agar plates or in DG-2 broth without subcultures or dilutions. The frequency of successful spiroplasma isolations was increased by centrifuging samples, after passing through a 0.45-μm filter, at 25,000 × g for 1 h. Spiroplasmas were obtained from peach, cherry, Madagascar periwinkle, and celery with typical symptoms of the Green Valley strain of X disease (GVX), from peach with typical symptoms of the peach yellow leaf roll strain of X disease (PYLR), from Madagascar periwinkle with typical symptoms of aster yellows (AY), from celery with atypical symptoms of GVX (mild GVX), from plantago with atypical symptoms of aster yellows (mild AY), and from stubborn-diseased citrus. Isolations were consistent (>90%) from plants with mild GVX, mild AY, and citrus stubborn, while isolations were inconsistent (0–9%) from plants with typical symptoms of GVX, PYLR, and AY. The role of the isolated spiroplasmas in plant disease was not determined in this study. All spiroplasma isolates were serologically indistinguishable fromSpiroplasma citri. Spiroplasmas were seen in electron micrographs of 8 out of 9 examined plants from which spiroplasmas were isolated. However, electron micrographs of all 13 examined plants from which no spiroplasmas were isolated contained mycoplasma-like organisms (MLOs) but no, spiroplasmas. These results indicate that there is a correlation between helical MLOs in vivo and successful isolation of spiroplasmas, and that plants may be infected with bothS. citri and nonhelical mycoplasmas.     

Direct isolation in cell-free medium of a spiroplasma fromHaemaphysalis leporispalustris (Acari: Ixodidae) in Maryland

A spiroplasma isolate, was obtained from rabbit ticks (Haemaphysalis leporispalustris) taken from cottontail rabbits in Maryland by inoculation of tick suspensions into SP-4 medium. The isolate was indistinguishable from an experimental vertebrate pathogen (suckling mouse cataract agent spiroplasma) when tested with other plant and tick spiroplasmas in growth inhibition, deformation, and metabolism inhibition tests. The isolated organism had a pathogenic profile for suckling rats and embryonated chicken eggs that differed significantly from that of other suckling mouse cataract agent strains. This is the first report of a direct spiroplasma isolation from ticks in cell-free medium, and confirms the specific association of spiroplasmas of the suckling mouse cataract agent serogroup with rabbit ticks.     

Infection densities of three Spiroplasma strains in the host Drosophila melanogaster

Maternally transmitted endosymbiotic bacteria of the genus Spiroplasma associate with numerous insect species, including the genus Drosophila. Among the Spiroplasma strains associated with Drosophila, several manipulate their host??s reproduction by killing the male offspring of the infected females. Although the male-killing mechanism is not well understood, previous studies of non-native strains transferred to D. melanogaster (strain Oregon-R) indicate that the male-killing strain achieves higher densities than two non-male-killing strains. Whether this pattern of higher male-killing strain densities occurs in other host-symbiont strain combinations is not known. Herein, we used quantitative PCR to examine infection densities of one non-male-killing strain native to D. hydei (Hyd1), and two male-killing strains; one native to D. nebulosa (NSRO), and one native to D. melanogaster (MSRO; recently discovered), upon artificial transfer to D. melanogaster (strain Canton-S). Infection densities were examined at four weekly intervals in adult flies, across three consecutive generations following artificial transfer. Infection densities of the non-male-killing strain were significantly lower than those of the two male killers immediately after adult emergence. At later time points, however, the non-male-killing strain (Hyd1) is capable of proliferating to densities similar to those of the two male-killing strains (NSRO and MSRO) in D. melanogaster (Canton-S). We also examined the effect of co-infection by the heritable bacterium Wolbachia, on Spiroplasma densities and male-killing ability. Wolbachia had little to no effect of Spiroplasma densities, but the male-killing ability of MSRO was lower in the presence of Wolbachia. Generation post-infection had little effect on Spiroplasma densities, but affected the male-killing ability.     

Naturally occurring single and double infection with Wolbachia strains in the butterfly Eurema hecabe: transmission efficiencies and population density dynamics of each Wolbachia strain

Wolbachia belonging to Alphaproteobacteria are transovarially transmitted bacteria responsible for reproductive alterations in a wide range of arthropods. In natural populations of the butterfly Eurema hecabe, there are two different types of Wolbachia-infected individuals. Individuals singly infected with Wolbachia strain wHecCI exhibit strong cytoplasmic incompatibility, whereas those doubly infected with wHecCI and wHecFem exhibit feminization. Here, we examined the infection frequencies and population densities of each Wolbachia strain in different host tissues (ovary, testis, fat body, midgut, Malpighian tubule and leg), and the cost of infection in offspring produced by single-infected and double-infected mothers of E. hecabe. The vertical transmission rate of wHecCI was nearly 100%, and that of wHecFem was c. 80%. The wHecCI densities were 10(3)-10(4)-fold higher than the wHecFem densities. In most tissues, the wHecCI densities were significantly higher in offspring of single-infected mothers than in offspring of double-infected mothers. In offspring of double-infected mothers, however, the wHecCI densities were not affected by the presence of wHecFem, suggesting a lack of interaction between the wHecCI and wHecFem densities. The offspring development time was dependent on the infection status of the mothers. These results imply that the maternal infection status affects the Wolbachia densities and fitness of offspring.     

Morphology and ultrastructure of helical and nonhelical strains of Spiroplasma citri.

Cells of the nonhelical strain of Spiroplasma citri underwent changes of morphology comparable to those which occurred in the normal helical strain. Cells of the nonhelical strain had the same ultrastructural features as helical cells and released long flexible fibrils similar to those seen in other spiroplasmas. Nonhelical organisms showed an increased tendency to aggregate, forming cell clusters of an unusual annular form. The cytoplasmic membrane of the nonhelical strain lacked a single protein present in all helical strains. Loss of helicity associated with the senescence of spiroplasma cells was not accompanied by the disappearance of this protein. Differences in colony morphology were shown to be a consequence of motility, and a technique was developed which facilitated the identification of nonmotile organisms.