The Drosophila paulistorum complex contains six semispecies that do not normally interbreed. In the laboratory, crosses between semispecies produce fertile daughters and sterile sons. Microbial endosymbionts have been observed in all D. paulistorum flies that display this male sterility.Streptococcal L-forms have been isolated from the AndeanBrazilian (Mesitas) and Transitional (Santa Marta) semispecies and cultured in artificial medium. Transfer of these Lforms from their native hosts into reciprocal semispecies resuited in sterile male progeny. When L-forms were inoculated into the semispecies from which they had been isolated, most of the male progeny were fertile. Control streptococcal Lforms did not show this sterility pattern.The term "semispecies" refers to subgroups derived from a single species and implies that the process of speciation is in progress. The six semispecies constituting the neotropical Drosophila paulistorum complex have been separated and designated by their geographical distribution. In nature these semispecies do not normally interbreed (Fig. la), but among hybrids produced in the laboratory, the males are sterile and the females are fertile (Fig. lb). When these hybrid females are backcrossed to males of either parental semispecies, their male progeny are still sterile and their female progeny are fertile (2).Factors responsible for this sterility can be artificially transmitted (3). When extracts of male flies from one semispecies were injected into females of another, the male progeny from these females were sterile. Similarly, if the extracts injected were of hybrid males that had a mother of a different semispecies than that of the recipient females, the same sterility pattern occurred (Fig. lc).Electron microscopy has revealed that all semispecies of the D. paulistorum complex contain cytoplasmic endosymbionts (2, 4)-i.e., each member harbors a semispeciesspecific symbiont with which it coexists. A hybrid fly inherits its chromosomes from both parents. It inherits its cytoplasmic endosymbiont only from its mother. The transmissibility of sterility by injection can be explained as a result of the transfer of an endosymbiont from its native semispecies to a different one. For example, in Fig. 1 b and c, the endosymbiont a contained in semispecies A (and in the extract of sterile hybrid males), when passed by mating or injection into females of semispecies B (containing symbiont 0), causes sterility of the male progeny.In our earlier studies, the host range of the sterility producing agent had been extended to larvae of Ephestia kuehniella, the Mediterranean meal moth (5). Studies in this alternate host suggested that the agent was most likely a cell wall-deficient microbe, perhaps a mycoplasma or similar organism (1).In this publication, we report the isolation on artificial media of cell wall-deficient organisms from two of the D. paulistorum semispecies. These organisms, commonly called Lforms, demonstrated a semispecies-specific male sterility pattern. METHODSInsects. We...

Strain 831-C4* (T = type strain), isolated from the surface of lettuce plants (Lactuca sativa) obtained from a retail food market, was shown to be a sterol-requiring mollicute. Morphological examination of this organism by electron and dark-field microscopic techniques showed that it consists of small, nonhelical, nonmotile, pleomorpbic coccoid cells, with individual cells surrounded by a single cytoplasmic membrane. No evidence of a cell wall was observed. The organism grew rapidly in all conventional culture medium formulations for mollicutes in either aerobic or anaerobic environments. The optimum temperature for growth was 30°C, but multiplication occurred at 18 to 37°C. Strain 831-C4T catabolized glucose, but hydrolysis of arginine or urea could not be demonstrated. The genome size of strain 831-C4T was determined to be about 569 megadaltons, while the base composition (guanine-plus-cytosine content) of the DNA was 30.0 mok % . Recent studies in which we compared the 16s rRNA sequences of strain 831-C4T with those of more than 40 other mollicutes indicated that this organism is phylogenetically related to the Spiroplasma-Mycoplasma mycoides clade. Strain 831-C4T was serologically unrelated to the type strains of previously described Mycoplasma species and to 18 other unclassified sterol-requiring isolates cultivated from various animal, plant, or insect sources. Strain 831-C4T (= ATCC 49193) is the type strain of MycopZasma lactucae sp. nov.The presence of nonhelical, wall-less procaryotes (class Mollicutes) on plant surfaces was first well documented in 1979 (8,15) when organisms with the general features of both Acholeplasma and Mycoplasma species were isolated from the surfaces of several tropical floral plants. Some of the strains described in these studies were serologically related to certain previously established Acholeplasma species that had at that time been found only in vertebrate hosts (e.g., Acholeplasma axanthum, Acholeplasma oculi, and Acholeplasma laidlawii). Other isolates discovered on plant surfaces eventually were found to represent distinct new Acholeplasma species having no obvious association with vertebrates (e.g., Acholeplasma Jorum) (14,28). Shortly thereafter, other acholeplasmas, representing both vertebrate-associated and new putative plant-associated species, were reported to be present on the surfaces of vegetables (17; also see the review in reference 21).The natural occurrence of acholeplasmas in the guts and hemolymphs of insects was established by Clark and colleagues in work carried out in the early 1980s (5, 25). These observations indicated that a wide variety of unclassified acholeplasmas can be found in insect gut fluids and on plant (especially flower) surfaces, leading to speculation that an exchange of these mollicutes occurs between insect and plant hosts during feeding excursions. We recently characterized one of these new acholeplasmas, Acholeplasma entomophilum, and identified it as an important part of the gut flora of a variety of insects and a frequent res...

The isolation of Mollicutes from food has not been reported. To isolate Mollicutes in the presence of high levels of unwanted bacteria, we first incubated fresh vegetables in liquid culture media containing lysozyme, ampicillin, and thallous acetate. Culture fluids were than separated from the vegetable samples, subjected to one freeze-thaw cycle, and passed through a filter of 0.4-micron porosity. Filtered samples were cultured in SP4 medium and in a conventional medium containing horse serum. With this procedure 21 acholeplasma isolations representing three species were obtained from endive, broccoli, and kale. Of 35 food samples tested, 11 were positive for acholeplasmas; acholeplasmas isolated from 6 of these samples were recovered only in SP4 medium. In seven single vegetable samples, two or more Acholeplasma spp. were isolated. A. laidlawii was isolated from all three vegetables and A. axanthum was found in broccoli and kale. Four isolates were serologically identified as A. oculi. Mycoplasma verecundum was the only Mycoplasma species recovered. Several isolates could not be typed serologically, as they reacted with antisera to both A. morum and A. hippikon. these isolates may include new Acholeplasma spp.

Intracellular symbionts are common among insects, and all semispecies of the neotropical Drosophila paulistorum complex contain them. Symbionts of D. paulistorum, identified as group D streptococcal L-forms (cell wall deficient), are involved in male sterility that results from crosses between semispecies. This is one of three reproductive isolating mechanisms that maintain the six semispecies. When extracts of Mesitas strain of the Andean-Brazilian semispecies are injected into females of the Santa Marta strain of the Transitional semispecies, the females, mated with Santa Marta males, produce male progeny that are sterile.

Crosses among the six semispecies of Drosophila paulistorum produce sterile male hybrids. This sterility is caused by an agent which has characteristics of a microorganism. It is pathogenic in a secondary host, the larvae of the Mediterranean meal moth, Ephestia kuehniella, and can be serially passaged in Ephestia, where it is lethal. The agent was passaged back into D. paulistorum, where it induced sterility in males of a semispecies different from that of origin of the agent. Infectious particles were obtained from an extract of infected Ephestia by ultracentrifugation in a sucrose-Ficoll-metrizamide gradient. Both crude and purified extracts were lyophilized and stored indefinitely without loss of killing power. The agent was destroyed by low pH, lipid solvents, ultraviolet light, and exposure to a temperature of 56°C for 30 min. It appeared to be sensitive to tetracycline and insensitive to penicillin, suggesting that the agent is not a virus, but more likely a cell wall-deficient bacterium or mycoplasma-like organism.