jeanflop/post_ocr_correction2 huggingface dataset:
more than 50 % of the eggs do not hatch. A wide range of incompatibility levels has been observed for different isofemale lines from the same population: 31-63 % for the four infected isofemale lines from Brazzaville (see legend of Table II). Similarly, some variability has been observed in the duplicates of 11 experiments (Table II). In addition, between the F2 and the F5, incompatibility of males Mooréa fell from 74 % to 44 % with Nasrallah females (and to 27 %, as an average, for their crosses to various treated lines). Partial incompatibility appears to be subject to large fluctuations within and between strains. This relatively low level of incompatibility is probably the reason why, given the high proportion of infected flies in almost all populations, its importance was not previously detected. Indeed, if 34 % of the flies are infected, 22 % of crosses between randomly chosen individuals will be incompatible. Assuming that 47 % of the eggs do not hatch due to partial incompatibility, the average of non-hatched eggs would be only about 10 %, or even less with male ageing. The same value is expected in a mass culture of a single strain, polymorphic for infection. This is probably the reason why this widespread incompatibility was previously detected in D. melanogaster only between two Australian populations in spite of intensive genetical studies on this species, two of them specifically devoted to this research. Given its high frequency, Wolbachia could be responsible for some cytoplasmic effects observed in D. melanogaster. It has to be noticed that this infection is not likely to be a laboratory culture by-product. We have observed infection in various other drosophila species and almost each of them harbors its own infective agent (Rousset and Solignac, unpublished results). Infected individuals have been observed in cultures from diverse laboratory stocks and infection was also observed in freshly captured flies. Partial incompatibility and crossing types The level of infection observed in testes cysts of D. melanogaster is very low when compared to D. simulans. In infected strains, D. melanogaster exhibits an average of 4.6 % cysts
This is probably the reason why this widespread incompatibility was previously detected in D. melanogaster only between two Australian populations in spite of intensive genetical studies on this species, two of them specifically devoted to this research. Given its high frequency, Wolbachia could be responsible for some cytoplasmic effects observed in D. melanogaster. It has to be noticed that this infection is not likely to be a laboratory culture by-product. We have observed infection in various other drosophila species and almost each of them harbors its own infective agent (Rousset and Solignac, unpublished results). Infected individuals have been observed in cultures from diverse laboratory stocks and infection was also observed in freshly captured flies. Partial incompatibility and crossing types The level of infection observed in testes cysts of D. melanogaster is very low when compared to D. simulans. In infected strains, D. melanogaster exhibits an average of 4.6 % cysts with DAPI positive granules whereas 87 % were observed in a strain of D. simulans from Brazzaville. However, intermediate values have been observed for the D. simulans stocks from Hawaii. Partial incompatibility found in D. melanogaster can be correlated to this low level of infection, as compared to D. simulans where incompatibility is higher or complete in relation with a higher cyst infection. A significant correlation is also observed within D. melanogaster between the level of male infection and incompatibility strength (Fig. 3). After experimental injection of infected cytoplasm into eggs, a similar observation has been made. Partial incompatibility may be the consequence of a low number of sperm modified by the bacteria since, in the testes, two populations of cysts exist: some are infected whereas others are not. However, in spite of this correlation, there is no direct numerical correspondence between the number of infected cysts (average 4.6 % in males two days after emergence) and unhatched eggs (average 46.7 %). According to the fact that infection decreases with male ageing, the simplest way to interpret this observation is to assume that infection is heavier during pupal stages and that young males transfer sperm which became mature during this
where incompatibility is higher or complete in relation with a higher cyst infection. A significant correlation is also observed within D. melanogaster between the level of male infection and incompatibility strength (Fig. 3). After experimental injection of infected cytoplasm into eggs, a similar observation has been made. Partial incompatibility may be the consequence of a low number of sperm modified by the bacteria since, in the testes, two populations of cysts exist: some are infected whereas others are not. However, in spite of this correlation, there is no direct numerical correspondence between the number of infected cysts (average 4.6 % in males two days after emergence) and unhatched eggs (average 46.7 %). According to the fact that infection decreases with male ageing, the simplest way to interpret this observation is to assume that infection is heavier during pupal stages and that young males transfer sperm which became mature during this stage. This low level of infection probably results from a control of the number of symbionts by the host species. Strains of D. melanogaster, transinfected through cytoplasm injection by the Riverside symbiont of D. simulans possess a number of symbionts lower than the donor strain and express only partial cytoplasmic incompatibility, like naturally infected ones. This result is not surprising since comparison of 16S rDNA sequences has suggested that the same bacterial strain infected both D. melanogaster and D. simulans Riverside. Thus the same bacterial strain may induce different levels of incompatibility, according to the host species, but different levels of incompatibility could be obtained in the same host species infected by different bacterial lineages (see Introduction). In spite of the great variability of hatchability in incompatible crosses, the results of crosses between females of various strains and infected males from Moorea gave a low level of unhatched eggs (see Fig. 2 and two exceptions below). Consequently, if the number of bacteria in females is variable, as it is in males, it is always sufficient in the eggs to repair the damage caused to the sperm by the bacteria in the most heavily infected males. Two intrastrain crosses showed a
incompatibility, like naturally infected ones. This result is not surprising since comparison of 16S rDNA sequences has suggested that the same bacterial strain infected both D. melanogaster and D. simulans Riverside. Thus the same bacterial strain may induce different levels of incompatibility, according to the host species, but different levels of incompatibility could be obtained in the same host species infected by different bacterial lineages (see Introduction). In spite of the great variability of hatchability in incompatible crosses, the results of crosses between females of various strains and infected males from Moorea gave a low level of unhatched eggs (see Fig. 2 and two exceptions below). Consequently, if the number of bacteria in females is variable, as it is in males, it is always sufficient in the eggs to repair the damage caused to the sperm by the bacteria in the most heavily infected males. Two intrastrain crosses showed a very high level of unhatched eggs (Villeurbanne and Harkerville, respectively 43 % and 63 %). The same is observed, at a lesser extent, in compatible crosses of the females of these strains to Nasrallah males (24 % and 33 % of unhatched eggs respectively) and to Moorea males (16 % and 41 % respectively) but not in reciprocal crosses. A high level of unhatched eggs was also observed in intrastrain crosses in the F5 (23 % and 19 %) and after tetracycline treatment (11 % and 42 %). Although these strains are infected by Wolbachia, unidentified additional problems of egg fertility interfere with cytoplasmic incompatibility. It is not easy to identify several crossing types when partial incompatibility is involved. However, the existence of a single incompatibility type for infected flies emerges from two lines of evidence: all the flies tested are infected by a single bacterial strain (judged from their 16S rDNA sequences) and females of all infected stocks were compatible with Moorea infected males. History of the infection In relation with cytoplasmic inheritance of both Wolbachia and mtDNA and lack of association of infection with mtDNA haplotypes, the initial event of infection occurred probably only once, early in the
and 41 % respectively) but not in reciprocal crosses. A high level of unhatched eggs was also observed in intrastrain crosses in the F5 (23 % and 19 %) and after tetracycline treatment (11 % and 42 %). Although these strains are infected by Wolbachia, unidentified additional problems of egg fertility interfere with cytoplasmic incompatibility. It is not easy to identify several crossing types when partial incompatibility is involved. However, the existence of a single incompatibility type for infected flies emerges from two lines of evidence: all the flies tested are infected by a single bacterial strain (judged from their 16S rDNA sequences) and females of all infected stocks were compatible with Moorea infected males. History of the infection In relation with cytoplasmic inheritance of both Wolbachia and mtDNA and lack of association of infection with mtDNA haplotypes, the initial event of infection occurred probably only once, early in the evolution of the species, prior to the coalescence time of the mtDNA tree. It is more likely that polymorphism for infection results from subsequent independent losses of the bacteria rather than from recent independent infection events of different mtDNA lineages by the same bacterial strain. In addition, because the symbiont of D. melanogaster belongs to the same lineage as those of its closest related species, D. simulans, a single event of infection could be suspected, anticipating their speciation, several million years ago. However, in D. Simulans, infection is associated with a mtDNA subtype of the haplotype s/Il [10, 30] and this haplotype itself belongs to an internal branch in the mtDNA tree, other bacteria being associated with other mtDNA types emerging deeper in the tree [11]. Moreover, the transfer through specific barriers by hybridization is not likely to have occurred, female hybrids being either inviable or sterile and mtDNA analyses providing no evidence of cytoplasmic introgression. Consequently, an independent infection of the two species by the same bacteria is more probable. The polymorphism of infection observed in D. melanogaster can be an equilibrium or not. A model of polymorphic equilibrium between loss of infection and selection resulting from incompatibility has
of D. melanogaster belongs to the same lineage as those of its closest related species, D. simulans, a single event of infection could be suspected, anticipating their speciation, several million years ago. However, in D. Simulans, infection is associated with a mtDNA subtype of the haplotype s/Il [10, 30] and this haplotype itself belongs to an internal branch in the mtDNA tree, other bacteria being associated with other mtDNA types emerging deeper in the tree [11]. Moreover, the transfer through specific barriers by hybridization is not likely to have occurred, female hybrids being either inviable or sterile and mtDNA analyses providing no evidence of cytoplasmic introgression. Consequently, an independent infection of the two species by the same bacteria is more probable. The polymorphism of infection observed in D. melanogaster can be an equilibrium or not. A model of polymorphic equilibrium between loss of infection and selection resulting from incompatibility has been proposed by Fine [39] but there is no stable equilibrium in this model for an infection frequency lower than 0.5. A second possibility would be that D. melanogaster populations are a stable mixture of infected and uninfected populations. This would require a strong population structuration which is not in agreement with the existence of polymorphism for infection observed in this work in 11 local populations and the absence of structuration of mtDNA polymorphism [30]. A third possibility is that Wolbachia is presently spreading or disappearing. In the laboratory, infection seems to be tenacious for at least 15 generations. A high rate of frequency change is not apparent in the observations covering 80 years of capture. In addition, the time of coalescence of mtDNA haplotypes could be about 0.5 million years, value calculated from the maximum nucleotide distance between molecules [30] and evolutionary rates of mtDNA in drosophila [40]. If our statement that infection occurred before this coalescence time is correct, this means that the rate of loss of the bacteria is quite low. On the contrary, if it was spreading, it would be associated to a particular mtDNA haplotype, as it is in D. simulans [33, 41]. In conclusion,
agreement with the existence of polymorphism for infection observed in this work in 11 local populations and the absence of structuration of mtDNA polymorphism [30]. A third possibility is that Wolbachia is presently spreading or disappearing. In the laboratory, infection seems to be tenacious for at least 15 generations. A high rate of frequency change is not apparent in the observations covering 80 years of capture. In addition, the time of coalescence of mtDNA haplotypes could be about 0.5 million years, value calculated from the maximum nucleotide distance between molecules [30] and evolutionary rates of mtDNA in drosophila [40]. If our statement that infection occurred before this coalescence time is correct, this means that the rate of loss of the bacteria is quite low. On the contrary, if it was spreading, it would be associated to a particular mtDNA haplotype, as it is in D. simulans [33, 41]. In conclusion, if this situation is not the result of a stable equilibrium, it corresponds to a change at a very low rate. It remains to discover the factor which limits the penetration of this character, because incompatibility is the mechanism which promotes the spreading of the bacteria and its maintenance in maternal lineages [4]. Acknowledgments: we thank Karin Ekström, Jean R. David, Yves Carton, Dominique Anxolabéhère, Jean-Claude Brégliano and Georges Periquet for providing flies, Aurore Galli, Marie-Thérèse Chassagnard, Christophe Bressac, Eric de Stordeur for their help in PCR amplification, DAPI observations and mtDNA analyses. VERSION ABRÉGÉE En utilisant en PCR des amorces spécifiques il a été montré que 34 % des 234 lignées analysées de Drosophila melanogaster, provenant du monde entier, étaient infectées par Wolbachia pipientis, une Protéobactérie endosymbiotique transmise maternellement. Plusieurs populations ont été trouvées polymorphes pour l'infection et le taux de cytoplasmes infectés est pratiquement constant sur tous les continents et sous toutes les latitudes. Le séquençage d'un long fragment (plus de 600 pb) de l'ARN ribosomique 16S des bactéries de 4 souches de D. melanogaster d'origine géographique différente suggère que toutes les populations véhiculent la même lignée d'endosymbionte. Celle-ci est indiscernable de la bactérie responsable du type R
maternal lineages [4]. Acknowledgments: we thank Karin Ekström, Jean R. David, Yves Carton, Dominique Anxolabéhère, Jean-Claude Brégliano and Georges Periquet for providing flies, Aurore Galli, Marie-Thérèse Chassagnard, Christophe Bressac, Eric de Stordeur for their help in PCR amplification, DAPI observations and mtDNA analyses. VERSION ABRÉGÉE En utilisant en PCR des amorces spécifiques il a été montré que 34 % des 234 lignées analysées de Drosophila melanogaster, provenant du monde entier, étaient infectées par Wolbachia pipientis, une Protéobactérie endosymbiotique transmise maternellement. Plusieurs populations ont été trouvées polymorphes pour l'infection et le taux de cytoplasmes infectés est pratiquement constant sur tous les continents et sous toutes les latitudes. Le séquençage d'un long fragment (plus de 600 pb) de l'ARN ribosomique 16S des bactéries de 4 souches de D. melanogaster d'origine géographique différente suggère que toutes les populations véhiculent la même lignée d'endosymbionte. Celle-ci est indiscernable de la bactérie responsable du type R d'incompatibilité dans l'espèce jumelle D. simulans. Cette bactérie détermine une incompatibilité partielle dans les croisements entre femelles non infectées et mâles infectés, qui se traduit par une mortalité embryonnaire pouvant atteindre 77 %, la moyenne s'établissant à 46,7 % (contre 6,2 % dans les croisements réciproques). La compatibilité des croisements entre souches infectées suggère que toutes appartiennent au même type d'incompatibilité, résultat en accord avec la détection d'une seule lignée bactérienne par séquençage. L'observation d'un étalement de spermatozoïdes en cours de différenciation sous éclairement ultraviolet après coloration au DAPI, montre que le pourcentage de cystes infectés est faible (comparé à celui de D. simulans de type R chez qui l'incompatibilité est à peu près complète). Il montre en outre une corrélation significative avec le degré d'incompatibilité observé dans les croisements. Quel que soit le degré de réactivité d'une souche, ses femelles sont compatibles avec les mâles les plus lourdement infectés. Cette observation suggère que si le niveau d'infection est variable chez les femelles, comme il l'est chez les mâles, le nombre de bactéries présente dans les ovocytes est toujours suffisant pour réparer les modifications subies par les spermatozoïdes chez le parent mâle et assurer ainsi la compatibilité du croisement. L'analyse
entre souches infectées suggère que toutes appartiennent au même type d'incompatibilité, résultat en accord avec la détection d'une seule lignée bactérienne par séquençage. L'observation d'un étalement de spermatozoïdes en cours de différenciation sous éclairement ultraviolet après coloration au DAPI, montre que le pourcentage de cystes infectés est faible (comparé à celui de D. simulans de type R chez qui l'incompatibilité est à peu près complète). Il montre en outre une corrélation significative avec le degré d'incompatibilité observé dans les croisements. Quel que soit le degré de réactivité d'une souche, ses femelles sont compatibles avec les mâles les plus lourdement infectés. Cette observation suggère que si le niveau d'infection est variable chez les femelles, comme il l'est chez les mâles, le nombre de bactéries présente dans les ovocytes est toujours suffisant pour réparer les modifications subies par les spermatozoïdes chez le parent mâle et assurer ainsi la compatibilité du croisement. L'analyse de la variabilité de l'ADN mitochondrial a été effectuée sur une partie des souches infectées et non infectées, pour rechercher une association éventuelle entre les haplotypes mitochondriaux et la présence/absence de l'infection. L'information historique inscrite dans la phylogénie de l'ADNmt peut en effet apporter des informations sur l'origine et l'évolution de l'infection puisque mitochondries et micro-organismes sont tous deux cotransmis maternellement. Wolbachia est associée aux quatre haplotypes mitochondriaux détectés par cette analyse. Chez D. simulans au contraire, les souches infectées par cette bactérie sont associées à un sous-type d'une branche terminale de l'arbre mitochondrial. Aussi, bien que D. melanogaster possède une lignée bactérienne apparemment identique à celle de son espèce jumelle D. simulans, une infection indépendante des deux espèces est probable, l'hybridation entre les deux espèces ne donnant que des produits stériles. Survenue très tôt dans l'évolution de D. melanogaster, antérieurement au temps de coalescence de l'ADNmt, l'infection aurait ensuite été perdue dans de nombreuses lignées maternelles. Le pourcentage de cytoplasmes infectés ne semble pas avoir changé significativement au cours des dernières décennies et l'existence d'un polymorphisme d'infection qui paraît relativement stable est difficile à interpréter en fonction des modèles existants. REFERENCES 1. Ghelelovitch S. 1952. Sur le déterminisme génétique
de l'infection puisque mitochondries et micro-organismes sont tous deux cotransmis maternellement. Wolbachia est associée aux quatre haplotypes mitochondriaux détectés par cette analyse. Chez D. simulans au contraire, les souches infectées par cette bactérie sont associées à un sous-type d'une branche terminale de l'arbre mitochondrial. Aussi, bien que D. melanogaster possède une lignée bactérienne apparemment identique à celle de son espèce jumelle D. simulans, une infection indépendante des deux espèces est probable, l'hybridation entre les deux espèces ne donnant que des produits stériles. Survenue très tôt dans l'évolution de D. melanogaster, antérieurement au temps de coalescence de l'ADNmt, l'infection aurait ensuite été perdue dans de nombreuses lignées maternelles. Le pourcentage de cytoplasmes infectés ne semble pas avoir changé significativement au cours des dernières décennies et l'existence d'un polymorphisme d'infection qui paraît relativement stable est difficile à interpréter en fonction des modèles existants. REFERENCES 1. Ghelelovitch S. 1952. Sur le déterminisme génétique de la stérilité dans les croisements entre différentes souches de Culex pipiens Roubaud. C. R. Acad. Sci. Paris 234: 2386-8. 2. Hertig M. 1936. The rickettsia, Wolbachia pipientis (gen. et sp. n.) and associated inclusions of the mosquito, Culex pipiens L. ### CORRECTION ### Simulans, infection is associated with a mtDNA subtype of the haplotype s/Il [10, 30] and this haplotype itself belongs to an internal branch in the mtDNA tree, other bacteria being associated with other mtDNA types emerging deeper in the tree [11]. Moreover, the transfer through specific barriers by hybridization is not likely to have occurred, female hybrids being either inviable or sterile and mtDNA analyses providing no evidence of cytoplasmic introgression. Consequently, an independent infection of the two species by the same bacteria is more probable. The polymorphism of infection observed in D. melanogaster can be an equilibrium or not. A model of polymorphic equilibrium between loss of infection and selection resulting from incompatibility has been proposed by Fine [39] but there is no stable equilibrium in this model for an infection frequency lower than 0.5. A second possibility would be that D. melanogaster populations are a stable mixture of infected and uninfected populations. This would