jeanflop/post_ocr_correction2 huggingface dataset:
S. Y. et Robert J. M. 1987. La production micro-algale des claires à huîtres : particularités nutritionnelles, importance de l'azote organique dissous. IFREMER Ad. Coll. 5: 185-214. 12. Delmas D., Frikha M. G., Linley E. A. S. 1990. Dissolved primary amine measurement by flow injection analysis with o-phthaldialdehyde : comparison with high-performance liquid chromatography. Mar. Chemistry 29: 145-54 13. Brooks S. P. J. 1922. A simple computer program with statistical tests for the analysis of enzyme kinetics. Biotechniques 13: 906-11. 14. Garland C. D., Nash G. V., McMeekin T. A. 1982. Absence of surface-associated microorganisms in adult oysters (Crassostrea gigas). Appl. environ. Microbiol. 1205-11. 15. Manahan D. T., Richardson K. 1983. Competition studies on the uptake of dissolved organic nutrients by bivalve larvae (Mytilus edulis) and marine bacteria. Mar. Biology 75: 241-7. 460 C. R. Acad. Sci. Paris, Sciences de la vie/Life sciences, 1994; 317: 457-60 C. R. Acad. Sci. Paris, Sciences de la vie/Life sciences, 1994; 317: 461-70 Evolution/Evolution Widespread occurrence of the proteobacteria Wolbachia and partial cytoplasmic incompatibility in Drosophila melanogaster Infection by the protobacteria Wolbachia and cytoplasmic incompatibility are widespread in Drosophila melanogaster MICHEL SOLIGNAC (1), DOMINIQUE VAUTRIN (1), FRANÇOIS ROUSSET (2) 1 Laboratoire de Biologie et Génétique évolutives, CNRS, 91198 Gif-sur-Yvette Cedex, France. 2 Institut des Sciences de l'Évolution, Génétique et Environnement, Case postale 065, 34095 Montpellier Cedex 05, France. Tirés à part : M. Solignac Résumé La plupart des populations de Drosophila melanogaster sont polymorphes pour l'infection par la Protéobactérie Wolbachia pipientis (le taux moyen d'infection étant de 34 %), qui est responsable d'une incompatibilité cytoplasmique partielle (le pourcentage d'œufs non éclos peut atteindre 77 %, avec une moyenne de 46,7 %). Des séquences partielles d'ARN ribosomique 16S de la bactérie, établies à partir de mouches provenant de localités éloignées, n'ont pas montré de différences entre elles et se sont avérées identiques à celle de la bactérie qui infecte le type R d'incompatibilité dans l'espèce voisine D. simulans. En relation avec cette observation, les résultats des croisements entre souches infectées s'interprètent par l'existence d'un seul type d'incompatibilité cytoplasmique chez D. melanogaster. En outre, les cytoplasmes
Génétique évolutives, CNRS, 91198 Gif-sur-Yvette Cedex, France. 2 Institut des Sciences de l'Évolution, Génétique et Environnement, Case postale 065, 34095 Montpellier Cedex 05, France. Tirés à part : M. Solignac Résumé La plupart des populations de Drosophila melanogaster sont polymorphes pour l'infection par la Protéobactérie Wolbachia pipientis (le taux moyen d'infection étant de 34 %), qui est responsable d'une incompatibilité cytoplasmique partielle (le pourcentage d'œufs non éclos peut atteindre 77 %, avec une moyenne de 46,7 %). Des séquences partielles d'ARN ribosomique 16S de la bactérie, établies à partir de mouches provenant de localités éloignées, n'ont pas montré de différences entre elles et se sont avérées identiques à celle de la bactérie qui infecte le type R d'incompatibilité dans l'espèce voisine D. simulans. En relation avec cette observation, les résultats des croisements entre souches infectées s'interprètent par l'existence d'un seul type d'incompatibilité cytoplasmique chez D. melanogaster. En outre, les cytoplasmes infectés se retrouvent dans divers haplotypes mitochondriaux de D. melanogaster alors que cette lignée bactérienne n'est présente que dans une branche terminale de l'arbre mitochondrial chez D. simulans. Par conséquent, l'infection s'est probablement produite indépendamment dans les deux espèces. Elle n'est survenue qu'une seule fois chez D. melanogaster, tôt dans l'évolution de l'espèce, et le symbionte a été perdu ensuite à plusieurs reprises. Le niveau d'incompatibilité trouvé dans les diverses souches est significativement corrélé au niveau d'infection des mâles (révélé par les colorations de testicules au DAPI) mais, quel que soit le niveau d'infection de la souche, les femelles infectées sont compatibles avec les mâles les plus lourdement infectés. Le pourcentage de cytoplasmes infectés ne semble pas avoir changé significativement au cours des dernières décennies et le mécanisme de maintien du polymorphisme d'infection est problématique. Mots clés : Drosophila melanogaster, Wolbachia, incompatibilité cytoplasmique, hérédité maternelle, ADNmt. Abstract Most populations of Drosophila melanogaster over the world are polymorphic for infection by the maternally inherited Proteobacteria Wolbachia pipientis (average rate of infection 34 %) responsible in the host species for partial cytoplasmic incompatibility (the percentage of unhatched eggs varies from negligible up to 77 %, with an average of 46,7 %). Partial
l'évolution de l'espèce, et le symbionte a été perdu ensuite à plusieurs reprises. Le niveau d'incompatibilité trouvé dans les diverses souches est significativement corrélé au niveau d'infection des mâles (révélé par les colorations de testicules au DAPI) mais, quel que soit le niveau d'infection de la souche, les femelles infectées sont compatibles avec les mâles les plus lourdement infectés. Le pourcentage de cytoplasmes infectés ne semble pas avoir changé significativement au cours des dernières décennies et le mécanisme de maintien du polymorphisme d'infection est problématique. Mots clés : Drosophila melanogaster, Wolbachia, incompatibilité cytoplasmique, hérédité maternelle, ADNmt. Abstract Most populations of Drosophila melanogaster over the world are polymorphic for infection by the maternally inherited Proteobacteria Wolbachia pipientis (average rate of infection 34 %) responsible in the host species for partial cytoplasmic incompatibility (the percentage of unhatched eggs varies from negligible up to 77 %, with an average of 46,7 %). Partial 16S rDNA sequences of bacteria offlies from four populations were identical and indistinguishable from the D. simulans symbiont type R. In crosses to an infected reference strain, all infected individuals seemed to belong to the same incompatibility type. In addition, in D. melanogaster, cytoplasm infection is irrespective of mtDNA haplotypes, but not in D. simulans where this bacterial lineage is only présent in a terminal branch in the mtDNA haplotype tree. It can be inferred that the cytoplasm of D. melanogaster was infected probably once, independently of D. simulans, early in the evolution of the species, and was subsequently lost several times. The level of incompatibility is related to the level of infection of the sperm cysts observed in young males, but infected females, whatever the strength of incompatibility determined by the males of their strains, are compatible with the most heavily infected males. The percentage of infected cytoplasms has not changed during the last decades and the maintenance of infection polymorphism is problematic. Key words : Drosophila melanogaster, Wolbachia, cytoplasmic incompatibility, maternal inheritance, mtDNA. Voir Version abrégée (p.468) The existence of incompatible crosses between strains of the mosquito Culex pipiens was originally reported by Ghelelovitch in 1952 [1]. Although
but not in D. simulans where this bacterial lineage is only présent in a terminal branch in the mtDNA haplotype tree. It can be inferred that the cytoplasm of D. melanogaster was infected probably once, independently of D. simulans, early in the evolution of the species, and was subsequently lost several times. The level of incompatibility is related to the level of infection of the sperm cysts observed in young males, but infected females, whatever the strength of incompatibility determined by the males of their strains, are compatible with the most heavily infected males. The percentage of infected cytoplasms has not changed during the last decades and the maintenance of infection polymorphism is problematic. Key words : Drosophila melanogaster, Wolbachia, cytoplasmic incompatibility, maternal inheritance, mtDNA. Voir Version abrégée (p.468) The existence of incompatible crosses between strains of the mosquito Culex pipiens was originally reported by Ghelelovitch in 1952 [1]. Although a rickettsia-like micro-organism, Wolbachia pipientis, was described in the cells of C. pipiens a long time ago [2], the relationships between Wolbachia and cytoplasmic incompatibility was established only in 1971 [3]. Cytoplasmic incompatibility, is now known in several orders of insects and in terrestrial isopods. In crosses between infected males and uninfected females, the sperm enters the egg but fails to perform syngamy and haploid embryos die at diverse stages of development. All the other crosses are compatible (review in [4]). During the last years, Drosophila simulans has been shown to exhibit cytoplasmic incompatibility in relation to the presence of the symbiotic micro-organism. Unidirectional incompatibility between males of infected strains and females of uninfected strains was first documented [5]. This finding was followed by the discovery of bidirectional incompatibility between infected strains from different geographical origin [6]. The two infected cytotypes have been found to be associated with different mitochondrial types [7]. A third infected cytotype, responsible for partial incompatibility, has been detected in a D. simulans line from Madagascar [8], which belongs to the third mitochondrial type known in this species [9, 10]. A molecular analysis of the diversity of the endosymbionts of D. simulans, using 16S rDNA sequences,
and uninfected females, the sperm enters the egg but fails to perform syngamy and haploid embryos die at diverse stages of development. All the other crosses are compatible (review in [4]). During the last years, Drosophila simulans has been shown to exhibit cytoplasmic incompatibility in relation to the presence of the symbiotic micro-organism. Unidirectional incompatibility between males of infected strains and females of uninfected strains was first documented [5]. This finding was followed by the discovery of bidirectional incompatibility between infected strains from different geographical origin [6]. The two infected cytotypes have been found to be associated with different mitochondrial types [7]. A third infected cytotype, responsible for partial incompatibility, has been detected in a D. simulans line from Madagascar [8], which belongs to the third mitochondrial type known in this species [9, 10]. A molecular analysis of the diversity of the endosymbionts of D. simulans, using 16S rDNA sequences, revealed that the different incompatibility types correspond to different bacterial lineages [11]. The only complication arose from the existence of two bacterial lineages associated with a single incompatibility type (Rousset and Solignac, unpublished results). The existence of rickettsia-like micro-organisms has long been documented in D. melanogaster. They have been referred to as Epsilon granules [12] in the pole cells, A bodies [13, 14] in an Oregon stock, "DNA and RNA containing bodies" [15] in salivary glands and imaginal disks and ovaries, commensal parasites in eggs [16], identified as rickettsiae in spermatocytes of pupae [17,18], in cell lines from inviable embryos [19]. Their maternal inheritance was demonstrated [17-19] and some were identified as Wolbachia [20]. The discovery of cytoplasmic incompatibility in D. simulans has prompted experimental analyses in order to investigate the existence of a similar phenomenon in D. melanogaster. A partial incompatibility, maternally inherited and tetracycline sensitive, was detected in Australian populations in crosses between males from Townsville and females from Melbourne [21], but not in Californian populations. The other studies failed to detect any cytoplasmic incompatibility in the strains examined [20, 22], probably because they used fly fecundity instead of egg hatchability to detect it. We provide evidence here
to as Epsilon granules [12] in the pole cells, A bodies [13, 14] in an Oregon stock, "DNA and RNA containing bodies" [15] in salivary glands and imaginal disks and ovaries, commensal parasites in eggs [16], identified as rickettsiae in spermatocytes of pupae [17,18], in cell lines from inviable embryos [19]. Their maternal inheritance was demonstrated [17-19] and some were identified as Wolbachia [20]. The discovery of cytoplasmic incompatibility in D. simulans has prompted experimental analyses in order to investigate the existence of a similar phenomenon in D. melanogaster. A partial incompatibility, maternally inherited and tetracycline sensitive, was detected in Australian populations in crosses between males from Townsville and females from Melbourne [21], but not in Californian populations. The other studies failed to detect any cytoplasmic incompatibility in the strains examined [20, 22], probably because they used fly fecundity instead of egg hatchability to detect it. We provide evidence here for the frequent occurrence of Wolbachia in D. melanogaster populations and its association with cytoplasmic incompatibility. Using PCR amplification with primers specific for Wolbachia, we have found that 34 % of the lines of D. melanogaster are infected. DAPI staining of spermatocytes showed, that in this species, males are infected to a lesser extent than most D. simulans individuals [23]. This low level of infection is accompanied by partial cytoplasmic incompatibility. All the D. melanogaster lines examined so far by 16S rDNA sequencing harbor the same bacterial strain and crosses between strains of different geographical origin suggest the existence of a single infected cytotype in this species. Material and methods Drosophila melanogaster: lines and strains We have used a total of 266 isofemale or plurifemale lines (listed in the legend of the Fig. 7) from the collections of the drosophila stock center of Umeå, from the laboratory of Gif-sur-Yvette and a few other sources. This includes 33 strains from Nasrallah, Tunisia, for which a detailed control of non-infection has been performed but is considered as a single strain in the calculations. Strains were maintained at 21-22 °C on laboratory medium. Incompatible and control crosses Crosses have been performed using 25
to a lesser extent than most D. simulans individuals [23]. This low level of infection is accompanied by partial cytoplasmic incompatibility. All the D. melanogaster lines examined so far by 16S rDNA sequencing harbor the same bacterial strain and crosses between strains of different geographical origin suggest the existence of a single infected cytotype in this species. Material and methods Drosophila melanogaster: lines and strains We have used a total of 266 isofemale or plurifemale lines (listed in the legend of the Fig. 7) from the collections of the drosophila stock center of Umeå, from the laboratory of Gif-sur-Yvette and a few other sources. This includes 33 strains from Nasrallah, Tunisia, for which a detailed control of non-infection has been performed but is considered as a single strain in the calculations. Strains were maintained at 21-22 °C on laboratory medium. Incompatible and control crosses Crosses have been performed using 25 selected isofemale lines: Nasrallah as uninfected reference, Morena as infected reference and 23 additional isofemale lines (of which 22 were infected), each extracted from an isofemale or plurifemale infected strain. Six virgin females and eight young males (one day old) were introduced into a plastic box containing a disk of standard medium changed every day. Eggs were transferred to a black medium (containing Chinese ink) and unhatched eggs were counted 24 and 48 hours after the transfer. The percentage of unhatched eggs, from a minimum of 150 eggs, gave a measure for the level of compatibility. Eggs were collected during a few days and occasional virgin females would have retained the eggs they produced. Antibiotics treatment Infected males were reared on standard medium supplemented with tetracycline-hydrochloride (Boehringer, 0.03 %) during three generations. DAPI fluorescent granules in testes After fixation and incubation in DAPI (4',6-Diamidine-2-phenylindole dihydrochloride, Boehringer), testes were observed on a Zeiss microscope equipped for epifluorescence as described [23]. The number of infected cysts was established for 10 males in each line. PCR conditions For each line or strain, total DNA was extracted from the ovaries of six females according to [24]. 16S rDNA has been amplified using primers specific
standard medium changed every day. Eggs were transferred to a black medium (containing Chinese ink) and unhatched eggs were counted 24 and 48 hours after the transfer. The percentage of unhatched eggs, from a minimum of 150 eggs, gave a measure for the level of compatibility. Eggs were collected during a few days and occasional virgin females would have retained the eggs they produced. Antibiotics treatment Infected males were reared on standard medium supplemented with tetracycline-hydrochloride (Boehringer, 0.03 %) during three generations. DAPI fluorescent granules in testes After fixation and incubation in DAPI (4',6-Diamidine-2-phenylindole dihydrochloride, Boehringer), testes were observed on a Zeiss microscope equipped for epifluorescence as described [23]. The number of infected cysts was established for 10 males in each line. PCR conditions For each line or strain, total DNA was extracted from the ovaries of six females according to [24]. 16S rDNA has been amplified using primers specific for Wolbachia as described [25]. Presence/absence of PCR products was controlled on agarose gels. 16S rDNA sequences PCR products were sequenced with the dideoxynucleotide chain termination method [26] either directly after precipitation [27] or after cloning in pBluescript KS+ (Stratagene) using alkaline denatured plasmid DNA [28] with the primers described [11, 25]. mtDNA haplotypes Total DNA, extracted from ovaries in 24 infected and 23 non-infected strains from various geographical origin, was digested with HaeIII and Aval. Restriction DNA was transferred onto nylon membranes and hybridized to highly purified mitochondrial DNA extracted from D. simulans [9], labeled with 32P by nick translation, and autoradiographed. Results Frequency of infected males A total of 266 lines or strains of Drosophila melanogaster from all continents have been analyzed. For plurifemale strains we have not attempted to analyze individual flies. However, for some populations, several isofemale lines were available and they appeared to either be all non-infected (Nasrallah, Tunisia: 32 isofemale lines and a plurifemale line) or polymorphic for infection (Brazzaville, Congo: 14 isofemale lines infected from 38; Medvasto, Finland: 3 infected isofemale lines from 5, and 9 additional populations, see legend Fig. 7). The total percentage of infected strains is 34 % (79/234, counting
[11, 25]. mtDNA haplotypes Total DNA, extracted from ovaries in 24 infected and 23 non-infected strains from various geographical origin, was digested with HaeIII and Aval. Restriction DNA was transferred onto nylon membranes and hybridized to highly purified mitochondrial DNA extracted from D. simulans [9], labeled with 32P by nick translation, and autoradiographed. Results Frequency of infected males A total of 266 lines or strains of Drosophila melanogaster from all continents have been analyzed. For plurifemale strains we have not attempted to analyze individual flies. However, for some populations, several isofemale lines were available and they appeared to either be all non-infected (Nasrallah, Tunisia: 32 isofemale lines and a plurifemale line) or polymorphic for infection (Brazzaville, Congo: 14 isofemale lines infected from 38; Medvasto, Finland: 3 infected isofemale lines from 5, and 9 additional populations, see legend Fig. 7). The total percentage of infected strains is 34 % (79/234, counting Nasrallah for a single strain) in our sample. Results have been pooled according to country or large geographical area (Fig. 1). Wolbachia is présent in D. melanogaster in almost all regions examined. Its absence in some places, especially in Northern Africa, would require further investigations to be confirmed. The frequency of infected individuals is very similar for every continent (34 % for America, 30 % for Europe, 41 % for Asia, 39 % for Africa and Indian Océan, 13 % for Oceania, P = 0.228 ± 0.010, exact test, M. Raymond and F. Rousset in préparation), for every latitude (35 %, 33 %, 28 %, 42 % and 23 % for the five latitudinal bands of the figure 1, from North to South, P = 0.438 ± 0.013), for every laboratory of origin (27 % for Umeâ, 35 % for Gif and 42 % for Paris, P = 0.170 ±0.010), for every date of collection (P = 0.693 ± 0.010, see 7afj/e /). However, this last result must be taken with some care, particularly for very old strains. A loss of infection or a mixture of strains is possible after one or two thousand générations in the laboratory. As an
of infected individuals is very similar for every continent (34 % for America, 30 % for Europe, 41 % for Asia, 39 % for Africa and Indian Océan, 13 % for Oceania, P = 0.228 ± 0.010, exact test, M. Raymond and F. Rousset in préparation), for every latitude (35 %, 33 %, 28 %, 42 % and 23 % for the five latitudinal bands of the figure 1, from North to South, P = 0.438 ± 0.013), for every laboratory of origin (27 % for Umeâ, 35 % for Gif and 42 % for Paris, P = 0.170 ±0.010), for every date of collection (P = 0.693 ± 0.010, see 7afj/e /). However, this last result must be taken with some care, particularly for very old strains. A loss of infection or a mixture of strains is possible after one or two thousand générations in the laboratory. As an example, we have examined five "Canton-S" strains : three were infected and two were not. Séquences of the 16S rDNA Partial séquences of the bacterial rRNA small subunit (617 bp between positions 74 and 729 of Escherichia coli) have been obtained for four D. melanogaster strains from various geographical origin : Moorea (Society Islands), Uman (Russia), Sevilla (Spain) and Texas (USA). A few singular nucleotides observed in the first two séquences (one clone per strain) have not been confirmed after direct sequencing of the PCR product of the région containing them. They are conservatively attributed to possible Taq polymerase errors. The same is probably true for two singular nucleotides (C instead of T in E. coli position 435 in Sevilla and A instead of G position 445 for Texas). Consequently, we consider that all these partial séquences of the D. melanogaster endosymbiont 16S are probably identical. They are indistinguishable from that of the microorganism found in the Riverside population of D. simulans (see [11, 29]) and not to the bacteria of Hawaiian stocks of D. simulans as inferred [20] from a shorter séquence. Reciprocal crosses between infected and non infected individuals The Nasrallah strain was used as a non infected