jeanflop/post-ocr-correction huggingface dataset:
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
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arose from the existence of two bacterial lineages associated with a single incompatibility type (Rousset and Solignac, unpublished results).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
and Solignac, unpublished results).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
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
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
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
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
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
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,
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
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
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
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
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
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 référence because 32 isofemale lines as well as 10 males
référence because 32 isofemale lines as well as 10 males and 10 females of a plurifemale line from this Tunisian population were négative for infection in PCR tests. Twenty four PCR positive strains from various origins have been retained. Experiments were performed with one isofemale line reextracted from each of them (23 were infected and one, Brazzaville 3, was uninfected), in order to avoid problems linked to the possible polymorphism for infection in laboratory strains. For five strains, tests performed with individual females showed that the variance of unhatched eggs is overdispersed compared to the binomial one. Results of crosses
is overdispersed compared to the binomial one. Results of crosses are given in Table II : intraline crosses and reciprocal crosses to Nasrallah (N) strain were performed using the F1 progeny of the isolated females. Reciprocal crosses of each isofemale line was performed between the F2 flies and the Moorea (M) strain which exhibited one of the strongest level of incompatibility in the F1 génération and crosses appeared to be compatible (Fig. 2). In addition, reciprocal crosses of the F5 of some lines treated during three générations with tetracycline were performed with both Nasrallah and Moorea untreated strains. The percentage
performed with both Nasrallah and Moorea untreated strains. The percentage of unhatched eggs in incompatible crosses (female from Nasrallah crossed to males from infected strains) varies from 10 to 77 %, with an average of 46,7 %. It has to be noticed that it is always higher or far higher than that observed number in the reciprocal Hawaii : 7+/; Society Islands : Tahiti 1+, Moorea 7+/; USA : Oregon 2-, Lausanne 7-, Canton S 3+ 2-, Tempe 1-, Tucson 1+, Texas 1+, Amherst 7-, Miami 1-, Florida 7-, South Carolina 7-/; Mexico : Mapimi 1-/; West Indies : Guadeloupe
Carolina 7-/; Mexico : Mapimi 1-/; West Indies : Guadeloupe 1-, San Thomas 7-, Petit Bourg 1; Bermudas : 1+/; French Guiana 7/; Colombia : Bogota 7-/; Brazil : Brazil 7+, Salvador 1+, Boa Esperança 1+, Curitiba 1-/; Argentina : Gruta 7-, San Miguel 1+/; Sweden : Umeå 3+ 2-, Bygdeå 1+, Karsnäs 2-, Ståket 1-, Strängnäs 7-, Strömsvretén 7-, Falsterbo 7-, Swedish 7-, Valdemarsvik 7-/; Finland : Turku 1-, Naantali 7-, Nokia 1+, Hameenlinna 1-, Medvastö 3+ 2-/; Great Britain : Billingham 1+, Bacup 1-/; Germany : Berlin 7-/; Czechoslovakia : Crkvenica 1+, Moravsky Pisek 7-, Suchumi 1-, Zabcice
Czechoslovakia : Crkvenica 1+, Moravsky Pisek 7-, Suchumi 1-, Zabcice 7; Austria : Wien 1-/; France : Draveil 2-, Champigny 1+ 7-, Charolles 2-, Villeurbanne 7+, Moulis 7-, Porquerolles 7-, Luminy 7-, Marseillan 7+ 5-, Tostes 7-, Brouilly 1-, Pérols 1-, Châtelet 7-, Mèze 3-, Tautavel 7-, Marchant 7-, Colmar 1+, Banyuls 7-, Chevreuse 1+, Mesnil Jourdain 1+, Paris 7-, Pyrénées 7+/; Spain : Oviedo 1+, Barcelona 1-, Sevilla 1+/; Yugoslavia : Slankamen 1-, Italy : Moltrasio 1-, Chieti 7-, Aspra 1-, Sicilia 1-/; Greece : Athens 2-/; Israël : Israël 7-, Qiryat-Anavim 2+ 1-/; Madeira : 1+/; Morocco :
Israël 7-, Qiryat-Anavim 2+ 1-/; Madeira : 1+/; Morocco : Rabat 1+, Casablanca 1+/; Algeria : Algeria 1-, Tizi Ouzou 1-/; Tunisia : Bizerte 1-, Nasrallah 3+ 3-/; Egypt : Alexandria 1+, Gianachis 1+, Aswan 7-/; Sierra Leone : 1; Ivory Coast : Taï 3+ 3-, Lamto 1; Bénin : Cotonou 1-/; Gabon : Makokou 1-; Congo : Brazzaville 1+ 2-, Dimonika 1/; Kenya : Nimba 7-; Tanzania : 7-/; Rhodesia : 1+/; Swaziland 1-; South Africa : Kruger 1-, Cape Town 7+, Harkerville 1+/; Seychelles : 2+ 2-/; Mauritius : Port Louis 1+/; Ukraine : Uman 2+ 7-, Magarach
: Port Louis 1+/; Ukraine : Uman 2+ 7-, Magarach 1-, Gurzuf 7-, Kishinev 1+/; CEI : Birsk 1+, Krasnodar 1+, Anapa 2+, Gelendzhik 7-, Siniy Gay 1; Armenia : Ashtarak 1; Azerbaijan : Kurdamir 1-/; Uzbekistan : Tashkent 1-; Kazakstan : Alma-Ata 1-; Uzbek Republic : Samarkand 2+ 7-/; Iraq : Baghdad 1+/; India : Delhi 1+ 5-, Bhubaneswar 1-/; China : Peking 2-, Chaogong 2+/; Korea : Chongju 7-/; Japan : Nagasaki 1+, Fukuoka 1-, Sapporo 1+, Hikone 2+, Akayu 1-, Chichijima 7-/; New Zealand 1-/; New Caledonia 7-/; Australia : Cardwell 1-/; Coffs Harbour 7+, Melbourne 7-,
7-/; Australia : Cardwell 1-/; Coffs Harbour 7+, Melbourne 7-, Château Tahbilk 4; Fairfield 1+ 5-/; Tasmania : Avondale 7-/ Table I Date of collection for 209 of the 234 stocks used in this study and number of infected (+) and uninfected (-) strains observed. The two distributions are not statistically different (P = 0.693 ± 0.010) Décade 1910 1920 1930 1940 1950 1960 1970 1980 1990 total J+) 3 6 2 4 15 40 7 75 (_) 2 2 3 3 4 14 28 65 13 134 464 C. R. Acad. Sci. Paris, Sciences de la vie/Life sciences, 1994;
R. Acad. Sci. Paris, Sciences de la vie/Life sciences, 1994; 317 : 461-70 Cytoplasmic incompatibility in <i>D. melanogaster</i> cross (23 comparisons, average 6.2 %) and consequently the effect of infection is highly significant, P="(1/2) 23, on egg hatchability. This effect disappeared after tetracycline treatment and treated (aposymbiotic) females henceforth showed a partial incompatibility with untreated Moorea males. The number of males which appeared to be infected after DAPI staining and fluorescence observation (over the 10 males observed per line) and the percentage of infected cysts per individual is given in parallel (Table II). Infection is detected in 70 % of
parallel (Table II). Infection is detected in 70 % of the males by this method. The level of infection observed by DAPI staining is significantly correlated to the level of cytoplasmic incompatibility (Fig. 3). Virgin and previously mated Moorea males, fifteen days old, were crossed to virgin Nasrallah females. These Table II Partial cytoplasmic incompatibility in D. melanogaster measured by the percentage of unhatched eggs. Isofemale lines of Moorea (M, first line) and Nasrallah (N, last line) have been used as infected and uninfected reference strains respectively. The lines are collectively called L Geographical PCR DAPI % unhatched eggs origin
collectively called L Geographical PCR DAPI % unhatched eggs origin N Cl CT IS 9Lx<?N SN<?L 5Lx<?M 5Mx<L 5LT<?N 5Nx<?LT ÎLT<?M ÎM<?LT Moorea + S> 12 2 2 74 2 2 A 2 V 1 Tahiti +843 21 3 19 13 2 Texas + 10 3 3 14 4 58 2 5 4 4 26 7 Canton S + 5 1 1 22 4 33 2 3 Billingham + 10 13 13 30 7 77 4 3 Villeurbanne + 6 ND ND 43 24 71 16 10 12 3 48 ND Oviedo + 3 5 2 12 1 39 38
ND Oviedo + 3 5 2 12 1 39 38 2 Sevilla + 10 10 10 2 2 76 ND 7 2 3 14 4 Casablanca +12 0 1 1 39 4 4 Rabat +9331 3 59 2 1 Alexandrie + 2 4 1 3 5 10 6 1 Gianochis + 9 ND ND 5 2 27 7 1 Brazzaville 1 + 10111184 54 8 9 Brazzaville2 + 107713 1 31 6 3 Brazzaville 3-000 23 2 13 29 3 Brazzaville 4 +105574 54 3 5 2 7 32 4 Brazzaville 5 +88774 53 3 7 Harkerville + 9 2
Brazzaville 5 +88774 53 3 7 Harkerville + 9 2 2 63 33 42 41 5 31 2 48 5 CapeTown +1010 8 23 2 52 3 12 Seychelles +1044 6 10 21 4 7 Port Louis +2 ND ND 2 6 18 3 2 Uman +53116 6 59 9 7 4 ND 18 17 Kishinev + 4 2 1 2 3 68 1 4 Baghdad +19 1 9 11 31 4 2 Nasrallah 0 0 0 11 Average 7.0 5.9 4.7 13.6 6.2 46.7 8.2 4.5 PCR : lines positive (+) and negative (-) by PCR analysis. DAPI :
positive (+) and negative (-) by PCR analysis. DAPI : number of two days old males over 10 showing positive granules in testes : N ; percentage of infected cysts in males positive : Cl ; percentage of infected cysts for the ten males observed : CT. Percentage of unhatched eggs: Intrastrain crosses: 15; reciprocal crosses between males and females of reference strains Nasrallah (N) and Mooréa (M); reciprocal crosses between strains treated by tetracycline (LT) and untreated N or M strains. ND: not determined. Bold characters for crosses SL x SM where more than 50% of unhatched eggs. Average
x SM where more than 50% of unhatched eggs. Average calculated only for infected strains. Several percentages for incompatible crosses are weighted values of two replicates: the lower range was observed for Uman (57-60 %), the higher one for Mooréa (60-87 %). C. R. Acad. Sci. Paris, Sciences de la vie/Life sciences, 1994; 317: 461-70 Figure 2. Percentage of unhatched eggs in compatible crosses (CC) between infected females of various populations with Mooréa males compared to incompatible crosses (IC) between infected males of these different populations with uninfected females from Nasrallah. Two strains, Villeurbanne and Harkerville, are atypical (see text).
Nasrallah. Two strains, Villeurbanne and Harkerville, are atypical (see text). Figure 3. Percentage of unhatched eggs (UH) on percentage of infected cysts (IC, this percentage includes the number of cysts observed in apparently uninfected males) for the different infected strains analyzed. There is a significant relationship between proportions of infected cysts and unhatched eggs (Spearman rank correlation, n=20, rs = 0.4872, P<0.05). Maternal transmission of the bacteria has been controlled in the progeny (four males and four females) of reciprocal crosses between Nasrallah and Mooréa. PCR products were observed only in the progeny of infected mothers (all individuals infected). Crosses
in the progeny of infected mothers (all individuals infected). Crosses have also confirmed maternal inheritance of cytoplasmic incompatibility (Table III). In addition, it appears that infection is tenacious since 21 females of the Mooréa line were still infected (PCR positive) after 15 generations of culture. Table III Maternal inheritance of cytoplasmic incompatibility in D. melanogaster. Percentage of unhatched eggs in crosses between uninfected individuals from Nasrallah (N), infected individuals from Mooréa (M) and their reciprocal hybrids (MN and NM, female parent indicated first). Results of a second experiment are indicated in bold characters. Females of two strains, Villeurbanne and Harkerville,
in bold characters. Females of two strains, Villeurbanne and Harkerville, gave atypical results in all crosses. mtDNA haplotypes of infected and uninfected cytoplasms In order to investigate the origin of the infection and its subsequent evolution, mtDNA polymorphism has been analyzed in 24 infected and 23 uninfected strains. As mitochondria and bacteria are cotransmitted by the cytoplasm, they do share a common history, only complicated by occasional horizontal transfers and losses of Wolbachia. Each of the two enzymes used (Aval and HaeIII, known to produce several restriction profiles in the species) had a single polymorphic site: site c or d
species) had a single polymorphic site: site c or d for Aval and site a for HaeIII, according to [30]. The substitutions inferred to account for this polymorphism are among the most primitive in the D. melanogaster mtDNA tree [30]. Four composite haplotypes have been obtained; this implies homoplasy for at least one of the two polymorphic sites. Coding 1 for presence and 0 for absence, the distribution of infected/non infected stocks for the different composite haplotypes is as follows: 00: 1/2, 01: 6/0, 10: 7/12, 11: 10/9. Infection being observed in all four haplotypes, it must predate the deepest
observed in all four haplotypes, it must predate the deepest node in the mtDNA phylogenetic tree. Discussion D. melanogaster exhibits, in most populations, partial cytoplasmic incompatibility, associated to the presence of the Proteobacteria Wolbachia, the only microorganism known to produce this symptom in a variety of insects and in crustacea [11, 25, 29, 31]. The presence of this bacterium has been detected by PCR amplifications of ovary DNA extracts and its nature has been confirmed by 16S rDNA sequencing. Partial incompatibility (intermediate percentages of hatchability) between infected males and uninfected females is rarely observed in other known cases [32, 33],
females is rarely observed in other known cases [32, 33], except with male ageing [5, 34]. The other features are all characteristic of infection by Wolbachia; namely the location of the microbe in some sperm cysts of the males, the incompatibility of crosses between infected males and uninfected females, the correlation between unhatched eggs and the percentage of infected cysts, the decrease of incompatibility with male ageing, the sensitivity of the symptom to tetracycline and the maternal inheritance of both cytoplasmic incompatibility and its causative agent. These lines of evidence argue in favor of the interpretation that egg inviability is
argue in favor of the interpretation that egg inviability is attributable to Wolbachia and not to other agents. Widespread occurrence of infected individuals and of partial cytoplasmic incompatibility The frequency of infection of Drosophila melanogaster by Wolbachia is about one third. This result is in agreement with frequent observations of infected lines in this species by electron microscope analysis (see Introduction) although some of them may be other rickettsiae or other bacteria. Infection is widespread in all continents, under all latitudes and there is no strong evidence of regions where the flies are less or more infected than other ones.
the flies are less or more infected than other ones. This distribution was obtained from a pool of stocks collected during 80 years (most of them after 1960) but there is no significant difference in the percentage of infection for stocks collected at different periods (Table I). Cytoplasmic incompatibility is sometimes rather low but, in one strain, it is as high as 77 % and in half of the strains 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
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