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Cytology of Wolbachia-induced parthenogenesis in Leptopilina clavipes (Hymenoptera: Figitidae) Bart A. Pannebakker, Laas P. Pijnacker, Bas J. Zwaan, and Leo W. Beukeboom

Abstract: Parthenogenesis induced by cytoplasmatically inherited Wolbachia bacteria has been found in a number of arthropod species, mainly Hymenoptera. Previously, two different forms of diploidy restoration have been reported to underlie parthenogenesis induction in Hymenoptera by Wolbachia. Both are a form of gamete duplication, but each differs in their timing. We investigated the cytology of the early embryonic development of a Wolbachia-infected strain of the parasitoid wasp Leptopilina clavipes and compared it with that of an uninfected sexual strain. Both strains have a similar meiosis. In the infected parthenogenetic strain, diploidy is restored by anaphase restitution during the first somatic mitosis, similar to Trichogramma, but not to Muscidifurax. Our results confirm the occurrence of different cytological mechanisms of diploidy restoration associated with parthenogenesis-inducing Wolbachia in the order Hymenoptera. Key words: gamete duplication, Leptopilina clavipes, parthenogenesis, thelytoky, Wolbachia. Résumé : La parthénogenèse induite par des bactéries Wolbachia, lesquelles sont transmises via le cytoplasme, a été rapportée chez nombre d’espèces d’arthropodes, principalement chez les hyménoptères. Précédemment, deux formes différentes de restauration de la diploïdie sous-tendant la parthénogenèse induite par Wolbachia avaient été rapportées chez les hyménoptères. Les deux reposent sur la duplication gamétique, mais à des moments différents. Les auteurs ont examiné la cytologie du développement embryonnaire précoce chez une souche de la guêpe parasitoïde Leptopilinia clavipes infectée par le Wolbachia et ils ont l’ont comparée à celle observée chez une souche sexuée non-infectée. La méiose était semblable chez les deux souches. Chez la souche infectée, la diploïdie était restaurée par suite d’une restitution en anaphase lors de la première mitose somatique, semblable à ce qui se produit chez le Trichogramma mais non le Muscidifurax. Ces résultats confirment l’existence de différents mécanismes cytologiques menant à la restauration de la diploïdie suite à une infection avec des Wolbachia chez les hyménoptères. Mots clés : duplication gamétique, Leptopilinia clavipes, parthénogenèse, thélytoquie, Wolbachia. [Traduit par la Rédaction]

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Introduction Parthenogenesis can be caused by a number of cytological mechanisms (Suomalainen et al. 1987; White 1973). A special case of parthenogenesis is that induced by intracellular bacteria of the genus Wolbachia (α-proteobacteria). Parthenogenesis-inducing (PI) Wolbachia are mainly found in the insect order Hymenoptera (Stouthamer 1997), but have recently been detected in a predatory thrips species (Thysanoptera; Arakaki et al. 2001) and in two phytophagous mite species (Acari: Tetranychidae; Weeks and Breeuwer 2001). PI Wolbachia seem to be restricted to

species with haplodiploid sex determination, in which males are haploid and females are diploid. The most common mode of haplodiploid reproduction is arrhenotoky, where males develop from unfertilized eggs and females from fertilized eggs. Another mode of reproduction is thelytoky, where females develop from unfertilized eggs and there are no males (Luck et al. 1993; White 1973). Wolbachia is involved in many cases of thelytoky studied in haplodiploids (e.g., Stouthamer 1997). Thus far in only a few cases, i.e., three Trichogramma species (Stouthamer and Kazmer 1994), Diplolepis rosae (Stille and Dävring 1980), and Muscidifurax uniraptor

Received 17 September 2003. Accepted 17 November 2003. Published on the NRC Research Press Web site at http://genome.nrc.ca on 13 March 2004. Corresponding Editor: W. Traut. B.A. Pannebakker.1 Sections of Animal Ecology and Evolutionary Biology, Institute of Biology, Leiden University, P.O. Box 9516, 2300 RA Leiden, The Netherlands. L.P. Pijnacker. Department of Genetics, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, P.O. Box 14, 9750 AA Haren, The Netherlands. B.J. Zwaan. Section of Evolutionary Biology, Institute of Biology, Leiden University, P.O. Box 9516, 2300 RA Leiden, The Netherlands. L.W. Beukeboom. Evolutionary Genetics Group, Centre for Ecological and Evolutionary Studies, University of Groningen, P.O. Box 14, 9750 AA Haren, The Netherlands. 1

Corresponding author (e-mail: [email protected]).

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doi: 10.1139/G03-137

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(Gottlieb et al. 2002), the cytological mechanism of parthenogenesis has been clarified. In general, PI Wolbachia cause diploidization of the unfertilized haploid eggs, which develop as diploid females. This occurs through different forms of gamete duplication, which results in the production of fully homozygous progeny (Suomalainen et al. 1987). In Trichogramma spp., diploidization is caused by segregation failure in the first mitotic anaphase (Stouthamer and Kazmer 1994). In Diplolepis rosae and Muscidifurax uniraptor, the first mitotic division is normal and diploidization occurs through a fusion of the two mitotic nuclei in the second prophase (Gottlieb et al. 2002; Stille and Dävring 1980). Although no cytological study was done, genetic analysis suggests that the mechanism of parthenogenesis in the Wolbachia-infected mite Bryobia praetiosa is not gamete duplication, as heterozygous progeny is produced (Weeks and Breeuwer 2001). Thus, although the number of studies is limited, they suggest that Wolbachia can cause parthenogenesis in multiple ways. Leptopilina clavipes (Hartig) (Hymenoptera: Figitidae) is a parasitoid wasp of fungi-breeding Drosophila larvae that occurs in western Europe (Nordlander 1980). In northwestern Europe, populations of L. clavipes are thelytokous (Driessen et al. 1990), which is Wolbachia induced (Werren et al. 1995; Schidlo et al. 2002). Curing Wolbachia infection with antibiotics can restore male production, but permanent sexual lines could not be established in culture (B.A. Pannebakker, unpublished data). Recently, uninfected arrhenotokous individuals were found in Spain (Pannebakker et al. 2004). Here we investigate the cytological mechanism of Wolbachia-induced parthenogenesis in thelytokous L. clavipes and compare it to that of uninfected arrhenotokous individuals. Knowing the exact mechanism of diploidy restoration is important, since it has important implications for the evolution of sexual reproduction (Haccou and Schneider 2004).

Materials and methods Insect cultures The infected thelytokous line examined, GBW-NL00, originated from Groot Buunderkamp, Wolfheze, The Netherlands (lat 52°00.85′N, long 05°45.71′E), and was collected in the spring of 2000. The uninfected arrhenotokous line DC-E00 originated from Duna Continental, El Montgrí, Spain (lat 42° 04.84′N, long 03° 08.66′E), and was also collected in the spring of 2000. The cultures were maintained in the laboratory on Drosophila phalerata larvae at 20 °C, 16 h light : 8 h dark and 65% relative humidity. Drosophila phalerata were reared on a medium containing mushroom (Agaricus bisporus), water, dry yeast (Saccharomyces cerevisiae Hansen), and agar. The fungicides nipagin and propionic acid were added to prevent molding of the medium. Egg collection and cytological techniques A surplus of L. clavipes females was allowed to oviposit in second instar D. phalerata larvae. The wasp eggs were left to develop, after which the host larvae were fixed in Carnoy’s fixative (3:1, ethanol – glacial acetic acid) at specific time intervals varying between 0 and 4 h. Moreover, embryos in the process of early segmentation of the body

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(2–3 days old) were fixed to obtain metaphases. Eggs were dissected from a larva on a microscopic slide and, after removing most of the larval debris, processed for cytological investigation. They were stained with DAPI (4′,6-diamidino2-phenylindole) and meiosis and early embryonic development were photographed, as described by Beukeboom and Pijnacker (2000). Photographs were processed into digital pictures using Adobe Photoshop 6.0 (Adobe Systems, San Jose, Calif.) and Corel Draw 9.0 (Corel, Ottawa, Ont.).

Results Arrhenotokous strain Meiosis, from the first metaphase onwards from oviposition, takes place in the periphery of the yolk somewhere between 10% and 40% of the length of the egg from the anterior end and normally finishes within 2 h. Five bivalents lie compact together in first metaphase in a spindle perpendicular or somewhat skew to the surface (Fig. 1a). The first division is reductional and results in a first polar body with five chromosomes in the periphery and a set of five chromosomes of the second meiotic division predestined to form the pronucleus, somewhat inward from the surface (Fig. 1b and 1c). Neither set despiralizes and both start mitotic division simultaneously (Fig. 1d). The polar body divides parallel to the periphery and results in two groups of five chromosomes. Only later on can these products be distinguished from those of the other division when they remain, as chromosomes, in situ and do not divide again. The spindle of the second division is rather skew or even parallel to the surface. This division results in one set of five chromosomes that remains in situ, as the second polar body, close to the two sets of the first polar body, and another set of five chromosomes that forms the female pronucleus (Fig. 1e). This pronucleus takes on the appearance of interphase and migrates away from the polar bodies without a particular direction, but remains in the anterior part. It increases in size considerably and becomes filled with finely despiralized chromatin (Fig. 1e). In unfertilized eggs, the pronucleus starts with the synchronous somatic divisions parallel to the surface typically from 2 h after oviposition onwards (Fig. 1f). The chromosomes in the eggs could not be caught in one level for a photograph owing to their small size and the rigidity of the yolk. Notwithstanding, their number (n = 5) could be counted for over 100 times from the meiotic divisions. From metaphases in haploid embryos of 2–3 days old it could be established that the chromosomes differ in length, but the exact location of the centromeres remains to be resolved (Figs. 1g and 1h). In fertilized eggs, only one sperm was ever found in the periphery of the yolk of the anterior part early after oviposition (Fig. 2a). The longdrawn sperm head shortens until it is a small ovoid interphase nucleus at the end of the first meiotic division (Figs. 2b and 2c). Before the first somatic division is expected to take place, two similar-looking large interphase nuclei may be found close together somewhere around the region of the polar bodies (Fig. 2d). The small male pronucleus thus increases also in size before nearing the female pronucleus. Paired prophase nuclei were not found and 3 × 2 groups of five chromosomes were observed in one prometaphase configuration (Fig. 2e). Fusion of both © 2004 NRC Canada



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Fig. 1. Meiosis and mitosis in unfertilized eggs of an arrhenotokous strain of Leptopilina clavipes. (a) First metaphase. (b) First metaanaphase. (c) First telophase. (d) Second metaphase and first polar body (at right). (e) Second polar body (out of focus), divided first polar body, and female pronucleus. (f) Haploid telophase of first somatic mitosis. (g and h) Haploid metaphase in embryo. Bar represents 5 µm.

Fig. 2. Meiosis and fertilization in an arrhenotokous strain of Leptopilina clavipes. (a) Spermatozoon in newly laid egg. (b) First telophase and despiralized spermatozoon (bottom). (c) Second telophase, telophase of first polar body and despiralized spermatozoon. (d) Paired female and male pronuclei, one at early prophase and one at interphase. (e) Paired prometaphases of male and female pronuclei. Bar represents 5 µm.

pronuclei thus takes place quickly and the diploid complement is restored during spindle organization. Normally, the first diploid somatic division takes place between 2 and 3 h after oviposition. In both fertilized and unfertilized eggs, the polar bodies remain visible as three groups of five chromosomes that show signs of degeneration only when at least eight somatic nuclei are present. Thelytokous strain Meiosis is similar to the arrhenotokous strain. It always

results in one large haploid female pronucleus and three polar bodies with five chromosomes at the same ultimate location. The pronucleus enters a prophase of which the number of chromosomes is still haploid, when compared with haploid and diploid prophases from the arrhenotokous strain (Figs. 3a and 3h). A spindle is formed parallel to the surface and the haploid metaphase (Figs. 3b and 3c) is followed by an aberrant anaphase. At first sight this anaphase shows a compact group of separating chromatids that are held together by their stretched ends (Fig. 3d). After squashing, di© 2004 NRC Canada



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Fig. 3. Restitution of diploidy in a thelytokous strain of Leptopilina clavipes. (a) Haploid prophase. (b) Haploid metaphase. (c) Haploid metaphase, second polar body (top) and meta-anaphase of first polar body (bottom). (d) Meta-anaphase of first somatic division. (e and f) Irregular segregation of chromatids at anaphase. (g) Restitution nucleus. (h) Diploid prophase after restitution, polar bodies out of focus. Bar represents 5 µm.

vided and undivided chromosomes can be seen among the dividing chromosomes (Figs. 3e and 3f). Instead of a haploid telophase, like in the arrhenotokous strain (Fig. 1f), a single interphase nucleus with irregularly divided chromatin is formed (Fig. 3g). In the thelytokous strain, diploidy is thus restored by anaphase restitution during the first somatic mitosis as caused by asynchronously dividing chromosomes, which generally takes place less than three hours after oviposition. This is substantiated by the following observations: one diploid mitosis takes place later on (Fig. 3h); the aberrant anaphases were never seen in degeneration; and fusion of two interphase nuclei, in a “figure eight” configuration during early fusion, was not observed, nor was fusion of two mitotic haploid nuclei. Subsequent mitoses are synchronous and parallel to the surface. It is important to note that complete separation of the homologous chromosomes during first meta-anaphase can be accomplished within 30 min after oviposition. However, a delay occurred regularly and eggs of both strains could still be in first meta-anaphase after 3.5 h. The frequency of delayed eggs varied from sample to sample without an obvious cause. Maximum numbers were 8 out of 16 eggs (50%) of the arrhenotokous strain collected after 2 h and 14 out of 22 eggs (64%) of the thelytokous strain collected after 2.5 h. Consequently, eggs of one sample collected at a certain time were in different phases of early embryonic development and many eggs had to be investigated to establish its exact course; i.e., 233 and 1087 eggs of the arrhenotokous and thelytokous strains, respectively.

Discussion The cytological mechanism of diploidy restoration leading to thelytoky in Leptopilina clavipes is gamete duplication by failure of the first mitotic anaphase division. Our conclusions are in line with those drawn for thelytokous Trichogramma by Stouthamer and Kazmer (1994). Based on cytological observations and segregation patterns of allozymes

they concluded that gamete duplication is the cytological mechanism underlying Wolbachia-induced parthenogenesis in Trichogramma. Gamete duplication leads to the generation of a completely homozygous progeny (Suomalainen et al. 1987). Recently Gottlieb et al. (2002) found a different mechanism of diploidy restoration in the PI Wolbachia-infected wasp Muscidifurax uniraptor. Their observations show a normal first mitosis, after which the two haploid nuclei duplicate their DNA to enter the second mitosis. Based on DNA content analysis of the microscopic images, the authors conclude that the two diploid (equal DNA value) nuclear products then fuse into a single tetraploid (equal DNA value) nucleus in the prophase of the second mitosis. This nucleus undergoes a normal mitotic division resulting in two diploid (equal number of chromosomes) nuclei. Gamete duplication in M. uniraptor hence occurs in the second mitotic division instead of in the first mitotic division. The same mechanism was presumably also found in the thelytokous Wolbachiainfected wasp Diplolepis rosae by Stille and Dävring (1980). The Wolbachia in Trichogramma, M. uniraptor, D. rosae, and L. clavipes are of different phylogenetic groups (respectively B, A, B, and B) (Werren et al. 1995; Zhou et al. 1998). The existence of distinct mechanisms of gamete duplication by Wolbachia in different phylogenetic groups suggests a polyphyletic origin of diploidy restoration in Wolbachia. Another possibility explaining the different mechanisms is a strong host–Wolbachia interaction, such as found for the effects of cytoplasmic incompatibility (CI) Wolbachia in the wasp Nasonia vitripennis (Bordenstein et al. 2003) and for feminizing Wolbachia in the moth Ostrinia scapulalis, which causes male killing after transfer to the moth Ephestia kuehniella (Fujii et al. 2001). The karyotype of L. clavipes (n = 5) was established for the first time. It deviates from the only other record for the Leptopilina genus, L. heterotoma, which has a haploid chromosome number of n = 10 (Jungen cited in Crozier 1975). This contrasts with the considerable uniformity in chromo© 2004 NRC Canada



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some numbers found within genera of parasitic Hymenoptera (Gokhman and Quicke 1995). Differences in chromosome number do appear within parasitoid genera, but generally not doubling of chromosome numbers. More cytological studies on the Leptopilina genus are needed to clarify this issue.

Acknowledgements Henk Mulder is acknowledged for preparing the figures, Jacques van Alphen and Paul Brakefield for fruitful discussion, and Richard Stouthamer and two anonymous reviewers for valuable comments on the manuscript. L.W.B. was supported by a PIONIER grant of the Netherlands Organization for Scientific Research (NWO).

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