W&M ScholarWorks Undergraduate Honors Theses Theses, Dissertations, & Master Projects 7-2012 A Comparative Study of Feminine Reproduction in a Rhabditis Clade Kathryn Rehain College of William and Mary Follow this and additional works at: https://scholarworks.wm.edu/honorstheses Part of the Biology Commons Recommended Citation Rehain, Kathryn, "A Comparative Study of Feminine Reproduction in a Rhabditis Clade" (2012). Undergraduate Honors Theses. Paper 470. https://scholarworks.wm.edu/honorstheses/470 This Honors Thesis is brought to you for free and open access by the Theses, Dissertations, & Master Projects at W&M ScholarWorks. It has been accepted for inclusion in Undergraduate Honors Theses by an authorized administrator of W&M ScholarWorks. For more information, please contact [email protected]. A Comparative Study of Feminine Reproduction in a Rhabditis Clade A thesis submitted in partial fulfillment of the requirements for the degree of Bachelor of Science with Honors in Biology at the College of William and Mary. Kathryn Rehain Accepted for: ______________________________ ______________________________ Advisor: Dr. Diane Shakes ______________________________ Dr. Jonathan Allen ______________________________ Dr. Oliver Kerscher ______________________________ Dr. Rowan Lockwood Williamsburg, Virginia May 2012 Table of Contents Abstract.…………………………………………………………………………………..5 Background.……………………………………………………………………………...7 An Introduction to Nematodes ……………………………………………………7 C. elegans: A Classic Model Organism ………………………………………….8 Beyond C. elegans: Other Nematodes in the Wild and in the Lab …………..…10 Rhabditis sp. SB347: An Unusual Lab Sustainable Nematode ………………....12 Studying the Diversity of Nematodes …………………………………………...15 Elements of a Reproductive Strategy ……………………………………………16 The Goal of This Study ………………………………………………………….19 Methods and Materials ………………………………………………………………...26 Nematode Strains and Culturing Conditions ……………………………………26 Brood Count Experiments ……………………………………………………….27 DIC of Whole Worms …………………………………………………………...27 DIC and Hoechst Staining of Isolated Gonads ………………………………….28 Whole Worm DAPI Staining ……………………………………………………28 Preparation for Immunofluorescence ……………………………………………29 Labeling with Anti-Phospho-Histone-H3(ser10) Antibody ……………………..30 Labeling with Anti-nucleolar Antibody …………………………………………31 Results …………………………………………………………………………………..33 Verifying that Self-fertile SB347 and SB372 Hermaphrodites Produce an Unexpected Proportion of Male Offspring …………….…………...33 Examining Intact Gonad Size Relative to Overall Body Size …………….…….34 Defining the Morphology of the Gonad …………………………………………35 2 Characterizing the Distal and Proximal Portions of the Gonad …………………36 Correlation of Chromosome Morphology and Nucleolus Size in Maturing Oocytes …………………………...……………………………….37 Mitotic Divisions of Germline Cells …………………………………………….39 Re-initiation of Meiosis in the Most Proximal Oocyte ………………………….40 Differences in Ovulation ………………………………………………………...41 Discussion ………………………………………………………………………………55 Moving Forward ………………………………………………………………...62 References ………………………………………………………………………………65 Figures and Tables Figure 1: C. elegans ……………………………………………………………..……...20 Figure 2: Caenorhabditis Phylogeny……………………………………………………21 Figure 3: Phylogeny Including C. elegans and Members of the Rhabditis Clade…………………………………………………..……..……..22 Figure 4: R. sp. SB347 ………………………………………………………...………...23 Figure 5: Developmental Diversity ………………………………….………………….24 Figure 6: Oogenesis and Oocyte Maturation in C. elegans …………………………….25 Table 1: Dilution of Levamisole in Dissecting Solution ……………………………….32 Table 2: Brood Size …………………………………………………………………….44 Table 3: Number of Male Progeny per Worm ………………………………………….44 Table 4: Percentage of Males in Total Brood …………………………………………..44 Figure 7: Gonad Morphology in Intact Worms …………………………….…...……...45 Figure 8: Isolated Gonadal Arms ……………………………………………………….47 3 Figure 8A: Differences in Germ-Cell Arrangement ……………………………………48 Figure 8B: Unidentified Somatic Cells …………………………………………………49 Figure 9: DNA Morphology and Nucleolus Size ………………………………………50 Figure 10: Actively Dividing Cells of the Mitotic Zone …………….………………….51 Figure 11: Re-initiation of Meiosis in Proximal Oocyte ……………………………….52 Figure 12: C. elegans Ovulation ………………………………………………………..53 Figure 13: SB347 Ovulation ……………………………………………………………54 4 Abstract Here I studied the reproductive strategies of nematodes in a Rhabditis clade. The identified members of this clade include Rhabditella axei and the Rhabditis species R. sp. SB347, R. sp. SB372, R. sp. JU1782, R. sp. JU1783, and R. sp. JU1809. To better define the reproductive strategies of the hermaphrodites/females within this clade the morphology of the gonad as well as other elements of reproductive strategies were compared, with the well-studied Caenorhabditis elegans hermaphrodite reproductive strategy used as a standard. It was found that the reproductive strategies of hermaphrodites/females of the Rhabditis clade differ from that of C. elegans hermaphrodites in a number of ways. In at least one, if not more, species within this clade, clear differences were documented in distal gonad morphology, the proportion of mitotically dividing cells in the distal gonad, chromosome morphology, the size of the nucleoli, spermatheca morphology, and offspring sex-ratios. However, a number of similarities were found as well. C. elegans, R. axei, and SB372 gonads have comparable numbers of mitotically dividing cells. In C. elegans, R. axei, and SB347 meiosis resumes in the most proximal oocyte. Furthermore, C. elegans, SB347, and SB372 hermaphrodites all produce broods of a similar size. Lastly, the hermaphrodites/females of all species examined had didelphic gonads containing oocytes in the proximal region, and these gonads occupied a large portion of the total worm volume in all species. In the future, these findings will be combined with molecular phylogenetic studies to examine the evolution of diverse reproductive strategies within closely related nematode species. Importantly, the reproductive features of one or more of the trioecious 5 species within this clade suggests that it could be an excellent model organism for the study of parasitic nematodes. Moreover, comparative studies within the Rhabditis clade provide an informative parallel to comparative studies within the genus Caenorhabditis. 6 Background An Introduction to Nematodes The phylum Nematoda is rich in history, number and diversity. Described in ancient times by the famous philosopher and early scientist Aristotle, nematodes are outnumbered only by arthropods in terms of the number of species and the number of individuals present on Earth (as reviewed in Croll and Matthews, 1977; Wharton, 1986). While many species share a morphologically similar small, thin, non-segmented “worm” body-form, nematodes as a whole utilize a surprisingly wide assortment of life-strategies (as reviewed in Malakhov and Hope, 1994). Some species are parasites of animals or plants, while others are free-living in soil, salt water, fresh water, and many other environments. Additionally, nematodes can be bacterial-feeders, fungal-feeders, algal- feeders, herbivores, omnivores, or carnivores (as reviewed in Wharton, 1986). Nematodes are in fact so numerous and diverse that Dr. Nathan Cobb, a famous nematologist, once wrote, “If all the matter in the universe except the nematodes were swept away, our world would still be dimly recognizable... we would find its mountains, hills, valleys, rivers, lakes and oceans represented by a film of nematodes,” (Cobb, 1915). One of the most studied nematode species is Caenorhabditis elegans, a renowned model organism for biological research. It was Dr. Sydney Brenner who voiced the need for a model organism that had three important traits. First, the organism had to be easily kept in the lab. Second, it needed to have tractable genetics. Third, the organism had to be easily observed. The nematode C. elegans fit the criteria, and in 1974 Dr. Brenner proposed using C. elegans to study genetic regulation of development (Brenner, 1974). Over time and with further study, it has become clear that C. elegans is an excellent 7 model not only to study development, but a wide range of subjects. It has since been used to study diverse topics ranging from gerontology, Parkinson’s Disease, and infectious diseases to programmed cell death and the function of small RNAs (as reviewed in Ellis and Horvitz, 1986; Gami and Wolkow, 2006; Harrington et al, 2010; Abbot, 2011; Glavis-Bloom et al, 2012). Further, as the first fully sequenced multi-cellular organism, it was instrumental to the development of larger genome sequencing projects (Sulston et al, 1992). C. elegans: A Classic Model Organism As previously stated, C. elegans possesses the three traits stipulated by Dr. Brenner as necessary for a good model organism: easy to grow; simple genetics; easy to observe (Brenner, 1974). While many other nematode species are quite difficult or impractical to cultivate in a lab setting, C. elegans is remarkably easy to grow and maintain (as reviewed in Croll and Matthews, 1977). Naturally a free-living soil nematode, C.elegans can be raised in the lab either on agar plates or in liquid culture with the bacterium Escherichia coli as a food source. Additionally, because these worms are only about one millimeter length, large numbers can be raised in a limited space. C. elegans is a dioecious species, meaning it has two genders, male and self-fertile hermaphrodite (Figure 1). The