Rapid diversification of mating systems in ciliates

Ciliates are a diverse group of microbial eukaryotes that exhibit tremendous variety in several aspects of their mating systems. To understand the evolutionary forces driving mating system diversification in ciliates, we use a comparative approach synthesizing data from many ciliate species in light of recent phylogenetic analyses. Specifically, we investigate the evolution of number of mating types, mode of mating type inheritance, and the molecular determinants of mating types across the taxonomic diversity of ciliates, with an emphasis on three well-studied genera: Tetrahymena , Paramecium , and Euplotes . We find that there have been many transitions in the number of mating types, and that the requirement of nuclear reorganization may be a more important factor than genetic exchange in determining the optimum number of mating types in a species. We also find that the molecular determinants of mating types and mode of inheritance are evolving under different constraints in different lineages of ciliates. Our results emphasize the need for further detailed examination of mating systems in understudied ciliate lineages.  © 2009 The Linnean Society of London, Biological Journal of the Linnean Society , 2009, 98 , 187–197.     

Actinomycin D-induced change of mating type in the ciliate Dileptus anser

The effects of actinomycin D on the expression and inheritance of mating types (MTs) were studied in mature laboratory clones of the ciliate Dileptus anser. In these ciliates, each mature clone isolated from the natural population belongs to one of three complementary MTs, i.e., MT I, MT II, or MT III. In the course of further cultivation of the clone under laboratory conditions, in a series of vegetative generations, its MT remained unchanged. However, treatment with actinomycin D (15 μg/ml, 3 days) causes these clones to transition to a state that is hereditarily unstable for their MTs. At weekly testing for MT for at least 15 weeks after this treatment (which corresponds to more than 100 cell divisions), many subclones of the treated clone were observed to reversibly exchange their MT for another one; a temporary state of immaturity and/or partial maturity was also revealed. These data confirm our hypothesis about epigenetic MT determination in D. anser. Taking into account that actinomycin D also induces the inheritable destabilization of some characters in amoebas Amoeba proteus, which obviously has an epigenetic nature, this antibiotic might be considered an epimutagen.     

Exploration of Genetic Variations through Single‐cell Whole‐genome Sequencing in the Model Ciliate Tetrahymena thermophila

Ciliates are unicellular eukaryotes with separate germline and somatic genomes and diverse life cycles, which make them a unique model to improve our understanding of population genetics through the detection of genetic variations. However, traditional sequencing methods cannot be directly applied to ciliates because the majority are uncultivated. Single‐cell whole‐genome sequencing (WGS) is a powerful tool for studying genetic variation in microbes, but no studies have been performed in ciliates. We compared the use of single‐cell WGS and bulk DNA WGS to detect genetic variation, specifically single nucleotide polymorphisms (SNPs), in the model ciliate Tetrahymena thermophila. Our analyses showed that (i) single‐cell WGS has excellent performance regarding mapping rate and genome coverage but lower sequencing uniformity compared with bulk DNA WGS due to amplification bias (which was reproducible); (ii) false‐positive SNP sites detected by single‐cell WGS tend to occur in genomic regions with particularly high sequencing depth and high rate of C:G to T:A base changes; (iii) SNPs detected in three or more cells should be reliable (an detection efficiency of 83.4–97.4% was obtained for combined data from three cells). This analytical method could be adapted to measure genetic variation in other ciliates and broaden research into ciliate population genetics.     

Discovery and Functional Study of a Novel Genomic Locus Homologous to Bα-Mating-Type Sublocus of Lentinula edodes

The interaction of mating pheromone and pheromone receptor from the B mating-type locus is the first step in the activation of the mushroom mating signal transduction pathway. The B mating-type locus of Lentinula edodes is composed of Bα and Bβ subloci, each of which contains genes for mating pheromone and pheromone receptor. Allelic variations in both subloci generate multiple B mating-types through which L. edodes maintains genetic diversity. In addition to the B mating-type locus, our genomic sequence analysis revealed the presence of a novel chromosomal locus 43.3 kb away from the B mating-type locus, containing genes for a pair of mating pheromones (PHBN1 and PHBN2) and a pheromone receptor (RCBN). The new locus (Bα-N) was homologous to the Bα sublocus, but unlike the multiallelic Bα sublocus, it was highly conserved across the wild and cultivated strains. The interactions of RcbN with various mating pheromones from the B and Bα-N mating-type loci were investigated using yeast model that replaced endogenous yeast mating pheromone receptor STE2 with RCBN. The yeast mating signal transduction pathway was only activated in the presence of PHBN1 or PHBN2 in the RcbN producing yeast, indicating that RcbN interacts with self-pheromones (PHBN1 and PHBN2), not with pheromones from the B mating-type locus. The biological function of the Bα-N locus was suggested to control the expression of A mating-type genes, as evidenced by the increased expression of two A-genes HD1 and HD2 upon the treatment of synthetic PHBN1 and PHBN2 peptides to the monokaryotic strain of L. edodes.     

Prevalent Ciliate Symbiosis on Copepods: High Genetic Diversity and Wide Distribution Detected Using Small Subunit Ribosomal RNA Gene

Toward understanding the genetic diversity and distribution of copepod-associated symbiotic ciliates and the evolutionary relationships with their hosts in the marine environment, we developed a small subunit ribosomal RNA gene (18S rDNA)-based molecular method and investigated the genetic diversity and genotype distribution of the symbiotic ciliates on copepods. Of the 10 copepod species representing six families collected from six locations of Pacific and Atlantic Oceans, 9 were found to harbor ciliate symbionts. Phylogenetic analysis of the 391 ciliate 18S rDNA sequences obtained revealed seven groups (ribogroups), six (containing 99% of all the sequences) belonging to subclass Apostomatida, the other clustered with peritrich ciliate Vorticella gracilis. Among the Apostomatida groups, Group III were essentially identical to Vampyrophrya pelagica, and the other five groups represented the undocumented ciliates that were close to Vampyrophrya/Gymnodinioides/Hyalophysa. Group VI ciliates were found in all copepod species but one (Calanus sinicus), and were most abundant among all ciliate sequences obtained, indicating that they are the dominant symbiotic ciliates universally associated with copepods. In contrast, some ciliate sequences were found only in some of the copepods examined, suggesting the host selectivity and geographic differentiation of ciliates, which requires further verification by more extensive sampling. Our results reveal the wide occurrence and high genetic diversity of symbiotic ciliates on marine copepods and highlight the need to systematically investigate the host- and geography-based genetic differentiation and ecological roles of these ciliates globally.     

On the nature of species: insights from <Emphasis Type="Italic">Paramecium</Emphasis> and other ciliates

The multiple species concepts currently in use by the scientific community (e.g. Morphological, Biological, Phylogenetic) are united in that they all aim to capture the process of divergence between populations. For example, the Biological Species Concept defines a species as a natural group of organisms that is reproductively isolated from other such groups. Here we synthesize nearly a century of research on the ciliate genus Paramecium that highlights the shortcomings of our prevailing notions on the nature of species. In this lineage, there is discordance between morphology, mating behavior, and genetics, features assumed to be correlated, at least after sufficient time has passed, under all species concepts. Intriguingly, epigenetic phenomena are well documented in ciliates where they influence features such as germline/soma differentiation and mating type determination. Consequently, we hypothesize that divergence within ciliate populations is due to a dynamic interaction between genetic and epigenetic factors. The growing list of examples of epigenetic phenomena that potentially impact speciation (i.e. by influencing the dynamics of sex chromosomes, fate of hybrids, zygotic drive and genomic conflicts) suggests that interactions between genetics and epigenetics may also drive divergence in other eukaryotic lineages.     

Disentangling signaling gradients generated by equivalent sources

Yeast cells approach a mating partner by polarizing along a gradient of mating pheromones that are secreted by cells of the opposite mating type. The Bar1 protease is secreted by a-cells and, paradoxically, degrades the α-factor pheromones which are produced by cells of the opposite mating type and trigger mating in a-cells. This degradation may assist in the recovery from pheromone signaling but has also been shown to play a positive role in mating. Previous studies suggested that widely diffusing protease can bias the pheromone gradient towards the closest secreting cell. Here, we show that restricting the Bar1 protease to the secreting cell itself, preventing its wide diffusion, facilitates discrimination between equivalent mating partners. This may be mostly relevant during spore germination, where most mating events occur in nature.     

Mating-Type Polymorphic Variation in Euplotes minuta (Ciliophora: Hypotrichida)1

An investigation was carried out to probe into the mating-type structure of a local population of the marine ciliate, Euplotes minuta. From this population, nine different mating types belonging to a unique set were isolated. The nine type-representative wild stocks analyzed were found to be heterozygous at the mating-type (mat) locus and provided, together with their sexual progeny, a total of 15 pure mating types. In E. minuta, the high-multiple nature of the basic mating system controlled by a series of peck-order alleles at a single locus should be considered a virtual certainty. The relationships among the genetic economies of the similar bottom-dwelling marine ciliates of the genus Euplotes, the E. vannus-crassus-minuta group, are discussed.     

Pheromone communication in the fission yeast Schizosaccharomyces pombe

Conjugation between two haploid yeast cells is generally controlled by the reciprocal action of diffusible mating pheromones, cells of each mating type releasing pheromones that induce mating-specific changes in cells of the opposite type. Recent studies into pheromone signalling in the fission yeast Schizosaccharomyces pombe have revealed significant parallels with processes in higher eukaryotes and could provide the opportunity for investigating communication in an organism that is amenable to both biochemical and genetic manipulation.     

Phylogeny of Intestinal Ciliates,Including Charonina ventriculi,and Comparison of Microscopy and 18S rRNA Gene Pyrosequencing for Rumen Ciliate Community Structure Analysis

The development of high-throughput methods, such as the construction of 18S rRNA gene clone or pyrosequencing libraries, has allowed evaluation of ciliate community composition in hundreds of samples from the rumen and other intestinal habitats. However, several genera of mammalian intestinal ciliates have been described based only on morphological features and, to date, have not been identified using molecular methods. Here, we isolated single cells of one of the smallest but widely distributed intestinal ciliates, Charonina ventriculi, and sequenced its 18S rRNA gene. We verified the sequence in a full-cycle rRNA approach using fluorescence in situ hybridization and thereby assigned an 18S rRNA gene sequence to this species previously known only by its morphology. Based on its full-length 18S rRNA gene sequence, Charonina ventriculi was positioned within the phylogeny of intestinal ciliates in the subclass Trichostomatia. The taxonomic framework derived from this phylogeny was used for taxonomic assignment of trichostome ciliate 18S rRNA gene sequence data stemming from high-throughput amplicon pyrosequencing of rumen-derived DNA samples. The 18S rRNA gene-based ciliate community structure was compared to that obtained from microscopic counts using the same samples. Both methods allowed identification of dominant members of the ciliate communities and classification of the rumen ciliate community into one of the types first described by Eadie in 1962. Notably, each method is associated with advantages and disadvantages. Microscopy is a highly accurate method for evaluation of total numbers or relative abundances of different ciliate genera in a sample, while 18S rRNA gene pyrosequencing represents a valuable alternative for comparison of ciliate community structure in a large number of samples from different animals or treatment groups.     

Purification and characterization of new mating pheromones of the ciliate Euplotes raikovi

Cell union in mating pairs in the ciliate Euplotes raikovi is controlled by a system of multiple mating types which are inherited with alleles codominant at the genetic locus mat and expressed via diffusible mating pheromones. The mating pheromones Er-2, Er-3, and Er-11 were purified from cells homozygous for the mat-2, mat-3, and mat-11 alleles, respectively. These pheromones are proteins of similar Mr (11,000-12,000) and acidity (pI 3.7-4.0) and are active at a concentration that varies from 2.9 X 10(-12) to 1.2 X 10(-11) M. Data on amino acid composition revealed that an unusually high amount of cysteine (12-15.7%) and poor contents of basic amino acids are common to every pheromone. On the basis of this uniformity in the main biochemical traits, which also holds for the previously purified pheromone Er-1, it was concluded that E. raikovi mating pheromones are members of a family of proteins structurally diversified from each other to varying extents.     

The genetic basis for mating-induced sex differences in starvation resistance in Drosophila melanogaster

Multiple genetic and environmental factors interact to influence starvation resistance, which is an important determinant of fitness in many organisms, including Drosophila melanogaster. Recent studies have revealed that mating can alter starvation resistance in female D. melanogaster, but little is known about the behavioral and physiological mechanisms underlying such mating-mediated changes in starvation resistance. In the present study, we first investigated whether the effect of mating on starvation resistance is sex-specific in D. melanogaster. As indicated by a significant sex × mating status interaction, mating increased starvation resistance in females but not in males. In female D. melanogaster, post-mating increase in starvation resistance was mainly attributed to increases in food intake and in the level of lipid storage relative to lean body weight. We then performed quantitative genetic analysis to estimate the proportion of the total phenotypic variance attributable to genetic differences (i.e., heritability) for starvation resistance in mated male and female D. melanogaster. The narrow-sense heritability (h²) of starvation resistance was 0.235 and 0.155 for males and females, respectively. Mated females were more resistant to starvation than males in all genotypes, but the degree of such sexual dimorphism varied substantially among genotypes, as indicated by a significant sex × genotype interaction for starvation resistance. Cross-sex genetic correlation was greater than 0 but less than l for starvation resistance, implying that the genetic architecture of this trait was partially shared between the two sexes. For both sexes, starvation resistance was positively correlated with longevity and lipid storage at genetic level. The present study suggests that sex differences in starvation resistance depend on mating status and have a genetic basis in D. melanogaster.     

Mating behaviour and chemical communication in the invasive Chinese mitten crab Eriocheir sinensis

Mating in the Chinese mitten crab (Eriocheir sinensis) was examined; in particular the nature of mating and the role of sex pheromones. A semi-lunar periodicity (16.8 days and 14.5 days, respectively) was observed in the mating frequencies in two consecutive breeding seasons (2001-2002 and 2002-2003). This semi-lunar rhythm coincided with spring tides (full and new moon), and activity peaked in November. Observation of the progression of specific behaviour types in mating and non-mating pairs revealed that pairs which would go on to complete mating progressed from fighting to mating behaviour significantly faster than non-mating pairs. These findings indicate that mate recognition occurs only after physical contact. Reproductively active pairs (ascertained from mating experiments) were then used for several bioassays aiming to assess under which conditions pheromones may be released by females. Firstly, male E. sinensis were exposed to female smell in an actograph experiment and secondly, male antennule flick rate was recorded before and after exposure to the urine of a sexually active female. In both cases no change in male E. sinensis behaviour was observed. Both experiments used females which had not had immediately prior exposure to males. However, in further experiments using water where mating had occurred, a significant response in antennule flick rate was triggered. Finally, a sponge assay was used in order to test the male attraction to a sponge injected with a water sample of varying concentrations (0.5×, 1×, 3×, 4.5×, 9×) of female smell. These samples were conditioned using a female immediately following a mating attempt. Males tried to grasp the sponge at 3× increased concentrations or higher. In conclusion, this study found no indication that E. sinensis females release a distance pheromone, but instead that mate recognition occurs after physical contact between male and female, most likely via a contact pheromone.     

Evolution of pre- and post-copulatory traits in female Drosophila melanogaster as a correlated response to selection for resistance to cold stress

Exposure to low temperatures reduces gamete viability and fecundity in females of insect species like Drosophila. Hence, adaptation to cold stress can in principle involve modifications in reproductive traits in females. Studies on resistance to cold stress have mostly addressed the evolution of adult survivorship post cold shock. Very few studies have addressed the evolution of reproductive traits in females in response to cold stress. We have successfully selected replicate populations of Drosophila melanogaster for resistance to cold shock. After 50 generations of selection, we investigated pre- and post-copulatory traits i.e. mating latency, copulation duration, mating frequency and progeny production in female flies exposed to cold shock or control conditions. Post cold shock, females from the selected populations were better at recovery in terms of mating latency, mating success, and progeny production relative to females from the control populations. Performance of the two types of females was not different under control conditions. These findings clearly indicate that adaptation to cold stress involves rapid modification of the reproductive traits.     

The sixth transmembrane region of a pheromone G-protein coupled receptor,Map3, is implicated in discrimination of closely related pheromones in Schizosaccharomyces pombe

Most sexually reproducing organisms have the ability to recognize individuals of the same species. In ascomycete fungi including yeasts, mating between cells of opposite mating type depends on the molecular recognition of two peptidyl mating pheromones by their corresponding G-protein coupled receptors (GPCRs). Although such pheromone/receptor systems are likely to function in both mate choice and prezygotic isolation, very few studies have focused on the stringency of pheromone receptors. The fission yeast Schizosaccharomyces pombe has two mating types, Plus (P) and Minus (M). Here, we investigated the stringency of the two GPCRs, Mam2 and Map3, for their respective pheromones, P-factor and M-factor, in fission yeast. First, we switched GPCRs between S. pombe and the closely related species Schizosaccharomyces octosporus, which showed that SoMam2 (Mam2 of S. octosporus) is partially functional in S. pombe, whereas SoMap3 (Map3 of S. octosporus) is not interchangeable. Next, we swapped individual domains of Mam2 and Map3 with the respective domains in SoMam2 and SoMap3, which revealed differences between the receptors both in the intracellular regions that regulate the downstream signaling of pheromones and in the activation by the pheromone. In particular, we demonstrated that two amino acid residues of Map3, F214 and F215, are key residues important for discrimination of closely related M-factors. Thus, the differences in these two GPCRs might reflect the significantly distinct stringency/flexibility of their respective pheromone/receptor systems; nevertheless, species-specific pheromone recognition remains incomplete.     

Stop codon recognition in ciliates: Euplotes release factor does not respond to reassigned UGA codon

In eukaryotes, the polypeptide release factor 1 (eRF1) is involved in translation termination at all three stop codons. However, the mechanism for decoding stop codons remains unknown. A direct interaction of eRF1 with the stop codons has been postulated. Recent studies focus on eRF1 from ciliates in which some stop codons are reassigned to sense codons. Using an in vitro assay based on mammalian ribosomes, we show that eRF1 from the ciliate Euplotes aediculatus responds to UAA and UAG as stop codons and lacks the capacity to decipher the UGA codon, which encodes cysteine in this organism. This result strongly suggests that in ciliates with variant genetic codes eRF1 does not recognize the reassigned codons. Recent hypotheses describing stop codon discrimination by eRF1 are not fully consistent with the set of eRF1 sequences available so far and require direct experimental testing.     

Use of Tetrahymena thermophila To Study the Role of Protozoa in Inactivation of Viruses in Water

The ability of a ciliate to inactivate bacteriophage was studied because these viruses are known to influence the size and diversity of bacterial populations, which affect nutrient cycling in natural waters and effluent quality in sewage treatment, and because ciliates are ubiquitous in aquatic environments, including sewage treatment plants. Tetrahymena thermophila was used as a representative ciliate; T4 was used as a model bacteriophage. The T4 titer was monitored on Escherichia coli B in a double-agar overlay assay. T4 and the ciliate were incubated together under different conditions and for various times, after which the mixture was centrifuged through a step gradient, producing a top layer free of ciliates. The T4 titer in this layer decreased as coincubation time increased, but no decrease was seen if phage were incubated with formalin-fixed Tetrahymena. The T4 titer associated with the pellet of living ciliates was very low, suggesting that removal of the phage by Tetrahymena inactivated T4. When Tetrahymena cells were incubated with SYBR gold-labeled phage, fluorescence was localized in structures that had the shape and position of food vacuoles. Incubation of the phage and ciliate with cytochalasin B or at 4°C impaired T4 inactivation. These results suggest the active removal of T4 bacteriophage from fluid by macropinocytosis, followed by digestion in food vacuoles. Such ciliate virophagy may be a mechanism occurring in natural waters and sewage treatment, and the methods described here could be used to study the factors influencing inactivation and possibly water quality.