Mating type differentiation in tetrahymena thermophila: Characterization of the delayed refeeding effect and its implications concerning intranuclear coordination

Mating type determination in Tetrahymena thermophila involves developmentally programmed, heritable alterations of the macronucleus, localized to the mtd locus. This determination can be predictably controlled by the environmental conditions during macronuclear development, eg, temperature and time of refeeding. In this article we have further characterized the effects of delayed refeeding on mating type determination, as revealed by the frequency of mating types among the progeny of a cross. Our results show that 1) the magnitude of this starvation effect decreases with temperature of conjugation and becomes undetectable at 18°C; 2) starvation during the interval 14 to 22 hr (after conjugation is induced at 30°C) is a necessary and sufficient condition for the induction of starvation effects; 3) relative mating type frequencies vary monotonically with nutrient concentration present during this critical period; and 4) sister macronuclei, developing under starvation conditions in the same cytoplasm, differentiate majority mating types characteristic of early or late refeeding; sister macronuclei show no apparent correlation with each other. On the basis of our observations on early and late refed cells, we propose that the composition of the newly developed macronucleus is the outcome of two key events: 1) mating type determination at the mtd locus and 2) differential molecular cloning of generally one or two autonomously replicating fragments (ARFs) of the macronuclear DNA bearing the mtd locus.     

Perturbance Analysis of Nuclear Determination in Tetrahymena

Mating type frequencies were ascertained among the progeny of crosses of strains A × B, Tetrahymena thermophila under a number of different circumstances. The frequencies are different if the parents are severely starved than if they are well-fed at the time of conjugation; severe starvation of the progeny before the first post-zygotic division has an effect similar to that of starving the parents. Mating type frequencies may also be modified by isolating conjugating pairs into cell extracts before the new macronuclei begin to develop; the changes do not appear to be related in a meaningful way to the mating type of the cells used as a source of the cell extracts. A third means of changing the mating type frequencies involves the exposure of conjugating pairs to CaCl₂ solutions. Finally, changed frequency patterns may appear "spontaneously", and reflect either some as yet unsuspected environmental variable, or else an intrinsic metastable state that conditions the probabilities of mating type fixation. With the exception of the starvation effects, the pattern variations seem to fall into two groups. No satisfactory mechanism to account for these results is yet available.     

Probable somatic DNA rearrangements in mating type determination in Tetrahymena thermophila: A review and a model

Experimental data on mating type determination in T. thermophila, collected by Nanney, Allen, and their collaborators over a period of 25 years, are reinterpreted in the light of our current understanding of macronuclear genetics. A strong case is developed supporting the idea that mating type determination involves the developmental alteration of somatic DNA that occurs regularly in developing macronuclei in conjugating pairs. A. testable DNA deletion/splicing model is developed that although based on a few simple, plausible assumptions, explains the observations remarkably well. The model is in (at least) superficial analogy to the mechanism that must be involved to explain the somatic differentiation and alteration of DNA sequences that ultimately constitute an expressed vertebrate immunoglobulin gene. Because of the genetic, biochemical, and micromanipulative versatility of Tetrahymena, it may well turn out to be a uniquely suitable microbial eukaryotic experimental system for the study of developmental alterations of somatic DNA.     

Developmental regulation of gene expression in Tetrahymena

The onset of gene expression in Tetrahymena thermophila during macronuclear differentiation was investigated by assay of galactokinase in conjugating deoxygalactose-resistant heterokaryons. Our results distinguish three successive states of galactokinase gene expression for cells developing a new macronucleus: stage 0, refractory to induction; stage 1, inducible by refeeding; and stage 2, induced. The refractory period ends at 12 to 13 hr after the onset of conjugation; this corresponds to the time of pair separation, and occurs several hours after the new macronuclei have become morphologically distinguishable. Stage 1 cells behave indistinguishably from mature starved cells. Inhibitor studies suggest that galactokinase synthesis is induced coincidentally with the induction of bulk protein synthesis during conjugation: thus it behaves developmentally like a typical protein; and that galactokinase mRNA is probably transcribed within 1 hr prior to its translation. Thus, when conjugating cells are refed during the refractory period, some developmental condition prevents the swift induction of protein (and galactokinase) synthesis observed upon refeeding starved (nonmating) cells. The possible nature of this developmental phenomenon is discussed.     

Karyonidal inheritance of mating types in tetrahymena malaccensis

Mating type determination in Tetrahymena malaccensis is karyonidal, ie, the four new macronuclei developing in a single conjugating pair are independently determined as to which of the six known mating types they will express. Occasional selfing clones are similar to those in T thermophila, in that any one is capable of stabilizing at a restricted range of mating types. The genetic basis of mating type potentialities is incompletely resolved. T malaccensis may, like T thermophila and T canadensis, have a single multiallelic locus that controls the array of types. Quantitative considerations suggest, however, that other loci may be involved.     

Genetic and environmental factors affecting mating type frequency in natural isolates of Tetrahymena thermophila

In Tetrahymena thermophila mating type alleles specify temperature sensitive frequency distributions of multiple mating types. A-like alleles specify mating types I, II, III, V and VI, whereas B-like alleles specify mating types II through VII. We have characterized the mating type distributions specified by several A- and B-like genotypes segregated by genomic exclusion from cells isolated from a pond in northwestern Pennsylvania. The B-like genotypes are alike in specifying very low frequencies of mating type III, but differ with respect to the frequencies of other mating types, particularly II and VII. An A-like genotype specifies a high frequency of mating type III and is unstable in successive generations for the expression of mating type II, suggesting a possible modifier. Inter se crosses performed at 18 degrees C, 28 degrees C and 34 degrees C showed that each genotype specifies a frequency distribution that is uniquely affected by temperature. No mating type was affected the same way by temperature in all genotypes. In A/B heterozygotes, the B-like genotype exhibited partial dominance. The genotypes described here differ significantly from previously described genotypes from the same pond, indicating that there are numerous mating type alleles. For frequency-dependent selection to equalize mating type frequencies, it must act not only on complex multiple alleles but also on the response of mating type alleles to temperature.     

Mapping the mating type locus of Tetrahymena thermophila: meiotic linkage of mat to the ribosomal RNA gene.

Tetrahymena thermophila has a multiple mating type system. While a sexually mature cell usually expresses only one mating type, its germline (micronucleus) carries the genetic potential for 5 to 7 mating types. The set of allowed mating types is specified by the mat locus. The choice of which particular mating type is expressed by a cell reflects a somatically inherited, developmentally programmed differentiation of the somatic nucleus (macronucleus). In this work we report that the mat locus maps to the left arm of chromosome 2, as determined by nullisomic deletion mapping. We also report a distance of 29 cM between the mat locus and the ribosomal RNA gene, previously mapped to chromosome 2L. This represents another (rare) case of meiotic linkage in Tetrahymena.     

Mating Behavior and Species Status of Host-Associated Populations of the Polyphagous Thrips, <Emphasis Type="Italic">Frankliniella schultzei</Emphasis>

The thrips taxon, Frankliniella schultzei Trybom, could possibly comprise a complex of unrecognised cryptic species. We therefore made a range of reproductive behavioral observations on F. schultzei individuals from different host-associated populations to investigate their species status. We first described the mating behavior of F. schultzei taken from flowers of a primary host species, Malvaviscus arboreus. The pattern of male-female interaction was complex and protracted, with several behavioral steps being performed. These steps were similar for all mating pairs. Most females responded to male presence before physical contact, suggesting that male pheromones may be involved. We then compared mating behaviors, brood sizes and sex ratios of F. schultzei pairs derived from different host plant species. All pairs in crosses regardless of their original host plant species produced female offspring, indicating successful mating. Frequencies of behaviors were similar for all pairs. Only those crosses involving F. schultzei individuals from Erythrina crista-galli flowers differed from the general pattern in pre-copulation and copulation times. The females from E. crista-galli also produced significantly fewer offspring than females from other crosses. Finally we used traps baited with males from two populations, but set among flowers of one host plant species, to test for differential attraction of females. We found no evidence that the different host-associated populations investigated differ from one another functionally in their sex pheromone system. However, these results do not yet discount the possibility of cryptic species within the taxon F. schultzei. Populations of F. schultzei on many other host species need to be investigated from this perspective.     

Developmental and genetic effects of alcian blue in conjugating Tetrahymena thermophila: doublet formation and macronuclear retention

Genetic, kinetical and cortical effects of treatment with the inducer of mucocyst release, alcian blue (AB), on conjugating pairs of Tetrahymena thermophila are reported. AB induces the formation of doublet cells from pairs, and the majority of them are homopolar doublets. We present a model in order to explain the origin of these cells. Macronuclear retention (MR) is the most important genetic effect observed. Two kinds of MR can be obtained: prezygotic-MR (uniparental micronucleus) and postzygotic-MR (cross-fertilized micronucleus). Within the first group, both homokaryon and heterokaryon cells are obtained. From some abnormal conjugational configurations and the results of conjugational kinetic analysis we propose an explanation for the origin of MR cells induced by AB. Genetic effects obtained after AB treatment at different conjugational times are independent of the cortical ones. The utility of these different effects in genetical and physiological studies is discussed.     

The mating system of the true fruit fly Bactrocera tryoni and its sister species,Bactrocera neohumeralis

The frugivorous “true” fruit fly, Bactrocera tryoni (Queensland fruit fly), is presumed to have a nonresourced‐based lek mating system. This is largely untested, and contrary data exists to suggest Bactrocera tryoni may have a resource‐based mating system focused on fruiting host plants. We tested the mating system of Bactrocera tryoni, and its close sibling Bactrocera neohumeralis, in large field cages using laboratory reared flies. We used observational experiments that allowed us to determine if: (i) mating pairs were aggregated or nonaggregated; (ii) mating system was resource or nonresource based; (iii) flies utilized possible landmarks (tall trees over short) as mate‐rendezvous sites; and (iv) males called females from male‐dominated leks. We recorded nearly 250 Bactrocera tryoni mating pairs across all experiments, revealing that: (i) mating pairs were aggregated; (ii) mating nearly always occurred in tall trees over short; (iii) mating was nonresource based; and (iv) that males and females arrived at the mate‐rendezvous site together with no evidence that males preceded females. Bactrocera neohumeralis copulations were much more infrequent (only 30 mating pairs in total), but for those pairs there was a similar preference for tall trees and no evidence of a resource‐based mating system. Some aspects of Bactrocera tryoni mating behavior align with theoretical expectations of a lekking system, but others do not. Until evidence for unequivocal female choice can be provided (as predicted under a true lek), the mating system of Bactrocera tryoni is best described as a nonresource based, aggregation system for which we also have evidence that land‐marking may be involved.     

Programmed autophagocytosis accompanying conjugation in the ciliate Stylonychia mytilus

Conjugation and postconjugant development in Stylonychia is accompanied by a period of approximately 80 hr during which the cells are unable to ingest food. This period is one of considerable synthetic activity, encompassing important portions of the development of the new macronucleus. Light- and electron-microscopic observations of conjugating pairs and exconjugant cells reveal a process of autophagocytosis that may provide the supplies of energy (and precursors) necessary for postconjugant developmental events. Small autophagosomes (AP) form in conjugants; degenerating mitochondria are prominent among the inclusions observed in these bodies. Soon after separation of pairs, large dense AP appear, apparently by coalescence and condensation of the smaller AP. These “mature” AP give only a slight acid phosphatase reaction. The number of AP slowly declines during postconjugant development; about 60 hr after separation all the AP have been digested. Several observations suggest that this autophagocytosis is part of the developmental program initiated soon after mating begins: (1) The first indications of AP formation occur while conjugating pairs are still able to feed, and thus cannot be attributed to the stress of starvation; (2) formation of large numbers of AP is rather abrupt, whereas their dissolution is very gradual, covering most of the nonfeeding period; and (3) the pattern of AP formation and dissolution is similar in cells whose new macronuclear development has been prevented by brief hydroxyurea treatment.     

利用铁葡聚糖分离纤毛虫接合对的方法

介绍一种制备微小铁葡聚糖颗粒以及利用微小铁葡聚糖颗粒分离纤毛虫接合对的方法,通过本方法可以获得接合率达95%以上,且发育基本同步的实验材料。     

Traumatic Induction of Early Maturity in Tetrahymena

Exposure of conjugating pairs of Tetrahymena thermophila to high temperature (37°) during macronuclear development causes an abortion of many macronuclei, but it also often induces an early appearance of sexual maturity in clones completing macronuclear development. Lines become mature after about 15 cell divisions rather than after 50 or more cell divisions in untreated pairs. The phenotype resembles that associated with Em (early maturity) mutants but, because it is not transmitted to the progeny in the next generation, it must be considered a phenocopy. The hypothesis is developed that an early genotype-environment incompatibility, whether associated with an abnormal genotype or an unusual environment, activates a shunt mechanism permitting the organisms to undertake quickly an ordinarily forbidden sexual lottery.     

A proteomics approach to cloning fenestrin from the nuclear exchange junction of tetrahymena

ABSTRACT. We set out to find the " fenestrin " gene, a gene whose protein is associated with numerous cellular apertures, including the nuclear exchange junction in mating Tetrahymena thermophila . First we developed protocols for imaging and isolating intact nuclear exchange junctions from conjugating cells. Proteins from these junctions were purified using SDS-PAGE, subjected to limited proteolysis, and precise molecular weights were determined by mass spectrometry. Using Protein Prospector^® software and the published Tetrahymena Genome Database, genes for 15 of the most abundant proteins found in our extracts were identified. The most promising candidate was cloned by PCR, fused to yellow fluorescent protein (YFP), and placed under the control of an inducible metallothionein promoter. YFP-localization within live Tetrahymena transformants strongly suggested that one of these genes encoded the fenestrin protein, a result that was subsequently confirmed by Western blotting.     

Mapping the mating type locus of Tetrahymena thermophila: Meiotic linkage of mat to the ribosomal RNA gene

Tetrahymena thermophila has a multiple mating type system. While a sexually mature cell usually expresses only one mating type, its germline (micronucleus) carries the genetic potential for 5 to 7 mating types. The set of allowed mating types is specified by the mat locus. The choice of which particular mating type is expressed by a cell reflects a somatically inherited, developmentally programmed differentiation of the somatic nucleus (macronucleus). In this work we report that the mat locus maps to the left arm of chromosome 2, as determined by nullisomic deletion mapping. We also report a distance of 29 cM between the mat locus and the ribosomal RNA gene, previously mapped to chromosome 2L. This represents another (rare) case of meiotic linkage in Tetrahymena. © 1992 Wiley-Liss, Inc.     

Perturbance analysis of nuclear determination in Tetrahymena I: background, rationale, and illustrative example, employing temperature responses

A model based on comparative considerations, is presented for the nuclear determination of mating type in Tetrahymena thermophila. The model proposes a system of three binary control elements, each capable of stable persistence in one of two states. A general method is proposed for evaluating the model and for assigning particular mating types to particular compound states. Preliminary assignments of mating types are made from the responses of nuclei to temperature differentials.     

A novel method for estimating the strength of positive mating preference by similarity in the wild

Mating preference can be a driver of sexual selection and assortative mating and is, therefore, a key element in evolutionary dynamics. Positive mating preference by similarity is the tendency for the choosy individual to select a mate which possesses a similar variant of a trait. Such preference can be modelled using Gaussian‐like mathematical functions that describe the strength of preference, but such functions cannot be applied to empirical data collected from the field. As a result, traditionally, mating preference is indirectly estimated by the degree of assortative mating (using Pearson's correlation coefficient, r) in wild captured mating pairs. Unfortunately, r and similar coefficients are often biased due to the fact that different variants of a given trait are nonrandomly distributed in the wild, and pooling of mating pairs from such heterogeneous samples may lead to “false–positive” results, termed “the scale‐of‐choice effect” (SCE). Here we provide two new estimators of mating preference (C_rough and C_scaled) derived from Gaussian‐like functions which can be applied to empirical data. Computer simulations demonstrated that r coefficient showed robust estimations properties of mating preference but it was severely affected by SCE, C_rough showed reasonable estimation properties and it was little affected by SCE, while C_scaled showed the best properties at infinite sample sizes and it was not affected by SCE but failed at biological sample sizes. We recommend using C_rough combined with the r coefficient to infer mating preference in future empirical studies.     

Food,shelter or competitors? Overlapping of life stages and host plant selection in a Neotropical stink bug species

Phytophagous insects may choose host plants based on conditions that enhance offspring performance. However, some insect species may also select plants based on attributes that enhance their own performance regardless of the consequences for offspring survival. An approach evaluating both hypotheses could provide a more comprehensive understanding of the host plant selection by phytophagous insects. In this study, we described the life stages of a Neotropical stink bug, Edessa contermina, co-occurring on Byrsonima verbascifolia plants in a conservation area of the Brazilian Savannah. We also empirically evaluated how food supply, shelter availability and competitors’ density on the host plants affected the densities of nymphs, adults and mating pairs. We identified and described five life stages of E. contermina. The amount of plant resources did not explain the nymph, adult and mating pairs’ density. However, adults and mating pairs chose plants with a low density of nymphs, probably because egg laying on the host plants with a high density of competitors may negatively affect offspring performance.     

Reconstitution of mating active membrane vesicles in Paramecium

Mating-active membrane vesicles isolated from cilia of Paramecium caudatum by the urea-EDTA method were dissolved in 9 mM lithium diiodosalicylate (LIS). Membrane vesicles were reconstituted from the LIS-soluble fraction by dialysis. They showed an ability to induce conjugating pairs without prior occurrence of mating agglutination. The same kind of membrane vesicles was obtained when cilia were treated with 4 mM LIS and stored after removing LIS for two weeks.