MATING PATTERN AND FITNESS-COMPONENT ANALYSIS ASSOCIATED WITH INVERSION POLYMORPHISM IN A NATURAL POPULATION OF DROSOPHILA BUZZATII

Direct studies of mating success or mating pattern associated with Mendelian factors rarely have been carried out in nature. From the samples taken for the standard analyses of selection components, it is not usually possible to obtain the mating table, and only directional selection for male mating success can be detected. Both processes, mating pattern and differential mating probability, together with other fitness components, have been investigated for the inversion polymorphism of a natural population of the cactophilic species Drosophila buzzatii. Two independent samples of adult flies were collected: nonmating or single individuals (base population) and mating pairs (mating population). All individuals were karyotyped for the second and fourth chromosomes. A sequence of models with increasing simplicity was fitted to the data to test null hypotheses of no selection and random union of gametes and karyotypes. The main results were (1) no deviations from random mating were found; (2) differential mating probability was nonsignificant in both sexes; (3) inversion and karyotypic frequencies did not differ between sexes; and (4) karyotypic frequencies did not depart from Hardy-Weinberg expectations. These results are discussed in light of complementary evidence showing the need for interpreting with caution no-effect hypotheses such as the ones tested here. The use of complementary selective tests in these studies is suggested.     

Mating Types in Stentor coeruleus

SYNOPSIS. Conjugation in Stentor coeruleus was investigated, using a standardized culture technic which yielded large numbers of mating pairs within a single culture. Spontaneous bursts of selfing occurred during a definite interval in the development of a culture. Structurally distinct preconjugator cells appeared immediately before as well as during the initial stages of a burst of conjugation. Mating pairs were formed by the union of 2 preconjugators.
Mixing 8 stocks in all possible combinations of pairs and observing their subsequent response revealed they were separable into 2 complementary mating types. The majority of mating pairs formed in mixtures of stocks consisted of individuals of different mating types.
It is suggested that control of mating types in the ciliate order Heterotrichida may be of a somewhat different nature from that found in other ciliates.     

G1 arrest and the division/conjugation decision in Tetrahymena.

The transformation from the asexual proliferative stage of Tetrahymena to the sexual stage, during which cells of complementary mating types pair and nuclear fertilization occurs, provides an opportunity to study the relationship between the division cycle and differentiation. Conjugation is induced in cells starved for at least 2 hr by mixing complementary mating types. To determine the effect of starvation on the cell cycle, dividing cells were selected from a log growth culture and stepped down to non-nutrient conditions. The G₁ stage is operationally divisible into two sectors, A and B. In the A stage, cells arrest in nutrient-free medium. In the B stage, they proceed through the division cycle. Arrested G₁A cells may conjugate directly when challenged with similar cells of a complementary mating type. It is thereby demonstrated that Tetrahymena cells in G₁A can be directed to divide (nutrient conditions) or can be directed to differentiate (non-nutrient conditions plus complementary mating type) without an intervening division cycle. This rules out a requirement for reprogramming via chromosomal replication or cell division and suggests that G₁A is a stage during which the division/differentiation decision is made in direct response to ambient conditions.     

Copulation, the dynamics of sperm transfer and female refractoriness in the leafhopper Balclutha incisa (Hemiptera: Cicadellidae: Deltocephalinae)

Abstract.  Mature sperm of the leafhopper Balclutha incisa (Matsumara) (Cicadellidae: Auchenorrhyncha: Hemiptera) are stored as a series of sperm bundles within seminal vesicles prior to ejaculation. During transfer, sperm are pumped from the vesicles into the ejaculatory duct to the complex aedeagus. Sperm transfer is marked by a c . 30-fold expansion of the spermatheca to accommodate both sperm and seminal fluid. Sperm number increases exponentially with male age, reaching a maximum of 700 000 after 14 days, while the number of sperm available on days 2–5 is between 70 000 and 100 000. During mating, maximum sperm transfer occurs after 7 min and mating is complete after about 10 min. Ejaculate size, defined by both sperm and associated accessory gland fluid, is influenced by male mating status and the interval since the previous mating. There is a positive correlation between duration of copulation and both ejaculate and the time to subsequent mating. Sperm are more likely to be retained in the testes during mating by males of 2–5 days post-emergence than older males. The number of sperm received by the female can be manipulated experimentally by mating males once (medium ejaculate) or twice (small ejaculate) immediately after their first mating. Females that receive small ejaculates from sperm-depleted males have a far shorter refractory period than females receiving medium to large ejaculates. Both ejaculate size and the time after males have mated influence the female post-mating refractory period as measured by the female's responsiveness to male sexual signalling.     

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.     

Cell surface interactions in conjugation: Tetrahymena ciliary membrane vesicles.

Tetrahymena ciliary membrane vesicles are shown to interact with preconjugant cells in a mating type-specific way. When cells are treated with vesicles of a different mating type before mixing for conjugation, cell pairing is enhanced, and the normal prepairing period is partially eliminated. This enhancement is mating type specific since it is not observed after pretreatment of cells with vesicles of their own mating type. In contrast, when vesicles are added at the time of mixing of two starved cultures, cell pairing is delayed in a concentration-dependent manner. By varying the conditions, we demonstrated enhancement or inhibition, or both. These results are interpreted in terms of two independent interactions of cells with vesicles. We suggest that first, vesicles substitute for another cell in cell-cell prepairing interaction and second, vesicles compete for adhesion sites produced during the prepairing period. Finally, the data presented are summarized within a speculative framework that calls attention to potential analogies with hormone-receptor signaling in mammalian cells.     

Genetics of Chemical Induction of Conjugation in PARAMECIUM AURELIA

Certain stocks of P. aurelia, syngen 8, could not be induced to conjugate in a solution (KCl + acriflavine + calcium-poor conditions) which was effective in inducing conjugation in other species of Paramecium as well as in other stocks of syngen 8. Both stocks could conjugate by interaction with cells of complementary mating type. Breeding analysis shows that each of the two stocks is homozygous for a recessive gene that blocks induction of conjugation by the KCl-acriflavine solution. These two genes are neither allelic nor linked. Analyses of the phenotypes of the two uninducibles and the wild type were carried out by attempting to induce mating in cells of a single mating type by exposing them to detached mating-reactive cilia from cells of complementary mating type and to the KCl-acriflavine solution, either sequentially or simultaneously. The results confirm the conclusions of others that there is at least one unique step in chemical induction not shared with induction by interaction of complementary mating types. But the results also indicate that there is more than one unique step in chemical induction and that the effects of the two genes described here operate during different periods of the hour required for chemical induction.     

Effects of age and repeated mating on male sperm supply and paternity in a parasitoid wasp

Post-copulatory paternity biases after female multiple mating are major constraints on both male and female reproductive systems. The outcome of paternity in certain situations is only controlled directly by male sperm stock. This was tested experimentally in the parasitoid wasp Anisopteromalus calandrae (Howard) (Hymenoptera: Pteromalidae), in which sperm stocks are small (several hundred) and the fertilizing efficiency of stored sperm is high (the ratio of sperm stored/fertilized eggs is about 0.75). Sperm in seminal vesicles and paternity of males of different status (virgin young, virgin old, or young previously mated) were measured after female single and double mating. The amount of sperm in the seminal vesicle differed according to male status (increasing from previously mated males to old males), but there was no difference in sperm stored by females after a single mating. In double mating experiments with two males of different status, paternity increased linearly with the relative amount of sperm in seminal vesicles. Paternity distribution conforms to 'a fair raffle' of sperm from both donors following complete mixing of sperm prior to fertilization. Thus, in a female multiple mating context, male fitness depends principally on their sperm stock, which in turn depends on life history parameters, such as age and previous mating.     

Mating agglutination of cilia detached from complementary mating types of Paramecium

Cell-free preparations of mating-reactive cilia were obtained from complementary mating types of Paramecium caudatum with the treatment of the cells in a Triton X-100 solution and then in a Ca solution. When the cilia from both mating types thus obtained were mixed, strong agglutination was observed.     

CHLOROPLAST INHERITANCE IN COSMARIUM TURPINII BRÉB.

Chloroplast inheritance was studied in Cosmarium turpinii Bréb. with respect to both vegetative and zygotic transmission. Analyses were carried out on (1) reciprocal haploid x diploid crosses with respect to the number and size of the zygotic chloroplasts, (2) differential survival of chloroplasts in hypnospores of different mating type strains, and (3) the position of the chloroplasts in diploid zygotes. Morphological evidence indicates that the chloroplasts in the mature zygote are derived from the (+) mating type gamete with an infrequent contribution from the (–) mating type gamete. Additional evidence suggests that the chloroplast material of each of 2 Bones arising from the zygote is derived from the plastid material of a semicell of a gamete. Differential survival of the chloroplasts is interpreted as a result of physiological differences between cells of complementary mating types.     

Costimulation in Tetrahymena. II. A nonspecific response to heterotypic cell--cell interactions

Costimulation, a developmental interaction requiring cell-to-cell contact, is the second stage in a linear sequence of events leading from vegetative growth to mating pairs in the ciliate Tetrahymena thermophila. This paper uses the kinetics of appearance of genetically marked progeny to measure accurately the duration of normal costimulation and then to examine the role of mating type in costimulation. Although diverse mating types are required for costimulation to occur, the costimulated cell's response is not specific to the mating type of the cell contacting it; costimulation by one mating type appears to prepare a cell for mating with any complementary mating type.     

Sex-specific growth responses in yeasts

Growing cells of complementary mating types from two genera of yeasts,Saccharomyces andHansenula, exhibited sex-specific responses. Two kinds of responses were observed. Either one of the sexes grew preferentially toward its mate which ceased to bud and increased in size, or both mating types grew preferentially toward each other. A growth response was observed between mating types ofSaccharomyces cerevisiae andHansenula anomala presumably complementary.     

Direct observation of histone H2B-YFP fusion proteins and transport of their mRNA between conjugating Paramecia

Cytoplasmic exchange between conjugating cells of Paramecium caudatum has been implicated by mating experiments using wild-type and behavioral mutant cells. To observe macromolecular transport between mating cells, we cloned and expressed the P. caudatum histone H2B gene as a fusion protein attached to an enhanced yellow fluorescent protein (YFP) named PcVenus. Significant fluorescent signals derived from histone H2B-PcVenus were detected throughout the macro- and micronuclei of transformant cells after microinjection of the expression vector. The normal growth and high mating reactivity of the transformants indicated that H2B-PcVenus functioned normally. Seven hours after a transformant cell expressing histone H2B-PcVenus was mated with an untransformed complementary mating-type cell, fluorescence derived from histone H2B-PcVenus was emitted from the macronuclei of the untransformed cell. About 48 h later, the fluorescent signal was detected not only in the macro- and micronuclei of untransformed cells but also in the macronuclear anlagen of both mating cells. This suggests that conjugant cells share parental histones during meiosis and subsequent DNA rearrangement. Single-cell RT-PCR analysis demonstrated the presence of H2B-PcVenus mRNA in untransformed cells 15 and 24 h after conjugation. We concluded that at least the mRNA of histone H2B-PcVenus was transferred from the transformed, to the untransformed cell during conjugation.     

Some properties ofSaccharomyces kluyveri

Four isolates of aSaccharomyces species which differed fromS. kluyveri by their ability to use cellobiose were analyzed genetically in relation to the latter species. Isolated single spores had low viability. Spore tetrads segregated mating types 2 2, with sexual agglutination occurring between complementary mating types. All single-spore isolates assimilated cellobiose indicating that these isolates were not naturally occurring hybrids betweenS. kluyveri and a cellobiose assimilatingSaccharomyces species.Two cell types were exhibited by single-spore cultures ofS. kluyveri, one granulated (G-type) and one vacuolated (g-type). G-type cultures formed fertile hybrids with complementary mating types of both G- and g-type cultures. Hybrids between two g-type cultures were sterile. They would, however, give fertile hybrids when mixed with G-type cultures.Sporulating hybrids betweenSaccharomyces sp. andS. kluyveri were produced. However the percentage spore germination was low. Single-spore cultures examined had cell types atypical of either parent. The ability to assimilate cellobiose was dominant and appeared to segregate with mating type and cell type.Weak mating reactions occurred when the (+) and (-) mating types ofSaccharomyces sp. were mixed with (a) and () mating types ofS. cerevisiae, respectively.The species ofSaccharomyces isolated from the Pacific Coast are designated as strains ofS. kluyveri.     

Decapacitation and recapacitation of rabbit spermatozoa treated with membrane vesicles from seminal plasma

The article presents 2 experiments to determine whether a dense vesicle fraction concentration (Fr.1) of seminal plasma membrane vesicles could decapacitate rabbit spermatozoa and whether spermatozoa decapacitated by this method could recapacitate. It was found that 9k to 1 mg. concentration of Fr.1/ml. impaired spermatozoic fertility in dose 2 hours after ovulation. Epididymal fluid, free of sperm cells, impaired the fertility of spermatozoa capacitated in utero. This result can be explained by the high content of Fr.1-type vesicles in the epididymis. Fertilization was achieved in 19 of 28 (68%) does with the deposit of decapacitated spermatozoa obtained 7 hours after mating in the uterus about 6 hours before ovulation. This is not significantly lower than the fertilization rate obtained with untreated spermatozoa (80%). Spermatozoa obtained 13 hours after mating showed a poor fertilization capacity when deposited 6 hours before ovulation.     

Biochemical studies of the excitable membrane of Paramecium tetraurelia. III. Proteins of cilia and ciliary membranes

As a first step in the biochemical analysis of membrane excitation in wild-type Paramecium and its behavioral mutants we have defined the protein composition of the ciliary membrane of wild-type cells. The techniques for the isolation of cilia and ciliary membrane vesicles were refined. Membranes of high purity and integrity were obtained without the use of detergents. The fractions were characterized by electron microscopy, and the proteins of whole cilia, axonemes, and ciliary membrane vesicles were resolved by SDS polyacrylamide gel electrophoresis and isoelectric focusing in one and two dimensions. Protein patterns and EM appearance of the fractions were highly reproducible. Over 200 polypeptides were present in isolated cilia, most of which were recovered in the axonemal fraction. Trichocysts, which were sometimes present as a minor contaminant in ciliary preparations, were composed of a very distinct set of over 30 polypeptides of mol wt 11,000--19,000. Membrane vesicles contained up to 70 polypeptides of mol wt 15,000--250,000. The major vesicle species were a high molecular weight protein (the "immobilization antigen") and a group of acidic proteins with mol wt similar to or approximately 40,000. These and several other membrane proteins were specifically decreased or totally absent in the axoneme fraction. Tubulin, the major axonemal species, occurred only in trace amounts in isolated vesicles; the same was true for Tetrahymena ciliary membranes prepared by the methods described in this paper. A protein of mol wt 31,000, pI 6.8, was virtually absent in vesicles prepared from cells in exponential growth phase, but became prominent early in stationary phase in good correlation with cellular mating reactivity. This detailed characterization will provide the basis for comparison of the ciliary proteins of wild-type and behavioral mutants and for analysis of topography and function of membrane proteins. It will also be useful in future studies of trichocysts and mating reactions.     

ASYMMETRY OF EYESPOT AND MATING STRUCTURE POSITIONS IN ULVA COMPRESSA (ULVALES,CHLOROPHYTA) REVEALED BY A NEW FIELD EMISSION SCANNING ELECTRON MICROSCOPY METHOD1

Gametes of the marine green alga Ulva compressa L. are biflagellate and pear shaped, with one eyespot at the posterior end of the cell. The species is at an early evolutionary stage between isogamy and anisogamy. In the past, zygote formation of green algae was categorized solely by the relative sizes of gametes produced by two mating types (+ and ?). Recently, however, locations of cell fusion sites and/or mating structures of gametes have been observed to differ between mating types in several green algae (asymmetry of cell fusion site and/or mating structure positions). To use this asymmetry for determining gamete mating type, we explored a new method, field emission scanning electron microscopy (FE‐SEM), for visualizing the mating structure of U. compressa. When gametes were subjected to drying stress in the process of a conventional critical‐point‐drying method, a round structure was observed on the cell surfaces. In the mating type MGEC‐1 (mt⁺), this structure was located on the same side of the cell as the eyespot, whereas it was on the side opposite the eyespot in the mating type MGEC‐2 (mt^?). The gametes fuse at the round structures. TEM showed an alignment of vesicles inside the cytoplasm directly below the round structures, which are indeed the mating structures. Serial sectioning and three‐dimensional construction of TEM micrographs confirmed the association of the mating structure with flagellar roots. The mating structure was associated with 1d root in the MGEC‐1 gamete but with 2d root in the MGEC‐2 gamete.     

Membrane glycoproteins of Chlamydomonas eugametos flagella

The flagellar glycoproteins exposed on Chlamydomonas eugametos gametes were labeled by means of lactoperoxidase, diiodosulfanilic acid and chloramine T, and characterised in SDS-electrophoresis gels. The medium from gamete cultures contains particles (isoagglutinins) that agglutinate gametes of the opposite mating type. When crude preparations of these particles were subjected to isopycnic centrifugation in a caesium chloride gradient, two bands of particles were found. The lighter, active band consisted of membrane vesicles. The denser, inactive band consisted of cell wall material. The active band had the same glycoprotein composition as membrane vesicles artificially made from isolated flagella. Preparations of glagella were also separated on a caesium chloride cushion into pure flagella and cell wall material. The flagella, but not the cell wall material, isoagglutinated opposite gametes. Again the glycoprotein composition of pure flagella was similar to that of pure isoagglutinin vesicles. No difference was detected between the protein and glycoprotein compositions of flagella and isoagglutinins from both mating types.Abbreviations LPO lactoperoxidase - PB phosphate buffer - DISA diazotized ¹²⁵I-iodo-sulfanilic acid - SDS sodium dodecyl sulphate - CBD coomassie Brilliant Blue - PAS periodic acid Schiff     

The structure of the bursa copulatrix in virgin and mated snails, Helisoma duryi (Mollusca): role of acid phosphatase in reproduction

Abstract. The fine structure of the bursa copulatrix of the virgin snails has been compared with that of mated snails. One of the noticeable changes after mating is an increase in the number of the Golgi and the secretory vesicles. Since some of the vesicles react positively for acid phosphatase it is suggested that this enzyme activity increases following mating. The bursa lumen of the virgin snail contains gel-like materials devoid of spermatozoa, however, following mating, the lumen is full of semen containing live spermatozoa and bacteria. The source of bacteria in the lumen is not known. Acid phosphatase activity is significantly higher in the luminal content of mated snails than in the virgin snails. The activity is higher in the lumen than in the epithelial cells, suggesting that the enzyme is secreted into the lumen where it is utilized for extracellular degradation of spermatozoa. Following mating, the spermatozoa are motile in the lumen of the bursa for ∼3–7 d, but become immobile and finally undergo extracellular digestion so that intact spermatozoa are not recognizable by day 10. The use of castrated snails in mating experiments suggest that individuals of Helisoma duryi reproduce by cross fertilization and that the bursa may act as the holding organ from where the spermatozoa are periodically transported to the carrefour over ∼7 d. At day 10 following mating, however, autosperms appear in the hermaphroditic duct awaiting the next mating.     

Effect of host size on male fitness in the parasitoid wasp Dinarmus basalis

The effect of host size on male fitness was tested in the parasitoid wasp Dinarmus basalis (Hymenoptera, Pteromalidae) using hosts of different fresh weight. Fitness was measured as the sperm stock in seminal vesicles, and the ability to access females in single or competition situations. Both body size and sperm in seminal vesicles increased with host fresh weight. Males from small hosts had a reduced size and sperm stock compared to those from larger hosts. In single situations, males from both small and large hosts had similar reproductive capacities, whereas in multiple mating or competition situations, males from small hosts were at a disadvantage, inseminating fewer females and copulating less frequently. However, females did not appear to choose between males, and no effect on sperm stored in the spermatheca was observed. Being small does not prevent a D. basalis male mating and producing progeny in single situations, although more offspring could be expected from larger males because of their better competitive abilities.