THE SEXUAL LIFE CYCLES OF PFIESTERIA PISCICIDA AND CRYPTOPERIDINIOPSOIDS (DINOPHYCEAE)1

Sexual life cycle events in Pfiesteria piscicida and cryptoperidiniopsoid heterotrophic dinoflagellates were determined by following the development of isolated gamete pairs in single‐drop microcultures with cryptophyte prey. Under these conditions, the observed sequence of zygote formation, development, and postzygotic divisions was similar in these dinoflagellates. Fusion of motile gamete pairs each produced a rapidly swimming uninucleate planozygote with two longitudinal flagella. Planozygotes enlarged as they fed repeatedly on cryptophytes. In <12 h in most cases, each planozygote formed a transparent‐walled nonmotile cell (cyst) with a single nucleus. Zygotic cysts did not exhibit dormancy under these conditions. In each taxon, dramatic swirling chromosome movements (nuclear cyclosis) were found in zygote nuclei before division. In P. piscicida, nuclear cyclosis occurred in the zygotic cyst or apparently earlier in the planozygote. In the cryptoperidiniopsoids, nuclear cyclosis occurred inthe zygotic cyst. After nuclear cyclosis, a single cell division occurred in P. piscicida and cryptoperidiniopsoid zygotic cysts, producing two offspring that emerged as biflagellated cells. These two flagellated cells typically swam for hours and fed on cryptophytes before encysting. A single cell division in these cysts produced two biflagellated offspring that also fed before encysting for further reproduction. This sequence of zygote development and postzygotic divisions typically was completed within 24 h and was confirmed in examples from different isolates of each taxon. Some aspects of the P. piscicida sexual life cycle determined here differed from previous reports.     

NUCLEAR FEATURES AND EFFECT OF NUTRIENTS ON GYMNODINIUM CATENATUM (DINOPHYCEAE) SEXUAL STAGES1

Gymnodinium catenatum Graham is an unarmored, cyst‐forming dinoflagellate species responsible for outbreaks of paralytic shellfish poisoning. The nuclear development of the cells during the sexual cycle and the effect of different nitrate and phosphate external levels on sexual stages were studied. Nuclear fusion of gametes occurred before or at the same time as cytoplasmic fusion. During this process, either both nuclei migrated to a central area in the sulcal region, or only one of them migrated to the other nucleus. The motile and longitudinally biflagellated zygote presented a large, pear‐shaped nucleus, and either divided or encysted. Planozygotes and germlings underwent similar division processes, which suggested an uncoordinated meiosis in both encysting and non‐encysting zygotes. Encystment in culture was greater under low nitrate and phosphate limitation (L/15) than when only one or neither of these nutrients were added (L‐N, L‐P, and ‐N‐P). However, planozygotes individually monitored achieved the maximum encystment (40%) in a medium with no phosphate or nitrate added (‐N‐P), while most of them divided (70%–90%) in replete (L1) or half‐replete (L‐N and L‐P) media. Low levels of nitrate in the medium of cyst formation promoted a deficient development of the cyst wall. On the other hand, low phosphate levels in the medium of germination prevented both planozygote and germling division and lowered the final germination frequencies of cysts. The minimum dormancy, with an average value of 13.7±5.5 days, was not affected by any of the nutritional conditions studied.     

Comparative study of the life cycles of Alexandrium tamutum and Alexandrium minutum (Gonyaulacales,Dinophyceae) in culture1

The microalgal genus Alexandrium includes species known to produce paralytic shellfish poisoning (PSP). Due to the importance of discriminating between HAB‐forming species, we compared the undescribed life‐cycle pattern of Alexandrium tamutum Montresor, Beran et U. John and of its toxic relative Alexandrium minutum Halim. Sexual stages, asexual and sexual division, mating type, and nuclear morphology were studied in both species. Sexual cysts are known to be morphologically identical. However, the relative size of the U‐shaped nucleus may be used to differentiate between the cysts of these species since DNA packaging in the resting cysts was lower in A. tamutum than in A. minutum, species in which the planozygote nucleus was reduced to half its volume prior to encystment. The dormancy period of the cysts was <20 d for A. tamutum, but longer than 1 month for A. minutum. In both species, cyst appearance needed to be explained by the existence of more than two sexual types (+/–), which indicates a complex heterothallic mating type. However, planozygotes of both species may divide instead of encysting. This characteristic was used for nutritional and heritage studies. Isolated planozygotes of both species encysted in larger percentages in medium deficient in both nitrates and phosphates (L/15) than in medium without phosphates added (L‐P), a medium in which most planozygotes neither divide nor encyst. Parental strains of A. minutum with and without the ventral pore formed planozygotes and, later, offspring with the ventral pore, although apparently smaller than usual. A synchronization–flow cytometry method for discriminating diploids formed by sexual fusion (planozygotes) from cells with 2C DNA content resulting from self‐duplication of DNA (dividing cells) was described. The results indicated that the maximum percentage of A. minutum planozygotes (20%) was achieved only 3 to 5 d after crossing the parental strains, and that light might not be needed for the sexual fusion and formation of planozygotes.     

The life history of the toxic marine dinoflagellate Protoceratium reticulatum (Gonyaulacales) in culture

Asexual and sexual life cycle events were studied in cultures of the toxic marine dinoflagellate Protoceratium reticulatum. Asexual division by desmoschisis was characterized morphologically and changes in DNA content were analyzed by flow cytometry. The results indicated that haploid cells with a C DNA content occurred only during the light period whereas a shift from a C to a 2C DNA content (indicative of S phase) took place only during darkness. The sexual life cycle was documented by examining the mating type as well as the morphology of the sexual stages and nuclei. Gamete fusion resulted in a planozygote with two longitudinal flagella, but longitudinally biflagellated cells arising from planozygote division were also observed, so one of the daughter cells retained two longitudinal flagella while the other daughter cell lacked them. Presumed planozygotes (identified by their longitudinally biflagellated form) followed two life-cycle routes: division and encystment (resting cyst formation). Both the division of longitudinally biflagellated cells and resting cyst formation are morphologically described herein. Resting cyst formation through sexual reproduction was observed in 6.1% of crosses and followed a complex heterothallic pattern. Clonal strains underwent sexuality (homothallism for planozygote formation and division) but without the production of resting cysts. Ornamental processes of resting cysts formed from the cyst wall under an outer balloon-shaped membrane and were fully developed in <1 h. Obligatory dormancy period was of ∼4 months. Excystment resulted in a large, rounded, pigmented, longitudinally biflagellated but motionless, thecate germling that divided by desmoschisis. Like the planozygote, the first division of the germling yielded one longitudinally biflagellated daughter cell and another without longitudinal flagella. The longitudinal biflagellation state of both sexual stages and of the first division products of these cells is discussed.     

VEGETATIVE REPRODUCTION AND SEXUAL LIFE CYCLE OF THE TOXIC DINOFLAGELLATE GYMNODINIUM CATENATUM FROM TASMANIA,AUSTRALIA1

The toxic, chain-forming dinoflagellate Gymnodinium catenatum Graham was cultured from vegetative cells and benthic resting cysts isolated from estuarine waters in Tasmania, Australia. Rapidly dividing, log phase cultures formed long chains of up to 64 cells whereas stationary phase cultures were composed primarily of single cells (23-41 pm long, 27-36 pm wide). Vegetative growth (mean doubling time 3-4 days) was optimal at temperatures from 14.5-20° C, salinities of 23-34% and light irradiances of 50-300 μE·m^?2·s^?1. The sexual life cycle of G. catenatum was easily induced in a nutrient-deficient medium, provided compatible opposite mating types were combined (heterothallism). Gamete fusion produced a large (59-73 μm long, 50-59 μm wide) biconical, posteriorly biflagellate planozygote (double longitudinal flagellum) which after several days lost one longitudinal flagellum and gradually became subspherical in shape. This older planozygote stage persisted for up to two weeks before encysting into a round, brown resting cyst (42-52 μm diam; hypnozygote) with microreticulate surface ornamentation. Resting cysts germinated after a dormancy period as short as two weeks under our culture conditions, resulting in a single, posteriorly biflagellate germling cell (planomeiocyte). This divided to form a chain of two cells, which subsequently re-established a vegetative population. Implications for the bloom dynamics of this toxic dinoflagellate, a causative organism of paralytic shellfish poisoning, are discussed.     

MULTIPLE ROUTES OF SEXUALITY IN ALEXANDRIUM TAYLORI (DINOPHYCEAE) IN CULTURE1

Alexandrium taylori Balech is a cyst‐forming dinoflagellate species responsible for recurrent blooms in Mediterranean coastal waters. The nuclear development of the cells during the sexual cycle and the effect of different external nitrate and phosphate levels were studied. Nuclear fusion of gametes occurred 6–12 h after the complete cytoplasmic fusion. The U‐shaped nuclei fused through the end of one nucleus and the mid‐area of the other. The mobile and biflagellated zygote had a large, U‐shaped nucleus and may follow three different fates: direct division, short‐term encystment (ecdysal), and long‐term encystment (resting). Ecdysal cysts may divide in >24–96 h into two, four, six, or eight cells before germinating. Meiosis presumably occurred in three locations: in the planozygote, within the ecdysal cyst, and in the planomeiocyte (germling) liberated either from ecdysal or resting cysts. The effects of nutrients on these routes were studied in individually isolated sexual stages. (1) Direct divisions occurred mainly under replete conditions (L1), whereas no direct planozygote divisions were recorded in media with no phosphate added (L‐P). (2) Short‐term encystment was larger in media lacking phosphate (L‐P and L/30) than in medium with no nitrate added (L‐N) or under replete conditions (L1). (3) Long‐term encystment was only observed in medium with no nitrate added (L‐N). The long‐lived resting cyst, not previously described for this species, had a clear double wall, an irregular shape, a flat morphology, and a middle orange spot. No cysts germinated in 1–2 months, whereas 86% of the cysts germinated 2–3 months after being formed. A flow cytometry analysis showed that sexual induction and zygote formation were very fast and highly common processes, zygotes being nearly half of the population at days 3 and 5 after the induction of sexuality in the cultures.     

The significance of sexual versus asexual cyst formation in the life cycle of the noxious dinoflagellate Alexandrium peruvianum

Alexandrium peruvianum (Balech et Mendiola) is a noxious phototrophic marine dinoflagellate. During the life cycle of this species, two kinds of cysts are produced: resting cysts, which are long-lasting and double-walled, and temporary cysts, which are short-lasting and thin-walled. In addition, short-lasting, but resting-like cysts can also be formed. Although it is crucial to identify sexual events in a dinoflagellate population, sexual and asexual cysts are morphologically very similar in this species. Therefore, we studied the complete life cycle and the nature of the cyst-like stages formed after individual isolation of specimens and crossing of clonal cultures established from germination of wild resting cysts. Asexual division in A. peruvianum takes place either in the motile stage by sharing of the theca (desmoschisis), or inside a vegetative cyst (temporary cyst), from which two, or at times four or six naked daughter cells can originate. The daughter cells completely synthesize new cell walls (eleutheroschisis). Sexuality was confirmed by the presence of fusing gamete pairs and longitudinally biflagellated planozygotes after out-crossing of compatible clonal strains. However, the clonal cultures had low levels of self-compatibility, since a flow cytometry analysis showed that synchronized self-crosses produced few zygotes (<5%). After isolation of individual cells, it was proved that the fate of the planozygotes depended on the nutritional status of the isolation media. Most of the planozygotes isolated to replete medium (L1) divided, whereas in medium lacking nitrates (L-N) or phosphates (L-P) they formed temporary, thin-walled cysts. Temporary cysts formed in L1 were always uninucleated and gave rise to one cell, while those formed in L-N or L-P produced 1–6 small cells. In addition, resting cysts were formed in culture, but never after individual planozygote isolation. Resting cysts were uninucleated and needed maturation time before entering dormancy. The resting cysts were considered sexual products, since longitudinally biflagellate germlings were liberated after germination in all cases studied. Mature resting cysts (52.3 ± 3.0 μm) had a dormancy period of 1–3 months, whereas temporary asexual cysts (32.5 ± 5.4 μm) germinated in less than 7 days.     

Role of resting cysts in Chilean Alexandrium catenella dinoflagellate blooms revisited

The detection of sparse Alexandrium catenella-resting cysts in sediments of southern Chilean fjords has cast doubts on their importance in the recurrence of massive toxic dinoflagellate blooms in the region. The role of resting cysts and the existence of different regional Chilean populations was studied by culturing and genetic approaches to define: (1) cyst production; (2) dormancy period; (3) excystment success; (4) offspring viability and (5) strain mating compatibility. This study newly revealed a short cyst dormancy (minimum 69 days), the role of key abiotic factors (in decreasing order salinity, irradiance, temperature and nutrients) controlling cyst germination (max. 60%) and germling growth rates (up to 0.36–0.52 div. day⁻¹). Amplified fragment length polymorphism (AFLP) characterization showed significant differences in genetic distances (GD) among A. catenella populations that were primarily determined by the geographical origin of isolates and most likely driven by oceanographic dispersal barriers. A complex heterothallic mating system pointed to variable reproductive compatibility (RCs) among Chilean strains that was high among northern (Los Lagos/North Aysén) and southern populations (Magallanes), but limited among the genetically differentiated central (South Aysén) populations. Field cyst surveys after a massive 2009 bloom event revealed the existence of exceptional high cyst densities in particular areas of the fjords (max. 14.627 cysts cm⁻³), which contrast with low cyst concentrations (<221.3 cysts cm⁻³) detected by previous oceanographic campaigns. In conclusion, the present study suggests that A. catenella resting cysts play a more important role in the success of this species in Chilean fjords than previously thought. Results from in vitro experiments suggest that pelagic–benthic processes can maintain year-round low vegetative cell concentrations in the water column, but also can explain the detection of high cysts aggregations after the 2009-bloom event. Regional drivers that lead to massive outbreaks, however, are still unknown but potential scenarios are discussed.     

HIGH ENCYSTMENT SUCCESS OF THE DINOFLAGELLATE SCRIPPSIELLA CF. LACHRYMOSA IN CULTURE EXPERIMENTS 1

Close to 100% encystment efficiency and a yield above 10⁵ cysts·mL ^{? 1} were routinely achieved in full strength f/2 medium‐based batch cultures (883 μM NO₃ ^? and 36 μM PO₄ ^{? 3}) of the marine dinoflagellate Scrippsiella cf. lachrymosa Lewis. Increases in cell density led to nutrient depletion in this enriched medium, which was the most likely cause for initiation of cyst formation. Lowering the concentration of either nutrient to 1/10 the initial levels decreased the encystment efficiency, whereas use of ammonium as the N source resulted in both low cell yield and low encystment efficiency. The mandatory dormancy period was ca. 60 days and was not affected by cold dark storage of the cysts. Cysts produced in the initial phase of sexual reproduction were relatively large (length 47 μm, width 31 μm) with a heavy calcareous cover. Cysts produced thereafter lacked apparent calcareous cover and were smaller (length 29 μm, width 19 μm). The decrease of cyst volume (by a factor of 0.24–0.4) suggested strong resource limitation during the course of encystment. However, after the mandatory dormancy period, germination success of the smaller cysts was higher (80%), compared with the larger cysts that had been produced initially (50%). Germling survival (74%) was independent of cyst type but was enhanced by higher nutrient concentration during incubation. The ratio of initial nutrient concentration in the medium to the cyst yield was used as a proxy to estimate the cellular nutrient quota. The conservative estimates of 9 pmol N·cyst ^{? 1} and 0.4 pmol P·cyst ^{? 1} obtained in this manner are at the low end of the range of previous published estimates for other dinoflagellate cysts. Given the high encystment observed in laboratory experiments, we have no reason to assume an inherently lower encystment success in dinoflagellate field populations. Our results do not challenge the low nutrient paradigm for dinoflagellate sexuality. We believe that the high encystment success and cyst yield of this particular species is at least partly due to its ability to achieve very high cell densities in cultures, which evidently leads to nutrient depletion even in f/2 medium.     

A STUDY OF THE SEXUAL REPRODUCTION AND DETERMINATION OF MATING TYPE OF GYMNODINIUM NOLLERI (DINOPHYCEAE) IN CULTURE1

Sexual reproduction of Gymnodinium nolleri ( Ellegaard & Moestrup 1999 ) was studied by intercrossing experiments in all combinations of six clonal strains and backcrossing of five clonal F1 offspring. The results indicated that the conjugation of G. nolleri responded to the existence of more than two sexual types (complex heterothallism) and that compatibility between progeny of one cyst (inbreeding) was the rule. Sexual fusion, planozygote formation and development, cyst formation, and germination and planomeiocyte division were followed using time‐lapse photography. This study revealed many similarities between the sexual stages and life cycle pattern of G. nolleri and the related G. catenatum and the existence under culture conditions of an alternative cycle between vegetative cells and zygotes without a hypnozygote stage. The fate of zygotes, division or encystment, was influenced by the nutritional status of the external medium. The division of G. nolleri planozygotes was promoted by high levels of external nutrients, whereas the maximum percentage of encystment was recorded when phosphates were reduced in the isolation medium. The division of zygotes might be different from both vegetative and planomeiocyte division because it resulted in two‐cell chains with the cells not oriented in parallel.     

EVIDENCE FOR ASEXUAL RESTING CYSTS IN THE LIFE CYCLE OF THE MARINE PERIDINOID DINOFLAGELLATE,SCRIPPSIELLA HANGOEI1

Scrippsiella hangoei (Schiller) Larsen is a peridinoid dinoflagellate that grows during winter and spring in the Baltic Sea. In culture this species formed round, smooth cysts when strains were mixed, indicating heterothallic sexuality and hypnozygote production. However, cysts of the same morphology were also formed in clonal strains exposed to slightly elevated temperature. To better understand the role of cysts in the life cycle of S. hangoei, cyst formation and dormancy were examined in culture experiments and the cellular DNA content of flagellate cells and cysts was compared in clonal and mixed strains using flow cytometry. S. hangoei exhibited a high rate of cyst formation in culture. Cysts produced in both clonal and mixed strain cultures were thick‐walled and underwent a dormancy period of 4 months before germinating. The S. hangoei flagellate cell population DNA distributions consisted of 1C, intermediate, and 2C DNA, indicative of respective eukaryotic cell cycle phases G1, S, and G2M. The majority (>95%) of cysts had a measured DNA content equivalent to the lower 1C DNA value, indicating a haploid nuclear phase and an asexual mode of cyst formation. A small percentage (<5%) of cysts produced in the mixed strain culture had 2C DNA, and thus could have been diploid zygotes. These findings represent the first measurements of dinoflagellate resting cyst DNA content, and provide the first quantitative evidence for dinoflagellate asexual resting cysts. Asexual resting cysts may be a more common feature of dinoflagellate life cycles than previously thought.     

LIFE CYLE AND SEXUALITY OF THE FRESHWATER RAPHIDOPHYTE GONYOSTOMUM SEMEN (RAPHIDOPHYCEAE)1

Previously unknown aspects in the life cycle of the freshwater flagellate Gonyostomum semen (Ehrenb.) (Raphidophyceae) are described here. This species forms intense blooms in many northern temperate lakes, and has increased in abundance and frequency in northern Europe during the past decades. The proposed life cycle is based on observations of life cycle stages and transitions in cultures. Viable stages of the life cycle were individually isolated and monitored by time‐lapse photography. The most common processes undertaken by the isolated cells were: division, fusion followed by division, asexual cyst formation, and sexual cyst formation. Motile cells divided by two different processes. One lasted between 6 and 24 h and formed two cells with vegetative cell size and with or without the same shape. The second division process lasted between 10 and 20 min and formed two identical cells, half the size of the mother cell. Planozygotes formed by the fusion of hologametes subsequently underwent division into two cells. Asexual cyst‐like stages were spherical, devoid of a thick wall and red spot, and germinated in 24–48 h. Heterogamete pairs were isogamous, and formed an angle of 0–90° between each other. Planozygote and sexual cyst formation were identified within strains established from one vegetative cell. The identity of these strains, which was studied by an amplified fragment length polymorphism analysis, was correlated with the viability of the planozygote. Resting cyst germination was described using cysts collected in the field. The size and morphology of these cysts were comparable with those formed sexually in culture. The excystment rate was higher at 24°C than at 19 or 16°C, although the cell liberated during germination (germling) was only viable at 16°C. The placement of G. semen within the Raphidophyceae family was confirmed by sequence analysis of a segment of the 18S ribosomal DNA.     

The effect of penicillin on the encystment of Bdellovibrio sp. strain W

When penicillin, and other inhibitors of peptidoglycan synthesis were added to encysting cultures of Bdellovibrio strain W, the encysting process continued, resulting in the production of cysts which were spherical in shape. Transmission electron micrographs of these spherical bdellocysts revealed the absence of an outer cyst wall. These cysts, devoid of cyst wall, were capable of germination under appropriate condition with the emergence from the prey ghost of highly motile spheroplasts. Withdrawl of the antibiotics after encystment had begun led to the production of spherical cysts that were surrounded by an outer cyst wall.     

Recognition of Young in A Colonially Nesting Bird

Parents ought to restrict costly parental care to their genetic offspring and, particularly when the risk of misdirecting care is high, parent‐offspring recognition may evolve. I tested whether adult cave swallows, which nest in dense colonies and feed fledglings in mixed‐family groups, discriminate against unrelated young, using temporary chick transfers at two nestling ages and a cross‐fostering experiment. Temporary chick transfers indicated that parents bias feedings toward their own offspring near fledging (18 d) but not at about halfway through the nesting period (10 d). I also examined how parents learn to identify their offspring by cross‐fostering young 3 d after hatching and testing parental response 2 wks later. Adults did not favor their own offspring over unrelated nestlings when both were unfamiliar to the focal parents. However, when parents encountered two of their own offspring, one of which was reared by foster parents, they preferentially fed the familiar nestling. By recognizing young, cave swallow parents reduce some risks of misdirected parental investment (mobile fledglings) but not others (extra‐pair young and intraspecific brood parasitism).     

High-resolution immunogold localization of Giardia cyst wall antigens using field emission SEM with secondary and backscatter electron imaging

We describe here the ultrastructural localization of Giardia cyst antigens in the filaments associated with the outer portion of intact cysts and on developing cyst wall filaments in encysting trophozoites. Post-embedding immunogold labeling of thin sections of intact Giardia cysts with polyclonal and monoclonal antibodies specific for cyst wall antigens (major protein bands of approximately 29, 75, 88, and 102 KD on Western blots) showed strong labeling of the filamentous cyst wall, whereas no labeling was seen on the membranous portion. High-resolution field emission scanning electron microscopy (FESEM) of Giardia cysts revealed that the cyst wall-specific polyclonal rabbit antisera and monoclonal mouse antibody produced gold labeling of 20-nm filaments in the cyst wall as detected with secondary electron imaging (SEI) and backscatter electron imaging (BEI) at 10 kV, despite coating of the cells with platinum by ion sputtering. FESEM studies of encysting Giardia trophozoites demonstrated that immunostaining with antibodies to cyst wall antigens produced colloidal gold labeling of developing cyst wall filaments on the cell surface; however, the intervening membrane domains were unlabeled. Substitution of normal serum for cyst wall-specific antibodies, or preabsorption of specific antibodies with Giardia cysts, eliminated immunolabeling of the filaments.     

Effect of parental ageing on offspring developmental and survival attributes in an aphidophagous ladybird,Cheilomenes sexmaculata

The present study is the first attempt to investigate the effect of parental ageing of Cheilomenes sexmaculata (Fabricius) on total developmental period, developmental rate, adult weight on emergence, longevity, egg to adult survival and age‐specific survivorship of the offspring. Young parents (10–20 day old) produced offspring with the shortest total developmental period, highest development rate, highest weight on emergence, greater longevity and highest survival. Age‐specific survivorship of the offspring of younger parents declined later than the offspring of middle (30–40 day old) and old (50–60 day old) aged parents. This study would help in understanding the effect of parental ageing on future generations of predaceous ladybird beetles and would be helpful in designing mass multiplication programme of the bioagent, C. sexmaculata, in the laboratory.     

SEX CHROMOSOME LINKAGE OF MATE PREFERENCE AND COLOR SIGNAL MAINTAINS ASSORTATIVE MATING BETWEEN INTERBREEDING FINCH MORPHS

Assortative mating is a key aspect in the speciation process because it is important for both initial divergence and maintenance of distinct species. However, it remains a challenge to explain how assortative mating evolves when diverging populations are undergoing gene flow (e.g., during hybridization). Here I experimentally test how assortative mating is maintained with frequent gene flow between diverged head‐color morphs of the Gouldian finch (Erythrura gouldiae). Contrary to the predominant view on the development of sexual preferences in birds, cross‐fostered offspring did not imprint on the phenotype of their conspecific (red or black morphs) or heterospecific (Bengalese finch) foster parents. Instead, the mating preferences of F₁ and F₂ intermorph‐hybrids are consistent with inheritance on the Z chromosomes, which are also the location for genes controlling color expression and the genes causing low fitness of intermorph‐hybrids. Genetic associations between color signal and preference loci on the sex chromosomes may prevent recombination from breaking down these associations when the morphs interbreed, helping to maintain assortative mating in the face of gene flow. Although sex linkage of reproductively isolating traits is theoretically expected to promote speciation, social and ecological constraints may enforce frequent interbreeding between the morphs, thus preventing complete reproductive isolation.     

31P NMR studies of metabolism in Acanthamoebacastellanii: Polyphosphate release from encysted cells

${}^{31}\text{P NMR}$ studies of $\text{Acanthamoeba}\text{castellanii}$ have shown that encysting cells release polyphosphate into the encystment medium. Mature cysts contain low levels of polyphosphate, as do vegetative cells. Young cysts (7 days) show detectable levels of nucleotide diphosphates and triphosphate similar to those observed in vegetative cells. Mature cysts (90 days) show only excreted polyphosphate as well as a component which has a chemical shift of a phosphodiester. The inorganic phosphate peak in the cyst shows that the cyst milieu is liquid-like and that the intracellular environment maintains a pH between 6 and 7.5 in the presence of extracellular values from 4 to 9.     

Contrasting Parental Tasks Influence Parental Roles for Paired and Single Biparental Cichlid Fish

Biparental care of young occurs when both parents provide some sort of care for offspring and can include a wide variety of behaviors, yet often studies focus on single aspects of parental care when trying to determine how each parent contributes. Here, we presented the biparental convict cichlid fish (Amatitlania nigrofasciata) with two conflicting parental behaviors: retrieval of non‐swimming offspring that have been displaced from the nest and defense against a conspecific intruder, a potential brood predator. We examined single males, single females, and pairs of parents to determine how males and females each contributed to overall parental care. Traditionally in this species, parents exhibit a division of labor (in which each parent contributes to all parental activities), but also exhibit a division of roles (in which males tend to favor the role of defense and females tend to favor more direct care, spending more time with offspring). We hypothesized that single parents would compensate for their absent mate, but that males and females would still favor preferred roles. Additionally, we hypothesized that there would be an asymmetrical expansion of roles, with females being more flexible. Our results show that the preferred roles of both parents were evident even when parents were without their mates and that males and females differed in their compensatory levels, at least when compared to the behaviors of the intact pair. Contrary to our prediction, females seem unable to fully compensate for the defensive behaviors usually exhibited by males, while males shifted completely to retrieve displaced offspring.