Kristin E. Gribble

Dinoflagellate cysts in recent marine sediments from the east coast of Russia

Abstract Forty-two different dinoflagellate cyst types were found in recent sediment samples collected between July 1999–September 2002 from 44 stations along the eastern coast of Russia. This represents the first survey of recent dinoflagellate cysts in Russian marine waters. Forty cysts were identified to the species level, representing 17 genera. The most common cysts were those of ellipsoidal Alexandrium spp., Protoceratium reticulatum, Gonyaulax spp., Polykrikos kofoidii, P. schwartzii, Protoperidinium americanum, P. minutum, P. conicoides, P. subinerme, P. conicum and Scrippsiella trochoidea. Fifteen of the dinoflagellate species have not previously been recorded as motile cells in Russian marine waters: Alexandrium cf. minutum, Cochlodinium cf. polykrikoides, Diplopsalis cf. lebourae, Fragilidium mexicanum, Gonyaulax elongata, G. membranaceae, Gymnodinium cf. catenatum, Pentapharsodinium dalei, P. tyrrhenicum, Protoperidinium americanum, P. cf. avellanum, Scrippsiella cf. lachrymosa, S. cf. precaria, S. cf. rotunda and Warnowia cf. rosea. Cysts of the potentially toxic species Alexandrium cf. minutum, A. tamarense and Gymnodinium cf. catenatum were also found in this survey. Ellipsoidal Alexandrium tamarense type cysts were widely distributed and dominated many localities in the study area. These data suggest that additional cyst surveys should be conducted in areas of the eastern Russian coastline not yet investigated, and that the potential for paralytic shellfish poisoning toxicity as a result of blooms of toxic species may be more widespread than previously documented.

High intraindividual, intraspecific, and interspecific variability in large-subunit ribosomal DNA in the heterotrophic dinoflagellates Protoperidinium, Diplopsalis, and Preperidinium (Dinophyceae)

K.E. Gribble and D.M. Anderson. 2007. High intraindividual, intraspecific, and interspecific variability in large-subunit ribosomal DNA in the heterotrophic dinoflagellates Protoperidinium, Diplopsalis, and Preperidinium (Dinophyceae). Phycologia 46: 315–324. DOI: 10.2216/06-68.1 Large-subunit ribosomal DNA (LSU rDNA) gene sequences are frequently used to infer the phylogeny and species identity of organisms. The many copies of the LSU rDNA found in the genome are thought to be kept homogeneous by concerted evolution. In this study, we found high intraindividual, intraspecific and interspecific diversity in the D1-D6 region of the LSU rDNA in eight species of thecate, heterotrophic dinoflagellates using single-cell PCR, cloning, and sequencing of many clones. For each species, the clone library was usually comprised of one highly represented copy and many unique sequences. Sequence differences were primarily characterized by single base pair substitutions, single base pair insertion/deletions (indels), and/or large indels. Phylogenetic analysis of all sequence clones gave strong support for monophyly of the polymorphic copies of each species and recovered the same species tree as an analysis using just one sequence per species. Analysis of LSU rDNA expression in three species by RT-PCR indicated that expressed copies have fewer substitutions and fewer and smaller indels and that 50% or more of the copies are pseudogenes. High intraspecific and intraindividual LSU rDNA sequence variability could lead to inaccurate species phylogenies and overestimation of species diversity in environmental sequencing studies.

Life-Span Extension by Caloric Restriction Is Determined by Type and Level of Food Reduction and by Reproductive Mode in Brachionus manjavacas (Rotifera)

We measured life span and fecundity of three reproductive modes in a clone of the monogonont rotifer Brachionus manjavacas subjected to chronic caloric restriction (CCR) over a range of food concentrations or to intermittent fasting (IF). IF increased life span 50%-70% for all three modes, whereas CCR increased life span of asexual females derived from sexually or asexually produced eggs, but not that of sexual females. The main effect of CR on both asexual modes was to delay death at young ages, rather than to prevent death at middle ages or to greatly extend maximum life span; in contrast CR in sexual females greatly increased the life span of a few long-lived individuals. Lifetime fecundity did not decrease with CCR, suggesting a lack of resource allocation trade-off between somatic maintenance and reproduction. Multiple outcomes for a clonal lineage indicate that different responses are established through epigenetic programming, whereas differences in life-span allocations suggest that multiple genetic mechanisms mediate life-span extension.

Genetic determinants of mate recognition in Brachionus manjavacas (Rotifera)

BackgroundMate choice is of central importance to most animals, influencing population structure, speciation, and ultimately the survival of a species. Mating behavior of male brachionid rotifers is triggered by the product of a chemosensory gene, a glycoprotein on the body surface of females called the mate recognition pheromone. The mate recognition pheromone has been biochemically characterized, but little was known about the gene(s). We describe the isolation and characterization of the mate recognition pheromone gene through protein purification, N-terminal amino acid sequence determination, identification of the mate recognition pheromone gene from a cDNA library, sequencing, and RNAi knockdown to confirm the functional role of the mate recognition pheromone gene in rotifer mating.ResultsA 29 kD protein capable of eliciting rotifer male circling was isolated by high-performance liquid chromatography. Two transcript types containing the N-terminal sequence were identified in a cDNA library; further characterization by screening a genomic library and by polymerase chain reaction revealed two genes belonging to each type. Each gene begins with a signal peptide region followed by nearly perfect repeats of an 87 to 92 codon motif with no codons between repeats and the final motif prematurely terminated by the stop codon. The two Type A genes contain four and seven repeats and the two Type B genes contain three and five repeats, respectively. Only the Type B gene with three repeats encodes a peptide with a molecular weight of 29 kD. Each repeat of the Type B gene products contains three asparagines as potential sites for N-glycosylation; there are no asparagines in the Type A genes. RNAi with Type A double-stranded RNA did not result in less circling than in the phosphate-buffered saline control, but transfection with Type B double-stranded RNA significantly reduced male circling by 17%. The very low divergence between repeat units, even at synonymous positions, suggests that the repeats are kept nearly identical through a process of concerted evolution. Information-rich molecules like surface glycoproteins are well adapted for chemical communication and aquatic animals may have evolved signaling systems based on these compounds, whereas insects use cuticular hydrocarbons.ConclusionOwing to its critical role in mating, the mate recognition pheromone gene will be a useful molecular marker for exploring the mechanisms and rates of selection and the evolution of reproductive isolation and speciation using rotifers as a model system. The phylogenetic variation in the mate recognition pheromone gene can now be studied in conjunction with the large amount of ecological and population genetic data being gathered for the Brachionus plicatilis species complex to understand better the evolutionary drivers of cryptic speciation.

Rotifers as experimental tools for investigating aging

Comparative biogerontology has much to contribute to the study of aging. A broad range of aging rates have evolved to meet environmental challenges, and understanding these adaptations can produce valuable insights into aging. The supra Phylum Lophotrochozoa is particularly understudied and has several groups that have intriguing patterns of aging. Members of the Lophotrochozoan phylum Rotifera are particularly useful for aging studies because cohort life tables can be conducted with them easily, and biochemical and genomic tools are available for examining aging mechanisms. This paper reviews a variety of caloric restriction (CR) regimens, small molecule inhibitors, and dietary supplements that extend rotifer lifespan, as well as important interactions between CR and genotype, antioxidant supplements, and TOR and jun-N-terminal kinase (JNK) pathways, and the use of RNAi to identify key genes involved in modulating the aging response. Examples of how rapamycin and JNK inhibitor exposure keeps mortality rates low during the reproductive phase of the life cycle are presented, and the ease of conducting life table experiments to screen natural products from red algae for life extending effects is illustrated. Finally, experimental evolution to produce longer-lived rotifer individuals is demonstrated, and future directions to determine the genetic basis of aging are discussed.

Maternal caloric restriction partially rescues the deleterious effects of advanced maternal age on offspring

While many studies have focused on the detrimental effects of advanced maternal age and harmful prenatal environments on progeny, little is known about the role of beneficial non‐Mendelian maternal inheritance on aging. Here, we report the effects of maternal age and maternal caloric restriction (CR) on the life span and health span of offspring for a clonal culture of the monogonont rotifer Brachionus manjavacas. Mothers on regimens of chronic CR (CCR) or intermittent fasting (IF) had increased life span compared with mothers fed ad libitum (AL). With increasing maternal age, life span and fecundity of female offspring of AL‐fed mothers decreased significantly and life span of male offspring was unchanged, whereas body size of both male and female offspring increased. Maternal CR partially rescued these effects, increasing the mean life span of AL‐fed female offspring but not male offspring and increasing the fecundity of AL‐fed female offspring compared with offspring of mothers of the same age. Both maternal CR regimens decreased male offspring body size, but only maternal IF decreased body size of female offspring, whereas maternal CCR caused a slight increase. Understanding the genetic and biochemical basis of these different maternal effects on aging may guide effective interventions to improve health span and life span.

Patterns of intraspecific variability in the response to caloric restriction

Caloric restriction (CR) is cited as the most robust means of increasing lifespan across a range of taxa, yet there is a high degree of variability in the response to CR, both within and between species. To examine the intraspecific evolutionary conservation of lifespan extension by CR, we tested the effects of chronic caloric restriction (CCR) at multiple food levels and of intermittent fasting (IF) in twelve isolates from the Brachionus plicatilis species complex of monogonont rotifers. While CCR generally increased or did not change lifespan and total fecundity, IF caused increased, unchanged, or decreased lifespan, depending upon the isolate, and decreased total fecundity in all but one isolate. Lifespan under ad libitum (AL) feeding varied among isolates and predicted the lifespan response to CR: longer-lived isolates under AL were less likely to have a significant increase in lifespan under CCR and were more likely to have a significantly shortened lifespan under IF. Lifespan under AL conditions and the response to CR were not correlated with hydroperiodicity of native habitat or with time in culture. Lack of trade-off between lifespan and fecundity under CCR, and differences in lifespan and fecundity under CCR and IF, even when average food intake was similar, suggest that longevity changes are not always directly determined by energy intake and that CCR and IF regimens extend lifespan through diverse genetic mechanisms.

The mate recognition protein gene mediates reproductive isolation and speciation in the Brachionus plicatilis cryptic species complex

BackgroundChemically mediated prezygotic barriers to reproduction likely play an important role in speciation. In facultatively sexual monogonont rotifers from the Brachionus plicatilis cryptic species complex, mate recognition of females by males is mediated by the Mate Recognition Protein (MRP), a globular glycoprotein on the surface of females, encoded by the mmr-b gene family. In this study, we sequenced mmr-b copies from 27 isolates representing 11 phylotypes of the B. plicatilis species complex, examined the mode of evolution and selection of mmr-b, and determined the relationship between mmr-b genetic distance and mate recognition among isolates.ResultsIsolates of the B. plicatilis species complex have 1–4 copies of mmr-b, each composed of 2–9 nearly identical tandem repeats. The repeats within a gene copy are generally more similar than are gene copies among phylotypes, suggesting concerted evolution. Compared to housekeeping genes from the same isolates, mmr-b has accumulated only half as many synonymous differences but twice as many non-synonymous differences. Most of the amino acid differences between repeats appear to occur on the outer face of the protein, and these often result in changes in predicted patterns of phosphorylation. However, we found no evidence of positive selection driving these differences. Isolates with the most divergent copies were unable to mate with other isolates and rarely self-crossed. Overall the degree of mate recognition was significantly correlated with the genetic distance of mmr-b.ConclusionsDiscrimination of compatible mates in the B. plicatilis species complex is determined by proteins encoded by closely related copies of a single gene, mmr-b. While concerted evolution of the tandem repeats in mmr-b may function to maintain identity, it can also lead to the rapid spread of a mutation through all copies in the genome and thus to reproductive isolation. The mmr-b gene is evolving rapidly, and novel alleles may be maintained and increase in frequency via asexual reproduction. Our analyses indicate that mate recognition, controlled by MMR-B, may drive reproductive isolation and allow saltational sympatric speciation within the B. plicatilis cryptic species complex, and that this process may be largely neutral.

Measurement of Survival Time in Brachionus Rotifers: Synchronization of Maternal Conditions

Rotifers are microscopic cosmopolitan zooplankton used as models in ecotoxicological and aging studies due to their several advantages such as short lifespan, ease of culture, and parthenogenesis that enables clonal culture. However, caution is required when measuring their survival time as it is affected by maternal age and maternal feeding conditions. Here we provide a protocol for powerful and reproducible measurement of the survival time in Brachionus rotifers following a careful synchronization of culture conditions over several generations. Empirically, poor synchronization results in early mortality and a gradual decrease in survival rate, thus resulting in weak statistical power. Indeed, under such conditions, calorie restriction (CR) failed to significantly extend the lifespan of B. plicatilis although CR-induced longevity has been demonstrated with well-synchronized rotifer samples in past and present studies. This protocol is probably useful for other invertebrate models, including the fruitfly Drosophila melanogaster and the nematode Caenorhabditis elegans, because maternal age effects have also been reported in these species.