Comparison of Two Pearl Sacs Formed in the Same Recipient Oyster with Different Genetic Background Involved in Yellow Pigmentation in <Emphasis Type="Italic">Pinctada fucata</Emphasis>

Color is one of the most important factors determining the commercial value of pearls. Pinctada fucata is a well-known pearl oyster producing high-quality Akoya pearls. Phenotypic variation in amount of yellow pigmentation produces white and yellowish pearls. It has been reported that polymorphism of yellow pigmentation of Akoya pearls is genetically regulated, but the responsible gene(s) has remained unknown. Here, we prepared pearl sac pairs formed in the same recipient oyster but coming from donor oysters that differ in their color. These two pearl sacs produced pearls with different yellowness even in the same recipient oyster. Yellow tone of produced pearls was consistent with shell nacre color of donor oysters from which mantle grafts were prepared, indicating that donor oysters strongly contribute to the yellow coloration of Akoya pearls. We also conducted comparative RNA-seq analysis and retrieved several candidate genes involved in the pearl coloration. Whole gene expression patterns of pair sacs were not grouped by pearl color they produced, but grouped by recipient oysters in which they were grown, suggesting that the number of genes involved in the yellow coloration is quite small, and that recipient oyster affects gene expression of the majority of genes in the pearl sac.     

Freshwater pearl mussel in Finland—current status and future prospects

Information on the present distribution and status of the threatened freshwater pearl mussel Margaritifera margaritifera populations in Finland was updated. The status of the populations was classified into seven categories which are viable, maybe viable, non-viable/partly viable, dying-out, almost extinct, probably extinct, and extinct. The main criteria for judging the viability class were the population size and proportion of juvenile mussels. According to calculated estimate 1.7% populations were viable, 8.5% maybe viable, 40.2% non-viable/partly viable, 14.5% dying-out, 30.8% almost extinct, and 4.3% probably extinct. The present number of rivers with M. margaritifera in Finland is 117.     

The relationship between the freshwater pearl mussel (<Emphasis Type="Italic">Margaritifera margaritifera</Emphasis>) and its hosts

Endangered freshwater pearl mussels Margaritifera margaritifera reveal a complex life cycle with an obligate host-dependent phase. Only two species, Atlantic salmon Salmo salar and brown trout S. trutta, are important hosts in Europe, indicating a high degree of specialization. Whilst freshwater pearl mussels with their filtering activity can provide important ecosystem services and indirectly improve the habitat quality for their salmonid hosts, their direct effects on physiological stress, reduced swimming performance, and increased mortality at high rates of infestation all support a parasitic character of the mussel during its hostdependent phase. From an evolutionary perspective, both the much greater generation time of the parasite compared to the host, as well as the great distribution ranges of M. margaritifera and its hosts should favour local adaptation patterns. The variable suitability of different salmonid strains and species as hosts for M. margaritifera and the resulting differences in the performance of larvae during the host-dependent phase indicate that host-management strategies should focus on maintaining high quality hosts at a regional scale to avoid selection or genetic drift effects which could erode the genetic and evolutionary potential for adaptation to changing environmental conditions.     

Rising the Persian Gulf Black-Lip Pearl Oyster to the Species Level: Fragmented Habitat and Chaotic Genetic Patchiness in <Emphasis Type="Italic">Pinctada persica</Emphasis>

Marine organisms with long pelagic larval stages are expected to exhibit low genetic differentiation due to their potential to disperse over large distances. Growing body of evidence, however, suggests that marine populations can differentiate over small spatial scales. Here we focused on black-lip pearl oysters from the Persian Gulf that are thought to belong to the Pinctada margaritifera complex given their morphological affinities. This species complex includes seven lineages that show a wide distribution ranging from the Persian Gulf (Pinctada margaritifera persica) and Indian Ocean (P. m. zanzibarensis) to the French Polynesia (P. m. cumingii) and Hawai’i (P. m. galtsoffi). Despite the long pelagic larval phase of P. m. persica, this lineage is absent from continental locations and can only be found on a few islands of the Persian Gulf. Mitochondrial COI-based analyses indicated that P. m. persica belongs to a clearly divergent ESU and groups with specimens from Mauritius (P. m. zanzibarensis). Microsatellite data, used here to assess the spatial scale of realized dispersal of Persian Gulf black-lip pearl oysters, revealed significant genetic structure among islands distant of only a few dozen kilometres. The scantiness of suitable habitats most likely restricted the distribution of this lineage originating the observed chaotic genetic patchiness. The hatchery-based enhancement performed in one of the sampled islands may also have affected population genetic structure. The long-term accumulation of genetic differences likely resulted from the allopatric divergence between P. m. persica and the neighbouring Indian Ocean black-lip pearl oysters.     

The populations of the freshwater pearl mussel, <Emphasis Type="Italic">Margaritifera margaritifera</Emphasis> (Linnaeus, 1758), and the thick shelled river mussel, <Emphasis Type="Italic">Unio crassus</Emphasis> Philipsson, 1788, in Latvia

The populations of two endangered species—the freshwater pearl mussel Margaritifera margaritifera and the thick shelled river mussel Unio crassus in Latvia were studied. The specimens were counted, measured, population density and age structure were calculated. The possible host fish presence was found.     

Irregularity of the linear growth of the <Emphasis Type="Italic">Margaritifera margaritifera</Emphasis> (Bivalvia: Margaritiferidae) population of the Nemina River,Karelia

The dependence of shell growth in length and height during ontogeny has been studied in the pearl mussel Margaritifera margaritifera inhabiting the Nemina River (basin of Lake Onega, Karelia). It has been shown that the population is heterogenous based on the height-to-length ratio. It has been found that during ontogeny M. margaritifera from the studied population undergoes a constant change in the relative growth of the shell leading to either lengthening or rounding of the shell.     

Variation in the <Emphasis Type="Italic">COI</Emphasis> gene of the freshwater pearl mussel <Emphasis Type="Italic">Margaritifera margaritifera</Emphasis> from River Vuokkijoki

The freshwater pearl mussel Margaritifera margaritifera L. is one of the most endangered freshwater mussels in the world. Effective conservation of threatened species requires not only ecological, but also genetic information from the target species and populations. Since low genetic diversity can reduce the ability of a species to adapt to environmental changes, maintaining genetic diversity has been identified as one of the key elements in successful conservation programs. We examined genetic variation of the freshwater pearl mussel from the River Vuokkijoki, Karelia, Russia. We sequenced a fragment of the cytochrome c oxidase subunit I gene (COI) from 22 individuals and compared the data to 32 previously published COI sequences available in GenBank. We identified 10 different COI haplotypes in the sequenced samples, three of which had not been previously reported. Our results show that the River Vuokkijoki has high genetic diversity and suggest that the colonization of this northern freshwater pearl mussel population might have occurred from multiple and even distant refugia. Therefore, the freshwater pearl mussel population of the River Vuokkijoki is valuable for the conservation of the whole species.     

Irregularity of the linear growth of the <Emphasis Type="Italic">Margaritifera margaritifera</Emphasis> (Bivalvia: Margaritiferidae) population of the Syskyanjoki River,Karelia

The interdependence of shell growth in length and height during ontogeny has been studied in the pearl mussel Margaritifera margaritifera, inhabiting the Syskyanjoki River (basin of Lake Ladoga, Karelia). It was shown that by the height-to-length ratio of the shell the population is heterogeneous. The question of whether this is due to the heterogeneity of genetic variability or due to variations in the environmental conditions remains unclear. It was found that during the ontogeny of the M. margaritifera from the population there is a constant change in the relative growth of the shell, leading to lengthening or rounding of the shell. Conventionally, all mussels can be divided into two groups, different by the periods of change in the relative growth. A comparison of the results with the data obtained previously for pearl mussels of the Varzuga River (basin of the White Sea, Murmansk oblast) was performed.     

A Sperm Spawn-Inducing Pheromone in the Silver Lip Pearl Oyster (<Emphasis Type="Italic">Pinctada maxima</Emphasis>)

Pheromones are considered to play an important role in broadcast spawning in aquatic animals, facilitating synchronous release of gametes. In oysters, the sperm has been implicated as a carrier for the spawn-inducing pheromone (SIP). In hatchery conditions, male pearl oysters (Pinctata maxima) can be stimulated to spawn through a variety of approaches (e.g. rapid temperature change), while females can only be induced to spawn through exposure to conspecific sperm, thus limiting development of targeted pairing, required for genetic research and management. The capacity for commercial production and improvement of genetic lines of pearl oysters could be greatly improved with access to a SIP. In this study, we prepared and sequenced crude and semi-purified P. maxima sperm extracts that were used in bioassays to localise the female SIP. We report that the P. maxima SIP is proteinaceous and extrinsically associated with the sperm membrane. Bioactivity from pooled RP-HPLC fractions, but not individual fractions, suggests that the SIP is multi-component. We conclude that crude sperm preparations, as described in this study, can be used as a sperm-free inducer of female P. maxima spawning, which enables for a more efficient approach to genetic breeding.     

The freshwater pearl mussel <Emphasis Type="Italic">Margaritifera margaritifera</Emphasis> L.: Metamorphosis,growth, and development dynamics of encysted glochidia

The growth and morphogenesis of glochidia of the freshwater pearl mussel Margaritifera margaritifera on the gills of the Atlantic salmon Salmo salar in the Syuskyuyanyoki River (Karelia) are studied. Comparative analysis of histological features of glochidia depending on the age of the cyst is made, and the results of studies of the relationship and the influence of seasonal changes in water temperature on growth and morphogenesis of glochidia, which is essential for adaptation of living organisms and ensuring the sustainability of the participants of the parasite–host relationship, are presented.     

Combination of newly developed SNP and InDel markers for genotyping the <Emphasis Type="Italic">Cf-9</Emphasis> locus conferring disease resistance to leaf mold disease in the tomato

The Cf-9 gene in the tomato is known to confer resistance against leaf mold disease caused by Cladosporium fulvum, and a gene-based marker targeted to the Cf-9 allele has been widely used as a crop protection approach. However, we found this marker to be misleading in genotyping. Therefore, we developed new single-nucleotide polymorphism (SNP) and insertion and deletion (InDel) markers targeted to the Cf-9 allele in order to increase genotyping accuracy and facilitate high-throughput screening. The DNA sequences of reported Cf-9, cf-9, Cf-0, and closely related Cf-4 alleles were compared, and two functional and non-synonymous SNPs were found to distinguish the Cf-9 resistance allele from the cf-9, Cf-0, and Cf-4 alleles. An SNP marker including these two SNPs was developed and applied to the genotyping of 33 tomato cultivars by high-resolution melting analysis. Our SNP marker was able to select all three Cf-9 genotypes (resistant, heterozygous, and susceptible alleles). Interestingly, two cultivars were grouped separately from these three genotypes. To further examine this outgroup, we preformed polymerase chain reaction (PCR) on two InDel regions identified by sequence comparison of the Cf-9 and Cf-4 genes. The band patterns revealed that these two cultivars carried Cf-4 rather than Cf-9 alleles and that three cultivars classified in the Cf-9 resistance group actually carried both Cf-9 and Cf-4 genes. To determine whether these genotyping results were consistent with disease resistance phenotypes, we examined the induction of a hypersensitive response by transiently expressing the corresponding effector genes, and found that the results matched perfectly with the genotyping results. These findings indicate that the combination of our SNP and InDel markers allows resistant Cf-9 alleles to be distinguished from cf-9 and Cf-4 alleles, which will be useful for marker-assisted selection of tomato cultivars resistant to C. fulvum.     

Effects of nonnative invertebrates on two life stages of the native eastern oyster <Emphasis Type="Italic">Crassostrea virginica</Emphasis>

Since their recent introductions into Florida waters, three sessile invertebrates [Perna viridis (Asian green mussel), Mytella charruana (charru mussel) and Megabalanus coccopoma (pink titan acorn barnacle)] have expanded their range along the Atlantic coast in estuarine waters. Little research has been done to understand how these nonnative species interact with the ecologically and economically important eastern oyster Crassostrea virginica. To assess the potential effects of P. viridis, M. charruana and M. coccopoma on C. virginica, the following questions were addressed in manipulative experiments. (1) Does the presence of nonnative species decrease oyster larval settlement? (2) Do oyster larvae avoid settling on nonnative species? (3) Do nonnative species decrease survival of juvenile oysters (spat)? (4) Do nonnative species hinder spat growth? We included two controls: absence of nonnative species and presence of the native mussel Geukensia demissa. The nonnative species influenced settlement, growth and survival of C. virginica in different ways. M. coccopoma and P. viridis negatively influenced larval settlement, whereas M. charruana had no influence on the total number of settled larvae. Oyster larvae avoided settling on all three nonnative species and the native G. demissa. Both nonnative mussels negatively affected survival of juvenile oysters but only M. charruana also reduced spat growth. The native mussel, G. demissa, had no negative impacts on total settlement, survival and growth of C. virginica; in fact, it increased larval settlement in some trials. These three nonnative species should be classified as invasive because all had negative effects on native C. virginica.     

Strong genetic differentiation and low genetic diversity of the freshwater pearl mussel (<Emphasis Type="Italic">Margaritifera margaritifera</Emphasis> L.) in the southwestern European distribution range

Genetic diversity of European freshwater pearl mussel, Margaritifera margaritifera (L.), appears exceptional with highest genetic variability found in the northernmost European populations of Scandinavia and lower genetic variability in central and southern Europe. The objective of this study was to investigate genetic diversity and differentiation of 14 southernmost populations on the Iberian Peninsula which greatly differ in terms of life span and habitat conditions from the rest of central and northern European populations. The analyses of ten microsatellite loci revealed a pronounced level of genetic divergence and very low genetic diversity. These results match the expectations of geographically peripheral populations with respect to their genetic composition. The life history strategy, the narrow ecological niche of the species, and anthropogenic habitat modifications have most likely shaped the genetic pattern of Iberian pearl mussel populations. The peripheral position with less optimal habitat conditions may increase the extinction risk of these populations and thus effective conservation strategies for the Iberian M. margaritifera are needed. The successful conservation of the species at its southwestern margin requires inclusion of genetically different conservation units which may reveal local adaptation.     

DNA Fingerprinting of Pearls to Determine Their Origins

We report the first successful extraction of oyster DNA from a pearl and use it to identify the source oyster species for the three major pearl-producing oyster species Pinctada margaritifera, P. maxima and P. radiata. Both mitochondrial and nuclear gene fragments could be PCR-amplified and sequenced. A polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay in the internal transcribed spacer (ITS) region was developed and used to identify 18 pearls of unknown origin. A micro-drilling technique was developed to obtain small amounts of DNA while maintaining the commercial value of the pearls. This DNA fingerprinting method could be used to document the source of historic pearls and will provide more transparency for traders and consumers within the pearl industry.     

Integrated gene mapping and synteny studies give insights into the evolution of a sex proto-chromosome in <Emphasis Type="Italic">Solea senegalensis</Emphasis>

The evolution of genes related to sex and reproduction in fish shows high plasticity and, to date, the sex determination system has only been identified in a few species. Solea senegalensis has 42 chromosomes and an XX/XY chromosome system for sex determination, while related species show the ZZ/ZW system. Next-generation sequencing (NGS), multi-color fluorescence in situ hybridization (mFISH) techniques, and bioinformatics analysis have been carried out, with the objective of revealing new information about sex determination and reproduction in S. senegalensis. To that end, several bacterial artificial chromosome (BAC) clones that contain candidate genes involved in such processes (dmrt1, dmrt2, dmrt3, dmrt4, sox3, sox6, sox8, sox9, lh, cyp19a1a, amh, vasa, aqp3, and nanos3) were analyzed and compared with the same region in other related species. Synteny studies showed that the co-localization of dmrt1-dmrt2-drmt3 in the largest metacentric chromosome of S. senegalensis is coincident with that found in the Z chromosome of Cynoglossus semilaevis, which would potentially make this a sex proto-chromosome. Phylogenetic studies show the close proximity of S. senegalensis to Oryzias latipes, a species with an XX/XY system and a sex master gene. Comparative mapping provides evidence of the preferential association of these candidate genes in particular chromosome pairs. By using the NGS and mFISH techniques, it has been possible to obtain an integrated genetic map, which shows that 15 out of 21 chromosome pairs of S. senegalensis have at least one BAC clone. This result is important for distinguishing those chromosome pairs of S. senegalensis that are similar in shape and size. The mFISH analysis shows the following co-localizations in the same chromosomes: dmrt1-dmrt2-dmrt3, dmrt4-sox9-thrb, aqp3-sox8, cyp19a1a-fshb, igsf9b-sox3, and lysg-sox6.     

Morphological and physiological changes,and the functional analysis of <Emphasis Type="Italic">PdSPL9</Emphasis> in the juvenile-to-adult phase transition of <Emphasis Type="Italic">paeonia delavayi</Emphasis>

Tree peony has a long juvenile stage, which has limited breeding efforts. To date, very little information is available regarding the juvenile stage of tree peony. In the present study, Paeonia delavayi plants of varying ages were used to investigate the juvenile phase, juvenile-to-adult phase transition, and adult phase via morphological, physiological, and molecular genetic analysis. Micro-observation of buds of different ages and flower induction experiments suggested that the juvenile-to-adult phase transition of P. delavayi occurred at around 2-years of age. Plant height, crown width, leaf length, and the soluble sugar and starch content were positively correlated with plant age. The juvenile gene PdSPL9 contained miR156 targeted sites and a typical SBP domain was cloned from P. delavayi. Expression analysis showed that the expression levels of PdSPL9, PdmiR172d, and PdLFY increased with plant age, while the inverse pattern was observed for PdmiR156a. Function of PdSPL9 was further characterized in Arabidopsis plant. According to function characterization of PdSPL9 in Arabidopsis and expression patterns of PdSPL9 in P. delavayi, it was suggested that PdSPL9 plays a role in controlling juvenile-to-adult phase transition by promoting miR172d, and PdLFY expression in P. delavayi plants, while the expression of PdSPL9 is repressed by miR156a.