Pearl Mussel Research Articles

Expression and functional characterization of the CUB domain-containing protein from the triangle sail mussel (Hyriopsis cumingii) in response to pathogenic infection.

The complement C1r/C1s, Uegf, and Bmp1 (CUB) domains, which are most exclusively found in extracellular and plasma membrane-related proteins, are involved in various biological processes. In this study, a CUB domain-containing protein (designed as HcCDCP) was cloned and characterized from freshwater pearl mussel (Hyriopsis cumingii). The 2280bp complete cDNA of the HcCDCP contained a 1002bp open reading frame, which encoded a protein with 333 amino acids. The predicted HcCDCP protein contained a typical CUB domain and a transmembrane region. The tissue distribution analysis indicated that the HcCDCP was detected in all tissues, and the highest expression was found in hepatopancreas followed by gills. After infection with bacteria (i.e., Staphylococcus aureus and Vibrio parahaemolyticus), virus (white spot syndrome virus) and virus analogs (poly[I:C]), the mRNA level of the HcCDCP was significantly upregulated, suggesting that the HcCDCP might be involved in host immune defense response. The RNA interference revealed that the silencing of the HcCDCP could evidently inhibit the expression levels of lysozyme and tumor necrosis factor. Moreover, the recombinant protein of the CUB domain (rCUB) possessed binding capacity to eight different kinds of bacteria. The polysaccharide binding assay showed that the rCUB specifically bound to lipopolysaccharide, peptidoglycan, and D-mannose. This study provided valuable information for exploring the biological roles of CDCPs in the host defense system of mollusks.

Genotype by environment interactions for inner shell color and growth traits in the purple freshwater pearl mussel, Hyriopsis cumingii, reared with different water depths and mud substrates

A breeding selection program for improving inner shell color and growth was developed for Hyriopsis cumingii, to enhance purple pearl production. In freshwater pearl culture, adjusting culture water depth and mud substrate supply are routinely carried out to improve pearl quality. To improve selection decisions in different conditions, we estimated heritabilities and genotype by environment (G × E) interactions on inner shell color and growth traits in 1571 17-month-old animals from 8 half sib family of purple selective strain of H. cumingii reared in different water depths (WD1, 32 cm; WD2, 48 cm) and mud substrates (MS, mussel pond mud substrate; SS, shrimp pond mud substrate) were analyzed. With the same mud substrate, H. cumingii reared at WD1 showed increased performance in shell color and growth. Heritability estimates for growth traits (shell length, shell height, shell width, body weight and shell weight) were higher at WD1 and higher at WD2 for inner shell color parameters (L, a, band dE⁎) when rearing in MS, while showed opposite pattern in SS. At the same water depth, H. cumingii reared with MS showed better performance in shell color and growth traits. Heritability estimates for growth traits were higher in SS and higher in MS for inner shell color parameters when rearing at WD1, while showed opposite pattern at WD2. Heritability estimates for L, a and all growth traits were moderate and high (h2 > 0.15), while were low for b and dE⁎ (h2 < 0.15). Genetic correlations for inner shell color parameters and growth traits within the four conditions were less than 0.8, except that some combinations of depth and mud substrate with over 0.9 for shell growth traits and shell color traits showed significant G × E correlations. The rank of individuals with comprehensive breeding value was different in four conditions, and the percentage of individual family sources shared in some pair conditions were small, which indicated re-ranking during selective breeding program. This study provides guidelines for a selective breeding strategy for improving inner shell color and growth in H. cumingii when mussels are reared under different conditions

Growth Parameters of the Freshwater Pearl Mussel Margaritifera margaritifera (Bivalvia, Margaritiferidae), Vuokinjoki River Population (Karelia)

Individual linear growth rates were studied in the freshwater pearl mussel Margaritifera margaritifera from the Vuokinjoki River. Growth deceleration coefficients were shown to vary widely and differ significantly among individuals. The average value of the growth deceleration coefficient for the population is 0.069. The growth of mussels in the Vuokinjoki River is accompanied by three regular biorhythms. These biorhythm periods were roughly constant both through an individual's ontogeny and among mussels. Their average periods were 13.3, 6.4, and 4.0 years. The growth deceleration coefficient a depends on the age of the mussels. We discuss the possibility that the hydrological regime disturbance caused by the Kem hydropower plants' cascade construction could influence the growth rate.

Assessing Evolutionary Significant Units (ESU) of the Endangered Freshwater Pearl Mussel (Margaritifera margaritifera) in Southeast Norway on the Basis of Genetic Analysis.

A total of 312 specimens of freshwater pearl mussel (Margaritifera margaritifera) were sampled from 11 populations, located in four different river systems in Southeast Norway, and analyzed for 11 simple sequence repeat (SSR) (microsatellite) markers. All study populations have landlocked brown trout (Salmo trutta) as the only possible host. Several populations had experienced recruitment failure, probably due to low pH (about 6.0) and calcium concentration. STRUCTURE clustering analysis revealed two genetic clusters, of which one cluster occurred mainly in the western river systems, and totally dominated in one population (Fallselva (A-FAL)) that had higher genetic diversity than the others. Cluster 2 completely dominated in the populations of the eastern river systems, and all of them had low genetic diversity. Bottleneck events were indicated in all populations and the inbreeding coefficient FIS was significant in all populations, except for the southernmost population (Sørkedalselva (B-SØR)), which was the only population with genotypes in Hardy–Weinberg equilibrium. FIS were especially high in the populations of the eastern river systems, and maximum shell length was negatively correlated to FIS. If artificially breeding and stocking should become necessary for future preservation, it should be based on single populations; alternatively, the eastern populations should be based on cross-breeding of populations within the cluster to increase their genetic diversity.

The impact of publication on freshwater pearl mussel conservation: To conceal or to reveal the location of threatened habitats?

Abstract The freshwater pearl mussel, Margaritifera margaritifera, is a threatened species. One of the threats faced by this species is direct extermination by poachers, and therefore some researchers conceal information on the locations of pearl mussel habitats. For example, some researchers do not publish the names of the rivers where pearl mussels occur, whereas other researchers do. Concealing the names of rivers containing pearl mussels makes them unknown not only to potential offenders but also to administrators, nature conservation practitioners, and ordinary citizens. As a result, no protection measures are undertaken, which is all the more regrettable as damage to pearl mussel habitats can occur accidentally. Concealing details of the location of pearl mussel habitats in publications has an adverse impact on the work of specialists, as they do not have access to enough information to study and analyse the biology and distribution of these molluscs. One river with pearl mussels in the Gulf of Finland was initially described anonymously (as ‘River B’). Unfortunately, the river was subject to severe human impact, and the largest aggregation of pearl mussels was almost exterminated in the process of constructing a new electric supply line. Concealing information on pearl mussel habitats in the Russian section of the Baltic Sea basin was also shown to have been a poor decision. Although the threat from potential poachers was insignificant because of the low commercial value of pearl mussels, the authorities remained uninformed about the existence of valuable habitats. The question of whether the locations of pearl mussel habitats should be concealed or revealed in scientific publications does not have an unambiguous answer and should be considered on a case‐to‐case basis; however, the cases for which disclosure is reasonable seem to be more numerous.

Demonstrating the practical impact of studies on biotic interactions and adaptation of a threatened unionoid mussel (Margaritifera margaritifera) to its host fish (Salmo trutta)

Abstract To understand the ecological factors behind the decline of functionally important threatened species with complex life cycles, many different life‐cycle stages need to be investigated. The highly threatened unionoid freshwater mussels, with their complex life cycle, including a parasitic stage on host fish, often have a large influence on biodiversity and ecosystem functioning. The overall aim of the present article is to summarize and discuss the impact of two articles published in Aquatic Conservation: Marine and Freshwater Ecosystems (AQC) on biotic interactions and adaptation of a threatened unionoid mussel (Margaritifera margaritifera) to its host fish (Salmo trutta). The two AQC publications described research on the influence of population size and density of mussels and host fish, and host–parasite interactions between mussels and their host fish, on the recruitment of juvenile mussels. The results from these publications filled gaps in knowledge and resulted in recommendations and incentives for conservation. The results and method development have been used in practical conservation work with threatened mussel species and have been implemented and cited in management handbooks. The outcome of the publications has been implemented in large conservation and restoration projects, and in several recent scientific publications. Specifically, the results from one publication showed that ecological parameters such as mussel and host fish density and population size influenced recruitment of the threatened freshwater pearl mussel. The results from the second publication showed that understanding host–parasite interactions is important for comparing the suitability of host fish strains, and that host fish strains differ in their suitability for mussel infestations. In combination, the articles show that integrating ecological parameters of threatened mussels and their host fish with host–parasite interaction experiments can be an important influence on conservation recommendations, adaptive management and national management programmes for threatened species.

Population genetics of freshwater pearl mussel (Margaritifera margaritifera) in central Massachusetts and implications for conservation

Abstract The freshwater pearl mussel Margaritifera margaritifera is an ecologically important globally endangered species, yet little is known about its biodiversity and population genetics in North American populations. This study focused on M. margaritifera from six locations within two stream systems in central and eastern Massachusetts, USA, to understand the historical impact of damming and habitat fragmentation on local population structure and genetic diversity. Approximately 300 informative single nucleotide polymorphisms were generated from 59 individuals across six geographical locations, using the restriction‐site‐associated DNA sequencing approach. Genotypes were also gleaned from publicly available RNA sequencing data of 23 French M. margaritifera samples. Predictions of population structure using discriminant analysis of principal components and fastStructure both indicate the existence of a single genetic population in central and eastern Massachusetts, and a clear separation from French individuals. This observation is further supported by frequency‐based calculations of population genetics, which indicate near‐complete admixture and a high number of migrants between Massachusetts sites. These results suggest that the effects of habitat fragmentation from damming in the past century have not radiated through the Massachusetts population of M. margaritifera, potentially because of the long lifespan and generation time of the species. However, the Massachusetts population has similar levels of genetic diversity to the endangered populations in Europe, and these factors should be considered by conservationists when creating conservation and management plans. The findings of this study suggest that populations in North America are reasonably healthy and diverse and that, as one population, the study area represents one conservation unit. This indicates that dam removal, habitat restoration, and captive breeding programmes may be viable options to increase the small population numbers and retain a substantial portion of genetic diversity without sacrificing local adaptation.

Identification of a uroporphyrinogen III synthetase gene and characterization of its role in pearl sac formation in Hyriopsis cumingii

Uroporphyrinogen III synthetase (UROS) is an important enzyme involved in the structural formation of porphyrin, which is responsible for color intensity. A UROS homolog was isolated and characterized from the pearl mussel, Hyriopsis cumingii (HcUros). The HcUros cDNA was 858 bp in length with an open reading frame that encoded a 285 amino acid protein, which contained a conserved HemD domain. HcUros exhibited widespread tissue distribution, displaying particular enrichment in the mantle and haemolymph of purple mussels, compared to white mussels. In situ hybridization of HcUros in mantle tissue demonstrated that it was specifically expressed in the dorsal epithelial cells of the mantle pallia. Additionally, HcUros expression was upregulated following implantation of the pearl sac to produce pearls. The data suggest that HcUros contributes to shell pigmentation, and nacre formation and coloring in H. cumingii.

Growth Biorhythms of the European Pearl Mussel Margaritifera margaritifera (Bivalvia, Margaritiferidae) of the Varzuga River Population (Murmansk Oblast)

The individual linear growth of 90 specimens of the freshwater pearl mussel Margaritifera margaritifera was studied by measuring the successive annual rings on the surface of the shell. It was found that the growth of each mollusk can be described using a united equation, a particular case of which is the Bertalanffy equation with an average growth retardation coefficient a = 0.048 year–1 and the coefficient of the initial conditions d = 5.3 mm/year. It was noted that the growth of mollusks is accompanied by three regular biorhythms with periods of 13.4, 6.8, and 4.0 years, with the first two biorhythms fading, the latter having a constant amplitude. It was found that a decrease in the amplitude of fading biorhythms can be described by a power equation with the same coefficients. Questions related to the nature of the biorhythms identified are discussed.

Acute Toxicity of Sodium Chloride, Nitrates, Ortho-Phosphates, Cadmium, Arsenic and Aluminum for Juveniles of the Freshwater Pearl Mussel: Margaritifera Margaritifera (L.1758)

The freshwater pearl mussel (FWPM) Margaritifera margaritifera (L.1758) is critically endangered in Europe and ecotoxicological studies on the species are scares. Here, 96 h acute toxicity tests were conducted at 16 °C with sodium chloride (NaCl), nitrates (NO3−), ortho-phosphates (PO43−), cadmium (Cd), aluminum (Al) and arsenic (As) on 13- to 28-month-old post-parasitic juveniles. The experimental protocol was developed according to conditions described in a standard guide and was modified in order to assess toxicity thresholds for the Dronne River. Results showed that juveniles were tolerant to concentrations of Al, Cd, As, PO43−, NO3− and NaCl, largely higher than those found in the Dronne river, since 96 h EC50s (effective concentrations) were &gt;954 µg/L for Al; &gt;110 µg/L for Cd; &gt;127 µg/L for As; &gt;5.01 mg/L for PO43−; between 1000 and 1500 mg/L for NO3− and were 1.19 and 1.33 g/L for NaCl. Moreover, the use of a substrate in experiments was found not to affect juvenile responses and younger juveniles seemed more sensitive than older individuals. This study thus provides new data about the tolerance of FWPM to environmental pollution and suggests that pollutant concentrations in the river are significantly lower than levels affecting them.

"Artistic Research on Socially and Environmentally Engaged Art - Ethics of Gathering

Art symposiums and similar gatherings in which international artists come together to collaborate are a longstanding tradition of the global art world. In 2019, artists and environmental researchers and experts were invited to work with a local school on environmental issues to create place-specific art and scientific collaborations. Three interdisciplinary teams focused on locally current topics. In this article, we present the process of one of them: the freshwater pearl mussels sub-project. In addition, we discuss the research practice of the International Socially Engaged Art Symposium and the ethics related to the gathering. The research practice included various forms of dialogue, such as presentations, structured group discussions, reflections, mentoring and art-based practice with community members. Tight living together in a humble, small house supported the dialogue. Environmental issues, critical environmentalist thinking and the ethics of the practices were discussed in the process. The ethical elements of the symposium were considered during the processes of defining the themes and aims, curating and producing the event, respecting peer artists and researchers, interacting respectfully with the surrounding community such as school children and community members in countryside villages, and non-human nature, considering the environmental footprint of the symposium and aiming for a meaningful ecological handprint. The documentation, data collection and research reporting also considered ethical issues. The research is part of a cyclic development of art symposiums as socially and environmentally engaged events.

Time travelling through local ecological knowledge regarding an endangered species

European freshwater pearl mussel (Margaritifera margaritifera) populations are declining despite a growing effort to conserve them. Here we used a combination of local ecological knowledge (LEK) and field sampling to assess past and current distribution and conservation status of this endangered species in Cávado and Neiva Rivers (Portugal). We performed face-to-face interviews in both rivers and sampled the entire area where the respondents confirmed the historical presence of this species. Abiotic characterization, water quality and fish diversity were also assessed in both rivers. We found that freshwater pearl mussels are now possibly extinct in Cávado River but almost 50% of the respondents confirm its presence in the past, especially elders that lived in villages near its historical distribution. To the contrary, and although the species is still present in Neiva River, only 3.8% of the respondents remembered its presence in the past. In both rivers, respondents suggested pollution as the most important explanation for the freshwater pearl mussels decline. However, nowadays both rivers present excellent water quality and trout Salmo trutta (the freshwater pearl mussel fish host) is still abundant. Since we identified the areas where the species was present in a recent past, this information is vital for possible management actions with the aim of re-introduce or increase the abundance of M. margaritifera populations and/or for the rehabilitation of habitats in both rivers. We also highlight the vital importance of getting LEK, mainly from elders, in order to avoid shifting baseline syndromes and to get qualitative accurate information of past references and/or experience with historical conditions. Results reported here reinforce concern about the conservation status of freshwater pearl mussel populations in Portugal and can be used to guide future research and management initiatives to better conserve this species.

Occurrence depth of juvenile freshwater pearl mussels (Margaritifera margaritifera) in a river bed tested by experimental mesh tubes

Sediment depth preference was tested on 1-year-old freshwater pearl mussel juveniles. Mesh tubes allowing vertical migration with a possibility to leave hyporheic space, filled with 1–2-mm granulometry size substrate and populated with groups of juveniles, were placed in the natural river bed of the Tepla Vltava River, Czech Republic. The depth position of the juveniles was recorded after 1- and 2-month exposure during the summer and after 8 months including wintering. The juveniles showed a tendency not to stay on the sediment surface, but to penetrate into a very shallow depth, and to accumulate and stay 2–3 cm deep in the substrate in the summer period. No juvenile was found below 8 cm sediment depth, neither in the summer nor after wintering. In the experimental tubes, various survival rates (0–100%) were recorded in close relation to substrate oxygenation development at the tested sites. Oxygen decrease was accompanied by juveniles trying to escape the hyporheal.

Pulsed flow-through cultivation of Margaritifera margaritifera: Effects of water source and food quantity on the survival and growth of juveniles

Conservation of the endangered freshwater pearl mussel (FPM) includes artificially rearing juveniles, but the pulsed flow-through (PFT) method, enabling the continuous renewal of water and food in culture containers, has not been applied to FPM. This study tested the PFT method in culture of FPM juveniles, and the effect of water source (tap vs well water) and food concentration (mixture of commercial phytoplankton products) on the survival and growth of juveniles. Beaker-specific survival rates varied from 0 to 100% (mean: 34%) and from 0 to 58% (mean: 16%) in the 1st (2-week) and 2nd (10-week) experiment, respectively. In the 1st experiment, juveniles attained statistically significantly bigger sizes and more than two times higher survival in well water than they did in tap water. In both experiments, the food concentration of 0.250 µl/l resulted in the highest survival (compared to other concentrations ranging from 0.125 to 0.500 µl/l). However, food concentration did not have a significant effect on size of juvenile FPM in either of the experiments. Results indicate that the PFT system is suitable for FPM rearing experiments, but its applicability to mass culture of FPM juveniles requires further investigation.

Small hydropower plants as a threat to the endangered pearl mussel Margaritifera margaritifera

Freshwater mussels are undergoing rapid global declines due to habitat loss and fragmentation, among other factors, but little is known about the effects of small hydropower plants. Here we assessed the impact of small hydropower plants on the abundance and size structure of the imperilled pearl mussel Margaritifera margaritifera. For this, we sampled 66 sites in three Portuguese rivers (Mente, Rabaçal and Tuela) located upstream and downstream of dams and within the reservoirs. Pearl mussels were significantly more abundant upstream than downstream of dams (97.4% more) or within reservoirs (98.5% more). In addition, juveniles were mostly found upstream of dams. The most significant environmental alterations that explained the observed patterns were related to changes in sediment characteristics (accumulation of fine sediments and organic matter in reservoirs) and water chemistry, most notably suspended solids (highest values in reservoirs) and dissolved oxygen (lowest values in reservoirs). Overall, results show that small hydropower plants can deeply affect pearl mussel populations: specimens almost disappeared from the areas within the reservoirs and sites located downstream only retained adults without signs of recent recruitment. Future management measures devoted to the conservation of pearl mussels should take into account the results reported here to avoid the construction of new dams in pearl mussel rivers; improve management of the river flow in downstream areas; and consider the decommissioning of dams in pearl mussel rivers with a particular attention devoted to the re-naturalization of river sections under the influence of the reservoir and downstream areas.

Conservation status of two endangered freshwater mussel species in Bavaria, Germany: Habitat quality, threats, and implications for conservation management

Abstract The freshwater pearl mussel (Margaritifera margaritifera) and the thick‐shelled river mussel (Unio crassus) are relatively widespread across Europe, but are strongly declining and are now protected by the European Habitats Directive. In the course of this study, 20 pearl mussel and 14 thick‐shelled river mussel streams in Bavaria, Germany, were investigated. The mussel populations were mapped to determine population size and age structure. For the assessment of habitat quality, host fish abundance and physicochemical parameters were investigated, e.g. substratum quality, water chemistry, redox potential, and turbidity. Furthermore, potential risks for the populations such as predation or river maintenance were also recorded and assessed. The average population size and recruitment rates of M. margaritifera populations were lower than in U. crassus populations, with 3517 (2.2% juveniles) compared with 5566 (41.4% juveniles) individuals, respectively. On average, 22.3% of particles were smaller than 0.85 mm in diameter at M. margaritifera sites, whereas the mean proportion of fine particles at U. crassus sites was twice as high, at 41.3%. Other parameters such as redox potential or electric conductivity also indicated more favourable habitat conditions in M. margaritifera streams. Unio crassus seems to be less vulnerable to adverse substratum texture and increased nutrient levels than M. margaritifera. The main threats for U. crassus were physical habitat destruction, predation by muskrat, or a lack of host fish, whereas M. margaritifera mainly suffered from siltation leading to a lack of oxygen supply to the interstitial zone, affecting recruitment. Consequently, conservation strategies need to be species‐specific and address stream‐specific reasons for decline. As a basis, accurate and comparable monitoring data are necessary, which implies the standardization of monitoring protocols.

Freshwater pearl mussels from northern Sweden serve as long-term, high-resolution stream water isotope recorders

Abstract. The stable isotope composition of lacustrine sediments is routinely used to infer Late Holocene changes in precipitation over Scandinavia and, ultimately, atmospheric circulation dynamics in the North Atlantic realm. However, such archives only provide a low temporal resolution (ca. 15 years), precluding the ability to identify changes on inter-annual and quasi-decadal timescales. Here, we present a new, high-resolution reconstruction using shells of freshwater pearl mussels, Margaritifera margaritifera, from three streams in northern Sweden. We present seasonally to annually resolved, calendar-aligned stable oxygen and carbon isotope data from 10 specimens, covering the time interval from 1819 to 1998. The bivalves studied formed their shells near equilibrium with the oxygen isotope signature of ambient water and, thus, reflect hydrological processes in the catchment as well as changes, albeit damped, in the isotope signature of local atmospheric precipitation. The shell oxygen isotopes were significantly correlated with the North Atlantic Oscillation index (up to 56 % explained variability), suggesting that the moisture that winter precipitation formed from originated predominantly in the North Atlantic during NAO+ years but in the Arctic during NAO− years. The isotope signature of winter precipitation was attenuated in the stream water, and this damping effect was eventually recorded by the shells. Shell stable carbon isotope values did not show consistent ontogenetic trends, but rather oscillated around an average that ranged from ca. −12.00 to −13.00 ‰ among the streams studied. Results of this study contribute to an improved understanding of climate dynamics in Scandinavia and the North Atlantic sector and can help to constrain eco-hydrological changes in riverine ecosystems. Moreover, long isotope records of precipitation and streamflow are pivotal to improve our understanding and modeling of hydrological, ecological, biogeochemical and atmospheric processes. Our new approach offers a much higher temporal resolution and superior dating control than data from existing archives.

Sertoli cells in the freshwater pearl mussel Margaritifera laevis (Bivalvia: Margaritiferidae): A histological and ultrastructural study

The developmental changes of Sertoli cells were examined and described in the freshwater pearl mussel Margaritifera laevis using light and transmission electron microscopy. Sertoli cells, which are located on the basal lamina of acini in the testis, include a large number of glycogen granules, electron-dense globules, lipid droplets, and sperm morulae. Electron-dense globules are the vacuoles into which the electron-dense material is condensed. In aging Sertoli cells, the content of the globules leaks out to the extracellular area. Large lipid droplets are formed by the deposition of smaller lipid droplets into a vacuole. After the disruption of the Sertoli cell, the lipid droplets are discharged to the extracellular area and fuse with to form a larger mass. The spermatogonia which were engulfed by the Sertoli cells begin to condense their chromatin and transform themselves into sperm morulae. The constituent cells of the sperm morulae proliferate and finally differentiate into the spermatozoa. After the disruption of the Sertoli cell, the spermatozoa produced from the sperm morulae are released into the acinus lumen. Numerous matured spermatozoa in the acini gather around the large lipid droplet, to form the sperm sphere. The completed sperm spheres are subsequently released through the exhalant siphon into the stream.

Evaluation of Genetic Breeding for the Color of Shell Nacre in the Freshwater Pearl Mussel Hyriopsis cumingii (Lea, 1852) by Raman Spectroscopy

The color of pearl, one of the important quality traits, is determined by the shell nacre color of host mussel; however, the effective method to evaluate the effect of genetic breeding on the purity and uniformity of the nacre color was still limited to the freshwater mussel Hyriopsis cumingii. In this study, the characteristics of the υ1 band of polyene pigments in the colorful shell nacre and pearl samples from different selective generations of H. cumingii were detected by Raman spectroscopy. The results of this study showed that the number of peaks at the υ1 band of polyene pigments decreased gradually from 6–8 in the shell nacre and pearls with the color scheme of purple for wild mussels to 1 in those from the mussels of the F4 generation by genetic breeding. Similar results were also found in the samples with the color scheme of orange. But, no obvious peak of the υ1 band was detected in the samples with white color after four generations of selective breeding. Thus, it could be speculated that the single peak of the υ1 band in the Raman spectroscopy of shell nacre could be used as the sign of color uniformity to reflect the degree of purification in shell nacre color by genetic breeding. Meanwhile, the results showed that the samples with the same color scheme have a similar main peak at the υ1 band (ca. 1,508 cm–1 for the purple color scheme and ca. 1,528 cm–1 for the orange color scheme), suggesting that the polyene pigments in shell nacre and pearl with the same color scheme have the same length of C = C double bond. The length of C = C double bond were 12 and 9 in purple and orange samples, respectively. In summary, the results of this study support the conclusion that Raman spectroscopy is an efficient method to evaluate the genetic breeding for nacre color in H. cumingii.

Effects of suppressing Smads expression on wound healing in Hyriopsis cumingii

As a specific pearl mussel in China, Hyriopsis cumingii has enormous economic value. However, the organism damage caused by pearl insertion is immeasurable. TGF-β/Smad signal transduction pathways are involved in all phases of wound healing. We have previously reported on two cytoplasmic signal transduction factors, Smad3 and Smad5 in mussel H. cumingii (named HcSmads), suggesting their involvements in wound healing. Here, Smad4 was cloned and described. The full length cDNA of HcSmad4 was 2543 bp encoded 515 amino acids. Deduced HcSmad4 protein possessed conserved MH1 and MH2 domains, nuclear location signals (NLS), nuclear exput signals (NES) and Smad activation domain (SAD). Transcripts of Smad3, 4 and 5 were constitutively expressed in all detected tissues, at highest levels in muscles. Furthermore, HcSmad4 mRNA levels were significantly increased at incision site post wounding, and expression of downstream target genes of Smad4, such as HcMMP1, HcMMP19, HcTIMP1 and HcTIMP2 were upregulated to a certain extent. Whatever knocked down HcSmad3/4 or treated by specific inhibitors of Smad 3 (SIS3), expression levels of these genes displayed a significantly downregulated tendency compared with the wound group. In addition, histological evaluation suggested that Smad3 knockdown or SIS3 treatment was accelerated wound healing, and then Smad4 knockdown delayed the process of wound healing in mussels. These data implicate that Smad3/4 play an important role in tissue repair in mollusks.