Pinctada Research Articles

Casting light on formation of the pearl oyster shell with nanobeam X-ray total scattering

Casting light on formation of the pearl oyster shell with nanobeam X-ray total scattering

Immunomodulatory effects of one novel microRNA miR-63 in pearl oyster Pinctada fucata martensii

Novel microRNA miR-63 (novel-miR-63) from pearl oyster Pinctada fucata martensii (Pm-novel-miR-63) is a species-specific miRNA. Our previous research has shown that the expression of Pm-novel-miR-63 was significantly downregulated at 24 h after nucleus transplantation. In this study, we analyzed the function and regulatory role of Pm-novel-miR-63 in the immune response of pearl oysters. The results showed that Pm-novel-miR-63 expression increased after the stimulation of pathogen associated molecular patterns at 6–12 h, and the activity of immune and antioxidant enzymes in the serum decreased after Pm-novel-miR-63 overexpression. Transcriptome analysis revealed that Pm-novel-miR-63 participated in regulating transplantation immunity through the Notch and mRNA surveillance signaling pathways. Target prediction and dual luciferase analysis revealed that Pm-GDP-FucTP, Pm-CysLTR2, and Pm-RLR were the target genes of Pm-novel-miR-63. These results suggested that Pm-novel-miR-63 participated in regulating the immune response in pearl oysters and can serve as a new interference target to reasonably control excessive immune rejection in pearl culture.

Pearl shape classification using deep convolutional neural networks from Tahitian pearl rotation in Pinctada margaritifera

Tahitian pearls, artificially cultivated from the black-lipped pearl oyster Pinctada margaritifera, are renowned for their unique color and large size, making the pearl industry vital for the French Polynesian economy. Understanding the mechanisms of pearl formation is essential for enabling quality and sustainable production. In this paper, we explore the process of pearl formation by studying pearl rotation. Here we show, using a deep convolutional neural network, a direct link between the rotation of the pearl during its formation in the oyster and its final shape. We propose a new method for non-invasive pearl monitoring and a model for predicting the final shape of the pearl from rotation data with 81.9% accuracy. These novel resources provide a fresh perspective to study and enhance our comprehension of the overall mechanism of pearl formation, with potential long-term applications for improving pearl production and quality control in the industry.

Integrated transcriptomic and metabolomic analysis reveals the response of pearl oyster (Pinctada fucata martensii) to long-term hypoxia

The frequency at which organisms are exposed to hypoxic conditions in aquatic environments is increasing due to coastal eutrophication and global warming. To reveal the effects of long-term hypoxic stress on metabolic changes of pearl oyster, commonly known as Pinctada (Pinctada fucata martensii), the present study performed the integrated analysis of transcriptomics and metabolomics to investigate the global changes of genes and metabolites following 25 days hypoxia challenge. Transcriptome analysis detected 1108 differentially expressed genes (DEGs) between the control group and the hypoxia group. The gene ontology (GO) analysis of DEGs revealed that they are significantly enriched in functions such as “microtubule−based process”, “histone (H3–K4, H3–K27, and H4–K20) trimethylation”, “histone H4 acetylation”, “kinesin complex”, and “ATPase activity”, and KEGG pathway functions, such as “DNA replication”, “Apoptosis”, and “MAPK signaling pathways”. Metabolome analysis identified 68 significantly different metabolites from all identified metabolites, and associated with 25 metabolic pathways between the control and hypoxia groups. These pathways included aminoacyl−tRNA biosynthesis, arginine and proline metabolism, and phenylalanine metabolism. Our integrated analysis suggested that pearl oysters were subject to oxidative stress, apoptosis, immune inhibition, and neuronal excitability reduction under long-term hypoxic conditions. We also found a remarkable depression in a variety of biological functions under long-term hypoxia, including metabolic rates, biomineralization activities, and the repression of reorganization of the cytoskeleton and cell metabolism. These findings provide a basis for elucidating the mechanisms used by marine bivalves to cope with long-term hypoxic stress.

The first reporting of prevalence Vibrio species and expression of HSP genes in rayed pearl oyster (Pinctada radiata) under thermal conditions.

The main goal of the present study was to evaluate the influence of thermal exposure on Vibrio population and HSP genes expression (HSP 90, HSP70, and HSP20) in rayed pearl oyster (P. radiata). To this end, the oysters were reared for 30 days at temperatures of 22°C (control), 25°C, 27°C, and 29°C. The results showed that five dominate Vibrio strains including Vibrio hepatarius, V. harveyi, V. alginolyticus, V. parahaemolyticus, and V. rotiferianus were identified. The highest population of V. parahaemolyticus, V. alginolyticus, and V. harveyi, was found in 29οC group. According to real-time PCR, mantle exhibited the highest expression levels of HSP20, HSP70, and HSP90 genes. A higher level of HSP20 expression was observed at high temperatures (25°C, 27°C, and 29°C) in the gonad and mantle compared to the control group (22°C) while decrease in HSP90 expression level was recorded in 25°C, 27°C, and 29°C groups. HSP20 expression level in adductor muscle was remarkably down-regulated in 27°C and 29°C groups. In this tissue, HSP70 was detected at highest levels in the 29οC group. In mantle, HSP90 gene expression was lowest at 22°C water temperature. Several Vibrio strains have been identified from pearl Gulf oyster that haven't been previously reported. The identification of dominant Vibrio species is essential for epidemiological management strategies to control and prevent Vibrio outbreaks in pearl oyster farms. The expression pattern of HSP genes differs in rayed pearl oyster tissues due to differences in their thermal tolerance capability and physiological and biological characteristics. The present study provides useful molecular information for the ecological adaptation of rayed pearl oysters after exposure to different temperature levels.

Purification and Characterization of the Lecithin-Dependent Thermolabile Hemolysin Vhe1 from the Vibrio sp. Strain MA3 Associated with Mass Mortality of Pearl Oyster (Pinctada fucata).

In a previous study, we isolated a Vibrio sp. strain MA3 and its virulence factor, a hemolysin encoded by vhe1. This strain is associated with mass mortalities of the pearl oyster Pinctada fucata. In the present study, the vhe1 gene from strain MA3 was cloned and its encoded product was purified and characterized. Our results show that the vhe1 gene encodes a protein of 417 amino acids with an estimated molecular mass of 47.2kDa and a pI of 5.14. The deduced protein, Vhe1, was found to contain the conserved amino acid sequence (GDSL motif) of the hydrolase/esterase superfamily and five conserved blocks characteristic of SGNH hydrolases. A BLAST homology search indicated that Vhe1 belongs the lecithin-dependent hemolysin/thermolabile hemolysin (LDH/TLH) family. In activity analyses, the optimal temperature for both the hemolytic and phospholipase activities of Vhe1 was 50°C. Vhe1 hemolytic activity and phospholipase activity were highest at pH 8.5 and pH 8.0, respectively. However, both enzymatic activities sharply decreased at high temperature (> 50°C) and pH < 7.0. Compared with previously reported hemolysins, Vhe1 appeared to be more thermal- and pH-labile. Both its hemolytic activity and phospholipase activity were significantly inhibited by CuCl2, CdCl2, ZnCl2, and NiCl2, and slightly inhibited by MnCl2 and CoCl2. Vhe1 showed higher phospholipase activity toward medium-chain fatty acids (C8-C12) than toward shorter- and longer-chain fatty acids. These results accumulate knowledge about the LDH/TLH of V. alginolyticus, which detailed characterization has not been reported, and contribute to solving of the mass mortality of pearl oyster.

Zrakasta bisernica Pinctada radiata (Leach, 1814) (Bivalvia: Pteriidae) u istočnom dijelu Jadrana – novija opažanja

Adriatic Sea as a part of the Mediterranean is settled by many introduced species which extend their range northward. Among them is the rayed pearl oyster Pinctada radiata, a mollusk of Indo-Pacific origin that colonises habitats throughout the Mediterranean. P. radiata established a population in a fish farm on the island of Mljet in the Adriatic Sea, which was observed in 2017. To determine the status of the population, 48 specimens of P. radiata were randomly sampled from the ropes of a fish farm in 2019. Morphometric parameters were determined using a digital calliper. The recorded parameters showed that shell height ranged from 50.55 mm to 72.98 mm, shell length from 46.66 mm to 71.15 mm, and shell width from 19.98 mm to 58.54 mm. During additional visual survey in 2020 the presence of the spat within population at Mljet fish farm was not observed. It appears that only adult specimens are present, and it is unclear if the population is reproducing. In addition, P. radiata spat was observed in the shallow waters of Lokrum Island in 2021 and 2022, indicating the possibility of survival of larvae naturally dispersing from pre-existing populations. An adult specimen of P. radiata was also observed in Mali Ston Bay on two occasions, in 2009 and 2021. P. radiata, a highly adaptable species that tolerates a wide range of environmental factors, has successfully colonised new habitats.

Red Seaweed Porphyra spp. (Bangiales) from Urimessing Waters of Ambon Island - Maluku

A study on the cultivation of Porphyra spp. has been carried out in the waters of Ambon Island. This plant is in great favor with local communities but has not been able to meet broader demands. The purpose of this research was to look into the future of mass culture. In the splashing zone, which is about 10 m above sea level, samples of gametophyte foliose or thalli were taken. The thallus was brought to the laboratory and maintained in a 100-liter container. During the rearing period, the thallus released spermatia and fertilized carpogonia. Carpogonia develop into carposporangia and release zygotospores outside the thallus. A small zygotospore develops into a large carpospore. Carpospores are the forerunners of new sporophytes if they find the correct substrate. In the laboratory, the nacre layer of pearl oyster shells was used to replace the natural substrates. Carpospores attached to the nacre layer germinated and grew into conchocelis. The conchocelis then develops the conchosporangia outward, followed by the release of the conchospores. Conchospores are a source of new thalli grown on polyethylene nylon nets. Conchospores that grew into new thalli on nylon nets were then moved to their natural environments so they could keep growing.

Influence of the chemical content of sawdust on the levels of important macronutrients and ash composition in Pearl oyster mushroom (Pleurotus ostreatus).

Influence of chemical composition of sawdust on the nutritional profile of oyster mushrooms (Pleurotus ostreatus) has yet to receive significant research attention. This information will help mushroom growers to select specific sawdust for the production of mushroom with desired dietary preferences. This study assessed the influence of the chemical composition of sawdust on the macronutrients and ash content of the pearl oyster mushrooms. The American Standard for Testing Materials and other widely accepted protocols were used to determine the C-N ratio, pH, lignin, hemicellulose and cellulose contents of mixed sawdust from tropical wood species. The study evaluated the fat, crude fibre, crude protein, carbohydrate, and ash content of the oyster mushroom cultivated on the sawdust. Cellulose constituted the largest component of the sawdust (47.82%), followed by lignin (33.29%). The yield of the mushroom (on 0.05 kg of sawdust) ranged from 490.1 to 540.9 g (biological efficiency: 44-50%); the average carbohydrates constituent in the mushroom was 56.28%. pH of the sawdust influenced the crude protein, carbohydrate, fat and ash content of oyster mushrooms (p<0.05) most significantly. The hemicelluloses also had a significant effect (p<0.05) on the mushroom's minerals, fat and crude fiber content. The study revealed that the mushroom producers would likely obtain high protein content using sawdust with low pH (slightly acidic to slightly basic) in the oyster mushroom. Mushrooms grown on substrates, rich in hemicelluloses, had low fat and high crude fiber content.

THE EFFECT OF THE INTERVAL TIME IMMERSION IN THE NATURAL FEED TANK OF Chaetoceros simplex ON GROWTH AND SURVIVAL RATE OF PEARL OYSTER (Pinctada Maxima)

Pearl oysters are a fishery commodity with a high economic value because all parts of its body have a sale value. However, many problems of pearl oyster hatcheries are low survival rates and pearl oyster growth is not uniform. The research objective is to determine the effect of the interval time immersion in the natural feed tank in the feed concentrate of Chaetoceros simplex and to determiinterval time best immersion is duced the grey growth and survival rate of (Pinctada maxima). The method in this research is the experimental method and with a completely randomised design (CRD), with five treatments andthree3 replications. The A treatment) : control/without immersion, B (Interval time 24 hours immersion every one day, C (Interval time 48 hours immersion every two days, D (Interval time 72 hours): Immersion every three days and E treatment (Interval time 92 hours): immersion-furry for four days. The research result is interval time immersion spat of pearl oysters in the natural feed tank significantly affects the shell growth of the dorso ventral of pearl oyster Pinctada maxima (p&lt;0,05). The highest growth was achieved in treatment B with an absolute growth value of 1.68 mm, a relative growth of 122,06% from the initial size, and a daily specific growth rate of 0,59% per day.

Short-term in vitro exposure of Pinctada imbricata’s haemocytes to quaternium-15: Exploring physiological and cellular responses

Since the 2000 s, the pearl oyster Pinctada imbricata (Röding, 1798) has become established along the transitional waterways of the “Capo Peloro Lagoon” natural reserve, where it is now abundant due to its adaptability to different hydrological, climatic, environmental, and pollution conditions. This study aims to evaluate haemocyte immune-mediated responses in vitro to quaternium-15, a common pollutant in aquatic ecosystems. Cell viability and phagocytosis activity decreased when exposed to 0.1 or 1 mg L− 1 of quaternium-15. Moreover, decreasing phagocytosis was confirmed by gene expression modulation of actin, involved in cytoskeleton rearrangement. Effects on oxidative stress-related genes were also assessed (Cat, MnSod, Zn/CuSod, GPx). The qPCR data revealed alterations in antioxidant responses through gene dose- and time-dependent modulation. This study presents insights into the physiological responses and cellular mechanisms of P. imbricata haemocytes to environmental stressors, indicating that this species is useful as a novel bioindicator for future toxicological studies.

The Effect of Pearl Oyster (Pinctada maxima L.) Biofouling Waste Organic Fertilizer and Watering Interval on The Growth and Results of Mustard Greens (Brassica juncea L.)

This research was conducted in Pelita Jaya Village, Pohon Batu Hamlet, West Seram Regency from June to July 2022. The research was conducted to study the effect of the interaction of doses of organic fertilizer from pearl oyster biofouling waste and intervals of water administration on the growth and yield of mustard plants. The design used in this study was a factorial randomized group trial design (RAK) with two treatments as follows: Factor I: Pearl Oyster Biofouling Waste Fertilizer (L) with 4 levels, namely L0 = Control, L1 = Dosage of 50 gr/tan, L = 100 g/tan and L3 = 150 gr/tan Factor II : Watering Time Interval (W) namely W1 = 1 day, W2 = 2 days and W3 = 3 days. Each treatment was combined to obtain 12 treatments with 3 replications, resulting in 36 experimental plots. In each plot, 4 sample plants were taken so that in total there were 144 sample plants. The results of this study indicate that the interaction of organic fertilizer from pearl oyster biofouling waste with intervals of watering has a very significant effect on the growth and yield of mustard plants (number of leaves, leaf area, plant fresh weight and plant dry weight). The best treatment interaction was the combined dose of pearl oyster biofouling waste fertilizer 150 g/tan with an interval of watering once every three days for mustard greens (Brassica juncea L.)

Identification of Growth-Related SNPs and Genes in the Genome of the Pearl Oyster (Pinctada fucata) Using GWAS

Pinctada fucata, the pearl oyster, is a bivalve primarily cultivated for the production of saltwater pearls. In this study, the genome-wide association study (GWAS) for the growth-related traits and a principal components analysis (PCA) in P. fucata were performed. Genomic parameters of 6 growth-related traits in 60 individuals were estimated by using 4,937,162 single-nucleotide polymorphisms (SNPs). A total of 45 SNPs associated with growth traits were thus identified. Furthermore, 165 candidate genes were identified, including collagen alpha-3 (VI), serine/threonine-protein kinase mos-like harboring significant markers, and histidine-rich protein PFHRP-III-like, which may influence growth-related traits associated with various biological processes. The results of this study can facilitate marker-assisted selection and breeding programs designed to enhance growth and also offer a theoretical foundation for the further development and utilization of genomic resources in P. fucata.

The Puzzle of the Thinly Coated Pearl

This article takes animal materials as contested elements of ecological knowledge production. The focus is on Ago Bay, a Japanese inlet at the mid-twentieth-century global epicenter of demand for “cultured” pearls that formed inside surgically manipulated shellfish. In 1950s Ago, long-established pearl cultivators complained that their pearls had thinner outer coatings than they expected. Tracing shifting ideas about shellfish stocking densities, smallholder aquaculture, rates of pearl formation, and the accumulation of organic wastes in water over time, this article reconsiders the puzzle of the thinly coated pearl. In its guise as host to thousands of working pearl farms and a network of researchers studying the effects of intensive pearl cultivation, Ago Bay is a rich site from which to think about aquaculture’s ecological and infrastructural limits. The bay was not simply a natural receptacle that housed pearl cultivation. The shore, water, seafloor, and floating pearl oyster raft-and-cage systems could be—and were—defined as infrastructure that could undergo regulation and rearrangement. Pearl cultivation did not just happen in the bay; it was part of the bay—and it reshaped ideas about the bay. This paper is part of a special issue entitled “Making Animal Materials in Time,” edited by Laurence Douny and Lisa Onaga.

FABRICATION OF CHITOSAN BIOPOLYMER FROM PEARL OYSTER SHELLS (Pinctada maxima) FOR MEDICAL APPLICATIONS

Chitosan is one of the biopolymers that has recently been developed in the medical field. Chitosan is biocompatible, biodegradable, and non-toxic, so it is safe for the human body. This study aimed to identify the characteristics of chitosan isolated from pearl oyster shells. Chitosan is obtained through three stages: deproteination, demineralization, and deacetylation. Characterization of chitosan is done physically and chemically including organoleptic test, yield calculation in each step of isolation, FTIR, and XRD. Isolated chitosan identified deacetylation degree and functional group with FTIR. While the crystal structure was determined using XRD. The results showed that chitosan powder has a beige color with a final yield of 7,06%. The characterization of FTIR shows that synthesized pearl oyster shells have successfully formed chitosan compounds with a deacetylation degree of 81,50%. Another characteristic is the crystal structure obtained; chitosan has orthorhombic unit cells with a degree of chitosan crystallinity of 36,94%. Based on the result, chitosan has met several standards in medical applications as a biomaterial.

MiR-10a-3p Participates in Nacre Formation in the Pearl Oyster Pinctada fucata martensii by Targeting NPY.

MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression via the recognition of their target messenger RNAs. MiR-10a-3p plays an important role in the process of ossification. In this study, we obtained the precursor sequence of miR-10a-3p in the pearl oyster Pinctada fucata martensii (Pm-miR-10a-3p) and verified its sequence by miR-RACE technology, and detected its expression level in the mantle tissues of the pearl oyster P. f. martensii. Pm-nAChRsα and Pm-NPY were identified as the potential target genes of Pm-miR-10a-3p. After the over-expression of Pm-miR-10a-3p, the target genes Pm-nAChRsα and Pm-NPY were downregulated, and the nacre microstructure became disordered. The Pm-miR-10a-3p mimic obviously inhibited the luciferase activity of the 3' untranslated region of the Pm-NPY gene. When the interaction site was mutated, the inhibitory effect disappeared. Our results suggested that Pm-miR-10a-3p participates in nacre formation in P. f. martensii by targeting Pm-NPY. This study can expand our understanding of the mechanism of biomineralization in pearl oysters.

Larval dispersal of pearl oysters Pinctada margaritifera in the Gambier Islands (French Polynesia) and exploring options for adult restocking using in situ data and numerical modelling

Black pearl farming is the second source of French Polynesia income after tourism, and Gambier Islands are the main farming sites. Gambier main lagoon contains several sub-lagoons critical for pearl oyster rearing and spat collecting (SC). The Rikitea lagoon, traditionally had good SC rates in the warm season which ensured steady supplies of oysters for black pearl production. However, since 2018, SC has abruptly decreased. To assess the factors affecting SC, Gambier lagoon hydrodynamics was investigated in 2019–2020 to calibrate a hydrodynamic model and simulate larval dispersal around the SC areas. The model shows the strong wind influence on larval dispersal and accumulation patterns and suggests that windy months in the warm season as it can occur during La Niña episodes can explain recent poor SC. Larval dispersal scenarios also informed on best locations to perform adult oyster restocking, a practice that can also enhance SC on the long term.

A comparative study of fatty acid compositions of three cultivated edible mushroom species of Bangladesh

A comparative assessment of three cultivated edible mushroom species namely Bottom mushroom (Agaricus bisporus), Pearl Oyster (Pleurotus ostreatus), Shiitake Mushroom (Lentinul aedodes) on the cumulative fatty acid compositions through fatty acids methyl esters (FAMEs) was devised. About Thirty-six fatty acids were distinguished in the selected mushroom species to interpret descriptive and analytical information by deriving all free fatty acids to their corresponding FAMEs utilizing gas chromatography-mass spectrometry (GC-MS/MS). Cis-linoleic acid was the predominant fatty acid in the fruit bodies of all studied mushrooms. The range of cis-linoleic acid content in the species was 40.33% to 65.59%. Cis-oleic and heptadesenoic acids, respectively, were the other two main fatty acids. More than 32 fatty acids were found and quantified in addition to the three primary fatty acids that are already mentioned. The sequences of Polyunsaturated fatty acids (PUFA), monounsaturated fatty acids (MUFA) and saturated fatty acids (SFA) within all saturated and unsaturated fatty acids for three mushroom species were PUFA > MUFA > SFA for Agaricus bisporus and Lentinula edodes while the sequence was PUFA > SFA > MUFA for Pleurotus ostreatus. Lentinula edodes presented the highest fatty acids amounts which were followed by Pleurotus ostreatus and Agaricus bisporus respectively. The fatty acid composition of the mushrooms revealed that the unsaturated fatty acids are present in higher amounts than the saturated fatty acids in the analyzed mushrooms.

The role of Bcl-2 family genes in the physiology and immune response of the Pinctada fucata martensii

In the process of pearl production, Pinctada fucata martensii has a severe immune response, even leading to death. To effectively improve pearl production, researchers are committed to reducing immunological rejection through molecular biology methods. In previous studies, the apoptosis pathways were found to play an important function in the immune response of P. f. martensii. Bcl-2 family genes, the key genes in the apoptosis pathway, play a key role in regulating apoptotic pathways in cells. In the study, we cloned two genes of the P. f. martensii Bcl-2 family and named them respectively as PmBak/PmBcl-2. Fluorescence quantification of the pearl oysters' tissues showed that PmBak expression was highest in the hepatopancreas while PmBcl-2 expression was highest in the gill; nevertheless, different developmental stages showed that Bcl-2 family genes were highest in the blastula. Immune response assay using LPS, PGN, and poly I: C revealed that two genes were involved in the immune response; however, they showed relatively consistent expression patterns. To gain insight into the mechanism of action of the two genes, it was demonstrated by yeast two-hybrid experiments that PmBak and PmBcl-2 protein can interact with each other, while a subcellular localization assay revealed that Pmbak acts on mitochondria after apoptosis induction. In this study, we demonstrated the involvement of the Bcl-2 family of genes in the P. f. martensii immune response and explored the mechanism of the two genes. The study therefore provides insight into the mechanism of the apoptotic pathway in the immune response process of P. f. martensii.

Comparative analysis of anesthetic agents used in pre-operative therapy for pearl culture in the red abalone Haliotis rufescens (Swainson, 1822)

In pearl culture, the proper selection of anesthetic agents may be a key step to reduce stress and facilitate handling of the organisms, either bivalves (pearl oysters) or gastropods (abalone). This study evaluated the visual (rate of relaxation and recovery, selection of organisms suitable for bead-nucleation) and physiological (glycogen and mucopolysaccharide indices) responses in the red abalone Haliotis rufescens exposed to different types and concentrations of anesthetics. The treatments were: Eugenol at 0.15 mL L−1 (EUA) and 0.25 mL L−1(EUB); Phenoxyethanol at 1.5 mL L−1 (PEA) and 3.0 mL L−1(PEB); Benzocaine at 0.15 g L−1 (BZA) and 0.30 g L−1(BZB); and CO2 to saturation. A different treatment with abalone maintained in fresh seawater without anesthetics was included as the control group. EUB, PEB and CO2 promoted higher proportions of anesthetized abalone (>80%) and abalone suitable for seeding surgery. Mortalities only occurred with the use of Eugenol at both concentrations. With CO2 in particular, 90% of the abalone were suitable for seeding, fully recovered in 168 min, and showed no side effects. The greatest significant decrease in the glycogen index occurred in the abalone treated with EUB, compared to the control group and the rest of treatments. The same trend occurred with the mucopolysaccharide index, where a maximum increase occurred in those abalone anesthetized with EUB, whereas PEB and CO2 showed similar values to the control. Based on these results, the therapy with CO2 is recommended to anesthetize H. rufescens prior to pearl culture; conversely, we suggest avoiding Eugenol as anesthetic due to its various side effects.