Reproduction of an alien Ruditapes clam (Bivalvia: Veneridae) on recreational clam-gathering grounds in Tokyo Bay,Japan

The Manila clam Ruditapes philippinarum is an important fisheries resource and popular recreational native species in Japan. To counter the domestic shortage of Manila clams caused by depleted catches, similar clams were introduced into Japan from China and the Korean peninsula. The introduced clam is an unnamed morph closely related to R. philippinarum that originates from southern China. Its shell is morphologically different from that of the typical Manila clam and there are small but distinct genetic differences. There is evidence to suggest that the Chinese morph may hybridize with the Manila clam in the Ariake Sea, but not in Tokyo Bay. We carried out studies to determine the degree of overlap of the breeding seasons of both taxa by collecting samples of the Manila clam and the alien clam released onto recreational clam-gathering grounds in Tokyo Bay and their gonadal maturity was examined histologically. Spawning-stage individuals of the Manila clam appeared from April to August and those of the southern Chinese morph from July to August, indicating overlapping spawning periods and thus the potential for hybridization. We discussed the forming condition of the hybridized clam population in terms of reproduction and recruitment process of the clams.     

Brown muscle disease: Impact on Manila clam Venerupis (=Ruditapes) philippinarum biology

This study assessed the effect of Brown Muscle Disease (BMD) on Manila clam Venerupis philippinarum fitness. BMD was discovered in 2005. It affects the posterior adductor muscle and leads to clam gaping and eventually death. Three statuses of clams were compared: buried individuals with no signs of BMD (BUR); clams at the surface of the sediment with no signs of BMD (SURF) and clams at the surface of the sediment exhibiting signs of brown muscle disease (BMD). Physiological (condition index), immune (hemocyte parameters) and molecular (gene expressions) parameters collected seasonally were analyzed and compared.Results demonstrated a seasonal pattern in condition index (CI) with peaks in spring/summer and decreases in autumn/winter. At each season, the highest CI was observed in BUR and the lowest CI was observed in BMD.In terms of immune response, phagocytosis rate and capacity were higher in clams with BMD whereas the health status of the clams did not influence the total hemocyte count. Genes involved in the immune system (comp, tnf, inter) were upregulated in clams with BMD. The molecular analysis of gill and posterior muscle showed higher mitochondrial metabolism (cox-1, 16S) in cells of infected clams, suggesting a stronger energetic demand by these cells. Finally, genes involved in oxidative stress response (cat, sod), detoxification (mt) and DNA repair (gadd45) were also overexpressed due to reactive oxygen species production.Most of the studied parameters underlined a cause–effect correlation between Manila clam health status (BUR, SUR, BMD) and physiological parameters. An important stress response was observed in BMD-infected clams at different scales, i.e. condition index, immune parameters and stress-related gene expression.     

Experimental challenges of wild Manila clams with Perkinsus species isolated from naturally infected wild Manila clams

Manila clams, Ruditapes philippinarum, are widely harvested in the coastal waters in Japan. However, there have been significant decreases in the populations of Manila clams since the 1980s. It is thought that infection with the protozoan Perkinsus species has contributed to these decreases. A previous study demonstrated that high infection levels of a pure strain of Perkinsus olseni (ATCC PRA-181) were lethal to hatchery-raised small Manila clams, however, the pathogenicity of wild strain Perkinsus species to wild Manila clam is unclear. To address this, we challenged large (30-40mm in shell length) and small (3-15mm in shell length) wild Manila clams with Perkinsus species isolated from naturally infected wild Manila clams. We report high mortalities among the small clams, but not among the large ones. This is the first report to confirm the pathogenicity of wild isolate of Perkinsus species to wild Manila clams.     

Impact of the infaunal Manila clam, <Emphasis Type="Italic">Ruditapes philippinarum</Emphasis>, on sediment stability

The aim of this study was to examine the impact of bioturbation by the Manila clam, Ruditapes philippinarum, on sediment stability. A laboratory benthic annular flume system (AFS) was deployed to evaluate the relationship between sediment stability of a subtidal mudflat and density of the infaunal clam under the influence of different current velocities. There was a significant correlation between mean erosion rate and current velocities in all treatments with clams (p < 0.001). There was also a significant correlation between mean erosion rate and R. philippinarum density (p < 0.001), reflecting bioturbation-enhanced sediment erosion. The effects of clam density on sediment erodability were more marked at the lower current velocities. In the control, the critical erosion velocity (Ū_crit) was about 32 cm s⁻¹. With increasing R. philippinarum density, Ū_crit decreased down to the minimum value of about 20 cm s⁻¹ at a density of 206 clams m⁻². This study demonstrated that the burrowing activity of R. philippinarum reduces sediment stability, particularly at relatively low current velocities (25 cm s⁻¹) and at densities below those found in the clam cultivation areas within the Sacca di Goro lagoon.     

Quantitative assessment of reproductive effort of the Manila clam Ruditapes philippinarum in a lagoon on Jeju Island (Korea) using enzyme-linked immunosorbent assay

We investigated gonad development and reproductive effort (RE) of the Manila clam Ruditapes philippinarum at Jeju Island, Korea. Gonad maturation and RE were determined using histology and an indirect enzyme-linked immunosorbent assay (ELISA). In June 2006, most of the clams (80%) in the lagoon were in the resting stage. Spawning clams first appeared in late July, and most clams spawned from early August to mid-September. The condition index increased gradually from early July to late August, then declined from early to mid-September, suggesting that spawning occurred during this period. The gonadosomatic index assessed by ELISA also increased dramatically from June (0.9), peaked in early August (19.7) then declined from late August to mid-September, indicating that clams at the study site had only one spawning pulse during the spawning period. Spawning at Jeju Island was one month later than Manila clams on the west coast of Korea. The delayed spawning and low RE of the clams could be in part, be explained by lower food availability, as the level of chlorophyll-a recorded in this study was much lower than that found in water from the west and south coast.     

First report of three protozoan parasites (a haplosporidian, Marteilia sp. and Marteilioides sp.) from the Manila clam, Venerupis (=Ruditapes) philippinarum in Japan

Recently, natural stocks of the Manila clam, Venerupis (=Ruditapes) philippinarum, have been drastically reduced in Japan. To clarify the reason for this decline in number, clams were sampled monthly from Yamaguchi and processed for histological observations, during which three protozoan parasites were discovered. Transmission electron microscopy revealed that these parasites were unidentified haplosporidian in the connective tissues, Marteilia sp. in the digestive gland and Marteilioides sp. in the oocytes. Histopathological observations suggest that Marteilia sp. and Marteilioides sp. were not pathogenic to the host. However, infection with a haplosporidian may have a negative impact on the clams. The prevalence of these parasites was low and further investigations should be undertaken to clarify their taxonomic status and establish any pathogenicity to clams.     

文蛤的生物沉积和呼吸排泄过程及其在双台子河口水层-底栖系统中的耦合作用

为揭示河口埋栖性双壳贝类优势种类在水层——底栖系统中的生态耦合作用,利用生物沉积物捕集器和封闭式代谢瓶,于双台子河口现场研究了文蛤主要生理生态过程如生物沉积速率、耗氧率、排氨率和排磷率的季节变化。结果表明,文蛤的生物沉积速率、耗氧率、排氨率及排磷率均具有明显的季节变化:夏季最高,冬季最低。二龄及三龄文蛤个体的生物沉积速率周年变化分别为0.02—0.30 g-1个-1d-1、0.06—0.60 g-1个-1d-1;耗氧率变化分别为0.45—16.64 mg-1个-1d-1、1.03—30.51 mg-1个-1d-1;排氨率季节变化分别为0.001—0.14 mg-1个-1d-1、0.002—0.28 mg-1个-1d-1;排磷率季节变化分别为0.002—0.069 mg-1个-1d-1、0.003—0.16 mg-1个-1d-1。文蛤的生物沉积速率及呼吸排泄速率均受龄期制约:在同一季节,文蛤的单位个体生物沉积速率及呼吸和排泄速率均表现为二龄三龄。方差分析显示,季节、龄期及两者交互作用对文蛤生物沉积速率、耗氧率、排氨率及排磷率均有显著影响。基于不同季节双台子河口文蛤生物量(0.67个/m2、2.4 g/m2),估算出文蛤种群每年向该河口排放大约5321.90 t生物沉积物(干重)、1.43 t NH+4-N和0.93 t PO3-4-P,并且消耗大约221.59 t O2。研究结果表明,文蛤通过生物沉积及呼吸排泄作用,大大加强了双台子河口沉积物-水界面的物质交换通量,在双台子河口水层-底栖系统耦合作用中扮演着重要生态角色。     

The susceptibility of Irish-grown and Galician-grown Manila clams, Ruditapes philippinarum, to Vibrio tapetis and Brown Ring Disease

Brown Ring Disease (BRD), which affects the Manila clam in Europe, is caused by the bacterium, Vibrio tapetis. BRD has been diagnosed in Ireland on only one occasion (1997) although the aetiological agent has recently been detected in apparently healthy Manila clams from a number of sites around the Irish coast. The present work investigated the susceptibilities to BRD of two stocks of Manila clams, one from Ireland and the second from Galicia, north-western Spain, where BRD has been reported on a number of occasions. Exposure of the clams was by addition of V. tapetis to the holding waters. Development of BRD was assessed by the appearance of brown ring signs on the host shells, by bacterial isolation and characterization, and by detection of the bacterium by PCR. The pathogen was recovered from infected individuals and confirmed as V. tapetis by biochemical tests and a slide agglutination test. Galician clams experienced significantly higher mortalities, BRD prevalences and V. tapetis levels than Irish clams. Background infection with V. tapetis in the control stocks prevented conclusions being drawn on comparative susceptibility of the two stocks. Irish clams were significantly affected by the experimental challenge, as demonstrated by the development of BRD and an increase in V. tapetis levels. Results illustrate the vulnerability of Irish clams to BRD and have implications for the movement and transfer of clam seed in Ireland.     

亚热带养殖海湾皱瘤海鞘生物沉积的现场研究

皱瘤海鞘是亚热带海域分布最广和数量最多的附着生物种类之一.2012年1月-7月在典型的亚热带养殖海湾-大亚湾大鹏澳海域,利用沉积物捕集器现场测定了皱瘤海鞘(Styela plicata)的生物沉积速率,并测定了生物沉积物中有机物(OM)、总碳(TC)、总氮(TN)、有机碳(OC)和有机氮(ON)含量.结果显示:皱瘤海鞘的生物沉积速率变化范围为每天145.5-1011.8m/个,平均每天516.0mg/个,海鞘的生物沉积速率变化范围为每天154.8-1065.8 mg/g干重,平均每大463.3 mg/g干重.海鞘生物沉积物中OM、TC、OC、TN和ON含量分别为14.38％、10.80％、2.87％、3.06％和0.86％,高于自然沉积物中的含量,分别为13.39％、7.36％、2.32％、2.29％和0.67％.其中TC和ON含量要显著高于自然沉积物(P＜0.05).皱瘤海鞘的OM、TC、OC、TN和ON的生物沉积速率分别为每天74.20,55.73,14.80,15.79和4.43 mg/个.实验期间附着在浮筏养殖设施和养殖牡蛎壳上的海鞘密度变化范围为54.9-222.1个/m2,平均147.5个/m2,养殖海域单位面积的海鞘生物沉积速率平均为每天76.1 g/m2,是自然沉积速率(平均每天62.7 g/m2)的1.21倍,其中OM、TC、OC、TN和ON的平均沉积速率分别为每天10.94,8.21,2.18,2.32和0.65 g/m2.据此可推算,大鹏澳筏式牡蛎养殖海区(约103 hm2)皱瘤海鞘的年生物沉积物负荷为29000 t,其中OM,TC,OC,TN和ON分别4100,3100,820,870和240 t.研究结果说明,海鞘等附着生物在大规模浅海贝类养殖中对养殖生态环境的影响也不容忽视.     

The effects of water depth and density on the growth of a unionid clam*

SUMMARY. 1. Unionid clams from Narrow Lake, Alberta, were collected to quantify the natural variation in growth, to assess the natural variation in abundance, age and size distribution, and growth with water depth in the lake, and to conduct in situ experiments to directly test the effects of water depth (temperature) and clam abundance on clam growth. 2. The unionid clam, Anodonta grandis simpsoniana, showed wide variation in length at a given age. There were no significant differences in growth between clams collected at 1,3, 5, and 7m depths in the lake despite marked differences in water temperature. The wide variation in clam biomass within each depth zone may have masked possible effects of water depth. 3. The effect of water depth and variation in clam density on clam growth was tested directly by stocking clams into small enclosures at densities equivalent to 50, 150, 250, 350 and 450g m-2 (live weight) at each of 1, 3, 5 and 7 m depths in Narrow Lake (each depth and abundance treatment in triplicate). A uniform sandy substrate was used in all enclosures to eliminate any possible effect of substrate type on growth. 4. Mortality was negligible (0.9%) during the experiment. Clam density had no significant effect on clam growth which suggests that clam growth was not food limited in the lake. 5. Clams reared at 7 m grew more slowly than clams reared at 1, 3 and 5 m. Clams reared at 5 m grew more slowly than clams reared at 1 and 3m. Growth of clams reared at 1 and 3m did not differ. These differences in growth were strongly correlated with the measured differences in water temperature between depths. 6. Migration between depths probably accounts for the lack of a depth effect on clams growing in the natural habitat.     

DGGE-based detection method for Quahog Parasite Unknown (QPX)

Quahog Parasite Unknown (QPX) is a significant cause of hard clam Mercenaria mercenaria mortality along the northeast coast of the United States. It infects both wild and cultured clams, often annually in plots that are heavily farmed. Subclinically infected clams can be identified by histological examination of the mantle tissue, but there is currently no method available to monitor the presence of QPX in the environment. Here, we report on a polymerase chain reaction (PCR)-based method that will facilitate the detection of QPX in natural samples and seed clams. With our method, between 10 and 100 QPX cells can be detected in 1 l of water, 1 g of sediment and 100 mg of clam tissue. Denaturing gradient gel electrophoresis (DGGE) is used to establish whether the PCR products are the same as those in the control QPX culture. We used the method to screen 100 seed clams of 15 mm, and found that 10 to 12% of the clams were positive for the presence of the QPX organism. This method represents a reliable and sensitive procedure for screening both environmental samples and potentially contaminated small clams.     

Bioavailability of sediment-bound Cd, Cr and Zn to the green mussel Perna viridis and the Manila clam Ruditapes philippinarum

We assessed the degree to which Cd, Cr and Zn bound with sediment were assimilated by the green mussel Perna viridis and the Manila clam Ruditapes philippinarum. The influences of the metal concentration in the sediment, the presence of phytoplankton, and the oxidation condition of the sediment on metal assimilation were examined. No major difference was found for metal assimilation efficiency (AE) in sediment with different metal concentrations, except for Cd in the green mussels, in which the AE increased by 1.7x when the Cd concentration in sediment was elevated to 15x the natural background level. The higher assimilation of Cd with increasing Cd load in ingested sediment may be due to the higher desorption of Cd in the acidic gut of the bivalves. Both mussels and clams assimilated metals at a higher efficiency from the diatom diet (Thalassiosira pseudonana) than from inorganic sediment particles. The presence of algal particles had little influence on metal assimilation from ingested sediment, and conversely, the presence of sedimentary particles had little effect on metal assimilation from ingested diatom (except for Cd in the mussels). In the mussels, AEs were higher from oxic sediment than from anoxic sediment by 3.1x for Cd, 2.0x for Cr, and 1.4x for Zn, and in the clams AEs were higher from oxic sediment by 2.8x for Cd, 2.0x for Cr, and 2.0x for Zn. Our study suggested that metals associated with anoxic sediment can be potentially available to marine bivalves, and that metal AEs determined for a single diet were probably not affected by the presence of other food particles.     

Seasonal Variation of Metals in Seawater, Sediment, and Manila Clam Ruditapes philippinarum from China

Concentrations of trace metals (Cu, Pb, Zn, Cd, Cr, Hg, and As) were determined for the first time in seawater, sediment, and Manila clam from Deer Island, Liaoning Province, China. The seawater, sediment, and clam samples were collected seasonally at three clam farming sites around Deer Island during 2010–2011. The average concentrations of Cu, Pb, Zn, Cd, Cr, Hg, and As in the seawater samples were 4.16, 0.72, 5.88, 0.45, 2.51, 0.03, and 1.02 μg/l, respectively. The seasonal variations of trace metals in seawater showed a significant difference in the concentrations of Cu, Pb, Zn, Hg, and As among seasons. The average concentrations of Cu, Pb, Zn, Cd, Cr, Hg, and As in the sediment samples were 6.43, 13.80, 53.08, 1.10, 36.40, 0.05, and 4.78 mg/kg dry weight, respectively. Trace metal concentrations in sediment seasonally varied significantly except for Cd and Hg. The average concentrations of Cu, Pb, Zn, Cd, Cr, Hg, and As in the clam samples were 11.28, 0.61, 92.50, 0.58, 3.98, 0.03, and 1.98 mg/kg dry weight, respectively. Concentrations of Cu, Zn, Cd, Cr, and As in Manila clam showed marked seasonal fluctuations with significant difference. Cu and Zn were the metals with the highest mean biosediment accumulation factor values in Manila clam. Besides, significant correlations for the concentrations of Cu and Zn relative to their concentrations in sediment were also found. Such differences in regression analyzes may be explained by differential bioaccumulation of essential and xenobiotic metals. Concentrations of trace metals in Manila clam did not exceed the maximum established regulatory concentrations for human consumption. Moreover, the calculations revealed that the estimated daily intake values for the examined clam samples were below the internationally accepted dietary guidelines and the calculated hazard quotient values were well less than 1, thus strongly indicating that health risk associated with the intake studied metals through the consumption of Manila clam from Deer Island was absent.     

Isolation and identification of Perkinsus olseni from feces and marine sediment using immunological and molecular techniques

Molecular and immunological probes were used to identify various life stages of Perkinsus olseni, a protozoan parasite of the Manila clam Ruditapes philippinarum, from a marine environment and decomposing clam tissue. Western blotting revealed that the antigenic determinants of the rabbit anti-P. olseni antibody developed in this study were peptides with molecular masses of 55.9, 24.0, and 19.2 kDa. Immunofluorescent assay indicated that the rabbit anti-P. olseni IgG was specific to all life stages, including the prezoosporangium, trophozoite, and zoospore. Perkinsus olseni prezoosporangium-like cells were successfully isolated from marine sediment collected from Hwangdo on the west coast of Korea, where P. olseni-associated clam mortality has recurred for the past decade. Purified cells were positively stained with the rabbit anti-P. olseni antibody in an immunofluorescence assay, confirming for the first time the presence of P. olseni in marine sediment. Actively replicating zoospores inside the prezoosporangia were observed in the decomposing clam tissue collected from Hwangdo. P. olseni was also isolated from the feces and pseudofeces of infected clams and confirmed by PCR. The clams released 1-2 prezoosporangia per day through feces. The data suggested that the fecal discharge and decomposition of the infected clam tissue could be the two major P. olseni transmission routes.     

Experimental evaluation of the pathogenicity of Perkinsusolseni in juvenile Manila clams Ruditapes philippinarum

We evaluated the pathogenicity of Perkinsus olseni towards the Manila clam, Ruditapes philippinarum, by an experimental challenge. For production of prezoosporangia of P. olseni, we injected uninfected Manila clams with cells of a pure strain of P. olseni and reared them for 7 d. Prezoosporangia were isolated from the soft tissue of the injected clams after culturing in Ray’s fluid thioglycollate medium. Hatchery-reared, uninfected juvenile clams (3-10 mm shell length) were challenged by immersion in one of two concentrations of a prezoosporangial suspension of P. olseni for 6 d. The challenged clams had significantly higher mortality at both the concentrations than the unchallenged clams. The mortality due to infection dose-dependently began approximately 4 weeks and 7 weeks after challenge in the higher and lower concentrations, respectively. This is the first experimental evidence that P. olseni causes direct mortality in Manila clams. The lethal level of infection was estimated at approximately 10⁷ pathogen cells/g soft tissue weight.     

Isolation of the pathogen Vibrio tapetis and defense parameters in brown ring diseased Manila clams Ruditapes philippinarum cultivated in England

The Manila clam Ruditapes philippinarum was introduced for aquacultural purposes to Europe in the 1970s. In 1987, brown ring disease (BRD), caused by Vibrio tapetis, appeared in clams cultivated in Brou?nou (Finistère, France) and later became increasingly widespread and was reported in cultivated and wild clams existing on the Atlantic coasts of France and Spain. The present study reports, for the first time, the presence of BRD in clams cultivated in England. The etiologic bacterium was isolated and identified using bacteriological and serological techniques. The defence response of affected clams was also studied and significant changes in the hematological and biochemical characteristics of hemolymph and extrapallial fluids were demonstrated. Significant mobilization of hemocytes toward the extrapallial fluids, in contact with the main site of infection (mantle-periostracal lamina area), was observed, suggesting a role for these pseudo-internal compartments in the preservation of clam health.     

Gymnophallid digenean Parvatrema duboisi uses Manila clam as the first and second intermediate host

To identify the metacercariae of a gymnophallid trematode in the Manila clam Ruditapes philippinarum from the Ariake Sea, experimental infection and molecular analysis were conducted. Based on the morphology of adult worms obtained from experimentally infected mice, the parasite was identified as Parvatrema duboisi. Comparison of internal transcribed spacer (ITS) region of ribosomal DNA (rDNA) sequences for metacercariae and sporocysts from Manila clams and adult worms collected from wild wigeon Anas penelope showed minor differences ranging from 0 to 0.8%. These data strongly suggest that in the Ariake Sea, the parasite has a lifecycle using the Manila clam as the first and second intermediate hosts and wigeon as the definitive host.     

Impact of Brown Ring Disease on the energy budget of the Manila clam Ruditapes philippinarum

Brown Ring Disease (BRD) is a bacterial disease caused by the pathogen, Vibrio tapetis. The disease induces formation of a brown deposit on inner shell of the Manila clam, Ruditapes philippinarum. Development of this disease is correlated with a decrease in the condition index of infected clams. Experiments were conduced in order to assess the effect of the development of BRD on two parameters affecting the energy balance of the clams: the clearance and the respiration rates. Experiments were performed in a physiological measurement system that allowed simultaneous measures of clearance and respiration rates. During both acclimation and measurements clams were fed with cultured T-iso and temperature was close to seasonal field temperature (10°C). Our results showed that severely diseased clams (conchiolin deposit stage, CDS ≥ 4) are subject to weight loss in comparison to uninfected ones, indicating that BRD induces a disequilibrium in the energy balance. We demonstrated a reduction of the clearance rate of severely diseased clams which led to a decrease in energy acquisition. Respiration rate showed a significant decrease with BRD symptoms, but evidence in the literature allowed us to hypothesize that energy mobilised for an immune response and lesion repair increases overall organism maintenance costs. Both factors should thus contribute to the degradation of the energy balance of diseased clams. Because effects of BRD on naturally infected clams only appears significant for CDS ≥ 4, when brown ring assumes a significant place on the inner shell, we consider that the Manila clam is tolerant of low disease levels.     

Perkinsosis in the Manila clam Ruditapes philippinarum affects responses to the harmful-alga, Prorocentrum minimum

The dinoflagellate Prorocentrum minimum is increasingly recognized as a harmful algal bloom (HAB) species that affects filter-feeding shellfish. An experiment was done to investigate possible interactions between parasitic diseases and exposure to P. minimum in Manila clams, Ruditapes philippinarum. Manila clams, with variable levels of infection with Perkinsus olseni, were exposed for three or six days to the benign phytoplankton species Chaetoceros neogracile or a mixed diet of C. neogracile and P. minimum. After three or six days of exposure, clams were assessed individually for condition index, parasite status, and plasma and hemocyte parameters (morphological and functional) using flow-cytometry. Histological evaluation was also performed on individual clams to assess prevalence and intensity of parasitic infection, as well as other pathological conditions.Prorocentrum minimum caused several changes in Manila clams, especially after six days of exposure, such as decreased hemocyte phagocytosis and size and clam condition index. Pathological conditions observed in Manila clams exposed to P. minimum were hemocyte infiltration in the intestine and gonad follicles, myopathy, and necrosis of the intestine epithelial cells. The parasite P. olseni alone had no significant effect on Manila clams, nor did it modulate the hemocyte variables in clams exposed to P. minimum; however, the parasite did affect the pathological status of Manila clams exposed to the P. minimum culture, by causing atrophy and degeneration of residual ova in the gonadal follicles and hyaline degeneration of the muscle fibers, indicating synergistic effects of both stressors on the host over a short period of time. Additionally, an in vitro experiment also demonstrated detrimental effects of P. minimum and exudates upon P. olseni cells, thus suggesting HAB antagonistic suppression of transmission and proliferation of the parasite in the natural environment over a longer period of time. The results of this experiment demonstrate the complexity of interactions between host, parasite, and HAB.