Bivalve farming plays a dominant role in mariculture in China.Paralytic shellfish toxins(PSTs)can be accumulated in bivalves and cause poisoning the consumers.A sensitive detection of PSTs can provide early warning to...Bivalve farming plays a dominant role in mariculture in China.Paralytic shellfish toxins(PSTs)can be accumulated in bivalves and cause poisoning the consumers.A sensitive detection of PSTs can provide early warning to decrease poisoning events in bivalve consuming.PSTs are traditionally examined using the whole soft-tissues.However,PSTs accumulation varies dramatically in different tissues of bivalves.Some tough tissues/organs(such as mantle),which account for a large proportion of the total soft body,exhibit a lower accumulation of PSTs and make the toxin extraction time-and reagent-consuming,potentially decreasing the accuracy and sensitivity of PSTs monitoring in bivalves.To develop a sensitive and cost-effective approach for PSTs examination in massively farmed bivalves,we fed three commercially important bivalves,Yesso scallop Patinopecten yessoensis,Pacific oyster Crassostrea gigas,and blue mussel Mytilus edulis with PSTs-producing dinoflagellate Alexandrium catenella,and detected PSTs concentration in different tissues.For all three bivalve species,the digestive gland accumulated much more PSTs than other tissues,and the digestive gland’s toxicity was significantly correlated with the PSTs toxicity of the whole soft-tissues,with r^(2)=0.94,0.92,and 0.94 for Yesso scallop,Pacific oyster,and blue mussel,respectively.When the toxicity of the whole soft-tissues reached 80μgSTXeq(100g)^(−1),the regulatory limit for commercial shellfish,the digestive gland’s toxicity reached 571.48,498.90,and 859.20μgSTXeq(100g)^(−1) in Yesso scallop,Pacific oyster,and blue mussel,respectively.Our results indicate that digestive gland can be used for the sensitive and cost-effective monitoring of PSTs in bivalves.展开更多
Objective To study the transfer of paralytic shellfish toxins （PST） using four simulated marine food chains： dinoflagellate Alexandrium tamarense→Artemia Artemia salina→Mysid shrimp Neomysis awatschensis; A. tama...Objective To study the transfer of paralytic shellfish toxins （PST） using four simulated marine food chains： dinoflagellate Alexandrium tamarense→Artemia Artemia salina→Mysid shrimp Neomysis awatschensis; A. tamarense→N. awatschensis; A. tamarense→A, salina→Perch Lateolabrax japonicus; and A. tamarense→L, japonicus. Methods The ingestion of A. tamarense, a producer of PST, by L. japonicus, N. awatschensis, and A. salina was first confirmed by microscopic observation of A. tamarense cells in the intestine samples of the three different organisms, and by the analysis of Chl.a levels in the samples. Toxin accumulation in L. japonicus and N. awatschensis directly from the feeding on A. tamarense or indirectly through the vector of A. salina was then studied, The toxicity of samples was measured using the AOAC mouse bioassay method, and the toxin content and profile of A. tamarense were analyzed by the HPLC method. Results Both A. salina and N. awatschensis could ingest A. tamarense cells. However, the ingestion capability of A. salina exceeded that of N. awatschensis. After the exposure to the culture of A. tamarense （2 000 cells·mL^-1） for 70 minutes, the content of Chl.a in A. salina and N. awatschensis reached 0.87 and 0.024 μg.mg^-1, respectively. Besides, A. tamarense cells existed in the intestines of L. japonicus, N. awatschensis and A. salina by microscopic observation. Therefore, the three organisms could ingest A. tamarense cells directly. A. salina could accumulate high content of PST, and the toxicity of A. salina in samples collected on days 1, 4, and 5 of the experiment was 2.18, 2.6, and 2.1 MU.g^-1, respectively. All extracts from the samples could lead to death of tested mice within 7 minutes, and the toxin content in anemia sample collected on the 1st day was estimated to be 1.65×10 ^5 μg STX equal/individual. Toxin accumulation in L japonicus and N. awatschensis directly from the feeding on A. tamarense or indirectly from the vector ofA. salina was also studied. The mice injected with extracts from L japonicus and N. awatschensis samples that accumulated PST either directly or indirectly showed PST intoxication symptoms, indicating that low levels of PST existed in these samples. Conclusion Paralytic shellfish toxins can be transferred to L. japonicus, N. awatschensis, and A. salina from A. tamarense directly or indirectly via the food chains.展开更多
Paralytic shellfish toxins(PSTs) are notorious neurotoxins that threaten public health and food safety worldwide.Although PST monitoring programs have recently been established throughout China, the profiles and varia...Paralytic shellfish toxins(PSTs) are notorious neurotoxins that threaten public health and food safety worldwide.Although PST monitoring programs have recently been established throughout China, the profiles and variation of PSTs in important commercial clams(e.g., Mactra veneriformis, Ruditapes philippinarum, and Meretrix meretrix) along the Jiangsu Province coastline remain largely unexplored. In this study, a validated hydrophilic interaction liquid chromatography–tandem mass spectrometry(HILIC-MS/MS) method was used to examine PST profiles and levels in 540 clam samples from natural production areas along Jiangsu Province coastline during2014–2016. Although the PST levels(≤6.38 μg saxitotoxin equivalents(eq)/kg) were consistently below European Union regulatory limits(≤800 μg saxitotoxin eq/kg) during this time period, saxitotoxin, decarbamoylsaxitotoxin,and gonyautoxins 1 and 4 were detected, and nearly 40% of the samples were saxitotoxin-positive. The PST levels also varied significantly by seasons, with peak values observed in May during 2014–2016. This is the first systematic report of PSTs in clams from Jiangsu Province, and additional research and protective measures are needed to ensure the safety of clams harvested in this area.展开更多
The current testing for paralytic shellfish poisoning(PSP) in shellfish is based on the mouse bioassay(MBA).To alleviate animal welfare concerns,we evaluated the utility of using sublethal indicators of toxicity as an...The current testing for paralytic shellfish poisoning(PSP) in shellfish is based on the mouse bioassay(MBA).To alleviate animal welfare concerns,we evaluated the utility of using sublethal indicators of toxicity as an alternative to measuring time to death.Live mice were injected with a PSP congener and the changes in neurotransmitter levels were measured 60,90,and 120 min after injection.Acetylcholine(ACh) was the most sensitive marker for PSP toxicity.The changes in neurotransmitter levels were most pronounced in the blood.Thus,measurement of Ach levels in the blood may serve as a sensitive predictor for PSP that would not require sacrifice of the mice.This method was relatively simple,sensitive(less than 1 μg/kg weight,equivalent to 20 ng/mL),low maintenance,and rapid(less than 60 min).展开更多
Dissected tissues of three shellfish species, the Chinese scallop, Chlamys farreri, Manila clam, Ruditapes philippinarurn, and Razor shell, Solen strictu were evaluated for in vitro transformation of paralytic shellfi...Dissected tissues of three shellfish species, the Chinese scallop, Chlamys farreri, Manila clam, Ruditapes philippinarurn, and Razor shell, Solen strictu were evaluated for in vitro transformation of paralytic shellfish poisoning （PSP） toxins. Tissue homogenates were incubated with extraction from toxic algae Alexandriurn rninutura to determine toxin conversion. The effects of heating and addition of a natural reductant （glutathione） on toxin conversion were also assessed. The toxin profile was investigated through high performance liquid chromatography with fluorescence detection （HPLC-FLD）. The evident variations in the toxin content were observed only in Chinese scallop viscera homogenates. The concentration of GTX4 was reduced by 45% （approximately 0.8 μmol/dm^3） and 25% （approximately 1 μmol/dm^3） for GTX1, while GTX2 and GTX3 increased by six times （approximately 1 μmol/dm^3） and 3 times （approximately 0.3μmol/dm^3） respectively. Simultaneously, the total toxicity decreased by 38% during the 48 h incubation period, the final toxicity was 20.4 nmol STXeq/g. Furthermore, heated Chinese scallop viscera homogenates samples were compared with non-heated samples. The concentration of the GTX4 and GTX1 was clearly 28% （approximately 0.53 μmol/dm^3） and 17% （approximately 0.69μmol/dm^3） higher in heated samples, GTX2 and GTX3 were four times （0.66 μmol/dm^3） and two times （0.187 μmol/dm^3） lower respectively. GSH （＋） Chinese scallop viscera homogenates samples were compared with GSH （-） samples, the concentration in the GTX4 and GTX1 was 9% （approximately 0.12 μmol/dm^3） and 11% （approximately 0.36 μmol/dm^3） lower respectively, GTX2 and GTX3 was 17% （approximately 0.14 μmol/dm^3） and 19% （approximately 0.006 μmol/dm^3） higher respectively. In contrast,there was a little change in the concentration of PSP toxins of Manila clam and Razor shell tissue ho- mogenates. These observations on three shellfish tissues confirmed that there were species-specific differences in PSP toxins transformation. PSP toxins transformation was more pronounced in viscera tissue than in muscle tissue. PSP toxins was possibly interfered by some carbamoylase enzyme, and the activity in Chinese scallop viscera tissue is more remarkable than in the other two species.展开更多
An inter-laboratory comparison of the AOAC mouse bioassay for paralytic shellfish poisoning (PSP) toxicity in shellfish was carried out among 25 Chinese laboratories to examine the overall performance for PSP testing ...An inter-laboratory comparison of the AOAC mouse bioassay for paralytic shellfish poisoning (PSP) toxicity in shellfish was carried out among 25 Chinese laboratories to examine the overall performance for PSP testing in China, and to analyze the main factors affecting the performance of this method. The toxic scallop Patinopecten yessoensis collected from coast of Bohai Sea, China, was used as a test sample in the comparison study. The results were reported and evaluated using robust statistical methods. The z scores showed that 80%, 8%, and 12% of laboratories reported satisfactory results, unsatisfactory results, and questionable results, respectively. This evaluation demonstrates that the PSP mouse bioassay is an appropriate method for screening and testing PSP toxicity in shellfish. However, it was found that the experience and skill of technicians, as well as the body weight and health status of mice being used significantly affected the accuracy of the method.展开更多
To study the paralytic shellfish toxins（PSTs） depuration in Japanese scallop Patinopecten yessoensis in natural environment, Japanese scallops naturally contaminated with paralytic shellfish poisoning（PSP） toxins ...To study the paralytic shellfish toxins（PSTs） depuration in Japanese scallop Patinopecten yessoensis in natural environment, Japanese scallops naturally contaminated with paralytic shellfish poisoning（PSP） toxins in the Dayao Bay in the northern Huanghai Sea are transited to Qipanmo waters in the Bohai Sea of no reported PSTs incidents. The levels and profile of PSTs during 30-day depuration are detected by the high performance liquid chromatography with fluorescence detection（HPLC-FLD）. The results show that the toxicity of the PSTs in soft tissues decreases to a relatively low level at Day 9. Moreover, the depurated ratio at the early stage of the PSTs depuration is higher than that at the later stage. The toxicity analysis of dissected organs reveals that the digestive gland is the most contaminated PSTs part, which is of important implication for the human health and scallop aquiculture. The mortality of Japanese scallops during PSTs depuration experiment is relevant to PSTs level in the soft tissue.展开更多
Alexandrium pacificum(A.pacificum)is a typical paralytic shellfish poisonous dinoflagellate.Harmful algal blooms(HABs)caused by this species can bring serious environmental problems and economic losses to the aquacult...Alexandrium pacificum(A.pacificum)is a typical paralytic shellfish poisonous dinoflagellate.Harmful algal blooms(HABs)caused by this species can bring serious environmental problems and economic losses to the aquaculture industry.In this study,transcriptome sequencing and analyses were performed on the neural tissue of Litopenaeus vannamei(L.vannamei)after acute exposure to A.pacificum disrupted solution for 72 h,and differentially expressed genes(DEGs)were identified.The results showed that,compared with the control samples,300 DEGs were identified in the experimental group,of which 194 were up-regulated,and 106 down-regulated.The gene ontology(GO)functional enrichment analysis showed that DEGs were significantly enriched in the cortical cytoskeleton organization,troponin complex,amylo-alpha-1,6-glucosidase and thymidine phosphorylase.Kyoto encyclopedia of genes and genomes(KEGG)enrichment analysis found that DEGs were mainly enriched in the oxidative phosphorylation process,intercellular tight junctions and mitophagy.The results showed that the proteoglycans,signaling pathways,and various metabolic processes that regulate cell proliferation,differentiation,and apoptosis all played an essential role in the response of L.vannamei to A.pacificum toxins.展开更多
基金funded by the National Key R&D Project(No.2019YFC1605704)the Taishan Industry Leading Talent Project(No.LJNY201816)supported by Sanya Yazhou Bay Science and Technology City(No.SKJCKJ-2019KY01).
文摘Bivalve farming plays a dominant role in mariculture in China.Paralytic shellfish toxins(PSTs)can be accumulated in bivalves and cause poisoning the consumers.A sensitive detection of PSTs can provide early warning to decrease poisoning events in bivalve consuming.PSTs are traditionally examined using the whole soft-tissues.However,PSTs accumulation varies dramatically in different tissues of bivalves.Some tough tissues/organs(such as mantle),which account for a large proportion of the total soft body,exhibit a lower accumulation of PSTs and make the toxin extraction time-and reagent-consuming,potentially decreasing the accuracy and sensitivity of PSTs monitoring in bivalves.To develop a sensitive and cost-effective approach for PSTs examination in massively farmed bivalves,we fed three commercially important bivalves,Yesso scallop Patinopecten yessoensis,Pacific oyster Crassostrea gigas,and blue mussel Mytilus edulis with PSTs-producing dinoflagellate Alexandrium catenella,and detected PSTs concentration in different tissues.For all three bivalve species,the digestive gland accumulated much more PSTs than other tissues,and the digestive gland’s toxicity was significantly correlated with the PSTs toxicity of the whole soft-tissues,with r^(2)=0.94,0.92,and 0.94 for Yesso scallop,Pacific oyster,and blue mussel,respectively.When the toxicity of the whole soft-tissues reached 80μgSTXeq(100g)^(−1),the regulatory limit for commercial shellfish,the digestive gland’s toxicity reached 571.48,498.90,and 859.20μgSTXeq(100g)^(−1) in Yesso scallop,Pacific oyster,and blue mussel,respectively.Our results indicate that digestive gland can be used for the sensitive and cost-effective monitoring of PSTs in bivalves.
基金The work was supported by National Basic Research Project No. 2001 CB409700, NNSFC KZCX2-YW-208.
文摘Objective To study the transfer of paralytic shellfish toxins （PST） using four simulated marine food chains： dinoflagellate Alexandrium tamarense→Artemia Artemia salina→Mysid shrimp Neomysis awatschensis; A. tamarense→N. awatschensis; A. tamarense→A, salina→Perch Lateolabrax japonicus; and A. tamarense→L, japonicus. Methods The ingestion of A. tamarense, a producer of PST, by L. japonicus, N. awatschensis, and A. salina was first confirmed by microscopic observation of A. tamarense cells in the intestine samples of the three different organisms, and by the analysis of Chl.a levels in the samples. Toxin accumulation in L. japonicus and N. awatschensis directly from the feeding on A. tamarense or indirectly through the vector of A. salina was then studied, The toxicity of samples was measured using the AOAC mouse bioassay method, and the toxin content and profile of A. tamarense were analyzed by the HPLC method. Results Both A. salina and N. awatschensis could ingest A. tamarense cells. However, the ingestion capability of A. salina exceeded that of N. awatschensis. After the exposure to the culture of A. tamarense （2 000 cells·mL^-1） for 70 minutes, the content of Chl.a in A. salina and N. awatschensis reached 0.87 and 0.024 μg.mg^-1, respectively. Besides, A. tamarense cells existed in the intestines of L. japonicus, N. awatschensis and A. salina by microscopic observation. Therefore, the three organisms could ingest A. tamarense cells directly. A. salina could accumulate high content of PST, and the toxicity of A. salina in samples collected on days 1, 4, and 5 of the experiment was 2.18, 2.6, and 2.1 MU.g^-1, respectively. All extracts from the samples could lead to death of tested mice within 7 minutes, and the toxin content in anemia sample collected on the 1st day was estimated to be 1.65×10 ^5 μg STX equal/individual. Toxin accumulation in L japonicus and N. awatschensis directly from the feeding on A. tamarense or indirectly from the vector ofA. salina was also studied. The mice injected with extracts from L japonicus and N. awatschensis samples that accumulated PST either directly or indirectly showed PST intoxication symptoms, indicating that low levels of PST existed in these samples. Conclusion Paralytic shellfish toxins can be transferred to L. japonicus, N. awatschensis, and A. salina from A. tamarense directly or indirectly via the food chains.
基金The Public Science and Technology Research Funds Projects of Ocean under contract Nos 201305007 and 201405017
文摘Paralytic shellfish toxins(PSTs) are notorious neurotoxins that threaten public health and food safety worldwide.Although PST monitoring programs have recently been established throughout China, the profiles and variation of PSTs in important commercial clams(e.g., Mactra veneriformis, Ruditapes philippinarum, and Meretrix meretrix) along the Jiangsu Province coastline remain largely unexplored. In this study, a validated hydrophilic interaction liquid chromatography–tandem mass spectrometry(HILIC-MS/MS) method was used to examine PST profiles and levels in 540 clam samples from natural production areas along Jiangsu Province coastline during2014–2016. Although the PST levels(≤6.38 μg saxitotoxin equivalents(eq)/kg) were consistently below European Union regulatory limits(≤800 μg saxitotoxin eq/kg) during this time period, saxitotoxin, decarbamoylsaxitotoxin,and gonyautoxins 1 and 4 were detected, and nearly 40% of the samples were saxitotoxin-positive. The PST levels also varied significantly by seasons, with peak values observed in May during 2014–2016. This is the first systematic report of PSTs in clams from Jiangsu Province, and additional research and protective measures are needed to ensure the safety of clams harvested in this area.
基金Supported by the National High Technology Research and Development Program of China(863 Program)(No.2007AA092001-15)the Key Laboratory of Marine Integrated Monitoring and Applied Technologies of Harmful Algal Blooms,Chinese State Oceanic Administration(No.MATHAB20120101)the Shanghai Municipal Oceanic Bureau(Nos.2011-02,2012-02)
文摘The current testing for paralytic shellfish poisoning(PSP) in shellfish is based on the mouse bioassay(MBA).To alleviate animal welfare concerns,we evaluated the utility of using sublethal indicators of toxicity as an alternative to measuring time to death.Live mice were injected with a PSP congener and the changes in neurotransmitter levels were measured 60,90,and 120 min after injection.Acetylcholine(ACh) was the most sensitive marker for PSP toxicity.The changes in neurotransmitter levels were most pronounced in the blood.Thus,measurement of Ach levels in the blood may serve as a sensitive predictor for PSP that would not require sacrifice of the mice.This method was relatively simple,sensitive(less than 1 μg/kg weight,equivalent to 20 ng/mL),low maintenance,and rapid(less than 60 min).
基金The International cooperation programs of the Ministry of Science and Technology of China under contract No.2007DFA30710the Society commonweal programs of the Ministry of Science and Technology of China under contract No.2005DIB2J116
文摘Dissected tissues of three shellfish species, the Chinese scallop, Chlamys farreri, Manila clam, Ruditapes philippinarurn, and Razor shell, Solen strictu were evaluated for in vitro transformation of paralytic shellfish poisoning （PSP） toxins. Tissue homogenates were incubated with extraction from toxic algae Alexandriurn rninutura to determine toxin conversion. The effects of heating and addition of a natural reductant （glutathione） on toxin conversion were also assessed. The toxin profile was investigated through high performance liquid chromatography with fluorescence detection （HPLC-FLD）. The evident variations in the toxin content were observed only in Chinese scallop viscera homogenates. The concentration of GTX4 was reduced by 45% （approximately 0.8 μmol/dm^3） and 25% （approximately 1 μmol/dm^3） for GTX1, while GTX2 and GTX3 increased by six times （approximately 1 μmol/dm^3） and 3 times （approximately 0.3μmol/dm^3） respectively. Simultaneously, the total toxicity decreased by 38% during the 48 h incubation period, the final toxicity was 20.4 nmol STXeq/g. Furthermore, heated Chinese scallop viscera homogenates samples were compared with non-heated samples. The concentration of the GTX4 and GTX1 was clearly 28% （approximately 0.53 μmol/dm^3） and 17% （approximately 0.69μmol/dm^3） higher in heated samples, GTX2 and GTX3 were four times （0.66 μmol/dm^3） and two times （0.187 μmol/dm^3） lower respectively. GSH （＋） Chinese scallop viscera homogenates samples were compared with GSH （-） samples, the concentration in the GTX4 and GTX1 was 9% （approximately 0.12 μmol/dm^3） and 11% （approximately 0.36 μmol/dm^3） lower respectively, GTX2 and GTX3 was 17% （approximately 0.14 μmol/dm^3） and 19% （approximately 0.006 μmol/dm^3） higher respectively. In contrast,there was a little change in the concentration of PSP toxins of Manila clam and Razor shell tissue ho- mogenates. These observations on three shellfish tissues confirmed that there were species-specific differences in PSP toxins transformation. PSP toxins transformation was more pronounced in viscera tissue than in muscle tissue. PSP toxins was possibly interfered by some carbamoylase enzyme, and the activity in Chinese scallop viscera tissue is more remarkable than in the other two species.
基金Supported by a thesis research project of General Administration of Quality Supervision, Inspection and Quarantine of China (No. 2010IK168)
文摘An inter-laboratory comparison of the AOAC mouse bioassay for paralytic shellfish poisoning (PSP) toxicity in shellfish was carried out among 25 Chinese laboratories to examine the overall performance for PSP testing in China, and to analyze the main factors affecting the performance of this method. The toxic scallop Patinopecten yessoensis collected from coast of Bohai Sea, China, was used as a test sample in the comparison study. The results were reported and evaluated using robust statistical methods. The z scores showed that 80%, 8%, and 12% of laboratories reported satisfactory results, unsatisfactory results, and questionable results, respectively. This evaluation demonstrates that the PSP mouse bioassay is an appropriate method for screening and testing PSP toxicity in shellfish. However, it was found that the experience and skill of technicians, as well as the body weight and health status of mice being used significantly affected the accuracy of the method.
基金The National Natural Science Foundation of China under contract No.30470275the National Special Grant of China under contract Nos 908-01-ZH3 and 908-ZC-I-15the National Basic Research Grant of China under contract No.2010CB428706
文摘To study the paralytic shellfish toxins（PSTs） depuration in Japanese scallop Patinopecten yessoensis in natural environment, Japanese scallops naturally contaminated with paralytic shellfish poisoning（PSP） toxins in the Dayao Bay in the northern Huanghai Sea are transited to Qipanmo waters in the Bohai Sea of no reported PSTs incidents. The levels and profile of PSTs during 30-day depuration are detected by the high performance liquid chromatography with fluorescence detection（HPLC-FLD）. The results show that the toxicity of the PSTs in soft tissues decreases to a relatively low level at Day 9. Moreover, the depurated ratio at the early stage of the PSTs depuration is higher than that at the later stage. The toxicity analysis of dissected organs reveals that the digestive gland is the most contaminated PSTs part, which is of important implication for the human health and scallop aquiculture. The mortality of Japanese scallops during PSTs depuration experiment is relevant to PSTs level in the soft tissue.
基金supported by the Modern Seed Industry Park for Whiteleg Shrimp of Guangdong Province(No.K22226)the National Natural Science Foundation of China(No.32102796)+3 种基金the Natural Science Foundation of Guangdong Province(No.2020A1515110086)the Program for Scientific Research Start-up Funds of Guangdong Ocean University(Nos.060302022102,060302022201)the Program of Shrimp Aquaculture Talent Development(No.B22424)the Undergraduate Innovation Team of Guangdong Ocean University(No.CXTD2023002).
文摘Alexandrium pacificum(A.pacificum)is a typical paralytic shellfish poisonous dinoflagellate.Harmful algal blooms(HABs)caused by this species can bring serious environmental problems and economic losses to the aquaculture industry.In this study,transcriptome sequencing and analyses were performed on the neural tissue of Litopenaeus vannamei(L.vannamei)after acute exposure to A.pacificum disrupted solution for 72 h,and differentially expressed genes(DEGs)were identified.The results showed that,compared with the control samples,300 DEGs were identified in the experimental group,of which 194 were up-regulated,and 106 down-regulated.The gene ontology(GO)functional enrichment analysis showed that DEGs were significantly enriched in the cortical cytoskeleton organization,troponin complex,amylo-alpha-1,6-glucosidase and thymidine phosphorylase.Kyoto encyclopedia of genes and genomes(KEGG)enrichment analysis found that DEGs were mainly enriched in the oxidative phosphorylation process,intercellular tight junctions and mitophagy.The results showed that the proteoglycans,signaling pathways,and various metabolic processes that regulate cell proliferation,differentiation,and apoptosis all played an essential role in the response of L.vannamei to A.pacificum toxins.
