Identification of Monitoring Organ in Bivalves for Early Warning of Paralytic Shellfish Toxins Accumulation

MENG Deting, SHI Jiaoxia, LI Moli, WEI Zhongcheng, WANG Yangrui,XU Yiqiang, LI Yubo, BAO Zhenmin, 2), 3), and HU Xiaoli, 2), *

Identification of Monitoring Organ in Bivalves for Early Warning of Paralytic Shellfish Toxins Accumulation

MENG Deting1), #, SHI Jiaoxia1), #, LI Moli1), WEI Zhongcheng1), WANG Yangrui1),XU Yiqiang1), LI Yubo1), BAO Zhenmin1), 2), 3), and HU Xiaoli1), 2), *

1),,,266003,2),,266237,3),,,572000,

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 ac- curacy and sensitivity of PSTs monitoring in bivalves. To develop a sensitive and cost-effective approach for PSTs examination inmassively farmed bivalves, we fed three commercially important bivalves, Yesso scallop, Pacific oyster,and blue musselwith PSTs-producing dinoflagellate, and detected PSTs concen- tration 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, with2=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.

paralytic shellfish toxins; monitoring; bivalve; early warning; digestive gland

1 Introduction

Paralytic shellfish toxins (PSTs), which are one of themost potent neurotoxins produced by phytoplankton or bac-teria, can be enriched through food chains (Etheridge, 2010;Hodgson, 2012). Bivalves, a dominant aquaculture species in China, can accumulate PSTs in a short period of time byfilter-feeding on phytoplankton(Cembella and Todd, 1993; Bricelj and Shumway, 1998; Haberkorn., 2011). The consumption of contaminated bivalves may consequently pose risks to local fisheries, seafood security and humanhealth (Berdalet., 2016). During the last decades, col-lapses in local fisheries associated with PSTs have been widely recorded in East Asia (Sakamoto., 2021). To ensure the security and quality of farmed bivalves, PSTs to- xicity in bivalves are evaluated to determine whether the bi- valves can be sent to the market (Dorner., 2016). Mostcountries set the regulatory limit of PSTs at 80µgSTXeq(100g)−1, which is the standard specified and recommend- ed by World Health Organization (WHO) and Food and Agriculture Organization of the United Nations (FAO) (An-dersen., 2004). Bivalves from natural grounds or aqua- culture farms in coastal and estuarine waters will be tem- porarily prohibited from harvesting once the toxicity of bi- valves exceeds the regulatory limit (Council, 2004). More- over, the production or breeding area will be closed when the toxicity of bivalves exceeds 80µgSTXeq(100g)−1.To decrease the risk of consuming toxic bivalve and the loss of bivalve farming caused by PSTs accumulation, more strin- gent vigilance standards have been developed in some coun- tries, such as 60µgSTXeq(100g)−1 in Philippines(Andres., 2019), and 40µgSTXeq(100g)−1 in Ireland and Ne- therlands (Wright, 1995). For both bivalve farming indus- try and marketing, early warning of PSTs contamination in commercial bivalves is required to avoid the problems caused by PSTs poisoning (Fernandes-Salvador., 2021), which needs sensitive detection of PSTsin bivalve. Cur- rently, the whole soft-tissues are subject to examination to assess PSTs toxicity in bivalves (Bricelj and Shumway, 1998; Escobedo-Lozano., 2012). While this method can provide an overall evaluation on toxins in bivalves, there are two major deficiencies. First, the capacity of accumu- lating toxins varies among tissues in bivalves, so the subtle accumulation of toxins in some tissues may lower the over-all level of toxins in a whole bivalve and thus potentially decrease the accuracy and sensitivity of PSTs monitoring in bivalves. Second, some tough tissues/organs (such as mantle) make toxin extraction time- and reagent-consum- ing. Therefore, it is necessary to develop a sensitive and cost-effective approach for PSTs examination in massive- ly farmed bivalves.

Tissues/organs of bivalves vary in their capacity in PSTsaccumulation, with digestive gland and kidney bearing muchhigher level of toxicity than other organs (Kwong., 2006; Estrada., 2007; Ryoji., 2015; Li., 2017;Alvarez., 2019), so they can be potential organs forearly warning of PSTs accumulation. In this study, we ex- amined the profiles of PSTs in different tissues/organs inthree massively cultured bivalve, Yesso scallop, Pacific oyster, and blue mussel, during the exposure to PSTs-produ- cing dinoflagellateusing liquid chro-matography-mass spectrometry (LC-MS). We found thatdigestive gland can be used for sensitive monitoring of PSTslevel in all the three bivalves, which provides a new method for early warning of PSTs contamination in farmed bivalves.

2 Materials and Methods

2.1 A. catenellaCulturing

We maintained the toxin-producing algae,cultures, in 5L Erlenmeyer flasks using f/2-Si culture me- dium prepared with autoclaved natural seawater (pH 7.9±0.1, salinity 30±1) filtered through 0.45mm glassfiber for years. All cultures were maintained at 23℃±1℃, with the cool white fluorescent illumination (90μmolphotonm−2s−1) and a 14:10 (light:dark) cycle. A fraction of theculture was collected every day for cell-counting using Countstar® BioTech Automated Cell Counter and PSTs ana- lysis during the experiment.

2.2 Exposure of Bivalves to PSTs-Producing Algae

To set up toxin accumulation trials, we first acclimated Yesso scallops (; 83.4mm±3.3mm in shelllength), the Pacific oysters (;90.3mm±11.5mm inshell length) and blue mussels (; 80.5mm±8.8mmin shell length) for two weeks in three aquarium tanks (300mm×300mm×500mm) with static aerated seawater at 12℃±1℃, 18℃±1℃, and 18℃±1℃ respectively. Then, werandomly selected40 scallops, 30 oysters and 40 mussels, and cultured them in three new aquarium tanks (300mm×300mm×500mm).Each tank contained 10L static aerated seawater.They were fed with toxic alga(den- sity: 3×103cellsmL−1) at 1:00 p.m every day. Lastly, we randomly collected eight Yesso scallops at days 0 (con- trol), 1, 3 and 5 of the experiment, shucked them immedi- ately, and dissected them into digestive gland, kidney and other soft tissues. Similarly, we also randomly collected six Pacific oysters and eight mussels at days 0 (control), 1, 3 and 5 of the experiment, and dissected them into digestive gland and other soft tissues. We weighed tissues carefully and stored them in the freezer at −20℃ for the subsequent toxin analyses.

2.3 PSTs Detection

2.3.1 PSTs extraction

We freeze-dried the tissue samples for 24h and manual- ly ground the tissues using a stainless medicine spoon. Weadded 2mL 0.1% formic acid to 1g different tissues, mixed them thoroughly and let them set for 48h. Then, we centri- fuged the mixture at 12000for 10min under 4℃, collect- ed the supernatant, cleaned up the supernatant over an Oa- sis® HLB Extraction Cartridge treated with 6mL methanol and 6mL water, mixed the filter liquor with an equal amountof acetonitrile, centrifuged the mixture at 12000for 10min under 4℃, filtered the supernatant through a 0.22µm mem- brane, and decanted the filtered supernatant into a 2mL brown vial for the following LC-MS analysis.

2.3.2 LC-MS analysis

We performed the LC-MS analysis according to the na- tional standard GB 5009.213-2016 (Determination of Para-lytic Shellfish Toxins in Shellfish)with minor modifications. PSTs were determined by using a 4500 QTrap mass spec- trometer (AB SCIEX, USA), equipped with an ESI source, and an ExionLC AC HPLC. The chromatographic separa- tion was performed on a TSK-gel Amide-80 column (3.0μm, 2.0mm×250mm) at 30℃, eluted at 0.25mLmin−1. Mo- bile phase A was water and B was 95% acetonitrile aque- ous solution; both A and B contained 2mmolL−1ammoniumformate and 0.1% formic acid. The elution time program was as follows: gradient elution from 60% solvent B to 40%solvent B for 15min and hold 40% solvent B for 5min; from 40% solvent B to 60% solvent B for 1min and hold 60% solvent B for 4 min. The MS parameters were as fol- lows: the curtain gas of 35psi, Gas 1 of 50psi, Gas 2 of 55psi and probe gas temperature of 500℃. The toxins were detected in the MRM mode and other ion source parame- ters and MRM channels are summarized in Table 1.

Table 1 The ion source parameters and MRM channels used in this study

Notes: *, quantitative ion; +/−, positive or negative ionization mode.

2.3.3 Statistical analysis

We used toxicity (Eq. (1)), the overall toxicity of toxins in a specific tissue (µgSTXeq(100g)−1), and toxin concen-tration (X) (Eq. (2)), the amount of each toxin per unit weight of a specific tissue (ngg−1), of PSTs to evaluate the degree of toxin accumulation in different tissues of three bivalves. The calculation of toxicity and toxin concentra- tion refer to the national standard GB 5009.213-2016 (De- termination of Paralytic Shellfish Poison in Shellfish).

Toxicity was determined by formula

whereCwas toxin content (μmol) of toxinin a specific tissue,was the weight (g) of a specific tissue,Fwas the relative toxicity of toxinto saxitoxin and 372.2 was mo- lecular weight of saxitoxin dihydrochloride (C10H19N7O4Cl2, gmol−1) (Table 2).

Table 2 Relative toxicity of different paralytic shellfish toxins

Xwas determined by formula

wherecrepresents the concentration (μmolL−1) of toxinin the extracted solution,was the weight of a specific tissue (g),extractwas the total volume (L) of the extracted solution andrwas the molecular weight of toxin.

In order to explore if the level of toxicity of digestivegland or kidney could represent that ofthe whole soft-tissues, we further analyzed the correlation between the to- xicity of these two tissues and that of the whole soft-tis- sues,respectively.

3 Results

3.1 Identification of Sensitive PSTs-Monitoring Or- gan in Yesso ScallopPatinopecten yessoensis

It was worth noting that the toxicity of scallops had in- creased rapidly after scallops were exposed to.The toxicity of both digestive gland and kidney was dra- matically higher than that of the whole soft-tissues (Fig.1). After 24h of exposure, the toxicity of digestive gland was 307.15µgSTXeq(100g)−1and the toxicity of kidney was 297.32µgSTXeq(100g)−1, which were 6.1 and 5.9 times higher than the toxicity of the whole soft-tissues (50.39µgSTXeq(100g)−1), respectively. The maximum values of to- xicity of both digestive gland and kidney were recorded at day 5, which were 943.30 and 1094.11µgSTXeq(100g)−1, respectively, and 7.8 and 9.0 times higher than that of the whole soft-tissues (120.95µgSTXeq(100g)−1), respectively.

Fig.1 Toxicity of paralytic shellfish toxins (PSTs) in diges- tive gland, kidney and the whole soft-tissues of Yesso scal- lop Patinopecten yessoensis exposed to dinoflagellate Ale- xandrium catenella. Vertical lines represent mean±standarderror (n=8). * represent significant difference (P<0.05) be- tween experimental group and control group (day 0).

The toxicity of digestive gland was well correlated withthat of the whole soft-tissues (Fig.2a,2=0.9387). When the toxicity of the whole soft-tissues reached the regulatory li-mit for commercial shellfish (80µgSTXeq(100g)−1), di- gestive gland’s toxicity reached 571.48µgSTXeq(100g)−1. Meanwhile, 80µgSTXeq(100g)−1 in digestive gland cor- responding to 13.7µgSTXeq(100g)−1 in the whole soft-tis-sues. The toxicity of digestive gland had changed from 400to 800µgSTXeq(100g)−1while the toxicity of the whole soft-tissues went from 60 to 100µgSTXeq(100g)−1, sug- gesting that subtle changes in toxicity may be detected in digestive gland but not in the whole soft-tissues. Although the weight of digestive gland accounts for only 6% of that of the whole soft-tissues, its maximum toxin contribution rate was up to 48% (Table3).

Fig.2Correlations between the toxicity of digestive gland (a) or kidney (b) and that of the whole soft-tissues in Yesso scal- lopexposed to dinoflagellate. Dotted line indicates the curve of fitting.

Table 3 The weight and PSTs contribution of digestive gland in three bivalve species

Note: PSTs contribution represents the ratio of the amount of PSTs in the digestive gland to that in the whole soft-tissues of bivalve exposed to toxic algae.

The toxicity level of kidney was also high (Fig.1), whichreached 686.22µgSTXeq(100g)−1when the toxicity of thewhole soft-tissues reached the regulatory limit 80µgSTXeq(100g)−1. However, the toxicity of kidney showed poor cor-relation with that of the whole soft-tissues (Fig.2b,2=0.7128).

3.2 Identification of Sensitive PSTs-Monitoring Organ in Pacific Oyster Crassostrea gigas

In the Pacific oyster, the toxicity of digestive gland in- creased dramatically after oysters was exposed toduring the first 24h and it was greatly higher than that of the whole soft-tissues (Fig.3a). The toxicity of di- gestive gland (244.58µgSTXeq(100g)−1) was three times higher than that of the whole soft-tissues (80.60µgSTXeq(100g)−1). Both toxicity(Fig.3a)and toxin concentration(Fig.4)of digestive gland increased continually during the entire experiment. The maximum values of toxicity in di- gestive gland was recorded at day 5, which was 2564.88µgSTXeq(100g)−1. The toxicity of digestive gland was 10.7 times higher than the toxicity of the whole soft-tissues (239.38µgSTXeq(100g)−1).

The weight of digestive gland in the Pacific oysterac- counts for only 9% of the whole soft-tissues weight, but its toxin contribution rate was up to 68% after 5 days of ex-posure(Table3). Moreover, the toxicity of digestive glandwas well correlated with that of the whole soft-tissues (Fig.3b;2=0.9178). When the toxicity of the whole soft- tissues reached 80µgSTXeq(100g)−1, digestive gland’s toxicity reached 498.90µgSTXeq(100g)−1. The toxicity of 80µgSTXeq(100g)−1in digestive gland, corresponding to 45.60µgSTXeq(100g)−1in the whole soft-tissues.

Fig.3 Toxicity of paralytic shellfish toxins (PSTs) in Pacific oyster Crassostrea gigas exposed to dinoflagellateAlexan- drium catenella. (a), PSTs toxicity in digestive gland and the whole soft-tissues. Vertical lines represent mean±standard error (n=6). (b),Correlation between the toxicity of digestive gland and that of the whole soft-tissues. Dotted line indicates the curve of fitting. * represent significant difference (P<0.05) between experimental group and control group (day 0).

3.3 Identification of Sensitive PSTs-Monitoring Organ in Blue Mussel Mytilus edulis

In mussels, both toxicity (Fig.5a) and toxin concentra- tion (Fig.6) in digestive gland increased continually dur- ing the entire experiment. It was worth noting thatless to- xins were detected in other soft tissues, almost all toxins were accumulated in digestive gland (Table 3). There were no high-potency toxins such as STX in digestive gland of mussels (Fig.6) and the composition of toxins was similar to that in the toxicthat bivalves fed on (Fig.7).Moreover, the toxicity of digestive gland was well corre- lated with that of the whole soft-tissues (Fig.5b,2=0.9431), with the toxicity of digestive gland exceeding 859.20µgSTXeq(100g)−1when the toxicity of the whole soft-tissues broke the regulatory limit for commercial shellfish, 80µgSTXeq(100g)−1. Correspondingly, when digestive gland’s toxicity exceeded 80µgSTXeq(100g)−1, the toxicity of the whole soft-tissues was only 5.93µgSTXeq(100g)−1, more than 15 times lower than the regulatory limit.

4 Discussion

Although many countries have implemented various mea- sures on monitoring PSTs for commercial bivalves, the outbreak of PSTs still persists and PSTs-related illness oc- curs frequently in bivalve consumers (Callejas., 2015).Therefore, it is essential to establish an early warning sys- tem to forecast the accumulation of PSTs in bivalve so as to avoid PSTs-associated food security issues. A recent sum- mary by the European Food Safety Authority (EFSA) statesthe opinion that 7.50µgSTXeq(100g)−1should be suggest-ed as the regulatory limit for PSTsrather than current 80µgSTXeq(100g)−1(Etheridge, 2010). Therefore, moresensi- tive detection for PSTs monitoring is required.

Fig.5 Toxicity of paralytic shellfish toxins (PSTs) in blue mussels Mytilus edulis exposed to dinoflagellate Alexandrium catenella. (a), PSTs toxicity in digestive gland and the whole soft-tissues. Vertical lines represent mean±standard error (n=8). (b), Correlation between the toxicity of digestive gland and that of the whole soft-tissues. Dotted line indicates the curve of fitting. * represent significant difference (P<0.05) between experimental group and control group (day 0).

Fig.7 Composition of paralytic shellfish toxins (PSTs) inthe dinoflagellate Alexandrium catenella used for bivalve exposure in this study. GTX1 to GTX4 and GTX6, gonyau- toxins; C1 and C2, C-toxins..

Kidney is one of the tissues with a high accumulation of PSTs (Liu., 2020). While the toxin profile in digestive gland of bivalves was similar to that of the toxic(Fig.8) which bivalves feed on, some highly toxic to- xins, such as STX and NEO that were not present in toxic, were detected in kidney, which might be the result ofPSTs biotransformation in kidney (Li., 2017; Liu., 2020). Although its toxicity level was higher thanthat of the whole soft tissues, the toxicity of kidney and the whole soft tissues were poorly correlated (Fig.2). The rea- son might be that kidney accounts for a lower proportion (0.9%±0.2%) compared to digestive gland (6.0%±0.5%) of the total soft body in scallop, and the proportion of scal- lop PSTs in kidney (4.9%±1.0%) is much lower than that in the digestive gland (48.0%±7.9%) (Table 3). Thus kid- ney is not suitable for PSTs monitoring.

Fig.8 Concentration of paralytic shellfish toxins (PSTs) in the digestive gland (a) and kidney (b) of Yesso scallop Pati- nopecten yessoensis exposed totoxic dinoflagellate Alexandrium catenella. STX, saxitoxin; NEO, neosaxitoxin; GTX1 to GTX4 and GTX6, gonyautoxins; C1 and C2, C-toxins.

Digestive gland has been shown to play a crucial role in accumulating and concentrating PSTs in bivalves (Shum- way., 1994; Chen and Chou, 2001; Li., 2005;Jean-Luc., 2012;Garcia., 2015). In this study, a huge amount of toxins (PSTs) were rapidly accumulated in digestive gland during the first 24h of exposure, demon-strating digestive gland was asensitive organfor PSTs de- tection. Wang. (2011) also described high toxicity of digestive gland in noble scallops () and Green mussels (), showing four and eight times higher than that of the whole soft-tissues, respectively. Qiu. (2018) even found that PSTs were mainly accumu-lated in digestive gland of Zhikong scallop (), andno PSTs were detected in the adductor muscle, gill and bloodthroughout scallops that were exposed toThese findings suggest that digestive gland might be a suit- able organ for assessing PSTs toxicity, especially for early warning. In this study, we further found that the toxicity of digestive gland is highly correlated with that of the whole soft-tissues in all the three bivalve species we examined, indicating that detecting PSTs in digestive gland could be a sensitive method for bivalve PSTs examination. Mean-while, only sampling digestive gland for PSTs extractionand the easiness to extract toxins from digestive gland fur- ther decrease the time- and reagent-consuming in PSTs de- tection.Therefore, digestive gland could be used for sen- sitive and cost-effective PSTs monitoring in bivalve.

5 Conclusions

By conducting toxin accumulation trials in three com- mercially important bivalves, Yesso scallop, Pacific oysterand blue mus- sel, we demonstrate that a large amount of toxins can be rapidly accumulated in digestive gland of bi- valves. Moreover, the toxicity of digestive gland and that of the whole soft-issues are well correlated with each other.Plus the easiness to extract toxins from digestive gland, we suggest that digestive gland can be used in the sensitive and cost-effective monitoring of PSTs in bivalves.

Acknowledgements

This research was funded by the National Key R&D Pro-ject (No. 2019YFC1605704), the Taishan Industry Leading Talent Project (No. LJNY201816), and was supported by Sanya Yazhou Bay Science and Technology City (No. SKJC- KJ-2019KY01). We would like to thank Dr. Xiaoshen Yin for manuscript editing and revision.

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(April 13, 2022; revised May 6, 2022; accepted September 6, 2022)

© Ocean University of China, Science Press and Springer-Verlag GmbH Germany 2023

#The two authors contributed equally to this work.

Corresponding author. E-mail: hxl707@ouc.edu.cn

(Edited by Qiu Yantao)