The microbial carbon pump and climate change.

The ocean has been a regulator of climate change throughout the history of Earth. One key mechanism is the mediation of the carbon reservoir by refractory dissolved organic carbon (RDOC), which can either be stored in the water column for centuries or released back into the atmosphere as CO2 depending on the conditions. The RDOC is produced through a myriad of microbial metabolic and ecological processes known as the microbial carbon pump (MCP). Here, we review recent research advances in processes related to the MCP, including the distribution patterns and molecular composition of RDOC, links between the complexity of RDOC compounds and microbial diversity, MCP-driven carbon cycles across time and space, and responses of the MCP to a changing climate. We identify knowledge gaps and future research directions in the role of the MCP, particularly as a key component in integrated approaches combining the mechanisms of the biological and abiotic carbon pumps for ocean negative carbon emissions.

Haff disease associated with consumption of buffalofish (Ictiobus spp.) in the United States, 2010-2020, with confirmation of the causative species.

BACKGROUND: In the United States, buffalofish (Ictiobus spp.) are sporadically associated with sudden onset muscle pain and weakness due to rhabdomyolysis within 24 h of fish consumption (Haff disease). Previous genetic analyses of case-associated samples were unable to distinguish the three species of buffalofish that occur in the US, Ictiobus cyprinellus (bigmouth buffalo), Ictiobus bubalus (smallmouth buffalo), and Ictiobus niger (black buffalo). METHODS: Ten events were investigated between 2010 and 2020 and demographic and clinical information was collected for 24 individuals. Meal remnants were collected from 5 of 10 events with additional associated samples (n = 24) collected from another five of 10 events. Low-coverage whole-genome sequencing (genome skimming) was used to identify meal remnants. RESULTS: Patients (26-75 years of age) ranged from 1-4 per event, with 90% involving ≥2 individuals. Reported symptoms included muscle tenderness and weakness, nausea/vomiting, and brown/tea-colored urine. Median incubation period was 8 h. Ninety-six percent of cases were hospitalized with a median duration of four days. The most commonly reported laboratory finding was elevated creatine phosphokinase and liver transaminases. Treatment was supportive including intravenous fluids to prevent renal failure. Events occurred in California (1), Illinois (2), Louisiana (1), New York (1), Mississippi (1), Missouri (2), New Jersey (1), and Texas (1) with location of harvest, when known, being Illinois, Louisiana, Mississippi, Missouri, Texas, and Wisconsin. Meal remnants were identified as I. bubalus (n = 4) and I. niger (n = 1). Associated samples were identified as I. bubalus (n = 16), I. cyprinellus (n = 5), and I. niger (n = 3). DISCUSSION: Time course, presentation of illness, and clinical findings were all consistent with previous domestic cases of buffalofish-associated Haff disease. In contrast to previous reports that I. cyprinellus is the causative species in US cases, data indicate that all three buffalofish species are harvested but I. bubalus is most often associated with illness.

Ciguatoxin in Hawai'i: Fisheries forecasting using geospatial and environmental analyses for the invasive Cephalopholis argus (Epinephelidae).

Invasive species can precede far-reaching environmental and economic consequences. In the Hawai'ian Archipelago Cephalopholis argus (family Serranidae) is an established invasive species, now recognized as the dominant local reef predator, negatively impacting the native ecosystem and local fishery. In this region, no official C. argus fishery exists, due to its association with Ciguatera seafood poisoning (CP); a severe intoxication in humans occurring after eating (primarily) fish contaminated with ciguatoxins (CTXs). Pre-harvest prediction of CP is currently not possible; partly due to the ubiquitous nature of the microalgae producing CTXs and the diverse bioaccumulation pathways of the toxins. This study investigated the perceived risk of CP in two geographically discrete regions (Leeward and Windward) around the main island of Hawai'i, guided by local fishers. C. argus was collected and investigated for CTXs using the U.S. Food and Drug Administration (FDA) CTX testing protocol (in vitro neuroblastoma N2a-assay and LC-MS/MS). Overall, 76% of fish (87/113) exceeded the FDA guidance value for CTX1B (0.01 ng g-1 tissue equivalents); determined by the N2a-assay. Maximum CTX levels were ≅2× higher at the Leeward vs Windward location and, respectively, 95% (64/67) and 54% (25/46) of fish were positive for CTX-like activity. Fisher persons and environmental understandings, regarding the existence of a geographic predictor (Leeward vs Windward) for harvest, were found to be (mostly) accurate as CTXs were detected in both locations and the local designation of C. argus as a risk for CP was confirmed. This study provides additional evidence that supports the previous conclusions that this species is a severe CP risk in the coastal food web of Hawai'i, and that ocean exposure (wave power) may be a prominent factor influencing the CTX content in fish within a hyperendemic region for CP.

An Outbreak Investigation of Scombrotoxin Fish Poisoning Illnesses in the United States Linked to Yellowfin Tuna Imported from Vietnam-2019.

ABSTRACT: Scombrotoxin fish poisoning (SFP) is caused by the ingestion of certain fish species with elevated concentrations of histamine due to decomposition. In fall 2019, the U.S. Food and Drug Administration (FDA) was notified of 51 SFP cases including two hospitalizations from 11 states through the FDA consumer complaint system or directly from state partners. A case patient was defined as an individual who experienced a histamine-type reaction after consumption of tuna imported from Vietnam and an illness onset between 14 August and 24 November 2019. A traceback investigation was initiated at 19 points of service to identify a common tuna source. The FDA and state partners collected 34 product samples throughout the distribution chain, including from a case patient's home, points of service, distributors, and the port of entry. Samples were analyzed for histamine by sensory evaluation and/or chemical testing. Case patients reported exposure to tuna imported from Vietnam. The traceback investigation identified two Vietnamese manufacturers as the sources of the tuna. Twenty-nine samples were confirmed as decomposed by sensory evaluation and/or were positive for elevated histamine concentrations by chemical testing. Both Vietnamese companies were placed on an import alert. Seven U.S. companies and one Vietnamese company initiated voluntary recalls. The FDA released public communication naming the U.S. importers to help suppliers and distributors identify the product and effectuate the foreign company's recall. This SFP outbreak investigation highlights the complexities of the federal outbreak response, specifically related to imported food. Cultural considerations regarding imported foods should be addressed during outbreak responses when timing is critical. Collaboration with countries where confidentiality agreements are not in place can limit information sharing and the speed of public health responses.

Analysis of Chloramphenicol and Two Metabolites in Crab and Shrimp Following Waterborne Exposure.

The use of chloramphenicol (CAP) in aquaculture products is banned in many countries, including the United States, due to human health issues. Very few depletion and metabolism studies of CAP in seafood have been performed. Current detection methods for CAP residues in food are directed toward the parent drug molecule, but rapid elimination following treatment suggests the need for an alternative marker residue. We identified, characterized, and determined the persistence of two CAP metabolites, CAP-base (CAP-B) and CAP-alcohol (CAP-OH), in crab and shrimp. Interday recoveries of CAP, CAP-B, and CAP-OH in muscle fortified ( n = 9) at levels of 0.15 to 0.60 ng/g ranged from 95 to 127% and 101 to 119% for crab and shrimp, respectively, with repeatability ranging from 4 to 19%. The limit of detection for CAP and metabolites in crab and shrimp ranged from 0.05 to 0.11 ng/g. We also monitored the depletion of CAP, CAP-B, and CAP-OH in crab following waterborne exposures. To our knowledge, we present the first CAP depletion and metabolite study following waterborne exposure in crabs, with the aim of identifying alternative marker residues.

Mixing it up in the ocean carbon cycle and the removal of refractory dissolved organic carbon.

A large quantity of reduced carbon is sequestered in the ocean as refractory dissolved molecules that persist through several circuits of global overturning circulation. Key aspects of the cycling of refractory dissolved organic carbon (DOC) remain unknown, making it challenging to predict how this large carbon reservoir will respond to climate change. Herein we investigate mechanisms that remove refractory DOC using bioassay experiments with DOC isolated from surface, mesopelagic and deep waters of the Atlantic Ocean. The isolated DOC was refractory to degradation by native microbial communities, even at elevated concentrations. However, when the refractory DOC was introduced to a series of novel environmental conditions, including addition of a labile substrate, a microbial community from coastal waters and exposure to solar radiation, a substantial fraction (7-13%) was removed within 1.5 years. Our results suggest that while refractory molecules can persist in the ocean for millennia, removal is rapid when they encounter their fate. The observed and projected climate-induced slowdown of global overturning circulation could reduce the exposure of refractory molecules to disparate removal processes. Assuming a constant rate of production, the reservoir size of refractory DOC could increase as overturning circulation slows, providing a negative feedback to rising atmospheric CO2.

An Updated Review of Ciguatera Fish Poisoning: Clinical, Epidemiological, Environmental, and Public Health Management.

Ciguatera Fish Poisoning (CFP) is the most frequently reported seafood-toxin illness in the world. It causes substantial human health, social, and economic impacts. The illness produces a complex array of gastrointestinal, neurological and neuropsychological, and cardiovascular symptoms, which may last days, weeks, or months. This paper is a general review of CFP including the human health effects of exposure to ciguatoxins (CTXs), diagnosis, human pathophysiology of CFP, treatment, detection of CTXs in fish, epidemiology of the illness, global dimensions, prevention, future directions, and recommendations for clinicians and patients. It updates and expands upon the previous review of CFP published by Friedman et al. (2008) and addresses new insights and relevant emerging global themes such as climate and environmental change, international market issues, and socioeconomic impacts of CFP. It also provides a proposed universal case definition for CFP designed to account for the variability in symptom presentation across different geographic regions. Information that is important but unchanged since the previous review has been reiterated. This article is intended for a broad audience, including resource and fishery managers, commercial and recreational fishers, public health officials, medical professionals, and other interested parties.

Validation of the Biofish-300 HIS Enzymatic Biosensor for the Detection of Histamine in Fishery Products.

The Biofish-300 HIS method is a simple, reliable, and specific enzymatic biosensor for the detection of histamine. This technology is highly specific and selective and allows quantification of histamine in fishery products (fresh/frozen and processed) in a short time frame (2-3 min). Histamine in raw tuna, raw mackerel, raw sardine, raw anchovy, boiled tuna, canned tuna in water, canned tuna in oil, canned mackerel in tomato sauce, canned pickled sardine, and canned salted anchovy was analyzed using a water-based extract. Matrix-specific assay procedures and calibration curves were used to enable analyses to be carried out across multiple sample types. The performance of this assay was examined using samples that were naturally contaminated (reference materials and interlaboratory studies) and spiked with histamine. All data were judged against previously established acceptance criteria. Performance measures were evaluated for linearity, selectivity, matrix, lot consistency, and robustness. Results produced in all performance measures, except robustness, were within acceptable ranges. Out-of-range robustness results reflected deviation in sample volume compared to the standard assay procedures. Positive interferences from the presence of agmatine were shown.

Draft Genome Sequences of Histamine- and Non-Histamine-Producing Photobacterium Strains.

Histamine-producing bacteria (HPBs) have recently been identified from the marine environment. The identification and characterization of HPBs is important to developing effective mitigation strategies for scombrotoxin fish poisoning. We report here the draft genomes of seven histamine-producing and two non-histamine-producing marine Photobacterium strains.

Draft Genome Sequences of Histamine-Producing Morganella psychrotolerans Strains.

Histamine-producing bacteria are responsible for scombrotoxin (histamine) fish poisoning, a leading cause of fish poisoning in the United States. We report here the first draft genomes of three histamine-producing Morganella psychrotolerans strains, isolated from tuna and mahi-mahi.

Prevalence and Characterization of High Histamine-Producing Bacteria in Gulf of Mexico Fish Species.

Recent developments in detection and enumeration of histamine-producing bacteria (HPB) have created powerful molecular-based tools to better understand the presence of spoilage bacteria and conditions, resulting in increased risk of scombrotoxin fish poisoning. We examined 235 scombrotoxin-forming fish from the Gulf of Mexico for the presence of high HPB. Photobacterium damselae subsp. damselae was the most prevalent HPB (49%), followed by Morganella morganii (14%), Enterobacter aerogenes (4%), and Raoultella planticola (3%). The growth characteristics and histamine production capabilities of the two most prevalent HPB were further examined. M. morganii and P. damselae had optimum growth at 35°C and 30 to 35°C and 0 to 2% and 1 to 3% NaCl, respectively. P. damselae produced significantly (P < 0.001) higher histamine than M. morganii in inoculated mahimahi and Spanish mackerel incubated at 30°C for 24 h, but histamine production was not significantly different between the two HPB in inoculated tuna, possibly due to differences in muscle composition and salt content. Results in this study showed that P. damselae was the most prevalent high HPB in Gulf of Mexico fish. In addition, previously reported results using the traditional Niven's method may underreport the prevalence of P. damselae. Molecular-based methods should be used in addition to culture-based methods to enhance detection and enumeration of HPB.

Storage Time and Temperature Effects on Histamine Production in Tuna Salad Preparations.

Scombrotoxin fish poisoning (SFP), also known as histamine (Hst) poisoning, has been associated with consumption of scombroid-type fish, including tuna and tuna fish products. Preparation of commercial tuna salad contaminated with Hstproducing bacteria (HPB), combined with time-temperature abuse, can present a food safety hazard. A potential source of HPB is raw ingredients, such as celery and onions. The objectives of this study were to determine whether raw ingredients can be a source of HPB and to ascertain the effects of storage time (up to 4 days or 4 weeks) and temperature (4, 10, 18, 25, 30°C) on growth and Hst production by high-HPB (>1,000 ppm of Hst) in tuna salad preparations. Pantoea-Erwinia, Erwinia persicinus, Erwinia spp., and Enterobacter pyrinus isolated from celery in this study were used to inoculate tuna salad and tuna salad with celery or onion. HPB numbers were 0.7 to 4.3 log most probable number per g higher in the presence of celery or onion versus plain tuna salad (3:1 tuna:mayonnaise). E. pyrinus-inoculated plain tuna salad and tuna salad with celery and onion had >500 ppm of Hst after 2 days at 30°C and 4 days at 25°C. E. pyrinus-inoculated salad with celery and onion had >500 ppm of Hst after 4 days at 18°C and 2 weeks at 10°C. Raw celery can introduce HPB into tuna salad, which can cause SFP if the product is time-temperature abused. Tuna salad products must be refrigerated at ≤4°C to prevent growth and Hst production by the HPB used in this study, to protect consumers from potential SFP.

Modification and Single-Laboratory Validation of AOAC Official Method 977.13 for Histamine in Seafood to Improve Sample Throughput.

Histamine is the main causative agent in scombrotoxin fish poisoning, the most frequently reported illness related to fish consumption. The AOAC official method for histamine determination in fish is the fluorometric method AOAC 977.13, which is sensitive and reproducible but somewhat labor intensive and time consuming. We investigated multiple modifications to this method in an attempt to reduce assay time and increase sample throughput while maintaining the performance of the original method. Some of the attempted modifications negatively affected the performance characteristics of the method. However, omitting the heating step during extraction and replacing the cuvette style fluorometer with a microplate reader retained method performance while increasing sample throughput. Therefore, we adopted these modifications and conducted a single-laboratory validation. The recovery, precision (RSD), and LOD of the modified method assessed by the single-laboratory validation ranged from 92 to 105%, 1 to 3%, and 0.2 to 0.5 ppm, respectively, in tuna, mahi-mahi, and Spanish mackerel samples. We conclude that the AOAC 977.13 fluorometric method, modified as described, will improve assay time and sample throughput efficiency cumulatively, as the number of sample units analyzed increases. We anticipate that this modified method could be used by regulatory agencies and other laboratories following successful multilaboratory validation.

Draft Genome Sequences of Histamine-Producing Photobacterium kishitanii and Photobacterium angustum, Isolated from Albacore (Thunnus alalunga) and Yellowfin (Thunnus albacares) Tuna.

Histamine-producing bacteria are responsible for scombrotoxin (histamine) fish poisoning, a leading cause of fish poisoning in the United States. We report here the draft genome sequences of four histamine-producing (HP) Photobacterium kishitanii strains and nine HP Photobacterium angustum strains isolated from tuna.

Marine sequestration of carbon in bacterial metabolites.

Linking microbial metabolomics and carbon sequestration in the ocean via refractory organic molecules has been hampered by the chemical complexity of dissolved organic matter (DOM). Here, using bioassay experiments and ultra-high resolution metabolic profiling, we demonstrate that marine bacteria rapidly utilize simple organic molecules and produce exometabolites of remarkable molecular and structural diversity. Bacterial DOM is similar in chemical composition and structural complexity to naturally occurring DOM in sea water. An appreciable fraction of bacterial DOM has molecular and structural properties that are consistent with those of refractory molecules in the ocean, indicating a dominant role for bacteria in shaping the refractory nature of marine DOM. The rapid production of chemically complex and persistent molecules from simple biochemicals demonstrates a positive feedback between primary production and refractory DOM formation. It appears that carbon sequestration in diverse and structurally complex dissolved molecules that persist in the environment is largely driven by bacteria.

Screening for petrochemical contamination in seafood by headspace solid-phase microextraction gas chromatography-mass spectrometry.

A headspace solid-phase microextraction gas chromatography-mass spectrometry (SPME GC-MS) method is described, to screen seafood for volatile organic compounds (VOCs) associated with petrochemical taint. VOCs are extracted from the headspace of heated sample homogenates by adsorption onto a SPME fiber and desorbed for analysis by GC-MS. Targeted compounds are determined semi-quantitatively using representative calibration standards for the various classes (alkanes, alkylbenzenes, indanes/tetralins, and naphthalenes) of VOCs analyzed. Sample preparation is minimal, and the analyses are rapid and automated with a capacity of 50 samples per day. The method was optimized in terms of headspace temperature, sample heating time, extraction time, and desorption time using oyster samples fortified with target compounds. Calibrations for hydrocarbon components were linear in the range of 8.3-167 ng/g; the limit of detection ranged between 0.05 and 0.21 ng/g, and the limit of quantitation between 0.16 and 0.69 ng/g. Good precision (RSD < 10 % at 16.7 ng/g for individual VOCs) and accuracy (recovery range 89-118 % at 25 ng/g) were obtained in oyster, crab, shrimp, and finfish matrices. The trueness of the method was demonstrated by quantifying VOCs at 1-2-ppb levels in oyster fortified with certified reference material NIST SRM 1491a. Following single laboratory validation, the method was employed for the determination of VOCs in seafood exposed to oil contaminated seawater and for the determination of background VOC levels in seafood species from the Gulf of Mexico and local food stores. The method as described can be used to supplement human sensory testing for petrochemical taint in seafood.

The size-reactivity continuum of major bioelements in the ocean.

Most of the carbon fixed in primary production is rapidly cycled and remineralized, leaving behind various forms of organic carbon that contribute to a vast reservoir of nonliving organic matter in seawater. Most of this carbon resides in dissolved molecules of varying bioavailability and reactivity, and aspects of the cycling of this carbon remain an enigma. The size-reactivity continuum model provides a conceptual framework for understanding the mechanisms governing the formation and mineralization of this carbon. In the seawater bioassay experiments that served as the original basis for this model, investigators observed that larger size classes of organic matter were more bioavailable and more rapidly remineralized by microbes than were smaller size classes. Studies of the chemical composition and radiocarbon content of marine organic matter have further indicated that the complexity and age of organic matter increase with decreasing molecular size. Biodegradation processes appear to shape the size distribution of organic matter and the nature of the small dissolved molecules that persist in the ocean.

Importance of histamine-producing Clostridium perfringens in scombrotoxin-forming fish.

It has been suggested that anaerobic histamine-producing bacteria (HPB) are important contributors to scombrotoxin fish poisoning (SFP). In order to assess the role of Clostridium perfringens in SFP, we developed a real-time PCR method for rapid detection of histamine-producing (HP) C. perfringens. The real-time PCR assay was 100% inclusive for detecting 23 HP C. perfringens and did not detect any of the other 116 HP or non-HP isolates examined. The efficiency of the assay with or without internal amplification control DNA was 102%; in the presence of background flora and inhibitory matrices, it was 90 to 99%. To investigate the importance of HP C. perfringens in SFP, we examined histamine production by C. perfringens in inoculated fish samples incubated under anaerobic conditions. C. perfringens produced low histamine levels in tuna (19 ppm) and Spanish mackerel (3 ppm), whereas gram-negative HPB produced high histamine levels (6,345 ppm in tuna; 1,223 ppm in Spanish mackerel) under the same conditions. When one bonito, two bigeye tuna, nine mahi-mahi, and five yellowfin tuna were examined for the presence of HPB, none (0 of 17) of the samples contained HP C. perfringens or other gram-positive HPB, whereas 86% of the samples contained gram-negative HPB. Our study indicates that histamine production by C. perfringens in scombrotoxin-forming fish was minimal compared with that by gram-negative HPB and that C. perfringens may not be an important bacterial species associated with SFP.

Pan-Arctic distributions of continental runoff in the Arctic Ocean.

Continental runoff is a major source of freshwater, nutrients and terrigenous material to the Arctic Ocean. As such, it influences water column stratification, light attenuation, surface heating, gas exchange, biological productivity and carbon sequestration. Increasing river discharge and thawing permafrost suggest that the impacts of continental runoff on these processes are changing. Here, a new optical proxy was developed and implemented with remote sensing to determine the first pan-Arctic distribution of terrigenous dissolved organic matter (tDOM) and continental runoff in the surface Arctic Ocean. Retrospective analyses revealed connections between the routing of North American runoff and the recent freshening of the Canada Basin, and indicated a correspondence between climate-driven changes in river discharge and tDOM inventories in the Kara Sea. By facilitating the real-time, synoptic monitoring of tDOM and freshwater runoff in surface polar waters, this novel approach will help understand the manifestations of climate change in this remote region.