Evidence for a Novel Marine Harmful Algal Bloom: Cyanotoxin (Microcystin) Transfer from Land to Sea Otters

“Super-blooms” of cyanobacteria that produce potent and environmentally persistent biotoxins (microcystins) are an emerging global health issue in freshwater habitats. Monitoring of the marine environment for secondary impacts has been minimal, although microcystin-contaminated freshwater is known to be entering marine ecosystems. Here we confirm deaths of marine mammals from microcystin intoxication and provide evidence implicating land-sea flow with trophic transfer through marine invertebrates as the most likely route of exposure. This hypothesis was evaluated through environmental detection of potential freshwater and marine microcystin sources, sea otter necropsy with biochemical analysis of tissues and evaluation of bioaccumulation of freshwater microcystins by marine invertebrates. Ocean discharge of freshwater microcystins was confirmed for three nutrient-impaired rivers flowing into the Monterey Bay National Marine Sanctuary, and microcystin concentrations up to 2,900 ppm (2.9 million ppb) were detected in a freshwater lake and downstream tributaries to within 1 km of the ocean. Deaths of 21 southern sea otters, a federally listed threatened species, were linked to microcystin intoxication. Finally, farmed and free-living marine clams, mussels and oysters of species that are often consumed by sea otters and humans exhibited significant biomagnification (to 107 times ambient water levels) and slow depuration of freshwater cyanotoxins, suggesting a potentially serious environmental and public health threat that extends from the lowest trophic levels of nutrient-impaired freshwater habitat to apex marine predators. Microcystin-poisoned sea otters were commonly recovered near river mouths and harbors and contaminated marine bivalves were implicated as the most likely source of this potent hepatotoxin for wild otters. This is the first report of deaths of marine mammals due to cyanotoxins and confirms the existence of a novel class of marine “harmful algal bloom” in the Pacific coastal environment; that of hepatotoxic shellfish poisoning (HSP), suggesting that animals and humans are at risk from microcystin poisoning when consuming shellfish harvested at the land-sea interface

A New Quinoline-Based Chemical Probe Inhibits the Autophagy-Related Cysteine Protease ATG4B

The cysteine protease ATG4B is a key component of the autophagy machinery, acting to proteolytically prime and recycle its substrate MAP1LC3B. The roles of ATG4B in cancer and other diseases appear to be context dependent but are still not well understood. To help further explore ATG4B functions and potential therapeutic applications, we employed a chemical biology approach to identify ATG4B inhibitors. Here, we describe the discovery of 4–28, a styrylquinoline identified by a combined computational modeling, in silico screening, high content cell-based screening and biochemical assay approach. A structure-activity relationship study led to the development of a more stable and potent compound LV-320. We demonstrated that LV-320 inhibits ATG4B enzymatic activity, blocks autophagic flux in cells, and is stable, non-toxic and active in vivo. These findings suggest that LV-320 will serve as a relevant chemical tool to study the various roles of ATG4B in cancer and other contexts

Microcystin-LR concentrations (ppb) at the top, middle, and bottom of seawater tanks at two exposure concentrations (Tank 2 and Tank 3¹) and varying postexposure intervals.

1<p>All samples from Tank 1 (seawater control) tested negative for microcystin-LR. All 3 tanks were flushed with fresh seawater starting 96 H postexposure).</p>2<p>nd  =  microcystin concentration was below minimum detection limits on liquid chromatography-tandem mass spectrophotometry.</p

Microcystin LR concentrations (ppb wet weight) in marine invertebrate gastrointestinal tissues collected from Tank 3 (high microcystin exposure tank) at various time intervals post-exposure¹.

1<p>All tanks were flushed continually with clean seawater beginning at 96 H post-exposure.</p>2<p>n = 1 or 2 pooled invertebrates of each species at each sample point, except snails, where n = 7.</p>3<p>nd  =  microcystin concentration was below minimum detection limits on liquid chromatography-tandem mass spectrophotometry.</p>4<p>---  =  not tested.</p>5<p>Average microcystin-LR concentration across the top, middle and bottom of Tank 3 at each time point.</p

Variation in microcystin detection between conventional “grab” samples and Solid Phase Adsorption Toxin Tracking (SPATT).

<p>Comparison of microcystin (MCY-LR) detection in fresh water using intermittent “grab” sampling (sample periods indicated by black circles) and SPATT (solid line indicating weekly averaged toxin values) in Pinto Lake, demonstrating the higher sensitivity of SPATT for microcystin detection. Grab samples were collected at the beginning of each weekly SPATT deployment, and from the same sample location, so each 7-day integrated SPATT deployment is bracketed by two grab samples.</p

Stranding information and microcystin (MCY) concentrations (ppb wet weight) for wild, microcystin-positive sea otters and captive controls.

1<p>nd  =  microcystin concentration was below minimum detection limits on liquid chromatography-tandem mass spectrophotometry.</p

Map of Monterey Bay showing distribution of sea otters dying due to microcystin intoxication (yellow circles).

<p>Note spatial association of sea otter strandings with coastal locations of river mouths, harbors, coastal ponds and embayments. Habitat utilization distributions for 4 radio-tagged, microcystin-poisoned otters are plotted as kernel density distributions fit to daily re-sighting locations (red shading, with regions of most intense shading corresponding to the habitats most frequently utilized by affected animals). Locations of freshwater samples collected during a “Super-bloom” of <i>Microcystis</i> in 2007 are indicated by green circles, with numbers that correspond with the microcystin concentrations listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012576#pone-0012576-g001" target="_blank">Figure 1</a>.</p