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Variable substrate preference among phospholipase D toxins from sicariid spiders

Venoms of the sicariid spiders contain phospholipase D enzyme toxins that can cause severe dermonecrosis and even death in humans. These enzymes convert sphingolipid and lysolipid substrates to cyclic phosphates by activating a hydroxyl nucleophile present in both classes of lipid. The most medically relevant substrates are thought to be sphingomyelin and/or lysophosphatidylcholine. To better understand the substrate preference of these toxins, we used 31 P NMR to compare the activity of three related but phylogenetically diverse sicariid toxins against a diverse panel of sphingolipid and lysolipid substrates. Two of the three showed significantly faster turnover of sphingolipids over lysolipids, and all three showed a strong preference for positively charged (choline and/or ethanolamine) over neutral (glycerol and serine) headgroups. Strikingly, however, the enzymes vary widely in their preference for choline, the headgroup of both sphingomyelin and lysophosphatidylcholine, versus ethanolamine. An enzyme from Sicarius terrosus showed a strong preference for ethanolamine over choline, whereas two paralogous enzymes from Loxosceles arizonica either preferred choline or showed no significant preference. Intrigued by the novel substrate preference of the Sicarius enzyme, we solved its crystal structure at 2.1 Å resolution. Lastly, the evolution of variable substrate specificity may help explain the reduced dermonecrotic potential of some natural toxin variants, because mammalian sphingolipids use primarily choline as a positively charged headgroup; it may also be relevant for sicariid predatory behavior, because ethanolamine-containing sphingolipids are common in insect prey.

59 BASIC BIOLOGICAL SCIENCES↗

Bioaugmentation of an Engineered Metal Treatment Wetland with Cupriavidus SRS

Cupriavidus SRS is a metal resistant, rod shaped, gram negative bacteria that was isolated from a location with heavy metal contamination at the Department of Energy’s Savannah River Site in Aiken, South Carolina. This isolate has also been able to demonstrate antibiotic resistance and predatory behavior to other microorganisms, giving it a competitive advantage. The effect of bioaugmentation with Cupriavidus SRS and copper (Cu) uptake is not well understood, although multiple studies have analyzed this effect with other metal resistant bacteria. In this study, sediment from the AL1 outfall area will be inoculated with Cupriavidus SRS to analyze the effectiveness of the degradation of Cu.

12 MANAGEMENT OF RADIOACTIVE AND NON-RADIOACTIVE W↗

Human fatalities resulting from wild pig attacks worldwide – 2000-2019

Although reported to be rare, human fatalities resulting from wild pig (Sus scrofa) attacks do occur. Toward a better understanding of patterns in fatal wild pig attacks, we synthesized worldwide reports of wild pig attacks on humans between 2000 and 2019. We documented 163 separate reports of fatal wild pig attacks that resulted in 172 human deaths. On average, 8.6 human deaths occurred annually due to wild pig attacks during those 2 decades. The majority of fatal attacks resulted in a single human death; however, there were 6 cases in which an individual fatal attack resulted in 2–4 human deaths. These fatal wild pig attacks occurred in 29 countries, mostly within the wild pig’s native global range. Fatal attacks primarily occurred under non-hunting circumstances and involved seemingly unprovoked wild pigs. Under hunting circumstances, fatal attacks primarily involved provoked or wounded wild pigs. Fatal attacks typically involved a solitary wild pig, with 12% involving multiple pigs. Solitary pigs involved in fatal attacks were typically large boars that in most attacks exhibited defensive behaviors, although we discovered 7 attacks during which the pig’s behaviors appeared to be predatory. Three fatal attacks were initially investigated as homicides. Overall, victims of fatal wild pig attacks were between 3 and 85 years old and were traveling on foot when the attack occurred. The majority of victims of fatal attacks were adult (20–59 years old), male, traveling on foot, and working in isolation. Among all fatal attacks, 50% identified the cause of death, which included exsanguination/hemorrhagic shock, severe injury, heart attack, craniocerebral injury, severe injury/disembowelment/intestinal prolapse, and toxemia/septicemia. Fatal wild pig attacks occurred primarily in rural areas, with fatal attacks 390% more likely to occur in rural areas with large populations and at least 45% forested and agricultural cover. The greater the rural human population size within a country is, the greater the number of fatal wild pig attacks.

60 APPLIED LIFE SCIENCES↗

Active predation, phylogenetic diversity, and global prevalence of myxobacteria in wastewater treatment plants

The operation of modern wastewater treatment plants (WWTPs) is driven by activated sludge microbiota, a complex assemblage of trophically interacting microorganisms. Microbial predation is crucial to fundamental understanding of how biological interactions drive microbiome structuring and functioning of WWTPs. However, predatory bacteria have received little attention regarding their diversity, activity, and ecological function in activated sludge, limiting the exploitation of food web interactions for wastewater microbiome engineering. Here, by using rRNA-stable isotope probing of activated sludge microbiota with 13 C-labeled prey bacteria, we uncovered diverse as-yet-uncultivated putative predatory bacteria that actively incorporated 13 C-biomass. Myxobacteria, especially Haliangium and the mle1-27 clade, were found as the dominant active predators, refreshing conventional views based on a few predatory isolates of Bdellovibrionota from WWTPs. The identified predatory bacteria showed more selective predation on prey compared with the protists dominated by ciliates, providing in situ evidence for inter-domain predation behavior divergence in activated sludge. Putative predatory bacteria were tracked over a two-year microbiome monitoring effort at a local WWTP, revealing the predominance of Myxococcota (6.5 ± 1.3%) over Bdellovibrionota (1.0 ± 0.2%) lineages. Phylogenetic analysis unveiled highly diverse myxobacteria inhabiting activated sludge and suggested a habitat filtering effect in global WWTPs. Further mining of a global activated sludge microbiome dataset revealed the prevalence of Myxococcota (5.4 ± 0.1%) species and potential impacts of myxobacterial predation on process performance. Collectively, our findings provided unique insights into the predating activity, diversity, and prevalence of Myxococcota species in activated sludge, highlighting their links with wastewater treatment processes via trophic regulation of enteric and functional bacteria.

59 BASIC BIOLOGICAL SCIENCES↗

Ecologically Driven Ultrastructural and Hydrodynamic Designs in Stomatopod Cuticles

Ecological pressures and varied feeding behaviors in a multitude of organ-isms have necessitated the drive for adaptation. One such change is seen in the feeding appendages of stomatopods, a group of highly predatory marine crustaceans. Stomatopods include “spearers,” who ambush and snare soft bodied prey, and “smashers,” who bludgeon hard-shelled prey with a heavily mineralized club. The regional substructural complexity of the stomatopod dactyl club from the smashing predator Odontodactylus scyllarus represents a model system in the study of impact tolerant biominerals. The club consists of a highly mineralized impact region, a characteristic Bouligand architec-ture (common to arthropods), and a unique section of the club, the striated region, composed of highly aligned sheets of mineralized fibers. Detailed ultrastructural investigations of the striated region within O. scyllarus and a related species of spearing stomatopod, Lysiosquillina maculate show consistent organization of mineral and organic, but distinct differences in macro-scale architecture. Evidence is provided for the function and substructural exaptation of the striated region, which facilitated redeployment of a rapto-rial feeding appendage as a biological hammer. Furthermore, given the need to accelerate underwater and “grab” or “smash” their prey, the spearer and smasher appendages are specifically designed with a significantly reduced drag force.

36 MATERIALS SCIENCE↗