Over the last decade, a number of USA aquaculture facilities have experienced periodic mortality events of unknown aetiology in their clownfish (Amphiprion ocellaris). Clinical signs of affected individuals included lethargy, altered body coloration, reduced body condition, tachypnea, and abnormal positioning in the water column. Samples from outbreaks were processed for routine parasitological, bacteriological, and virological diagnostic testing, but no consistent parasitic or bacterial infections were observed. Histopathological evaluation revealed individual cell necrosis and mononuclear cell inflammation in the branchial cavity, pharynx, oesophagus and/or stomach of four examined clownfish, and large basophilic inclusions within the pharyngeal mucosal epithelium of one fish. Homogenates from pooled external and internal tissues from these outbreaks were inoculated onto striped snakehead (SSN-1) cells for virus isolation and cytopathic effects were observed, resulting in monolayer lysis in the initial inoculation and upon repassage. Transmission electron microscopy of infected SSN-1 cells revealed small round particles (mean diameter=20.0–21.7 nm) within the cytoplasm, consistent with the ultrastructure of a picornavirus. Full-genome sequencing of the purified virus revealed a novel picornavirus most closely related to the bluegill picornavirus and other members of the genus Limnipivirus. Additionally, pairwise protein alignments between the clownfish picornavirus (CFPV) and other known members of the genus Limnipivirus yielded results in accordance with the current International Committee on Taxonomy of Viruses criteria for members of the same genus. Thus, CFPV represents a proposed new limnipivirus species. Future experimental challenge studies are needed to determine the role of CFPV in disease.

Cells of Serratia liquefaciens were grown on trypticase soy agar (TSA) for 28 d under Martian conditions of 7 mbar, 0 °C, and CO₂-enriched anoxic atmospheres (called Mars low-PTA conditions). Earth controls were maintained for 24 h at 1013 mbar, 30 °C, and a standard pN₂/pO₂ gas composition. Cells were harvested at either 24 h or 28 d from TSA surfaces and processed for SEM and TEM imaging. Cells of S. liquefaciens grown under Earth conditions were uniform in shape and size, averaging approximately 1.25 µm in length and 0.5 µm in width. Fimbriae were observed on 10–20% of cells grown under Earth conditions. Key features of low-PTA grown cultures were (1) cells exhibited swollen blunt ends at sites of cell division tapering to unusually constricted points on the distal ends of progeny cells, (2) cell division appeared disrupted with division planes occurring at odd angles often forming right-angle oriented daughter cells, (3) some cells failed to form divisional planes resulting in long spiral and oddly shaped cells measuring up to 6–8 µm in length, and (4) fimbriae were lacking. Cell walls were found to be approx. 17% thinner when cells were grown in low-PTA environments compared to lab-standard conditions.

A major unknown in the field of planetary protection is the degree to which natural atmospheric processes remove terrestrial microorganisms from robotic and crewed spacecraft that could potentially contaminate Mars (i.e., forward contamination). We present experiments in which we measured the removal rate of Bacillus subtilis HA101 spores from aluminum surfaces under the bombardment of naturally rounded sand grains. To simulate grain impacts, we constructed a pneumatic sand-feed system and gun to accelerate grains to a desired speed, with independent control of impacting grain mass, flux, and angle. Spore counts of the resulting bombarded surfaces when using scanning electron microscopy indicate that although spores directly impacted by sand grains would likely be killed, those immediately adjacent to grain impacts might be released into the environment intact. The experiments demonstrate a linear relationship between the fractional dislodgement rate of spores and grain impact speed, which can be used to estimate input to microbial transport models (e.g., using numerical models of saltation). Even the slowest grain impacts (∼2.7 m/s) dislodged spores. Such slow events may be common and widespread on Mars, which suggests that microbial dislodgement by slow saltation near the surface is largely unavoidable.

We present experiments demonstrating that B. subtilis HA101 endospores (henceforth spores) may remain intact after impact by, and subsequent transport on the surfaces of, sand grains. To simulate the ricochet of windblown sand grains on a spore-contaminated surface, we constructed a pneumatic gun that accelerated sand grains to 5 m/s. The grains then impacted a spore-laden aluminum surface. They were captured after a single bounce and then imaged with scanning electron microscopy to determine whether any spores adhered to the sand grains and whether they remained intact or were crushed by the impact. Of the 187 identified spores, 74 (40%) appeared intact, which suggests that bacterial spores were not only protected during saltation impact but were also flung onto the retreating sand grain during elastic rebound. Experiments with different aluminum coatings suggested that impacts on uncoated aluminum were more likely to crush spores, whereas anodized and chromate conversion coatings better protected spores. These findings suggest that natural sand transport is a mechanism that could contaminate the environment in the vicinity of a landed spacecraft or in the process of human exploration of a remote aeolian environment, such as that on Mars.