Polar tube firing dynamics and ultrastructure from the shrimp microsporidian Ecytonucleospora hepatopenaei (EHP)

Ecytonucleospora (Enterocytozoon) hepatopenaei or EHP is an obligate intracellular parasite that belongs to phylum Microsporidia. EHP infection in shrimp results in growth retardation and size variation, leading to severe economic loss to shrimp aquaculture worldwide. Similar to other microsporidian species, EHP utilizes a harpoon-like invasion apparatus called the polar tube in order to infect host cells. The polar tube typically coils inside the spore and rapidly transits to a long, extended tube after being activated with proper stimuli. However, the mechanism and physical conditions affecting the polar tube firing in EHP are poorly understood. Here, we screened several germination buffers and found that a buffer containing potassium hydrogen phthalate (KHP) at pH 3.0 yielded the highest germination rate. The optimal temperatures for EHP germination range between 25-28{degrees}C, similar to the shrimps body temperature. The spores require at least 30 seconds to be activated, suggesting that the stimuli could rapidly move across the spore wall. We utilized high-speed live-cell imaging to study the dynamics of the EHP polar tube firing and compared the dynamics when the spores were treated with KHP and a previously reported stimulus, phloxine B. The polar tube firing dynamics between these two conditions are different. The total firing time was [~]100 milliseconds with the maximum velocity of [~]300 m*s-1 in KHP condition. Further investigation on the EHP polar tube ultrastructure using cryo-electron microscopy revealed that the EHP polar tube was composed of a membrane layer with an additional repetitive protein-array on the outermost surface. The distance between each repetitive unit ranges between 52-66 [A]. These repetitive units could possibly be polar tube proteins (PTPs). Altogether, this study provides insights into EHP polar tube firing dynamics and its architecture which are important foundations for understanding the biophysical factors governing EHP pathogenesis and in developing EHP control strategies. Author summaryThe microsporidian Ecytonucleospora hepatopenaei (EHP) poses a significant threat to global shrimp aquaculture, causing substantial economic losses. Many studies on EHP have been focused on development of detection methods, and farm management. Little is known about EHP pathogenesis and how it infects shrimp cells. Here, we identified laboratory conditions to activate the parasites infection process, which occurs on a millisecond timescale. We utilized a high-speed live-cell imaging to further characterize the rapid EHP infection process. The architecture and ultrastructure of EHP invasion organelle were also investigated using cryo-electron microscopy. Our findings provide a better understanding of the EHP infection mechanisms that are crucial for developing effective control strategies.