Figure 1.
Field data on growth of Scenedesmus dimorphus.
Ash-free dry weights (AFDW) of S. dimorphus ponds indicate culture productivity is reduced over time when pests are present (dashed lines − −). Ponds without pests demonstrate a continued increase in productivity (solid lines −). Error bars are standard deviations of three replicates.
Figure 2.
Laboratory data on growth of Scenedesmus dimorphus.
Axenic cultures of S. dimorphus infected with outdoor pond culture show a decrease in cell titer over time (solid line with ▪, left Y axis [titer × 10 000]) and an increase in target pest genomic DNA (dashed line with ▪, right Y axis [Ct (cycle threshold) value]). Uninfected controls show an increase in cell titer (solid line with ▴, left Y axis [titer × 10 000]). Error bars are standard deviations of three replicates.
Figure 3.
Phylogenetic placement of isolate FD01, Amoeboaphelidium protococcarum (star), in Cryptomycota based on multi-gene rDNA analysis.
To place A. protococcarum, sequences from other fungal phyla and Microsporidia were included, with a Holozoa outgroup. Comparative Bayesian and ML support values are indicated. ML –lnL = 38006.62.
Figure 4.
Light microscopy of life cycle of Amoeboaphelidium protococcarum.
A. Motile aplanospore (arrow) proximal to host. B. Aplanospore (arrow) encounters Scenedesmus dimorphus cells and attaches. C. Aplanospore has completed attachment and has formed an epibiotic cyst (AplC). D. Parasite protoplasm (PP) has been injected into the host cell and clearly visible within the cell. E. Clear view of the parasite penetration tube (PT); progeny begin to mature within parasite sporangium. F. Dehiscence of aplanospore cyst occurs and progeny (arrow) are released from the infected cell.
Figure 5.
Ultrastructural features of Amoeboaphelidium protococcarum parasitizing Scenedesmus dimorphus.
A. A healthy, uninfected S. dimorphus cell, surrounded by a cell wall (HCW) and containing lipid globules (L), starch granules (S), a nucleus (N), and a pyrenoid (Py). B. Three motile aplanospores; aplanospores have multiple filose pseudopodia (FP) and contain a single nucleus, multiple lipid globules backed by endoplasmic reticulum (ER), microbodies (Mb), multiple mitochondria (M), a Golgi apparatus (G), and dispersed ribosomes (R). C. Mitochondrion, illustrating lamellar cristae. D. Microbody (Mb) (arrow) appressed to two lipid globules. E. Multi-layered endoplasmic reticulum (arrow) backing microbody in the microbody-lipid globule complex. F. Golgi apparatus. G. Motile aplanospore with pseudopodium. H. Magnification of pseudopodium in G; putative actin microfilaments indicated by arrows. I-K. Aplanospore cysts. I. Cyst containing a nucleus and lipid globule, and subtended by an appressorium (Apr). J. Cyst penetrating host cell wall with a penetration tube. K. An aplanospore cyst surrounded by a rigid cell wall (CCW) and containing a vacuole (Vac) and a multivesicular body (Mvb); the penetration tube penetrates the host cell wall; regions indicated by single and double arrows are magnified in Figs. 3L and M. L, M. Single arrow (L) and double arrows (M) illustrate continuity of parasite plasma membrane around parasite protoplasm as cyst contents are injected into host (Fig. 3K). Bars: H = .025 µm; C, F, L, M = 0.1 µm; D, E = 0.25 µm; B, G, I, J = 0.5 µm; A, K = 1.0 µm.
Figure 6.
Ultrastructural features of Amoeboaphelidium protococcarum parasitizing Scenedesmus dimorphus (cont.).
A. Early infection of host by A. protococcarum; aplanospore cyst subtended by an appressorium attached to host cell wall, and a penetration tube that penetrates host cell wall. B. Intermediate infection of host; remnant of aplanospore cyst contains myelin-like material, and developing parasite protoplasm contains ribosomes, lipid globules, and a primary nucleus; arrows indicate parasite plasma membrane. C. Longitudinal section of algal cell with three infections. Bars: A-C = 1.0 µm.
Figure 7.
Ultrastructural features of Amoeboaphelidium protococcarum parasitizing Scenedesmus dimorphus (cont.).
A. Advanced infection of host (H) by parasite (P), illustrating the host/parasite interface (HPI). Area indicated by arrow is enlarged in B. B. Host and parasite interface, illustrating opposing host plasma membrane (HPM) and parasite plasma membrane (PPM); chloroplast (Cp) indicates host location. C. Entire interior of infected algal cell is filled with parasite protoplast that contains a primary nucleus and multiple lipid globules. Remnant of aplanospore cyst persists on host cell wall. D. Multiple nuclei in mature parasite sporangium indicate the onset of mitosis. Bars: B = 0.1 µm; A, C, D = 1.0 µm.
Figure 8.
Ultrastructural features of Amoeboaphelidium protococcarum parasitizing Scenedesmus dimorphus (cont.).
A. As indicated by cleavage furrows (CF), contents of parasite sporangium have cleaved into aplanospores (Apl); plasma membranes surrounding the sporangium have fused and fragmented (arrows). B. Aplanospores in host cell surround a central food vacuole. C. An empty host cell (HC) with an attached, empty remnant of an aplanospore cyst, of which a sub-apical portion (arrow) has dissolved. D. A host cell 26 d after infection, containing two large lipid globules and an unreleased aplanospore (arrow). E. Two unreleased aplanospores, each developing a thick wall. Bars: D = 0.5 µm; A, B, C = 1.0 µm; E = 0.25 µm.
Figure 9.
Schematic of amoeboid, filose pseudopodiate aplanospore of Amoeboaphelidium protococcarum, illustrating endoplasmic reticulum, Golgi apparatus, lipid globules, mitochondria, microbody, nucleus, ribosomes, vacuole, and vesicles.
Figure 10.
Life cycle of Amoeboaphelidium protococcarum.
The life cycle of A. protococcarum is completed 3–4 days following infection. Stages of the life cycle generally correspond with Days 2–5 following infection. At Day 2: A. Abundant healthy algal cells (arrow) and abundant amoeboid, filose pseudopodiate aplanospores (Sp) are evident. B. A minority of algal cells indicate infection by the presence of a cyst (arrow) attached via an appressorium to the algal cell wall. Day 3: C. The walled, stalked aplanospore cyst has enlarged, a penetration tube penetrates the host cell wall, and a host reaction at the site of infection is evident. D. In some algal cells a membrane-bound, developing parasite protoplasm occupies a portion of the interior of the host cell, and a host/parasite interface is evident; the empty remnant of the aplanospore cyst persists. Day 4: E. Cleaved aplanospores within the host cell are surrounded by fused and fragmented plasma membrane; the empty remnant of the aplanospore cyst persists. Day 5: F. A minority of host cells retain one or a few unreleased aplanospores, a sub-apical portion of the empty remnant of the aplanospore cyst has dissolved, and abundant aplanospores occur outside host cells. G. Most infected host cells are empty, but retain the remnant of the aplanospore cyst.