Fig. 1. The life cycle of Diplostomum spp (“eye flukes”). A. Sexual reproduction of adult parasites within the intestine of an avian definitive host. B. Fertilized egg. C. Miracidium, seeking the first intermediate host, where it will give rise to sporocysts. D. Asexual reproduction of sporocysts in a lymnaeid snail. E. Cercaria, released from snail, seeking the second intermediate host, where it will shed its tail and develop as the second larval stage. F. Metacercaria residing within the fish eye. G. The life cycle is completed when an avian definitive host eats an infected fish.

Fig. 2. Eye fluke metacercariae present in eyes of threespine stickleback fish. A. View of paraformaldehyde-fixed stickleback eye (from Hvita) following removal of lens, showing flukes (arrows) visible within the eyecup. B. View of lens (L) removed from a fixed stickleback eye (from Hvita), showing fluke that was recently released from the eyecup along with the lens (arrow). C. Differential interference contrast (DIC/Nomarski) image of two flukes that were released from the eye (stickleback from Hofdi LS) during dissection. Ventral views show oral (O) and ventral (V) suckers and excretory pore (EP). D. Hematoxylin and eosin-stained section (7 μm thickness) of stickleback eye (from Hofdi LS), showing a fluke present within and disrupting the retinal pigmented epithelium (RPE), with the ventral sucker apparently clinging to a fragment of RPE (asterisk in center of image), and possibly some RPE within the oral cavity (middle of white circle at anterior of metacercaria). Fluke shown in 2D is the same as that shown in Fig. 3C. Scale bars: A, 1.0 mm; B, 1.0 mm; C, 200 μm; D, 50 μm.

Fig. 3. Histology (hematoxylin and eosin staining) of sectioned (7 μm thickness) stickleback retina and retinal pigmented epithelium (RPE) A. Retina and RPE of uninfected eye (from Hrauny), showing normal retinal lamination and RPE histology. B. Region of fluke-infected eye (from Hofdi LS) showing normal retinal lamination and RPE histology. Flukes were located >100 μm from region shown. C. Region of fluke-infected eye (from Hofdi LS) showing presence of a metacercaria (same as in Fig. 2D, but rotated orientation) surrounded by disrupted and damaged retina and RPE. D. Region of fluke-infected eye (from Galta) showing major damage to retinal organization. Numerous flukes were located <100 μm from retina, within subretinal space (but not in imaged area). E. Low-magnification view of fluke-infected eye (from Galta) showing region of major damage to retina and RPE, and the presence of numerous metacercariae; one fluke appears to occupy the choroid (CH). F. Region of fluke-infected stickleback eye (from Hofdi LS) showing a “laminar fusion” (LF), a histological feature typical of regenerated retina, in which nuclei are present in the inner plexiform layer (IPL). G. An additional example of a laminar fusion in a stickleback retina (from Hofdi LS). H. Region of fluke-infected stickleback eye (from Hofdi LS), in which disorganized, supernumerary nuclei appear within the ganglion cell layer (GCL). For panels F, G, and H, flukes were located >100 μm from regions shown. ONL, outer nuclear layer; INL, inner nuclear layer. Scale bars: A (applies to all others except E), 20 μm; E, 50 μm.

Fig. 4. Indirect immunofluorescence of Müller glia and cone photoreceptor staining within sectioned (7 μm thickness) stickleback retina. All sections were counterstained with DAPI to show positions of retinal nuclei. A. Anti-glutamine synthetase (GS) staining of a retina of an uninfected eye (from Berserk) showing normal distribution of the GS antigen throughout Müller glia, extending from the outer limiting membrane (OLM) to the inner limiting membrane (ILM). B. A region of a fluke-infected eye (from Hofdi LS) also showing normal distribution of the GS antigen. C. Anti-GS staining of a region of a fluke-infected eye (from Pristi), with fluke metacercaria positioned in the interface between retina and RPE (arrow), showing possibly reduced GS antigen in the OLM. D. Zpr1 staining (targets arrestin3a) of a retina of an uninfected eye (from Thanga) showing normal distribution of the antigen within double cones in outer segments (OS), inner segments (IS), and synaptic terminals within the outer plexiform layer (OPL). E. A region of a fluke-infected eye (from Galta) showing somewhat disorganized distribution of the antigen. F. Zpr1 staining of a region of a fluke-infected eye (from Grimsa), with fluke metacercaria positioned in the interface between retina and RPE (arrow), showing shortened cones and fewer nuclei within the outer nuclear layer (ONL). INL, inner nuclear layer, GCL, ganglion cell layer. Scale bars: A (applies to B-E), 20 μm.

Fig. 5. Cell proliferation in retinas of fluke-infected eyes of threespine stickleback. Sections (7 μm thickness) were counterstained with DAPI to show positions of retinal nuclei. A-C. Anti-PCNA (A) and DAPI (B) staining of a region of a fluke-infected eye (from Grimsa) showing PCNA + nuclei (C; merged image) within the outer nuclear layer (ONL), possibly representing a response by rod precursors to photoreceptor damage. Inset to C is a merged image of a region an uninfected eye (from Berserk) showing no damage and very few PCNA-positive nuclei (arrows). D-F. Anti-PCNA (D) and DAPI (E) staining of a region of a fluke-infected eye (from Galta) showing PCNA + nuclei (F; merged image) within the ONL, along with clusters of nuclei and individual nuclei within the inner nuclear layer (INL), possibly representing a more robust regenerative response. Inset to F is a merged image of a region from the same eye showing no damage and no PCNA-positive nuclei. AF, autofluorescence of photoreceptor inner segments. Scale bars: A (applies to B–F), 20 μm; Insets to C and F, 20 μm.

Fig. 6. Cell proliferation in damaged RPE of fluke-infected eyes of threespine stickleback. Sections (7 μm thickness) were counterstained with DAPI to show positions of nuclei associated with the RPE. A-D. Region of healthy RPE (from Grimsa): brightfield (BF) image (A), PCNA (B), and DAPI (C) staining. No PCNA-positive nuclei are present in this region of RPE (D; merged image; three DAPI-positive nuclei are indicated with arrows). E-H. Region of damaged RPE (from Grimsa): brightfield image (E), PCNA (F), and DAPI (G) staining. Several PCNA-positive nuclei are associated with the damaged RPE (H; merged image), two are indicated with arrows. Scale bar: A (applies to all), 20 μm.

Fig. 7. Neuronal and synaptic markers in retinal regions of laminar fusions (LF) of fluke-infected eyes of threespine stickleback. Sections (7 μm thickness) were counterstained with DAPI to show positions of retinal nuclei. A. Anti-HuC/D and DAPI staining of uninfected retina (from Hrauny) showing anti-HuC/D staining within neurons of the inner nuclear layer (INL) and ganglion cell layer (GCL). B. A region of a fluke-infected eye (from Pristi) showing HuC/D+ neurons within a laminar fusion (LF) joining the INL to the GCL. C. Anti-SV2 and DAPI staining of retina of uninfected eye (from Hofdi), with labeling of the outer plexiform layer (OPL) and inner plexiform layer (INL). D. A region of a fluke-infected eye (from Pristi) showing tissue in which the SV2+ OPL and IPL are interrupted by the presence of cell nuclei. Scale bar: A (applies to all), 20 μm.