blocks embedded with twigs and toothpicks for climbing and biting, as well as a four-chambered Petri dish (100 mm à 15 mm;. VWR), which was lined with sand ...
Supporting Information Fredericksen et al. 10.1073/pnas.1711673114 SI Materials and Methods Colony Maintenance. Camponotus castaneus colonies in the laboratory were housed in plastic cages with metal mesh lids and containing a plastic tube covered with aluminum foil for housing, as well as two feeding tubes: one filled with water and the other with 10% sugar water. The colonies were kept in an insectary with a temperature range from 23 °C to 28 °C, humidity from 50 to 70%, and a strict light:dark cycle (L:D 12:12). Fungal hyphae (Ophiocordyceps unilateralis sensu lato) used for infection were grown on potato dextrose agar and liquid medium supplemented with 10% FBS, as described in detail previously (11). The culture used in this study was grown from ascospores that were collected from an infected C. castaneus from the field site in South Carolina in July 2014. Preparation of Fungal Tissue. Three infections were performed with O. unilateralis s.l. For the first infection, ants were injected with fungal tissue obtained from fresh C. castaneus cadavers (K-4 and X-2) that had been collected from the field site in South Carolina in September 2014. Tissue was extracted from the fungal stalk and from the ant’s gaster and placed in separate sterile 2-mL Eppendorf tubes containing two 8/32-inch metal balls (Wheels Manufacturing Inc.) and 100 μL Grace’s medium (Sigma). Ascospores were collected on a PDA plate, which was placed in an incubator at 26 °C to allow the spores to germinate. A piece of agar containing germinating ascospores was cut with a razor blade and again placed in a sterile 2-mL tube with metal balls and 100 μL Grace’s medium. The tube was then vortexed and centrifuged. Hemocytometer counts revealed that spores were present at a concentration of ∼1 × 105 spores per milliliter. For the second and third infections, O. unilateralis s.l. hyphae were obtained from culture SC09B, which had been grown in the laboratory since July 25, 2014. Approximately 1 cm2 of the fungal colony was added to 500 μL Grace’s medium and disrupted in a TissueLyser II (Qiagen) for 30 s at 30 freq/s. This suspension was diluted 4× and precipitate was removed to yield the final suspension used for infections. Ants were injected with 1 μL of this suspension on the ventral side of the thorax, underneath one of the forelegs where the cuticle is naturally thinned. Dry spores of Beauveria bassiana (PSU53, PCK 5/5/14) were kindly provided by Nina Jenkins, Department of Entomology, Pennsylvania State University, University Park, PA. Spores were stored in a glass bottle at 4 °C before use. Dry spores were suspended in 0.05% Tween/water solution at a concentration of 4.1 × 108 spores per milliliter. Spore concentration was determined using a hemocytometer. Spore viability was determined using a germination test: spore suspension (80 μL, 100× diluted from infection suspension) was placed on a PDA plate and stored in an incubator at 28 °C for 22 h, after which 300 spores were counted and the percent of germinating spores determined. More than 90% of the spores were germinated, indicating sufficient viability. Infections. All experimental ants were collected from uninfected colonies. Ants were color-coded using paint pens (Edding) according to randomly assigned treatment groups. For the first O. unilateralis s.l. infection, one colony (Fleming 12) was used. Treatment groups included those infected with stalk tissue (30 ants), gaster tissue (30 ants), ascospores (40 ants), and control (14 ants). For the second infection, three colonies (KFM 1, KFM 22, KFM X) were used, and for the third infection, one colony (KFM 15-11) was used. Infections were performed using Fredericksen et al. www.pnas.org/cgi/content/short/1711673114
a laser pulled 10-μL micropipette (Drummond) and aspirator tube (Drummond). Ants infected with B. bassiana were also obtained from two separate infections. The first occurred on March 31, 2015 and ants were obtained from another colony (KFM 11), collected at the same time and location as the others. Thirty ants were surface-infected (no injection) by placing 2 μL of spore suspension on their ventral side. Each ant was then isolated overnight in a cell culture dish (60 mm × 15 mm; Corning) with filter paper (47 mm; GE) that had received 200 μL of spore suspension. Control ants (11 ants) were placed in cell culture dishes with filter paper that had been inoculated with a 0.05% TWEEN/ water solution. Following isolation, ants were combined in a single cage and given water and 10% sugar water ad libitum. Ants were monitored daily for mortality and hourly following the first death. Dead ants were collected and processed (fixed or flash-frozen in liquid nitrogen) just before or within 1 h after death. The second infection occurred on April 21, 2015, and ants were obtained from colony KFM 1. The infection procedures were similar to those described above, but the ants were monitored every 15 min following the first death. Sample Collection. For the first infection, infected ants were separated into two groups based on colony of origin. One group was kept on a windowsill in the laboratory and received a natural L:D cycle (yielded one sample: Ant #1, infected with ascospores), and the other was kept in an incubator at 26 °C and D: D, constant darkness (yielded three samples: Ant #2, infected with gaster tissue and kept in incubator for 23 d; Ant #3 and Ant #4, infected with ascospores and kept in incubator for 17 d). Each group was housed in a 750-cm2 cage containing foam blocks embedded with twigs and toothpicks for climbing and biting, as well as a four-chambered Petri dish (100 mm × 15 mm; VWR), which was lined with sand and darkened with red filter paper and served as a nest. For the second and third infections, ant cages were placed on a laboratory bench by a large window. In addition, two heat lamps were suspended above the cages and set on a timer to approximately match the time of sunrise and sunset. Cages were placed in a glass aquarium (46 cm × 92 cm), which was lined on the bottom with sand. Cages contained a foam block embedded with toothpicks for climbing and biting, two Petri dishes (100 mm × 15 mm; VWR) filled with plaster, and a plastic nest made from a pipette tip box filled with plaster and covered with red plastic filter film. Water was added daily to the sand in the aquarium and to the plaster dishes inside the nests to regulate the temperature and humidity. Infected ants were checked daily for abnormal behavior or biting. For the first infection, ants were collected within 12 h of biting. Three samples were postmortem and one was premortem at time of collection. Ants were dissected immediately using a scalpel (size 10; Miltex) and placed in 0.5 mL fixative (2.5% glutaraldehyde, 2% formaldehyde, 2 mM calcium chloride in 0.15 M cacodylate buffer, pH 7.4) for SEM. For the second and third infections, all collected ants were still alive and actively biting at the time of collection. These ants were flash frozen in liquid nitrogen, transported to a −80 °C freezer, and then dissected at a later date by thawing them on ice and then separating the parts using a scalpel and placed in fixative (for EM: 2.5% glutaraldehyde, 2% formaldehyde, 2 mM calcium chloride in 0.15 M cacodylate buffer, pH 7.4; for immunohistochemistry: 4% paraformaldehyde in PBS). For those ants used for immunofluorescence, whole sections of cuticle 1 of 4
were removed from the caudal-most region of the head moving upwards between the eyes to allow for adequate fixation. We dissected 12 ants to check for the presence of anastomosis between hyphal bodies. One femur from each ant was dissected by squeezing hemolymph and muscle tissue onto a slide using an insect pin. Slides were stained using Lacto-fuchsin and viewed at 40× under a light microscope. We searched for anastomoses between hyphal bodies by systematically examining each slide from top left to bottom right over ∼10 min. Histology. Samples were obtained from the left half of the head, anterior half of the thorax, and anterior half of the gaster. Samples were then embedded in paraffin wax and sectioned at 8 μm using a microtome (Shandon Finesse), dewaxed using a slide stainer (Shandon Gemini), and stained using methylene blue. Serial Block-Face SEM. Briefly, samples were postfixed in ferrocyanidereduced osmium tetroxide followed by thiocarbohydrazide-osmium liganding (OTO) and subsequent uranyl acetate and en bloc lead aspartate staining, dehydrated in a graded ethanol series, and
embedded in Durcupan resin (Fluka). Resin-embedded specimens were trimmed with a razor blade to expose muscle tissue. Data Collection. Fungus/muscle interaction. A total of 30 muscle fibers were examined
from five to eight images per stack, with images spaced at least 20 μm apart when possible to avoid repeated sampling of fungal cells. In each image, five muscle fibers were randomly chosen for analysis, and we counted the number of fungal cells (hyphal bodies or hyphae) that were invading (inside cell membrane) and contacting (outside cell membrane) each muscle fiber. Fungus/fungus interaction. A total of ∼60 O. unilateralis s.l. hyphal bodies were examined from three slices per image stack. From each slice, approximately 20 fungal cells within the muscle fiber matrix were randomly chosen for analysis. Stacks of 300 images were opened in Gatan Digital Micrograph software and the slice player function used to scroll through the stack to determine the number of connections for each fungal cell (hyphal body). We also recorded the number of muscle fibers contacted by each hyphal body.
Fig. S1. Distribution of O. unilateralis s.l. in the host body. Lab-infected ant biting showing manipulated biting behavior (substrate: toothpick) and light micrographs from the head, thorax, and gaster of three different ants (all infected with O. unilateralis s.l.). (A) Transverse section of head at 4× magnification. (Scale bar, 200 μm.) (B) Same region at 100× magnification, showing individual hyphal bodies and connections between them. (Scale bar, 10 μm.) (C) Frontal section of alitrunk, 4×. (Scale bar, 100 μm.) (D) Same region at 40× magnification, showing hyphal bodies and muscle fibers. (Scale bar, 20 μm.) (E) Section through gaster, magnification 4×. (Scale bar, 200 μm.) (F) Same region at 60× magnification showing individual hyphal bodies. (Scale bar, 10 μm.) Paraffinembedded samples were sliced at 8 μm and stained with methylene blue. Ant image credit: Raquel Loreto. Microscopy and figure credit: João Araújo.
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Fig. S2. Leg dissections reveal connected hyphal bodies under light microscopy. Light micrograph showing hyphal bodies dissected from the leg of an ant infected with O. unilateralis s.l. Connections are clearly present between several hyphal bodies (e.g., arrowhead). Slides were stained using Lacto-fuchsin. Magnification: 40×.
Fig. S3. Interactive 3D reconstruction of muscle fiber surrounded by fungal network. A single fiber of an ant mandible adductor muscle (red) surrounded by a partial network of 25 hyphal bodies (yellow). (Adobe Reader 8.1 or higher required for interactive 3D view). Colored xyz arrows indicate the angle at which the figure is rotated. This reconstruction was created using Avizo software from a stack of 1,000 images, each with a thickness of 50 nm. Final image smoothing and 3D pdf credit: Thomas van de Kamp.
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Movie S1. Raw images (1,000 images) through 50 μm of head tissue followed by a 3D reconstruction of one muscle fiber (red) surrounded by 25 connected O. unilateralis s.l. hyphal bodies (yellow) (see Fig. 3A for the size of the structures). Images from Ant #1. 3D reconstruction and video created using Avizo software. Movie S1
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