Entamoeba Clone-Recognition Experiments: Morphometrics, Aggregative Behavior, and Cell-Signaling Characterization

Abstract

Studies on clone- and kin-discrimination in protists have proliferated during the past decade. We report clone-recognition experiments in seven Entamoeba lineages (E. invadens IP-1, E. invadens VK-1:NS, E. terrapinae, E. moshkovskii Laredo, E. moshkovskii Snake, E. histolytica HM-1:IMSS and E. dispar). First, we characterized morphometrically each clone (length, width, and cell-surface area) and documented how they differed statistically from one another (as per single-variable or canonical-discriminant analyses). Second, we demonstrated that amebas themselves could discriminate self (clone) from different (themselves vs. other clones). In mix-cell-line cultures between closely-related (E. invadens IP-1 vs. E. invadens VK-1:NS) or distant-phylogenetic clones (E. terrapinae vs. E. moshkovskii Laredo), amebas consistently aggregated with same-clone members. Third, we identified six putative cell-signals secreted by the amebas (RasGap/Ankyrin, coronin-WD40, actin, protein kinases, heat shock 70, and ubiquitin) and which known functions in Entamoeba spp. included: cell proliferation, cell adhesion, cell movement, and stress-induced encystation. To our knowledge, this is the first multi-clone characterization of Entamoeba spp. morphometrics, aggregative behavior, and cell-signaling secretion in the context of clone-recognition. Protists allow us to study cell-cell recognition from ecological and evolutionary perspectives. Modern protistan lineages can be central to studies about the origins and evolution of multicellularity.

Keywords: Active cell death; Entamoeba proliferation activating factors; cell migration; chemical cues; inclusive fitness; social ameba.

Figure 1

Schematic phylogeny based on ssrRNA sequences of the Entamoeba clones discussed in this article. The gray circles indicate two relatively-close related clones (E. invadens IP-1 and E. invadens VK-1:NS) used in the aggregation experiments described in Fig. 4. The black circles indicate two relatively-distant related clones (E. terrapinae and E. moshkovskii Laredo) used in the aggregation experiments described in Fig. 5. Accession numbers NCBI: E. invadens IP-1 AF149905.1; E. terrapinae AF149910.1; E. moshkovskii Laredo AF149906.1; E. histolytica X56991; and E. dispar Z49256.1. The ssrRNA sequence of E. invadens VK-1:NS was provided by Graham C. Clark. The placement of E. moshkovskii Snake in the phylogeny (dashed-line branching) is tentative because its ssrRNA sequence is under review. Numbers indicate statistical branch support based on Approximate Likelihood-Ratio Test.

Figure 2

Morphometric analyses of Entamoeba clones. A. Length: data-point distributions per clone (each point corresponds to an individual ameba) with flat diamonds representing confidence intervals; the line in the center of a diamond depicts the group’s mean (the mean of all groups is marked by the horizontal line across groups); the vertical span of the diamond represents the 95% confidence interval for the mean in each group (inside are the standard errors, barely visible); the standard deviations are marked by two small lines above and below each diamond; the extent of the diamond in the x axis is proportional to the number of amebas per clone (E. invadens IP-1, N = 399; E. invadens VK-1:NS, N = 399; E. moshkovskii Laredo, N = 400; E. histolytica, N = 396; E. terrapinae, N = 400; E. moshkovskii Snake, N = 400, and E. dispar, N = 383); one-way ANOVA, F_{6, 2,776} = 229.51, P < 0.0001; lowercase letters indicate Tukey-Kramer HSD test pair-wise comparisons, P < 0.05. B. Width: caption analogous to A; one-way ANOVA, F_{6, 2,776} = 76.91, P < 0.0001. C. Area: caption analogous to A; one-way ANOVA, F_{6, 2,776} = 114.50, P < 0.0001.

Figure 3

Canonical plot depicting the spatial dimensions for maximum morphometric separation among seven Entamoeba clones. The labeled rays (length and width) show the directions of the covariates in the canonical space; the rays branch out from the point 0,0, which corresponds to the grand mean (N = 2,777); each multivariate mean is denoted by a + symbol, inside a small circle, as per Entamoeba clone (the small circle corresponds to the 95% confidence level area for each mean); the large circles depict the regions that contain 50% of the data points per clone. Note how E. invadens IP-1 (right-bottom circle) is the most elongated ameba in respect to the other taxa; E. dispar is the smallest (short and thin; left-bottom circle), and E. histolytica is the widest (top-left circle). Wilks Lambda = 0.572, F_{12, 5,538} = 148.51, P < 0.0001; the full model explains 43% of the variance shared between the variables length and width, with length alone capturing most of the association (34%) between extent-of-elongation and ameba-clone identity; width alone captured 14% of the association. Number of amebas per clone is provided in caption of Fig. 2.

Figure 4

Clone-aggregation preference shown by Entamoeba invadens IP-1 and E. invadens VK-1:NS in mixed- (top) or single-cell-line (bottom) cultures. A. Fluorescent micrograph of E. invadens IP-1 labeled green and E. invadens VK-1:NS labeled red, each clone aggregates in distinct clusters. B. Reverse-color labeling of trophozoites of E. invadens IP-1 (red) and E. invadens VK-1:NS (green), the clones aggregate in distinct clusters. C. Entamoeba invadens IP-1 labeled with both green and red dyes; trophozoites mix equally and look yellow under the microscope. D. Entamoeba invadens VK-1:NS labeled with both green and red dyes. In all trials, cells were labeled with CellTracker Green CMFD and Red (Invitrogen, Carlsbad, CA). All images taken at 36-h, scale bar = 100-μm, 10× magnification.

Figure 5

Clone-aggregation preference shown by E. terrapinae and Entamoeba moshkovskii Laredo in mixed- (top) or single-cell-line (bottom) cultures. A. Fluorescent micrograph of E. terrapinae labeled green and E. moshkovskii Laredo labeled red, each clone aggregates in distinct clusters. B. Reverse-color labeling of trophozoites of E. terrapinae (red) and E. moshkovskii Laredo (green), the clones aggregate in distinct clusters. C. Entamoeba terrapinae labeled with both green and red dyes; trophozoites mix equally and look yellow under the microscope. D. Entamoeba moshkovskii Laredo labeled with both green and red dyes. In all trials, cells were labeled with CellTracker Green CMFD and Red (Invitrogen, Carlsbad, CA). All images taken at 36-h, scale bar = 100-μm, 10× magnification.

Figure 6

Quantification of aggregative behavior of distinctive Entamoeba clones in fresh or aged culture-media at 12-h, 18-h and 36-h of growth. As shown in Fig. 4 C–D, trophozoites of single-cell lines, labeled with both green and red dyes, aggregated and looked yellow under the microscope (an indication that cells had fully mixed). When equal green-/red-dyed mixes of 3-day-old amebas were inoculated in fresh (Fr) or sterile but aged media (1, 2 or 3), in which same-clone trophozoites had previously grown and released their chemosignals (Entamoeba Proliferation Activating Factors, EPAFs), the amebas grew and aggregated faster as function of media age (1 = 12-h; 2 = 24-h, and 3 = 48-h). A. Entamoeba invadens IP-1, Chi-square = 1,692.50, df = 6, P < 0.0001. B. Entamoeba invadens VK-1:NS, Chi-square = 5,653.42, df = 6, P < 0.0001. Surface area of ameba aggregates was determined by Image-Pro software (Micro-Tech Optical New England Inc.). Measurements were averaged from three replicate wells and three separate experiments (methods followed Espinosa and Paz-y-Mino-C 2012). Standard errors are shown.