Structure and functions of the ventral tube of the clover springtail Sminthurus viridis (Collembola Sminthuridae)

YesSpringtails (Collembola) are unique in Hexapoda for bearing a ventral tube (collophore) on the first abdominal segment. Although numerous studies have been conducted on the functions of the ventral tube, its fine structure has not been thoroughly elucidated to date. In this paper, we observed the jumping behavior of the clover springtail Sminthurus viridis (Linnaeus, 1758) and dissected the ventral tube using light microscopy to elucidate the fine structure and the possible function of the ventral tube. The results show that a pair of eversible vesicles can be extended from the apical opening of the ventral tube. The eversible vesicles are furnished with numerous small papillae, and can be divided into a basal part and a distal part. The eversible vesicles have a central lumen connected to the tiny papillae and leading to the body cavity. The eversible vesicles can reach any part of the body, and may serve as following functions: (a) absorbing moisture; (b) uptaking water; (c) cleaning the body surface; and (d) fastening the body on a smooth surface

CD14-Dependent Monocyte Isolation Enhances Phagocytosis of Listeria monocytogenes by Proinflammatory, GM-CSF-Derived Macrophages

Macrophages are an important line of defence against invading pathogens. Human macrophages derived by different methods were tested for their suitability as models to investigate Listeria monocytogenes (Lm) infection and compared to macrophage-like THP-1 cells. Human primary monocytes were isolated by either positive or negative immunomagnetic selection and differentiated in the presence of granulocyte macrophage colony-stimulating factor (GM-CSF) or macrophage colony-stimulating factor (M-CSF) into pro- or anti-inflammatory macrophages, respectively. Regardless of the isolation method, GM-CSF-derived macrophages (GM-Mφ) stained positive for CD206 and M-CSF-derived macrophages (M-Mφ) for CD163. THP-1 cells did not express CD206 or CD163 following incubation with PMA, M- or GM-CSF alone or in combination. Upon infection with Lm, all primary macrophages showed good survival at high multiplicities of infection whereas viability of THP-1 was severely reduced even at lower bacterial numbers. M-Mφ generally showed high phagocytosis of Lm. Strikingly, phagocytosis of Lm by GM-Mφ was markedly influenced by the method used for isolation of monocytes. GM-Mφ derived from negatively isolated monocytes showed low phagocytosis of Lm whereas GM-Mφ generated from positively selected monocytes displayed high phagocytosis of Lm. Moreover, incubation with CD14 antibody was sufficient to enhance phagocytosis of Lm by GM-Mφ generated from negatively isolated monocytes. By contrast, non-specific phagocytosis of latex beads by GM-Mφ was not influenced by treatment with CD14 antibody. Furthermore, phagocytosis of Lactococcus lactis, Escherichia coli, human cytomegalovirus and the protozoan parasite Leishmania major by GM-Mφ was not enhanced upon treatment with CD14 antibody indicating that this effect is specific for Lm. Based on these observations, we propose macrophages derived by ex vivo differentiation of negatively selected human primary monocytes as the most suitable model to study Lm infection of macrophages

Fluorescence microscopy of GM-Mφ (GM) and M-Mφ (M) generated from monocytes isolated by negative (neg.) or positive (pos.) selection and infected with <i>Lm</i> at an MOI of 10 (A).

<p>Nuclei of macrophages are stained with DAPI (blue) and <i>Lm</i> was stained with a specific antibody (red). Scale bar is 10 µm. Infection of GM-Mφ (-GM or +GM, black bars) and M-Mφ (-M or +M, white bars) with <i>Lm</i> was determined at different multiplicities of infection (MOI) and fluorescence microscopy images were analysed either for the percentage of infected cells (B) or the number of bacteria per infected cell (C). For each MOI and macrophage phenotype, infected cells and <i>Lm</i> per infected cell were counted in random microscopic fields of view of three donors. For each donor, three independent fields of view with at least 100 cells were analysed. Statistical analysis was performed on the means of different donors by pairwise comparison of GM-Mφ vs. M-Mφ at the different MOIs using Student's <i>t</i>-test (n.s.: not significant).</p

Phagocytosis of <i>Lactococcus lactis</i> (A), <i>Escherichia coli</i> (B), <i>Leishmania major</i> (C) and HCMV (D) by GM-Mφ (GM, black bars) and M-Mφ (M, white bars) generated from monocytes isolated by negative (neg.) or positive (pos.) selection.

<p>Cells were infected with all microorganisms at an MOI of 1. Phagocytosis was determined as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066898#s2" target="_blank">Materials and Methods</a> expressed as CFU/well or infection rate (%). Results from one representative donor measured in triplicate are shown and similar results were obtained with cells of at least three different donors. Statistical analysis was performed by pairwise comparison of GM-Mφ vs. M-Mφ using Student's <i>t</i>-test.</p

Phagocytosis of fluorescent latex beads by GM-Mφ and M-Mφ generated from monocytes isolated by negative (neg.) or positive (pos.) selection as measured by flow cytometry.

<p>Histogram plots show macrophages incubated with latex beads (hatched area) and cells alone (black lines) as control. Percentage of macrophages which are positve for latex beads are indicated in each histogram plot. Results from one representative donor are shown and similar results were obtained with cells of at least three different donors.</p

Surface expression of CD14, CD163 and CD206 on untreated THP-1 cells (mock) or after differentiation into macrophage-like cells by stimulation with PMA alone (PMA) or in combination with 50 ng ml⁻¹ M-CSF (M+PMA) or GM-CSF (GM+PMA).

<p>All experiments were performed in triplicates (n = 3). Histogram plots show surface marker staining as hatched areas and isotype controls as black lines.</p

Phagocytosis of <i>Lm</i> by GM-Mφ (GM, black bars) or M-Mφ (M, white bars) generated from monocytes isolated by negative (A) or positive (B) selection or THP-1 macrophages (C) at different multiplicities of infection (MOI).

<p>Phagocytosis is measured as colony forming units (CFU) per well. Results from one representative donor (A and B) or experiment (C) measured in triplicates are shown and similar results were obtained with cells of at least three different donors or in three independent experiments. Statistical analysis was performed by pairwise comparison of GM-Mφ vs. M-Mφ at the different MOIs using Student's <i>t</i>-test (n.s.: not significant).</p