Identification and ultrastructural characterization of the Wolbachia symbiont in Litomosoides chagasfilhoi

Abstract

Background: Filarial nematodes are arthropod-transmitted parasites of vertebrates that affect more than 150 million people around the world and remain a major public health problem throughout tropical and subtropical regions. Despite the importance of these nematodes, the current treatment strategies are not efficient in eliminating the parasite. The main strategy of control is based on chemotherapy with diethylcarbamazine, albendazole and ivermectin. In the 1970s, it was found that some filarids possess endosymbiotic bacteria that are important for the development, survival and infectivity of the nematodes. These bacteria belong to the genus Wolbachia, which is a widespread and abundant intracellular symbiont in worms. Knowledge about the structure of the bacteria and their relationship with their nematode hosts may allow new perspectives for the control of filarial nematodes.

Methods: In this study, we used transmission electron microscopy combined with three-dimensional approaches to observe the structure of the endosymbiont of the filarial nematode Litomosoides chagasfilhoi, an experimental model for the study of lymphatic filariasis. In addition, the bacterium was classified based on PCR analyses.

Results: The bacterium was mainly found in the hypodermis and in the female reproductive system in close association with host cell structures, such as the nucleus and endoplasmic reticulum. Our ultrastructural data also showed that the symbiont envelope is composed of two membrane units and is enclosed in a cytoplasmic vacuole, the symbiosome. Molecular data revealed that the bacterium of L. chagasfilhoi shares 100% identity with the Wolbachia endosymbiont of Litomosoides galizai.

Conclusions: Here we described ultrastructural aspects of the relationship of the Wolbachia with the filarial nematode Litomosoides chagasfilhoi and the findings lead us to consider this relationship as a mutualistic symbiosis.

Figure 1

Radial phylogenetic tree of 12 nematode endosymbionts species based on the analysis of 705 nucleotides of 16S rDNA. Bootstrap values >90 are indicated. Endosymbiont of Litomosoides chagasfilhoi (this work); DQ408758, Gammaproteobacterium endosymbiont of Astomonema sp. clone LSI-A2; DQ408757, Gammaproteobacterium endosymbiont of Astomonema sp. clone LSI-A1; DQ314214, Candidatus Paenicardinium endonii; AY278355, Wolbachia endosymbiont of Mansonella perstans isolate M02-052 N; AJ548800, Wolbachia pipientis, specific host Litomosoides galizai; AJ548799, Wolbachia pipientis, specific host Litomosoides brasiliensis; AJ279034, Wolbachia endosymbiont of Mansonella ozzardi; AJ276500, Wolbachia endosymbiont of Dirofilaria repens; AJ276499, Wolbachia endosymbiont of Onchocerca gibsoni; AJ012646, Wolbachia endosymbiont of Brugia pahangi; AF069068, Endosymbiont of Litomosoides sigmodontis.

Figure 2

Transmission electron microscopy revealing that the symbiotic bacterium is present in different tissues of Litomosoides chagasfilhoi. A – General view of the body wall showing the cuticle (C) and a bacterium (black arrowhead) present on the hypoderm (H) (Bar 2 μm). B – At high magnification, it is possible to observe a dividing bacterium on the hypoderm. It is worth noting that a classical septum is absent in the central constriction region (arrow) (Bar 5 μm). C and D – Transversal section showing the body wall composed of the cuticle (C), hypoderm (H), muscle layer (m) and proximal region of the ovary showing the ovary wall (Ow) and oocytes (Oc). At higher magnification, the endosymbiotic bacteria (arrowheads) are observed in the cytoplasmic oocytes and nucleus (N) (Bar = 50 μm in C and 20 mm in D). E – Longitudinal view of microfilariae (M) showing the nucleus (N) of sub-cuticular cells and endosymbiotic bacteria (arrowheads). Bar = 2 μm. F – At higher magnification, it is possible to note that in the microfilariae (M), the endosymbiont is inside a vacuole (arrowhead) close to the host cell nucleus (N) (Bar 0.5 μm.)

Figure 3

Transmission electron microscopy showing the Litomosoides chagasfilhoi symbiont ultrastructure. A - D: symbionts observed in early-embryo stages. (A) Endosymbiotic bacteria (arrowheads) were observed inside a vacuole (V) and close to the nucleus (N). (B) The symbiont matrix is composed of an electron-dense region rich in ribosomes (small white arrow), and an electron-lucid area that contains the DNA fibers (f – open arrowhead) (Bar 0.5 μm). C – At high magnification, it is possible to observe the symbiont in close proximity to the host cell nucleus (N). An electron-dense region is indicative that the symbiont vacuole touches the nuclear envelope (white arrow). It is also interesting to note the association (small black arrow) between the symbiont and the endoplasmic reticulum (RE) containing ribosomes (R). (Bar 0.5 μm). D – The endosymbiont is enclosed by two membrane units: an outer membrane (OM) that faces the vacuole (V) and an inner membrane (IM) that faces the matrix. Note that the symbiont electron-lucid area in the matrix contains DNA fibers (open arrowhead) and that the bacteria are associated with the endoplasmic reticulum (R) with associated ribosome (Bar 0.5 μm).

Figure 4

Ultrastructural cytochemistry of Litomosoides chagasfilhoi by OsKI staining. A – General view of the body wall showing the cuticle (C) and hypoderm (H) that present the endosymbionts (E). There is a positive reaction specific to regions near the bacteria (thin arrow) (Bar 20 μm). B – Note the electron-dense and reticular structures close to the endosymbiont (thin arrow) (Bar 20 μm). C – Detail showing the intimate association between the bacterial membrane and the electron-dense structures that resemble the membranes of the endoplasmic reticulum (thin arrow) (Bar 10 μm).

Figure 5

Electron tomography of endosymbiotic bacteria of Litomosoides chagasfilhoi. A-C – Tomographic series of the bacteria present in the hypodermis, showing the vacuole membrane (V), outer membrane (OM) and regions that present DNA fibers (large arrow) and ribosomes (R) (Bar 5 μm).

Figure 6

Electron tomography and 3-D model of endosymbiotic bacteria of Litomosoides chagasfilhoi. A: Tomographic series showing a bacterium in an embryo cell. The bacterium is located in a vacuole (V) near the nucleus (N), and it was possible to observe the outer membrane (OM) and some association with the endoplasmic reticulum (ER) (Bar 5 μm). B: 3-D model obtained from a tomographic series (yellow: nucleus, green: symbiont outer membrane, light blue: vacuole membrane and pink: endoplasmic reticulum). C: Tomographic series showing a bacterium and the vacuole membrane (V) in close association with the nucleus (N) of the nematode host (Bar 5 μm). D: Detail of the vacuole (V) and nucleus (N) proximity (Bar 5 μm). E: 3-D model obtained from a tomographic series (yellow: nucleus, green: symbiont outer membrane and light blue: vacuole membrane).