Morphological Diversity of Buccopharyngeal Armatures in Susceptible and Refractory Sandflies to Leishmania major

Citation: Egypt. Acad. J. Biolog. Sci. (A. Entomology) Vol. 10(2)pp: 1524 (2017) Egyptian Academic Journal of Biological Sciences is the official English language journal of the Egyptian Society for Biological Sciences, Department of Entomology, Faculty of Sciences Ain Shams University. Entomology Journal publishes original research papers and reviews from any entomological discipline or from directly allied fields in ecology, behavioral biology, physiology, biochemistry, development, genetics, systematics, morphology, evolution, control of insects, arachnids, and general entomology. www.eajbs.eg.net Provided for non-commercial research and education use. Not for reproduction, distribution or commercial use.


INTRODUCTION
Leishmania are digenetic protozoa which inhabit two highly specific hosts, the sandfly where they grow as motile, flagellated promastigotes in the gut, and the mammalian macrophage where they grow intracellularly as non-flagellated amastigotes.According to the World Health Organization there are 2 million new cases each year and 1/10 of the world's population is at risk of infection (Gravino, 2004).The vectors of leishmaniasis are phlebotomine sandflies belonging to the genera Phlebotomus.In Egypt, P. papatasi is proved to be the vector of L. major (Wahba et al., 1990 andHanafi et al., 1998) from rodents to human (El Hossary et al., 2000).Like other blood-sucking nematoceran insects, the cuticle-lined foregut of P. papatasi comprises the biting mouthparts, the cibarium and the pharynx.The latter two are modified into pumps flanked by the cibarial and stomodaeal valves, which regulate blood flow into the midgut (Jobling, 1987).
The pharyngeal armature of sandfly consisted of pointed-teeth with various shape, number and arrangement among different species (Guo et al., 2004).The topology of the internal surfaces of the sandfly female's alimentary tract is directly relevant to the development of Leishmania promastigotes within it (Killick-Kendrick, 1999).Accordingly, the present work aims to study the structure diversity of armatures in susceptible and refractory sandflies to Leishmania parasite.

MATERIALS AND METHODS
Sandflies: Phlebotomine sandflies were colonized at the Research and Training Center (RTC), Ain Shams University.Phlebotomus papatasi and sergenti colonies were set up from gravid females caught in CDC light traps from Suez and southern Sinai, governorates, respectively while Sergentomyia spp.were collected from North Sinai.Colonies were maintained at 27±2°C and 80% R.H. Adults had permanent access to cotton pad soaked in saturated sucrose solution.
Females were allowed to feed on anesthetized (Thiopental) golden hamsters twice per week.

Morphological Diversity of Armatures in Susceptible and Refractory Sandflies to Leishmania:
For light microscopy, the adult females were narcotized with carbon dioxide and then the head is removed from the body and mounted ventral side uppermost in a thin film of medium, thus allowing examination at high magnification.Photographs were taken by interference microscopy connected with computer.Variations between the cibarium and the pharynx of Phlebotomus as susceptible species and Sergentomyia as refractory species were examined under objectives with phasecontrast or interference illumination.

RESULTS AND DISCUSSION
Leishmania parasites develop as extracellular promastigotes that are attached to the alimentary canal wall of the sand fly vector (Killick-Kendrick, 1979).The cibarium and the pharynx, which function as pumping organs, are lined with cuticle (Warburg, 2008).The author found also that the cibarium and the pharynx bear diverse types of cuticular spines and appendages.He added that sand fly pharyngeal armatures consist of pointed-teeth with a variety of shape, number and arrangement among different species.To evaluate the relationship between Leishmania development and the presence of sandfly foregut armatures, the foregut of females of P. papatasi, P. sergenti, S. squamipleuris and S. christophersi were examined using light microscope for the structure of pharyngeal pump and cibarium in each of them.
In the present study, light microscope photos showed the features of pharyngeal pump in the four selected sandfly species.Large variations were detected in the size, shape and numbers of the pharyngeal armatures in the various tested species.Phlebotomus papatasi females have fleshy pharynx, tapering after posterior bulge, armature consisting of many small scales with fringe of fine backward-pointing teeth (Fig. 1A) and without cibarial armature or pigment patch (Fig. 1B).Phlebotomus sergenti females have a pharynx with large scales anteriorly, some produced into long broad spines, scales becoming broader and flatter posteriorly with hind margin serrated with fine teeth (Fig. 2A).Oppositely, S. squamipleuris females have tapering pharynx abruptly posteriorly, with rows of angular teeth (Fig. 3A).The size and shape of the cibarial pump varied from one species to another being markedly larger and more pointed in S. squamipleuris (Fig. 3B) and S. christophersi (Fig. 4B) than the two Phlebotomus tested species (Fig. 1B, 2B).Cibarial armatures are arranged in two rows of tiny cones and rods and sharp long pointed spines (Fig 3B ,  4B).Structure of the pharyngeal armatures in these Leismania refractory species are characterized by developing three to four rows of small weak warts with flattened edges (Fig 3A).In S. squamipleuris, cibarium with convex rows of fine, parallel horizontal teeth, undulating row of vertical teeth and pigment patch small, tapering anteriorly, broad, chitinised, transverse band are present (Fig. 3B).
Pharyngeal armatures in S. christophersi reflect its refractoriness to Leishmania by having minute scattered spines (Fig. 4A), few scale-like folds anteriorly and a series of vague ridges posteriorly.Moreover, cibarium with well developed large sharp strong blades with pointed spines arranged in two rows of eight blades in the first row and five to six small spiny blades are present (Fig. 4B).Such differences may provide the morphological proof for identification of species (Randrianambinintsoa et al., 2013 andMéndez-de Daboín et al., 2015).Léger et al. (2005) described S. goodmani, the pharynx being narrow, and the cibarial armature comb-like.The presence of anterolateral teeth of the pharyngeal armature in P. chabaudi, never observed in P. riouxi helped to differentiate between the two closely related species (Bounamous et al., 2008).The inclusion of P. barguesae n. sp. in the subgenus Euphlebotomus is acceptable on the basis of characters of the male genitalia and of female pharyngeal armature (two kinds of teeth) (Depaquit et al., 2009).
In the context of large size parasite transmission, it has been suggested that the shape and density of the cuticular spines can influence the capacity to kill microfilaria.Thus, Culex pipiens that lack developed pharyngeal or cibarial pumps is more competent vector of Wuchereria bancrofti than Anopheles multicolor and Aedes caspius having respectively well developed cibarial and pharyngeal armatures that kill microfilariae (Shoukry and Soliman, 1995).Cuticular pharyngeal armature in mosquitoes has also been shown to provoke haemolysis (Chadee et al., 1996).However, in the case of Leishmania and other protozoan parasites, it is doubtful that mechanical disruption plays a role in preventing infection.Leishmania promastigotes frequently colonize the pharynx, cibarium and mouthparts of infected sand fly females by attaching to the cuticular surfaces (Killick-Kendrick et al., 1988 andWalters et al., 1989).
In related studies, P. fengi, a suspected visceral leishmaniasis vector in China, had distinct pharyngeal armature (Leng and Zhang, 1994).Within the fallax-group, S. fallax was distinguished readily by its heart-shaped pharynx and the variation in armature between the dorsal and lateral plates (Benabdennbi et al., 1996).For comparison, females of P. sergenti and P. similes differ by the armature morphology of the pharynx and by the rings number of the spermatheca (Depaquit et al., 1998).Females of P. fertei and P. huberti have a characteristic pharyngeal armature bearing about ten cibarial teeth (Depaquit et al., 2002).The female of P. fertei was described from Madagascar (Depaquit et al., 2004), having pharyngeal armature with small teeth and cibarium with 15 to 30 denticles.

CONCLUSION
The present study supports the hypothesis that armature structures of sand flies are directly related to their vectorial capacity to Leihmania parasite.