Details of Digestive Organ of Bemisia tabaci Biotype B, the Vector of Begomoviruses by Electron Microscopy

Document Type : Research Article

Authors

1 Isfahan Agricultural and Natural Resources Research and Education Center

2 Fars Agricultural and Natural Resources Research and Education Center, (AREEO), Shiraz, Iran

3 , Khorasan Razavi Agricultural and Natural Resources Research and Education Center, (AREEO), Mashhad, Iran

Abstract

Introduction: Whitefly, Bemesia tabaci Bemisia (Gennadius) (Hemiptera: Aleyrodidae) is the key pest of many crops worldwide, directly damages the plant by feeding and indirectly by transmitting the virus. The Begomoviruses in the family Geminiviridae are transmitted by B. tabaci through a circulative and persistent manner. For some insect vectors of plant viruses like aphids, information has been collected regarding their digestive tract anatomy and cell structure in relation to the formation of virus receptor for successful transmission. In B. tabaci, such structural details are poorly understood. This study was performed to determine the details of the digestive organs of B. tabaci.
Materials and Methods: Ultrathin sections from important regions of the digestive tract of the insect were prepared and studied by electron microscopy according to a conventional procedure (Cristofoletti et al., 2003). After dissection of the alimentary tract, the tissue was fixed in Karnovsky solution and Osmium tetraoxide, 8 and 1.5 hours, respectively. After rinsing in sodium cacodylate buffer, the tissue was dehydrated in successive alcohols grades. Then the tissue was embedded in resin and ultrathin sections (50 nm) were prepared using a U3 ultra-microtome. These sections were stained in Uranyl acetate and Lead Citrate then were examined by a Philips CM10 electron microscope.
Alimentary canal of B. tabaci was dissected in a few drops of 260 mM NaCl buffer located on a microscope slide on top of an ice block. Dissected tissues were homogenized in a motorized potter-elvehjem homogenizer (Teflon pestle, 0.1 mm clearance) for 3 minutes at 500 rpm. Enzyme assay methods were according to (Allahyari et al., 2010). Briefly, α and β-glucosidase activity were measured using 5 mM α and β-D, 4-nitrophenyl glucopyranoside in 50 mM citrate– phosphate buffer pH 5.0, respectively, based on the appearance of p-nitrophenol in the solution. Protein concentration was measured according to the method of Bradford (1976), using bovine serum albumin (Bio-Rad, Munchen, Germany) as a standard.
Results and Discussion: Observations showed that ascending and descending midgut are composed of thick epithelial cells with microvilli (MV) extending into the large lumen. These microvilli appeared to join each other and tighten by trabeculae. Furthermore, the Modified Premicrovillar Membranes (MDPMV) are sent into the lumen through the inter-lamellar space in the form of small vesicles between microvilli (fig. 2, A). These membranes are synthesized in Golgi cisternae and then fused whit with the microvillar membrane at the base of microvilli. Based on our knowledge this is the first report describing MPMV in B. tabaci. The receptor position for begomoviruses is probably on microvillar membrane as reported by other vectors however, MDPMV may have some roles in virus transmission processes. In order to study the possibility of isolating these membranes, we measured the activity of membrane-bound enzymes, α and β- glycosidase reported in other Hemipetran insects (Silva et al., 1996). Assessing specific activities of these enzymes showed that they were not present or active in B. tabaci gut. The specific activity of α and β- glycosidase in the gut homogenate of B. tabaci were 0.34 and 0.053 mU/mg protein whereas in Eurygaster integriceps first part of midgut homogenate mean specific activity of these enzymes were 316.97 and 27.93, respectively (Allahyari et al., 2010). Previous research also found that other enzymes may be involved in trehalose formation and decreasing osmotic pressure due to high sucrose concentration in phloem sap (Salvucci, 2000).
Conclusion: Binding sites of the virus particles on an inner surface of the alimentary canal, which most probably has a role in the absorbing region of this organ, the midgut and the inner surface of the midgut are covered by microvilli. Thus the virus particles firstly must be bind to the microvillar membrane. Glycoproteins and other transmembrane proteins are possible binding sites. Modified perimicrovillar membranes also may have an important role in virus transmission and digestion. In the process of digestion free amino acids that are abundant in phloem sap are trapped in the MDPMV and in this way making amino acid absorption easier by increasing the concentration of amino acids (Cristofoletti et al., 2003). In order to study the physiology of virus interaction with midgut, it is necessary to isolating microvillar and perimicrovillar membrane. Investigations using other candidate enzymes like aminopeptidase and also lectin binding properties are powerful markers to isolate these membranes for future studies.

Keywords


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