پديد آورندگان :
رستگار، مريم سازمان تحقيقات آموزش و ترويج كشاورزي - مركز تحقيقات و آموزش كشاورزي و منابع طبيعي استان اصفهان - بخش تحقيقات گياه پزشكي، اصفهان , الهياري، مرتضي سازمان تحقيقات آموزش و ترويج كشاورزي - مركز تحقيقات و آموزش كشاورزي و منابع طبيعي استان فارس - بخش تحقيقات گياه پزشكي، شيراز , قاروني كارداني، سارا سازمان تحقيقات آموزش و ترويج كشاورزي - مركز تحقيقات و آموزش كشاورزي و منابع طبيعي استان خزاسان رضوي - بخش تحقيقات گياه پزشكي، مشهد
كليدواژه :
بافت‌شناسي حشرات , ساختار معده , سفيدبالك , ويروس پيچيدگي برگ زرد گوجه‌فرنگي
چكيده فارسي :
سفيدبالك Bemisia tabaci از آفات مهم محصولات زراعي و باغي، به‌صورت مستقيم با تغذيه و به‌طور غيرمستقيم با انتقال ويروس به گياه خسارت مي‌زند. ويروس‌هاي جنس Begomovirus از تيره Geminiviridae توسط B. tabaci به حالت گردشي و پايا انتقال مي‌يابند. مطالعه آناتومي داخلي ناقلين، سلول‌ها و ارگان‌هاي درگير در انتقال، يك پيش‌نياز براي بررسي برهم‌كنش تعيين كننده‌هاي ويروسي و سلولي در تداوم گيرش و انتقال ويروس است. در بعضي ناقل‌ها مانند شته‌ها، مطالعاتي در اين زمينه صورت گرفته است، ولي در B. tabaci اطلاعات موجود اندك مي‌باشد. به‌منظور تعيين جزئيات دستگاه گوارش اين حشره و تعيين بخش‌هاي درگير در انتقال ويروس، از قسمت‌هاي مختلف دستگاه گوارش بيوتيپ B اين حشره، مقاطع نازك تهيه و با ميكروسكوپ الكتروني تصويربرداري گرديد. مشخص شد كه در سلول‌هاي ديوارۀ معده مياني، جلويي و عقبي ميكروويلي‌هايي (MV) وجود دارد، كه كاملاً به هم چسبيده‌اند. به‌علاوه غشاهاي تغيير‌يافته پريميكروويلار (MDMV) به‌صورت وزيكل‌هاي كوچك از لابه‌لاي ميكروويلي‌ها به درون فضاي معده فرستاده مي‌شوند. در اين مطالعه MPMV براي اولين بار در مورد B. tabaci توصيف مي‌شود. غشاهاي MV و MDMV ممكن است محل اتصال ويروس‌ها و در واقع جايگاه گيرنده بگوموويروس‌ها باشد. در راستاي جداسازي اين غشاها و مطالعات بيشتر روي آن‌ها، لازم است از آنزيم‌هاي متصل به غشاها، به‌عنوان ماركر استفاده شود. براي اين منظور ميزان فعاليت ويژه دو آنزيم آلفا و بتا گلوكوزيداز مورد بررسي قرار گرفت، كه قابل توجه نبود؛ لذا احتمالا اين آنزيم‌ها متصل به غشاهاي مذكور در B. tabaci نمي‌باشند. به دليل اهميت موضوع، آنزيم‌هاي ديگري مانند آمينوپپتيداز نيز بايد مورد بررسي و مطالعه قرار گيرند.
چكيده لاتين :
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 pnitrophenol 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.
