Morphological regional differences of epithelial cells along the midgut in Diatraea saccharalis Fabricius (Lepidoptera: Crambidae) larvae / Diferenças morfológicas regionais das células epiteliais ao longo do intestino médio de larvas de Diatraea saccharalis Fabricius (Lepidoptera: Crambidae)

The sugarcane borer, Diatraea saccharalis Fabricius, is a pest to sugarcane and many other crops. This work aims to characterize morphological variability in the epithelial cells (columnar, goblet and regenerative) along the midgut of D. saccharalis larvae. Fragments of the midgut (anterior, middle and posterior regions) were fixed and processed by light and scanning electron microscopy. There are both cytochemical and ultrastructural differences in the morphology of the epithelial cells, depending on their localization along the midgut. The apical surface of columnar cells shows an increase in both number and size of the apical protrusions from the anterior to the posterior midgut regions. There is an increase in the amount of PAS-positive (Periodic Acid-Schiff Reaction) granules detected in the cytoplasm of both the columnar and regenerative cells, from the anterior to the posterior region. The goblet cell apical surface is narrow in the anterior region, and enlarged in the posterior midgut; the chamber's cytoplasm extrusion are small and thin at the apical cavity surface, being thicker, longer and more numerous at the basal portion of the cavity. Our results suggest that the sugarcane borer midgut has two morphologically different regions, the anterior and the posterior; the middle region is a transitional region.

A broca da cana, Diatraea saccharalis Fabricius, é uma praga da cana-de-açúcar e outras plantações. O objetivo deste trabalho foi caracterizar variações morfológicas nas células epiteliais (colunares, caliciformes e regenerativas) ao longo do intestino médio de larvas de D. saccharalis. Fragmentos do intestino médio (anterior, mediano e posterior) foram fixados e processados para microscopia de luz e eletrônica de varredura. Existem diferenças morfológicas citoquímicas e ultra-estruturais nas células epiteliais, dependendo da sua localização no intestino médio. A superfície apical de algumas células colunares exibe projeções citoplasmáticas que aumentam em número e volume da região anterior para a posterior do intestino médio. Existe aumento dos grânulos PAS-positivos (Reação de Schiff) no citoplasma apical das células colunares e regenerativas, da região anterior para a posterior. A câmara das células caliciformes, na região anterior do intestino médio, mostra seu ápice estreito, enquanto que na posterior essa porção da câmara é alargada; as evaginações citoplasmáticas da câmara são pequenas e finas no ápice, sendo numerosas, longas e mais espessas na porção basal. Os resultados sugerem que o intestino médio da broca da cana apresenta duas regiões morfologicamente distintas, a anterior e posterior; a região mediana é uma região de transição.

Characterization of a newly established potato tuber moth (Phthorimaea operculella Zeller) cell line.

BACKGROUND & OBJECTIVE: Potato tuber moth (PTM), Phthorimaea operculella Zeller is a widely distributed, devastating pest of potatoes attacking the foliage and infest the tubers in both field and store causing serious economic damage. As application of PTM granulovirus (PTM-GV) has shown significant reduction in damage, attempts were made to develop a new cell line from this insect to grow PTM-GV for use as a biopesticide. METHODS: Approximately 100 mg of insect eggs were collected, surface sterilized and crushed gently in a boiling tube aseptically. The tissues were washed with physiological saline, suspended in growth medium and incubated stationary at 28 degrees C. Morphology of cells was studied after staining with Giemsa. Besides karyological and growth curve studies, PCR amplification was also done for rapid amplified polymorphic DNA pattern. RESULTS: A new cell line from the embryonic tissue of PTM was maintained in Mitsuhashi Maramorosch medium supplemented with 10 per cent foetal bovine serum. It is in the 78th passage level and designated as NIV-PTM-1095. Random amplified polymorphic DNA profile analysis indicated this as a new cell line from potato tuber moth and differed from the profiles of two other lepidopteran cell lines maintained in the laboratory. Three different cell types were observed at the 40th passage level and comprised of epithelial-like cells (77%), fibroblast-like cells (20%) and giant cells (3%). The chromosome number varied from 54-176. The cell line had a cell doubling time of approximately 42 h during the logarithmic phase of growth. The cell line did not support the multiplication of any of the baculoviruses used in the study. INTERPRETATION & CONCLUSION: Since the new cell line is found to replicate PTM-GV, it may be useful for the propagation of PTM-GV in large scale. Studies to scale up the production of the GV in the cell line and field trials may lead to its widespread use as an eco-friendly biopesticide.

Baculovirus studies in new, indigenous lepidopteran cell lines.

Eight lepidopteran cell lines were established recently and their susceptibility to different insect viruses was studied. Two Spodoptera litura cell lines from the larval and pupal ovaries, were found highly susceptible to S. litura nuclear polyhedrosis virus (SLNPV, 5-6 x 10(6) NPV/ml). The Helicoverpa armigera cell line from the embryonic tissue was highly susceptible to H. armigera NPV (HaNPV, 6.3 x 10(6) NPV/ml). These in vitro grown SLNPV and HaNPV caused 100% mortality to respective 2nd instar larvae. The susceptibility of the cryo-preserved cell lines to respective baculoviruses (SLNPV/HaNPV) was studied and no significant difference in their susceptibility status was observed. The cultures could grow as suspension culture on shakers and may find application for in vitro production of wild type/recombinant baculoviruses as bio-insecticides. S. litura and Bombyx mori cell lines from larval ovaries, were highly susceptible to Autographa californica NPV (5.5 x 10(6) NPV/ml) and Bombyx mori NPV (BmNPV, 6.1 x 10(6) NPV/ml) respectively. These cell lines may find application in baculovirus expression vector studies for the production of recombinant proteins, useful in the development of diagnostic kits or as vaccines.

New Helicoverpa armigera Hbn cell line from larval hemocyte for baculovirus studies.

A new cell line from the larval hemocytes of H. armigera was established in Grace's medium modified by adding lactalbumin hydrolysate and yeastolate (3.3g/l), and supplemented with fetal bovine serum (20%). The cell line was designated as NIV-HA-1195. The cell population at P-78 consisted mainly of epithelial-like cells (89.36%), fibroblast-like cells (8.31%) and giant cells (2.13%). The population doubling time was 96hr at P-8, 60hr at P-43. The chromosome number ranged from 45 to 200. The cell line is susceptible to the baculoviruses, Autographa californica nucleopolyhedrovirus (AcNPV), Spodoptera litura NPV and the homologous HaNPV. Isoenzyme profile and results of 16S rRNA heteroduplex analysis clearly indicated the species specificity of the new cell line.

Changes in haemolymph constituents of American bollworm, Helicoverpa armigera (Hübner), infected with nucleopolyhedrovirus.

Six types of haemocytes viz., prohaemocytes, plasmatocytes (round, fusiform, vermiform and spindle shaped), granular cells, spherule cells, oenocytoids and adipohaemocytes were found in the haemolymph of larvae of American bollworm H. armigera. The total and differential haemocyte counts (THC and DHC) in H. armigera haemolymph were affected by nucleopolyhedrovirus (NPV) treatment. There was a general decrease in THC in response to NPV treatment in both young and old larvae. However the decrease was more apparent in 5 and 8 day old larvae than in 10 day old larvae. The differential haemocytes showed less of granular cells and more of spherule cells and prohaemocytes in the old larvae. Plasmatocytes and granular cells in 10 day old larvae initially phagocytosed polyhedra; however, disintegrated after 3 to 4 hr. The haemolymph of NPV treated larvae melanized slowly particularly in old larvae. Phenoloxidase (PO) activity decreased positively with granular cells and oenocytoids in 10 day old treated larvae. Cellular fraction had high level of PO activity, which was transferred to plasma in response to NPV infection in the older larvae. The role of NPV pathogenesis vis-à-vis immunity in insect is discussed.