Print version ISSN 1519-566X
Neotrop. Entomol. vol.32 no.3 Londrina July/Sept. 2003
SYSTEMATICS, MORPHOLOGY AND PHYSIOLOGY
Morphometric study of the midgut epithelium in larvae of Diatraea saccharalis Fabricius (Lepidoptera: Pyralidae)
Estudo morfométrico do epitélio do intestino médio em larvas de Diatraea saccharalis Fabricius (Lepidoptera: Pyralidae)
Daniela O. PinheiroI; Reinaldo J. SilvaII; Irani Quagio-GrassiottoI; Elisa A. GregórioI
IDepto. Morfologia, Instituto de Biociências, Universidade Estadual Paulista - UNESP Campus de Botucatu, 18618-000, Botucatu, SP
IIDepto. Parasitologia, Instituto de Biociências, Universidade Estadual Paulista - UNESP Campus de Botucatu, 18618-000, Botucatu, SP
The sugarcane borer, Diatraea saccharalis Fabricius, has great economical interest as it affects the culture and industrial use of the sugarcane. However, there are few studies concerning the internal morphology of this insect. This work aims to study morphometrically the midgut and the epithelium along their lenght, trying to characterize different regions. Midgut of last instar larvae was divided in three regions: anterior, middle and posterior, and the fragments were processed for light microscopic observation. Histological sections were analyzed in a computerized system concerning the length, width and area of the epithelium, their cells, and the midgut lumen. The obtained data were statistically analyzed by the Kruskal-Wallis test and by multivariate analysis. Our results showed that the midgut has two different regions, the anterior and the posterior; the middle region presents values that are coincident with the ones of either the anterior and the posterior portions, suggesting that there is an intermediate region between the other two ones. The epithelial cells (columnar, goblet and regenerative cells), when evaluated by multivariate analysis, do not present significant morphometric differences in the different midgut regions. However, the analysis of variance for separate variables show that the regenerative cells present wide morphometric variability along the midgut.
Key words: Morphometry, epithelial cell, insect, sugarcane borer, mesenteron
A larva de Diatraea saccharalis Fabricius (broca-da-cana) tem grande interesse econômico, pois afeta o cultivo e aproveitamento industrial da cana-de-açúcar. Entretanto, poucos são os estudos sobre a morfologia interna desse inseto. O objetivo deste trabalho foi estudar, morfometricamente, o seu epitélio intestinal, ao longo de seu comprimento, visando caracterizar regiões estruturalmente diferentes. O intestino médio de larvas no último instar foi subdividido em três regiões: proximal, mediana e distal e os fragmentos foram processados para observação em microscopia de luz. Os cortes histológicos foram analisados em sistema computadorizado de análise de imagens para medir comprimento, largura e área do epitélio, das diferentes células epiteliais, dos seus respectivos núcleos e do lúmen intestinal. Os dados obtidos foram submetidos ao teste estatístico de Kruskal-Wallis e à análise multivariada. Nossos resultados mostraram que o intestino médio apresentou-se constituído, morfometricamente, por duas diferentes regiões, proximal e distal; a região mediana apresentou valores coincidentes tanto com a região proximal quanto com a distal, sugerindo ser região intermediária. As células epiteliais (colunares, caliciformes e regenerativas), quando avaliadas pela análise estatística multivariada, não apresentaram diferença morfométrica nas diferentes regiões do intestino médio. Entretanto, a análise de variância, realizada para variáveis isoladas, mostrou que as células regenerativas apresentaram maior variabilidade morfométrica.
Palavras-chave: Morfometria, célula epitelial, inseto, broca da cana, mesêntero
In Lepidoptera four cell types mainly compose the midgut epithelium: columnar, goblet, regenerative and endocrine cells (Lehane & Billingsley 1996). The predominant columnar cells are responsible for processing the diet, secretion of the digestive enzymes and the uptake of the final products (Lehane & Billingsley 1996). The goblet cells co-operate with the columnar cells in ionic homeostasis and metabolite absorption (Lello et al. 1984, Chiang et al. 1986). Scattered throughout the epithelium there are relatively undifferentiated regenerative cells found singly, paired or in group (Turbeck 1974, Cavalcante & Cruz-Landim 1999). The scarce endocrine cells are variable in shape being pyramidal, bowl-shaped, oval or fusiform (Andries & Beauvillain 1988, Cavalcante & Cruz-Landim 1999).
Although all cell types can be found in the midgut extension, many works suggest that the distribution, morphology and function of these cells may be variable along the midgut length (Cioffi 1979, Lello et al. 1984, Santos et al. 1984, Chiang et al. 1986, Lehane & Billingsley 1996, Cristofoletti et al. 2000). These morphological differences are mainly detected at ultrastructural level for all the cell types (Cioffi 1979, Lello et al. 1984, Santos et al. 1984, Lehane & Billingsley 1996).
The sugarcane borer, Diatraea saccharalis Fabricius, is a serious pest of sugarcane and many others crops including maize, sorghum, corn and rice (Long & Hensley 1972). Preliminary study showed that their columnar epithelial cells present differences in the ultrastructural organization that can be related with their localization in the midgut (Pinheiro & Gregório 2001). This finding has been previously described for other Lepidoptera, such as Manduca sexta L. (Cioffi 1979), Spodoptera frugiperda Smith (Jordão et al. 1999) and Erinnyis ello L. (Santos et al. 1984).
However, there are few morphometric studies of the epithelial cells along insect midgut, most of them in Diptera (Nopanitaya & Misch 1974, Wood & Lehane 1991, Andrade-Coeho et al. 2001), few of them in Heteroptera (Ranjini & Mohamed 2000) and Hemiptera (Billingsley 1988), but none in Lepidoptera.
This work aims to study morphometrically the midgut of the D. saccharalis larvae. Measurements of the midgut, their lumen and epithelium, as well as of the columnar, goblet, and regenerative cells were obtained along their lenght, in an attempt to distinguish differential regions in this organ.
Material and Methods
D. saccharalis larvae were reread on artificial diet (Hensley & Hammond 1968) and maintained under controlled temperature (25-27ºC) and humidity (70%). Fifty insects at the beginning the last larval instar (12-17 days of development) were dissected under stereomicroscope and the midgut were immediately fixed in 2% glutaraldehyde-4% paraformaldehyde solution in 0.1 M phosphate buffer (pH 7.3) for 24h.
The midgut was equally divided in three fragments, named anterior, middle and posterior regions. These fragments were processed and embedded in Historesin (JB4-Polysciences); tissue sections were stained either with Toluidine blue (Pearse 1972) or with Schiff's reagent-Bromophenol blue-Ehrlich's haematoxylin (Coello 1989).
Histological cross sections were morphometrically analyzed in computerized system for analysis of images (QWin Lite 2.5 - Leica), adapted in a DMLB light microscope (Leica). The measurement obtained were: 1 - the larger (LAx) and smaller (SAx) axis, and the area (A) of the a) lumen; b) each epithelial cell type and their nucleus; and c) midgut (epithelium + lumen); 2 - the height (H) of the epithelium, expressed as the mean, as well as the maximum (HMax) and the minimum (HMin) values of 20 measurements throughout the midgut perimeter. All measurements were taken in the central region of either the cell or the midgut lumen.
The obtained data were statistically analyzed by the Kruskal-Wallis test, and the difference among the groups was determined by the Dunn's method, using the Sigma-Stat 2.0 software. Significance level was established at 5%. The multivariate analysis, accomplished by the MVSP 3.2 software, was also used to compare the lumen and the different epithelial cell types in the different regions of the midgut.
The D. saccharalis midgut is a simple tube composed by a single layered epithelium delimiting the lumen (Fig. 1A); different cell types, mainly the columnar, goblet, and regenerative cells constitute the midgut epithelium (Fig. 1B). The height of the epithelium is quite variable in each of the three studied regions (Table 1); there are differences as much as twice between the HMax and HMin obtained in the same region. However, there is no significant difference among the different regions concerning the H and the HMax values, but the HMin of the anterior and middle regions are significantly different. For the intestinal lumen, the LAx and SAx of the middle and posterior regions are similar amongst themselves, but these measurements of both regions differ significantly from the one of the anterior region; the same differences among the three regions are detected for the midgut (epithelium + lumen).
The multivariate analysis of the data concerning the measurements of the midgut lumen (Fig. 2) shows that the midgut has two different regions, the anterior and the posterior. The middle region presents values that are coincident with the ones of either the anterior region or the posterior region, as well as different values from these two regions.
The analysis of variance (Table 2) shows that some of the separate measurements taken from the different epithelial cells are variable along the midgut. In the columnar cells, most of the variables are not significantly different between the anterior and middle regions; however, the LAx of the cell, and the SAx and the A of their nucleus from both of the anterior and middle regions were different of the ones from the posterior region. The goblet cells show little variability among the midgut regions; the LAx of the cell and their nucleus in the anterior region are significantly different from the ones of both the middle and posterior regions. The regenerative cells are morphometric variable along the midgut; the SAx and the A of the cell and of their nucleus in the anterior and posterior regions are significantly different, and these values for the middle region do not differ from the ones of the anterior and posterior regions.
The multivariate analysis of the data concerning all the measurements of the epithelial cells in the midgut (Figs. 3-5) shows that the columnar, goblet, and regenerative cells do not constitute morphometrically different cellular populations in the three analyzed midgut regions.
The morphometrical evaluation of the D. saccharalis midgut (the epithelium and the lumen) showed that the anterior and the posterior regions are distinct; the middle region present intermediate values between the anterior and the posterior one, suggesting that this portion of the midgut may be considered a transition. Studies on the insect midgut morphometry, at light microscopy level, do not exist in the literature. Our results confirmed the general idea that the long midgut of the insect larvae are structurally differentiate along their lenght, as suggested by ultrastructural observations (Cioffi 1979, Lello et al. 1984, Santos et al. 1984).
However, when each individual epithelial cell type was morphometrically analyzed in the three midgut regions, it was not possible to characterize differences in the cells along the midgut. The multivariate analysis compares two or more parameters, using as much as possible numeric variables for these entities. Moço et al. (2002) used this analysis in the identification and characterization of Hepatozoon, a protozoa that parasites snake erythrocytes; measuring cellular and nuclear variables (length, width and area), these authors were able to identify different populations of this blood parasites.
Preliminary report on the ultrastructure of the columnar cells in the sugarcane borer (Pinheiro & Gregório 2001) showed differences in their subcellular organization that could be related with the localization along the midgut lenght; these differences were mainly related with the apical cytoplasmic projections, the amount of mitochondria and of the basal membrane infoldings. However, our present results suggest that these ultrastructural characteristics did not interfere in the morphometric parameters analyzed for this cell and their nucleus (LAx, SAx and A) in the same regions.
Morphometric data concerning the ultrastructural organization of the midgut epithelial cells were reports for many insect order, as in Diptera (Nopanitaya & Misch 1974, Wood & Lehane 1991, Andrade-Coeho et al. 2001), Orthoptera (Srivastava 1997), Heteroptera (Ranjini & Mohamed 2000), and Hemiptera (Billingsley 1988). However, they did not compare the cells along the midgut; they were mainly related with experimental aspects during the digestion process (Billingsley 1988, Wood & Lehane 1991, Ranjini & Mohamed 2000, Andrade-Coeho et al. 2001), sex differences (Rudin & Hecker 1976) and insect development (Nopanitaya & Misch 1974). The application of multivariate analysis, presented in this paper, may also be an important tool for a better interpretation of the physiologic, sexual and ontogenetic differences, as showed by the authors above.
The endocrine cells were not considered in our study, as they are scarce and very difficult to be identified at light microscopy level, mainly using conventional staining techniques (Endo et al. 1983, Montuenga et al. 1989). The small number of endocrine cells in the midgut epithelium were also described for many insect species (Andries & Beauvillain 1988, Montuenga et al. 1989).
The regenerative cells presented wide morphometric variability in the different midgut regions. Our data showed that there are difference among these cells comparing the anterior and the posterior regions. The middle region presented an intermediate pattern for the regenerative cells parameters, supporting the idea that there are a transition portion in the midgut between the anterior and the posterior ones.
We gratefully acknowledge the Entomological Laboratory of Usina Barra Grande, Lençóis Paulista, SP, for rearing the insects, and to Ricardo André Santos Teixeira and Antônio Vicente Salvador for technical assistance. This work was partially supported by FAPESP (Proc. 01/10868-0).
Andrade-Coeho, C.A., J. Santos-Mallet, N.A. Souza, U. Lins, M.N.L. Meirelles & E.F. Rangel. 2001. Ultrastructural features of the midgut epithelium of females Lutzomia intermedia (Lutz and Neiva, 1912) (Diptera: Psychodidae: Phlebotominae). Mem. Inst. Oswaldo Cruz 96: 1141-1151. [ Links ]
Andries, J.C. & J.C. Beauvillain. 1988. Ultrastructural study of cholecystokininlike immunoreactivity in endocrine cells of the insect midgut of Nepa cinerea (Insecta, Heteroptera): ultrastructure and genesis. Biol. Cell. 46: 195-202. [ Links ]
Billingsley, P.F. 1988. Morphometric analysis of Rhodnius prolixus Stal (Hemiptera: Reduviidae) midgut cells during blood digestion. Tissue Cell 20: 291-301. [ Links ]
Cavalcante, V.M. & C. Cruz-Landim. 1999. Types of cells present in the midgut of the insects: a review. Naturalia 24: 19-40. [ Links ]
Chiang, A.S., D.F. Yen & W.K. Peng. 1986 Defense reaction of midgut epithelial cells in the rice moth larva (Corcyra cephalonica) infected with Bacilus thuringiensis. J. Invertebr. Pathol. 47: 333-339. [ Links ]
Cioffi, M. 1979. The morphology, fine structure of the larval midgut of a moth (Manduca sexta) in relation to active ion transport. Tissue Cell 11: 467-479. [ Links ]
Coello, S. 1989. A new staining schedule for formalin-fixed, glycol methacrylate-embedded fish ovaries. J. Fish Biol. 34: 329-330. [ Links ]
Cristofoletti, P.T., A.F. Ribeiro & W.T. Terra. 2000. Apocrine secretion of amylase, exocytosis of trypsin along the midgut of Tenebrio molitor larvae. J. Insect Physiol. 47: 143-155. [ Links ]
Endo, Y., H. Sugihara, S. Fujita & J. Nishiitsutsuji-Uwo. 1983. Kinetics of columnar and endocrine cells in the cockroach midgut. Biomedical Res. 4: 51-60. [ Links ]
Hensley, S.D. & A.M. Hammond Jr. 1968. Laboratory techniques for rearing the sugarcane borer on an artificial diet. J. Econ. Entomol. 61: 1742-1743. [ Links ]
Jordão, B.P., A.N. Capella, W.R. Terra, A.F. Ribeiro & C. Ferreira. 1999. Nature of the anchors of membrane-bound aminopeptidase, amylase, trypsin a secretory mechanism in Spodoptera frugiperda (Lepidoptera) midgut cells. J. Insect Physiol. 45: 29-37. [ Links ]
Lehane, M.J. & P.F. Billingsley. 1996. (eds.) Biology of the insect midgut. London, Chapman and Hall, 486p. [ Links ]
Lello, E., W.K. Hanton, S.T. Bishoff & D.W. Misch. 1984. Histopathological effects of tobacco hornworm larvae (Manduca sexta): Low doses compared with fasting. J. Invertebr. Pathol. 43: 169-181. [ Links ]
Long, W.H. & S.D. Hensley. 1972. Insect pests of sugarcane. Annu. Rev. Entomol. 17: 149-176. [ Links ]
Moço, T.C., L.H. O'Dwyer, F.C. Vilela, T.H. Barrella & R.J. Silva. 2002. Morphologic and morphometric analysis of Hepatozoon spp. (Apicomplexa, Hepatozoidae) of snakes. Mem. Inst. Oswaldo Cruz 97: 1169-1176. [ Links ]
Montuenga, L.M., M.A. Barrenechea, P. Sesma, J. López & J.J. Vázquez. 1989. Ultrastructure and immunocytochemistry of endocrine cells in the midgut of the desert locust, Schistocerca gregaria. Cell Tissue Res. 258: 577-583. [ Links ]
Nopanitaya, W. & D.W. Misch. 1974. Developmental cytology of the midgut in the Flesh-fly, Sarcophaga bullata (Parker). Tissue Cell 6: 487-502. [ Links ]
Pearse, A.G.E. 1972. (eds.) Histochemistry: theoretical an applied. Edinburgh, Churchill Livingstone, v.1/2, 1055p. [ Links ]
Pinheiro, D.O. & E.A. Gregório. 2001. Ultrastructure of the columnar epithelial cell along the midgut of the Diatraea saccharalis (Lepidoptera: Pyralidae) larvae. Acta Microscopica 2 (B): 171-172. [ Links ]
Ranjini, K.R. & U.V.K. Mohamed. 2000. Morphometric analysis of the midgut epithelium of Iphita limbata Stal (Heteroptera: Pyrrhocoridae) during digestion. J. Adv. Zool. 21: 7-11. [ Links ]
Rudin, W. & H. Hecker. 1976. Morphometric comparison of the midgut epithelial cells in male and female Aedes aegypti L. (Insecta, Diptera). Tissue Cell 8: 459-470. [ Links ]
Santos, C.D., A.F. Ribeiro, C. Ferreira & W.R. Terra. 1984. The larval midgut of the cassava hornworm (Erinnyis ello). Ultrastructure, fluid fluxes, the secretory activity in relation to the organization of digestion. Cell Tissue Res. 237: 565-574. [ Links ]
Srivastava, C.N. 1997. Morpho-histological studies on the digestive system of Gryllus domesticus (Linn.) (Orthoptera: Gryllidae). J. Entomol. Res. 21: 321-328. [ Links ]
Turbeck, B. l974. A study of the concentrically laminated concretions spherites in the regenerative cells of the midgut of Lepidopterous larvae. Tissue Cell 6: 627-40. [ Links ]
Wood, A.R. & M.J. Lehane. 1991. Relative contributions of apocrine and eccrine secretion to digestive enzyme release from midgut cells of Stomoxys calcitrans (Insecta: Diptera). J. Insect Physiol. 37: 161-166. [ Links ]
Received 13/12/02. Accepted 10/06/03.