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Ultrastructure of the Alabama argillacea (Hübner) (Lepidoptera: Noctuidae) midgut
Micron 40 (2009) 743–749
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Ultrastructure of the Alabama argillacea (Hu¨bner) (Lepidoptera: Noctuidae) midgut Maria Esmeralda C. de Sousa a, Vale´ria Wanderley-Teixeira b,*, A´lvaro A.C. Teixeira b, Herbert A.A. de Siqueira a, Fa´bio A.B. Santos c, Luiz C. Alves c a Departamento de Agronomia, Programa de Po´s-Graduaca˜o em Entomologia Agrı´cola, Universidade Federal Rural de Pernambuco, Av. Dom Manoel de Medeiros s/n, Dois Irma˜os CEP 52171-900, Recife, PE, Brazil b Departamento de Morfologia e Fisiologia Animal, Universidade Federal Rural de Pernambuco, Av. Dom Manoel de Medeiros s/n, Dois Irma˜os CEP 52171-900, Recife, Pernambuco, Brazil c Centro de Pesquisa Aggeu Magalha˜es (CPqAM), Laborato´rio de Imunopatologia Keizo Asami (LIKA) - Universidade Federal de Pernambuco, Av. Moraes Rego s/n, CEP 50670-420, Recife 50670-420, PE, Brazil
A R T I C L E I N F O
A B S T R A C T
Article history: Received 11 March 2009 Received in revised form 16 April 2009 Accepted 18 April 2009
The insect midgut has ultimately been the focus of researches tempting to control insect pests because alterations in the insect gut may affect not only its development, but also physiological events such as nutrient absorption and transformation. The objective of the present work was to describe morphologically, histochemically, and ultrastructurally the larva midgut of Alabama argillacea (Hu¨bner) (Lepidoptera: Noctuidae), a cotton key pest in Brazil. Light and electronic transmission microscopy was used to obtain images from midgut sections of late fourth-instar larvae of A. argillacea. In general, the morphology, histochemistry, and ultrastructure characteristics of A. argillacea midgut follow that described in the literature for other lepidopteran species. However, the results showed a mitochondrial polymorphism and branched microvilli, which suggest an ultrastrucutural and physiological modification possibly associated with a high absorption and secretion activity by the columnar cells of this species. This intense activity may favor a faster response related to the action of ingested microbial agents and/or toxins, and can explain the high susceptibility of A. argillacea to the agents of control such as the toxin of Bacillus thuringiensis. ß 2009 Elsevier Ltd. All rights reserved.
Keywords: Midgut Cotton leafworm Cotton pest Histology
1. Introduction The insect alimentary canal is differentiated in three regions with particular embryologic origins: foregut, midgut, and the hindgut. It represents one of the most important areas of contact between the insects and the environment, and therefore it has been the focus of researches aiming to develop environmentally sound methods of insect pests control (Chapman, 1998; Levy et al., 2004). Among the three regions, the midgut region has particularly been the most studied, because alterations on it affect the growth and development of insects as a result of changes in the physiological events that depend on meal provision, absorption and transformation (Mordue (Luntz) and Blackwell, 1993; Mordue (Luntz) and Nisbet, 2000). The epithelium of the midgut in Lepidoptera is composed of four types of cells, which are involved in the processes of absorption and enzymes secretion (columnar cells), ionic homeostasis (goblet cells), endocrine function (endocrine cells), and the
* Corresponding author. Tel.: +55 81 33206389. E-mail address: [email protected] (V. Wanderley-Teixeira). 0968-4328/$ – see front matter ß 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.micron.2009.04.008
epithelium renewal (regenerative cells) (Terra et al., 2006; Pinheiro et al., 2008). One of the characteristics of many insects is the presence of the peritrophic membrane in the midgut, which has a fundamental role of protection of the midgut. Because it is positioned between the gut lumen and the epithelial layer, it works protecting this epithelium from mechanical damage, also as a barrier against toxins and harmful chemicals to the insect Terra (2001). Results already available related to the morphological and ultrastructural description of insects midgut from Lepidoptera such as Diatraea saccharalis (Fabricius) (Lepidoptera: Pyralidae), Manduca sexta L. (Lepidoptera: Sphingidae), Spodoptera frugiperda (Lepidoptera: Noctuidae) (J.E. Smith), and Anticarsia gemmatalis (Hu¨bner) (Lepidoptera: Noctuidae) suggest that the distribution and morphology of the epithelial cells can vary along this region (Pinheiro et al., 2003; Levy et al., 2004; Pinheiro et al., 2006). These differences are usually observed at the ultrastructural level (Santos et al., 1984; Lehane and Billingsley, 1996). In Brazil, the main defoliator of cotton is the cotton leafworm, Alabama argillacea (Hu¨bner) (Lep.: Noctuidae). In the tropics, it occurs any time of the year and in most part of the crop phenology, depending on the climatic conditions of each region (Silva et al.,
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1980). The larvae feed on leaves and, at high population density, they usually heavy defoliate the cotton plants. In the Brazil Northeast, the population densities are more accentuated in the initial stages of crop development, while in the Central and South region it predominates during the boll stage, but in both periods they cause significant reduction on yield (Gravena and Cunha, 1991). A particular aspect of the cotton leafworm is its monophagous habits, feeding almost exclusively on cotton plants. In fact, this pest has a great importance for the cotton crops in Brazil (Azevedo et al., 2002). With the release of Bt Cry1Ac cotton in Brazil since, 2006, the importance of A. argillacea has increased because its biology may jeopardize the control by this recent available technology to its control. By studying the gut structure and biology of the A. argillacea might help to understand further interactions of potential xenobiotics and the insect. Therefore, the present study will give emphasis to the morphological, histological, and ultrastructural description of the midgut of the A. argillacea.
Laboratory of Histology of the Department of Morphology and Animal Physiology of the Federal Rural University of Pernambuco (UFRPE), and the Laboratory of Immunopathology Keizo Asami of the Federal University of Pernambuco (UFPE).
2. Material and methods
2.2. Light microscopy of A. argillacea midgut
The research was conducted in the Laboratory of InsectToxicant Interactions of the Department of Agronomy, the
Caterpillars of fourth instar were immobilized at low temperature ( 4 8C) for 5 min, dissected to excise the midgut, and
2.1. Insect rearing The caterpillars of A. argillacea used in the experiments were obtained from the stock in the Laboratory of Insect-toxicant Interactions (UFRPE) and were kept in a conditioned room at 25 2 8C, 60 10% UR and 12 h photoperiod, using the methodology of Oliveira et al. (2001). Briefly, the adults were kept in PVC cages (20 cm height 15 cm diameter) with the inner side covered with white sulfite paper for oviposition and the top closed with voile. The adults were fed with a 5% honey solution soaked in hydrophilic cotton. Eggs from adult cages were daily transferred to PVC cages containing cotton (Acala DP 90 isoline) leaves. After pupation, pupae were transferred to Petri dishes (150 mm 20 mm) until the adult emergence.
Fig. 1. Light microscopy: (A) Midgut of A. argillacea showing simple epithelium supported between two layers of muscles. Bar = 100 mm. (B) Detail of epithelial cells. Bar = 100 mm. (C) Notice several cytoplasmic protuberances in the columnar cells directed into the lumen. Bar = 200 mm. (D) Notice cells: goblet and regenerative. Bar = 25 mm. SE—simple epithelium, CM—circular muscle, LM—longitudinal muscle, CC—columnar cell, GC—goblet cell, Arrow—regenerative cell, N—nucleus, CP— cytoplasmic protuberance, C—globosa chamber, BB—brush border. Toluidine blue stain.
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immediately transferred to Bou¨in’s fixative (75 mL saturated solution of picric acid, 25 mL formaldehyde, and 5 mL acetic acid). Midguts were fixed for 24 h at 4 8C. Midguts dehydration was performed as follows: 70%, 90%, and 100% ethylic alcohol solutions in order for 20 min each, further 20 min in 100% ethylic alcohol + xylol (1/1), and a final step in xylol for 20 min (diaphanization). After this stage, dehydrated midguts section were embedded in 50% paraffin diluted in xylol at 58 8C for 1 h each and mounted in 2 cm3 moulds. The blocks were cut in a Minot microtome (LEICA RM 2035) adjusted to a thickness of 7 mm, the sections mounted on slides with Mayer albumin and kept at 37 8C for 24 h. Next, the slides were colored by Mallory’s trichrome and Schiff’s Periodic Acid (P.A.S.), using the methodology described in Michalany (1990). Histological analysis was performed in an OLYMPUS BX-49 microscope and photos taken in an OLYMPUS BX51 photomicroscope. 2.3. Transmission electron microscopy (TEM) of A. argillacea midgut Larvae of 4th instar were kept at low temperature ( 4 8C) for 5 min and the fragments of the midgut were excised after larva’s dissection. The fragments were fixed in 2.5% glutaraldehyde in
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0.1 M phosphate buffer at pH 7.2. After rinsing 3 times in this buffer, they were post-fixed in 2% osmium tetroxide (OsO4) in the same buffer for 1 h. After that, the dehydration of the fragments were carried out in raising series of acetone for 30 min each at room temperature and followed by embedding in Embed 812/ Araldite resin (Electron Microscopy Sciences, Hartfield, PA) at 70 8C for 48 h. Semi thin sections were stained with toluidine blue for morphological observation, while ultrathin sections were subsequently contrasted in uranyl acetate for 1 h and lead citrate for 10 min, and observations done in a Zeiss EM109 microscope. 3. Results Ultrathin sections of the 4th instar A. argillacea larvae midgut showed that it comprises a simple epithelium supported by an inner and outer muscle layers, both arranged circularly and longitudinally, respectively (Fig. 1A). Three types of cells distributed along the extension of the epithelium were identified in the light microscopy photos: columnar, goblet and regenerative (Fig. 1B). However, a fourth cell type, the endocrine cell, could be observed in the ultrastructural electron microscopy level (Fig. 4B). The most frequent cells were the columnar, tall cells with
Fig. 2. Transmission electron micrographs from the apical region of the columnar cell: (A) microvilli. Bar = 0.2 mm. (B) Notice the secretion process. Bar = 0.2 mm. (C) Mitochondria richness. Bar = 0.5 mm. (D) Mitochondrial polymorphism. Bar = 0.2 mm. BM—bifurcated microvillus, SV—secretion vesicle, 1—spherical mitochondria, 2— elongated mitochondria, 3—hoodlike mitochondria, 4—mitochondria storing electron lucent material.
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Fig. 3. Transmission electron micrographs: (A) detail of mitochondria containing electron lucent material. Bar = 0.3 mm. (B) Autophagic vacuole. Bar = 0.3 mm. (C) Tight junction between columnar cells. Bar = 0.5 mm. (D) Central region of the columnar cell Bar = 0.5 mm. Po—polysomes, SER—smooth endoplasmic reticulum, AV—autophagic vacuole, M—mitochondria, long arrow—zonula occludens, short arrow—intracellular spaces, LI—detail of columnar cell lateral region, showing lateral invaginations, RER, rough endoplasmic reticulum, N—nucleus.
characteristic brush borders, nuclei very heterochromatic with location varying from central to apical. In the apical region, it evidenced several cytoplasmatic protuberances, which detached from cells in the lumen direction (Fig. 1B and C). Detailed observations of these cells apical region showed numerous long microvilli, some branched, surrounding vesicles of secretion (Fig. 2A and B). In the microvilli subjacent region, it was noted that a richness of mitochondria of varying size such as spheric, long, and hoodlike form. Some of these mitochondria contained in their interiors electronlucent material, characterizing a mitochondrial polymorphism (Fig. 2C and D). The electronlucent material was composed by a variable number of polysomes and smooth endoplasmic reticulum (Fig. 3A). It was also observed that the presence of autophagic vacuoles surrounded by mitochondria (Fig. 3B). The columnar cells are intimately connected by scalariform junctions in the lateral region and close to the apical pole, which makes the spaces between these specializations very clear, additional to the invaginations observed in the basolateral region (Fig. 3C). The central region of the columnar cells was characterized by the presence of great amount of rough endoplasmic reticulum, Golgi apparatus, and some mitochondria (Fig. 3D). Numerous invaginations of the plasmatic membrane forming a labyrinth were observed in the basal region (Fig. 4A). The goblet
cell was particularly well characterized by the presence of a cavity in the form of a calyx, also limited by the plasmatic membrane projections, with mitochondria in the base of those projections, presence of basal labyrinth, besides heterochromatic basal nucleus and nucleolus very distinct (Figs. 1D and 4B). In both light and electron microscopy observations, the regenerative cells were easily characterized because of their pyramidal morphologies and isolation in the base of the epithelium. The cytoplasm was very dense with predominating free polysomes and central, voluminous nucleus showing disperse chromatin (Figs. 1D and 4C and D). The endocrine and regenerative cells did not extend in the lumen direction, hence, of basal localization. These cells presented long morphology, containing several granules, and generally concentrated in the basal region of the cells (Fig. 4B). Histochemically, the midgut showed the presence of mucous and glycoprotein secretions when stained by Mallory’s trichrome and Schiff’s periodic acid, respectively (Fig. 5A and B). 4. Discussion The epithelium of Lepidoptera midgut can be either simple (Jorda˜o et al., 1999; Cristofoletti et al., 2001; Pinheiro et al., 2003) or pseudo-stratified (Levy et al., 2004), and constituted by four
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Fig. 4. Transmission electron micrographs: (A) basal region of the columnar cell. Bar = 0.5 mm. (B) Cells: goblet and endocrine. Bar = 0.5 mm. (C and D) Regenerative cell. Bar = 0.5 and 0.25 mm, respectively. BL—basal labyrinth, GC—goblet cell, EC—endocrine cell, arrowhead—granule, N—nucleus, DC—dense cytoplasm, CM—circular muscle, LM—longitudinal muscle, Po—polysomes.
types of cells. The epithelium of A. argillacea is histologically a simple type and composed of columnar, goblet, endocrine, and regenerative cells, hence following the general pattern observed in other lepidopterans larvae. On the other hand, it was observed
numerous long microvilli, sometimes branched, and cytoplamatic protuberances besides the columnar cells along the extension of the epithelium. The observation of branched microvilli in these cells is the first report, to our best knowledge, and suggests a
Fig. 5. Histochemistry of the A. argillacea midgut: (A) Notice the mucous nature of goblet cells. Mallory’s trichrome. Bar = 25 mm. (B) Notice positive reaction by P.A.S. in the epithelial surface and secretion products. Bar = 25 mm. Arrow—peritrophic membrane, GC—goblet cell, SP—secretion product, DM—digested material.
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possible presence of stereocilia as these are projections in the apical region of the epithelial cells that can anastomose, having also an absorption role (Junqueira and Carneiro, 2008). However, Guo et al. (2000), studying the structural arrangement of microvilli, suggested that modifications in proteins can lead them to deformities without changing their functions. Cytoplasmic protuberances were also observed by Pinheiro et al. (2008) in the columnar cells of the midgut in D. saccharalis and, according to these authors, they would result from the process of apocrine secretion of digestive enzymes towards the lumen (Cruz-Landim et al., 1996; Jordao et al., 1996; Serra˜o and Cruz-Landim, 2000). Mitochondrial polymorphism in the apical region of columnar cells and autophagic vacuoles were observed through the ultrastructural microscopy. According to Abdalla and Cruz-Landim (2001, 2004), variations in mitochondria sizes and shapes commonly occur in the secretory cells of insects in general. However, as far as we know, no reports exist in the literature about the presence of mitochondrial polymorphism in mesenteric columnar cells from Lepidoptera. Abdalla and Cruz-Landim (2005), after analysis of this polymorphism in cells of Dufour’s glands from Scaptotrigona postica Latreille (Hymenoptera: Apidae), classified the mitochondria morphology into four types: the spherical as type 1, the long as type 2, the hoodlike shape or those containing polysomes and smooth endoplasmic reticulum in their matrixes as type 3, and those containing lipids in their matrixes as type 4. All of these types were found in the columnar cells of A. argillacea, except for the type 4. According to Caetano et al. (2002) the type 4 polymorphism results from the development cycle where morphological changes induce the mitochondria to increasingly accumulate lipid secretions in its matrix. Moyes and Hood (2003) and Devin and Rigoulet (2007) report that animal cell is able to remodel mitochondrial structure and function to better accommodate energetic needs. So mitochondrial polymorphism in columnar cell may suggest, a better accommodation of the energy requirements during the larval stages. The presence of vacuoles in the columnar cells represents a normal physiological condition in epithelial cells. The degradation of cytoplasmic components in the midgut is an important process to the cell physiology, particularly for the apoptosis or organelles recycling (Weissman, 2001). During the degradation process, the organelles are isolated from the cytoplasm in double-membrane vesicles called autophagosomes or autophagic vacuoles (Klionsky and Emr, 2000). Although the origin of the membranes of the autophagic vacuoles is a subject in debate, evidences indicate that they are derived from the rough endoplasmic reticulum (Dunn, 1990). The columnar cells showed the epithelial exclusive adjoining and interconnecting specializations of cell membranes close to cell apex, the scalariform junctions, similar related in midgut epithelium of Meliponinae larvae (Serra˜o et al., 2004), intercalated by intracellular spaces. These spaces can suggest the place of substance exchange between cells. Its because, according to Abdalla (2002), the presence of junctions delimiting these spaces does not allow substances found in this region getting into the lumen or the hemocoel. Invaginations were observed in the basolateral cell membrane. Their sizes may vary according to their functions in the basal region and location in the midgut (Terra et al., 2006). The labyrinth observed in the basolateral membrane is responsible for increasing the contact surface between the basolateral membrane and the hemolymph (Cavalcante and Cruz-Landim, 1999, Terra et al., 2006). The goblet cells observed in this study with A. argillacea were similar to those found in other Lepidoptera, which exhibit the typical cavity called chamber. These cells, located throughout the midgut epithelium and intercalated by columnar cells, and have as main function the transport of potassium from the hemolymph to
the lumen, keeping the ionic homeostasis and assisting the columnar cells with the metabolites absorption (Cavalcante and Cruz-Landim, 1999; Klowden, 2002). An intense secretory activity of mucous nature was observed from these cells, an indication of large amount of mucins present in the mucus, since mucins react with the Mallory’s trichrome (Behmer et al., 1976, Junqueira and Junqueira, 1983; Michalany, 1990). The negative reaction of the goblet cells with P.A.S. reinforce the reports of Pinheiro et al. (2008) that glycoprotein secretion is produced exclusively by columnar cells. The regenerative cells are relatively undifferentiated and responsible for the midgut epithelium renewal, not only substituting the senescent cells, but also contributing to the gut growth in every ecdysis. They are usually found isolated or in groups forming nests in the base of the midgut (Cavalcante and Cruz-Landim, 1999; Wanderley-Teixeira et al., 2006; Martins et al., 2006). The lack of endocrine cells in the midgut from A. argillacea after light microscopy analysis agrees with previous reports that these cells are not easily identified through these routine technique, thus ultrastructural and immunohistochemical analysis are necessary (Montuenga et al., 1989, Jimenez and Gilliam, 1990; Neves et al., 2002; Pinheiro et al., 2008). However, after ultrastructural analysis, A. argillacea endocrine cells could be seen in the base of the epithelium, though without reach the lumen. According to Cavalcante and Cruz-Landim (1999), the endocrine cells that do not enter in contact with the lumen are classified as open type, and those with lumen direct contact are the closed type. In general, the morphological, histochemical and ultrastructural characteristics of the midgut of A. argillacea were similar to those described in the literature for most Lepidoptera. However, the presence of mitochondrial polymorphism and bifurcated microvilli suggest a physiological and ultrastructural modification, which may be associated with both higher absorption and secretory activities of the columnar cells in this species. Such increased activity may lead to a faster action of toxins and/or microbial agents, suggesting that this species may be highly susceptible to some of these agents. But, this hypothesis needs further investigation. Acknowledgements The authors would like to thank CAPES for the financial support of the first author and to Rafael Padilha (LIKA/UFPE) for his technical assistance during the material preparation and observation in the electron microscopies. References Abdalla, F.C., Cruz-Landim, C., 2001. Changes in the morphology of the Dufour gland of Apis mellifera L. (Hymenoptera Apidae) during the life stages of the female castes. Rev. Bras. Entomol. 45, 123–129. Abdalla, F.C., Cruz-Landim, C., 2004. Occurrence, morphology and ultrastructure of the Dufour gland in Melipona bicolor Lepeletier, 1836 (Hymenoptera Meliponini). Rev. Bras. Entomol. 48, 1–19. Abdalla, F.C., Cruz-Landim, C., 2005. Ocorreˆncia, morfologia e ultra-estrutura da glaˆndula de Dufour de Scaptotrigona postica Latreille (Hymenoptera: Apidae). Neotrop. Entomol. 34, 47–57. Abdalla, F.C., 2002. Glaˆndula de Dufour. In: Cruz-Landim, C., Abdalla, F.C. (Eds.), Glaˆndulas exo´crinas das abelhas. FUNPEC, Ribeira˜o Preto, pp. 127–149. Azevedo, F.R., Mattos, K.O., Vieira, F.V., 2002. Comportamento alimentar de Alabama argillacea Hubner (Lep. Noctuidae) em algodoeiro. Cieˆn. Agron. 33, 5–9. Behmer, O.A., Tolosa, E.M.C., Freitas Neto, A.G., 1976. Manual de te´cnicas para histologia normal e patolo´gica. Edart, Sa˜o Paulo, 115 pp. Caetano, F.H., Zara, F.J., Grego´rio, E.A., 2002. The origin of lipid droplets in the postpharyngeal gland of Dinoponera australis (Formicidae: Ponerinae). Cytology 67, 301–308. Cavalcante, V.M., Cruz-Landim, C., 1999. Types of cells present in the midgut of the insects: a review. Nature 24, 19–39. Chapman, R.F., 1998. The Insects: Structure and Function, 4th ed. Cambridge University Press, Cambridge, 770 pp.
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Ultrastructure of the Alabama argillacea (Hu¨bner) (Lepidoptera: Noctuidae) midgut Maria Esmeralda C. de Sousa a, Vale´ria Wanderley-Teixeira b,*, A´lvaro A.C. Teixeira b, Herbert A.A. de Siqueira a, Fa´bio A.B. Santos c, Luiz C. Alves c a Departamento de Agronomia, Programa de Po´s-Graduaca˜o em Entomologia Agrı´cola, Universidade Federal Rural de Pernambuco, Av. Dom Manoel de Medeiros s/n, Dois Irma˜os CEP 52171-900, Recife, PE, Brazil b Departamento de Morfologia e Fisiologia Animal, Universidade Federal Rural de Pernambuco, Av. Dom Manoel de Medeiros s/n, Dois Irma˜os CEP 52171-900, Recife, Pernambuco, Brazil c Centro de Pesquisa Aggeu Magalha˜es (CPqAM), Laborato´rio de Imunopatologia Keizo Asami (LIKA) - Universidade Federal de Pernambuco, Av. Moraes Rego s/n, CEP 50670-420, Recife 50670-420, PE, Brazil
A R T I C L E I N F O
A B S T R A C T
Article history: Received 11 March 2009 Received in revised form 16 April 2009 Accepted 18 April 2009
The insect midgut has ultimately been the focus of researches tempting to control insect pests because alterations in the insect gut may affect not only its development, but also physiological events such as nutrient absorption and transformation. The objective of the present work was to describe morphologically, histochemically, and ultrastructurally the larva midgut of Alabama argillacea (Hu¨bner) (Lepidoptera: Noctuidae), a cotton key pest in Brazil. Light and electronic transmission microscopy was used to obtain images from midgut sections of late fourth-instar larvae of A. argillacea. In general, the morphology, histochemistry, and ultrastructure characteristics of A. argillacea midgut follow that described in the literature for other lepidopteran species. However, the results showed a mitochondrial polymorphism and branched microvilli, which suggest an ultrastrucutural and physiological modification possibly associated with a high absorption and secretion activity by the columnar cells of this species. This intense activity may favor a faster response related to the action of ingested microbial agents and/or toxins, and can explain the high susceptibility of A. argillacea to the agents of control such as the toxin of Bacillus thuringiensis. ß 2009 Elsevier Ltd. All rights reserved.
Keywords: Midgut Cotton leafworm Cotton pest Histology
1. Introduction The insect alimentary canal is differentiated in three regions with particular embryologic origins: foregut, midgut, and the hindgut. It represents one of the most important areas of contact between the insects and the environment, and therefore it has been the focus of researches aiming to develop environmentally sound methods of insect pests control (Chapman, 1998; Levy et al., 2004). Among the three regions, the midgut region has particularly been the most studied, because alterations on it affect the growth and development of insects as a result of changes in the physiological events that depend on meal provision, absorption and transformation (Mordue (Luntz) and Blackwell, 1993; Mordue (Luntz) and Nisbet, 2000). The epithelium of the midgut in Lepidoptera is composed of four types of cells, which are involved in the processes of absorption and enzymes secretion (columnar cells), ionic homeostasis (goblet cells), endocrine function (endocrine cells), and the
* Corresponding author. Tel.: +55 81 33206389. E-mail address: [email protected] (V. Wanderley-Teixeira). 0968-4328/$ – see front matter ß 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.micron.2009.04.008
epithelium renewal (regenerative cells) (Terra et al., 2006; Pinheiro et al., 2008). One of the characteristics of many insects is the presence of the peritrophic membrane in the midgut, which has a fundamental role of protection of the midgut. Because it is positioned between the gut lumen and the epithelial layer, it works protecting this epithelium from mechanical damage, also as a barrier against toxins and harmful chemicals to the insect Terra (2001). Results already available related to the morphological and ultrastructural description of insects midgut from Lepidoptera such as Diatraea saccharalis (Fabricius) (Lepidoptera: Pyralidae), Manduca sexta L. (Lepidoptera: Sphingidae), Spodoptera frugiperda (Lepidoptera: Noctuidae) (J.E. Smith), and Anticarsia gemmatalis (Hu¨bner) (Lepidoptera: Noctuidae) suggest that the distribution and morphology of the epithelial cells can vary along this region (Pinheiro et al., 2003; Levy et al., 2004; Pinheiro et al., 2006). These differences are usually observed at the ultrastructural level (Santos et al., 1984; Lehane and Billingsley, 1996). In Brazil, the main defoliator of cotton is the cotton leafworm, Alabama argillacea (Hu¨bner) (Lep.: Noctuidae). In the tropics, it occurs any time of the year and in most part of the crop phenology, depending on the climatic conditions of each region (Silva et al.,
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1980). The larvae feed on leaves and, at high population density, they usually heavy defoliate the cotton plants. In the Brazil Northeast, the population densities are more accentuated in the initial stages of crop development, while in the Central and South region it predominates during the boll stage, but in both periods they cause significant reduction on yield (Gravena and Cunha, 1991). A particular aspect of the cotton leafworm is its monophagous habits, feeding almost exclusively on cotton plants. In fact, this pest has a great importance for the cotton crops in Brazil (Azevedo et al., 2002). With the release of Bt Cry1Ac cotton in Brazil since, 2006, the importance of A. argillacea has increased because its biology may jeopardize the control by this recent available technology to its control. By studying the gut structure and biology of the A. argillacea might help to understand further interactions of potential xenobiotics and the insect. Therefore, the present study will give emphasis to the morphological, histological, and ultrastructural description of the midgut of the A. argillacea.
Laboratory of Histology of the Department of Morphology and Animal Physiology of the Federal Rural University of Pernambuco (UFRPE), and the Laboratory of Immunopathology Keizo Asami of the Federal University of Pernambuco (UFPE).
2. Material and methods
2.2. Light microscopy of A. argillacea midgut
The research was conducted in the Laboratory of InsectToxicant Interactions of the Department of Agronomy, the
Caterpillars of fourth instar were immobilized at low temperature ( 4 8C) for 5 min, dissected to excise the midgut, and
2.1. Insect rearing The caterpillars of A. argillacea used in the experiments were obtained from the stock in the Laboratory of Insect-toxicant Interactions (UFRPE) and were kept in a conditioned room at 25 2 8C, 60 10% UR and 12 h photoperiod, using the methodology of Oliveira et al. (2001). Briefly, the adults were kept in PVC cages (20 cm height 15 cm diameter) with the inner side covered with white sulfite paper for oviposition and the top closed with voile. The adults were fed with a 5% honey solution soaked in hydrophilic cotton. Eggs from adult cages were daily transferred to PVC cages containing cotton (Acala DP 90 isoline) leaves. After pupation, pupae were transferred to Petri dishes (150 mm 20 mm) until the adult emergence.
Fig. 1. Light microscopy: (A) Midgut of A. argillacea showing simple epithelium supported between two layers of muscles. Bar = 100 mm. (B) Detail of epithelial cells. Bar = 100 mm. (C) Notice several cytoplasmic protuberances in the columnar cells directed into the lumen. Bar = 200 mm. (D) Notice cells: goblet and regenerative. Bar = 25 mm. SE—simple epithelium, CM—circular muscle, LM—longitudinal muscle, CC—columnar cell, GC—goblet cell, Arrow—regenerative cell, N—nucleus, CP— cytoplasmic protuberance, C—globosa chamber, BB—brush border. Toluidine blue stain.
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immediately transferred to Bou¨in’s fixative (75 mL saturated solution of picric acid, 25 mL formaldehyde, and 5 mL acetic acid). Midguts were fixed for 24 h at 4 8C. Midguts dehydration was performed as follows: 70%, 90%, and 100% ethylic alcohol solutions in order for 20 min each, further 20 min in 100% ethylic alcohol + xylol (1/1), and a final step in xylol for 20 min (diaphanization). After this stage, dehydrated midguts section were embedded in 50% paraffin diluted in xylol at 58 8C for 1 h each and mounted in 2 cm3 moulds. The blocks were cut in a Minot microtome (LEICA RM 2035) adjusted to a thickness of 7 mm, the sections mounted on slides with Mayer albumin and kept at 37 8C for 24 h. Next, the slides were colored by Mallory’s trichrome and Schiff’s Periodic Acid (P.A.S.), using the methodology described in Michalany (1990). Histological analysis was performed in an OLYMPUS BX-49 microscope and photos taken in an OLYMPUS BX51 photomicroscope. 2.3. Transmission electron microscopy (TEM) of A. argillacea midgut Larvae of 4th instar were kept at low temperature ( 4 8C) for 5 min and the fragments of the midgut were excised after larva’s dissection. The fragments were fixed in 2.5% glutaraldehyde in
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0.1 M phosphate buffer at pH 7.2. After rinsing 3 times in this buffer, they were post-fixed in 2% osmium tetroxide (OsO4) in the same buffer for 1 h. After that, the dehydration of the fragments were carried out in raising series of acetone for 30 min each at room temperature and followed by embedding in Embed 812/ Araldite resin (Electron Microscopy Sciences, Hartfield, PA) at 70 8C for 48 h. Semi thin sections were stained with toluidine blue for morphological observation, while ultrathin sections were subsequently contrasted in uranyl acetate for 1 h and lead citrate for 10 min, and observations done in a Zeiss EM109 microscope. 3. Results Ultrathin sections of the 4th instar A. argillacea larvae midgut showed that it comprises a simple epithelium supported by an inner and outer muscle layers, both arranged circularly and longitudinally, respectively (Fig. 1A). Three types of cells distributed along the extension of the epithelium were identified in the light microscopy photos: columnar, goblet and regenerative (Fig. 1B). However, a fourth cell type, the endocrine cell, could be observed in the ultrastructural electron microscopy level (Fig. 4B). The most frequent cells were the columnar, tall cells with
Fig. 2. Transmission electron micrographs from the apical region of the columnar cell: (A) microvilli. Bar = 0.2 mm. (B) Notice the secretion process. Bar = 0.2 mm. (C) Mitochondria richness. Bar = 0.5 mm. (D) Mitochondrial polymorphism. Bar = 0.2 mm. BM—bifurcated microvillus, SV—secretion vesicle, 1—spherical mitochondria, 2— elongated mitochondria, 3—hoodlike mitochondria, 4—mitochondria storing electron lucent material.
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Fig. 3. Transmission electron micrographs: (A) detail of mitochondria containing electron lucent material. Bar = 0.3 mm. (B) Autophagic vacuole. Bar = 0.3 mm. (C) Tight junction between columnar cells. Bar = 0.5 mm. (D) Central region of the columnar cell Bar = 0.5 mm. Po—polysomes, SER—smooth endoplasmic reticulum, AV—autophagic vacuole, M—mitochondria, long arrow—zonula occludens, short arrow—intracellular spaces, LI—detail of columnar cell lateral region, showing lateral invaginations, RER, rough endoplasmic reticulum, N—nucleus.
characteristic brush borders, nuclei very heterochromatic with location varying from central to apical. In the apical region, it evidenced several cytoplasmatic protuberances, which detached from cells in the lumen direction (Fig. 1B and C). Detailed observations of these cells apical region showed numerous long microvilli, some branched, surrounding vesicles of secretion (Fig. 2A and B). In the microvilli subjacent region, it was noted that a richness of mitochondria of varying size such as spheric, long, and hoodlike form. Some of these mitochondria contained in their interiors electronlucent material, characterizing a mitochondrial polymorphism (Fig. 2C and D). The electronlucent material was composed by a variable number of polysomes and smooth endoplasmic reticulum (Fig. 3A). It was also observed that the presence of autophagic vacuoles surrounded by mitochondria (Fig. 3B). The columnar cells are intimately connected by scalariform junctions in the lateral region and close to the apical pole, which makes the spaces between these specializations very clear, additional to the invaginations observed in the basolateral region (Fig. 3C). The central region of the columnar cells was characterized by the presence of great amount of rough endoplasmic reticulum, Golgi apparatus, and some mitochondria (Fig. 3D). Numerous invaginations of the plasmatic membrane forming a labyrinth were observed in the basal region (Fig. 4A). The goblet
cell was particularly well characterized by the presence of a cavity in the form of a calyx, also limited by the plasmatic membrane projections, with mitochondria in the base of those projections, presence of basal labyrinth, besides heterochromatic basal nucleus and nucleolus very distinct (Figs. 1D and 4B). In both light and electron microscopy observations, the regenerative cells were easily characterized because of their pyramidal morphologies and isolation in the base of the epithelium. The cytoplasm was very dense with predominating free polysomes and central, voluminous nucleus showing disperse chromatin (Figs. 1D and 4C and D). The endocrine and regenerative cells did not extend in the lumen direction, hence, of basal localization. These cells presented long morphology, containing several granules, and generally concentrated in the basal region of the cells (Fig. 4B). Histochemically, the midgut showed the presence of mucous and glycoprotein secretions when stained by Mallory’s trichrome and Schiff’s periodic acid, respectively (Fig. 5A and B). 4. Discussion The epithelium of Lepidoptera midgut can be either simple (Jorda˜o et al., 1999; Cristofoletti et al., 2001; Pinheiro et al., 2003) or pseudo-stratified (Levy et al., 2004), and constituted by four
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Fig. 4. Transmission electron micrographs: (A) basal region of the columnar cell. Bar = 0.5 mm. (B) Cells: goblet and endocrine. Bar = 0.5 mm. (C and D) Regenerative cell. Bar = 0.5 and 0.25 mm, respectively. BL—basal labyrinth, GC—goblet cell, EC—endocrine cell, arrowhead—granule, N—nucleus, DC—dense cytoplasm, CM—circular muscle, LM—longitudinal muscle, Po—polysomes.
types of cells. The epithelium of A. argillacea is histologically a simple type and composed of columnar, goblet, endocrine, and regenerative cells, hence following the general pattern observed in other lepidopterans larvae. On the other hand, it was observed
numerous long microvilli, sometimes branched, and cytoplamatic protuberances besides the columnar cells along the extension of the epithelium. The observation of branched microvilli in these cells is the first report, to our best knowledge, and suggests a
Fig. 5. Histochemistry of the A. argillacea midgut: (A) Notice the mucous nature of goblet cells. Mallory’s trichrome. Bar = 25 mm. (B) Notice positive reaction by P.A.S. in the epithelial surface and secretion products. Bar = 25 mm. Arrow—peritrophic membrane, GC—goblet cell, SP—secretion product, DM—digested material.
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possible presence of stereocilia as these are projections in the apical region of the epithelial cells that can anastomose, having also an absorption role (Junqueira and Carneiro, 2008). However, Guo et al. (2000), studying the structural arrangement of microvilli, suggested that modifications in proteins can lead them to deformities without changing their functions. Cytoplasmic protuberances were also observed by Pinheiro et al. (2008) in the columnar cells of the midgut in D. saccharalis and, according to these authors, they would result from the process of apocrine secretion of digestive enzymes towards the lumen (Cruz-Landim et al., 1996; Jordao et al., 1996; Serra˜o and Cruz-Landim, 2000). Mitochondrial polymorphism in the apical region of columnar cells and autophagic vacuoles were observed through the ultrastructural microscopy. According to Abdalla and Cruz-Landim (2001, 2004), variations in mitochondria sizes and shapes commonly occur in the secretory cells of insects in general. However, as far as we know, no reports exist in the literature about the presence of mitochondrial polymorphism in mesenteric columnar cells from Lepidoptera. Abdalla and Cruz-Landim (2005), after analysis of this polymorphism in cells of Dufour’s glands from Scaptotrigona postica Latreille (Hymenoptera: Apidae), classified the mitochondria morphology into four types: the spherical as type 1, the long as type 2, the hoodlike shape or those containing polysomes and smooth endoplasmic reticulum in their matrixes as type 3, and those containing lipids in their matrixes as type 4. All of these types were found in the columnar cells of A. argillacea, except for the type 4. According to Caetano et al. (2002) the type 4 polymorphism results from the development cycle where morphological changes induce the mitochondria to increasingly accumulate lipid secretions in its matrix. Moyes and Hood (2003) and Devin and Rigoulet (2007) report that animal cell is able to remodel mitochondrial structure and function to better accommodate energetic needs. So mitochondrial polymorphism in columnar cell may suggest, a better accommodation of the energy requirements during the larval stages. The presence of vacuoles in the columnar cells represents a normal physiological condition in epithelial cells. The degradation of cytoplasmic components in the midgut is an important process to the cell physiology, particularly for the apoptosis or organelles recycling (Weissman, 2001). During the degradation process, the organelles are isolated from the cytoplasm in double-membrane vesicles called autophagosomes or autophagic vacuoles (Klionsky and Emr, 2000). Although the origin of the membranes of the autophagic vacuoles is a subject in debate, evidences indicate that they are derived from the rough endoplasmic reticulum (Dunn, 1990). The columnar cells showed the epithelial exclusive adjoining and interconnecting specializations of cell membranes close to cell apex, the scalariform junctions, similar related in midgut epithelium of Meliponinae larvae (Serra˜o et al., 2004), intercalated by intracellular spaces. These spaces can suggest the place of substance exchange between cells. Its because, according to Abdalla (2002), the presence of junctions delimiting these spaces does not allow substances found in this region getting into the lumen or the hemocoel. Invaginations were observed in the basolateral cell membrane. Their sizes may vary according to their functions in the basal region and location in the midgut (Terra et al., 2006). The labyrinth observed in the basolateral membrane is responsible for increasing the contact surface between the basolateral membrane and the hemolymph (Cavalcante and Cruz-Landim, 1999, Terra et al., 2006). The goblet cells observed in this study with A. argillacea were similar to those found in other Lepidoptera, which exhibit the typical cavity called chamber. These cells, located throughout the midgut epithelium and intercalated by columnar cells, and have as main function the transport of potassium from the hemolymph to
the lumen, keeping the ionic homeostasis and assisting the columnar cells with the metabolites absorption (Cavalcante and Cruz-Landim, 1999; Klowden, 2002). An intense secretory activity of mucous nature was observed from these cells, an indication of large amount of mucins present in the mucus, since mucins react with the Mallory’s trichrome (Behmer et al., 1976, Junqueira and Junqueira, 1983; Michalany, 1990). The negative reaction of the goblet cells with P.A.S. reinforce the reports of Pinheiro et al. (2008) that glycoprotein secretion is produced exclusively by columnar cells. The regenerative cells are relatively undifferentiated and responsible for the midgut epithelium renewal, not only substituting the senescent cells, but also contributing to the gut growth in every ecdysis. They are usually found isolated or in groups forming nests in the base of the midgut (Cavalcante and Cruz-Landim, 1999; Wanderley-Teixeira et al., 2006; Martins et al., 2006). The lack of endocrine cells in the midgut from A. argillacea after light microscopy analysis agrees with previous reports that these cells are not easily identified through these routine technique, thus ultrastructural and immunohistochemical analysis are necessary (Montuenga et al., 1989, Jimenez and Gilliam, 1990; Neves et al., 2002; Pinheiro et al., 2008). However, after ultrastructural analysis, A. argillacea endocrine cells could be seen in the base of the epithelium, though without reach the lumen. According to Cavalcante and Cruz-Landim (1999), the endocrine cells that do not enter in contact with the lumen are classified as open type, and those with lumen direct contact are the closed type. In general, the morphological, histochemical and ultrastructural characteristics of the midgut of A. argillacea were similar to those described in the literature for most Lepidoptera. However, the presence of mitochondrial polymorphism and bifurcated microvilli suggest a physiological and ultrastructural modification, which may be associated with both higher absorption and secretory activities of the columnar cells in this species. Such increased activity may lead to a faster action of toxins and/or microbial agents, suggesting that this species may be highly susceptible to some of these agents. But, this hypothesis needs further investigation. Acknowledgements The authors would like to thank CAPES for the financial support of the first author and to Rafael Padilha (LIKA/UFPE) for his technical assistance during the material preparation and observation in the electron microscopies. References Abdalla, F.C., Cruz-Landim, C., 2001. Changes in the morphology of the Dufour gland of Apis mellifera L. (Hymenoptera Apidae) during the life stages of the female castes. Rev. Bras. Entomol. 45, 123–129. Abdalla, F.C., Cruz-Landim, C., 2004. Occurrence, morphology and ultrastructure of the Dufour gland in Melipona bicolor Lepeletier, 1836 (Hymenoptera Meliponini). Rev. Bras. Entomol. 48, 1–19. Abdalla, F.C., Cruz-Landim, C., 2005. Ocorreˆncia, morfologia e ultra-estrutura da glaˆndula de Dufour de Scaptotrigona postica Latreille (Hymenoptera: Apidae). Neotrop. Entomol. 34, 47–57. Abdalla, F.C., 2002. Glaˆndula de Dufour. In: Cruz-Landim, C., Abdalla, F.C. (Eds.), Glaˆndulas exo´crinas das abelhas. FUNPEC, Ribeira˜o Preto, pp. 127–149. Azevedo, F.R., Mattos, K.O., Vieira, F.V., 2002. Comportamento alimentar de Alabama argillacea Hubner (Lep. Noctuidae) em algodoeiro. Cieˆn. Agron. 33, 5–9. Behmer, O.A., Tolosa, E.M.C., Freitas Neto, A.G., 1976. Manual de te´cnicas para histologia normal e patolo´gica. Edart, Sa˜o Paulo, 115 pp. Caetano, F.H., Zara, F.J., Grego´rio, E.A., 2002. The origin of lipid droplets in the postpharyngeal gland of Dinoponera australis (Formicidae: Ponerinae). Cytology 67, 301–308. Cavalcante, V.M., Cruz-Landim, C., 1999. Types of cells present in the midgut of the insects: a review. Nature 24, 19–39. Chapman, R.F., 1998. The Insects: Structure and Function, 4th ed. Cambridge University Press, Cambridge, 770 pp.
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