ISJ 12: 158-165, 2015 ISJ 12: 158-165, 2015 ISSN 1824-307X RESEARCH REPORT Effect of silicon on the morphology of the midgut and mandible of tomato leafminer Tuta absoluta (Lepidoptera: Gelechiidae) larvae MC dos Santos1, AM Resende Junqueira1, VG Mendes de Sá2, JC Zanúncio3, JE Serrão4 1Faculty of Agronomy and Veterinary Sciences, University of Brasilia, 70910-970, Brasília, Distrito Federal, Brazil 2Faculty of Engineering, University of the State of Minas Gerais, 35930-314, João Monlevade, Minas Gerais State, Brazil 3Department of Entomology, Federal University of Viçosa, 36.570-000, Viçosa, Minas Gerais State, Brazil 4Department of General Biology, Federal University of Viçosa, 36.570-000, Viçosa, Minas Gerais State, Brazil Accepted May 12, 2015 Abstract Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae) is an important insect pest causing serious losses to tomato plantations in Brazil. Some populations of T. absolute are reported to present insecticide resistance resulting in its control failure and the use of alternative control based on silicon, which is clean and sustainable, can reduce pesticide use, increasing fruit quality and protecting the environment. This study evaluated changes in the morphology of the midgut and the mandibles of T. absoluta larvae caused by feeding with compounds containing silicon. Larvae of T. absoluta were fed on tomato leaves with different compounds containing silicon and the histology of the midgut of fourth instar larvae was analyzed. The mandibles of all larval stages were dissected and analysed by scanning electron microscopy. There were no changes in the morphology of the mandibles of the larvae of T. absoluta fed on silicon compared to the control group. Larvae of T. absoluta from the control group and the treatments where the calcium silicate was applied to the soil had not differences in the morphology of the midgut epithelial cells, which had four cell types: digestive, goblet, regenerative and mycetocyte cells. In larvae of T. absoluta obtained from silicon-based treatments applied to the leaves, the midgut epithelium showed detachment of the basal membrane, which can characterize the possible effect of this toxic element to larvae of T. absoluta. Key Words: Lycopersicon esculetum; fertilization; morphology; pest control   Introduction The tomato (Lycopersicon esculentum) is widely cultivated in the world and it is one of the most produced vegetables in Brazil, which is currently among the top ten world producers (Vilela, 2009). The tomato crop in Brazil is expanding and modernizing, seeking greater productivity and quality to meet market demands, thereby generating more concern about pest control (Vivan et al., 2002) that can cause substantial reductions in fruit quality (Miranda et al., 1998; Kennedy, 2003; Mahanil et al., 2008). The tomato pinworm, Tuta absoluta (Lepidoptera: Gelechiidae), which attacks leaves and flowers causing mines and fruit drilling throughout the cycle of tomato, stands out as the most important insect ___________________________________________________________________________ Corresponding author: José Eduardo Serrão Department of General Biology Federal University of Viçosa 36.570-000, Viçosa, Minas Gerais State, Brazil E-mail: jeserrao@ufv.br pest of this crop and has usually been controlled by multiple applications of insecticides (Medeiros et al., 2009). However, the frequent use of insecticides may be harmful to the natural biological control, generates the production of foods with high levels of toxic waste, environmental contamination, besides the risk of selecting resistant insects streams (Vianna et al., 2009). Many insecticides have the midgut cells of the insects as a first target (Forkpah et al., 2014), which is the main organ where digestion and absorption take place (Lehane and Billingsley, 1996). The midgut wall has three layers: two muscular layers and an epithelial lining of the lumen, composed of a single layer of cells that, in most insects, has three cell types: columnar or digestive, regenerative, and endocrine, while a fourth cell type, goblet cells, occurs in Lepidoptera (Lehane and Billingsley, 1996). The epithelium surface is isolated from the food content by a non-cellular membrane, the peritrophic membrane or matrix (Lehane and Billingsley, 1996; Terra, 2001).   158 mailto:jeserrao@ufv.br Table 1 Treatments used to evaluate the effect of silicon on the morphology of the midgut and mandible of Tuta absoluta (Lepidoptera, Gelechiidae) Treatments Product Amount (ton ha-1) T1 0.45 (ton ha-1) T2 0.9 (ton ha-1) T3 1.35 (ton ha-1) T4 AgrosilícioTM Soil 1.8 (ton ha-1) T5 2.0 (ton ha-1) T6 4.0 (ton ha-1) T7 6.0 (ton ha-1) T8 AgrosilícioTM leaves 8.0 (ton ha-1) T9 0.5 (l ha-1) T10 1.0 (l ha-1) T11 2.0 (l ha-1) T12 Sili-KTM leaves 3.0 (l ha-1) T13 0.25 % T14 0.50 % T15 0.75 % T16 Silicic Acid leaves 1.0 % T17 0.25 % T18 0.50 % T19 0.75 % T20 Silicic Acid soil 1.0 % In Lepidoptera larvae, the peritrophic membrane is secreted by the midgut digestive cells (De Priester, 1971; Terra, 1988). This membrane, although it prevents direct contact of food with the digestive cells, allows the passage of digestive enzymes in the direction of the midgut lumen and the absorption of the products of digestion which are then eliminated with the faeces (Terra, 1988) avoiding mechanical damage, impeding or preventing the entry of pathogens and partition the digestion process (Terra, 1988; 2001). The digestive cells are the most abundant and are responsible for producing enzymes and absorbing products of digestion (Terra, 1988). Goblet cells are involved in the homeostasis and absorption of metabolites, a function performed in conjunction with the digestive cells (Lehane and Billingsley, 1996; Terra et al., 2006). The regenerative cells are seen alone, paired or in nidi at the base of the epithelium and they play a role in cell renewal (Serrão and Cruz-Landim, 1996a; Martins et al., 2006). The endocrine cells are located at the base of the epithelium and they are characterized by the presence of a large number of cytoplasmic granules, which produce peptide hormones (Serrão and Cruz-Landim, 1996b; Neves et al., 2002). Synthetic insecticides results in many ecological problems such as selection of resistance strains of insects, ecological unbalance and mammals intoxications. Thus, it is necessary the use of programs of integrated pest management, which aim the reduction of the use of pesticides compatible with natural enemies (Zanuncio et al., 2003). Although silicon is not essential for most plants (Savant et al., 1999), it is beneficial to improve plant resistance against some biotic and abiotic stresses (Do Gramaci et al., 2013; Haynes, 2014). The application of silicon has increased plant resistance to insect pests (Almeida et al., 2008; Cherry et al., 2012; Sidhu et al., 2013; Keeping et al., 2014; Pinto et al., 2014), mainly for its ability to accumulate in the plant cell wall (Costa and Moraes 2006), thereby increasing the synthesis of lignin and phenolic compounds (Chérif et al., 1992; Ghanmi et al., 2004), in addition to activating the endogenous chemical defenses of plants (Gomes et al., 2005). The use of silicon in controlling defoliator insects occurs due to the action of the mechanical barrier provided by the deposition of this mineral in the cell wall of leaves (Goussain et al., 2002; Massey et al., 2006; Kvedaras et al., 2009). Tuta absoluta reared on tomato plants accumulating silicon show decrease in larvae and pupae survival and male and female weight (Santos et al., 2012). The deleterious effects on insect larvae that feed on plants treated with calcium silicate have been suggested to occur due damages in the incisor teeth of the mandibles, affecting the   159 insect nourishment and development (Goussain et al., 2002; Kvedaras et al., 2009) but data about its effect on midgut remains unknown. This study evaluated whether treatment of tomato plants with silicon affects the morphology of the midgut and the mandibles of the tomato pinworm T. absoluta. Materials and Methods Planting, experimental design and treatments The planting of seedlings of tomato, variety Tospodoro obtained at Embrapa Vegetables, Brasília, DF, was carried out a greenhouse at the Federal University of Viçosa, Viçosa, MG, Brazil. The experimental plots of tomato plants grown in 3 L polyethylene pots, containing one plant in each with planting nitrogen-containing fertilizer (600 kg ha-1 ammonium sulfate), phosphorus (3300 kg ha-1 superphosphate) and potassium (330 kg ha-1 potassium chloride), whose amounts were calculated on the basis of soil analysis. Three compounds used as sources of silicon were: AgrosilícioTM (10.5 % Si), Sili-kTM (12.2 % Si) and silicic acid (100 % Si). The experimental design was randomized blocks with five replications, twenty treatments (Table 1) and control (without addition of any compound containing silicon). The AgrosilícioTM was added to the soil together to fertilizer planting, aiming to increase alcaline saturation to 70 % (Ribeiro and Guimarães 1999) in the treatments T1 to T4, and applied weekly by foliar sprays in treatments T5 to T8. The Sili-KTM product was applied weekly only to the leaves, in the treatments T9 to T12, due to the fact that compound is not available for soil application. The silicic acid solution was applied weekly, in foliar application, treatments T13 to T16 and in the soil around the stems of plants, treatments T17 to T20. The first foliar application of the products was made 30 days after planting of tomatoes, a total of three applications at weekly intervals in treatments where AgrosilícioTM was applied to the soil, since this product has a soil corrective effect (Sommer et al., 2006). In all treatments with AgrosilícioTM, Sili-KTM and silicic acid soil were corrected with dolomitic lime (0.8 t ha-1) in order to raise the alkaline saturation to 70 %. Release of T. absoluta eggs Tomato leaves with T. absoluta eggs of the same age, from the mass rearing of the Laboratory of Agricultural Entomology, Federal University of Viçosa, were sectioned in order to contain about 30 eggs approximately. Each of these sectioned areas was fixed with the aid of a pin in the branches of the tomato plant caged in organza bags 40x70 cm involving the entire plant, seven days after the last foliar application of products containing silicon. Light Microscopy To evaluate the silicon effect in the midgut of T. absoluta, fourth instar larvae obtained from the treatments were collected and transferred to flasks containing 2 mL of Zamboni fixative solution (Stefanini et al., 1967). Afterwards, the midguts of T. absoluta were dissected in insect saline solution (0.1 M NaCl + 0.1 M KH2PO4 + O.1M Na2HPO4), dehydrated in a graded ethanol series (70, 80, 90 and 95 %) and embedded in JB-4TM historesin for 24 h. The samples were sectioned at 5 μm slices, stained with hematoxyline and eosin, examined and photographed in light photomicroscope. Scanning electron microscopy To evaluate the silicon effect in the wear of the mandibles in all four larval stages, T. absoluta larvae, were collected each 12 h, two larvae per plant, and placed in flasks containing 2 mL of Zamboni fixative. Larvae were collected immediately before ecdysis, based on the duration of each larval instar (Giustolin et al., 2002) to ensure greater exposure to treatments. The mandibles were removed, dehydrated in a graded ethanol series (70, 80, 90 and 95 %), transferred to HMDS (Hexamethyldisilazane) and after five min air drying. The mandibles were gold covered (20 nm) and analyzed under a scanning electron microscope LEO VP1430 at the Center for Microscopy and Microanalysis at the Federal University of Viçosa. Results The midgut of T. absoluta larvae showed a single layered epithelium with four cell types: digestive, goblet, regenerative and micetocyte cells (Figs 1a - e). The digestive cells were tall, which apex showing a well-developed brush border and cytoplasm containing numerous vacuoles (Fig. 1c). In the middle portion of these cells was found an oval nucleus with a predominance of decondensed chromatin (Fig. 1b). The goblet cells were characterized by a cavity formed by invagination of the apical surface of these cells that showed a basal nucleus (Figs 1a - c). The regenerative cells were small, never reaching the midgut lumen, containing a relatively large nucleus in relation to the cytoplasm and were scattered between the base of the goblet and digestive cells isolated or forming nests till three cells (Fig 1a). Throughout the midgut epithelium of T. absoluta were found some large and globular cells with spherical nucleus and cytoplasm almost filled with small basophilic bacteria-like particles (Fig. 1e), which allowed the characterization of this cell type as mycetocytes. A well-developed peritrophic membrane line the midgut lumen (Fig. 1c). The midgut epithelium was seated on a homogeneous basal membrane and a layer of circular muscles was found externally, followed by an outer layer of longitudinal muscles. Larvae of T. absoluta from the control group and treatments where calcium silicate was applied to the soil showed no differences in the structural organization of the midgut that was described above. Larvae of T. absoluta obtained from silicon- based treatments applied to leaves showed changes in the morphology of the midgut, a characterized by the detachment of the basal membrane of the digestive epithelium (Figs 1a, 1b), presence of cytoplasmic protrusions with strongly   160 Fig. 1 Histological sections of the midgut of fourth instar larvae of Tuta absoluta (Lepidoptera, Gellechidae) fed on tomato plants treated with compounds containing silicon. a) Midgut epithelium of larvae fed on plants exposed to AgrosilícioTM leaves (40 t ha-1) showing digestive cells (DC), globet cells (GC) and regenerative cell nidus (RC) Note basal membrane detached (BM), well developed brush border (BB) and apoptotic bodies (arrows) released to lumen (L) Bar = 20 μm; b) Midgut epithelium of larvae fed on plants exposed to AgrosilícioTM leaves (40 t ha-1) showing basophilic apoptotic bodies (arrow) Bar = 10 μm; c) Midgut epithelium of larvae fed on plants exposed to Silicic Acid leaves (1,0 %) showing digestive (DC) and globet cells (GC) with vacuoles in the cytoplasm (V) Notice the peritrophic membrane (PM) Bar = 10 μm; d) Midgut epithelium of larvae fed on plants exposed to Sili-KTM leaves (30 l ha-1) showing detached basal membrane (BM) Bar = 10 μm; e) Midgut epithelium of larvae from plants the control group without any addition of compounds containing silicon and fed plants exposed to Sili-KTM leaves (05 l ha-1) showing mycetocyte (MY) characterized by basophilic granules bacteria-like N, nucleus with decondensed chromatin Bar = 10 μm. basophilic content, some them containing the cell nucleus, which are released into the midgut lumen, similar to apoptotic bodies (Figs 1a, 1b). The larvae of T. absoluta from all treatments including the control group, had six teeth in their mandibles without variation in the number or shape of them, and show no signs of damage from first to fourth instar (Figs 2a, 2b). Discussion The efficiency of silicon-containing products to control T. absoluta has been suggested to occur due its toxic and anti-feeding effect to the larval stage, playing a role as activator of tomato plants resistance (Rodrigues et al., 2004; Côté-Beaulieu et al., 2009). However, there were no studies proving   161 this mechanism of silicon action, which is accumulated in higher concentrations as silica in the leaf tissues of plants (Basagli et al., 2003; Correa et al., 2005; Gomes et al., 2005). In this study, one of the effects caused by the application of silicon in the leaves was the detachment of the midgut epithelium from the basal membrane, which leads to the reduction of digestive capacity in insects (Barbeta et al., 2008; Almeida et al., 2009). However, there are no significant difference in the structural organization of the midgut epithelium of T. absoluta between the larvae in the control group and those of silicon treatments applied to the soil, as previously described for other Lepidoptera larvae (Levy et al., 2004; Pinheiro et al., 2008; Sousa et al., 2009, 2010). The mechanisms of silicon action in the gut cells of insects are not yet fully understood. Cellular detachment from basal membrane in the midgut of larvae of T. absoluta may indicate that the anti- feeding action of the compounds containing silicon may be related to physiological effects resulting from their ingestion. Compounds derived from neem seeds applied in leaves for use in integrated pest management also showed similar effects by altering the epithelial cells of the midgut of insects and, consequently, any effect on their development (Mordue and Nisbest 2000; Ndione et al., 2007; Barbeta et al., 2008; Almeida et al., 2009, 2014). The midgut of Alabama argillacea (Lepidoptera: Noctuidae) caterpillar fed on Bacillus thuringiensis cotton leaves has morphological changes of goblet and digestive cells with increase in cytoplasm vacuoles amount, degeneration of muscle layer and absence of peritrophic membrane (Sousa et al., 2010). The presence of cell protrusions like apoptotic bodies being released in the midgut lumen of T. absoluta larvae treated with silicon applied to leaves, suggest a cytotoxic effect of silicon. Apoptosis is a morphological pattern of programmed cell death characterized by cell shrinkage (Ihara et al., 1998). The intake of silicon may have a toxic effect caused by damaging the epithelial cells of the midgut and, therefore, a response would be the elimination of these cells by programmed cell death. Toxins such as Cry1Ac from Bt have similar effect, providing a toxic effect and causing apoptosis in cells of the insects midgut (Zhang et al., 2005). The occurrence of small basophilic granules found in globular cells of midgut of T. absoluta larvae in this study are similar to structures called mycetocytes found in some insect that depend on the obligatory mutualism with microorganisms, mainly bacteria (Moran and Baumann, 2000). However, the occurrence of such cells accumulating microorganisms has not been reported to occur in Lepidoptera and the identification of the microorganisms found inside mycetocytes of T. absoluta must be conducted in the future, in order to understand their function in the physiology of the insect or disease transmission to plants. Our results showed no effect of silicon on the mandibles of T. absoluta, which have not wear signals in all larval stages. Silicon is beneficial to plants, acting as an inducing resistance agent against pest insects, and the silification of the Fig. 2 Mandibles of Tuta absoluta (Lepidoptera, Gellechidae) larvae; a) Fed on tomato plants exposed to Silicic Acid leaves (0.5 %) Bar = 20 μm; b) Control group without any addition of compounds containing silicon Bar = 20 μm epidermis prevents the penetration and chewing by insects due to the hardening of plant cell wall (Datnoff et al., 2001). In larvae of Spodoptera frugiperda (Lepidoptera: Noctuidae) fed on corn plants treated with calcium silicate occurs a wearing in the mandibles teeth (Goussain et al., 2002). However, in Eldana saccharina (Lepidoptera: Pyralidae) caterpillars fed on sugar cane treated with silicon, there was no significant effect in wearing in the mandibles (Kvedaras et al., 2009). Absence of wear in the mandibles of T. absoluta larvae fed on tomato plants treated with silicon, may be due to physiological characteristics of the plant and the feeding habits of these larvae. The tomato is a non-accumulating silicon plant because it absorbs little silicon through the roots (Ma and Yamaji 2006) and the increasing levels of this compound in tomato leaves is not proportional to their availability in the soil (Pereira et al., 2003). The effect of mechanical protection of silicon in plants is attributed to its storage as amorphous silica (SiO2nH2O) in the cell wall (Datnoff et al., 2001). The AgrosilicioTM (insoluble in water) applied to leaves forms only one layer of silica on the epidermis of the leaves while the Sili-KTM (silicon- soluble liquid) forms a silica layer evidenced by its   162 polymerization with cuticular compounds of the plant (unpublished data). The larvae of T. absoluta feed and develop in the mesophyll of tomato leaves (Medeiros et al., 2009) and, probably, due to the diet, to the compounds containing silicon does not penetrate the leaves of tomato plants and this plant not accumulate Si (Ma et al., 2001), the mandibles of T. absoluta have been not stressed. Conclusion The foliar application of silicon-containing compounds in tomato plants was effective against the attack of T. absoluta caterpillars causing detachment of midgut cells from the basal membrane, which may result in digestion difficulties and larval mortality. Acknowledgements This research was supported by Brazilian research agencies CAPES, CNPq and FAPEMIG. Authors are grateful to Center for Microscopy and Microanalysis (UFV) for technical assistance, Prof. Picanço M (UFV) for make available T. absoluta samples, Prof. Sedyama T (UFV) for greenhouse assistance and to EMBRAPA. 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