Ultrastructure of the Digestive Cells in the Midgut of the Predator Brontocoris Tabidus (Heteroptera: Pentatomidae) After Different Feeding Periods on Prey and Plants

MORPHOLOGY,HISTOLOGY, AND FINE STRUCTURE Ultrastructure of the Digestive Cells in the Midgut of the Predator Brontocoris tabidus (Heteroptera: Pentatomidae) After Different Feeding Periods on Prey and Plants

MARIA DO CARMO Q. FIALHO,1 JOSE´ C. ZANUNCIO,1 CLO´ VIS A. NEVES,2 3 2,4 FRANCISCO S. RAMALHO, AND JOSE´ EDUARDO SERRA˜ O

Ann. Entomol. Soc. Am. 102(1): 119Ð127 (2009) ABSTRACT Brontocoris tabidus (Signoret) (Heteroptera: Pentatomidae) is an obligate zoophytophagous predator because its population can be maintained in the laboratory when fed on both prey and plants. We evaluated ultrastructural changes in the midgut digestive cells of adult B. tabidus, subjected to different treatments (starvation or feeding on plant material and prey) for different periods. Their midguts were dissected, divided into anterior, medium and posterior sections, pro- cessed, and analyzed with light and transmission electron microscopy. The anterior region of the midgut of B. tabidus, starved or fed on eucalyptus leaves, contained no glycogen. B. tabidus fed on plant material showed multivesicular bodies in this region, and spherocrystals after6hoffeeding on prey. The microvilli of the medium midgut were longer than those of the anterior and posterior midgut. The posterior midgut differed from the other two regions by an abundance of mitochondria, rough endoplasmatic reticulum and double membrane vesicles in the apical region, 6 h after feeding. The ultrastructural features of the digestive cells in the anterior, medium and posterior regions of the midgut suggest that they play a role in digestive enzyme synthesis, ion and nutrient absorption, and storage and excretion of substances.

KEY WORDS midgut, ultrastructure, zoophytophagous, starvation

Phytophagy is common among Heteroptera predators studies of the midgut of predatory insects are important (Eubanks and Denno 1999, Gillespie and Mcgregor because this is the organ where digestion and absorption 2000, Coll and Guershon 2002, Zanuncio et al. 2004, occur (Terra and Ferreira 1994). Further studies may Lemos et al. 2005), and it has been deÞned as a type elucidate how pentatomid predators feed on different of omnivory called zoophytophagy (Coll and Guers- sources, and how ecological, evolutionary, and behav- hon 2002). This process supplies predators with es- ioral variations take place in omnivorous insects. sential compounds and water that helps extraoral di- To maintain its population, B. tabidus require plants gestion (Eubanks and Denno 1999, Sinia et al. 2004). in their diet (Barcelos et al. 1994, Zanuncio et al. 2000). Moreover, zoophytophagy facilitates these natural en- Nevertheless, this species is considered an obligate emies establishment in the Þeld before the emergence zoophytophage because they attain higher survival of the insect pests, allowing their survival during prey and reproductive rates when fed on both prey and shortage (Eubanks and Denno 1999). One such insect plants. is Brontocoris tabidus (Signoret) (Heteroptera: Pen- The midgut of B. tabidus was studied by Guedes et tatomidae), an important stink bug predator of defo- al. (2007), but they focused on gross histological as- liator pests (Zanuncio et al. 2000). pects. The objective of our study was to evaluate The digestive tract of insects is divided into three main ultrastructural changes in the midgut of B. tabidus regions: foregut and hindgut, that are derived from ec- starved or fed on prey or plants for different periods. toderm, and midgut that comes from endoderm Because these conditions occur in the natural envi- (Snodgrass 1935). The midgut of predators and phyto- ronment of this predator, such an evaluation may phagous Hemiptera differs in muscle structure and gut contribute to understanding the evolution of feeding cell types (Lehane and Billingsley 1996). Morphological habits in Pentatomidae.

1 Departamento de Biologia Animal, Universidade Federal de Vi- çosa, 36570-000 Viçosa, MG, Brazil. 2 Departamento de Biologia Geral, Universidade Federal de Viçosa, Materials and Methods 36570-000 Viçosa, MG, Brazil. Animals. Adults of B. tabidus were obtained from 3 Unidade de Controle Biolo´gico EMBRAPA-Algodaõ, 58107-720, Campina Grande, PB, Brazil. the colony in the Laboratory of Biological Control of 4 Corresponding author, e-mail: [email protected]. Insects of the Federal University of Viçosa (Viçosa,

0013-8746/09/0119Ð0127$04.00/0 ᭧ 2009 Entomological Society of America 120 ANNALS OF THE ENTOMOLOGICAL SOCIETY OF AMERICA Vol. 102, no. 1

MG, Brazil). They were fed on Tenebrio molitor L. (Coleoptera: Tenebrionidae) pupae and leaves of Eu- calyptus urophylla (S. T. Baker) at 25 Ϯ 2ЊC under a photophase of 12:12 (L:D) h. Twenty adults of B. tabidus were used of which four were included in each treatment. Treatments. Adults of B. tabidus were placed indi- vidually in 500-ml plastic cups with 2.5-ml cylindrical plastic tubes Þlled with water (Zanuncio et al. 1994), and were starved for 5 d. After this period, individuals were dissected according to the following treatments: starved, 2, 6, or 12 h after the beginning of feeding on T. molitor pupae and 2 h after feeding on eucalyptus leaves. Light Microscopy. Individuals of B. tabidus were cryoanesthetized, and the midguts were dissected in insect saline solution (0.1 M NaCl, 0.1 M KH2PO4, and 0.1MNa2HPO4). The midguts were divided into anterior, middle, and posterior regions and transferred to Zamboni Þxative solution (Stefanini et al. 1967) for 4 h. The samples were dehydrated in a graded ethanol series for 10 min in each bath, embedded in resin JB4 (Electron Microscopy Sciences, HatÞeld, PA) following the manufacturerÕs instructions, and 3-␮m slices were stained with 1% toluidine blue for 5 min. Transmission Electron Microscopy. Individuals of B. tabidus were cryoanesthetized, and the midguts were dissected in insect saline solution. The midguts were divided into anterior, middle, and posterior midgut regions, as described above, and transferred to 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer for 2 h. Samples were postÞxed in 1% osmium tetroxide in the same buffer for 2 h. The samples were dehydrated in a graded acetone series and embedded in Araldite 502/EMbed 812 kit (Electron Microscopy Sciences) following the manufacturerÕs instructions. Ultrathin sections were stained with 1% aqueous ura- nyl acetate and lead citrate and were analyzed with an EM 109 transmission electron microscope (Carl Zeiss, Jena, Germany).

Results The midgut wall of B. tabidus is formed by a single layer of digestive cells on a basal lamina, surrounded by a layer of inner circular and outer longitudinal muscles. The digestive cells of the three midgut areas Figs. 1–3. Midgut of B. tabidus. 1. Anterior midgut. 2. are columnar, containing an enlarged apex which Middle midgut. 3. Posterior midgut. Lumen (L), epithelium (Ep) and muscle (Mu). Bars ϭ 30 ␮m. shows a well developed brush border and cytoplasmic granules (Figs. 1, 2, and 3). These cells contain short microvilli (Figs. 4 and 5) with a thin glycolcalyx (Fig. 11 and 12). CellÐcell contact is maintained by smooth 5) and perimicrovillar membranes (Fig. 6). The apical septate junctions in the apical region of the cells (Fig. cytoplasm has granules with different electron densi- 13). The midgut epithelium rests on a thick noncel- ties, lysosomes, lipid droplets, and glycogen (Fig. 7). lular basal lamina, which may penetrate into the in- Lipid droplets, glycogen, Golgi complex, electron- tercellular space (Figs. 11 and 12). The three midgut dense granules, rough endoplasmic reticulum (RER), regions contain a perimicrovillar membrane in all and mitochondria occur in the perinuclear region of treatments (Fig. 6). Some apoptotic-like cells (Fig. 14) the digestive cells (Figs. 8 and 9), as does the elon- were found in the midgut epithelium. gated nucleus with an evident nucleolus (Fig. 10). Anterior Midgut. The digestive cells of the anterior The basal region of the digestive cells of B. tabidus midgut of B. tabidus showed ultrastructural differ- is characterized by plasma membrane infoldings as- ences among the treatments, with short microvilli sociated with mitochondria and RER proÞles (Figs. (0.63 Ϯ 0.28 ␮m). Glycogen storage and electron- January 2009 FIALHO ET AL.: ULTRASTRUCTURE OF B. tabidus MIDGUT 121

Figs. 4–8. Electron micrographs of the midgut of Brontocoris tabidus. 4. Anterior midgut (12 h after fed on prey) showing digestive cell (DC), lumen (L), microvilli (Mv), lipids (Li) and lysosome (Ls). Bar ϭ 2 ␮m. 5. Posterior midgut (fed on leaf) showing microvilli (Mv), glycocalyx (Gc). Bar ϭ 0.1 ␮m. 6. Middle midgut (12 h after fed on prey) showing lumen (L), perimicrovillar membrane (PmM) and microvilli (Mv). Bar ϭ 1 ␮m. 7. Anterior midgut (two hours after fed on prey) showing digestive cell (DC), microvilli (Mv), lysosome (Ls), glycogen (G) and lipid (Li). Bar ϭ 1 ␮m. 8. Posterior midgut (six hours after fed on prey) showing rough endoplasmic reticulum (RER) and mitochondria (M). Bar ϭ 1 ␮m. dense granules were not found in the predators, ing in a basal labyrinth. Multivesicular bodies (Fig. 15) starved or fed on plants. The basal region of the di- were found in the basal region of the cells in the gestive cells, in those treatments, had plasma mem- insects fed on plants. The basal lamina, in all treat- brane infoldings associated with mitochondria, result- ments, was 0.66 Ϯ 0.1 ␮m thick. 122 ANNALS OF THE ENTOMOLOGICAL SOCIETY OF AMERICA Vol. 102, no. 1

Figs. 9–13. Electron micrographs of the midgut of Brontocoris tabidus. 9. Middle midgut (two hours after fed on prey) showing digestive cell (DC), lipid (Li) and glycogen (G). Bar ϭ 1 ␮m. 10. Posterior midgut (fed on leaf) showing digestive cell (DC), mitochondria (M), nucleus (N) and chromatin (C). Bar ϭ 1 ␮ m. 11. Middle midgut of starved insect showing digestive cell (DC), rough endoplasmic reticulum (RER), glycogen (G), basal lamina (BL) and muscle (Mu). Bar ϭ 0.5 ␮m. 12. Posterior midgut (six hours after fed on prey) showing digestive cell (DC), glycogen (G), mitochondria (M), basal lamina (BL) and basal plasma membrane infoldings (If). Bar ϭ 0.5 ␮m. 13. Posterior midgut (fed on leaf) showing microvilli (Mv) and smooth septate junctions (SJ). Bar ϭ 0.5 ␮m. January 2009 FIALHO ET AL.: ULTRASTRUCTURE OF B. tabidus MIDGUT 123

Figs. 14–18. Electron micrographs of the midgut of Brontocoris tabidus. 14. Posterior midgut (six hours after fed on prey) showing digestive cell (DC), mitochondria (M), vacuole (V), apoptotic cell (AC) and nucleus (N). Bar ϭ 2 ␮m. 15. Anterior midgut (fed on leaf) showing glycogen (G) and multivesicular bodies (MB). Bar ϭ 1 ␮m. 16. Anterior midgut (two hours after fed on prey) showing spherocrystals (Sc). Bar ϭ 1 ␮m. 17. Posterior midgut (12 h after fed on prey) showing digestive cell (DC), perimicrovillar membrane (PmM), microvilli (Mv), lysosome (Ls) and lipid (Li). Bar ϭ 1 ␮ m. 18. Posterior midgut (6 h after fed on prey) showing microvilli (Mv) and vesicles with double membrane (*). Bar ϭ 0.1 ␮m. 124 ANNALS OF THE ENTOMOLOGICAL SOCIETY OF AMERICA Vol. 102, no. 1

The apical and perinuclear cytoplasm of the diges- tabidus showed differences in number and distribu- tive cells in the anterior midgut had many granules tion of lysosomes, RER and mitochondria among the with different electron-densities, lipid droplets and three midgut regions, as found in the hematophagous glycogen deposits, in the treatments of 2, 6 and 12 h Rhodnius prolixus Stål (Heteroptera: Reduviidae) after feeding on prey. Spherocrystals were found in (Billingsley and Downe 1983, 1989). Differences in the cytoplasm of the digestive cells 2 h after feeding microvilli length of the digestive cells among the mid- on prey (Fig. 16), and 6 h after feeding there was a gut regions of B. tabidus suggest a high absorption rate decrease in glycogen deposits and an increase in ly- in the middle midgut because in this region the mi- sosomes. The basal region of the digestive cells of crovilli are larger, thus increasing the cell surface. those insects fed on prey showed deeper basal plasma Glycogen in the digestive cells, in all midgut regions membrane infoldings associated with mitochondria, in of B. tabidus and all treatments, suggest that this gut comparison with those insects starved or fed on eu- region plays an important role in carbohydrate storage calyptus leaves. (Silva-Olivares et al. 2003), which provides necessary Middle Midgut. Ultrastructural differences among energy for the digestive cells to accomplish different treatments were little evident in the digestive cells of metabolic activities (Turunen and Crailsheim 1996). the middle midgut, but the microvilli were longer The presence of glycogen deposits in B. tabidus (2.00 Ϯ 0.26 ␮m) than those found in the anterior and starved and fed on leaves may be a source of energy posterior midgut. The apical cytoplasm in all treat- accumulation for enzyme synthesis, when these in- ments contained lipid droplets, electron-dense gran- sects are searching for prey. Predatory insects do not ules, and well-developed RER in the perinuclear re- have food constantly available, unlike phytophages. As gion. Six hour after feeding, these cells showed a result, they need to synthesize ready digestive en- lysosome accumulation and a decrease in glycogen zymes when they are searching for prey, which de- storage. The basal region of the digestive cells, besides mands energy that can be quickly obtained from gly- plasma membrane infoldings associated with mito- cogen deposits. Predatory beetles synthesize digestive chondria, showed lipid droplets and glycogen storage. enzymes immediately after feeding (Pradhan 1940), The basal lamina was 0.42 Ϯ 0.1 ␮m thick. and in newly emerged holometabolous insects the Posterior Midgut. The digestive cells of the poste- digestive cells use energy from glycogen in early adult rior midgut had short microvilli (0.62 Ϯ 0.40 ␮m) and life (Serraõ and Cruz-Landim 1996, Neves et al. 2003). ultrastructural differences compared with the cells of Glycogen deposits are also associated frequently with the anterior and middle midgut. The apical and pe- intense absorptive activity in digestive cells (Billings- rinuclear cytoplasm of the digestive cells, in all treat- ley 1990), which may also occur in B. tabidus because ments, showed lipid droplets, granules with different this predator has extraoral digestion and some sub- electron densities, RER, and many lysosomes and mi- stances may be absorbed without additional digestion tochondria. The basal region of these cells presented in the midgut. fewer plasma membrane infoldings in comparison to Multivesicular bodies in digestive cells of the ante- the others two parts of the midgut. Mitochondria and rior midgut of B. tabidus, fed on leaves, indicates the lipid droplets were associated with those infoldings. degradation of membrane receptors involved in the The basal lamina was 0.66 Ϯ 0.05 ␮m thick. absorption of amino acids contained in the sap or Ultrastructural differences, among treatments in storage of some proteins that should be used only after the posterior midgut, were evident. Digestive cells of feeding with prey. These structures, frequently found starved insects contained less glycogen. In those in- in the midgut cells of insects (Serraõ and Cruz-Landim sects fed on plants a large amount of glycogen was 1996), are formed from early endosomes due to an found distributed throughout the cytoplasm. Six hours inward budding of its membrane resulting in intralu- after feeding on prey, RER and mitochondria were menal vesicles whose main function is “collecting” found in abundance in the cytoplasm of the digestive plasma membrane receptors to be degraded into the cells (Fig. 16), including the apical cytoplasm. In con- lysosomes, although in some specialized mammalian trast with the anterior and middle midgut of the other cells they can temporarily store some proteins (Al- treatments, these organelles were absent in this re- berts et al. 2004, Piper and Katzmann 2007). gion. Besides those differences, in this treatment dou- The digestive cells of B. tabidus with spherocrystals ble membrane vesicles in the apical cell portion were in the anterior midgut and cytoplasmic granules with observed (Fig. 18). The digestive cells of B. tabidus calcium and iron in the mid-midgut of this insect 12 h after feeding presented numerous lysosomes in (Guedes et al. 2007) indicates the importance of this the apical cytoplasm (Fig. 17). organ for the absorption and storage of these ions. Similar granules were found in Apis mellifera L. (Hy- menoptera: Apidae) and Melipona quadrifasciata Discussion anthidioides (Lepeletier) (Hymenoptera: Apidae) Microvilli, large nuclei with evident nucleoli, cyto- (Cruz-Landim and Serraõ 1997). The morphological plasm rich in RER, Golgi complex, and vesicles in the and chemical similarities between the spherocrystals digestive cells B. tabidus midgut indicate that those of bees and those of B. tabidus suggest that spheroc- cells are involved in the synthesis and secretion of rystal accumulation in the cell apex and their release proteins, and absorption and transport of substances by apocrine secretion represents an excretory func- (Rudin and Hecker 1979). The digestive cells of B. tion of the midgut (Cruz-Landim and Serraõ 1997). January 2009 FIALHO ET AL.: ULTRASTRUCTURE OF B. tabidus MIDGUT 125

Droplet lipids in the three midgut regions of B. go´rio 2003). The cells of the posterior midgut of T. tabidus agree with results from R. prolixus, although molitor have higher metabolic activity than those cells the latter had a larger concentration in the anterior in the anterior and middle midgut (Ferreira et al. midgut (Billingsley 1988). Lipid accumulation in the 1990). This process can be explained by the higher anterior midgut is important for the survival of R. concentration of mitochondria in this midgut region prolixus during starvation (Billingsley and Downe found in B. tabidus, suggesting that the metabolic 1989). The droplet lipids in the midgut cells of B. processes in the posterior midgut require a larger tabidus may result in a coupled transport and also be amount of energy than other midgut regions. temporarily stored in the midgut for use during peri- In hematophagous insects, water absorptions take ods of starvation. place in the anterior midgut where basal plasma mem- Hemiptera have no peritrophic membrane. How- brane infoldings may extend through half of the cell, ever, they have a system of membranes covering the and they are associated with numerous mitochondria microvilli that form an outer microvillar membrane, (Chapman 1998). This also was observed in B. tabidus, called the perimicrovillar membrane (Andries and suggesting that the anterior midgut play an essential Torpier 1982, Silva et al. 1995, Terra 1988, Silva et al. role in water absorption. 2007). The perimicrovillar membrane was found in the The structure of the digestive tract of extant insects three midgut regions of B. tabidus in all treatments, cannot be attributed solely to the diet, but to adap- whereas in R. prolixus, the perimicrovillar membrane tations acquired by the ancestor (Ribeiro et al. 1990, was synthesized only some hours after the insect fed Schumaker et al. 1993). The ultrastructure of the B. (Billingsley and Downe 1983, 1986). This suggests that tabidus midgut is similar to that of R. prolixus, corrob- control of perimicrovillar membrane production orating the hypothesis that predatory and hematoph- differs between hematophagous and predatory agous Hemiptera arose from a phytophagous ancestor Hemiptera. The perimicrovillar membrane arose in that has lost the peritrophic membrane associated the Condylognatha ancestor (Paraneroptera that in- with the absence of initial digestion in the midgut cludes Hemiptera and Thysanoptera) that fed on lumen (Terra 1990). They acquired the perimicrovil- phloem. The Condylognatha ancestor lost enzymes lar membrane due to an efÞcient absorption process of involved in initial and intermediate digestion and the diluted diets (Terra 1988, Ribeiro et al. 1990). R. pro- peritrophic membrane (Terra 1988, Terra and Fer- lixus maintain the perimicrovillar membrane as a sub- reira 1994, Terra et al. 2006). Hematophagous stitute for the peritrophic membrane that is used in the Hemiptera and B. tabidus have the perimicrovillar compartmentalization of the digestive process (Terra membrane. In B. tabidus, the synthesis of this mem- 1990). The perimicrovillar membrane of B. tabidus brane is constitutive, suggesting similarity with the also substitutes for the peritrophic membrane in the sapsucker ancestor. This similarity may be explained accomplishment all its functions, because the diges- by the fact that phytophagous Hemiptera have constitutive synthesis of the perimicrovillar membrane tion of polymers occurs in the midgut of this predator (Silva et al. 2004) and B. tabidus has enzymes involved (Guedes et al. 2007), after extraoral digestion (Aze- in initial digestion (Azevedo et al. 2007, Guedes et al. vedo et al. 2007). 2007). The posterior midgut of Diptera has cells containing The perimicrovillar membrane arises from the inner more RER and mitochondria than in the anterior mid- membrane of the double membrane vesicles or mul- gut (Hecker 1977, Andrade-Coeˆlho et al. 2001), as timembrane vesicles that likely originated from the found in B. tabidus, suggesting that the posterior mid- Golgi complex (Silva et al. 1995, Cristofoletti et al. gut of this predator is the main location of enzyme 2003). The posterior midgut of B. tabidus has double secretion, even though polymerases exist in all three membrane vesicles, suggesting that these structures midgut regions of B. tabidus (Guedes et al. 2007). are responsible for the formation of the perimicrovil- Therefore, we suggest that digestive enzymes are re- lar membrane. However, mitochondria can be modi- leased in the posterior midgut and transferred to the Þed in secretory granules with double membranes in anterior regions of the midgut due to the ßux created termites (Sˇobotnõ´k and Weyda 2003, Costa-Leonardo by water absorption in the anterior midgut. A similar 2006) or undergo changes that precede cell death explanation was proposed for Lepidoptera and (Tettamanti et al. 2007). B. tabidus has the perimi- Diptera by Terra et al. (1985). crovillar membrane and because double membrane Mitochondria closely associated with the long basal vesicles are well documented in the perimicrovillar plasma membrane infoldings of the digestive cells in membrane formation in Hemiptera, we suggest that the anterior midgut of B. tabidus indicate water and these vesicles perform that function although an in- nutrient absorption in this region. Longer microvilli in dication of cell death cannot be discarded, because the middle midgut suggest higher nutrient absorption cell degeneration is a common event in the midgut of than in other midgut regions. Finally, the posterior insects (Rost 2006, Tettamanti et al. 2007). midgut can be characterized by a large number of A large number of mitochondria in the basal and mitochondria and RER in the cell apex indicating the apical regions of the digestive cells of the anterior and occurrence of synthesis of proteins and active trans- posterior midgut of B. tabidus were found. This cor- port in this region. These ultrastructural features of roborates the results observed in Diatraea saccharalis the midgut indicate that physiological differences ex- F. larvae (Lepidoptera: Pyralidae) (Pinheiro and Gre- ist among the three midgut regions of B. tabidus. 126 ANNALS OF THE ENTOMOLOGICAL SOCIETY OF AMERICA Vol. 102, no. 1

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