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Ultrastructural studies on the midgut of biting midge Forcipomyia nigra (Winnertz) (Diptera: Ceratopogonidae)
Accepted Manuscript Title: Ultrastructural studies on the midgut of biting midge Forcipomyia nigra (Winnertz) (Diptera: Ceratopogonidae) Author: Aleksandra Urbanek Magdalena M. Rost-Roszkowska PII: DOI: Reference:
S0968-4328(14)00194-2 http://dx.doi.org/doi:10.1016/j.micron.2014.11.003 JMIC 2141
To appear in:
Micron
Received date: Revised date: Accepted date:
4-9-2014 17-10-2014 7-11-2014
Please cite this article as: Urbanek, A., Rost-Roszkowska, M.M.,Ultrastructural studies on the midgut of biting midge Forcipomyia nigra (Winnertz) (Diptera: Ceratopogonidae), Micron (2014), http://dx.doi.org/10.1016/j.micron.2014.11.003 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Highlights
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Midgut of larvae and adults flies of Forcipomyia nigra was examined
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Larval digestive cells reveal different ultrastructure according to their age
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Females and larval digestive cells take part in storage reserve material
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Type diet influences on the function of digestive cells in species examined
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Ultrastructural studies on the midgut of biting midge Forcipomyia nigra (Winnertz) (Diptera: Ceratopogonidae)
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Aleksandra Urbanek*1, Magdalena M. Rost-Roszkowska2
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Department of Invertebrate Zoology and Parasitology, University of Gdańsk, Wita Stwosza 59, 80308 Gdańsk, Poland
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Department of Animal Histology and Embryology, Bankowa 9, University of Silesia, 40-007 Katowice, Poland
* corresponding author: [email protected], phone +48 58 523 61 84
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Abstract. Biting midges belonging to the genus Forcipomyia are known to be haematophagous, predatory or saprophagous. Different stages of Forcipomyia nigra midges were investigated to provide a description of midgut ultrastructure. Larvae feeding on decaying organic matter possess simple, straight alimentary tracts whose middle regions are the longest. TEM studies of the larval midgut epithelium reveal that digestive cells show different ultrastructure depending on their age. The older cells with electron-dense cytoplasm degenerate while the younger ones with electron-lucent cytoplasm remain active in digestion. In saprophagous females, the ultrastructure of midgut epithelium changes according to the age of flies. Oogenesis induces degeneration of digestive cells and utilization of reserve material accumulated by them. The midgut epithelia of male midges consists of digestive and regenerative cells that show no evidence of cell degeneration as observed in females. Our results demonstrate differences between midgut digestive cells of males and females.
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Key words: biting midge, Forcipomyia nigra, midgut, digestive cell, regenerative cell
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1.Introduction
Members of the family Ceratopogonidae, better known as biting midges, are small, about 1-5 mm, nematoceran flies developing in a great range of habitats. Observations of their trophic relations reveal that the biting habit is restricted to the females, which show diverse feeding behaviour. They are hematophagous (species from genera Culicoides, Leptoconops, Austroconops or Forcipomyia), predators on other insects (most species of the subfamily Ceratopogoninae) or pollinivorous (e.g., Atrochopogon pollinivorous) (Szadziewski et al., 1997). Protein-rich meal is required for initiation of oogenesis in the majority of species of 2
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2.1. Insects
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2.Material and methods
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biting midges (Szadziewski et al., 1997). Males feed usually on nectar or honeydew, but in subfamily Forcipomyiinae (genus Dasyhelea and some species of Forcipomyia) both sexes take nectar from flowers. The structure of the midgut of biting midges is simple: it is composed of a single layer of epithelial cells. It is divided into the anterior tubular region and posterior stomach (Megahed, 1956; Chaika, 1983; Filimonova, 2005). Ultrastructural observations of ceratopogonid alimentary tracts have mainly concentrated on hematophagous females of the genus Culicoides known as important vectors of arboviruses, protozoans or nematodes (Mellor et al., 2000; Carpenter et al., 2008; Seblova et al., 2012). Their peritrophic membrane and midgut epithelial cells come in direct contact with parasites (Campbell et al., 2004; Filimonova, 2004; Svobodová et al., 2007; León Villalba et al., 2011). However, most of these published data were combined with analysis of isolated microorganisms of infected midguts, whereas investigations on the ultrastructure of midgut epithelia have been very rare (Filimonova, 2005). The anatomy of the larval digestive tract was described by Saunders (1924), but further observations on the gut ultrastructure of preimaginal stages of biting midges were not continued. The object of this work was to study and compare the fine structure of the midgut epithelium in the last larval stage and adult midges of Forcipomyia nigra (Winnertz) using light and transmission electron microscopy. We have also compared the ultrastructures for both sexes of this ceratopogonid species. Our results are the basis of further research on feeding anatomy of biting midges.
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Forcipomyia nigra larvae pass through four instars and need about 22-30 days for total development. They live in moist habitats, usually under the tree bark or in rotten wood, and feed on decaying organic matter and fungi. Larvae were collected in the pine forest of Gdańsk-Górki Wschodnie from October to November 2013. Only IV instar larvae were found, because earlier stages, from I to III, were difficult to obtain. About 50 larvae were obtained from under the tree bark of dead trees, subsequently placed in plastic boxes containing wet lignin and fragments of bark. They were carried into the laboratory in the Department of Invertebrate Zoology and Parasitology, University of Gdańsk. Material was reared in the dark at T = 15°C±1°C and relative humidity of 75–85%. Larvae fed on decaying organic matter and their metamorphosis to adults occurred after about 32 days.
2.2. Transmission electron microscopy The alimentary tracts were isolated from 10 reared larvae, whereas adult flies (4 males and 4 females) were examined as whole specimens. Both, larval alimentary tracts and whole adult 3
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flies were fixed in 2.5% glutaraldehyde in phosphate buffer saline (PBS) (48 h, temp 4°C). After being rinsed in PBS, the material was post-fixed in 2% OsO4 in 0.1 M phosphate buffer for 2 h and subsequently dehydrated in a graded ethanol series of 75, 80, 85, 90, 99.9% and acetone (15 min each). Then samples were embedded in Spurr resin. Ultrathin sections (70nm) were stained with uranyl acetate and lead citrate and observed under a Philips CM100 transmission electron microscope. 2.3. Light microscopy
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For light microscopy, semithin sections (0,3µm) of the material embedded in Spurr resin, were stained with 1% toluidine blue in 1% borax and examined with Nikon Eclipse E800 microscope equipped with camera Nikon Digital Sight DS.-U1. 2.3.1. Histochemistry
3.1. Light microscopy
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3. Results
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For detection of glycogen, polysaccharides and proteins semi-thin sections were treated with a 2% solution of periodic acid to remove the osmium and to oxidize diols in sugars, and subsequently stained with Schiff’s reagent (Periodic acid-Schiff staining, PAS) or bromophenol blue (BPB) (24 h, 37°C). Semi-thin sections were also stained with Sudan Black B at 60°C (1h) for detection of lipids.
Histological examination of the midgut of Forcipomyia nigra larvae and adult specimens shows columnar epithelial cells (digestive cells) with easily distinguished nuclei and numerous granules (Fig. 1A, 1C, 1F). Food particles visible in the gut lumen of larvae are separated from the apical cell membranes of epithelial cells by the peritrophic membrane (Fig. 1A). Digestive cells accumulate lipids, which stain with Sudan Black B and occur throughout all regions of the cells (Fig. 1B), but PAS-positive granules were not found. Digestive cells of adult females contain a lot of vesicles (Fig. 1C) and lipid droplets (Fig. 1D). PAS-positive granules are localized in the apical region (Fig. 1E). However, in the cytoplasm of male digestive cells PAS- or SBB-positive and granules are absent. BPBpositive granules of reserve material were not detected in either sex.
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3.2. Transmission electron microscopy 3.2.1. Larvae
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3.2.2. Females
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Midgut epithelia is composed of digestive and regenerative cells that rest on a non-cellular basal lamina (Fig. 2A-B) is composed of the digestive and regenerative cells (Figs. 2A-C, 3A-F). The cytoplasm of the digestive cells in larval midgut epithelia reveals three distinct regions: basal, perinuclear and apical (Figs. 2A-C; 3A-D). Basal membrane forms conspicuous invaginations (Fig. 2A). Basal cytoplasm contains numerous multivesicular bodies, cisterns of the rough endoplasmic reticulum and some mitochondria (Fig. 2A). Perinuclear region has a lot of the reserve material and cisterns of the rough endoplasmic reticulum (Fig. 3A). The number of mitochondria appears to be high (Fig. 3A). Septate junctions joining adjacent digestive cells in their perinuclear region are present (Fig. 3A). The apical cytoplasm possesses numerous mitochondria and cisterns of the rough endoplasmic reticulum (Fig. 3B-D). Microvilli form small blebs within the midgut lumen suggesting microapocrine secretion (Fig. 3B). Smooth septate junctions are observed between the apical regions of adjacent digestive cells (Fig. 3B-C). Small regenerative cells, scattered among the basal regions of the digestive cells throughout the larval midgut, have electron-lucent cytoplasm (Figs. 2B-C, 3E-F). Regenerative cells have oval nuclei with small patches of heterochromatin and a distinct nucleolus. A few mitochondria and cisterns of the rough endoplasmic reticulum are observed in the cytoplasm of regenerative cells during interphase (Fig. 3E). The mitotic divisions of regenerative cells have not been observed. However, some of them undergo the differentiation into the digestive cells. During the differentiation, the regenerative cell elongates toward the midgut lumen. Mitochondria gather just above the nucleus and some reserve material starts to accumulate (Fig. 2C). When the differentiating cell reaches the midgut lumen, the microvilli of the apical membrane and regionalization of organelles appears (Fig. 3B).
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Adult females of different ages were examined during this ultrastructural study. Digestive cells of younger females have cylindrical shapes and demonstrate well-defined basal, perinuclear and apical regions (Fig. 4A-F). The basal region forms a membranous labyrinth (Fig. 4A), which expands into vesicular folds reaching the perinuclear region. Cytoplasm of the perinuclear region is rich in electron-lucent vesicles, mitochondria, cisterns of the rough endoplasmic reticulum and Golgi complexes (Figs. 4B-D). The apical cytoplasm is rich in cisterns of the rough endoplasmic reticulum and granules with the reserve material (Fig. 4E). Microvilli of the apical membrane release small vesicles into the midgut lumen (Figs. 4D-E). Between the adjacent digestive cells in the apical regions smooth septate junctions have been observed (Fig. 4F). Regenerative cells with electron-lucent cytoplasms are very rare and show the same ultrastructure as these described for larvae. Mature females were distinguished by having ovarioles filled with developing oocytes, which are absent in younger females of F. nigra (unpublished data). Midgut digestive cells demonstrate different shapes, because they appear to be shrunken with prominent deep invaginations of the basal cell membrane (Fig. 5A, C, D). Their cytoplasm is electron-dense, 5
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and distinct regionalization of organelles is not observed. The entire cytoplasm is rich in swollen cisterns of the rough endoplasmic reticulum (Fig. 5A), numerous vacuoles and vesicles with electron-lucent content, lamellar bodies (Figs. 5A-B, E) and only sporadic mitochondria (Figs. 5A-B). Cortex layer is not visible (Figs. 5A-B). The reserve material has not been observed in mature females. Smooth septate junctions connect adjacent digestive cells in the apical regions (Fig. 5B). Regenerative cells with distinct nuclei and electron-dense cytoplasms, that are poor in organelles, are easily recognized (Fig. 5C).
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3.2.3. Males
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Cylindrical-shaped digestive cells and regenerative cells form the midgut epithelium of F. nigra males (Figs. 6A, 7D-E). The entire cytoplasm of a digestive cell contains welldeveloped rough endoplasmic reticulum (Figs. 6B-D, 7A). Basal membrane folds and forms a labyrinth (Figs. 6A-B). Numerous autophagosomes occur in the perinuclear and apical regions of the cytoplasm (Figs. 6C-D, 7C). The reserve material is absent, but numerous lamellar bodies appear in the perinuclear region (Figs. 6C-D, 7C). In the apical cytoplasm some electron-lucent vesicles have been found (Fig. 7A-B). A cortex layer is not visible (Fig. 7A). Between apical regions of adjacent digestive cells smooth septate junctions occur (Fig. 6D). Regenerative cells are distributed individually among the digestive cells, and their cytoplasms are rich in rough endoplasmic reticulum and mitochondria (Fig. 7D-E).
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The midgut of insects plays a crucial role in digestion and absorption of acquired food. In Ceratopogonidae this part of the alimentary canal is a simple tube devoid of any pouches or diverticula (Megahed, 1956). We have studied larval and adult (both sexes) midgut of biting midges, Forcipomyia nigra and described the ultrastructure of digestive cells of all examined stages. Midgut epithelial cells of biting midges described here show similar fine structure to other nematoceran flies (i.e. Andrade-Coêlho et al., 2001; Ferreira et al., 2008; Nardi et al., 2009). Endocrine cells have not been observed in analysed material, but they have been found in blood-feeding females of the genus Culicoides (Filimonova, 2005). Larvae feed on decaying organic matter and fungi (usually Ascomycetes) that grow on such substrata (Urbanek et al., 2011, 2012). In these preimaginal stages, the straight and long midgut is composed of digestive and regenerative cells. Present studies reveal that digestive cells probably participate in synthesis and secretion of enzymes as they contain welldeveloped rough endoplasmic reticulum and numerous mitochondria in the cytoplasm (Okuda et al., 2002; Biagio et al., 2009; Fialho et al., 2012). If that is so, multivesicular bodies occupying the basal region of the cells may play a role in enzyme accumulation or protein sorting, recycling, transport and release (Piper and Katzmann, 2007; von Bartheld and Altick, 2011; Rost-Roszkowska et al., 2013). Basal membrane infoldings form a kind of extracellular compartment which has limited access to hemolymph. Solutes concentrate in this area and consequently generate the osmotic pressure between the compartment and the midgut lumen 6
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(Terra et al., 2006). Folded basal membrane with associated mitochondria, present in larval midgut cells, is evidently involved in transport of ions and water (Fialho et al., 2009; Gonçalves et al., 2014). Numerous spheres and lipid droplets that accumulate in the cytoplasm of digestive cells suggest their storage function. We speculate that lipids in midgut cells may be precursors of steroid hormones (ecdysone) whose concentration increases, at the end of IV-th larval stage, to initiate metamorphosis (Lafont et al., 2012). Some of the digestive cells have an electron-dense cytoplasm and more reserve material than cells with electron-lucent cytoplasm, perhaps indicating metabolic differences between these cells. Digestive cells with electron-lucent cytoplasms probably represent cells that have newly differentiated from regenerative cells. The dietary composition of adult biting midges in the genus Forcipomyia is diverse, especially for females, because males usually just drink nectar or honeydew (Szadziewski et al., 1997). Females can be haematophagous, subgenus Lasiohelea (Szadziewski et al., 1997; Cribb, 2000; Spinelli et al., 2005), ectoparasitic (sucking haemolmyph of other insects), subgenera Trichohela or Microhelea (Downes, 1958; Wirth 1971, 1972; Lane; 1977; Clastrier and Legrand, 1991; Clastrier and Wirth, 1995; Koptur et al., 2013), nectar feeders or pollinivorous (Bystrak and Wirth, 1978; Szadziewski et al., 1997; Urbanek et al., 2014). On the other hand, the diet of Forcipomyia nigra midges is poorly known. Females are usually collected from flowers, so they could be pollinivorous or just sucking haemolymph from insects that pollinate plants. However, the biting midges examined here were reared in the laboratory conditions on the same substratum as larvae. So males could just suck condensed water accumulated on the rotting wood, while females might have obtained food from decaying organic matter or relied on protein nutrients, stored during larval stage, which which support oogenesis (Arrese and Soulages, 2010). Midgut epithelium has been described as one of the organs which, together with the fat body, storage cells, trophocytes and oocytes, participates in the synthesis of yolk and chorion components in invertebrates – e.g., insects, annelids, arachnids or tardigrades (Kessel and Beams, 1980; Węglarska, 1987; Rosell and Coons, 1992; Poprawa, 2005, 2006; RostRoszkowska, et al. 2011). Females of biting midges live about 7-10 days and die soon after oviposition (Gustevich, 1973). In females of F. nigra the digestive cells, similarly to larval midgut cells, take part in water and ion transport (folded basal membrane) and storage of reserve material. The amount of reserve material in midgut cells of adult females decreases with age; reserves are probably absorbed by the haemolymph and taken up by fat body cells in preparation for reproduction (Arrese and Soulages, 2010.) Because of the fact that a protein-rich meal is required for initiation of oogenesis in the majority of species of biting midges (Szadziewski et al., 1997), the midgut epithelium of the species examined does not accumulate proteins (all cells are BPB-negative). Only lipids and polysaccharides can be gathered. Relatively poor nutrition of males (consisting mainly of water and carbohydrate food) influences the ultrastructure of their midgut digestive cells. Abundant rough endoplasmic reticulum occupied the entire cytoplasm; mitochondria, more numerous than in females midgut cells, and electron-lucent vesicles suggest a fast and active absorption process and transport of solutes (Fialho et al., 2009; Chajec et al., 2014). Basal membranes of male cells 7
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display deep infoldings involved in transport of ions and water. But in contrast to females, male digestive cells have no storage function. The regenerative cells are distributed as isolated cells among digestive cells, or form regenerative groups which are called regenerative nests or regenerative crypts (Silva-Olivares et al. 2003, Illa-Bochaca and Montuenga 2006; Wanderley-Teixeira et al. 2006, Baton and Ranford-Cartwright 2007, Mehrabadi et al., 2012; Fernandes et al., 2014). Regenerative nests or crypts were apparently not observed in ceratopogonid midgut epithelium. Only isolated regenerative cells were noted. In biting midges of the genus Culicoides regenerative cells are small and replace old vacuolated cells (Megahed, 1956; Sieburth, 1991). Therefore, here we present the first report on the fine ultrastructure of the regenerative cells in this group of insects. Because these cells are treated as the midgut stem cells, they should possess the potential for proliferation and differentiation (Loeb, 2010; Rost-Roszkowska et al., 2010). However, in F.nigra the mitotic divisions of the regenerative cells were not observed, and their differentiation was detected only in larvae. Failure to observe mitosis may be attributed to the fact that mitotic divisions of cells are governed by a circadian rhythm (Chajec et al. 2012, 2014), or are coupled to molt cycles. Damages caused by the food particles may also influence mitotic divisions (Fernandes et al., 2014). Based on results that we obtained, we conclude that: (1) the midgut epithelium of F. nigra is formed by digestive columnar cells and regenerative cells; (2) the differences in the ultrastructure of the digestive cells between males and females are associated with differences in meals fed and finally (3) digestive cells play an important role in storage of reserve material (larvae and females), secretion process, transport of ion/water, absorption and transport of nutrients.
Acknowledgements We thank professor J. Bohdanowicz from Department of Plant Cytology and Embryology (University od Gdańsk) for giving us the opportunity to examine our material with a Nikon Eclipse E800 light microscope. This work was supported by Young Researcher grant No. 538-L114-B080-13 from the University of Gdańsk.
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References
Andrade-Coêlho, C.A., Santos-Mallet, J., Souza, N.A., Lins, U., Meirelles, M.N., Rangel, E.F., 2001. Ultrastructural features of the midgut epithelium of females Lutzomyia intermedia (Lutz & Neiva, 1912) (Diptera: Psychodidae: Phlebotominae). Mem. Inst. Oswaldo Cruz 96, 1141-1151.
8
Page 8 of 22
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
Arrese, E.L., Soulages, J.L., 2010. Insect fat body: energy, metabolism, and regulation. Ann. Rev. Entomol. 55, 207-225.
22 23
Chaika, S.Yu.. 1983. On Analysis of Ultrastructure of Midgut of Some Blood-Sucking Flies (Diptera), Entomol. Obozr. 62, 470-477.
24 25 26
Chajec, Ł., Rost-Roszkowska, M.M., Vilimova, J., Sosinka, A., 2012. Ultrastructure and regeneration of midgut epithelial cells in Lithobius forficatus (Chilopoda, Lithobiidae). Invertebr. Biol. 131, 119-132.
27 28 29
Chajec, Ł., Sonakowska, L., Rost-Roszkowska, M.M.. 2014. The fine structure of the midgut epithelium in a centipede, Scolopendra cingulata (Chilopoda, Scolopendridae), with the special emphasis on epithelial regeneration. Arthropod. Struct. Dev. 43, 27-42.
30 31 32
Clastrier, J., Legrand, J., 1991. Forcipomyia (Trichohelea) araneivora n. sp. ectoparasite d'une araignée habitant les monts nimba en Guinée (Diptera, Ceratopogonidae; Araneae, Araneidae). Rev. Fr. Entomol. 13, 4155-4158.
33 34 35
Clastrier, J., Wirth, W.W., 1995. Révision des Forcipomyia du sous-genre Microhelea de la région néotropicale, parasites de phasmes (Diptera: Ceratopogonidae). Ann. Soc. Entomol. Fr. 31, 97-150.
36 37
Cribb, B.W., 2000. Oviposition and maintenance of Forcipomyia (Lasiohelea) townsvillensis (Diptera: Ceratopogonidae) in the laboratory. J. Med. Entomol. 37, 316-318.
ip t
Baton, L.A., Ranford-Cartwright, L.C., 2007. Morphological evidence for proliferative regeneration of the Anopheles stephensi midgut epithelium following Plasmodium falciparum ookinete invasion. J Invertebr. Pathol. 96, 244-254.
cr
Biagio, F.P., Tamaki, F.K., Terra, W.R., Ribeiro, A.F., 2009. Digestive morphophysiology of Gryllodes sigillatus (Orthoptera: Gryllidae). J. Insect Physiol. 55, 1125-1133.
us
Bystrak, P. G., Wirth, W.W., 1978. The North American species of Forcipomyia, subgenus Euprojoannisia (Diptera: Ceratopogonidae). United States Department of Agriculture Technical Bulletin number 1951. 51. pp.
an
Campbell, C.L., Mummey, D.L., Schmidtmann, E.T., Wilson, W.C., 2004. Cultureindependent analysis of midgut microbiota in the arbovirus vector Culicoides sonorensis (Diptera: Ceratopogonidae). J. Med. Entomol. 41, 340-348.
Ac ce p
te
d
M
Carpenter, S., Mellor, P.S., Torr, S.J., 2008. Control techniques for Culicoides biting midges and their application in the U.K. and northwestern Palaearctic. Med. Vet. Entomol. 22, 175187.
9
Page 9 of 22
Downes, J.A., 1958. The feeding habits of biting flies and their significance in classification. Ann. Rev. Entomol. 3, 249-263.
3 4
Fernandes, K.M., Neves, C.A., Serrão, J.E., Martins, G.F., 2014. Aedes aegypti midgut remodeling during metamorphosis. Parasitol. Int. 63, 506-512.
5 6 7
Ferreira, M.A.R., Lima, J.B., Rocha, R.M., Pimenta, P.F.P., 2008. Morphological Aspect of the Midgut of Anopheles aquasalis (Curry, 1932) Insecta: Diptera. Int. J. Morphol. 26, 577582.
8 9 10 11
Fialho, M.C.Q., Zanuncio, J.C., Neves, C.A., Ramalho, F.S. Serrão, J.E., 2009. Ultrastructure of the digestive cells in the midgut of the predator Brontocoris tabidus (Heteroptera: Pentatomidae) after different feeding periods on prey and plants. Ann. Entomol. Soc. Am. 102, 119-127.
12 13 14
Fialho, M.C.Q., Moreira, N.R., Zanuncio, J.C., Ribeiro, A.F., Terra, W.R., Serrão, J.E., 2012. Prey digestion in the midgut of the predatory bug Podisus nigrispinus (Hemiptera: Pentatomidae). J. Insect Physiol. 58, 850-856.
15 16
Filimonova, S.A., 2004. Formation of the peritrophic membrane in the gut of Culicoides punctatus (Diptera: Ceratopogonidae). Parazitologiia 38, 12-19. (in Russian)
17 18
Filimonova, S.A., 2005. Morphological Study of Digestive Cycle in Bloodsucking Biting Midges of Genus Culicoides. J. Evol. Biochem. Physiol. 41, 221-232.
19 20 21
Gonçalves, W.G., Fernandes, K.M., Barcellos, M.S., Silva, F.P., Magalhães-Junior, M.J., Zanuncio, J.C., Martins, G.F., Serrão, J.E., 2014. Ultrastructure and immunofluorescence of the midgut of Bombus morio (Hymenoptera: Apidae: Bombini). C R Biol. 337, 365-372.
22 23
Gustevich, A.V., 1973. The bloodsucking midges (Ceratopogonidae). Fauna SSSR, Dipterous Insects, III, 5, Nauka, Leningrad, 270 pp. (in Russian).
24 25
Illa-Bochaca, I., Montuenga, L.M., 2006. The regenerative nidi of locust as a model to study epithelial cell differentiation from stem cells. J. Exp. Biol. 209, 2215-2223.
26 27 28
Kessel, R.G., Beams, H.W., 1980. Cytodifferentiation and vitellogenesis during oogenesis in Arachnida: cytological studies on developing oocytes of harvestman. J. Morphol. 163, 175190.
29 30 31
Koptur, S., J. E. Pena, Grogan, Jr., W.L., 2013 The biting midge Forcipomyia (Microhelea) eriophora (Williston) (Diptera: Ceratopogonidae), an ecotoparasite of larval Phoebis sennae (Pieridae) in South Florida. J. Lepid. Soc. 67, 128-130.
32 33 34
Lafont, R., Dauphin-Viellemant, C., Warren, J.T., Rees, H., 2012. Ecdysteroid chemistry and biochemistry, in: Gilbert, L.I. (Ed.), Insect endocrinology, Academic Press, London, pp. 106156.
Ac ce p
te
d
M
an
us
cr
ip t
1 2
10
Page 10 of 22
Lane, R.P., 1977. Ectoparasitic adapatations in Forcipomyia from butterflies with two new African species (Ceratopogonidae). System. Entomol. 2, 305-312.
3 4 5
León Villalba, L.R., Romoser, W.S., Patrican, L., 2011. Meconial peritrophic matrix and meconial degradation in the biting midge, Culicoides variipennis (Coquillett) (Diptera: Ceratopogonidae). Open Access Insect Physiol. 3, 1-6.
6 7
Loeb, M.J., 2010. Factors affecting proliferation and differentiation of Lepidopteran midgut stem cells. Arch. Insect Biochem. Physiol. 74, 1-16.
8 9 10
Megahed, M.M., 1956. Anatomy and histology of the alimentary tract of the female of the biting midge Culicoides nubeculosus Meigen (Diptera: Heleidae=Ceratopogonidae). Parasitology 46, 22-47.
11 12 13
Mehrabadi, M., Bandani, A.R., Allahyari, M., Serrão, J.E., 2012. The Sunn pest, Eurygaster integriceps Puton (Hemiptera: Scutelleridae) digestive tract: histology, ultrastructure and its physiological significance. Micron 43, 631-637.
14 15
Mellor, P. S., J. Boorman, and M. Baylis. 2000. Culicoides biting midges: their role as arbovirus vectors. Annu. Rev. Entomol. 45, 307-340.
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Nardi, J.B., Miller, L.A., Bee, C.M., Lee, R.E. Jr, Denlinger, D.L., 2009. The larval alimentary canal of the Antarctic insect, Belgica antarctica. Arthropod. Struct. Dev. 38, 377389.
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Okuda, K., de Souza Caroci, A., Ribolla, P.E., de Bianchi, A.G., Bijovsky, A.T., 2002. Functional morphology of adult female Culex quinquefasciatus midgut during blood digestion. Tissue Cell 34, 210-219.
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Piper, R. C., Katzmann, D.J., 2007. Biogenesis and Function of Multivesicular Bodies. Annu. Rev. Cell. Dev. Biol. 23, 519-547.
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Poprawa, I., 2005. The ovary structure, previtellogenic and vitellogenic stages in parthenogenetic species Dactylobiotus dispar (Murray, 1097) (Tardigrada: Eutardigrada). Tissue Cell 37, 385-392.
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Poprawa, I., 2006. Ultrastructural changes of the storage cells during oogenesis in Dactylobiotus dispar (Murray, 1907) (Tardigrada: Eutardigrada). Zool. Pol. 51, 13-18.
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Rosell, R., Coons, L.B., 1992. The role of the fat body, midgut and ovary in vitellogenin production and vitellogenesis in the female tick, Dermacentor variabilis. Int. J. Parasitol. 22, 341-349.
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Rost-Roszkowska, M.M, Poprawa, I., Klag, J., Migula, P., Mesjasz-Przybylowicz, J., Przybyłowicz, W., 2008. Degeneration of the midgut epithelium in Epilachna cf. nylanderi (Insecta, Coccinellidae): apoptosis, autophagy and necrosis. Can. J. Zool. 86, 1179-1188.
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Rost-Roszkowska, M.M., Poprawa, I., Klag, J., Migula, P., Mesjasz-Przybyłowicz, J., Przybyłowicz, W., 2010. Differentiation of regenerative cells in the midgut epithelium of Epilachna cf. nylanderi (Mulsant 1850) (Insecta, Coleoptera, Coccinellidae). Fol. Biol. (Krakow) 58, 209-216.
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Rost-Roszkowska, M.M., Poprawa, I., Wójtowicz, M., Kaczmarek, L., 2011. Ultrastructural changes of the midgut epithelium in Isohypsibius granulifer granulifer Thulin, 1928 (Tardigrada: Eutardigrada) during oogenesis. Protoplasma 248, 405-414.
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Rost-Roszkowska, M.M., Poprawa, I., Hyra, M., Marek-Swędzioł, M., Kaczmarek, Ł., 2013. The fine structure of the midgut epithelium in Xerobiotus pseudohufelandi (Iharos, 1966) (Tardigrada, Eutardigrada, Macrobiotidae). J. Limnol. 72, 54-61.
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Saunders, L.G., 1924. On the life history and the anatomy of the early stages of Forcipomyia (Diptera, Nemat., Ceratopogonidae). Parasitology 16, 164-213.
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Seblova, V., Sadlova, J., Carpenter, S., Volf, P., 2012. Development of Leishmania Parasites in Culicoides nubeculosus (Diptera: Ceratopogonidae) and implications for screening vector competence. J. Med. Entomol. 49, 967-970.
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Sieburth, P.J., Nunamaker, C.E., Ellis, J., Nunamaker, R.A., 1991. Infection of the midgut of Culicoides variipennis (Diptera: Ceratopogonidae) with bluetongue virus. J. Med. Entomol. 28, 74-85.
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Silva-Olivares, A., E. Diaz, M. Shibayama, V. Tsutsmi, R. Cisneros, Zuniga, G., 2003. Ultrastructural study of the midgut and hindgut in eight species of the genus Dendroctonus Erichson (Coleoptera: Scolytidae). Ann. Entomol. Soc. Am. 96, 883-900.
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Spinelli, G.R., Ronderos, M.M., Díaz, F., Marino, P.I., 2005. The bloodsucking biting midges of Argentina (Diptera: Ceratopogonidae). Mem. Inst. Oswaldo Cruz 100, 137-150.
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Svobodová, M., Zídková, L., Čepička, I., Oborník, M., Lukeš, J., Votýpka, J., 2007. Sergeia podlipaevi gen. nov., sp. nov. (Trypanosomatidae, Kinetoplastida), a parasite of biting midges (Ceratopogonidae, Diptera). Int. J. Syst. Evol. Microbiol. 57, 423-432.
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Szadziewski, R., Krzywiński, J., Giłka, W., 1997. Diptera Ceratopogonidae, Biting Midges. In: Nilsson, A.N. (Eds), Aquatic Insects of North Europe - A taxonomic Handbook, Vol. 2, Apollo Books, Stenstrup, Denmark, pp. 243-263.
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Terra, W.R., Costa, R.H., Feirreira, C., 2006. Plasma membranes from insect midgut cells. Ann. Braz. Acad. Sci. 78, 255-269.
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Urbanek, A., Richert, M., Giłka, W. and Szadziewski, R., 2011. Morphology and histology of secretory setae in terrestrial larvae of biting midges of the genus Forcipomyia (Diptera: Ceratopogonidae). Arthropod. Struct. Dev. 40, 485-494.
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Urbanek, A, Szadziewski, R, Stepnowski, P, Boros-Majewska, J, Gabriel, I, Dawgul, M, Kamysz, W, Sosnowska, D, Gołębiowski, M., 2012 Composition and antimicrobial activity of fatty acids detected in the hygroscopic secretion collected from the secretory setae of larvae of the biting midge Forcipomyia nigra (Diptera: Ceratopogonidae). J. Insect. Physiol. 58, 12651276.
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Urbanek, A., Piotrowicz, M., Szadziewski, R. and Giłka, W., 2014. Sensilla coeloconica ringed by microtrichia in host-seeking biting midges. Med. Vet. Entomol. doi: 10.1111/mve.12057
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von Bartheld CS, Altick AL, 2011. Multivesicular bodies in neurons: distribution, protein content, and trafficking functions. Prog. Neurobiol. 93, 313-340.
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Wanderley-Teixeira, V., Teixeira, A.A.C., Cunha, F.M., Costa, M.K.C.M., Veiga, A.F.S.L., Oliveira, J.V., 2006. Histological description of the midgut and the pyloric valve of Tropidacris collaris (Stoll, 1813) (Orthopetera: Romaleidae). Braz. J. Biol. 66, 1045-1049.
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Węglarska, B., 1987. Yolk formation in Isohypsibius (Eutardigrada). Zoomorphology 107, 287-292.
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Wirth, W.W., 1971. A review of the “stick-ticks,” Neotropical biting midges of the Forcipomyia subgenus Microhelea parasitic on walking stick insects (Diptera: Ceratopogonidae). Entomol. News 82, 229-245.
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Wirth, W.W., 1972. Midges sucking blood of caterpillars (Diptera: Ceratopogonidae). J. Lepid. Soc. 26, 165.
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Fig. 1. Light micrographs of F. nigra larva, 4th instar (A, B) and adults (C-F). (A) Cross section of larval midgut stained with toluidine blue, bar = 50 µm, bar for insert = 10 µm. (B) Sudan black-positive droplets in larval midgut cells, bar = 50 µm. (C) Cross section of female midgut stained with toluidine blue, epithelial cells contain numerous vesicles (arrowhead), bar = 10 µm. (D) Dark lipid droplets (arrowheads) present in the female midgut epithelial cell stained with Sudan black B, bar = 10 µm. (E) PAS-positive granules (arrow) concentrate in apical region of female midgut epithelial cell, bar = 10 µm. (F) Longitudinal section of male midgut stained with toluidine blue, bar = 10 µm. e – epithelial cell, fb – fat body, g- granule, ld – lipid droplet, lu – lumen, sb – striated border, Mal – Malpighian tube, n – nucleus, pm – peritrophic membrane.
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Fig. 2. F. nigra larva, 4th instar, TEM. (A) Basal region of digestive cell, bar = 2 µm. (B) Digestive cell with electron-dense cytoplasm (dc) and large elongating regenerative cell (rc) bar = 5 µm. (C) Elongating regenerative cell (rc) located between two digestive cells (dc) bar = 5 µm. bl – basal lamina, bm – basal membrane, m – mitochondrion, mb – multivesicular
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body mc – circular muscle, n – nucleus, nu- nucleolus, rm – reserve material, RER – rough endoplasmic reticulum, SER – smooth endoplasmic reticulum.
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Fig. 3. F. nigra larva, 4th instar, TEM. (A) Perinuclear region of two digestive cells joined by septate junctions (arrowhead), bar = 1 µm . (B) Apical region of electron-dense digestive cell, newly differentiated from regenerative cell (dc1) and electron-lucent digestive cell (dc) with visible smooth septate junction (arrowhead); insert indicates on microapocrine secretion (arrow), bar = 2 µm, for insert 0,5 µm . (C) Smooth septate junctions (arrowhead) connected adjacent digestive cells, bar = 1 µm . (D) Apical region of digestive cell, below microvilli, bar = 1 µm. (E) Regenerative cell in interphase (rci), bar = 2 µm. (F) Regenerative cell (rc) bordered with digestive cell (dc), bar = 5 µm. AG – Golgi complex, au – autophagosome, bl – basal lamina, bm – basal membrane, m – mitochondrion, mv – microvilli, n – nucleus, nu – nucleolus, RER – rough endoplasmic reticulum, v – vesicle.
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Fig. 4. F. nigra female, TEM. (A) Basal membrane of digestive cell with different size vesicles (arrowheads), bar = 1 µm. (B) Perinuclear region of digestive cell, bar = 2 µm. (C) Nucleus of digestive cell surrounded by vesicular folds of basal membrane (white arrow), bar = 1 µm. (D) Midgut epithelial cells occupied by vesicular folds of basal membrane (arrowheads), bar = 5 µm. (E) Micorapocrine secretion (arrows) of digestive cells, bar = 2 µm. (F) Apical region of midgut digestive cell with septate junctions (arrow), bar = 1 µm. AG – Golgi complex , bf – basal fold, m – mitochondrion, mv – microvilli, n – nucleus, nu – nucleous, rm – reserve material, RER – rough endoplasmic reticulum, v – vesicle.
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Fig. 5. F. nigra female, TEM. (A) Midgut epithelial cells with deep basal folds (black arrows), bar = 5 µm. (B) Apical region of digestive cells with visible smooth septate junctions (arrowheads), bar = 2 µm. (C) Regenerative cell (rc), bar = 2 µm. (D) Basal membrane of digestive cell, bar = 1 µm. (E) Lamellar body in the cytoplasm of digestive cell, bar = 1 µm.. au – autophagosome, bf – basal fold, bl – basal lamina, lb – lamellar body, m – mitochondrion, mc – circular muscle, ml –longitudinal muscle, n – nucleus, RER – rough endoplasmic reticulum, v – vesicle, va – vacuole.
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Fig. 6. F. nigra male, TEM. (A) Fragment of midgut epithelium consisted of digestive (dc) and regenerative (rc) cells, bar = 5 µm. (B) Basal membrane of digestive cell, bar = 1 µm. (C) Perinuclear region of digestive cell, bar = 2 µm. (D) Perinuclear and apical region of digestive cells with visible septate junctions (arrowhead), bar = 2 µm. au – autophagosome, bf – basal fold, bl – basal lamina, lb – lamellar body, m – mitochondrion, mc – circular muscle, ml – longitudinal muscle, mv – microvilli, n – nucleus, RER – rough endoplasmic reticulum, v – vesicle, va – vacuole.
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Fig. 7. F. nigra male, TEM. (A) Apical region of digestive cell, bar = 0,5 µm. (B) Vacuoles present in the apical region of digestive cell, bar = 0,5 µm. (C) Septate junction with visible microfilaments (arrow) between digestive cells in their perinuclear region, bar = 0,5 µm. (D) Regenerative cell (rc), bar = 2 µm. (E) Fragment of regenerative cell (rc) borders with basal membrane, bar = 1 µm. AG – Golgi complex, au – autophagosome, bm – basal membrane, lb
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S0968-4328(14)00194-2 http://dx.doi.org/doi:10.1016/j.micron.2014.11.003 JMIC 2141
To appear in:
Micron
Received date: Revised date: Accepted date:
4-9-2014 17-10-2014 7-11-2014
Please cite this article as: Urbanek, A., Rost-Roszkowska, M.M.,Ultrastructural studies on the midgut of biting midge Forcipomyia nigra (Winnertz) (Diptera: Ceratopogonidae), Micron (2014), http://dx.doi.org/10.1016/j.micron.2014.11.003 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Highlights
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Midgut of larvae and adults flies of Forcipomyia nigra was examined
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Larval digestive cells reveal different ultrastructure according to their age
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Females and larval digestive cells take part in storage reserve material
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Type diet influences on the function of digestive cells in species examined
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Ultrastructural studies on the midgut of biting midge Forcipomyia nigra (Winnertz) (Diptera: Ceratopogonidae)
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Aleksandra Urbanek*1, Magdalena M. Rost-Roszkowska2
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Department of Invertebrate Zoology and Parasitology, University of Gdańsk, Wita Stwosza 59, 80308 Gdańsk, Poland
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Department of Animal Histology and Embryology, Bankowa 9, University of Silesia, 40-007 Katowice, Poland
* corresponding author: [email protected], phone +48 58 523 61 84
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Abstract. Biting midges belonging to the genus Forcipomyia are known to be haematophagous, predatory or saprophagous. Different stages of Forcipomyia nigra midges were investigated to provide a description of midgut ultrastructure. Larvae feeding on decaying organic matter possess simple, straight alimentary tracts whose middle regions are the longest. TEM studies of the larval midgut epithelium reveal that digestive cells show different ultrastructure depending on their age. The older cells with electron-dense cytoplasm degenerate while the younger ones with electron-lucent cytoplasm remain active in digestion. In saprophagous females, the ultrastructure of midgut epithelium changes according to the age of flies. Oogenesis induces degeneration of digestive cells and utilization of reserve material accumulated by them. The midgut epithelia of male midges consists of digestive and regenerative cells that show no evidence of cell degeneration as observed in females. Our results demonstrate differences between midgut digestive cells of males and females.
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Key words: biting midge, Forcipomyia nigra, midgut, digestive cell, regenerative cell
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1.Introduction
Members of the family Ceratopogonidae, better known as biting midges, are small, about 1-5 mm, nematoceran flies developing in a great range of habitats. Observations of their trophic relations reveal that the biting habit is restricted to the females, which show diverse feeding behaviour. They are hematophagous (species from genera Culicoides, Leptoconops, Austroconops or Forcipomyia), predators on other insects (most species of the subfamily Ceratopogoninae) or pollinivorous (e.g., Atrochopogon pollinivorous) (Szadziewski et al., 1997). Protein-rich meal is required for initiation of oogenesis in the majority of species of 2
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2.1. Insects
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biting midges (Szadziewski et al., 1997). Males feed usually on nectar or honeydew, but in subfamily Forcipomyiinae (genus Dasyhelea and some species of Forcipomyia) both sexes take nectar from flowers. The structure of the midgut of biting midges is simple: it is composed of a single layer of epithelial cells. It is divided into the anterior tubular region and posterior stomach (Megahed, 1956; Chaika, 1983; Filimonova, 2005). Ultrastructural observations of ceratopogonid alimentary tracts have mainly concentrated on hematophagous females of the genus Culicoides known as important vectors of arboviruses, protozoans or nematodes (Mellor et al., 2000; Carpenter et al., 2008; Seblova et al., 2012). Their peritrophic membrane and midgut epithelial cells come in direct contact with parasites (Campbell et al., 2004; Filimonova, 2004; Svobodová et al., 2007; León Villalba et al., 2011). However, most of these published data were combined with analysis of isolated microorganisms of infected midguts, whereas investigations on the ultrastructure of midgut epithelia have been very rare (Filimonova, 2005). The anatomy of the larval digestive tract was described by Saunders (1924), but further observations on the gut ultrastructure of preimaginal stages of biting midges were not continued. The object of this work was to study and compare the fine structure of the midgut epithelium in the last larval stage and adult midges of Forcipomyia nigra (Winnertz) using light and transmission electron microscopy. We have also compared the ultrastructures for both sexes of this ceratopogonid species. Our results are the basis of further research on feeding anatomy of biting midges.
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Forcipomyia nigra larvae pass through four instars and need about 22-30 days for total development. They live in moist habitats, usually under the tree bark or in rotten wood, and feed on decaying organic matter and fungi. Larvae were collected in the pine forest of Gdańsk-Górki Wschodnie from October to November 2013. Only IV instar larvae were found, because earlier stages, from I to III, were difficult to obtain. About 50 larvae were obtained from under the tree bark of dead trees, subsequently placed in plastic boxes containing wet lignin and fragments of bark. They were carried into the laboratory in the Department of Invertebrate Zoology and Parasitology, University of Gdańsk. Material was reared in the dark at T = 15°C±1°C and relative humidity of 75–85%. Larvae fed on decaying organic matter and their metamorphosis to adults occurred after about 32 days.
2.2. Transmission electron microscopy The alimentary tracts were isolated from 10 reared larvae, whereas adult flies (4 males and 4 females) were examined as whole specimens. Both, larval alimentary tracts and whole adult 3
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flies were fixed in 2.5% glutaraldehyde in phosphate buffer saline (PBS) (48 h, temp 4°C). After being rinsed in PBS, the material was post-fixed in 2% OsO4 in 0.1 M phosphate buffer for 2 h and subsequently dehydrated in a graded ethanol series of 75, 80, 85, 90, 99.9% and acetone (15 min each). Then samples were embedded in Spurr resin. Ultrathin sections (70nm) were stained with uranyl acetate and lead citrate and observed under a Philips CM100 transmission electron microscope. 2.3. Light microscopy
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For light microscopy, semithin sections (0,3µm) of the material embedded in Spurr resin, were stained with 1% toluidine blue in 1% borax and examined with Nikon Eclipse E800 microscope equipped with camera Nikon Digital Sight DS.-U1. 2.3.1. Histochemistry
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For detection of glycogen, polysaccharides and proteins semi-thin sections were treated with a 2% solution of periodic acid to remove the osmium and to oxidize diols in sugars, and subsequently stained with Schiff’s reagent (Periodic acid-Schiff staining, PAS) or bromophenol blue (BPB) (24 h, 37°C). Semi-thin sections were also stained with Sudan Black B at 60°C (1h) for detection of lipids.
Histological examination of the midgut of Forcipomyia nigra larvae and adult specimens shows columnar epithelial cells (digestive cells) with easily distinguished nuclei and numerous granules (Fig. 1A, 1C, 1F). Food particles visible in the gut lumen of larvae are separated from the apical cell membranes of epithelial cells by the peritrophic membrane (Fig. 1A). Digestive cells accumulate lipids, which stain with Sudan Black B and occur throughout all regions of the cells (Fig. 1B), but PAS-positive granules were not found. Digestive cells of adult females contain a lot of vesicles (Fig. 1C) and lipid droplets (Fig. 1D). PAS-positive granules are localized in the apical region (Fig. 1E). However, in the cytoplasm of male digestive cells PAS- or SBB-positive and granules are absent. BPBpositive granules of reserve material were not detected in either sex.
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3.2. Transmission electron microscopy 3.2.1. Larvae
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3.2.2. Females
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Midgut epithelia is composed of digestive and regenerative cells that rest on a non-cellular basal lamina (Fig. 2A-B) is composed of the digestive and regenerative cells (Figs. 2A-C, 3A-F). The cytoplasm of the digestive cells in larval midgut epithelia reveals three distinct regions: basal, perinuclear and apical (Figs. 2A-C; 3A-D). Basal membrane forms conspicuous invaginations (Fig. 2A). Basal cytoplasm contains numerous multivesicular bodies, cisterns of the rough endoplasmic reticulum and some mitochondria (Fig. 2A). Perinuclear region has a lot of the reserve material and cisterns of the rough endoplasmic reticulum (Fig. 3A). The number of mitochondria appears to be high (Fig. 3A). Septate junctions joining adjacent digestive cells in their perinuclear region are present (Fig. 3A). The apical cytoplasm possesses numerous mitochondria and cisterns of the rough endoplasmic reticulum (Fig. 3B-D). Microvilli form small blebs within the midgut lumen suggesting microapocrine secretion (Fig. 3B). Smooth septate junctions are observed between the apical regions of adjacent digestive cells (Fig. 3B-C). Small regenerative cells, scattered among the basal regions of the digestive cells throughout the larval midgut, have electron-lucent cytoplasm (Figs. 2B-C, 3E-F). Regenerative cells have oval nuclei with small patches of heterochromatin and a distinct nucleolus. A few mitochondria and cisterns of the rough endoplasmic reticulum are observed in the cytoplasm of regenerative cells during interphase (Fig. 3E). The mitotic divisions of regenerative cells have not been observed. However, some of them undergo the differentiation into the digestive cells. During the differentiation, the regenerative cell elongates toward the midgut lumen. Mitochondria gather just above the nucleus and some reserve material starts to accumulate (Fig. 2C). When the differentiating cell reaches the midgut lumen, the microvilli of the apical membrane and regionalization of organelles appears (Fig. 3B).
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Adult females of different ages were examined during this ultrastructural study. Digestive cells of younger females have cylindrical shapes and demonstrate well-defined basal, perinuclear and apical regions (Fig. 4A-F). The basal region forms a membranous labyrinth (Fig. 4A), which expands into vesicular folds reaching the perinuclear region. Cytoplasm of the perinuclear region is rich in electron-lucent vesicles, mitochondria, cisterns of the rough endoplasmic reticulum and Golgi complexes (Figs. 4B-D). The apical cytoplasm is rich in cisterns of the rough endoplasmic reticulum and granules with the reserve material (Fig. 4E). Microvilli of the apical membrane release small vesicles into the midgut lumen (Figs. 4D-E). Between the adjacent digestive cells in the apical regions smooth septate junctions have been observed (Fig. 4F). Regenerative cells with electron-lucent cytoplasms are very rare and show the same ultrastructure as these described for larvae. Mature females were distinguished by having ovarioles filled with developing oocytes, which are absent in younger females of F. nigra (unpublished data). Midgut digestive cells demonstrate different shapes, because they appear to be shrunken with prominent deep invaginations of the basal cell membrane (Fig. 5A, C, D). Their cytoplasm is electron-dense, 5
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and distinct regionalization of organelles is not observed. The entire cytoplasm is rich in swollen cisterns of the rough endoplasmic reticulum (Fig. 5A), numerous vacuoles and vesicles with electron-lucent content, lamellar bodies (Figs. 5A-B, E) and only sporadic mitochondria (Figs. 5A-B). Cortex layer is not visible (Figs. 5A-B). The reserve material has not been observed in mature females. Smooth septate junctions connect adjacent digestive cells in the apical regions (Fig. 5B). Regenerative cells with distinct nuclei and electron-dense cytoplasms, that are poor in organelles, are easily recognized (Fig. 5C).
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Cylindrical-shaped digestive cells and regenerative cells form the midgut epithelium of F. nigra males (Figs. 6A, 7D-E). The entire cytoplasm of a digestive cell contains welldeveloped rough endoplasmic reticulum (Figs. 6B-D, 7A). Basal membrane folds and forms a labyrinth (Figs. 6A-B). Numerous autophagosomes occur in the perinuclear and apical regions of the cytoplasm (Figs. 6C-D, 7C). The reserve material is absent, but numerous lamellar bodies appear in the perinuclear region (Figs. 6C-D, 7C). In the apical cytoplasm some electron-lucent vesicles have been found (Fig. 7A-B). A cortex layer is not visible (Fig. 7A). Between apical regions of adjacent digestive cells smooth septate junctions occur (Fig. 6D). Regenerative cells are distributed individually among the digestive cells, and their cytoplasms are rich in rough endoplasmic reticulum and mitochondria (Fig. 7D-E).
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The midgut of insects plays a crucial role in digestion and absorption of acquired food. In Ceratopogonidae this part of the alimentary canal is a simple tube devoid of any pouches or diverticula (Megahed, 1956). We have studied larval and adult (both sexes) midgut of biting midges, Forcipomyia nigra and described the ultrastructure of digestive cells of all examined stages. Midgut epithelial cells of biting midges described here show similar fine structure to other nematoceran flies (i.e. Andrade-Coêlho et al., 2001; Ferreira et al., 2008; Nardi et al., 2009). Endocrine cells have not been observed in analysed material, but they have been found in blood-feeding females of the genus Culicoides (Filimonova, 2005). Larvae feed on decaying organic matter and fungi (usually Ascomycetes) that grow on such substrata (Urbanek et al., 2011, 2012). In these preimaginal stages, the straight and long midgut is composed of digestive and regenerative cells. Present studies reveal that digestive cells probably participate in synthesis and secretion of enzymes as they contain welldeveloped rough endoplasmic reticulum and numerous mitochondria in the cytoplasm (Okuda et al., 2002; Biagio et al., 2009; Fialho et al., 2012). If that is so, multivesicular bodies occupying the basal region of the cells may play a role in enzyme accumulation or protein sorting, recycling, transport and release (Piper and Katzmann, 2007; von Bartheld and Altick, 2011; Rost-Roszkowska et al., 2013). Basal membrane infoldings form a kind of extracellular compartment which has limited access to hemolymph. Solutes concentrate in this area and consequently generate the osmotic pressure between the compartment and the midgut lumen 6
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(Terra et al., 2006). Folded basal membrane with associated mitochondria, present in larval midgut cells, is evidently involved in transport of ions and water (Fialho et al., 2009; Gonçalves et al., 2014). Numerous spheres and lipid droplets that accumulate in the cytoplasm of digestive cells suggest their storage function. We speculate that lipids in midgut cells may be precursors of steroid hormones (ecdysone) whose concentration increases, at the end of IV-th larval stage, to initiate metamorphosis (Lafont et al., 2012). Some of the digestive cells have an electron-dense cytoplasm and more reserve material than cells with electron-lucent cytoplasm, perhaps indicating metabolic differences between these cells. Digestive cells with electron-lucent cytoplasms probably represent cells that have newly differentiated from regenerative cells. The dietary composition of adult biting midges in the genus Forcipomyia is diverse, especially for females, because males usually just drink nectar or honeydew (Szadziewski et al., 1997). Females can be haematophagous, subgenus Lasiohelea (Szadziewski et al., 1997; Cribb, 2000; Spinelli et al., 2005), ectoparasitic (sucking haemolmyph of other insects), subgenera Trichohela or Microhelea (Downes, 1958; Wirth 1971, 1972; Lane; 1977; Clastrier and Legrand, 1991; Clastrier and Wirth, 1995; Koptur et al., 2013), nectar feeders or pollinivorous (Bystrak and Wirth, 1978; Szadziewski et al., 1997; Urbanek et al., 2014). On the other hand, the diet of Forcipomyia nigra midges is poorly known. Females are usually collected from flowers, so they could be pollinivorous or just sucking haemolymph from insects that pollinate plants. However, the biting midges examined here were reared in the laboratory conditions on the same substratum as larvae. So males could just suck condensed water accumulated on the rotting wood, while females might have obtained food from decaying organic matter or relied on protein nutrients, stored during larval stage, which which support oogenesis (Arrese and Soulages, 2010). Midgut epithelium has been described as one of the organs which, together with the fat body, storage cells, trophocytes and oocytes, participates in the synthesis of yolk and chorion components in invertebrates – e.g., insects, annelids, arachnids or tardigrades (Kessel and Beams, 1980; Węglarska, 1987; Rosell and Coons, 1992; Poprawa, 2005, 2006; RostRoszkowska, et al. 2011). Females of biting midges live about 7-10 days and die soon after oviposition (Gustevich, 1973). In females of F. nigra the digestive cells, similarly to larval midgut cells, take part in water and ion transport (folded basal membrane) and storage of reserve material. The amount of reserve material in midgut cells of adult females decreases with age; reserves are probably absorbed by the haemolymph and taken up by fat body cells in preparation for reproduction (Arrese and Soulages, 2010.) Because of the fact that a protein-rich meal is required for initiation of oogenesis in the majority of species of biting midges (Szadziewski et al., 1997), the midgut epithelium of the species examined does not accumulate proteins (all cells are BPB-negative). Only lipids and polysaccharides can be gathered. Relatively poor nutrition of males (consisting mainly of water and carbohydrate food) influences the ultrastructure of their midgut digestive cells. Abundant rough endoplasmic reticulum occupied the entire cytoplasm; mitochondria, more numerous than in females midgut cells, and electron-lucent vesicles suggest a fast and active absorption process and transport of solutes (Fialho et al., 2009; Chajec et al., 2014). Basal membranes of male cells 7
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display deep infoldings involved in transport of ions and water. But in contrast to females, male digestive cells have no storage function. The regenerative cells are distributed as isolated cells among digestive cells, or form regenerative groups which are called regenerative nests or regenerative crypts (Silva-Olivares et al. 2003, Illa-Bochaca and Montuenga 2006; Wanderley-Teixeira et al. 2006, Baton and Ranford-Cartwright 2007, Mehrabadi et al., 2012; Fernandes et al., 2014). Regenerative nests or crypts were apparently not observed in ceratopogonid midgut epithelium. Only isolated regenerative cells were noted. In biting midges of the genus Culicoides regenerative cells are small and replace old vacuolated cells (Megahed, 1956; Sieburth, 1991). Therefore, here we present the first report on the fine ultrastructure of the regenerative cells in this group of insects. Because these cells are treated as the midgut stem cells, they should possess the potential for proliferation and differentiation (Loeb, 2010; Rost-Roszkowska et al., 2010). However, in F.nigra the mitotic divisions of the regenerative cells were not observed, and their differentiation was detected only in larvae. Failure to observe mitosis may be attributed to the fact that mitotic divisions of cells are governed by a circadian rhythm (Chajec et al. 2012, 2014), or are coupled to molt cycles. Damages caused by the food particles may also influence mitotic divisions (Fernandes et al., 2014). Based on results that we obtained, we conclude that: (1) the midgut epithelium of F. nigra is formed by digestive columnar cells and regenerative cells; (2) the differences in the ultrastructure of the digestive cells between males and females are associated with differences in meals fed and finally (3) digestive cells play an important role in storage of reserve material (larvae and females), secretion process, transport of ion/water, absorption and transport of nutrients.
Acknowledgements We thank professor J. Bohdanowicz from Department of Plant Cytology and Embryology (University od Gdańsk) for giving us the opportunity to examine our material with a Nikon Eclipse E800 light microscope. This work was supported by Young Researcher grant No. 538-L114-B080-13 from the University of Gdańsk.
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References
Andrade-Coêlho, C.A., Santos-Mallet, J., Souza, N.A., Lins, U., Meirelles, M.N., Rangel, E.F., 2001. Ultrastructural features of the midgut epithelium of females Lutzomyia intermedia (Lutz & Neiva, 1912) (Diptera: Psychodidae: Phlebotominae). Mem. Inst. Oswaldo Cruz 96, 1141-1151.
8
Page 8 of 22
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
Arrese, E.L., Soulages, J.L., 2010. Insect fat body: energy, metabolism, and regulation. Ann. Rev. Entomol. 55, 207-225.
22 23
Chaika, S.Yu.. 1983. On Analysis of Ultrastructure of Midgut of Some Blood-Sucking Flies (Diptera), Entomol. Obozr. 62, 470-477.
24 25 26
Chajec, Ł., Rost-Roszkowska, M.M., Vilimova, J., Sosinka, A., 2012. Ultrastructure and regeneration of midgut epithelial cells in Lithobius forficatus (Chilopoda, Lithobiidae). Invertebr. Biol. 131, 119-132.
27 28 29
Chajec, Ł., Sonakowska, L., Rost-Roszkowska, M.M.. 2014. The fine structure of the midgut epithelium in a centipede, Scolopendra cingulata (Chilopoda, Scolopendridae), with the special emphasis on epithelial regeneration. Arthropod. Struct. Dev. 43, 27-42.
30 31 32
Clastrier, J., Legrand, J., 1991. Forcipomyia (Trichohelea) araneivora n. sp. ectoparasite d'une araignée habitant les monts nimba en Guinée (Diptera, Ceratopogonidae; Araneae, Araneidae). Rev. Fr. Entomol. 13, 4155-4158.
33 34 35
Clastrier, J., Wirth, W.W., 1995. Révision des Forcipomyia du sous-genre Microhelea de la région néotropicale, parasites de phasmes (Diptera: Ceratopogonidae). Ann. Soc. Entomol. Fr. 31, 97-150.
36 37
Cribb, B.W., 2000. Oviposition and maintenance of Forcipomyia (Lasiohelea) townsvillensis (Diptera: Ceratopogonidae) in the laboratory. J. Med. Entomol. 37, 316-318.
ip t
Baton, L.A., Ranford-Cartwright, L.C., 2007. Morphological evidence for proliferative regeneration of the Anopheles stephensi midgut epithelium following Plasmodium falciparum ookinete invasion. J Invertebr. Pathol. 96, 244-254.
cr
Biagio, F.P., Tamaki, F.K., Terra, W.R., Ribeiro, A.F., 2009. Digestive morphophysiology of Gryllodes sigillatus (Orthoptera: Gryllidae). J. Insect Physiol. 55, 1125-1133.
us
Bystrak, P. G., Wirth, W.W., 1978. The North American species of Forcipomyia, subgenus Euprojoannisia (Diptera: Ceratopogonidae). United States Department of Agriculture Technical Bulletin number 1951. 51. pp.
an
Campbell, C.L., Mummey, D.L., Schmidtmann, E.T., Wilson, W.C., 2004. Cultureindependent analysis of midgut microbiota in the arbovirus vector Culicoides sonorensis (Diptera: Ceratopogonidae). J. Med. Entomol. 41, 340-348.
Ac ce p
te
d
M
Carpenter, S., Mellor, P.S., Torr, S.J., 2008. Control techniques for Culicoides biting midges and their application in the U.K. and northwestern Palaearctic. Med. Vet. Entomol. 22, 175187.
9
Page 9 of 22
Downes, J.A., 1958. The feeding habits of biting flies and their significance in classification. Ann. Rev. Entomol. 3, 249-263.
3 4
Fernandes, K.M., Neves, C.A., Serrão, J.E., Martins, G.F., 2014. Aedes aegypti midgut remodeling during metamorphosis. Parasitol. Int. 63, 506-512.
5 6 7
Ferreira, M.A.R., Lima, J.B., Rocha, R.M., Pimenta, P.F.P., 2008. Morphological Aspect of the Midgut of Anopheles aquasalis (Curry, 1932) Insecta: Diptera. Int. J. Morphol. 26, 577582.
8 9 10 11
Fialho, M.C.Q., Zanuncio, J.C., Neves, C.A., Ramalho, F.S. Serrão, J.E., 2009. Ultrastructure of the digestive cells in the midgut of the predator Brontocoris tabidus (Heteroptera: Pentatomidae) after different feeding periods on prey and plants. Ann. Entomol. Soc. Am. 102, 119-127.
12 13 14
Fialho, M.C.Q., Moreira, N.R., Zanuncio, J.C., Ribeiro, A.F., Terra, W.R., Serrão, J.E., 2012. Prey digestion in the midgut of the predatory bug Podisus nigrispinus (Hemiptera: Pentatomidae). J. Insect Physiol. 58, 850-856.
15 16
Filimonova, S.A., 2004. Formation of the peritrophic membrane in the gut of Culicoides punctatus (Diptera: Ceratopogonidae). Parazitologiia 38, 12-19. (in Russian)
17 18
Filimonova, S.A., 2005. Morphological Study of Digestive Cycle in Bloodsucking Biting Midges of Genus Culicoides. J. Evol. Biochem. Physiol. 41, 221-232.
19 20 21
Gonçalves, W.G., Fernandes, K.M., Barcellos, M.S., Silva, F.P., Magalhães-Junior, M.J., Zanuncio, J.C., Martins, G.F., Serrão, J.E., 2014. Ultrastructure and immunofluorescence of the midgut of Bombus morio (Hymenoptera: Apidae: Bombini). C R Biol. 337, 365-372.
22 23
Gustevich, A.V., 1973. The bloodsucking midges (Ceratopogonidae). Fauna SSSR, Dipterous Insects, III, 5, Nauka, Leningrad, 270 pp. (in Russian).
24 25
Illa-Bochaca, I., Montuenga, L.M., 2006. The regenerative nidi of locust as a model to study epithelial cell differentiation from stem cells. J. Exp. Biol. 209, 2215-2223.
26 27 28
Kessel, R.G., Beams, H.W., 1980. Cytodifferentiation and vitellogenesis during oogenesis in Arachnida: cytological studies on developing oocytes of harvestman. J. Morphol. 163, 175190.
29 30 31
Koptur, S., J. E. Pena, Grogan, Jr., W.L., 2013 The biting midge Forcipomyia (Microhelea) eriophora (Williston) (Diptera: Ceratopogonidae), an ecotoparasite of larval Phoebis sennae (Pieridae) in South Florida. J. Lepid. Soc. 67, 128-130.
32 33 34
Lafont, R., Dauphin-Viellemant, C., Warren, J.T., Rees, H., 2012. Ecdysteroid chemistry and biochemistry, in: Gilbert, L.I. (Ed.), Insect endocrinology, Academic Press, London, pp. 106156.
Ac ce p
te
d
M
an
us
cr
ip t
1 2
10
Page 10 of 22
Lane, R.P., 1977. Ectoparasitic adapatations in Forcipomyia from butterflies with two new African species (Ceratopogonidae). System. Entomol. 2, 305-312.
3 4 5
León Villalba, L.R., Romoser, W.S., Patrican, L., 2011. Meconial peritrophic matrix and meconial degradation in the biting midge, Culicoides variipennis (Coquillett) (Diptera: Ceratopogonidae). Open Access Insect Physiol. 3, 1-6.
6 7
Loeb, M.J., 2010. Factors affecting proliferation and differentiation of Lepidopteran midgut stem cells. Arch. Insect Biochem. Physiol. 74, 1-16.
8 9 10
Megahed, M.M., 1956. Anatomy and histology of the alimentary tract of the female of the biting midge Culicoides nubeculosus Meigen (Diptera: Heleidae=Ceratopogonidae). Parasitology 46, 22-47.
11 12 13
Mehrabadi, M., Bandani, A.R., Allahyari, M., Serrão, J.E., 2012. The Sunn pest, Eurygaster integriceps Puton (Hemiptera: Scutelleridae) digestive tract: histology, ultrastructure and its physiological significance. Micron 43, 631-637.
14 15
Mellor, P. S., J. Boorman, and M. Baylis. 2000. Culicoides biting midges: their role as arbovirus vectors. Annu. Rev. Entomol. 45, 307-340.
16 17 18
Nardi, J.B., Miller, L.A., Bee, C.M., Lee, R.E. Jr, Denlinger, D.L., 2009. The larval alimentary canal of the Antarctic insect, Belgica antarctica. Arthropod. Struct. Dev. 38, 377389.
19 20 21
Okuda, K., de Souza Caroci, A., Ribolla, P.E., de Bianchi, A.G., Bijovsky, A.T., 2002. Functional morphology of adult female Culex quinquefasciatus midgut during blood digestion. Tissue Cell 34, 210-219.
22 23
Piper, R. C., Katzmann, D.J., 2007. Biogenesis and Function of Multivesicular Bodies. Annu. Rev. Cell. Dev. Biol. 23, 519-547.
24 25 26
Poprawa, I., 2005. The ovary structure, previtellogenic and vitellogenic stages in parthenogenetic species Dactylobiotus dispar (Murray, 1097) (Tardigrada: Eutardigrada). Tissue Cell 37, 385-392.
27 28
Poprawa, I., 2006. Ultrastructural changes of the storage cells during oogenesis in Dactylobiotus dispar (Murray, 1907) (Tardigrada: Eutardigrada). Zool. Pol. 51, 13-18.
29 30 31
Rosell, R., Coons, L.B., 1992. The role of the fat body, midgut and ovary in vitellogenin production and vitellogenesis in the female tick, Dermacentor variabilis. Int. J. Parasitol. 22, 341-349.
32 33 34
Rost-Roszkowska, M.M, Poprawa, I., Klag, J., Migula, P., Mesjasz-Przybylowicz, J., Przybyłowicz, W., 2008. Degeneration of the midgut epithelium in Epilachna cf. nylanderi (Insecta, Coccinellidae): apoptosis, autophagy and necrosis. Can. J. Zool. 86, 1179-1188.
Ac ce p
te
d
M
an
us
cr
ip t
1 2
11
Page 11 of 22
Rost-Roszkowska, M.M., Poprawa, I., Klag, J., Migula, P., Mesjasz-Przybyłowicz, J., Przybyłowicz, W., 2010. Differentiation of regenerative cells in the midgut epithelium of Epilachna cf. nylanderi (Mulsant 1850) (Insecta, Coleoptera, Coccinellidae). Fol. Biol. (Krakow) 58, 209-216.
5 6 7
Rost-Roszkowska, M.M., Poprawa, I., Wójtowicz, M., Kaczmarek, L., 2011. Ultrastructural changes of the midgut epithelium in Isohypsibius granulifer granulifer Thulin, 1928 (Tardigrada: Eutardigrada) during oogenesis. Protoplasma 248, 405-414.
8 9 10
Rost-Roszkowska, M.M., Poprawa, I., Hyra, M., Marek-Swędzioł, M., Kaczmarek, Ł., 2013. The fine structure of the midgut epithelium in Xerobiotus pseudohufelandi (Iharos, 1966) (Tardigrada, Eutardigrada, Macrobiotidae). J. Limnol. 72, 54-61.
11 12
Saunders, L.G., 1924. On the life history and the anatomy of the early stages of Forcipomyia (Diptera, Nemat., Ceratopogonidae). Parasitology 16, 164-213.
13 14 15
Seblova, V., Sadlova, J., Carpenter, S., Volf, P., 2012. Development of Leishmania Parasites in Culicoides nubeculosus (Diptera: Ceratopogonidae) and implications for screening vector competence. J. Med. Entomol. 49, 967-970.
16 17 18
Sieburth, P.J., Nunamaker, C.E., Ellis, J., Nunamaker, R.A., 1991. Infection of the midgut of Culicoides variipennis (Diptera: Ceratopogonidae) with bluetongue virus. J. Med. Entomol. 28, 74-85.
19 20 21
Silva-Olivares, A., E. Diaz, M. Shibayama, V. Tsutsmi, R. Cisneros, Zuniga, G., 2003. Ultrastructural study of the midgut and hindgut in eight species of the genus Dendroctonus Erichson (Coleoptera: Scolytidae). Ann. Entomol. Soc. Am. 96, 883-900.
22 23
Spinelli, G.R., Ronderos, M.M., Díaz, F., Marino, P.I., 2005. The bloodsucking biting midges of Argentina (Diptera: Ceratopogonidae). Mem. Inst. Oswaldo Cruz 100, 137-150.
24 25 26
Svobodová, M., Zídková, L., Čepička, I., Oborník, M., Lukeš, J., Votýpka, J., 2007. Sergeia podlipaevi gen. nov., sp. nov. (Trypanosomatidae, Kinetoplastida), a parasite of biting midges (Ceratopogonidae, Diptera). Int. J. Syst. Evol. Microbiol. 57, 423-432.
27 28 29
Szadziewski, R., Krzywiński, J., Giłka, W., 1997. Diptera Ceratopogonidae, Biting Midges. In: Nilsson, A.N. (Eds), Aquatic Insects of North Europe - A taxonomic Handbook, Vol. 2, Apollo Books, Stenstrup, Denmark, pp. 243-263.
30 31
Terra, W.R., Costa, R.H., Feirreira, C., 2006. Plasma membranes from insect midgut cells. Ann. Braz. Acad. Sci. 78, 255-269.
32 33 34
Urbanek, A., Richert, M., Giłka, W. and Szadziewski, R., 2011. Morphology and histology of secretory setae in terrestrial larvae of biting midges of the genus Forcipomyia (Diptera: Ceratopogonidae). Arthropod. Struct. Dev. 40, 485-494.
Ac ce p
te
d
M
an
us
cr
ip t
1 2 3 4
12
Page 12 of 22
Urbanek, A, Szadziewski, R, Stepnowski, P, Boros-Majewska, J, Gabriel, I, Dawgul, M, Kamysz, W, Sosnowska, D, Gołębiowski, M., 2012 Composition and antimicrobial activity of fatty acids detected in the hygroscopic secretion collected from the secretory setae of larvae of the biting midge Forcipomyia nigra (Diptera: Ceratopogonidae). J. Insect. Physiol. 58, 12651276.
6 7 8
Urbanek, A., Piotrowicz, M., Szadziewski, R. and Giłka, W., 2014. Sensilla coeloconica ringed by microtrichia in host-seeking biting midges. Med. Vet. Entomol. doi: 10.1111/mve.12057
9 10
von Bartheld CS, Altick AL, 2011. Multivesicular bodies in neurons: distribution, protein content, and trafficking functions. Prog. Neurobiol. 93, 313-340.
11 12 13
Wanderley-Teixeira, V., Teixeira, A.A.C., Cunha, F.M., Costa, M.K.C.M., Veiga, A.F.S.L., Oliveira, J.V., 2006. Histological description of the midgut and the pyloric valve of Tropidacris collaris (Stoll, 1813) (Orthopetera: Romaleidae). Braz. J. Biol. 66, 1045-1049.
14 15
Węglarska, B., 1987. Yolk formation in Isohypsibius (Eutardigrada). Zoomorphology 107, 287-292.
16 17 18
Wirth, W.W., 1971. A review of the “stick-ticks,” Neotropical biting midges of the Forcipomyia subgenus Microhelea parasitic on walking stick insects (Diptera: Ceratopogonidae). Entomol. News 82, 229-245.
19 20
Wirth, W.W., 1972. Midges sucking blood of caterpillars (Diptera: Ceratopogonidae). J. Lepid. Soc. 26, 165.
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Figure captions
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Fig. 1. Light micrographs of F. nigra larva, 4th instar (A, B) and adults (C-F). (A) Cross section of larval midgut stained with toluidine blue, bar = 50 µm, bar for insert = 10 µm. (B) Sudan black-positive droplets in larval midgut cells, bar = 50 µm. (C) Cross section of female midgut stained with toluidine blue, epithelial cells contain numerous vesicles (arrowhead), bar = 10 µm. (D) Dark lipid droplets (arrowheads) present in the female midgut epithelial cell stained with Sudan black B, bar = 10 µm. (E) PAS-positive granules (arrow) concentrate in apical region of female midgut epithelial cell, bar = 10 µm. (F) Longitudinal section of male midgut stained with toluidine blue, bar = 10 µm. e – epithelial cell, fb – fat body, g- granule, ld – lipid droplet, lu – lumen, sb – striated border, Mal – Malpighian tube, n – nucleus, pm – peritrophic membrane.
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Fig. 2. F. nigra larva, 4th instar, TEM. (A) Basal region of digestive cell, bar = 2 µm. (B) Digestive cell with electron-dense cytoplasm (dc) and large elongating regenerative cell (rc) bar = 5 µm. (C) Elongating regenerative cell (rc) located between two digestive cells (dc) bar = 5 µm. bl – basal lamina, bm – basal membrane, m – mitochondrion, mb – multivesicular
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body mc – circular muscle, n – nucleus, nu- nucleolus, rm – reserve material, RER – rough endoplasmic reticulum, SER – smooth endoplasmic reticulum.
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Fig. 3. F. nigra larva, 4th instar, TEM. (A) Perinuclear region of two digestive cells joined by septate junctions (arrowhead), bar = 1 µm . (B) Apical region of electron-dense digestive cell, newly differentiated from regenerative cell (dc1) and electron-lucent digestive cell (dc) with visible smooth septate junction (arrowhead); insert indicates on microapocrine secretion (arrow), bar = 2 µm, for insert 0,5 µm . (C) Smooth septate junctions (arrowhead) connected adjacent digestive cells, bar = 1 µm . (D) Apical region of digestive cell, below microvilli, bar = 1 µm. (E) Regenerative cell in interphase (rci), bar = 2 µm. (F) Regenerative cell (rc) bordered with digestive cell (dc), bar = 5 µm. AG – Golgi complex, au – autophagosome, bl – basal lamina, bm – basal membrane, m – mitochondrion, mv – microvilli, n – nucleus, nu – nucleolus, RER – rough endoplasmic reticulum, v – vesicle.
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Fig. 4. F. nigra female, TEM. (A) Basal membrane of digestive cell with different size vesicles (arrowheads), bar = 1 µm. (B) Perinuclear region of digestive cell, bar = 2 µm. (C) Nucleus of digestive cell surrounded by vesicular folds of basal membrane (white arrow), bar = 1 µm. (D) Midgut epithelial cells occupied by vesicular folds of basal membrane (arrowheads), bar = 5 µm. (E) Micorapocrine secretion (arrows) of digestive cells, bar = 2 µm. (F) Apical region of midgut digestive cell with septate junctions (arrow), bar = 1 µm. AG – Golgi complex , bf – basal fold, m – mitochondrion, mv – microvilli, n – nucleus, nu – nucleous, rm – reserve material, RER – rough endoplasmic reticulum, v – vesicle.
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Fig. 5. F. nigra female, TEM. (A) Midgut epithelial cells with deep basal folds (black arrows), bar = 5 µm. (B) Apical region of digestive cells with visible smooth septate junctions (arrowheads), bar = 2 µm. (C) Regenerative cell (rc), bar = 2 µm. (D) Basal membrane of digestive cell, bar = 1 µm. (E) Lamellar body in the cytoplasm of digestive cell, bar = 1 µm.. au – autophagosome, bf – basal fold, bl – basal lamina, lb – lamellar body, m – mitochondrion, mc – circular muscle, ml –longitudinal muscle, n – nucleus, RER – rough endoplasmic reticulum, v – vesicle, va – vacuole.
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Fig. 6. F. nigra male, TEM. (A) Fragment of midgut epithelium consisted of digestive (dc) and regenerative (rc) cells, bar = 5 µm. (B) Basal membrane of digestive cell, bar = 1 µm. (C) Perinuclear region of digestive cell, bar = 2 µm. (D) Perinuclear and apical region of digestive cells with visible septate junctions (arrowhead), bar = 2 µm. au – autophagosome, bf – basal fold, bl – basal lamina, lb – lamellar body, m – mitochondrion, mc – circular muscle, ml – longitudinal muscle, mv – microvilli, n – nucleus, RER – rough endoplasmic reticulum, v – vesicle, va – vacuole.
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Fig. 7. F. nigra male, TEM. (A) Apical region of digestive cell, bar = 0,5 µm. (B) Vacuoles present in the apical region of digestive cell, bar = 0,5 µm. (C) Septate junction with visible microfilaments (arrow) between digestive cells in their perinuclear region, bar = 0,5 µm. (D) Regenerative cell (rc), bar = 2 µm. (E) Fragment of regenerative cell (rc) borders with basal membrane, bar = 1 µm. AG – Golgi complex, au – autophagosome, bm – basal membrane, lb
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– lamellar body, m – mitochondrion, ml – longitudinal muscle, n – nucleus, RER – rough endoplasmic reticulum, v – vesicle, va –vacuole.
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