Turkish Journal of Zoology Turk J Zool (2017) 41: 379-389 http://journals.tubitak.gov.tr/zoology/ © TÜBİTAK Research Article doi:10.3906/zoo-1603-12

Fine structure of the Malpighian tubules in campestris (Linnaeus, 1758) (, )

Irmak POLAT, Nurcan ÖZYURT, Damla AMUTKAN, Zekiye SULUDERE*, Selami CANDAN Department of Biology, Faculty of Science, Gazi University, Ankara, Turkey

Received: 03.03.2016 Accepted/Published Online: 02.12.2016 Final Version: 23.05.2017

Abstract: The Malpighian tubules are responsible for excretion and osmoregulation in . The Malpighian system consists of tubules that are closed at the distal end and extend from the point of junction of the midgut and hindgut. This organ helps the water and electrolyte balance of the internal metabolism by filtration with absorption of water and soluble materials as mineral salts from the hemolymph. In this study, the histology of Malpighian tubules in Gryllus campestris (Orthoptera, Gryllidae) has been examined in detail by light microscopy, scanning electron microscopy, and transmission electron microscopy. The distal ends of the Malpighian tubules in G. campestris, which are long and wiggly in shape, extend freely into the body cavity. The other end is connected to the alimentary canal. In a cross-section of the tubules, it has been observed that the outer surface of the cells is covered by muscles and trachea. The lumen is lined with a single-layer epithelium. The nuclei (which are oval or spherical in shape), rough endoplasmic reticulum, mitochondria, and spherocrystals or mineral concretions are seen in the cells. Microvilli are found in the apical membrane. When the structure of the Malpighian tubules was compared with those of other species belonging to Orthoptera and other orders, similarities and differences were observed.

Key words: Excretory system, Malpighian tubule, ultrastructure, electron microscope

1. Introduction 12 to 150 in , 2 to 200 in Orthoptera, and The Malpighian tubules are the excretory and 50 to 60 in Odonata, this number is 6 in Lepidoptera and osmoregulatory organs of insects, which are specialized for 4 in Diptera. In many species, the tubules are separated organic solute transport, metabolism, immune defenses, into morphologically different regions and can contain and detoxification (Wigglesworth, 1965; Chapman, different cell types (Bradley et al., 1982; Chapman, 1998; 1998; Dow, 2009). They are homologous to the vertebrate Acar, 2009; Pacheco et al., 2014; Amutkan et al., 2015). nephron in the function of removing different chemical Most insects in the order Diptera have small stellate cells substances from the body fluids, and they are associated scattered among the principal tubule cells. In contrast with with water and ion transport from the hemolymph to this, insects in the Heteroptera order have only one type of the lumen (Wigglesworth, 1965; Chapman, 1998; Pal cells (Chapman, 1998). and Kumar, 2012). Thus, the Malpighian tubules serve The Malpighian tubules of insects consist of a long the functions of both generating the primary urine and tubular structure formed from a single layer of epithelial selectively reabsorbing the desirable solutes; in terrestrial cells and are closed at one end. However, apart from insects, the filtered fluid or primary urine is passed to the this general form of the Malpighian tubules, there is hindgut and rectum, where this filtered fluid is modified as considerable variation in the form of them throughout a result of reabsorption of certain substances and excretion the class of insects. Generally, the tubules are free in the of others to produce urine. This selective modification of hemocoel, which connect the alimentary canal at the primary urine occurs in the ileum, rectum, or even in junction of the midgut and hindgut (Roeder and College, the Malpighian tubules (Maddrell and Gardiner, 1974; 1953; Martini et al., 2007). Roeder and College (1953) Maddrell, 1981; Chapman, 1998). reported that the Malpighian tubules of insects generally The Malpighian tubules are present in variable numbers can be divided into four groups. They stated that the in different orders of insects. Although the number of the simplest of these groups is found in species of Orthoptera, Malpighian tubules can vary from 1 to 4 in Hemiptera, Dermaptera, Neuroptera, and some Coleoptera. The * Correspondence: [email protected] 379 POLAT et al. / Turk J Zool distal end of this group’s tubules is closed and free in the 2.3. Transmission electron microscopy hemocoel. In the Malpighian tubules that belong to the For transmission electron microscopy (TEM), tissues were second group (some families of Lepidoptera, Coleoptera, fixed in 2.5% glutaraldehyde with a 0.2 M phosphate buffer and Hymenoptera), the distal ends are found to be attached (pH 7.2). Postfixation was performed using 1% OsO4 with to the rectal structure (Nation, 2002). The third group of a phosphate buffer for 1 h. The OsO4 was then removed Malpighian tubules can be observed in some species of and the samples were rinsed three times with phosphate Hemiptera; the distal regions are free and attached to the buffer. Dehydration was performed using a series of gut with ampulla (Roeder and College, 1953). Finally, the alcohol solutions with increasing concentrations (70%, last group of Malpighian tubules can be observed as a 80%, 90%, 96%, and 100%). The tissues were embedded combination of the second and third groups. The variety in Araldite. The Araldite blocks were sectioned using in form observed in tubules of different species represents a Leica EM UC6 ultramicrotome, and the sections were different ways of achieving adequate excretory capacity. then stained with lead citrate and uranyl acetate. Analysis For instance, the tubules of species with high numbers of tubules are often short, whereas those from species with and imaging were performed using a JEOL JEM 1400 few tubules are often long and convoluted, as in Hemiptera transmission electron microscope at 80 kV in the Electron species, or smooth, as in Orthoptera (Denholm and Skaer, Microscopy Laboratory of the Faculty of Science at Gazi 2005; Acar, 2009; Amutkan et al., 2015). University, in Ankara, Turkey. In previous studies, the structure and morphology of the Malpighian tubules were examined in different 3. Results insect species in a comprehensive manner. However, the The Malpighian tubules of Gryllus campestris are relatively structure of the Malpighian tubules for Gryllus campestris long, smooth tubules that end blindly in the abdominal (Orthoptera, Gryllidae), which is a harmful insect species cavity and open into the alimentary canal immediately in Turkey, has not been studied. Consequently, we have behind the midgut. They are located at the point of junction described the Malpighian tubules of G. campestris in order of the midgut and hindgut. The number of Malpighian to contribute to a better understanding of this insect’s tubules of both males and females of the species displays morphological and histological features. variation between 90 and 150 tubules (Figures 1 and 2). The Malpighian tubules of G. campestris consist of a single- 2. Materials and methods layer cuboidal epithelium with a few cells encircling the Adult specimens of Gryllus campestris were collected in lumen (Figures 3 and 4). In SEM images, it is observed that Kazan and Kızılcahamam in Ankara from June to August the outer surface of the Malpighian tubules is covered with 2015. the trachea and muscles (Figure 5). In light microscopy 2.1. Light microscopy and TEM images, the cells have a close-packed structure of Male and female specimens of G. campestris were given microvilli on the lumen side (Figures 6 and 7). The length of anesthesia with ethyl acetate fumes, and the Malpighian the microvilli is approximately the same as the epithelium tubules were dissected in sodium phosphate buffer width in some regions of the Malpighian tubules (Figure under a stereomicroscope. Malpighian tubules were 7). The most important characteristic of the microvilli is fixed in alcoholic Bouin’s solution. After washing and the the enlargement. This bulblike enlargement is observed at dehydration process, samples were embedded in paraffin. the end of the microvilli. This structure and the vesicles Subsequently, sections stained with hematoxylin and eosin were prepared in permanent slides with Entellan. Sections are demonstrated to be an apocrine secretion (Figure 8). that belonged to the Malpighian tubules were examined There are no mitochondria in the microvilli of the cells in using light microscopy and were photographed digitally. the Malpighian tubules (Figure 8). The cell nuclei, which are oval or spherical in shape, are 2.2. Scanning electron microscopy found in the middle of the cells. The outer surface of the The Malpighian tubules were fixed in 2.5% glutaraldehyde (pH 7.2, phosphate-buffered), rinsed three times with nuclei has a regular shape. The heterochromatin and the phosphate buffer, dehydrated in ascending series of ethyl euchromatin regions are clearly observed in TEM (Figure alcohol (70%, 80%, 90%, 96%, and 100%), and dried with 7). critical point drying (Polaron CPD 7501 Critical Point The basement membrane, which is a thin structure, Dryer). The specimens were then mounted with double- completely ensheaths the outer surface of the tubule. sided tape onto scanning electron microscopy (SEM) stubs The basal plasma membrane comprises intensively and coated with gold in a Polaron SC 502 sputter coater. well-developed infoldings. The infoldings are more or They were examined with a JEOL JSM 6060 LV scanning less parallel and radially aligned, and they penetrate to electron microscope at an accelerating voltage of 5–10 kV varying depths in the transverse section. The numerous and photos were taken. mitochondria are organized between the basal infoldings

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Figure 1. Stereomicroscope image of a part of the alimentary canal and the Malpighian tubules in Gryllus campestris.

Figure 2. SEM micrograph of a part of the alimentary canal and the Malpighian tubules in Gryllus campestris.

(Figures 9 and 10). There is a thin basal lamina under reticulum is present in all regions of the cytoplasm. The the basal plasma membrane (Figure 9). There are many rough endoplasmic reticulum is especially organized organelles in the cytoplasm. Plenty of mitochondria are around the nucleus (Figure 12). There are many also found homogeneously in the cytoplasm, and the spherocrystals or mineral concretions that are spherical cytoplasm contains many bodies morphologically similar in shape and of different sizes. The early stages of the to lysosomes (Figure 11). Extensive rough endoplasmic developing spherocrystals are observed in lucent vesicles.

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Figure 3. Light microscopy image of the cross-section of the Malpighian tubules (400×). Nucleus (arrows); microvilli (encircled); L: lumen.

Figure 4. SEM micrograph of the cross-section of the Malpighian tubules. Mv: Microvilli; L: lumen; E: epithelial cell.

During development, they extend; the late stages of the 4. Discussion spherocrystals have two or more concentric structures that The insect excretory system includes a number of have different electron densities. These are found in the Malpighian tubules. Its proximal region opens into central cytoplasm and close to the basal plasma membrane the alimentary canal at the junction of the midgut and (Figure 13). hindgut, and the distal region lies free in the abdominal

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Figure 5. SEM micrograph of the cross-section of the Malpighian tubules. Tr: Trachea; Ms: muscle; Mv: microvilli; L: lumen.

Figure 6. TEM micrograph of the cross-section of the Malpighian tubules. Muscle (arrow); N: nucleus; Mv: microvilli; L: lumen.

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Figure 7. TEM micrograph of the cross-section of the Malpighian tubules. Trachea (arrow); N: nucleus; Mv: microvilli; and L: lumen.

Figure 8. The enlargements of the tips of the microvilli (Þ).

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Figure 9. TEM micrograph of the basal side of the Malpighian tubules. BL: Basal lamina; (→): basal plasma membrane infoldings.

Figure 10. TEM micrograph of the basal side of the Malpighian tubules. Ms: Muscle; M: mitochondria; (→): basal plasma membrane infoldings.

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Figure 11. TEM micrograph of the cross-section of the Malpighian tubules. M: Mitochondria; Ls: lysosome-like bodies; Mv: microvilli. cavity (Acar, 2009; Amutkan et al., 2015). The structure outer surface of the Malpighian tubules is surrounded by of the Malpighian tubules in Gryllus campestris displays muscle as in other species previously examined (Beams et significant similarities and differences compared with those al., 1955; Ramsay, 1955; Arab and Caetano, 2002; Martini of species investigated previously (Ramsay, 1954; Beams et et al., 2007). al., 1955; Taylor, 1971; Acar, 2009; Pal and Kumar, 2012; The basement membranes of the Malpighian tubules 2014; Gonçalves et al., 2014; Amutkan et al., 2015; Zhong are permeable. Uptake of colloids and proteins from the et al., 2015). While the numbers of the Malpighian tubules hemolymph is provided with the basement membranes. in Gryllus campestris range from 90 to 150, this number The thickness of the basement membrane varies in is 4 in Graphosoma lineatum (Heteroptera, Pentatomidae) different orders for the class Insecta. Some species, such and Sarcophaga ruficornis (Diptera, Sarcophagidae) (Pal as A. aegypti, Calpodes ethlius (Lepidoptera, Hesperiidae), and Kumar, 2014; Amutkan et al., 2015). Aedes aegypti and G. campestris, have thin basement membranes (Beams (Diptera, Culicidae) has 5 Malpighian tubules (de Sousa et al., 1955; Ryerse, 1979; Chapman, 2013). Whether the and Bicudo, 1999). In Melanogryllus desertus (Orthoptera, basement membrane of the Malpighian tubules is thick Gryllidae), this number varies between 100 and 150 (Acar, or not, it serves for structural support, permeability of 2009). proteins, and ion selectivity. Additionally, it protects the The Malpighian tubules are surrounded by muscles in tubules against pressure from muscular movement (Beams insects. They move in the hemolymph and can carry out et al., 1955; Chapman, 2013). peristaltic movements to move material from the distal end In G. campestris, cells contain numerous secretory to the opening in the hindgut. Contraction of the muscles granules, mitochondria, ribosomes, spherocrystals creates hydrostatic pressure across the Malpighian tubule or mineral concretions in different sizes, and other system for the movement of water (Denholm and Skaer, cytoplasmic organelles. Spherocrystals are characteristic 2005; Chapman, 2013). Therefore, in G. campestris, the features of the Malpighian tubule cells and have various

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Figure 12. TEM micrograph of the cross-section of the Malpighian tubules. N: Nucleus; M: mitochondria; GER: rough endoplasmic reticulum; Mv: microvilli; L: lumen. roles in insects’ excretory systems. They are generally of small substances by passive movement from the known to regulate the maintenance of the appropriate hemolymph into the tubule lumen (Pal and Kumar, 2013). mineral composition and the accumulation of nontoxic In Troglophilus neglectus (Orthoptera, Rhaphidophoridae) waste materials and of toxic metals. The spherocrystals and Sarcophaga ruficornis (Diptera, Sarcophagidae), a also store products of mineral and organic materials (Sohal few mitochondria extend into the microvilli (Delakorda and Lamb, 1979; Wessing and Zierold, 1992; Cruz-Landim et al., 2009; Pal and Kumar, 2013). In contrast, there and Serrao, 1997; Delakorda et al., 2009; Lipovsek et al., is a lack of mitochondria in the microvilli in Podisus 2012; Chapman, 2013; Pal and Kumar, 2014). Numerous nigrispinus (Hemiptera, Pentatomidae), Graphosoma secretory granules, which have the roles of synthesis and lineatum (Heteroptera, Pentatomidae), Locusta migratoria storage of abundant protein substances, are found in (Orthoptera, Acrididae), and Schistocerca gregaria the tubule cells. Spherocrystals and secretory granules (Orthoptera, Acrididae) (Prado et al., 1992; da Cunha et are comparably found in other species, since they have al., 2012; Amutkan et al., 2015). When compared with the different shapes and sizes (Pal and Kumar, 2013; Zhong et Malpighian tubules of other species, those of G. campestris, al., 2015). which have long microvilli (about 2–5 µm) on the apical The Malpighian tubule cells of G. campestris have long surface, do not have mitochondria in the microvilli. The and wide basal plasma membrane infoldings and extremely mitochondria are found in the cytoplasm, mainly in the long microvilli. The structures of the basal plasma apical region, and are associated with the basal plasma membrane infoldings and microvilli indicate that these are membrane infoldings. In our opinion, the well-developed involved in the active transport mechanism that requires basal membrane infoldings and abundant mitochondria energy (Delakorda et al., 2009; Pal and Kumar, 2012). They among them in G. campestris are involved in active ion also regulate an osmotic gradient and active transport transportation.

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Figure 13. TEM micrograph of the cross-section of the Malpighian tubules. M: Mitochondria; Sg: secretory granules; (◌): spherocrystals or mineral concretions.

In conclusion, comparison of the Malpighian tubules of information on this important organ, which has different G. campestris with those of other species in the Orthoptera fundamental functions for insects. order showed similarities. There were also morphological differences from the other insect orders. These differences Acknowledgments can clearly be observed in the morphological structure We express our thanks to Gazi University, BAP project of the Malpighian tubules and the number of tubules. 05/2011-69, which partially supported this research. We We suggest that our findings provide new insights for also thank Ellen Yazar, who revised the grammar of this understanding the tubule structure. Moreover, this article. This study was presented as an oral presentation study provides key information for future research on at the 22nd Electron Microscopy Congress, Sabancı the Malpighian tubules. Thus, this study gives useful University, 2–4 September 2015, İstanbul.

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