Turkish Journal of Zoology Turk J Zool (2017) 41: 203-208 http://journals.tubitak.gov.tr/zoology/ © TÜBİTAK Research Article doi:10.3906/zoo-1602-61

Oocyte development in Melanogryllus desertus (Pallas, 1771) (Orthoptera: Gryllidae): presence of Balbiani body*

Özlem ÇAKICI** Zoology Section, Department of Biology, Faculty of Science, Ege University, İzmir, Turkey

Received: 29.02.2016 Accepted/Published Online: 19.07.2016 Final Version: 04.04.2017

Abstract: Oocyte development in the ovary of the black cricket, Melanogryllus desertus, was investigated. In the panoistic ovary of M. desertus, oocyte developmental stages based on their histological properties were classified as previtellogenic, vitellogenic, and maturation. Oocytes were encircled by follicle cells through the developmental stages. In the previtellogenic stage, a few vacuoles were observed just beneath the oolemma. At the beginning of the vitellogenic stage, subdivided into early-, middle-, and late-vitellogenic stages, yolk granules within the oocyte were initially observed just beneath the oolemma. In addition, small lipid droplets were initially present under the yolk granules. As vitellogenesis proceeded, both yolk granules and lipid droplets gradually increased and moved inside the oocyte. The Balbiani body was detected around the nucleus. In the maturation stage, large yolk granules covering the whole of the oocytes were determined. Some atretic oocytes occurring as a result of resorption of oocytes (oosorption) by hypertrophied follicle cells were also detected. Apoptosis in atretic follicles was observed as well.

Key words: Melanogryllus desertus, Gryllidae, Balbiani body, atretic follicle, apoptosis, oosorption

1. Introduction Pentatomidae) (Lemos et al., 2010), Ischnopsyllus spp. ovaries are bilaterally located organs and made (Siphonaptera: Ischnopsyllidae) (Simiczyjew and Margas, up of a number of ovarioles classified as panoistic or 2001), Bactrocera dorsalis (Diptera: Tephritidae) (Chou meroistic (polytrophic and teletrophic). The most et al., 2012), Gryllus yemma (Orthoptera: Gryllidae) primitive ovariole is panoistic type and there are no (Matsuzaki, 1971), and Acheta domesticus (Orthoptera: trophocytes or nurse cells, while the meroistic ovariole Gryllidae) (Dennis and Bradley, 1989). includes both types of cells. Oocytes in panoistic ovariole The Balbiani body is a structure in spherical form are nourished only by the follicular epithelium, while adjacent to the oocyte nucleus in early oogenesis. It nourishment is provided by both follicular cells and disintegrates and disperses within the ooplasm in late trophocytes in the meroistic type (Mazzini, 1987; Singh, oogenesis. This structure has recently been detected in 2007). While panoistic-type ovarioles are present in some (Kloc and Etkin, 2005). Among cricket apterygotes, grasshoppers, crickets, termites, dragonflies, species, the Balbiani body has only been determined stoneflies, fleas, and some beetles (Bonhag, 1958; Singh, in the house cricket, Acheta domesticus (Bradley et 2007), meroistic ovarioles are found in flies, moths, and al., 2001). Melanogryllus desertus is a cricket species butterflies (Bradley et al., 2001). commonly called the black cricket. Apart from South There are a number of studies related to the Europe, North Africa, southern parts of Siberia, and ovary morphology in different species, i.e. Middle Asia, this species is also widely distributed in Amblyomma trieste (Acari: Ixodidae) (de Oliveira et middle and west Anatolia in Turkey (Lodos, 1975). The al., 2006), Rhipicephalus sanguineus (Acari: Ixodidae) present study was designed to determine whether the (de Oliveira et al., 2005), Rhipicephalus microplus Balbiani body is found in the oocytes of M. desertus. To (Acari: Ixodidae) (de Souza, 2013), Elasmucha grisea that end, detailed examinations were conducted using (Heteroptera: Acanthosomatidae) (Ogorzalek and both histochemical and histological methods on oocyte Trochimczuk, 2009), Brontocoris tabidus (: development in M. desertus. * A part of this study was presented at a conference (2nd ICSAE 2015: International Conference on Sustainable Agriculture and En vironment). ** Correspondence: [email protected] 203 ÇAKICI / Turk J Zool

2. Materials and methods with PAS were determined for the first time around the 2.1. Cricket culture periphery of the oocytes in the early vitellogenic stage Black crickets were cultured in laboratory conditions (Figure 2a). As vitellogenesis proceeds, PAS positive (temperature: 26 ± 2 °C; relative humidity: 45 ± 5%; yolk granules cover more and more the oocyte. In the photoperiod: natural). They were maintained in glass meantime, PAS negative lipid droplets were observed in jars by feeding twice a week on lettuce and chicken grain. the ooplasm of these oocytes (Figure 2b). Similarly, yolk Cotton-plugged glass tubes filled with water were put granules reacted with BpB were firstly detected around into these jars to supply the water needs of the crickets. the periphery of the oocytes in the early vitellogenic stage In addition, small petri dishes with their surfaces covered (Figure 3a). In addition, lipid droplets that do not react with moist cotton were placed in the jars for mating with BpB were clearly seen (Figure 3b). On the other hand, females to lay eggs in. The first nymphs emerged within some degenerated oocytes were determined (atresia). In this process, follicle epithelial cells became hypertrophic. 10–12 days. They became adult crickets nine nymphal These cells with many vacuoles resorbed the yolk. This stages later. clearly showed the follicle cells’ phagocytic activity (Figure 2.2. Light microscope analyses 4a). Follicle cells encircled a space formed as a result of Following the dissection of adult females, their ovaries digestion of yolk (oosorption). Moreover, a crescent- were fixed in Bouin’s solution for 24 h. Dehydration was shaped nucleus in the follicle epithelium was determined achieved with graded ethanol (70%, 96%, 100%). Then as an indicator of apoptosis. Some apoptotic bodies around the ovaries were put into xylene to achieve transparency these follicle cells engulfing the oocyte were clearly seen and embedded in paraffin. Thus, fixed tissues were serially (Figure 4b). sectioned at 5-µm thickness using a microtome (Baird & Tatlock). These tissues were stained by Harris Hematoxylin 4. Discussion and Eosin (HE) to reveal general histological organization. Female insects typically produce a large number of eggs to In addition, Periodic Acid Schiff (PAS) and Bromophenol transfer their genes and to that end they invest remarkable Blue (BpB) were used to mark neutral mucosubstances resources (Swevers et al., 2005). In the oogenesis period, (GAGs) and protein in the oocytes, respectively. All proteins, lipids, carbohydrates, and other components are sections were photographed with an Olympus CX 31 regularly deposited in the developing oocyte (Fruttero et photomicroscope. al., 2011). Vitellogenesis is an important process for the maturation of an insect egg. In this period, rapid oocyte 3. Results growth occurs due to yolk deposition (Valle, 1993). Histologic examinations showed that oocytes at different Vitellogenins, which are a group of proteins synthesized stages of oogenesis are found in the ovary of Melanogryllus outside the ovary, are the main component of the yolk and desertus. Oocytes complete their development through stored as vitellin in oocytes (Hagedorn and Kunkel, 1979). three main stages: previtellogenic, vitellogenic, and In the maturation process of oocytes, they become maturation. In the previtellogenic stage, the oocytes are markedly enlarged. Oocytes at different developmental encircled by follicle cells and there are a few vacuoles under stages are present simultaneously in the ovaries of the oolemma (Figure 1a). A large nucleus with prominent Melanogryllus desertus. On the other hand, in the ovaries of nucleolus was determined in these oocytes (Figure 1b). Ambylomma triste, which has panoistic ovaries, there was The vitellogenic stage is subdivided into early-, middle-, no great morphological variation between the oocytes. In and late-vitellogenic stages. In this stage, oocyte volume other words, oocytes develop synchronously (de Oliveira begins to increase. In the early vitellogenic stage, yolk et al., 2006). granules firstly appear just beneath the oolemma. Small Immature oocytes of some invertebrates such as lipid droplets that do not react with H&E were observed chelicerates, myriapods, and insects and of vertebrates such just below the yolk granules (Figure 1c). As vitellogenesis as fish, amphibians, birds, and mammals include a distinct proceeds, both occupy the inside of the oocyte. The Balbiani cytoplasmic organelle called the Balbiani body (Bradley body is a juxtanuclear spherical complex of organelles in et al., 2001; Tworzydlo et al., 2009). Ultrastructurally, developing oocytes of many species (Jaglarz et al., 2003). the Balbiani body is a structure that contains a dense In the middle vitellogenic stage, the Balbiani body was cloud of tubular mitochondria, rough ER, Golgi-like determined around the germinal vesicle (nucleus) (Figure membrane aggregates, and microtubules. Well-developed 1d). In the late vitellogenic stage, many yolk granules Balbiani bodies form adjacent to and on opposite sides of and lipid droplets larger than the previous stage were the germinal vesicle in the oocytes of the house cricket, observed within the oocyte (Figure 1e). In the maturation Acheta domesticus (Bradley et al., 2001). Similarly, a stage, oocytes were filled with larger yolk granules than well-developed Balbiani body is determined around lipid droplets (Figure 1f). Yolk granules stained positive the germinal vesicle in the black cricket, Melanogryllus

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Figure 1. Developmental stages of oocytes. a) A few vacuoles (asterisks) of oocytes encircled by follicle epithelium (FE) in previtellogenic stage. b) Large nucleus with prominent nucleolus (No) in previtellogenic stage. c) Yolk granules (YG) firstly appearing just beneath the oolemma in early vitellogenic stage. d) Balbiani body (yolk nucleus) (Bb) around germinal vesicle (nucleus) (N) in mid-vitellogenic stage. e) Many yolk granules (YG) within the oocyte in late-vitellogenic stage. f) Oocytes with large yolk granules (YG) in maturation stage. desertus. This is the first report detecting the Balbiani body progressed from columnar to cuboidal to squamous in in the oocyte of M. desertus. Acheta domesticus (Dennis and Bradley, 1989). This is Follicular epithelium covers the developing oocyte. also determined in the oocytes of Melanogryllus desertus. During vitellogenesis, the epithelium of individual follicles Columnar follicular epithelial cells in previtellogenic

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Figure 2. Histological sections of ovary including different stage of oocytes stained with Periodic Acid Schiff (PAS). a) PAS positive materials (asterisk) in the oocytes belonging to different developmental stages. b) PAS positive yolk granules covering the vitellogenic (V) and mature oocytes (M) and PAS negative lipid droplets within these oocytes’ ooplasm.

Figure 3. Histological sections of ovary including different stage of oocytes stained with Bromophenol Blue (BpB). a) BpB positive materials (asterisks) in the oocytes belonging to different developmental stages. Arrow: Balbiani body. b) BpB positive yolk granules covering the vitellogenic (V) and mature oocytes (M) and lipid droplets that do not react with BpB.

Figure 4. Oosorption of oocytes. a) Oocytes (O) resorbed by hypertrophied follicle cells (H) and space (asterisk) encircled by follicle cells. b) Hypertrophic (H) follicle cells engulfing the oocyte and apoptotic cell with crescent-shaped nucleus (triangled) and apoptotic bodies (encircled).

206 ÇAKICI / Turk J Zool oocytes gradually transformed from cubic to squamous as follicles are termed atretic follicles. Follicular atresia is vitellogenesis proceeded. accepted as a natural phenomenon and occurs during PAS staining is used to determine neutral normal oogenesis in many species (Czarniewska mucosubstances (GAGs). PAS-positive material was firstly et al., 2014). Atretic follicles were also determined in the determined in the periphery of the oocytes; later it moved ovaries of Melanogryllus desertus. Similarly, these type to the inside of the oocyte in Melanogryllus desertus. In follicles occurred as a result of oosorption. In this process, contrast, PAS-positive material appeared first around follicle cells became hypertrophic with vacuoles. These the germinal vesicle in the early-vitellogenic stage and hypertrophied follicle cells having phagocytic activity later it migrated towards the periphery in the oocytes of play an active role in the oosorption. They engulfed the Orthetrum chrysis Selys (Libellulidae: Odonata) (Tembhare yolk within the oocyte. Some follicle cells had a crescent- and Thakare, 1975). shaped nucleus. This was noted as a sign of apoptosis. All germline cells except those undergoing apoptosis Some apoptotic bodies were also detected around follicle develop into mature oocytes in the panoistic ovaries (Tworzydlo et al., 2009). Unsuitable physiological, cells. behavioral, or environmental factors such as starvation, In conclusion, the ovary type of Melanogryllys desertus mating delay, host deprivation, and lack of available is panoistic as reported in other Gryllid species. This study oviposition sites may cause the degeneration of some examining histological and histochemical properties of follicles. In other words, some oocytes undergo developing oocytes showed the presence of Balbiani body oosorption, the resorption of oocytes in the ovary (Bell in the oocytes of M. desertus. This study will be useful and Bohm, 1975; Aguirre et al., 2011; Guo et al., 2011). for further investigations on oocyte development in this This process is known as follicular atresia and these type cricket species.

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