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http://journals.tubitak.gov.tr/zoology/

Turkish Journal of Zoology Turk J Zool(2017) 41: 203-208© TÜBİTAKdoi: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

1. IntroductionInsect ovaries are bilaterally located organs and made up of a number of ovarioles classified as panoistic or meroistic (polytrophic and teletrophic). The most primitive ovariole is panoistic type and there are no trophocytes or nurse cells, while the meroistic ovariole includes both types of cells. Oocytes in panoistic ovariole are nourished only by the follicular epithelium, while nourishment is provided by both follicular cells and trophocytes in the meroistic type (Mazzini, 1987; Singh, 2007). While panoistic-type ovarioles are present in apterygotes, grasshoppers, crickets, termites, dragonflies, stoneflies, fleas, and some beetles (Bonhag, 1958; Singh, 2007), meroistic ovarioles are found in flies, moths, and butterflies (Bradley et al., 2001).

There are a number of studies related to the ovary morphology in different arthropod species, i.e. Amblyomma trieste (Acari: Ixodidae) (de Oliveira et al., 2006), Rhipicephalus sanguineus (Acari: Ixodidae) (de Oliveira et al., 2005), Rhipicephalus microplus (Acari: Ixodidae) (de Souza, 2013), Elasmucha grisea (Heteroptera: Acanthosomatidae) (Ogorzalek and Trochimczuk, 2009), Brontocoris tabidus (Hemiptera:

Pentatomidae) (Lemos et al., 2010), Ischnopsyllus spp. (Siphonaptera: Ischnopsyllidae) (Simiczyjew and Margas, 2001), Bactrocera dorsalis (Diptera: Tephritidae) (Chou et al., 2012), Gryllus yemma (Orthoptera: Gryllidae) (Matsuzaki, 1971), and Acheta domesticus (Orthoptera: Gryllidae) (Dennis and Bradley, 1989).

The Balbiani body is a structure in spherical form adjacent to the oocyte nucleus in early oogenesis. It disintegrates and disperses within the ooplasm in late oogenesis. This structure has recently been detected in some insects (Kloc and Etkin, 2005). Among cricket species, the Balbiani body has only been determined in the house cricket, Acheta domesticus (Bradley et al., 2001). Melanogryllus desertus is a cricket species commonly called the black cricket. Apart from South Europe, North Africa, southern parts of Siberia, and Middle Asia, this species is also widely distributed in middle and west Anatolia in Turkey (Lodos, 1975). The present study was designed to determine whether the Balbiani body is found in the oocytes of M. desertus. To that end, detailed examinations were conducted using both histochemical and histological methods on oocyte development in M. desertus.

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

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

Research Article

* A part of this study was presented at a conference (2nd ICSAE 2015: International Conference on Sustainable Agriculture and En vironment). ** Correspondence: ozlem.cakici@ege.edu.tr

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2. Materials and methods2.1. Cricket cultureBlack crickets were cultured in laboratory conditions (temperature: 26 ± 2 °C; relative humidity: 45 ± 5%; photoperiod: natural). They were maintained in glass jars by feeding twice a week on lettuce and chicken grain. Cotton-plugged glass tubes filled with water were put into these jars to supply the water needs of the crickets. In addition, small petri dishes with their surfaces covered with moist cotton were placed in the jars for mating females to lay eggs in. The first nymphs emerged within 10–12 days. They became adult crickets nine nymphal stages later. 2.2. Light microscope analysesFollowing the dissection of adult females, their ovaries were fixed in Bouin’s solution for 24 h. Dehydration was achieved with graded ethanol (70%, 96%, 100%). Then the ovaries were put into xylene to achieve transparency and embedded in paraffin. Thus, fixed tissues were serially sectioned at 5-µm thickness using a microtome (Baird & Tatlock). These tissues were stained by Harris Hematoxylin and Eosin (HE) to reveal general histological organization. In addition, Periodic Acid Schiff (PAS) and Bromophenol Blue (BpB) were used to mark neutral mucosubstances (GAGs) and protein in the oocytes, respectively. All sections were photographed with an Olympus CX 31 photomicroscope.

3. ResultsHistologic examinations showed that oocytes at different stages of oogenesis are found in the ovary of Melanogryllus desertus. Oocytes complete their development through three main stages: previtellogenic, vitellogenic, and maturation. In the previtellogenic stage, the oocytes are encircled by follicle cells and there are a few vacuoles under the oolemma (Figure 1a). A large nucleus with prominent nucleolus was determined in these oocytes (Figure 1b). The vitellogenic stage is subdivided into early-, middle-, and late-vitellogenic stages. In this stage, oocyte volume begins to increase. In the early vitellogenic stage, yolk granules firstly appear just beneath the oolemma. Small lipid droplets that do not react with H&E were observed just below the yolk granules (Figure 1c). As vitellogenesis proceeds, both occupy the inside of the oocyte. The Balbiani body is a juxtanuclear spherical complex of organelles in developing oocytes of many species (Jaglarz et al., 2003). In the middle vitellogenic stage, the Balbiani body was determined around the germinal vesicle (nucleus) (Figure 1d). In the late vitellogenic stage, many yolk granules and lipid droplets larger than the previous stage were observed within the oocyte (Figure 1e). In the maturation stage, oocytes were filled with larger yolk granules than lipid droplets (Figure 1f). Yolk granules stained positive

with PAS were determined for the first time around the periphery of the oocytes in the early vitellogenic stage (Figure 2a). As vitellogenesis proceeds, PAS positive yolk granules cover more and more the oocyte. In the meantime, PAS negative lipid droplets were observed in the ooplasm of these oocytes (Figure 2b). Similarly, yolk granules reacted with BpB were firstly detected around the periphery of the oocytes in the early vitellogenic stage (Figure 3a). In addition, lipid droplets that do not react with BpB were clearly seen (Figure 3b). On the other hand, some degenerated oocytes were determined (atresia). In this process, follicle epithelial cells became hypertrophic. These cells with many vacuoles resorbed the yolk. This clearly showed the follicle cells’ phagocytic activity (Figure 4a). Follicle cells encircled a space formed as a result of digestion of yolk (oosorption). Moreover, a crescent-shaped nucleus in the follicle epithelium was determined as an indicator of apoptosis. Some apoptotic bodies around these follicle cells engulfing the oocyte were clearly seen (Figure 4b).

4. DiscussionFemale insects typically produce a large number of eggs to transfer their genes and to that end they invest remarkable resources (Swevers et al., 2005). In the oogenesis period, proteins, lipids, carbohydrates, and other components are regularly deposited in the developing oocyte (Fruttero et al., 2011). Vitellogenesis is an important process for the maturation of an insect egg. In this period, rapid oocyte growth occurs due to yolk deposition (Valle, 1993). Vitellogenins, which are a group of proteins synthesized outside the ovary, are the main component of the yolk and stored as vitellin in oocytes (Hagedorn and Kunkel, 1979).

In the maturation process of oocytes, they become markedly enlarged. Oocytes at different developmental stages are present simultaneously in the ovaries of Melanogryllus desertus. On the other hand, in the ovaries of Ambylomma triste, which has panoistic ovaries, there was no great morphological variation between the oocytes. In other words, oocytes develop synchronously (de Oliveira et al., 2006).

Immature oocytes of some invertebrates such as chelicerates, myriapods, and insects and of vertebrates such as fish, amphibians, birds, and mammals include a distinct cytoplasmic organelle called the Balbiani body (Bradley et al., 2001; Tworzydlo et al., 2009). Ultrastructurally, the Balbiani body is a structure that contains a dense cloud of tubular mitochondria, rough ER, Golgi-like membrane aggregates, and microtubules. Well-developed Balbiani bodies form adjacent to and on opposite sides of the germinal vesicle in the oocytes of the house cricket, Acheta domesticus (Bradley et al., 2001). Similarly, a well-developed Balbiani body is determined around the germinal vesicle in the black cricket, Melanogryllus

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desertus. This is the first report detecting the Balbiani body in the oocyte of M. desertus.

Follicular epithelium covers the developing oocyte. During vitellogenesis, the epithelium of individual follicles

progressed from columnar to cuboidal to squamous in Acheta domesticus (Dennis and Bradley, 1989). This is also determined in the oocytes of Melanogryllus desertus. Columnar follicular epithelial cells in previtellogenic

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.

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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).

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oocytes gradually transformed from cubic to squamous as vitellogenesis proceeded.

PAS staining is used to determine neutral mucosubstances (GAGs). PAS-positive material was firstly determined in the periphery of the oocytes; later it moved to the inside of the oocyte in Melanogryllus desertus. In contrast, PAS-positive material appeared first around the germinal vesicle in the early-vitellogenic stage and later it migrated towards the periphery in the oocytes of Orthetrum chrysis Selys (Libellulidae: Odonata) (Tembhare and Thakare, 1975).

All germline cells except those undergoing apoptosis develop into mature oocytes in the panoistic ovaries (Tworzydlo et al., 2009). Unsuitable physiological, behavioral, or environmental factors such as starvation, mating delay, host deprivation, and lack of available oviposition sites may cause the degeneration of some follicles. In other words, some oocytes undergo oosorption, the resorption of oocytes in the ovary (Bell and Bohm, 1975; Aguirre et al., 2011; Guo et al., 2011). This process is known as follicular atresia and these type

follicles are termed atretic follicles. Follicular atresia is accepted as a natural phenomenon and occurs during normal oogenesis in many animal species (Czarniewska et al., 2014). Atretic follicles were also determined in the ovaries of Melanogryllus desertus. Similarly, these type follicles occurred as a result of oosorption. In this process, follicle cells became hypertrophic with vacuoles. These hypertrophied follicle cells having phagocytic activity play an active role in the oosorption. They engulfed the yolk within the oocyte. Some follicle cells had a crescent-shaped nucleus. This was noted as a sign of apoptosis. Some apoptotic bodies were also detected around follicle cells.

In conclusion, the ovary type of Melanogryllys desertus is panoistic as reported in other Gryllid species. This study examining histological and histochemical properties of developing oocytes showed the presence of Balbiani body in the oocytes of M. desertus. This study will be useful for further investigations on oocyte development in this cricket species.

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