Ultrastructure alterations induced by gamma ...

Environ Sci Pollut Res (2017) 24:22102–22110 DOI 10.1007/s11356-017-9869-5

RESEARCH ARTICLE

Ultrastructure alterations induced by gamma irradiation in spermiogenesis of the ground beetle, Blaps sulcata: reference to environmental radiation protection Dalia Kheirallah 1 & Lamia El-Samad 1 & Naglaa Fahmi 2 & Wafaa Osman 1

Received: 25 April 2017 / Accepted: 31 July 2017 / Published online: 8 August 2017 # Springer-Verlag GmbH Germany 2017

Abstract Ultrastructure alterations in spermiogenesis of the ground beetle, Blaps sulcata (Coleoptera: Tenebrionidae) were studied in normal adult males and in two male groups irradiated by gamma rays of 3 and 16 μSv/h dose rate. Ultrastructure examination of irradiated sperms revealed many alterations both in the head and in the flagellum regions of mature sperm. Alterations of the head region included nuclei with uncondensed chromatin materials and irregular nuclear envelope. Abnormal flagella contained malformed mitochondrial derivatives and damaged axonemes in addition to the absence of accessory bodies. Bi- and multi-flagellate sperms (with two, three, and four axonemes) were the most common alterations. Sperm cells with residual bodies were also obtained. Agglutinated sperms and sperms with enlarged and/or vacuolated cytoplasm were common. Sperm abnormalities were more pronounced in males irradiated by 16 μSv/h dose rate than those irradiated by 3 μSv/h. Spermiogenesis alterations induced by irradiation of B. sulcata may be used as a monitoring model for predicting the effects of environmental radioactivity.

Keywords Tenebrionid beetles . Monitoring model . Radiation effects . Ultrastructure alterations . Spermiogenesis

Responsible editor: Philippe Garrigues * Wafaa Osman [email protected] 1

Department of Zoology, Faculty of Science, Alexandria University, Alexandria 21568, Egypt

2

Department of Physics, Faculty of Science, Alexandria University, Alexandria 21568, Egypt

Introduction Radiation is energy traveling through space and is present in our life in a variety of forms. Cosmic radiation, terrestrial radiation, and internal radiation in our bodies are natural backgrounds of radiation that are inevitably present in our environments. Sunshine is the most familiar form of radiation (Hall 1984). Growing attention has been devoted to the effects of natural radioactivity on human health. Any release of radioactivity to the environment must be monitored for environmental protection. Moreover, natural radioactivity in soil must be measured to determine the amount of change in the natural background activity with time as a result of any radioactive release (Sroor et al. 2001). Gamma radiations offer solutions for the serious environmental problems caused by the overuse of pesticides. The future trends in controlling insect pests would be directed to biological control methods as sterile insect techniques (SIT) treatments (Dyck et al. 2005; Ayvaz and Yilmaz 2015) by irradiating the insects at doses sufficiently high to produce the desired effects. The technique is used to control insect pests by producing either mortality or sterility in the insects (Prabhakumary et al. 2011; Sengupta 2013; Mohamed et al. 2014). High doses of gamma radiation could inactivate insect sperms, producing, at least, dominant lethal mutations in cells and could immediately kill insects while low doses might affect normal growth, development, and reproduction of the insects by decreasing sperm quality and production, causing sterility (Hallman 2003; Helinski et al. 2009). This technique that was applied as a part of an area-wide integrated pestmanagement approach (AW-IPM) offers considerable potential and has been used with great success against major pests of agricultural importance. Insect irradiation is safe and reliable when the established safety and quality-assurance

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guidelines are followed. On the other hand, low doses of radiation may induce adaptive responses and have beneficial effects (Mitchel 2006; Koana and Tsujimura 2010). Biomonitoring is the use of biological variables to survey the environment (Gerhardt 2000). An indicator may be used for biomonitoring at many levels of organization, ranging from suborganismal (i.e., gene, cell, tissue) and organismal to population, community, and even ecosystem levels (Niemi and McDonald 2004). Tenebrionid insects (Coleoptera) are good examples for experimental study as they respond to environmental stimuli and activity rhythms maintaining themselves in favourable microhabitats most of the time (diurnal and seasonal rhythms of activity). Blaps species, among Tenebrionidae, exhibit several behavioural and morphological adaptations in relation to the environmental variability and thus represent valuable biological markers for conservation biology (De Los Santos et al. 2000; Fattorini 2006; Pardo et al. 2008). Few studies showed the effects of radiation on the ultrastructure of insects (Paoli et al. 2014; Hassan et al. 2017). However, there is no information available on using ultrastructure alterations on insects due to radiation as a biomonitoring tool. Hence, our present study was subjected to show the effects of gamma radiation on spermiogenesis of the adult ground beetle, Blaps sulcata, not only to evaluate the efficacy of radiation against insects but also to find new tools in biomonitoring by using ultrastructure alterations for evaluating environmental impact of radioactivity.

Material and methods The insect sampling Adult insects were collected from the garden of the Faculty of Science, Moharram Bey, Alexandria University, Egypt, where there is not any sign of pollution, and this site is considered as a clear environment (Osman et al. 2015). The insects, B. sulcata, aggregate in large numbers under stones and in crevices of the ground where the temperature and humidity are favorable. They were confined at the edges of the garden under shrubs and were available in early morning, so they were easily collected. They were sexed by examination of the abdomen, after which male insects were placed in jars and transferred to the laboratory. Experimental set-up In the laboratory, 30 adult male insects were divided into three groups, namely, group A, B, and C. Insects of group A (10 insects) were considered as a control group wherein they did not receive any radiation treatment and were housed at normal environmental conditions (20–25 °C). Groups B and C (10 insects each) were exposed to two different dose rates of gamma rays from Am241 source. At the end of the experiment, the

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testes of the three insect groups were dissected out of the insects for ultrastructure investigation. Irradiation procedure At the Cockcroft Laboratory of Physics Department, Faculty of Science, Alexandria University, insects of groups B and C were housed in a Perspex cage of dimensions 100 × 30 × 35 cm3 in front of Am241 source separated by 0.22-m distance from the center of the cage. Am241 decayed with a half-life of 432.7 years by emitting alpha particles having an average energy of 5.465 MeV and was accompanied by gamma rays with average energy of about 60 KeV. The source was calibrated by the National Institution of Standard (NIS) and by using track detector CR-39. The amount of radiation, or Bdose,^ received by the insect is measured in terms of the energy absorbed in the body tissue and is expressed in gray. One gray (Gy) is 1 J deposited per kilogram of mass, but when we talk about radiation effects, we therefore express the radiation as effective dose, in a unit called the sievert (Sv). At the area of exposure, Am241 gave gamma rays with an average dose rate of 3 and 16 μSv/h for groups B and C, respectively. The source was kept inside a well shielding to avoid any radiation escaping. For the exposure, a small window with a shutter is made to control the exposure period which was 8 h/day, 5 days/week for 20 days. So insects in our present research have been exposed to an accumulative dose of gamma rays (insects of group B received 2400 μSv after 20 days which is equivalent to 0.0024 Gy and insects of group C received 12,800 μSv after 20 days equivalent to 0.0128 Gy). For more caution, the cages were dismissed at time of unexposure to make sure it did not receive any radiations. Ultrastructure investigation For ultrastructure study, the testes of adult male insects of the three studied groups were dissected out and fixed by immersing them immediately in 4% formaldehyde and 1% gluteraldehyde (4F1G) in a phosphate buffer solution (pH 7.2) at 40 °C for 3 h. Specimens were then post fixed in 2% osmium tetroxide (OsO4) in the same buffer for 2 h. Samples were washed in the buffer and dehydrated at 40 °C through a graded series of ethanol, then they were embedded in Epon-Araldite mixture in labeled beam capsules. Ultra-thin sections (0.06–0.07 μm thick) were cut from the testes of the three studied insect groups for the transmission electron microscope (TEM). These ultrathin sections were of either pale gold or silver interference color and were picked upon 200-mesh naked copper grids. Grids were double-stained with uranyle for half an hour

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and lead citrate for 20–30 min (Reynolds 1963). Scoping and photographing the grids were achieved by Joel 100 CX TEM, at E.M. Unit, Faculty of Science, Alexandria University, Egypt.

Results Ultrastructure patterns in spermiogenesis of group A (control group) In the present study, normal mature sperm of group A appeared with a head and a flagellum. The head contained large a nucleus with a conical acrosome covering the anterior of the nucleus that appeared compact in an electron-dense material (Fig. 1a). The tail or flagellum contained an axoneme that elongated from the centriole during sperm formation (early spermatid) (Fig. 1b) and two mitochondrial derivatives that further extended to the posterior (Fig. 1b). The mitochondrial derivatives are elongated structures that ran parallel to the axoneme (Fig. 1b, c). The flagella of normal spermatozoa contained also two small accessory bodies located laterally between the axoneme and the two mitochondrial derivatives and appeared round in transverse sections (Fig. 1d). Mature spermatozoa had condensed nuclei. During chromatin condensation, the nucleus diminished in size and became a small rod between thread-like acrosome and a long flagellum. In transverse sections of the head region (Fig. 1e), the nucleus appeared round with double membrane and with normal condensation of chromatin materials. Transverse sections through the flagella of normal spermatozoa showed that the axoneme consists of nine accessory tubules in a circle around the doublets (Fig. 1d). The axoneme is hence described as having the familiar 9 + 9 + 2 pattern of microtubules (9, outer row of single microtubules; +9, intermediate row of double microtubules; +2, central single microtubules). No alterations were observed in the spermiogenesis of group A during ultrastructure examination. Ultrastructure patterns in spermiogenesis of group B (irradiated by 3 μSv/h) Ultrastructure examination of the sperms of group B pointed out many alterations of the general architecture. The nucleus of mature sperm showed irregular nuclear envelope (Fig. 2a). Malformed bi-flagellate sperms were observed in longitudinal (Fig. 2b) and in transverse sections (Fig. 2b, c), having two axonemes. They appeared with irregular and, sometimes, rupture of plasma membrane in transverse sections (Fig. 2c). Malformed mitochondrial derivatives (disintegrated and appeared with electron dense matrices) were also observed in transverse sections (Fig. 2a–c) in addition to agglutinated

Fig. 1 Transmission electron micrographs in spermiogenesis of normal B. sulcata males of group A showing a L.S. in the head of a normal mature sperm with large nucleus (N) and with anterior conical acrosome (ac); b L.S. in a normal spermatid with nucleus (N), axoneme (ax) elongated from the centriole (ce), and mitochondrial derivatives (md) extend to the posterior; c L.S. in the flagella of normal mature sperms with elongated mitochondrial derivatives (md) that ran parallel to the axoneme (ax); d T.S. in the flagella of normal mature sperms with axoneme (ax) having 9 + 9 + 2 arrangement of microtubules, two small rounded accessory bodies (ab), and two equal mitochondrial derivatives (md); e T.S. in the head of normal mature sperms (arrows) with round nuclei (N) and with condensed chromatin, T.S. in the flagella of normal mature sperms (arrowheads)

sperms (Fig. 2c). Some cells appeared empty without any structures (Fig. 2c). Ultrastructure patterns in spermiogenesis of group C (irradiated by 16 μSv/h) Transmission electron micrographs of sperms of group C showed that the abnormalities were more pronounced.


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became smaller in size with shrinkage wall (Fig. 3a), as compared with those of group A (Fig. 1d). Nuclei with uncondensed chromatin materials were observed in longitudinal (Fig. 3b, c) and in transverse sections (Fig. 3e). Irregular nuclear envelope appeared in longitudinal (Fig. 3d) and in transverse sections (Fig. 3e). Agglutinated sperms were common in transverse (Fig. 3e) and in longitudinal sections (Fig. 4a). In some mature sperm bundles, groups of double-tailed sperms with two axonemes appeared (Fig. 4b, c). Some of the cells fuse to form abnormal structures with multiple axial filaments and malformed mitochondrial derivatives. Hence, large numbers of bi-, tri-, and tetra-flagellate sperms with two, three, and four axonemes, respectively, were observed (Figs. 4d, e and 5e). The most obvious alterations were in the flagella of mature sperms where they appeared with degenerated (damaged) mitochondrial derivatives (Figs. 4b, c and 5a, b). Ultrastructure assessment of sperms of group C revealed the incidence of damaged axoneme (Fig. 5a) and agglutinated sperms (Fig. 5b). Some cells appeared without accessory bodies (Fig. 5a, b), while others appeared without axoneme or accessory bodies but with only two malformed mitochondrial derivatives (Fig. 5b). A characteristic alteration that appeared only in group C, not in either group A or B, is the enlarged cytoplasm around some sperms (Fig. 5c, d). Appearance of large vacuoles around some cells is another feature characterizing only group C (Fig. 5e). An important observation was in the cytoplasm of some cysts showing conspicuous vesicles similar to residual bodies (Figs. 4d, e and 5c).

Discussion

Fig. 2 Transmission electron micrographs in spermiogenesis of irradiated B. sulcata males of group B showing a T.S. in the head of an abnormal sperm (arrow) with nucleus (N) and with irregular nuclear envelop (Ne), T.S. in the flagella of abnormal mature sperms (arrowheads) with malformed mitochondrial derivatives (md); b malformed bi-flagellate sperm in longitudinal section (arrow) and in transverse section (arrowhead); c T.S. in malformed bi-flagellate sperms (arrows) with malformed mitochondrial derivatives (md) and with rupture of plasma membrane (arrowhead), agglutinated sperms (double head arrows), and empty cells (ec)

Although some spermatids became spermatozoa, many of them did not complete development and their numbers decreased and

In insects, susceptibility to gamma irradiation is known to be varied between and within developmental stages of the same species (Cogburn et al. 1973; Burditt et al. 1989; Ozyardimci et al. 2006). For many holometabolous species, a good time for irradiation is late in the pupal stage, or early in the adult stage, when germ tissues have formed (Anwar et al. 1971; Ohinata et al. 1971, 1977, 1978). In Coleoptera, pupal or adult irradiation has rather similar effects and histological damage is expected to be similar (Tilton and Brower 1983). However, the findings of Andreasen and Curtis (2005) emphasize the need to radiosterilize males as adults in order to minimize the fitness cost. Hence, adult insects were chosen for the present investigation. Insect spermiogenesis is a post-meiotic process where spermatids undergo a series of modifications and morphological alterations ended by the formation of highly differentiated spermatozoa which are able to fertilize oocytes (de Almeida and Cruz-Landim 2000; Fernandes et al. 2001). Morphological characteristics of the reproductive system,

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Fig. 3 Transmission electron micrographs in spermiogenesis of irradiated B. sulcata males of group C showing a T.S. in the flagella of shrinkage sperms; b, c L.S. in the head of an abnormal sperm with nucleus (N) having uncondensed chromatin (arrows), note multi-flagellate sperm with malformed mitochondrial derivatives (md) in c (arrowhead); d L.S. in an abnormal sperm with irregular nuclear envelop (Ne); e T.S. in the head of mature sperms with nuclei (N) having uncondensed chromatin (arrows) and irregular nuclear envelop (Ne), note also agglutinated sperms (double head arrows)

spermatogenesis, and spermiogenesis of many tenebrionid beetles have been investigated (Wolf and Joshi 1995; de Almeida and Cruz-Landim 2000; Dias et al. 2012, 2013a, b, 2015). The structure of mature spermatozoa of B. sulcata showed typical coleopteran characteristics, with the head having an acrosome and an elongated nucleus, and a flagellum with an axoneme having 9 + 9 + 2 microtubule pattern, with two mitochondrial derivatives, and with two equal-sized accessory bodies (Alzahrani et al. 2013; Khaled et al. 2015) confirming the phylogenetic affinity of the tenebrionid group with other coleopteran groups studied by Dallai et al. (1998) and Paoli et al. (2014). These characters are shared by other tenebrionids (e.g., Baccetti et al. 1973; Dias et al. 2015) except that B. sulcata showed two equal-sized mitochondrial derivatives. As it is the first time that spermiogenesis and mature spermatozoa of this insect were studied, so our data could be useful for further phylogenetic analyses.

Germinal cells are particularly radiosensitive, and because of their active division and differentiation, express radiation damage quickly (Banu et al. 2006). The reproductive organs of mature insects are sensitive to gamma radiation more than other tissues (Tilton and Brower 1983). Sperm nuclei with abnormal chromatin condensation and irregular nuclear envelope showed similarities to those reported by Nardi et al. (2013). Sperm nucleus development is characterized by chromatin conversion from a dispersed to a very condensed state. During spermiogenesis, the histones complexed to DNA are exchanged for specific arginine-rich basic proteins, the protamines, which are responsible for a high degree of condensation in the chromatin of the nucleus (McMaster-Kaye and Kaye 1976; Mello 1987; Quagio-Grassiotto and Dolder 1988). Radiation may affect the regulation of histone exchange causing abnormal chromatin condensation. It is well-known that condensation of the sperm nucleus chromatin is characteristic for


Fig. 4 Transmission electron micrographs in spermiogenesis of irradiated B. sulcata males of group C showing a L.S. in malformed agglutinated sperms (arrows); b, c T.S. in malformed bi-flagellate sperms with two axonemes (ax) and malformed mitochondrial derivatives (md); d T.S. in abnormal bi-, tri-, and tetra-flagellate sperms, some cysts having conspicuous vesicles similar to residual bodies (rb); e higher magnification of d

the differentiation stage and species (Cruz-Landim and Ferreira 1976). Although the different chromatin arrangement patterns may reflect specific intra-nuclear mechanisms (Báo and Hamú 1993), the obtained uncondensed

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chromatin may be due to the effect of radiation which needs further studies. In the present study, bi- and multi-flagellate spermatozoa with multiple axonemes and/or mitochondrial derivatives were observed and were more pronounced in group C irradiated by the higher dose, also observed by Paoli et al. (2014). Many investigations related the appearance of these malformed spermatozoa to the increase of cell fusion during gonial mitosis. A complex system of cytoplasmic bridges connects the germ cells throughout spermatogenesis (Suja et al. 1987, 1989; Wolf 1997).The cytoplasmic bridges fuse the cells with one another. Moreover, supernumerary chromosomes (B chromosomes), if present, would increase the incidence of cell fusion during spermatogenesis (Suja et al. 1987, 1989; Alzahrani et al. 2013). Another explanation for these alterations was reported by LaChance and Olstad (1988), that spermatogonia or spermatocytes, under the effect of radiation, failed to undergo cytokinesis, and organelles remained within undivided cytoplasmic masses. The axoneme, the two mitochondria derivatives, and the two accessory bodies are the distinct elements in the sperm flagella of B. sulcata, and any alterations in these organelles could affect sperm motility and may, consequently, affect the reproductive capacity of the male insects. Sperm axoneme, as a motile organelle (Werner and Simmons 2008), was found to be sensitive to gamma radiation, and the alterations that occurred after irradiation could affect the sperms motility and the male reproductive capacity (Hassan et al. 2017). Mitochondrial derivatives of insect sperm also participate in the control and regulation of flagellar movement by the storage and release of the energy required for flagellar motility (Phillips 1970; Yasuzumi 1974; Tokuyasu 1975). Hence, the presence of malformed mitochondrial derivatives in sperms of irradiated insects could affect flagellar motility and reproductive capacity. Absence of accessory bodies could also affect sperm motility as they contribute in supplying the energy required for the maintenance of the large waves of sperm movement due to their intense UTPase and ATPase activities (Baccetti et al. 1973). Another alteration that may affect sperm motility is the agglutination of sperms. Healthy sperms must not stick together. As this alteration was observed in groups B and C and was not observed in group A, hence, we attribute it to the effect of radiation. Ultrastructure alterations in irradiated testes of B. sulcata confirmed the inhibitory effect of gamma radiation at the cellular level of the insects, although it contradicts with the study of Paoli et al. (2014) on curculionid insects. They could not obtain any ultrastructural differences between non-irradiated and irradiated sperms except for some abnormalities in maturing spermatids that showed supernumerary

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Fig. 5 Transmission electron micrographs in spermiogenesis of irradiated B. sulcata males of group C showing a, b T.S. in the flagella of abnormal mature sperms (arrows) without accessory bodies, with degenerated mitochondrial derivatives (md), with a degenerated axoneme (ax) in a (arrowhead), and without axonemes in b, agglutinated sperms (double head arrows) in b, note the rest sections appeared normal; c, d T.S. in the flagella of abnormal mature sperms having enlarged cytoplasm (ecy) around some sperms, some cysts having conspicuous vesicles similar to residual bodies (rb) in c, sperm agglutination in d (arrow); e T.S. in the flagella of abnormal mature sperms having large vacuoles (v) around some cells, bi-flagellate sperms with two axonemes (arrows), and a tetra-flagellate sperm with four axonemes (arrowhead)

axonemes and mitochondria similar to our findings. This may be due to how susceptibility to gamma irradiation is varied between different insect families and even between and within developmental stages of the same species as we mentioned before (Cogburn et al. 1973; Burditt et al. 1989; Ozyardimci et al. 2006). In our present study, the presence of residual bodies in some sperm cysts after irradiation could be attributed to elimination of germ cells (Fernandes et al. 2001) and confirms the radio-sensitivity of these cells. Alterations due to radiation were dose-rate dependent as mentioned by Hassan et al. (2017), in which testes irradiated by 16 μSv/h dose rates displayed a well-pronounced sensitivity compared to those irradiated by 3 μSv/h. Moreover, radiation-induced abnormalities may affect the fertility and mating ability of the studied insects and could lead to genomic instability in mitotically dividing cells and interfere with the function of tissues and organs (Moskalev 2007). This transgenerational genomic instability leads to destabilization in the developmental pathways (Møller 2002) and consequently, could

adversely affect offspring development and fitness (Beasley et al. 2012). Such abnormalities seem to be irreversible and could be inherited (Bloem et al. 1999; Carpenter et al. 2009).

Conclusions The present investigation revealed, for the first time, that gamma radiation altered the structure of germ cells of B. sulcata by changing the maturation pathway during spermiogenesis. Ultrastructurally, irradiated sperms exhibited severe alterations which were dose-rate dependent. It is worth mentioning that ultrastructure changes in spermiogenesis of irradiated male B. sulcata may be further used as a monitoring model for predicting the effects of environmental radioactivity. To investigate transgenerational genomic instability of radiation, studies should be extended to the F1 progeny of this insect, which will be the subject of our future work.

Environ Sci Pollut Res (2017) 24:22102–22110 Acknowledgements The authors would like to thank Prof. Hedayet Abd Elghaphar, Department of Zoology, Faculty of Science, Alexandria University, Egypt, for proofreading the manuscript and for her valuable comments. We would also like to thank Prof. Ahmed M. El-Sabbagh, Department of Geology, Faculty of Science, Alexandria University, Egypt, for his help in creating figures. Special thanks to our reviewers for their very helpful comments and suggestions that greatly improved the manuscript.

Compliance with ethical standards The work described has not been published before. It is not under consideration for publication anywhere else. Its publication has been approved by all co-authors. The study is not split up into several parts to increase the quantity of submissions. No data have been fabricated or manipulated (including images). No data, text, or theories by others are presented as if they were the author’s own. Conflict of interest The authors declare that they have no conflict of interest.

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