Midgut cells alteration in gamma-irradiated beetles (Blaps polycresta, Coleoptera: Tenebrionidae)

This study was conducted to examine the effect of gamma radiation on biological specimens. Thus, our concept is to clarify that exposure to accumulated dose of 0.2 Gy gamma rays (0.66 rad/Sec. dose rate) from Cs137 source induces cellular perturbations in the midgut epithelium of the F1 progeny of Blaps polycresta, therefore affecting nutrition and growth. Beetles were reared in laboratory conditions and the newly emerged adults were irradiated with the aforementioned dose. Histological and ultrastructure anomalies of midgut cells (digestive and regenerative cells) were observed by 72 h after radiation exposure to ensure that the cells will not return to control state. Retardation in the development of the F1 progeny was also noticed and beetles died through two weeks. In the light of these observations, biological tissue act as an indicator to the continuous exposure to environmental radiation.


Introduction
Environmental afflicts of ionizing radiation arose recently as a dominated research field. Natural radiation exposures enhanced from many sources such as mineral processing and use, phosphate fertilizer (production and use) and fossil fuel combustion (UNSCEAR, 1988). Radiation that has been derived exogenously from the environment known to induce cellular alterations in higher eukaryotes (Daly, 2012). Ionizing radiation removes electrons from the orbital shells in the tissues they penetrate (Borek et al., 1993). Experimental evidence of insects exposed to different doses of gamma rays has confirmed cellular radiation damage (Kheirallah, 2016;Kheirallah et al., 2017). Low doses of radiation may elicit a broad range of biochemical and tissue responses as compared to high doses (Feinendegen et al., 2004). Experimental researches assessed the effects of low doses of ionizing radiation on biological tissues as a reference to environmental radiation protection (Feinendegen et al., 2004;Kheirallah et al., 2017).
Our previous studies cleared the effects of high and low doses of gamma radiation on the sperm ultrastructure of B. polycresta and B. Sulcata (Kheirallah, 2016;Kheirallah et al., 2017). In the current study, we are investigating cellular alterations in the midgut of a newly hatched adult of B. polycresta induced by accumulated dose of gamma radiation. Since the midgut is the site of digestion and absorption any disruptions to its cells will inhibit these processes and converge development retardation and mortality which have been observed in this study.

Sampling procedure
Beetles were collected from a non-contaminated area, the garden of the Faculty of Science Moharram Bek, Alexandria University, Alexandria, Egypt (Osman et al., 2015) during their breeding season in August. They were transported to the laboratory and sustained alive in domestic soil and plants in glass containers and held under a day/night and temperature regime that is similar to their place of origin. Specimens were then sexed to obtain the inseminated females.

Rearing procedure
Rearing procedure followed Burakowski (1993). About five inseminated females were moved to a square aquarium measuring 10 x 20 x 10 cm and covered by a glass lid to prevent crowding. A slanting layer of wet soil, 1-4 cm thick were placed on the bottom. As the soil dries out, moistening is applied to one-third of it. Females oviposit into the wet soil and then first instar larvae appear. Larvae were then placed into a larger container 10 x 30 x 10 cm with wet soil and ant workers as a diet. Here, the larvae were fed from time to time, they pupated and adults appeared. Room temperature ranged from 27-30°C to accelerate the development. The entire development from egg to adult is 4 weeks longer. Adult development approximately takes two weeks.

Radiation Treatments (Gamma Radiation)
The irradiation process was performed using Gamma Cs-137 in 10/9/2018, at the National Center for Radiation Research and Technology (NCRRT, Cairo). The dose rate of the Radiation Unit was 0.66 rad/s. Fifty newly emerged adults of B. polycresta were divided into two groups; namely group A, group B. Beetles of group A (25 insects) were used as a control group, didn't receive radiation treatment and housed in normal environmental conditions. Beetles of group B (25 insects) were irradiated with a dose rate of 0.2 Gy. The exposure period was 8hs/day, for two weeks. At the end of the experiment, ten beetles from each group were anesthetized and dissected to remove the midgut from the alimentary canal for histological investigations. The rest of the beetles in group B maintained alive in a glass container with soil and plants to observe their development and record mortality.

Dissection Procedures
Beetles were dissected under a dissecting microscope in a drop of Ringer's physiological solution on a wax-fixed Petri dish. Forceps were used to open the abdominal cavity, the alimentary canal was taken out and the midgut was cutoff.

Histological and Ultrastructure Preparations
The histological technique followed Anderson and Gordon (1996) methods of dehydration, clearing, and paraffin embedding. The clearing agent was the xylene. Fixation of the midgut was in paraffin wax (65-60 °C) and 5μm thick sections were stained with hematoxylin and eosin.
In ultrastructure assembly, midgut was fixed in 4 F 1 G in phosphate buffer solution (pH 7.2) at 4 °C for 3 hours and post-fixed in 2% OsO 4 in the same buffer for two hours. Samples were washed in the buffer and dehydrated at 4 °C through a series of ethanol. Specimens were submerged in Epon-Araldite mixture in labeled beam capsules. LKB ultramicrotome was used (1 µm thick) for semithin sections. Sections were mounted on a glass slide, stained with toluidine blue and examined with the light microscope. Ultrathin sections (0.06-0.07 µm thick) were cut for TEM and picked upon 200 mesh naked copper grids. Grids were stained with uranyl acetate for half an hour and lead citrate for 20-30 min.

Histological and ultrastructure observations of the control group's midgut (group A)
Histologically, the midgut of B. polycresta is surrounded by muscle layers, outer longitudinal and inner circular (Figure 1a and b). Two types of cells were distinguished in the midgut epithelium: columnar digestive cells with a brush border of microvilli, facing the gut lumen and regenerative cells which occur in "nidi" (Figure 1a-c).
In the electron micrographs, the midgut cells showed normal nuclei and cytoplasmic organelles. The regenerative cells evinced with an oval nucleus, patches of heterochromatin and a well-defined nuclear envelope (Figure 2a-c). In the cytoplasm, normally distributed mitochondria, rough and smooth endoplasmic reticulum and free ribosomes were observed (Figure 2 a-c). Septate junctions, the desmosomes were found between neighboring epithelial cells (Figure 2a-d). The columnar digestive cells possess numerous mitochondria, glycogen granules, lysosomes and free ribosomes (Figure 2a, d). Their brush border has a uniformly distributed microvilli (Figure 1a, d).

Histological and ultrastructure observations of the irradiated group's midgut (group B)
Histological disruption of the midgut epithelium encompassed sever vacuolations, dense vesicles, distorted microvilli (Figure 3a-d), abnormally looking regenerative cells (Figure 3b-d) and increased numbers of lysosomal vesicles (Figure 3b).  Electron micrographs revealed numerous anomalies in the regenerative and digestives cells. Nuclear divergent included: abnormal chromatin condensation, tubular and globular inclusions which are signs of necrosis, and intended nuclear envelope (Figures 4a-i). Apoptotic cells were also observed which resulted from nuclear and DNA fragmentations (Figures 4b, h). The cytoplasm exhibited severe vacuolation (Figures 4a, b, c, e, f, h, i) and many alterations in the cytoplasmic organelles were noted. These alterations included: swollen mitochondria (Figures 4b, c, d), dilated rough and smooth endoplasmic reticulum (Figure 4d), myelin figures (Figure 4g) electron-dense vesicles, and secretory vesicles (Figure 4c). Disruption of the brush border of the digestive cells was pronounced (Figure 4a).

Development and mortality observations in the irradiated group (group B)
Beetles in the irradiated group failed to develop further. No growing in their size was observed compared to the mature adult beetles. Non-developing insects survive for almost two weeks. 75% mortality was reported at the beginning of the third week. By the end of the third week, 100% mortality was achieved.

Discussion
Exposure of biological systems to different doses of ionizing radiation has been found to induce tissue and cell effects (Sugahara et al., 1992;UNSCEAR, 1994;Acad'emie des Sciences, 1995;Feinendegen, et al., 1999;Feinendegen and Neumann, 2000;Kheirallah, 2016;Kheirallah et al., 2017). These responses involve the disruption of DNA integrity and cellular damage including necrosis and apoptosis as a result of the formation of intracellular free radicals (Schmid and Schrader, 2007;Feinendegen et al., 2004;Simone et al., 2009;Faraj et al., 2011;Daly, 2012;Mohamed et al., 2016). The type and expanse of the damage vary with species, cell type, cell cycle, and metabolism (Alberts et al., 1994;Hall, 2000). This is consistent with the current results as 0.2 Gy of gamma radiation evokes midgut tissue and its cells damage. Feinendegen et al., (2004) stated that conflicting cellular response may be related to the hits of the average micro doses in an exposed micro masses as well as to different radiation qualities. It was found that 0-8 Gy of gamma irradiation induces metabolic changes in animal models, such as mice (Lanz et al., 2009;Tyburski et al., 2008;Tyburski et al., 2009), rats (Johnson et al., 2011), and nonhuman primates   (Johnson et al., 2012). Also, Faraj et al. (2011) found that low doses of Gamma Rays (0.25, 0.5, 1, 2, 4 Gy) caused DNA damage in Human Cells.
Histological and ultrastructure examination in the present study were observed after 72 h from radiation exposure to quit cell's adaptive protection against an array of exogenous attack (Kwon et al., 2014). Our major observed anomalies in the midgut epithelium of B. polycresta were in the nucleus of both the regenerative and the digestive cells. These anomalies include abnormal chromatin clumping, globular and tubular inclusions in the nucleoplasm, and indentation of the nuclear envelopes. Grewal and Jia (2007) suggested that chromatin clumping reduce transcription. Our results agreed with Pazir et al. (2011) who observed nuclear inclusions in the nuclei of shrimp infected with two viral diseases. They figured out that these inclusion bodies were the first sign in cellular degeneration and resulted in pyknosis. Engedal et al. (1977) stated that their formation arose by the unification of membranes in the nucleoplasm. Furthermore, the course of cell death was assigned by the indentation of the nuclear envelope (Trump et al., 1997;Pazir et al., 2011).
Another alteration which has been observed in the current study was the severe vacuolation in the cytoplasm due to the action of acid hydrolases that has been released from the lysosomes (Vandenbulcke et al., 1998;Polidori et al., 2018) which is accompanied by changes in the PH of the cytosol. Moreover, alterations in cytoplasmic organelles such as swallow of the mitochondria and dilation of the rough and smooth endoplasmic reticulum may be a result of the radiation which disrupts cytoplasmic membranes as reported earlier by Kheirallah (2016) and Kheirallah et al. (2017). Radiation induces mitochondrial malformations which lead to disruption in energy production (Coggins, 1973;Mahomud and Shoman, 2009;Kheirallah, 2016;Ibrahim et al., 2017;Kheirallah et al., 2017). The presence of myelin figures and electron dense vesicles in our electron micrographs may be attributed to the accumulation of lipoprotein macromolecules. Ibrahim et al. (2017) observed myelin bodies in the testes of the F1 progeny of irradiated parental males of cowpea weevil, Callosobruchus maculatus and reported that these myelin bodies are fatty insulating substance. It was obvious from our results that gamma irradiation promotes cell killing (necrosis and /or apoptosis) which is in agreement with Shinomiya et al. (2000) who reported that low-dose irradiation, 5 Gy X-ray, induces post-mitotic apoptosis in U937 cells. Additionally, the distortion of the brush border microvilli which was seen in our preparations may be responsible for the cell damages. The cessation of the cytoskeleton of the microvilli leads to the protrusion of the cytoplasmic substances into the midgut lumen (Seidman et al., 1986). These findings are in conformance with Stiles et al. (1989) worked on tsetse fly (Glossina spp.) populations subjected to 130 Gy gamma radiation. They reported that the first sign of damage was the deterioration of the microvillous border that resulted in cell degeneration. These compulsive changes in the cells may upset the normal physiology of insects (Rawi et al., 2011).
Our histological and ultrastructure investigations have confirmed that gamma irradiation has an inhibitory effect at cellular at subcellular levels and leads to obstruction in the development of the F1 progeny and finally to their death. Insect development is powered by hormones (Lee, 2012) which may be arrested due to the action of ionizing radiation (Mansour, 1987). Radiation disturbs the function of tissues and organs and produces anomalies (Wong et al., 2003;Moskalev, 2007). These anomalies may interrupt the developmental pathways (Møller, 2002;Natarajan, 2006). It also has an opposed effect on the development and health of the offspring (Beasley et al., 2012). Prabhakumary et al. (2011) reported that higher doses of gamma irradiation caused mortality to Tribolium castaneum while lower doses caused inhibition of development and sterility of the surviving insects. Aye et al. (2008) observed the inhibitory effects of increasing doses from 0.1 to 1.0 KGy gamma irradiation on the development and reproduction of Plodia interpunctella. Our previous recommendation (Kheirallah et al. 2017) pointed for further investigations on the effect of gamma radiation on the F1progeny. It is worth to mention that this is the first study that reported the inhibitory effect of accumulated low dose of gamma irradiation on the midgut structure, development and survival of the F1 progeny of B. polycresta.

Conclusion
The study validates the inhibitory effect of accumulated low dose of gamma irradiation on the F1 progeny of B. polycresta. It is also a good model to detect the ramifications of gamma radiation in biological specimens and can be utilized to speculate the consequences that may result from the exposure to environmental radiation.