Oogenesis Anomalies Induced by Heavy Metal Contamination in Two Tenebrionid Beetles (Blaps polycresta and Trachyderma hispida)

Histological and ultrastructure analysis of the ovaries of Blaps polycresta and Trachyderma hispida were performed to illuminate the effect of industrial pollution from a plastic factory located in Khorshed district, Alexandria, Egypt. Detection of heavy metals in the ovarian tissues was achieved by using X-ray microanalysis. The study revealed various anomalies in oocytes and trophocytes. These basic anomalies included exfoliation and vacuolation of follicular epithelium, vacuolated trophocyst, nuclear abnormalities and morphological changes in cytoplasmic organelles. Conclusively, the study was initiated to assess the extent of reproductive corruptions induced by industrial pollution in two insects as a biomarker of exposure which may impair reproduction in humans.

Toxic chemicals used by industries in processing and manufacturing are the main cause of industrial pollution.These toxic chemicals are released into the environment and are hazardous to human health (THOMPSON et al. 2009) and affect natural populations (OEHLMANN et al. 2009).Plastic has the potential to transfer toxic substances to the food chain if plastic debris are ingested (TEUTEN et al. 2009).Their additives can interfere with hormone function (HU et al. 2009).Some toxic chemicals in plastic such as phthalates and bisphenol-A (BPA) affect reproduction and impair development (in crustaceans, mollusks, and amphibians) (OEHLMANN et al. 2009).Hazardous emissions from plastic industries include bromide and color pigments that contain heavy metals (chromium, copper, cobalt, selenium, lead, and cadmium) (VERMA et al. 2016).Cytotoxic effects of heavy metals were proven to have an adverse effect on human health even at low concentrations (TCHOUNWOU et al. 2012).In addition, their accumulation causes reproductive and physiological oddities (BEDNARSKA & STACHOWICZ 2013;XIE et al. 2014;KHEIRALLAH et al. 2016).Insects have been used as ecological bioindicators that function as gauges of environmental alterations due to heavy metal pollution (NUMMELIN et al. 2007;AZAM et al. 2015;EL-SAMAD et al. 2015;KHEIRALLAH 2015;OSMAN et al. 2015;KHEIRALLAH et al. 2016;GHANNEM et al. 2017;OSMAN & SHONOUDA 2017;SHONOUDA & OSMAN 2018;EL-SAMAD et al. 2019).They give a quick and tactful response to the accumulation of heavy metals (CERVERA et al. 2004).The intake of heavy metals by insects could be through integument, respiration or ingestion (BALLAN-DUFRANÇAIS 2002).
Energy dispersive X-ray spectroscopy (EDX) is a very effective tool to specify the heavy metal load in tissues and for monitoring of heavy metal pollution (BISTRICKI & MUNAWAR 1982;KHEIRALLAH 2015;OSMAN & SHONOUDA 2017;SHONOUDA & OSMAN 2018).Intensification of heavy metals in insects causes cell injury and leads to pyknosis which could be investigated through histological and ultrastructure analyses (SUN et al. 2007;FONTANETTI et al. 2010;KHAN et al. 2012;KHEIRALLAH 2015;

Study sites
Insects were sampled from two sites.The reference site (site A) was the backyard of the Faculty of Science Moharram Bek, Alexandria University, Alexandria, Egypt, with cultivated plants (EL-SAMAD et al. 2015).The polluted site (site B) was a juxtaposition of the plastic factory in Khorshed district (Latitude: 31.203945,Longitude: 30.038928) which is a residential area at the eastern edge of Alexandria, Egypt.

Specimen identification
Blaps polycresta and Trachyderma hispida (Forskal, 1775) were the main coleopteran insects inhabiting the selected sites.Specimen identification was performed at the Entomology Department, Faculty of Agriculture, Alexandria University.Insects belong to Coleoptera: Tenebrionidea.
Sampling procedure 100 insects from both species were collected randomly from each site.After sexual differentiation of the specimens, about 20 females from each site and species were kept alive in domestic soil and plants in glass containers.Absolute ethanol (95%) was used to anesthetize the beetles.Dissection was performed under a dissecting microscope in a drop of Ringer's physiological solution and the ovaries were taken out from the abdominal cavity.Procedures for protection and use of laboratory animals were done in compliance with ethical guidelines.The methodology was approved by the Ethics Committee of Alexandria University (protocol approval number is 0302440).

Bioaccumulation of metals in ovarian tissues
Detection of heavy metals in ovaries was performed in un-coated specimens using a Jeol scanning electron microscope-5300 equipped with a Link-Isis energy dispersive X-ray micro- analyzer.A static spot (X500) was analyzed randomly for 110 sec.Due to the divergent distribution of trace metals, three samples of ovarian tissues (each from 3 females, with a total number of 9 insects) were analyzed from each site.The identity of each peak was assigned automatically by the SEM-EDX software.The line intensities were measured for each element in the sample and for the same elements in calibration standards of known composition.
Histological and ultrastructure analysis Histological analysis followed ANDERSON and GORDON's (1996) method of dehydration, clearing and paraffin embedding.Xylene was used as a cleaning agent.Ovaries were fixed in paraffin wax (65-60°C) and sectioned 5 ìm thick, then stained with hematoxylin and eosin.Ultrastructural assemblies for the ovaries started by the fixation in 4 F 1 G (4% formaldehyde and 1% glutaraldehyde) 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.The buffer was used to wash the samples, then they were dehydrated at 4°C through a series of ethanols.Specimens were submerged in Epon-Araldite mixture in labeled beam capsules.For semithin sections, an LKB ultramicrotome was used (5 ìm thick).Sections were mounted on a glass slide and stained with toluidine blue.Examination with the light microscope was performed to determine the orientation and the structural characteristic.Ultra-thin sections (6-7 nm thick) were cut for TEM then picked upon 200 mesh naked copper grids.Grids were stained with uranyl for half an hour and lead citrate for 20-30 min.(REYNOLDS 1963).

Statistical analysis of the data
Data were analyzed using IBM SPSS software package version 20.0.(Armonk, NY: IBM Corp) (KIRKPATRICK & FEENEY 2013).The Shapiro-Wilk test was used to verify the normality of distribution of variables.The Student t-test (SOKAL & ROHLF 1981) was used to compare the two studied groups for normally distributed quantitative variables.Significance of the obtained results was judged at the 5% level.

X-ray microanalysis
Accumulation of metals in the ovarian tissues of the two insects was determined by X-ray microprobe analysis collected from both sites.12 elements were detected by the X-ray spectra inclusive of Na, Mg, Al, P, S, K, Cd, Ca, Ni, Cu, Zn, and Pb.
In B. polycresta, a significant elevation in the percentages of Al, Cu, and Zn were noticed in the polluted site (site B) compared with those of the reference site (site A) (Table 1).High percentages of Cd and Pb in the polluted site (site B) were noticed (Table 1).In T. hispida, high and significant proportions of Al, Cu and Zn in the polluted site were observed.Moreover, Pb, Cd, and Ni were discerned in the polluted site (site B) (Table 1).Percentages of Mg, P, K and Ca were significantly lower when compared with controls of the two insects.
High percentages of heavy metals detected in the ovarian tissues of the two insects collected from the polluted site were evoked from industrial pollutant delivered to the medium.These metals cannot be broken down by an insect's metabolism (AZAM et al. 2015).Furthermore, concentrations of some heavy metals in the tissues were positively correlated with their concentrations in the soil (WAN et al. 2014;GHANNEM et al. 2018).Metals bind to metal-binding sites on the cell surface, and thus exert a toxifying mechanism that causes cell death (VENTER et al. 2017).NUMMELIN et al. (2007) andGHANNEM et al. (2016) studied the accumulation of heavy metals in different predatory insects located close to a steel factory and stated that all insect groups can be used as heavy metal indicators.Several researchers have shown that heavy metals are responsible for histological and ultrastructure anomalies (BEDNARSKA et al. 2009;KHEIRALLAH et al. 2006;TALARICO et al. 2014;KHEIRALLAH 2015;OSMAN et al. 2015;KHEIRALLAH et al. 2016;OSMAN & SHONOUDA 2017;SHONOUDA & OSMAN 2018).

Macroscopic findings
The reproductive system of female B. polycresta and T. hispida consist of a pair of telotrophic meroistic ovaries (BÜNING 1994;BÜNING 1998;BÜNING 2006;TRAUNER & BÜNING 2007;OSMAN & SHONOUDA 2017).The ovaries are composed of numerous ovarioles (Figs 1a, b).Each ovariole is composed of a germarium which contains the trophocyte (confined to the trophic chamber), and the vitellarium with immature and mature oocytes in a single row, asynchronously in development.Trophocytes which supply nutrients to oocytes are connected to pro-oocytes and oocytes by a nutritive cord (TELFER et al. 1982).Two lateral oviducts at the end of each ovary unite to form a common oviduct.The common oviduct leads to the vagina housing a spermatheca with a spermathecal gland opened in it (Figs 1a,b).A vast amount of mature oocytes in the ovaries of B. polycresta were observed.In old females, almost all ovarioles develop synchronously (NGERNSIRI et al. 2015).No morphological abnormalities were observed in the ovarian structures of the two insect species collected from the polluted site.d), in addition to the appearance of lipid and fat droplets (Fig. 4d).Our results were compatible with MOHAMED et al. (2015) and OSMAN & SHONOUDA (2017), who worked on the ovarian structure of the coleopteran insect Callosobruchus maculatus and Blaps polycresta, respectively.
The ultrastructure archetypes of insects collected from the reference site accentuated that the trophocytes appeared with a heterochromatic rounded nucleus with uniform nuclear envelope (Fig. 4a, & Fig. 5a).The cytoplasm contains cytoplasmic organelles and dense vesicles (Figs 4a, b  & Figs 5a, b) and appeared more electron lucent and granulated in T. hispida (Figs 5a,b).Lysosomes were also observed in the cytoplasm of the trophocytes in T. hispida (Fig. 5a).Follicular epithelial cells that ensheathed the vitellogenic oocyte in B. polycresta appeared with a euchromatic nucleus and regular nuclear envelope (Figs 4c, d).Rounded mitochondria, RER, SER and vesicular bodies occurred in the cytoplasm (Figs 4c, d).The plasma membrane of the follicular cells enfolded into finger-like microvilli which interlocked with the microvilli of the oocyte plasma membrane, thus forming channels.This region is called the brush border.Sometimes the ends of the long channels are filled with condensed materials and cut off forming dense granules in the ooplasm, the pinosomes (Fig. 4c).The pinosomes grow in size towards the interior of the oocyte forming a voluminous dark globule (Fig. 4c).The ooplasm was also filled with mitochondria and yolk granules.The vitellogenic oocyte in T. hispida contained a heterochromatic nucleus of the follicular cells and festoon brush border (Figs 5c, d).Cytoplasmic organelles are well recognized in the cytoplasm of the follicular epithelial cells (Figs 5c, d).Pinosomes, mitochondria, dark globules and yolk granules appeared in the ooplasm as well as fat and lipid droplets (Fig. 5c).2017), who worked on the ovarian archetypes of the beetles Callosobruchus maculatus and Blaps polycresta, respectively.Trophocytes supply RNA to the oocytes via trophic cords.At a late stage of vitellogenesis, they are broken down by the action of lysosomes (DE WILDE 1964), and their products are assimilated into oocytes to provide protein, RNA, and other materials to the oocytes (ENGELMANN 1970).In the vitellogenic oocytes, microvilli unite with those of the follicle cells indicating the active involvement of follicle cells during vitellogenesis (MOHAMED et al. 2015).The existence of pinosomes at the oocyte surface suggests the transfer of material from hemolymph through the follicle cells.Materials such as the yolk proteins, synthesized in the fat bodies and released toward the follicle cells, are then secreted into the oocyte (BRENNAN et al. 1982;ISAAC & BOWNES 1982;BUTTERWORTH et al. 1992).
Some authors have reported pinocytosis in insects (KAMEL et al. 2005;MOHAMED et al. 2015).The presence of extensive mitochondria in the oocyte surface reflect high energy production during the active transport of materials.Yolk granules resulted from the fusion of pinosomes (TELFER & SMITH 1970;KAMEL et al. 2005).Peripheral ooplasm of the oocyte is rich with microvilli, which Several histological and ultrastructure corruptions were noticed in both insects collected from the polluted site.Histopathological observations of the ovarian structure of B. polycresta showed a reduction of the trophic chamber and the vitellogenic oocyte (compare Fig. 6a, with Fig. 2a).The tropharium appeared with large vacuoles and morphologically altered trophocytes (Figs 6b, c).Thickening of the follicular epithelium, vacuolation in the epithelial cells and empty ooplasm were noticed in the vitellogenic oocyte (Fig. 6d).In T. hispida, signs of degeneration were detected in the trophocytes and in the follicular epithelial cells of the vitellogenic oocyte (Figs 7a, b, c).Also, exfoliation of the follicular wall (Fig. 7a) and an oocyte with empty ooplasm was observed (Fig. 7d).
These alterations may retard oocyte growth because of the impairment of the structure and function of trophocytes and follicular cells (OSMAN & SHONOUDA 2017).MC GEE et al. (1992) reported that vulnerable membranes are susceptible to toxic effects of xenobiotics by interacting with protein or lipid components of cell membranes.LAGISZ & LASKOWSKI (2008) stated that metal-pollution affects egg quality and unexposed offspring.
Disruptions at the subcellular level were observed in the trophocytes and the oocytes of the two insects.Abnormalities were observed in the nucleus and cytoplasmic organelles of both trophocytes and oocytes of the two insects.Trophocytes of both insects displayed irregularities of nuclear envelopes (Figs 8a, 9a) and karyolysed nucleus.Sometimes the nuclei appeared with patches of heterochromatin (Figs 9a, b) and globular inclusion bodies (Figs 9b, c).In addition, mitochondria with disintegrated cristae (Figs 9a, b), lysosomes and dense vesicles (Figs 8a, 9a, b, c) were observed.The vitellogenic oocyte in the two insects evinced with a lytic area in the follicular cells (Figs 8c,d,e,9d,e,f,g).Some cells contained a a pyknotic nucleus (Fig. 8d) and other cells appeared with karolysed ones (Figs 9e, f, g).Globular inclusion bodies were observed at the nuclear envelope (Fig. 8e).In the cytoplasm, mitochondria with lysed ma- trices and dilated smooth endoplasmic reticulum were also noted (Fig. 8e).Sometimes, the cytoplasm lacked cytoplasmic organelles (Fig. 9d).
The brush borders of the microvilli were distorted and/or attenuated (Figs 8c,d,e,9d,e,f,g).Degenerated yolk granules and lytic areas were observed in the ooplasm (Figs 8c, 9d, e, f, g).Fusion of yolk granules into patches was observed (Fig. 9g).
The results of the present study showed that tenebrionid beetles, B. polycresta, and T. hispida, living in the metal-contaminated environment have many ultrastructural alterations.The toxic effect of heavy metals leads to the accumulation of proteins and lipids which may disrupt vitellogene-sis (OEWI¥TEK 2005;BEN AHMED et al. 2010;BEN AHMED et al. 2013;KHALED et al. 2017).The majority of trophocytes and vitellogenic oocytes were affected by heavy metal pollution and underwent degenerative changes, such as cytoplasmic vacuolization and nuclear divergence.Nuclear abnormalities could be the first sign in the course of cell death.The irregularity of nuclear envelopes and the appearance of globular inclusion bodies which is a novelty in our study revealed cellular degeneration (PAZIR et al. 2011).Synthesis of membranes within the nucleoplasm leads to the formation of globular tubules (ENGEDAL et al. 1977).The appearance of lytic areas in the cytoplasm could be due to the activity of lysosomal hy- drolase (VANDENBULCKE et al. 1998).The dense vesicles which were detected in the ovarian cells of the polluted site denote the accumulation of metals in the lysosomes (LAUVERJAT et al. 1989;SUN et al. 2007).
Several authors reported that heavy metals interfere with cytoplasmic membranes and cause pathological consequences (HAWKINS et al. 1980;SEIDMAN et al. 1986;KAWAHARA et al. 1990;PAWERT et al. 1996;AU et al. 2003; KHEIRALLAH   Overall, our present study should increase the perception of environmental pollution resulting from the plastic industry, which may affect reproduction.

Conclusion
Industrialization leads to a high risk of exposure to heavy metals.Heavy metals cause many biological ramifications, particularly disturbances in reproduction.Insects are efficient biomarkers in detecting environmental pollution.An effort is needed in order to reduce the hazards resulting from the plastic industry.In our opinion, studying oogenesis alterations in a biomarker such as an insect enables a perfect explanation for reproduction retardation in humans.
Histological and ultrastructure archetypes observed in ovaries of B. polycresta and T. hispida collected from the reference and polluted sites (site A & B) In both insects collected from the reference site (site A), the histological archetypes revealed that the tropharium consists of trophocytes immersed in a loose net of interstitial cells (Figs 2a, b, c & Figs 3a, b, c) (TRAUNER & BÜNING 2007; OSMAN & SHONOUDA 2017).Trophocytes are spherical cells with a rounded nucleus, whereas the interstitial cells are small asymmetrical cells among them (Figs 2b, c & Figs 3b, c) (MOHAMED et al. 2015).The oocytes are enfolded by the follicular wall.The follicular epithelial cells are cuboidal cells with a rounded nucleus (Figs 2d, e & Figs 3a, d).The ooplasm is stacked with yolk granules and a distinctive germinal vesicle (Figs 2d, e & Figs 3a, Fig. 2. Semithin sections of the tropharium (a, b, c) and vitellogenic oocyte (d, e) of the control adult female of B. polycresta.Trophic chamber (arrow), oocyte (curved arrow), trophocytes (T), trophocyte in mitotic division (double head arrow), interstitial cell (IC), nucleus (N), follicular epithelium (FE), follicular epithelial cells (FEC), nutritive cord (NC), oocyte (OC), yolk granules (YG).

Table 1
Element percentages in ovarian tissues of B. polycresta and T. hispida collected from reference and polluted sites using energy dispersive X-ray micro-analysis (EDX) KHALED et al. 2017).Our results are in line with those of OSMAN