Ovaries and testes of Lithobius forficatus (Myriapoda, Chilopoda) react differently to the presence of cadmium in the environment

Proper reproduction depends on properly functioning gonads (ovaries and testes). Many xenobiotics, including heavy metals, can cause changes in somatic and germ line cells, thus damaging the reproductive capacity. The aim of this study was to investigate the effect of the heavy metal cadmium on the gonads, including germ line and somatic cells. It is important to determine whether cell death processes are triggered in both types of cells in the gonads, and which gonads are more sensitive to the presence of cadmium in the environment. The research was conducted on the soil-dwelling arthropod Lithobius forficatus (Myriapoda, Chilopoda), which is common for European fauna. Animals were cultured in soil supplemented with Cd for different periods (short- and long-term treatment). Gonads were isolated and prepared for qualitative and quantitative analysis, which enabled us to describe all changes which appeared after both the short- and long-term cadmium treatment. The results of our study showed that cadmium affects the structure and ultrastructure of both gonads in soil-dwelling organisms including the activation of cell death processes. However, the male germ line cells are more sensitive to cadmium than female germ line cells. We also observed that germ line cells are protected by the somatic cells of both gonads.

www.nature.com/scientificreports/ Flow cytometry-quantitative analysis. Preparation of cell suspension. The dissected organs (testes and ovaries) isolated from specimens of each experimental group (Table 1) were crushed mechanically (Bead Bug microtube homogenizer) and suspended in 100 μL of PBS (pH 7.4). Then, the organs were homogenized and centrifuged as described in our previous paper 30 . The cell suspension was used for the flow cytometry according to the methods described below.
Viability assessment of gonad cells. Quantitative measures of viable, early, and late apoptotic and necrotic cells in ovaries and testis were obtained with the Annexin V-FITC (fluorescein isothiocyanate) Apoptosis Detection Kit (Abcam, № ab14085). This method is used to detect the early stages of apoptosis when translocation of phosphatidylserine (PS) groups from the inner to the outer leaflet of the plasma membrane occurs. Green fluorescence origins form cells bounded to the FITC-labeled Annexin V, while the red fluorescence origins form propidium iodide (PI). Thus, the distinction between necrotic cells (Annexin V-FITC-/PI +) and apoptotic cells (early apoptotic cells: Annexin V-FITC + /PI-; late apoptotic cells: Annexin V-FITC + /PI +) was enabled. Labe-

Results
Both the ovary and the testis in Lithobius forficatus are unpaired organs located on the dorsal side of the body. The ovary is prolonged anteriorly as a terminal filament, while its posterior end continues into a short oviduct. The testis is a very long tube-shape structure that forms two loops. Its anterior part extends into a terminal filament while the posterior part ends in a short vas deferens.
Ultrastructural changes in ovaries of L. forficatus exposed to cadmium. Ovary in control animals. The ovary of the control animals is a sack-like structure filled with female germ cells accompanied by somatic cells (Fig. 2A, B). The ovary wall is composed of cubical or flattened somatic cells suspended by the basal lamina (Fig. 2C). The cytoplasm of these cells is rich in cisterns of the rough endoplasmic reticulum, mitochondria, and ribosomes (Fig. 2C). Sporadically autophagosomes and autolysosomes could be observed in somatic cells of the ovary wall (Fig. 2C). Each germ cell located in the ovary lumen is surrounded by a single layer of the flat somatic cells that rests on the basal lamina (Fig. 2D). The nuclei of these cells are flattened. Short cisterns of the rough endoplasmic reticulum, mitochondria, ribosomes, and Golgi complexes could be observed in their cytoplasm. The oolemma that surrounded the oocytes forms short microvilli (Fig. 2D). The cytoplasm of young oocytes (previtellogenic oocytes) is rich in ribosomes, mitochondria, short cisterns of the rough endoplasmic reticulum and Golgi complexes (Fig. 2E). In the cytoplasm of the vitellogenic oocytes the yolk material in the form of spheres of different electron density is accumulated (Fig. 2F,G). Sporadically the autophagosomes occurred in germ-line cells (Fig. 2F).
Ovary in animals after short-term Cd treatment (12Cd). The ovary of the animals from the 12Cd group has the same shape as in the control group and retains its integrity (Fig. 3A, B). The entire cytoplasm of the majority of ovary wall cells is electron lucent with a small number of organelles (Fig. 3C, D). Cisterns of rough endoplasmic reticulum are short and their amount is lower than in the control group (Fig. 3C, D). Some mitochondria lose cristae and their matrix vacuolizes (Fig. 3D). Autophagosomes, autolysosomes and single spherites are visible in the cell cytoplasm in the ovary wall (Fig. 3C, D). Similar changes are observed in the somatic cells surrounding oocytes (Fig. 3E). Numerous autophagosomes, autolysosomes and degenerating mitochondria can be observed in these cells. Moreover, some of these cells show the necrosis. Their cytoplasm is electron lucent and the number of organelles is reduced (Fig. 3E). The oocytes show the intensification of autophagy; thus, numerous autophagic structures appear in their cytoplasm (Fig. 3F, G). Moreover, some mitochondria degenerate. They lose cristae and their matrix vacuolizes or becomes electron lucent (Fig. 3F, H). Single spherites appear in the cytoplasm (Fig. 3G). The yolk material is accumulated in the cytoplasm of vitellogenic oocytes. The yolk spheres are slightly smaller compared to those observed in oocytes in the control group.
Ovary in animals after long-term Cd treatment (45Cd). The ovary of the animals from the 45Cd group also retains its integrity and is filled with germ cells accompanied by somatic cells (Fig. 4A-B). The ovary wall cells look like those observed in the 12Cd group; however, only a few mitochondria show ultrastructural changes (Fig. 4C, E). Several of the somatic cells surrounding oocytes show signs of necrosis. They possess electron lucent cytoplasm and a reduced number of organelles (Fig. 4C, E). The second fraction of these cells has similar ultrastructure to that observed in the control group (Fig. 4C, D). Few autophagosomes and autolysosomes are observed in all types of somatic ovarian cells (Fig. 4C, E). Autophagosomes and autolysosomes observed in the cytoplasm of the oocytes are less numerous than in the 12Cd group (Fig. 4F, G), and moreover more intense autophagy occurs in previtellogenic oocytes (Fig. 4G). Only a few mitochondria show signs of degeneration, whereas most of them have the typical ultrastructure of the control group (Fig. 4F, G). Numerous spherites appear in the cytoplasm of vitellogenic oocytes (Fig. 4H). We did not observe a difference in the accumulation of yolk material between Cd12 and 45Cd groups.
Ultrastructural changes in testis of L. forficatus exposed to cadmium. Testis in control animals. The testis of the control animals is a long, tube-like structure filled with male germ cells (Fig. 5A) www.nature.com/scientificreports/  www.nature.com/scientificreports/  www.nature.com/scientificreports/ testis wall is composed of an internal layer of epithelial cells, circular muscles, thick connective tissue layers and an outer epithelial layer (Fig. 5B). The cytoplasm of epithelial cells is rich in ribosomes, mitochondria, and short www.nature.com/scientificreports/ cisterns of the rough endoplasmic reticulum (Fig. 5B). The spermatocytes have quite elongated egg-like shape (Fig. 5A). Their cytoplasm contains numerous mitochondria, ribosomes, short cisterns of the rough endoplasmic reticulum, and many highly curved Golgi complexes (Fig. 5B, C). In this stage mitochondria are distributed evenly throughout the cell. However, in elongated spermatids they are in the tail region close to the cell membrane forming the sheath of the axial filament (Fig. 5D). In the spermatid, microtubules form an axoneme that is surrounded by additional microtubules (microtubular sheet) that do not adhere to the axoneme (Fig. 5D).
Testis in animals after short-term Cd treatment (12Cd). The testis of the animals from the 12Cd group retains its integrity and is filled with germ cells (Fig. 6A). Epithelial cells of the testis wall possess numerous vacuoles, autophagosomes and autolysosomes (Fig. 6B). Some mitochondria degenerate and lose cristae, and their matrix become electron lucent. Some autophagosomes, autolysosomes and vacuoles appear in the cytoplasm of spermatocytes. Sparse degenerating mitochondria can be observed in these cells (Fig. 6C). In the spermatids, numerous mitochondria show signs of degeneration. They partially lose their cristae, and vacuoles or lamellar bodies appear inside them (Fig. 6D, E). Some cisterns of the Golgi complexes inflate (Fig. 6C). Moreover, autophagosomes and autolysosomes are observed in the cytoplasm of spermatids (Fig. 6E). There are no disturbances in the structure of the axoneme but small gaps in the microtubular sheet are observed (Fig. 6E).
Testis in animals after long-term Cd treatment (45Cd). The testis of the animals from the 45Cd group also retains its integrity and is filled with germ cells (Fig. 7A). Some of the testis wall epithelial cells show signs of www.nature.com/scientificreports/ necrosis. They possess electron lucent cytoplasm and a reduced number of organelles (Fig. 7B). Autophagosomes and autolysosomes appear in their cytoplasm (Fig. 7B). Ultrastructure of the spermatocytes is like that observed in the control group, but more autophagosomes, autolysosomes and few structurally altered mitochondria appear in the 45Cd group (Fig. 7C). Moreover, the Golgi complexes are less developed than in control and 12Cd groups and some of their cisterns inflate (Fig. 7C). In the cytoplasm of spermatids numerous degenerating mitochondria could be observed. They show similar changes to those observed in the mitochondria of the 12Cd spermatids (Fig. 7D). Single autophagosomes and vacuoles are also observed (Fig. 7D). No disturbances in the structure of the axoneme are observed while the microtubular sheet is damaged and its microtubules adhere to the axoneme (Fig. 7D).
Autophagy in gonads of L. forficatus exposed to cadmium-qualitive analysis. Qualitative analysis using LysoTracker Red showed that the signals from acid organelles (autolysosomes, lysosomes) were Viability assessment of gonads in L. forficatus exposed to cadmium. The quantitative analysis using flow cytometry revealed that more than a two-fold and nearly a three-fold increase in the number of early apoptotic cells was observed in the ovaries of individuals treated with cadmium for a period of 12 days (p = 0.035) and 45 days (p = 0.0003), respectively (Fig. 9A). The degree of severity of late apoptosis and necrosis in this organ of females was similar in all periods of cadmium treatment and like the control. Regardless of the time of exposure to cadmium in males there was observed an over six-fold increase in the percentage of early apoptotic cells (12Cd, p = 0.005; 45Cd, p = 0.004) and nearly threefold increase in the percentage of necrotic cells (12Cd, p = 0.001; 45Cd, p = 0.04) in testis, compared to the value of this parameter in individuals from the control group (Fig. 9B). The percentage of late apoptotic cells in gonads of males from 12 and 45Cd groups was similar as in the control group. Only after 12 days of exposure to cadmium were there statistically significant intergender differences in the percentage of early apoptotic cells (p = 0.002) and necrotic cells (p = 0.006) (Fig. 9A, B). The qualitative analysis using confocal microscopy showed that the signals connected with the DNA fragmentation (the feature of apoptosis) in both gonads were stronger in specimens exposed to cadmium for 12 and 45 days compared to the control group. It enabled the somatic and germ cells to be distinguished. In animals from the control group, some of the apoptotic signals originated from the somatic and germ-line cells in testes, while in ovaries we managed to detect the apoptosis of the somatic cells (Fig. 9C, F). In animals from 12Cd experimental group we managed to detect the increase of signals emitted by apoptotic cells in both gonads (Fig. 9D, G). In animals exposed to cadmium for 45 days (45Cd), strong apoptotic signals were still emitted by both the somatic and germ-line cells in testes (Fig. 9H). The apoptosis concerned only somatic cells in ovaries of animals treated for 45 days with cadmium (Fig. 9E). www.nature.com/scientificreports/ ATP level and ADP to ATP ratio in gonads of L. forficatus exposed to cadmium. Regardless of gender and time of exposure to the metal, the ATP level in gonads of individuals intoxicated with cadmium was nearly three-fold lower compared to the control (12Cd, p = 0.039; 45Cd, p = 0.029) (Fig. 10A). There were no statistically significant differences in ATP concentration in ovaries and testes of individuals from complementary groups exposed to the metal. The ADP/ATP index in control group gonads was below 0.3. In females treated with cadmium for 45 days, the ADP/ATP ratio in ovary cells was more than seven-fold higher, compared to the control (p = 0.035), and over two-fold higher compared to the 12Cd group. In males the highest level of ADP/ ATP index was in testes of individuals exposed to cadmium for 12 days (p = 0.018), whereas after 45 days of exposure, it decreased by almost two-fold, to a value not significantly different from the control (Fig. 10B). Only after 12 days of exposure to cadmium were there statistically significant intergender differences in ADP/ATP index (p = 0.015) (Fig. 10B).

Discussion
Centipedes in their natural environment are exposed to various types of stressors, including heavy metals. One of them is cadmium, which is highly toxic to living organisms. It can enter the body directly from the environment through the body wall or food 35,36 . In our previous studies, we analyzed the effect of short-and long-term soil cadmium exposure on midgut, salivary glands, fat body, and mitochondria activity in somatic and germ cells in the centipede Lithobius forficatus [30][31][32] . Our research results indicated that different organs in the body might react differently to the same stressor at the same concentration and duration of exposure. Our latest research on the effect of cadmium in soil on L. forficatus gonads has shown that the ovaries and testes may react differently to the presence of this xenobiotic in the environment. Moreover, differences in the changes taking place were also observed between somatic and germ cells in male and female gonads. The somatic cells that build the gonadal wall in both the ovary and the testis of the animals treated with cadmium for 12 and 45 days showed signs of degeneration. Their cytoplasm was electron lucent and poor in  www.nature.com/scientificreports/ cell organelles. In addition, small vacuoles appeared in the cells of the testis wall. Similar, but on a smaller scale, changes were observed in the epithelial cells surrounding each oocyte. Such large degenerative changes in the cells of the gonad wall are related to the fact that these organs lie close to the body wall through which the xenobiotic enters the animal's body. Therefore, they are one of the first barriers to protect reproductive cells. Similar degenerative changes were observed in the somatic cells of the gonads of other invertebrates exposed to cadmium [37][38][39] .
Histological studies showed that in Blaps polycresta Tschinkel 1975 (Coleoptera, Tenebrionidae), the follicular epithelium in the ovary was shrinking and ruptured under cadmium's influence. The testes of this species also showed a histopathological structure after the cadmium treatment, with shrinkage of acini and peritoneal membrane disintegration 39 . In cadmium-treated earthworm Dendrobaena veneta (Rosa, 1886), somatic gonadal cells underwent degenerative changes consisting of cell vacuolation, the appearance of irregular intercellular spaces, the disintegration of intercellular connections, and disintegration of cell membranes. These changes intensified both with the increase in cadmium concentration in the soil and the prolongation of this xenobiotic exposure 38 . Somatic cells in the ovaries and testes of animals are responsible for many functions related to the normal course of gametogenesis, including the synthesis of regulatory and storage substances, and in particular for the protection of germline cells 18,19 . The results of our studies on soil invertebrates confirmed such a statement. Although protected by somatic gonadal cells, germ line cells are also exposed to the toxic effects of cadmium. The main organelles affected by this xenobiotic are the mitochondria. Both in oocytes and L. forficatus spermatocytes, changes in ultrastructure were observed only in a few mitochondria. In contrast, significant changes in their activity were observed, as described in detail in the previous paper 32 . Much greater ultrastructural changes were observed in the mitochondria of the spermatids of the studied species after both 12 and 45 days of exposure to cadmium. They partially lost their cristae, and vacuoles or lamellar bodies appeared inside them. Distension, vacuolation, and reduction of mitochondrial cristae are common changes in these organelles under the influence of cadmium. They were described in female and male germ cells of D. veneta 38,[40][41][42] , in oocytes of Palaemon serratus (Pennant, 1777) 43 , in spermatids and spermatozoids of sea urchins and mussels [44][45][46] as well as the somatic cells of different organs [30][31][32][37][38][39] .
Organelles of L. forficatus such as the rough endoplasmic reticulum and Golgi complexes showed signs of increased activity in oocytes of animals exposed to short-and long-term exposure to cadmium and in spermatocytes of animals treated with cadmium for 12 days. Degeneration of Golgi complexes occurred in spermatocytes after long-term xenobiotic exposure. On the one hand, the activity of these organelles in oocytes may be related to the process of yolk material accumulation (vitellogenesis) 47 . It may also be associated with the synthesis of metallothioneins and other Cd-binding proteins, which are cellular defense tools against cadmium [48][49][50] .
Single spherites were observed in the cytoplasm of oocytes and L. forficatus ovarian wall cells in the 12Cd group. Their number increases significantly in oocytes from the 45Cd group. Spherites are spherical granules in the form of concentric lamination that have been described in cells of the different invertebrate organs 14,15,17,[51][52][53][54][55] . These structures can accumulate non-toxic and toxic substances such as calcium, magnesium, heavy metals, and organic material 14,15,17,51,52 . In L. forficatus, spherites were observed in midgut cells in the control group and the experimental groups treated with cadmium 30 . These structures were not observed in the cells of the salivary glands, fat body 31 , or testes (present study). Considering that the spherites do not appear in the control group's ovarian cells, they appear in the 12Cd group, and their number increases significantly in the oocytes from the 45Cd group, we can assume that these structures accumulate cadmium in the studied species. Probably the accumulation of cadmium in spherites is one of the mechanisms that protect oocytes against the toxic activity of this heavy metal. Spherites are concentrically layered structures described in many organs of invertebrates through which the detoxification takes place. They contain both organic and non-organic compounds, including heavy metals. Thus, they are regarded as a barrier that inhibits the harmful influence of metal ions from reaching the entire organism 56 . They have also been described in soil myriapods belonging to centipedes and millipedes 13,15,27,[57][58][59] . Spherites that accumulate in the oocytes of L. forficatus ovaries resemble that of class B containing cadmium, copper, and/or mercury 13 . Thus, we can conclude that the formation of spherites in oocytes of L. forficatus could be a protective mechanism against the effect of cadmium. Because of the fact that these structures were formed only in ovaries, these gonads are more protected than testes.
Literature data show that cadmium affects the process of vitellogenesis, i.e., the accumulation of yolk material in oocytes. The yolk is synthesized in smaller amounts, or its synthesis is inhibited 60,61 . Cadmium inhibits vitellogenesis, probably through a reduction in vitellogenin polypeptide synthesis 61 . L. forficatus individuals treated with cadmium synthesized yolk material, which accumulated in the oocytes. However, the yolk spheres were smaller than those observed in the oocytes of the control animals. These results may suggest that cadmium limits the synthesis of vitellogenins, which will hurt the studied species' reproduction. Further studies, however, must be done to show how the mechanisms of vitellogenins synthesis are affected by exposure to cadmium. Figure 9. (A) Percentage of early apoptotic (Annexin + PI − ), late apoptotic (Annexin + PI + ) and necrotic (Annexin − PI + ) cells (x ± SE) in ovaries of L. forficatus from the control group and exposed to cadmium (12Cd, 45Cd). The different letters (a, b, c) indicate significant differences within each parameter (Tukey test, p < 0.05; n = 5-6). (B) Percentage of early apoptotic (Annexin + PI − ), late apoptotic (Annexin + PI + ) and necrotic (Annexin − PI + ) cells (x ± SE) in testis of L. forficatus from the control group and exposed to cadmium (12Cd, 45 Cd). The different letters (a, b, c) indicate significant differences within each parameter (Tukey test, p < 0.05; n = 5-6). (C-E) TUNEL assay and DAPI staining. Apoptosis in the ovary (C-E) and testis (F-H) of L. forficatus in control group (C), after 12 days of Cd treatment (12Cd) and after 45 days of Cd treatment (45Cd). Apoptotic nuclei (red signals), nuclei (blue signals). Scale bar: (C-D) 16  www.nature.com/scientificreports/ During analysis changes in the microtubular sheet surrounding an axoneme in the spermatids of the cadmium-treated animals were observed. In the group treated with xenobiotics for 12 days, there were small gaps Figure 10. (A) Concentrations of ATP (x ± SE) in ovaries and testis of individuals from the control group and exposed to cadmium. The different letters (a, b) indicate significant differences among groups within each gonad (Tukey test, p < 0.05; n = 5-6). (B) ADP/ATP ratio (x ± SE) in ovaries (A) and testis (B) of individuals from the control group and exposed to cadmium. The different letters (a, b) indicate significant differences among groups within each gonad (Tukey test, p < 0.05; n = 5-6). www.nature.com/scientificreports/ in the microtubular sheet, while 45-day exposure to cadmium destroyed this structure. The remaining microtubules adhered to the axoneme. Similar changes were noted in the microtubular cuff surrounding the nucleus of cadmium-treated earthworm D. veneta spermatids. In this case, these changes depended mainly on the time of exposure to a xenobiotic, and to a lesser extent on its concentration 42 . Changes within the microtubular sheet could be related to disturbances in microtubule polymerization leading to the formation of a damaged microtubular sheet or its absence 62 or were related to depolymerization and destruction of microtubules in the already existing structure 63 . Cell death can occur via several processes, such as apoptosis, autophagy and/or necrosis. Apoptosis is treated as an irreversible process connected with caspase activation and leading to dead cell removal without inflammation 64 . It is a common process responsible for the proper course of different developmental processes including germ-line cell functioning 65,66 . The apoptosis of male germ-line cells in mammalians has been reported to be involved in different steps of testicular development. The number of cells in their tubules is determined by a distinct balance between cell proliferation and apoptotic cell death. Thus, in vertebrates cells with genetic defects could be eliminated from the organ 67,68 . In the case of vertebrate and invertebrate ovaries, apoptosis has been described in ovarian follicles through embryonic and adult life. While during the vertebrate fetal life, this process concerns the oocytes, it has been detected in granulosa cells of secondary and antral follicles in adults 69,70 . Apoptosis of germline-derived cells is a common phenomenon observed in invertebrate ovaries and is necessary for the proper development of the oocyte 64,71-77 . Its role in female gonads is the removal of cells that are unable to differentiate into fertile eggs 64,77 or to supply nutrients to the oocyte 77,78 . However, oocyte apoptosis is involved in the depletion of germ cells from the ovary and has a distinctly negative impact on mammalian female fertility 79,80 . In L. forficatus gonads, apoptosis was commonly observed in somatic cells, and it increased according to the increasing duration of animals' exposure to cadmium. Apoptosis of germ-line cells occurred only in testes, while we did not manage to detect the apoptosis of oocytes. This suggests that somatic cells play a protective role in both gonads for the germ line cells, enabling them to survive. However, the apoptosis of male germ cells could relate to the fact that the majority of all male germ cells produced are discarded through the cell death process 66,68 . Cells that are damaged or badly developed are removed in this process 68,81 . The described degeneration of organelles in the cytoplasm of cells in both gonads of L. forficatus correlates with the previously described degenerative changes in mitochondria. We observed a decrease in the number of active mitochondria and an increase in non-active mitochondria 32 . Changes in the transmembrane mitochondrial potential together with ultrastructural changes of these organelles are alterations that lead to cell death [82][83][84][85] . Additionally, the mitochondrial swelling and vacuolization seemed to be typical for Cd treatment, suggesting an impediment of the oxidative metabolism 86 . It also correlates with the increasing level of necrotic cells in both gonads.
Autophagy being responsible for the degradation of organelles and cytoplasmic components, rather than being a type of cell death, can also protect cells against their death [87][88][89][90] . However, apoptosis and autophagy can coexist; thus, the distinct cross-talk between these two processes has been described 30,31,55,77,[91][92][93] . The increase in the intensity of autophagy in the cells of both gonads after short-term treatment of the animals with cadmium proves that protective processes are in progress. During autophagy different organelles, e.g., mitochondria, the cisterns of the endoplasmic reticulum, spherites, etc., are neutralized inside autophagosomes due to intracellular digestion, leading to cell survival 89,90 . Autophagy in the 45Cd group turns out to be a less active process compared to the short-term treatment of the animals with cadmium. This correlates with an increase in the intensity of apoptosis in the testis and ovary. As in the case of organs such as the midgut and salivary glands 30,31 , there is a clear cross-talk between these two processes. Our research confirms the observation that autophagy is a common process that enables the functioning of both types of cells (the somatic and germ cells) in testes and ovaries 64,76,77,94,95 . The concentration of ATP was significantly reduced in both gonads after short-and long-term exposure to Cd. We obtained similar results for midgut epithelium 30 . It confirms the possibility of intensification of degenerative processes in cells of both gonads. The highest level of the ADP/ATP index was detected in testes of individuals exposed to cadmium for 12 days. It could relate to the fact that after short-term exposure to cadmium significant differences in the percentage of early apoptotic cells and necrotic cells appeared. The significantly low ATP levels favor necrosis 96,97 .
Our research raised further questions: (1) Does cadmium ingested with food cause similar changes to those observed in soil? (2) Is the number of eggs laid by cadmium-treated females the same as in the control group? (3) Is the success at hatching eggs the same in cadmium-treated and untreated animals? (4) Do the offspring of females treated with cadmium survive puberty in a similar ratio as the offspring of females not treated with this heavy metal? Answering these questions will allow a better understanding of the effects of cadmium on the reproductive abilities of L. forficatus and other soil animals. Therefore, more research is needed to explore this problem.

Conclusions
Our studies showed that (1) cadmium causes damage to the gonad (ovary and testis) structure and affects gametogenesis; (2) male germ-line cells are more sensitive to cadmium than female germ-line cells what is probably related to the accumulation of spherites in ovaries; (3) somatic cells of both gonads play a protective role against heavy metals; (4) distinct cross-talk between autophagy and apoptosis exists in both gonads. www.nature.com/scientificreports/