Microplastic pollution is believed to be one of the most widespread and long-lasting changes on a global scale. Our understanding that microplastics significantly impact terrestrial systems and are a global change stressor continues to grow. In the present study, we investigated the negative effect of long-term (28 days of exposure in food) polystyrene particles of micro (1.0-1.9 µm, 0.4-0.6 µm) and nano (0.04-0.06 µm) scale, in low doses, on the fruit fly - representing a common, globally distributed terrestrial invertebrate, and a model species in many fields. Our observations involved such parameters as ingestion and transfer of particles, survival, reproduction, changes in ultrastructure and tissue and cell responses in midgut epithelium (the place of direct contact with plastic), ovary, and testis in adults, and transgenerational effects in larvae. These observations may indicate possible toxic effects of the tested substances, even in low doses, that can be expected in other taxa, in terrestrial ecosystems. We observed a negative impact of polystyrene particles on the fruit fly survival, midgut, ovary, and testis, involving ultrastructural alterations, such as autophagy and/or ultimately necrosis in the midgut, triggering oxidative stress and activating processes of antioxidative protection. Despite the changes, midgut function and reproduction were not altered - spermatogenesis and oogenesis proceeded normally. The effect was size-dependent - the smaller the polystyrene particles were, the more substantial was the impact they caused. Ultrastructural changes and studied parameters, i.e., generation of ROS (overproduction of which generates oxidative stress), total glutathione concentration (involved in defense against ROS, acting in distinct pathways), and total antioxidant concentration (the oxidative defense system) showed the highest levels after exposure to the smallest nanoparticles, and vice versa. The effect was also sex-dependent, with male flies being more sensitive. Negative effects in males were more substantial and more prominent, even after contact with larger particles, compared to females. The smaller particles (0.4-0.6 µm, 0.04-0.06 µm) were transferred to the ovary and accumulated in the oocytes. In this case, a transgenerational negative effect was detected in larvae. It was characterized by size-dependent alterations, with smaller particles triggering higher levels of ROS and cellular oxidative response. Only the largest particles (1.0-1.9 µm) did not pass into the gonad and did not alter the larvae. These observations together demonstrated that polystyrene particles of micro- and nanoscale, even in a low dose, can induce numerous negative effects on terrestrial invertebrates.

Recent studies on the ovary organization and oogenesis in Tubificinae have revealed that their ovaries are small polarized structures that are composed of germ cells in subsequent stages of oogenesis that are associated with somatic cells. In syncytial cysts, as a rule, each germ cell is connected to the central cytoplasmic mass, the cytophore, via only one stable intercellular bridge (ring canal). In this paper we present detailed data about the composition of germ-line cysts in Tubifex tubifex with special emphasis on the occurrence and distribution of the cytoskeletal elements. Using fixed material and live cell imaging techniques, we found that the entire ovary of T. tubifex is composed of only one, huge multicellular germ-line cyst, which may contain up to 2,600 cells. Its architecture is broadly similar to the cysts that are found in other clitellate annelids, i.e. a common, anuclear cytoplasmic mass in the center of the cyst and germ cells that are connected to it via intercellular bridges. The cytophore in the T. tubifex cyst extends along the long axis of the ovary in the form of elongated and branched cytoplasmic strands. Rhodamine-coupled phalloidin staining revealed that the prominent strands of actin filaments occur inside the cytophore. Similar to the cytophore, F-actin strands are branched and they are especially well developed in the middle and outermost parts of the ovary. Microfilaments are also present in the ring canals that connect the germ cells with the cytophore in the narrow end of the ovary. Using TubulinTracker, we found that the microtubules form a prominent network of loosely and evenly distributed tubules inside the cytophore as well as in every germ cell. The well-developed cytoskeletal elements in T. tubifex ovary seem to ensure the integrity of such a huge germ-line cyst of complex (germ cells - ring canals - cytophore) organization. A comparison between the cysts that are described here and other well-known female germ-line cysts is also made.