Can insecticide-free clean water regenerate the midgut epithelium of the freshwater shrimp after dimethoate treatment?

Insecticides such as dimethoate persist for a long time in freshwater environments, influencing the physiology of the animals inhabiting such environments. In aquatic organisms, toxic substances can enter the body through the epidermis and the digestive system. The midgut is part of this system in which intense processes constitute a barrier against the effects of toxic substances on the body. The aim of this study was to evaluate the toxic potential of dimethoate in the midgut epithelium of the freshwater shrimp Neocaridina davidi, emphasizing ultrastructural alterations. However, the additional and main purpose was to determine whether the midgut epithelium can regenerate after placing animals in insecticide-free clean water after various periods of exposure to dimethoate. N. davidi originates from Asia, but it has also been described in European rivers. This species is of particular interest among breeders worldwide due to its ease of breeding and reproduction. The animals were treated with dimethoate for 1, 2, and 3 weeks and then placed in clean water for 1, 2, and 3 weeks. The qualitative and quantitative analysis revealed different sensitivity of organs forming the midgut in freshwater crustaceans and the possibility for midgut regeneration after insecticide exposure. We concluded that different processes were triggered in the intestine and hepatopancreas to regenerate cells after damage, and mitochondria were the first organelles to respond to the appearance of a stressor in the living environment.

Enzymatic activities in the digestive tract of spirostreptid and spirobolid millipedes (Diplopoda: Spirostreptida and Spirobolida).

Millipedes represent a model for the study of organic matter transformation, animal-microbial interactions, and compartmentalisation of digestion. The activity of saccharidases (amylase, laminarinase, cellulase, xylanase, chitinase, maltase, cellobiase, and trehalase) and protease were measured in the midgut and hindgut contents and walls of the millipedes Archispirostreptus gigas and Epibolus pulchripes. Assays done at pH 4 and 7 confirmed activities of all enzymes except xylanase. Hydrolysing of starch and laminarin prevailed. The hindgut of E. pulchripes was shorter, less differentiated. Micro-apocrine secretion was observed only in the midgut of A. gigas. Merocrine secretion was present in midgut and hindgut of E. pulchripes, and in the pyloric valve and anterior hindgut of A. gigas. Alpha-polysaccharidases were mostly active in the midgut content and walls, with higher activity at pH 4. The low activity of amylase (A. gigas) and laminarinase (E. pulchripes) in midgut tissue may indicate their synthesis in salivary glands. Cellulases were found in midgut. Chitinases, found in midgut content and tissue (E. pulchripes) or concentrated in the midgut wall (A. gigas), were more active at an acidic pH. Polysaccharidases were low in hindguts. Protease shows midgut origin and alkaline activity extending to the hindgut in E. pulchripes, whereas in A. gigas it is of salivary gland origin and acid activity restricted to the midgut. Some disaccharidases, with more alkaline activity, showed less apparent midgut-hindgut differences. It may indicate an axial separating of the primary and secondary digestion along the intestinal pH gradient or the presence of enzymes of hindgut parasites.

Autophagy and Apoptosis in the Midgut Epithelium of Millipedes.

The process of autophagy has been detected in the midgut epithelium of four millipede species: Julus scandinavius, Polyxenus lagurus, Archispirostreptus gigas, and Telodeinopus aoutii. It has been examined using transmission electron microscopy (TEM), which enabled differentiation of cells in the midgut epithelium, and some histochemical methods (light microscope and fluorescence microscope). While autophagy appeared in the cytoplasm of digestive, secretory, and regenerative cells in J. scandinavius and A. gigas, in the two other species, T. aoutii and P. lagurus, it was only detected in the digestive cells. Both types of macroautophagy, the selective and nonselective processes, are described using TEM. Phagophore formation appeared as the first step of autophagy. After its blind ends fusion, the autophagosomes were formed. The autophagosomes fused with lysosomes and were transformed into autolysosomes. As the final step of autophagy, the residual bodies were detected. Autophagic structures can be removed from the midgut epithelium via, e.g., atypical exocytosis. Additionally, in P. lagurus and J. scandinavius, it was observed as the neutralization of pathogens such as Rickettsia-like microorganisms. Autophagy and apoptosis ca be analyzed using TEM, while specific histochemical methods may confirm it.

The role of autophagy in the midgut epithelium of Parachela (Tardigrada).

The process of cell death has been detected in the midgut epithelium of four tardigrade species which belong to Parachela: Macrobiotus diversus, Macrobiotus polonicus, Hypsibius dujardini and Xerobiotus pseudohufelandi. They originated from different environments so they have been affected by different stressors: M. polonicus was extracted from a moss sample collected from a railway embankment; M. diversus was extracted from a moss sample collected from a petrol station; X. pseudohufelandi originated from sandy and dry soil samples collected from a pine forest; H. dujardini was obtained commercially but it lives in a freshwater or even in wet terrestrial environment. Autophagy is caused in the digestive cells of the midgut epithelium by different factors. However, a distinct crosstalk between autophagy and necrosis in tardigrades' digestive system has been described at the ultrastructural level. Apoptosis has not been detected in the midgut epithelium of analyzed species. We also determined that necrosis is the major process that is responsible for the degeneration of the midgut epithelium of tardigrades, and "apoptosis-necrosis continuum" which is the relationship between these two processes, is disrupted.

Fine structure of the midgut epithelium in the millipede Telodeinopus aoutii (Myriapoda, Diplopoda) with special emphasis on epithelial regeneration.

The midgut of millipedes is composed of a simple epithelium that rests on a basal lamina, which is surrounded by visceral muscles and hepatic cells. As the material for our studies, we chose Telodeinopus aoutii (Demange, 1971) (Kenyan millipede) (Diplopoda, Spirostreptida), which lives in the rain forests of Central Africa. This commonly reared species is easy to obtain from local breeders and easy to culture in the laboratory. During our studies, we used transmission and scanning electron microscopes and light and fluorescent microscopes. The midgut epithelium of the species examined here shares similarities to the structure of the millipedes analyzed to date. The midgut epithelium is composed of three types of cells-digestive, secretory, and regenerative cells. Evidence of three types of secretion have been observed in the midgut epithelium: merocrine, apocrine, and microapocrine secretion. The regenerative cells of the midgut epithelium in millipedes fulfill the role of midgut stem cells because of their main functions: self-renewal (the ability to divide mitotically and to maintain in an undifferentiated state) and potency (ability to differentiate into digestive cells). We also confirmed that spot desmosomes are common intercellular junctions between the regenerative and digestive cells in millipedes.

Investigation of the midgut structure and ultrastructure in Cimex lectularius and Cimex pipistrelli (Hemiptera: Cimicidae).

Cimicidae are temporary ectoparasites, which means that they cannot obtain food continuously. Both Cimex species examined here, Cimex lectularius (Linnaeus 1758) and Cimex pipistrelli (Jenyns 1839), can feed on a non-natal host, C. lectularius from humans on bats, C. pipistrelli on humans, but never naturally. The midgut of C. lectularius and C. pipistrelli is composed of three distinct regions-the anterior midgut (AMG), which has a sack-like shape, the long tube-shaped middle midgut (MMG), and the posterior midgut (PMG). The different ultrastructures of the AMG, MMG, and PMG in both of the species examined suggest that these regions must fulfill different functions in the digestive system. Ultrastructural analysis showed that the AMG fulfills the role of storing food and synthesizing and secreting enzymes, while the MMG is the main organ for the synthesis of enzymes, secretion, and the storage of the reserve material. Additionally, both regions, the AMG and MMG, are involved in water absorption in the digestive system of both Cimex species. The PMG is the part of the midgut in which spherites accumulate. The results of our studies confirm the suggestion of former authors that the structure of the digestive tract of insects is not attributed solely to diet but to the basic adaptation of an ancestor.

Ultrastructure of the salivary glands in Lithobius forficatus (Myriapoda, Chilopoda, Lithobiidae) according to seasonal and circadian rhythms.

The salivary glands (mandibular epidermal glands) of adult males and females of Lithobius forficatus (Myriapoda, Chilopoda) were isolated during spring, summer and autumn. In addition, the organs were isolated at different times of the day - at about 12:00 (noon) and about 00:00 (midnight). The ultrastructure of these organs depending on seasonal and circadian rhythms was analyzed using transmission and scanning electron microscopy and histochemical methods. The paired salivary glands of L. forficatus are situated in the vicinity of the foregut and they are formed by numerous acini that are surrounded by the fat body, hemocytes and tracheolae. The salivary glands are composed of a terminal acinar component and a system of tubular ducts that are lined with a cuticle. The glandular part is composed of secretory epithelial cells that are at various stages of their secretory activity. The saliva that is produced by the secretory cells of the acini is secreted into the salivary ducts, which are lined with a simple epithelium that is based on the non-cellular basal lamina. The ultrastructural variations suggest that salivary glands function differently depending on seasonal rhythms and prepare the animal for overwintering. Therefore, the salivary glands of the centipedes that were analyzed participate in the accumulation of proteins, lipids and polysaccharides during the spring, summer and autumn. Subtle differences in the ultrastructure of the secretory cells of the salivary glands during the circadian cycle must be related to the physiological reactions of the organism. The salivary ducts showed no differences in the specimens that were analyzed during the day/night cycle or during the seasonal cycle.

Apoptosis and necrosis during the circadian cycle in the centipede midgut.

Three types of cells have been distinguished in the midgut epithelium of two centipedes, Lithobius forficatus and Scolopendra cingulata: digestive, secretory, and regenerative cells. According to the results of our previous studies, we decided to analyze the relationship between apoptosis and necrosis in their midgut epithelium and circadian rhythms. Ultrastructural analysis showed that these processes proceed in a continuous manner that is independent of the circadian rhythm in L. forficatus, while in S. cingulata necrosis is activated at midnight. Additionally, the description of apoptosis and necrosis showed no differences between males and females of both species analyzed. At the beginning of apoptosis, the cell cytoplasm becomes electron-dense, apparently in response to shrinkage of the cell. Organelles such as the mitochondria, cisterns of endoplasmic reticulum transform and degenerate. Nuclei gradually assume lobular shapes before the apoptotic cell is discharged into the midgut lumen. During necrosis, however, the cytoplasm of the cell becomes electron-lucent, and the number of organelles decreases. While the digestive cells of about 10 % of L. forficatus contain rickettsia-like pathogens, the corresponding cells in S. cingulata are free of rickettsia. As a result, we can state that apoptosis in L. forficatus is presumably responsible for protecting the organism against infections, while in S. cingulata apoptosis is not associated with the elimination of pathogens. Necrosis is attributed to mechanical damage, and the activation of this process coincides with proliferation of the midgut regenerative cells at midnight in S. cingulata.

Ultrastructural analysis of apoptosis and autophagy in the midgut epithelium of Piscicola geometra (Annelida, Hirudinida) after blood feeding.

Cell death in the endodermal region of the digestive tract of the blood-feeding leech Piscicola geometra was analyzed using light and transmission electron microscopes and the fluorescence method. Sexually mature specimens of P. geometra were bred under laboratory conditions and fed on Danio rerio. After copulation, the specimens laid cocoons. The material for our studies were non-feeding juveniles collected just after hatching, non-feeding adult specimens, and leeches that had been fed with fish blood (D. rerio) only once ad libitum. The fed leeches were prepared for our studies during feeding and after 1, 3, 7, and 14 days (not sexually mature specimens) and some weeks after feeding (the sexually mature). Autophagy in all regions of the endodermal part of the digestive system, including the esophagus, the crop, the posterior crop caecum (PCC), and the intestine was observed in the adult non-feeding and feeding specimens. In fed specimens, autophagy occurred at very high levels--in 80 to 90 % of epithelial cells in all four regions. In contrast, in adult specimens that did not feed, this process occurred at much lower levels--about 10 % (esophagus and intestine) and about 30 % (crop and PCC) of the midgut epithelial cells. Apoptosis occurred in the feeding adult specimens but only in the crop and PCC. However, it was absent in the non-feeding adult specimens and the specimens that were collected during feeding. Moreover, neither autophagy nor apoptosis were observed in the juvenile, non-feeding specimens. The appearance of autophagy and apoptosis was connected with feeding on toxic blood. We concluded that autophagy played the role of a survival factor and was involved in the protection of the epithelium against the products of blood digestion. Quantitative analysis was prepared to determine the number of autophagic and apoptotic cells.

Does autophagy in the midgut epithelium of centipedes depend on the day/night cycle?

The midgut epithelium of two centipedes, Lithobius forficatus and Scolopendra cingulata, is composed of digestive, secretory and regenerative cells. In L. forficatus, the autophagy occurred only in the cytoplasm of the digestive cells as a sporadic process, while in S. cingulata, it occurred intensively in the digestive, secretory and regenerative cells of the midgut epithelium. In both of the species that were analyzed, this process proceeded in a continuous manner and did not depend on the day/night cycle. Ultrastructural analysis showed that the autophagosomes and autolysosomes were located mainly in the apical and perinuclear cytoplasm of the digestive cells in L. forficatus. However, in S. cingulata, the entire cytoplasm was filled with autophagosomes and autolysosomes. Initially the membranes of phagophores surround organelles during autophagosome formation. Autolysosomes result from the fusion of autophagosomes and lysosomes. Residual bodies which are the last stage of autophagy were released into the midgut lumen due to necrosis. Autophagy in the midgut epithelia that were analyzed was confirmed using acid phosphatase and mono-dansyl-cadaverine stainings.

The ultrastructure of the midgut epithelium in millipedes (Myriapoda, Diplopoda).

The midgut epithelia of the millipedes Polyxenus lagurus, Archispirostreptus gigas and Julus scandinavius were analyzed under light and transmission electron microscopies. In order to detect the proliferation of regenerative cells, labeling with BrdU and antibodies against phosphohistone H3 were employed. A tube-shaped midgut of three millipedes examined spreads along the entire length of the middle region of the body. The epithelium is composed of digestive, secretory and regenerative cells. The digestive cells are responsible for the accumulation of metals and the reserve material as well as the synthesis of substances, which are then secreted into the midgut lumen. The secretions are of three types - merocrine, apocrine and microapocrine. The oval or pear-like shaped secretory cells do not come into contact with the midgut lumen and represent the closed type of secretory cells. They possess many electron-dense granules (J. scandinavius) or electron-dense granules and electron-lucent vesicles (A. gigas, P. lagurus), which are accompanied by cisterns of the rough endoplasmic reticulum. The regenerative cells are distributed individually among the basal regions of the digestive cells. The proliferation and differentiation of regenerative cells into the digestive cells occurred in J. scandinavius and A. gigas, while these processes were not observed in P. lagurus. As a result of the mitotic division of regenerative cells, one of the newly formed cells fulfills the role of a regenerative cell, while the second one differentiates into a digestive cell. We concluded that regenerative cells play the role of unipotent midgut stem cells.

Apoptotic and necrotic changes in the midgut glands of the wolf spider Xerolycosa nemoralis (Lycosidae) in response to starvation and dimethoate exposure.

In the present study, the intensity of degenerative changes (apoptosis, necrosis) in the cells of the midgut glands of male and female wolf spiders, Xerolycosa nemoralis (Lycosidae), exposed to natural (starvation) and anthropogenic (the organophosphorous pesticide dimethoate) stressors under laboratory conditions were compared. The spiders were collected from two differentially polluted sites, both located in southern Poland: Katowice-Welnowiec, which is heavily polluted with metals, and Pilica, the reference site. Starvation and dimethoate treatment resulted in enhancement of apoptotic and necrotic changes in the midgut glands of the spiders. The frequency of degenerative changes in starving individuals was twice as high as in the specimens intoxicated with dimethoate. The percentage of apoptotic and necrotic cells was higher in starving males than in starving females. A high intensity of necrotic changes, together with increased Cas-3 like activity and a greater percentage of cells with depolarized mitochondria, were typical of starving males from the polluted site. The cell death indices observed in females depended more strongly on the type of stressor than on previous preexposure to pollutants.

The role of autophagy in the midgut epithelium of Eubranchipus grubii (Crustacea, Branchiopoda, Anostraca).

Eubranchipus grubii (Crustacea, Branchiopoda, Anostraca) is an omnivorous filter feeder whose life span lasts no more than 12 weeks. Adult males and females of E. grubii were used for ultrastructural studies of the midgut epithelium and an analysis of autophagy. The midgut epithelium is formed by columnar digestive cells and no regenerative cells were observed. A distinct regionalization in the distribution of organelles appears - basal, perinuclear and apical regions were distinguished. No differences in the ultrastructure of digestive cells were observed between males and females. Autophagic disintegration of organelles occurs throughout the midgut epithelium. Degenerated organelles accumulate in the neighborhood of Golgi complexes, and these complexes presumably take part in phagophore and autophagosome formation. In some cases, the phagophore also surrounds small autophagosomes, which had appeared earlier. Fusion of autophagosomes and lysosomes was not observed, but lysosomes are enclosed during autophagosome formation. Autophagosomes and autolysosomes are discharged into the midgut lumen due to apocrine secretion. Autophagy plays a role in cell survival by protecting the cell from cell death.

The role of cell death in the midgut epithelium in Filientomon takanawanum (Protura).

Midgut epithelium in Filientomon takanawanum is composed of epithelial cells and single, sporadic regenerative cells. In 80% of analyzed specimens midgut epithelial cells, as fat body and gonads, are infected with rickettsia-like microorganism. In non-infected specimens young and completely differentiated epithelial cells are distinguished among epithelial cells. Characteristic for midgut epithelial cells regionalization in organelles distribution is not observed. Autophagy is the sporadic process, but if the cytoplasm of epithelium cells possesses numerous spherites and sporadic autophagosomes, the apoptosis begins. Necrosis is observed sporadically. In the midgut epithelium cells of about 80% of analyzed specimens rickettsia-like microorganisms are observed. The more rickettsia-like microorganisms occur in the cytoplasm, the more autophagosomes are formed, and the process of apoptosis proceeds intensively.

Ultrastructural studies of midgut epithelium formation in Lepisma saccharina L. (Insecta, Zygentoma).

At the end of embryogenesis of Lepisma saccharina L. (Insecta, Zygentoma), when the stomodaeum and proctodaeum are completely formed, the midgut epithelium is replaced by the primary midgut, a yolk mass is surrounded by a cell membrane. Midgut epithelium formation begins in the 1st larval stage. Energids migrate toward the yolk periphery and aggregate just beneath the cell membrane. They are gradually enclosed by cell membrane folds of the primary midgut. Single cells are formed. Succeeding energids join just formed cells. Thus, groups of cells, regenerative cell groups, are formed. Their number gradually increases. The external cells of the regenerative cell groups transform into epithelial cells and their basal regions spread toward the next regenerative cell groups. Epithelial cells of neighboring regenerative cell groups join each other to form the epithelium. At the end of the 2nd larval stage, just before molting, degeneration of newly the formed epithelium begins. Remains of organelles and basal membrane occur between the regenerative cell groups. The new epithelium is formed from the regenerative cell groups, which are now termed stem cells of the midgut epithelium.