The current study revealed that the liver of molly fish appeared as a compound organ in the form of hepatopancreas. The hepatic parenchyma was not organized into lobules and was composed of continuous fields of hepatocytes interrupted by islands of connective tissue that enclosed the hepatic vascular and biliary components. These results agreed with those of Rocha et al. [9, 10] who stated that fish species present two liver phenotypes: either associated with pancreatic tissue (hepatopancreas) or not. Moreover, the hepatic lobules in most fish species are not well defined compared to those of higher vertebrates due to the shortening or complete absence of connective tissue septa .
The present study showed that the hepatocyte-sinusoidal structures of molly fish livers had taken two different forms: cord-like form and tubular form. Akiyoshi and Inoue  reported that the hepatocyte-sinusoidal structures of fish livers are categorized into three forms: a cord-like form, a tubular form, and a solid form. In the cord-like form, most hepatocytes are arranged in simple layers and the hepatic sinusoids are enlarged with straight capillaries. In the tubular form, most hepatocytes are arranged in double-layers and the sinusoidal capillaries are narrow and irregularly shaped. In the solid form, most hepatocytes are arranged in several layers and the hepatic sinusoids are narrow and short tortuous capillaries. Akiyoshi and Inoue  added that the morphology and spatial distribution of the intrahepatic vascular and biliary systems show great variation in the different fish species. These differences are not only interspecific, but they can also be recognized within the same species, varying with gender, age, water temperature, or hormonal changes correlated to the life cycle.
Bile is synthesized by hepatocytes and streams through the intrahepatic bile canaliculi, bile ductules, and bile ducts . The biliary tract constructions were categorized into four types: (a) isolated type, (b) biliary-arteriolar tract (BAT) type, (c) biliary-venous tract (BVT) type, and (d) portal-tract type. The current study revealed that the biliary tract system in molly fish showed two different types: isolated type and BVT type. Akiyoshi and Inoue  mentioned that the BAT type was recorded in nearly all species, creating two passageways, which joined with either the isolated type or the portal-tract type. In addition, no correspondence between the bile duct morphological structures and phylogenic advancement has been reported. This indicated that fish livers have established a biliary system like that of other vertebrates.
Hepatocytes are the chief parenchymal cells in the liver, and they perform very essential roles in metabolism, detoxification, and protein synthesis [5, 17]. Hepatocytes also stimulate innate immunity against invading pathogens by synthesizing and releasing innate immunity proteins . The current study revealed that the morphological characteristics of hepatocytes in the liver of molly fish are typical of those in mammalian liver. Their cytoplasm showed abundant filamentous mitochondria, well-developed smooth endoplasmic reticulum and rough endoplasmic reticulum, many lysosomes, well-prominent Golgi complex, and numerous glycogen and lipid droplets of different sizes. Similar findings were reported by many authors for many fish species, such as Teleostei, Salmonidae, grass carp or Ctenopharyngodon idella and Atlantic salmon [4, 9, 11].
An interesting finding in this study is the expression of APG5 in hepatocytes, Kupffer cells, melanomacrophages, and telocytes, indicating the involvement of these cells in the process of autophagy in the liver of molly fish. Autophagy is a highly synchronized biological process that is associated with stress adaptation induced by nutritional or trophic deficiencies to preserve cellular hemostasis by reprocessing the damaged organelles. APG5 is one of the essential players of the autophagy process , and it is critical for multiple processes including autophagic vesicle formation, lymphocyte development and proliferation, mitochondrial quality control, and apoptosis . Autophagy is considered to be a protective system for the cells as it inhibits the accumulation of toxic proteins; it plays a vital role in innate and adaptive immunity, and fortification against some diseases, and aging . In addition, autophagy activates apoptosis through different apoptotic mechanisms .
Another interesting finding in this study is the expression of TGF-β in hepatocytes, telocytes, and macrophages. Transforming growth factor-β (TGF-β) is a well-recognized, multifunctional cytokine transforming growth factor. TGF-β mediates hepatic stellate cell and fibroblast activation resulting in generation of myofibroblasts and deposition of extracellular matrix . It is a pleiotropic cytokine produced by a wide variety of cells including immune cells and non-hematopoietic cells. It has significant impacts on cell proliferation, oncogenesis, and immune response suppression, in addition to suppression of intestinal inflammatory responses to bacterial antigens . TGF-β plays a critical immunoregulatory role in mammals both in the innate and adaptive immune pathways , and it has been reported to regulate the active and inactive states of macrophages and monocytes under specific conditions . Higher expression of TGF-β1 was detected in immune-associated tissues of fish, including the spleen, thymus, and head kidney .
One of the most striking findings in this study is the recognition of Kupffer cells, which were typical to those of mammalian liver in their morphological features, Kupffer cells have not been recognized in many teleosts. These polymorphous cells are rare and hardly recognized in fish liver. When present, they show a strong phagocytic activity . Kupffer cells were recognized in spotted pimelodus  and in Juvenile crocodile . However, they were not observed in the liver of Kareius bicoloratus  or Salmo trutta fario .
Intrahepatic macrophages, Kupffer cells, and melanomacrophage centers are all present in the liver of molly fish. They can be distinguished by their topographical location and their morphology. The intrahepatic macrophages were located between the hepatocytes and characterized by many cytoplasmic processes. Their cytoplasm characteristically displayed many small dense vesicles (lysosomes), phagocytic vacuoles, and huge dense bodies with a heterogeneous content that represented phagososomes. Kupffer cells were pleomorphic cells, situated in the hepatic sinusoids. They projected slightly to the sinusoidal lumen and established close contact with endothelial cells. They possessed irregular cell surfaces and contained lysosomes, phagosomes in the form of vacuoles, as well as a few fat droplets. The nuclei of the Kupffer cells were indented and frequently eccentrically located. Clumps of heterochromatin were distributed throughout the nuclei and formed a distinct rim along the nuclear envelope. Melanomacrophage centers (MCs) were heterogeneous in composition, and widely distributed in the liver of molly fish: between hepatocytes, around bile ducts, in the adventitia of blood vessels, and associated with pancreatic–venous complexes, like those found in other fish species [32,33,34]. Moreover, melanomacrophages were characterized by pseudopodia-like processes and eccentric nucleus, and their cytoplasm contained numerous melanin-like granules.
By TEM, Kupffer cells showed large vesicular nuclei and many cytoplasmic processes that projected into the lumen of hepatic sinusoids in the liver of molly fish. In addition, they expressed strong immunoreactivity to APG5, TGF-β, and NF-kB. NF-kB responds to inflammatory and immune stimuli and regulates cell proliferation, adhesion, invasion, apoptosis, and angiogenesis in multiple cell types . Moreover, its signaling within epithelial cells plays fundamental roles for maintaining immune homeostasis in barrier tissues . In the innate immune response, NF-κB is a critical transcription factor that mediates production of many pro-inflammatory cytokines and plays a fundamental role in various signaling pathways . The findings of this study support the notion that that Kupffer cells play a vital role in autophagy and phagocytosis. These suggestions are in agreement with van Wilpe and Groenewald , who stated that Kupffer cells play a critical role in elimination of degenerated blood cells, degradation of hemoglobin, and removal of toxic materials.
Aggregations of intrahepatic macrophages and melanomacrophages were extensively distributed in the liver of molly fish. TEM revealed that the cytoplasm of these cells exhibited numerous phagocytic vacuoles, melanin-like granules and large, dense bodies containing heterogeneous materials that represented phagosomes. In addition, they expressed APG5, TGF-β, IL-1β, NF-κB, and SOX9. IL-1β is a critical early response proinflammatory cytokine that mediates immune regulation in both innate and adaptive immunity. It could be secreted by activated endothelial cells, tissue macrophages, blood monocytes, activated T lymphocytes, granulocytes, and other cell types . It affects almost every cell type, where it plays an essential role in the initiation of systemic and local responses to infection or injury by activating macrophages, T and B lymphocytes, and natural killer cells [39, 40]. The Sox family plays essential roles in stem cell maintenance, embryonic development, and lineage commitment , where sox9 regulates stem and progenitor cells in adult tissues .
Aghaallaei et al. and Agius and Roberts [17, 43] added that these melano-macrophage aggregations resemble the morphology of Kupffer cells and show a clear phagocytic activity. Mokhtar  found that macrophage aggregations in the liver of grass carp are heterogeneous in composition and their cytoplasm contains iron, melanin, lipofuscin, lipid, and glycogen. Moreover, Agius and Roberts  suggested that these aggregates could be a site of melanin synthesis rather than storage. Recently, melano-macrophages have been reported as a histological indicator of immune function in fish and other poikilotherms . They are considered one of the potential biomonitoring tools for determining the impacts of minute concentrations of pesticide contaminants [45,46,47,48], where an increase of melano-macrophage aggregates can serve as a biomarker of toxic effect .
Telocytes (TCs) are identified as a peculiar cell type of interstitial cells that is characterized by extremely long and thin cellular processes that are called telopodes (Tp) . These cells were identified in many different tissues and organs in humans, animals, birds, and fish. It was proved that they perform a wide range of very important biological functions [51,52,53,54,55]. Telocytes’ distribution was first recognized in the fish liver by Mokhtar , who recorded them around the bile ductules and hepatic blood vessels of grass carp. In agreement with , the current study revealed that telocytes in fish shared the same morphological characteristics those in mammals. However, one of the most interesting observations in this study is that telocytes expressed a strong immunoreactivity to APG5, TGF-β, and Nrf2. Nrf2 has been shown to be involved in osmoregulation, antioxidation, and immunopotentiation in fish under salinity stress [56, 57]. These data suggest that telocytes can play a role in cellular differentiation and regeneration in addition to phagocytosis and autophagy.