The advent of single-cell RNA sequencing technology (scRNA-seq) has revolutionized our understanding of gene regulation at the single-cell level by scrutinizing the gene expression profiles of thousands of individual cells, thus facilitating the identification of previously unknown cell types and their unique physiological states (12). Moreover, it has provided a means to unravel the intricate developmental trajectories of cells, shedding light on their origins and evolutionary pathways (12). Recent transcriptomic investigations have unveiled different cell types in the midgut in Drosophila (2, 13) mosquitoes (14) and silkworms (15), and a compelling narrative, revealing that the insect midgut, exemplified by Drosophila and silkworms, closely related to the mammalian small intestine in terms of the cell types present and their associated marker gene expression patterns (2). Table 1 summarizes well-established cell type markers from scRNA-seq analysis of midgut tissues in Drosophila, mosquitoes, silkworms and mice. In the model insect Drosophila, the midgut undergoes renewal at regular intervals of one to two weeks, facilitated by specialized regenerative cells known as intestinal stem cells (ISCs) (17). These ISCs exhibit similarities with mammalian transit-amplifying cells (TACs) in the G2 phase of the cell cycle. Additionally, within the midgut milieu in insects, there are endocrine cells, enterocytes, and enteroblasts, all of which share akin marker gene expression patterns when compared with their counterparts in mammals (2). In Drosophila, enteroendocrine cells (EEs) mirror their mammalian counterparts, while goblet cells found in lepidopteran midguts resemble mammalian goblet cells. Furthermore, cardia cells within the Drosophila midgut exhibit marked similarities in marker expression profiles with the goblet cells of mammals (2). Similar observations were reported by Xia et al. in a recent study, wherein diverse cell types identified in the silkworm midgut exhibited nearly identical patterns of marker gene expression (15). This convergence extends to the functional domain, where homologous marker genes underscore numerous facets of midgut function. These findings shed light on the remarkable conservation and convergent evolution of midgut biology across diverse taxa, underscoring its significance in understanding insect physiology and potential implications for our comprehension of mammalian gastrointestinal biology.

Regarding the origin of different types of cells, recent studies have revealed that the self-renewable ISCs similar to TACs in mammals raise enteroblasts (EBs) or enteroendocrine progenitor cells (EEPs), and the notch signaling pathway controls this determination. Heightened notch signaling pathway directs ISCs toward a developmental trajectory culminating in the formation of absorptive enterocytes (ISC → EB → EC) (18, 19). In contrast, when notch signaling is maintained at a lower level, ISCs follow an alternative route, leading to the generation of hormone-secreting enteroendocrine cells (ISC → EEP → EEs) (20). This finely regulated process of cellular fate determination plays a critical role in regulating the composition and functional dynamics of the insect midgut.

This review will discuss a concise yet comprehensive overview of the cell-level functionalities of the insect midgut, with a particular focus on the silkworm, from the insights derived from advanced transcriptomic studies. Additionally, we discuss the role of various midgut cell types in the immune defense mechanisms against pathogens. This integrated approach aims to provide readers with a coherent and deep understanding of the immunologically active organ, the insect midgut.

Columnar cells (CCs) are pivotal among the four primary cellular phenotypes within the insect midgut (Figure 1) (7). They are referred to as “enterocytes” by the feature of their akin absorptive functionalities to their mammalian small intestinal counterparts (21–23). Beyond their absorptive nature, these CCs prominently orchestrate the synthesis and secretion of digestive enzymes, thereby underscoring their indispensable contribution to digestive processes within the insect midgut environment (24). The distinct anatomical feature of CCs includes the centrally located nucleus accompanied by a prominently convoluted apical membrane, giving rise to microvilli with an inherent actin cytoskeletal structure (Figure 1) (25). In addition to the common characteristics inherent to CCs across insect species, there exist specialized ultrastructural features that are discernible within distinct regions of the midgut and feeding behaviors of insects (26–29). The ultrastructural composition of CCs within the midgut exhibits a transition along the longitudinal axis, displaying distinct roles in the shifting anatomical context (21). Structural evidence highlights that CCs in the anterior midgut are responsible for secreting an array of digestive enzymes encompassing exopeptidases, maltases, endopeptidases, amylases, lipases, and lysozymes (Figure 1) (24, 30). These enzymes are secreted into the midgut lumen via conventional exocytosis and through mechanisms involving apocrine or micro-apocrine modes of secretion (30). Conversely, CCs within the posterior midgut region are characterized by their involvement in synthesizing serine proteases (31–33). Given that digestive enzymes play a direct and crucial role in facilitating nutrient accessibility and development (34, 35), the production and activities of these enzymes are regulated by a rigorous regulatory framework. Multiple factors, encompassing the composition of nutrients, signals from endocrine and neuronal sources, and interactions with gut microorganisms, collectively contribute to the intricate modulation of enzymatic processes underlying nutrient digestion and absorption (36–39). Nutrient absorption within CCs is executed through the activity of transport proteins, which exhibit distinct distribution patterns upon the regionalization of the midgut (Figure 1) (32, 40). The Na⁺/K⁺ ATPase channel for absorption is absent in CCs, and the cotransporters of CCs capitalize on the electrochemical gradient of K⁺ rather than that of Na⁺ as a driving ion. The amino acids from protein digestion are translocated from the lumen into the intracellular cytoplasm, marking the specialized functionality of CCs in nutrient absorption (41, 42).

Nutrient uptake and digestion are the critical functions performed by the insect midgut. Along with these vital physiological phenomena’s essential for growth and development, another critical function performed by the gut is the maintenance of gut homeostasis. The homeostasis is maintained through the complex gut-brain axis, forming a neurohumoral communication system. Thanks to midgut EEs, which secrete biologically active peptides responsible for the sophisticated interplay of communication within the organism (38, 53, 54). Table 2 summarize some important peptides secreted by the EEs. EEs discovered around three decades ago (64–66), have two distinct morphological forms, open and closed types (67), characterized by their peptidergic nature (64); the former have direct contact with the midgut lumen (67, 68), while the latter lacks such interaction as they do not extend through the epithelium (Figure 1) (33, 67). The functional application of recent genomic and proteomic approaches provides compelling evidence of the critical role of EEs within the insect midgut play in the physiology and developmental processes of insects (56, 69, 70). The EEs of silkworms were classified into four distinct subtypes according to their spatial distribution. Type I cells were identified across all three midgut regions, while type II, III and IV cells were localized in the anterior, middle, and posterior midgut, respectively (56). The spatial distribution of EEs across different midgut regions also depends on the specific peptide they produce (17, 71). The peptides are transformed into active states by cleavage and post-translation modification from protein precursors produced by EEs. In Drosophila, 9 major precursors were identified (58), whereas in silkworms, 18 distinct protein precursors were identified (56). In different insects, the peptides produced after cleavage are mainly involved in the regulation of food in the alimentary canal, from reduced ion transport to suppressed gut contractions (72, 73). In B. mori tachykinin peptides (Tk) secretions are regulated by B. mori gustatory receptor (BmGr4) expressed in some Tk-producing EEs in the anterior midgut when food and digestive products arrived after feeding began (60). The regulatory role of peptides secreted by EEs is evident in locusts and Drosophila. Notably, in the midgut of starved locusts, the levels of Tk diminish while their circulation in the hemolymph increases, suggesting a potential responsiveness to nutritional status for enhanced food uptake (74). Further insights have emerged from studies in Drosophila, demonstrating that the EE-produced Tks contributes to lipid metabolism through interaction with their receptor TKR99D situated in the brain (61). Targeted ablation of EEs expressing TKs resulted in the suppression of intestinal stem cell proliferation, indicating the developmental role of these peptides (37).

Lepidopteran insect’s midgut possesses a distinctive category of cells known as goblet cells (GCs) (21, 23, 79, 80). These GCs, in conjunction with the midgut’s CCs, play a pivotal role in the elevated pH of the midgut environment. These insects primarily feed on plant matter; their food encompasses secondary metabolites, such as tannins and phenols, which can be detrimental. The presence of these secondary metabolites diminishes nutrient accessibility, as they form insoluble complexes via cross-linking with enzymes and proteins. To counteract this constrained nutrient availability, the coordinated efforts of GCs and CCs come into play, preserving an elevated pH within the midgut lumen. GCs are characterized by a conspicuous chalice-shaped central cavity, which originates from the invagination of the apical membrane (Figure 1) (21, 22, 81, 82). Their distribution within the midgut varies according to their respective locations, resulting in heterogeneous concentrations (21). These GCs establish intricate connections with other midgut cells, employing septate and gap junction formations to facilitate intercellular communication and coordination (83). The chalice-shaped central cavity (Goblet cell cavity, GCC) is lined by small structures known as microvilli, and these microvilli are longer at the basal portion of GCC than the apical portion, providing extensive surface area. These microvilli are surrounded by actin. The GCC content is effectively separated from the intestinal lumen through a sophisticated apical structure called a valve. This valve consists of densely packed microvilli lacking mitochondria, which do not allow the free movement of the content (82). GCs play a pivotal role in preserving the distinctive characteristic of elevated alkaline pH within the midgut of these insects. This distinct milieu is upheld through the presence of the vacuolar-type proton pump (V-H⁺ ATPase) (82, 84, 85). This pump is actively engaged in the transportation of protons (H⁺) from the cytoplasm into the interior of GCCs (86, 87) at the expense of energy against the concentration gradient (82). Mitochondria located within the microvilli supply the requisite energy for this process. The active translocation of H⁺ is instrumental in the preservation of a transmembrane electrical potential of 150 mV (88). The V-H⁺ ATPase pump orchestrates an intricate exchange between H⁺ and K⁺, resulting in the net flow of K⁺ from the cytoplasm of GCs into the interior of the GCCs (81). This establishes an electrochemical gradient conducive to the efflux of K⁺ from the GCCs into the lumen of the midgut via a specialized valve. This transport phenomenon is augmented by the activity of GCs carbonic anhydrase, which facilitates the generation of a flux of HCO3^- (81) This flux-causing K⁺ transport is essential in maintaining the elevated pH within the midgut.

The gastrointestinal tract of insects faces numerous challenges, including the abrasive effects of ingested food’s movement along the alimentary canal interactions with gut microbiota, a primary barrier against ingested toxic compounds and invasive pathogens. ISCs of midgut play a pivotal role in these critical functions. ISCs are also critical in upholding gut integrity and homeostasis, even within the challenging environment of the digestive tract of insects (97). ISCs positioned along the basal side of the pseudostratified epithelial monolayer (Figure 1) exhibit resemblances to their mammalian counterparts as reported by the Huang et al. in Drosophila (2). Functioning as self-renewing and multipotent entities, these cells have the ability to give rise to diverse differentiated cell lineages constituting the intestinal tract (98, 99). ISCs have blast-like morphology characterized by fewer organelles and a cytoplasm of lighter density (22). Notably, recent research has unveiled the presence of storage entities, lipid droplets and glycogen granules within ISCs (100, 101). The division of ISCs depends upon the cellular function performed after division. Two types of cell divisions are observed in ISCs, i.e. asymmetric and symmetric. In the asymmetric division, to maintain the constant population of ISCs within the gastrointestinal milieu, ISCs generate a stem cell and a terminally differentiated counterpart, poised to undertake specific functional roles. In contrast, the symmetric division modality results in the formation of two daughter cells, both of which assume mature and functional phenotypes (40, 102, 103). Once the ISCs are divided and differentiated, they can’t revert to stem cells (104). This interplay between divisional strategies underscores the regenerative prowess of ISCs but also attests to their pivotal role in orchestrating tissue homeostasis and repair.