Lectins are carbohydrate-binding proteins that recognize and selectively bind to specific sugar structures. Interaction of lectin with sugars on cell surface can activate multiple cellular responses, including the immune response. Many insect lectins have been identified or predicted but without in-depth analysis.
Lectin Families | Insect Species | Gene/Protein | a | Lectin Functions | Experiment Verification |
Predicted by GO/Homology | References | |
---|---|---|---|---|---|---|---|---|
RNA | b | Protein | c |
CTL | Aedes aegypti | AaeCTLs; CTL-20; mosGCTL-7 | Pathogen recognition; interacts with phosphatase; reduces exogenous toxin toxicity | + | + | [9][67][68][69] | [9,67,68,69] | ||||
Tribolium castaneum | TcCTL6, TcCTL3 | Responds to pathogen infection; regulates AMP expression | + | [70][71] | [70,71] | ||||||
Spodoptera litura | SliCTLs | Responds to pathogen infection | + | [21] | |||||||
Mythimna separata | EPL | Promotes encapsulation | + | [72] | |||||||
Ostrinia furnacalis | OfCTLs, OfIMLs | + | [73] | ||||||||
Spodoptera exigua | Se-LLs, Se-BLLs | Responds to virus infection | + | [74] | |||||||
Thitarodes xiaojinensis | CTL-S | , | CTL-X | , | IMLs | Responds to pathogen infection | + | [75] | |||
Helicoverpa armigera | Ha-lectin, HaCTL | Regulates ecdysone and juvenile hormone signaling; regulates AMP expression; promotes phagocytosis | + | [76] | |||||||
Drosophila melanogaster | Slf, DL2-3 | Organizes the cuticle layers; enhances encapsulation | + | [77][78] | [77,78] | ||||||
Antheraea pernyi | Ap-CT | Binds PAMPs; activates PO | + | ||||||||
Bombyx mori | BmIML, BmMBP, CTL-S3, BmEL-1, 2, 3 | Recognizes PAMPs; activates PO; promotes melanization; | + | ||||||||
Hyphantria cunea | Hdd15 | + | |||||||||
Periplaneta americana | LPS-BP | Responds to | E. coli | + | |||||||
Heliothis virescens | MBL | + | Reviewed by [9] | ||||||||
Manduca sexta | MsIML-1, 2, 3, 4 | Responds to pathogens; binds PAMPs; activates PO; enhances encapsulation | + | ||||||||
Anopheles gambiae | AgamCTLs | Responds to pathogens | |||||||||
Nilaparvata lugens | n.d. | ||||||||||
Plutella xylostella | n.d. | ||||||||||
Apis mellifera | n.d. | ||||||||||
Acyrthosiphon pisum | n.d. | ||||||||||
Chitinase like | Acyrthosiphon pisum | AcypiCht1 | (IDGF homologue) | Expresses in bacteriocyte and midgut | + | [41] | |||||
Anopheles gambiae | AgIDGF2 | , | AgIDGF | 4 | Expresses in different developmental stages and tissues | + | [79] | ||||
Bombyx mori | BmIDGF | Expresses in eggs, hemocytes, fat body, and silk gland | + | [80][81] | [80,81] | ||||||
Drosophila melanogaster | IDGF1-6 | Participates in would healing and wing development | + | + | [38][39][82] | [38,39,82] | |||||
Nilaparvata lugens | NlIDGF | Expresses in female reproductive organs and fat body | + | [42] | |||||||
Tribolium castaneum | TcIDGF2, 4 | Acts in adult eclosion | + | [83] | |||||||
Plutella xylostella | PxIDGF | n.d. | + | [84] | |||||||
Manduca sexta | MsIDGF1 | n.d. | + | [85] | |||||||
Bemisia tabaci | BtIDGF1-3 | Highly abundant in adults | + | [86] | |||||||
Galectin | Drosophila melanogaster | Dmgal | Expresses in hemocytes and in different developmental stages | + | [59][87] | [59,87] | |||||
Phlebotomus papatasi | PpGalec | Strong expression in adult female; binds pathogen | [61] | ||||||||
Anopheles gambiae | Agalectin | , | GALE6-8 | Expresses in salivary gland; Responds to viral infection | + | + | [52][88] | [52,88] | |||
Bombyx mori | BmGalectin-4 | Responds to bacteria in fertilized eggs; binds bacteria | + | [89] | |||||||
Aedes aegypti | galectin-6, galectin-14 | Reduces exogenous toxin toxicity | + | [57][58] | [57,58] | ||||||
Anopheles darlingi | n.d. | ||||||||||
Anopheles stephensi | n.d. | ||||||||||
Culex quinquefasciatus | n.d. | ||||||||||
Drosophila ananassae | n.d. | ||||||||||
Drosophila mojavensis | n.d. | ||||||||||
Drosophila pseudoobscura | n.d. | ||||||||||
Drosophila virilis | n.d. | + | Predicted by [87] | ||||||||
Drosophila willistoni | n.d. | ||||||||||
Drosophila yakuba | n.d. | ||||||||||
Glossina morsitans | n.d. | ||||||||||
Malus domestica | n.d. | ||||||||||
malectin | Aedes aegypti | n.d. | + | [27][28] | [27,28] | ||||||
Drosophila melanogaster | n.d. | + | |||||||||
Calnexin/calreticulin | Bombyx mori | Calr/Canx; BmCNX | Responds to ER stress | + | + | [30][90] | [30,90] | ||||
Drosophila melanogaster | Cnx | Regulates the function of sodium channel paralytic | + | [32] | |||||||
F-type lectin | Drosophila melanogaster | Furrowed | Functions in planar cell polarity | + | [37] | ||||||
Anopheles gambiae | n.d. | Reviewed by [36] | |||||||||
I-type (immuno-globulin fold) | Drosophila melanogaster | hemolin | n.d. | + | Reviewed by [91] | ||||||
Manduca sexta | HEM | Recognizes PAMPs; promotes nodulation, hemocyte aggregation, and phagocytosis | [63] | ||||||||
Spodoptera exigua | SeHem | Acts as opsonin; regulates phagocytic activities and encapsulation | + | [62] | |||||||
Plodia interpunctella | PiHem | Function related to gut bacteria | + | [92] | |||||||
Bombyx mori | Hemolin | n.d. | + | [93] | |||||||
Actias selene | As-HEM | Mediates immune response | + | [94] | |||||||
Antheraea pernyi | Hemolin | Regulates innate immunity | + | [95] | |||||||
L-type | Drosophila melanogaster | ERGIC-53 homolog | n.d. | [48], reviewed by [96] | |||||||
Bombyx mori | ERGIC-53 | Responds to ER stress | + | [50] | |||||||
R-type (ricin B type) | Drosophila melanogaster | lectin domain of GalNAc Transferase | Binds glycopeptides | + | [97], reviewed by [65] |
a some publications have predicted lectins but did not assign names for these lectins; therefore, there are some blanks in the table. b RNA verification studies included RT-qPCR, dsRNA silencing, and transcriptome analysis. c Protein verification included immunoblotting, recombinant protein production, etc.
Before hemocytes can activate the immune response, the pathogen or immune target must be recognized. During pathogen invasion, pathogen associated molecular patterns (PAMPs), such as bacterial peptidoglycan or fungal β-1,3-glucan, are recognized by specialized proteins called pattern recognition receptors (PRRs) [98]. The Gram-negative binding proteins (GNBPs), and peptidoglycan recognition proteins (PGRPs), are the two major PRR families. GNBPs mainly recognize fungal and Gram negative bacterial PAMPs, while PGRPs mainly respond to Gram positive bacteria [98]. Since many PAMPs are carbohydrate structures, lectins constitute an important part of the membrane bound or extracellular PRRs of hosts.
Lectins have been reported to bind and aggregate pathogens such as bacteria because of their recognition of carbohydrate structures. CTLs of H. armigera and M. sexta, were shown to bind various PAMPs, such as lipopolysaccharide (LPS), fungal glucan and peptidoglycan, to activate the humoral and cellular immune defenses [9,76,99,100]. In Drosophila, CTLs such as DL2 and DL3 can either be secreted or bound to the plasma membrane of hemocytes and they were shown to bind some Gram-negative bacteria and agglutinate them [15]. While many insect PRRs belong to the C-type lectin family, lectins from other families can also function as PRRs. For example, galectins have been reported to recognize and bind pathogen surface glycans [53]. The silkworm, B. mori, possesses a dual-CRD galectin which can bind a series of PAMPs, such as LPS, LTA (lipoteichoic acid), peptidoglycan and laminarin, and was shown to agglutinate E. coli, Staphylococcus aureus and Bacillus subtilis [89,101].
Many hemocytes can engulf the invading pathogens as well as dead cells or other entities in a process called phagocytosis [13,102]. Upstream events of phagocytosis include the recognition of the targets by the PRRs, which activates downstream events including receptor cross-linking, membrane remodeling, phagosome formation and maturation, and finally phagosome fusion with the endosomes and lysosomes to kill the pathogens by the acidic environment, AMPs, digestive enzymes, etc. [103]. To increase the efficiency of phagocytosis, hemocytes sometimes rely on opsonins, molecules that can coat and aggregate the pathogens, such as bacteria and viruses, to limit their mobility and promote recognition [103]. Lectins have been proven to stimulate phagocytosis, by acting as PRRs to detect pathogens or as opsonins to coat the invaders. For example, rHa, a CTL-lectin obtained from H. armigera, has two CRDs which are both required for lectin agglutination of rabbit erythrocytes, but any single domain is sufficient for aggregation of Gram-negative bacteria, Gram-positive bacteria, and fungi. Injection of rHa-lectin together with Bacillus thuringiensis bacteria in the insects, efficiently decreases the B. thuringiensis number in vivo, and hemocytes of H. armigera engulfed more B. thuringiensis in the presence of rHa lectin [104]. CTL-mediated phagocytosis was also observed in mammalians and shrimps [105,106]. Besides the CTLs, the I-type lectin hemolin, SeHem, from S. exigua also helped the host cells to eliminate bacteria by enhancing phagocytosis [62]. Galectins from Crustaceans have been reported to enhance host phagocytosis [107] but there are no such reports for insect galectins.
When the invading targets are too large, such as parasitoids or nematodes, a group of hemocytes are recruited to surround the targets, forming a capsule-like structure in a process termed encapsulation. In Drosophila, lamellocyte precursor cells are activated upon infection with parasitic wasp eggs and will differentiate into mature forms [16,108]. These cells are recruited to the site of infection, attach to the surface of the parasites, and undergo morphological changes to spread around the parasitoids [16]. This process in which the lamellocytes are flattened is called cell spreading and relies on phosphatase/kinase mediated cytoskeleton rearrangement and activation of adhesion proteins [109–111]. Cell spreading is a very fast reaction, 20 min after stimulation most of the Drosophila S2 cells already have entered this spreading state [112]. The spreaded cells cover the parasite to form the capsule. Stabilization of the capsule depends first on intercellular septate junctions, these ladder-like structures are composed of multiple adhesion proteins such as contactin, neurexin, fibronectin, etc. [113]. Second, melanization follows to strengthen the capsule and to kill the parasites. Melanization is a process in which phenols are oxidized to quinones that can be polymerized to form melanin [114]. The deposition of melanin will darken the capsule [115]. Encapsulated targets are restricted in their movement and are finally killed directly by melanization derived toxic components, such as quinones, reactive oxygen intermediates, and AMPs [115], or indirectly by nutrient deprivation [116].
Insect lectins have been shown to be involved in both the encapsulation and the melanization. One common method to study the encapsulation in vitro is the use of synthetic beads incubated with isolated hemocytes. Synthetic beads such as agarose or Sephadex can attract hemocytes which form capsules around the beads that can be easily observed under the microscope [109]. Coating of these beads with stimulating proteins can accelerate and increase the encapsulation [78]. For example, recombinantly produced Drosophila CTLs, DL2 and DL3 were coated on Ni-NTA agarose beads which can attract hemocyte attachment. These hemocytes will aggregate to the bead surface to form capsules and become darker colored after longer incubation, and this process can be blocked by antibodies targeted against the recombinant proteins [78]. Besides the in vitro test, injecting the coated beads into the insect hemocoel can also validate the hypothesis. In H. armigera, a CTL, HaCTL3, was coated on Sephadex A-25 beads and injected into the H. armigera larval hemocoel. After 12h the beads were found to be extensively encapsulated and melanized [99]. Besides CTLs, the I-type lectin SeHem was also reported to coat non-self-targets for encapsulation [62]. While many lectins have been reported to participate in encapsulation and melanization [9], it is not very clear which receptors on the plasma membrane are responsible for the effect. One possible explanation is through interaction with integrins. Evidence suggests that silencing of β-integrin, a hemocyte membrane protein participating in cell-cell adhesion and signal transduction, can effectively decrease the encapsulation of beads [99]. In addition, CTL mediated melanization is suggested to be specific. In an in vitro test, the immune lectin MsIML from M. sexta was shown to be able to activate a protease cascade required for phenoloxidase activation, which only happens when this lectin binds to the LPS. Phenoloxidase is proven to be a key enzyme for melanization [114,117].
Besides the cellular response, the insect host can secrete a series of extracellular effector molecules that can kill foreign invaders. Among these effectors, AMPs are the major participants [118]. AMPs are positively charged small peptides, consisting of 15-45 amino acids, which can bind to the normally negatively charged surface of microbes leading to membrane rupture and cell lysis [119]. The healthy host cells are protected from AMP damage mainly due to the cholesterol rich plasma membrane which makes healthy cells positively charged to repulse cationic AMP attachment [119]. However, when host cells are not healthy, they can be attacked by the AMPs. In the study of Drosophila tumor genesis, tumor cells tend to have a negatively charged cell surface due to the phosphatidylserine turning inside-out, allowing the AMP Defensin to locate and attack these cells to limit the tumor growth [120].
Classification of AMPs can vary based on different criteria, such as the type of the target microbe (anti-fungal or anti-Gram-positive/negative-bacterial AMPs) or based on the pathway by which they are activated (such as Toll regulated or Imd regulated AMPs) [118]. But neither classification system can perfectly group different AMPs. While AMPs like Drosomycin (Drs) are highly specific against fungal infections, other AMPs have a broader pathogen specificity. For example, Metchnikowin (Mtk) can target all three groups of pathogens mentioned above. In addition, the AMP regulation can also be complex, for example, while Drs is regulated by the Toll pathway, many others, such as Defensin (Def), are coregulated by both pathways [118,119].
The insect fat body around the body cavity is the major tissue to secrete AMPs [121]. When stimulated by the pathogen, the AMP titers in the hemolymph can drastically increase within 30 min and the concentration can reach up to 300 µM (reviewed by [15]). In addition to the fat body cells, hemocytes can also produce AMPs. For example, isolated hemocytes from the blue blowfly, Calliphora vicina, show the same ability to produce AMPs such as defensin, cecropin, diptericins, and proline-rich peptides [111]. Drosophila hemocyte-like S2 cells can also produce all kinds of AMPs upon stimulation by E. coli or other protein stimuli [112,122].
Insect lectin mediated pathogen recognition can trigger production of AMPs. Insect lectins are commonly coregulated with AMPs [123], but a recent study gives more direct evidence that the insect lectin can regulate AMPs. After silencing of HaCTL3, a CTL from H. armigera participating in larval development, the fat body expressed far less AMPs than in the control group. HaCTL3 was found to regulate different AMPs, including lebocin, attacin, cecropin 1, pre-gloverin, pre-lebocin and cecropin, of which the antimicrobial activities were confirmed by in vitro assays. And more interestingly, the upstream PRRs, including PGRPs, β-1,3-GRPs and even a CTL4, were also downregulated, suggesting lectin-regulated AMP production might be initiated through affecting upstream recognition events [76].
Within the host immunity, insect lectins and AMPs can have complex interactions. Insect lectins can protect the beneficial host microbiome against the toxic effects of AMPs. For example, silencing of A. aegypti C-type lectins (mosGCTLs), which are coregulated with AMP through the Imd pathway, leads to failure of colonization and maintenance of the gut microbial flora [124]. In addition, the AMP toxicity significantly decreased when bacteria were pre-coated by mosGCTLs, which blocked AMP deposition on the bacterial surface [124]. Viruses are also reported to use host lectins. The West Nile virus (WNV), a pathogen causing West Nile fever and transmitted by mosquitos, can stimulate expression of an A. aegypti C-type lectin, mosGCTL-1, which can strongly interact with a mosquito phosphatase, mosPTP-1. WNV uses mosGCTL-1 to coat its surface and enters in the cell through interaction with mosPTP-1 [125].