AMPs are ubiquitous defense molecules found in all life forms; unicellular organisms are thought to employ them during interspecific interactions, whereas multicellular life forms use them as part of their innate immune systems [36]. Despite the economic importance of many crustaceans, AMPs have not been extensively studied in these organisms, and research efforts continue to be channeled into identifying novel AMPs in a range of crustacean species [37]. Such efforts are likely to lead to the discovery of new AMPs, which will help to elucidate patterns in the evolution of these peptides in this animal group. To date, six AMPs have been identified in shrimp (order Decapoda, family Penaeidae) and 16 chemically distinct AMPs within the subphylum Crustacea (Table 1).

Interestingly, not all of the AMPs are found in all the crustacean groups. While some are found in all Crustacean orders [46], hinting at an ancient origin of these molecules, others are limited to one or a few families, thereby suggesting a more recent evolution (Table 1). A good example of an ancient class of AMPs are the ALFs, which have undergone intense diversification. In contrast, penaeidins are a class of AMPs found in a single family of crustaceans, where they have diversified into four distinct subtypes. Therefore, there is a vast diversity of AMPs that could be elucidated through future bioinformatics and proteomic searches [59]. Clues as to the causes of the great diversification of some AMPs in the Crustacea subphylum can be found by looking at the amino acid composition. Many AMPs show a clear amino acid bias, being rich in Pro, Pro/Arg, or Gly. These amino acids cause the peptide to acquire certain structures and have a cationic nature (although a few anionic AMPs do exist). These properties predispose the peptides to interact in certain ways with the membrane of pathogens.

Thus, some AMPs may have resulted from nonsense mutations in large proteins that have domains rich in these amino acids. Pro-rich proteins are ubiquitous in eukaryotic genomes, and are thus common signaling molecules. In general, Pro/Arg-rich proteins that can provide a blueprint for AMPs are not as common, but they can be found throughout the genomes of several eukaryotic organisms and have diverse functions, such as C9ORF72 [60]. However, within the Crustacea subphylum, Pro-rich AMPs have a diverse evolutionary origin [61]. Gly-rich proteins are common throughout Eukaryotes and often have diverse functions [62]. Prior to the mutations that may have led to the creation of putative “pre-AMPs”, the gene in question would have had to undergo duplication to avoid the loss of functionality. These genes are molecular “hopeful monsters”, resulting in new lineages of selfish genes that confer an important immune advantage to the individual, vis a vis population, in response to pathogens.

The proposed model for the diversification of other AMPs would also hold for the truncation of proteins that preserve entire domains, such as the single whey acidic protein (WAP) which contains eight well-conserved cysteine residues forming a four-disulfide core [31]. Truncation of proteins is followed by a nonsense mutation in one or more orthologs, of which only those with antibacterial properties confer an advantage to the animal. These novel AMPs may then have further multiplied through gene duplication and mutation. The observation that the antibacterial activity of AMPs is due to the properties of a certain group of amino acids suggests that the de novo synthesis of artificial AMPs could stem from the search of amino acid sequences with a similar bias or the search for motifs or domains with a heavy bias in the aforementioned amino acids [59].

Crustaceans belong to the phylum Arthropoda, and despite not having the sophistication of an adaptive immune system, they have a highly effective innate immune system to fight infections [63]. The first lines of crustacean defense are internal and external physical barriers. A strong external cuticle protects the body and the stomach, and the peritrophic membrane covers the midgut and hindgut [64]. The circulatory system of crustaceans is open; thus, the circulatory fluid, called hemolymph, flows freely through the circulatory system, which has open endings into the lacunal system (the hemocoel), which is in direct contact with tissues. The hemolymph has two components: cellular and humoral. The former is composed of hemocytes, the main immune effectors of the crustacean immune system. In contrast, several proteins, namely the respiratory pigment hemocyanin, the clotting factor, and several AMPs, among others, make up the humoral component. The crustacean hemolymph performs the functions of both the lymph and blood, and thus its biochemical and cellular components reflect the dual function of this tissue. For example, hemocyanin transports oxygen and participles in the immune response [65,66,67].

The innate immunity of crustaceans consists of a cellular and humoral response. The former involves the hemocytes, classified into three major types: hyaline cells (HCs), large granular cells (LGCs), and small-granular cells (SGCs). These cells exert diverse roles (Wu et al., 2019) as they interact with pathogens. HCs are involved in phagocytosis and coagulation, while SGCs participate in phagocytosis, nodule and capsule formation, and the release of the molecules of the prophenoloxidase (proPO) system and AMPs. LGCs store immune effector molecules, AMPs, and enzymes of proPO [68,69,70]. The proportions of each type of hemocyte in the hemolymph depend on various factors, such as molting [71,72]. Hemocytes are produced by hemopoietic tissues. In shrimp, these tissues are located as various epigastric lobules, mainly latero-dorsally to the cardiac stomach and in the proximal region of the maxillipeds. Hemopoietic hemocyte growth and differentiation is likely to be mediated by cytokines [68,73,74].

Given the open nature of the shrimp circulatory system, hemocytes easily infiltrate tissues, and can respond in a localized way or migrate to specific tissues (gills, lymphoid organs, and connective tissue), where they trigger cellular reactions or release a range of compounds. The cellular reaction includes phagocytosis, which is performed mainly by HCs and SGCs, and is accompanied by degradative reactions via the generation of lytic enzymes or reactive oxygen or nitrogen species (ROS and RNS) generated during the respiratory burst [75]. In response to massive invasions of microorganisms or large microorganisms, hemocytes form cell aggregations called nodules. Denser cell aggregations made up of several layers of hemocytes are known as capsules. Sometimes, nodules or tubules of the hepatopancreas (invaded by Vibrio spp.) appear to be encapsulated by hemocytes. Melanin, a sticky and toxic dark pigment, is deposited in a very localized way in the central areas of nodules and capsules, or in wounds during the healing process. Melanin is generated by phenoloxidase, an enzyme that is produced via the activation of proPO system. The proPO system, one of the main immunoeffector responses of crustaceans, comprises a series of enzymatic cascade reactions [76] triggered by components of the cell walls of microorganisms, such as β-1,3-glucans from fungi, or lipopolysaccharide (LPS) and peptidoglycans from Gram-negative and Gram-positive bacteria, respectively [77]. Melanin formation generates intermediate reactive compounds (quinones and ROIS), which are more toxic than melanin. ProPO oxidase is regulated by several protease inhibitors, among them, pacifastina and α2-macroglobulin, produced by HCs. α2-macroglobulin inhibits serine proteases of the proPO cascade, and possibly pathogen proteases [78]. Hemocytes also participate in another cellular reaction, namely, extracellular traps (ETs). This process involves the formation of DNA structures and AMPs to trap microorganisms. In the shrimp Marsupenaeus japonicus, ETs associated with c-type lysozyme have been described [79].

In addition to hemocytes, other tissues of the penaeid shrimp are involved in the immune response, including the heart reserve of phagocytes the podocytes of the gills, antennal gland, and the lymphoid organ (LO). The latter, which is near the hepatopancreas, receives hemolymph from the subgastric artery that forms a vascular plexus within this structure. The LO appears to filter viruses from the hemolymph [80]. Very strong hemocyte infiltration is detected in the LO, contributing to the immune functions of this organ. Many genes and molecules associated with hemocytes have been reported in the LO [80,81]. In this regard, a strong signal of penaeidin and α2-macroglobulin has been detected in the edges of tubules of the LO, in the same zone that participates in virus filtration. During infection, mainly that of viral etiology, nodular hyperplasia, known as LO spheroids (LOS) appear in the LO. These hyperplasic cells show a positive signal toward penaeidin and α2-macroglobulin [80]. It has been outlined that LOS are involved in the phagocytosis and destruction of foreign particles [82]. Terminal type C LOS exhibits an apoptosis signal, thus indicating the fate of engulfed material. Ectopic spheroids have also been detected in the connective tissues of other organs (stomach and buccal appendages).

Initially, there was some controversy regarding whether apoptosis as a response to viral infections in shrimp was a defense mechanism or a pathogenicity mechanism triggered by the virus [83]. The literature has reported that in the early stages of white spot syndrome virus (WSSV) infection, the virus exerts a mechanism to prevent host cells from entering apoptosis [84]. This observation thus indicates that apoptosis is a mechanism associated with the host’s immune response.

Humoral effectors involve the synthesis and secretion of immune proteins such as AMPs from hemocytes into the hemocoel. Part of the humoral response is the clotting system, which is stimulated by injuries and by the binding of microbial stimulants, such as β-1,3-D-glucans, to receptors on hemocytes. These cells release Ca²⁺-dependent transglutaminases, which promote the polymerization of clotting proteins [85]. Finally, proteins derived from hemocyanin and hemocyanin-derived proteins have multiple functions in the hemolymph, from oxygen transport, antimicrobial activity, and agglutination, to playing a major role in the proPO system [65,66,67,85]. The multiple roles of hemocyanin-related proteins, as well as the diversity of AMP families, suggest that gene duplication and subsequent functional diversification are an important feature of the crustacean immune response. The open nature of the circulatory system of crustaceans, which is composed of vessel systems and a system of lacunae and sinuses surrounding the most important organs [86], facilitates the rapid migration and infiltration of hemocytes in different tissues, where they can then release their molecules in a localized manner.

Recognition molecules, including various types of lectins, galectins, β-1,3-glucanase-related protein, Toll-like receptors (TLR), Thioester-containing proteins (TEPs), scavenger receptors, Down syndrome adhesion molecules, and fibrinogen-related proteins, can circulate freely in the hemolymph or be bound to cell membranes. These molecules are able bind to pathogen-associated molecular patterns (PAMPs). Thus, signaling cascades in hemocytes are triggered, leading to the production and exocytosis of AMPs [87] and lysozymes, as well the activation of the proPO system.