Rethinking Disease Control in Aquaculture Invertebrates: Harnessing Innate Immunity in Molluscs and Crustaceans

Abstract

Aquaculture of molluscs and crustaceans represents an important and expanding sector within global food production. The intensification of these systems has been accompanied by an increased prevalence and severity of infectious diseases, which continue to constrain productivity and sustainability. Current disease management approaches include biosecurity measures, husbandry practices, therapeutics, and selective breeding, which have shown limited efficacy against many emerging pathogens affecting invertebrate species. Unlike finfish, aquatic invertebrates lack adaptive immunity and rely exclusively on innate immune mechanisms, limiting the effectiveness of traditional vaccine strategies. There is growing interest in immunostimulants that enhance innate defenses and support immune priming or trans-generational immune priming (TGIP). This review summarises the current understanding of immune defence mechanisms in molluscs and crustaceans and examines recent progress in the development of immunomodulators and prophylactic interventions aimed at improving health outcomes and disease resilience in invertebrate aquaculture.

1. Introduction

Invertebrate aquaculture, the cultivation of various species of shellfish including molluscs (abalone, conch, clams, oysters, mussels, scallops, and cockles) [1,2,3,4,5,6,7] and crustaceans (shrimps, prawns, lobsters, and crabs) [8,9,10,11] encompasses a diverse range of environments, including freshwater, marine, and coastal settings. The farming of molluscs and crustaceans plays a vital role in meeting global demands for seafood alongside popular finfish production at 65%, contributing approximately 23% and 11% in crustacean and molluscan products, respectively [12]. Demand for crustacean and molluscan products has increased in popularity, with aquaculture surpassing wild caught production in 2022, producing 94.4 million tonnes of aquatic animals, a 6.7 million-tonne increase from wild catch [12]. Crustaceans and molluscs make up 24.6% and 15.6% of this production, respectively, second only to finfish aquaculture at 58.1% [12]. Thus, crustacean and molluscan aquacultural farming is vital to meet global seafood consumption demands, where wild-caught fisheries fall short and fail to meet these demands due to strict fishing regulations to combat overfishing [13].

The intensification of invertebrate aquaculture has brought numerous challenges inhibiting production, most notably infection and disease such as viruses [14,15,16], bacteria [17,18,19], fungi [20,21], and parasites [22,23]. Moreover, as aquatic invertebrate species lack adaptive immunity, relying exclusively on nonspecific innate immune responses, traditional vaccine practices are not useful. Additionally, in contrast to vertebrate immunology, the mechanisms of innate immunity in non-model organisms remain inadequately characterised [24,25], making it challenging to design anti-infectives and placing an overreliance on strict biosecurity practices, which often prove to be inefficient as evidenced by ongoing outbreaks on farms.

Immunostimulants are becoming an attractive alternative to traditional vaccines for invertebrate aquaculture, as invertebrates lack the antibody-based adaptive immunity on which most conventional vaccines rely on and instead rely solely on innate immune systems [26]. These immunostimulants are described as substances or nutrients that can enhance a nonspecific immune response and protection against pathogens and disease [27,28]. They do not directly interact with the pathogen to reduce infection and disease; rather, they enhance the immune response of the host [29,30]. The extent of enhanced immune response can last for a long period of time, extending beyond treatment of the current infection or disease [31,32].

This review focusses on the application of immunostimulants within aquaculture as an alternative strategy for intensively farmed marine invertebrates and defines and explores immune priming and immune memory of invertebrates. Immunostimulants such as bacterial proteins (flagellins), nucleic acids (dsRNA and CpG), and carbohydrates (β-glucans, peptidoglycans and lipopolysaccharides) are critically compared, discussing the efficacy and effectiveness in protection against commonly occurring virulent diseases in marine animals. Furthermore, challenges to screening suitable immunostimulant candidates, synthesis, delivery, and toxicity are explored in connection with the current immunostimulants discussed. This review aims to provide overall progress into immunostimulant use for pathogen disease prevention and resilience for the aquatic invertebrate aquacultural farming systems by reviewing literature that has focussed on the use of immunostimulants for pathogen defence in pathogen challenge trials only.

2. Innate Immunity in Invertebrates of Aquaculture

The innate immune system of various aquatic invertebrates is composed of two different mechanisms: cellular and humoral. Cellular defence is mediated by haemocytes within the circulatory system that target and eliminate pathogens through cellular processes, such as phagocytosis, superoxide production, pathogen encapsulation, haemocyte nodulation, and enzyme release. Humoral defence targets and eliminates pathogens through processes such as melanisation, haemolymph agglutination, activation of prophenoloxidase (proPO), and the secretion of antimicrobial peptides (AMPs). Various immune responses are activated through the interaction of different pattern-associated molecular patterns (PAMPS) of different pathogens or immune activators and pathogen recognition receptors (PRRs). This interaction triggers various immune signalling pathways and effector molecules involved in cellular and humoral immunity for pathogen clearance (Figure 1).

Multiple signalling pathways, PRRs, and immune effectors have been described for both crustaceans and molluscs, with many of these genes encoding innate immune receptors such as toll-like receptors (TLRs) to be homologous to those identified in vertebrate and mammalian species. However, within invertebrates, the number of TLRs are far more immense and largely undefined than the well-annotated and described 10–15 TLR families of vertebrates [34,35]. An example of this is observed in the Pacific oyster (Crassostrea gigas) [29] and the purple sea urchin (Stronglyocentrotus purpuratus) [36], which have approximately 83 and 222 TLR-encoding genes, with the mRNAs of these TLRs widely expressed in different tissue and stimulation by different PAMPS of Gram-negative/positive bacteria [37,38,39], viruses [40], fungal, parasitic, and immune stimulators [41,42]. Thus, suggesting that although these aquatic invertebrate species lack a highly specialised adaptive immune response typical of vertebrate and mammalian species, their innate immune defence has likely evolved to become more specialised to combat ongoing pathogen pressures.

3. Immune Priming and Trans-Generational Immune Priming

Immune priming is a phenomenon first described in arthropods, whereby there is an enhanced immune response to a pathogen or immune activator following previous exposure to a PAMP, resulting in enhanced humoral and cellular immune responses [43,44,45,46]. The exact mechanisms of immune priming have been largely debated, with nonspecific immune priming in invertebrates easily supported by a short-term elevation in generalised immune responses, such as that observed in increased haemocyte count, transcriptional changes of innate immune cells, and molecules such as AMPs. Thus, the definition of immune priming has varied amongst literature to include either of the following: increased survival to pathogen following infection in vivo, cytokine production, immune gene expression, and the up/downregulation of surface markers in circulating haemolymph fluid or within haemocytes challenged after priming in vivo or in vitro, as accepted by previous reviews [30,31,47,48,49]. Regardless of the exact mechanisms for confirmation of immune priming, all true immune priming experiments must involve the following: increased immune response, survival, and decreased pathogen load following a homologous challenge with a pathogen rather than a heterologous challenge (Figure 2A) [32,50,51].

Trans-generational immune priming (TGIP) expands on the immune priming phenomenon through the parental transfer of immunological protection against pathogens to the progeny (Figure 2B), whereby different immunological factors are transferred from parents to offspring, either vertically or horizontally between adults and non-parental juveniles [52]. These transferred components, such as immune effectors, signalling molecules, or PAMPS previously described in Section 2 can prime the innate immune system of offspring to better respond to pathogens previously encountered by their parents [31,32,52,53]. TGIP is typically nonspecific and maternally mediated, for example, through the transfer of AMPs to eggs during oogenesis [54]. However, the mechanisms underlying specific TGIP remain poorly understood and are debated [52,53]. For aquatic invertebrates, the first evidence of TGIP was reported in the shrimp Penaeus monodon, where maternal exposure to the fungal immunostimulant β-glucan enhanced offspring resistance to white spot syndrome virus (WSSV) [55]. TGIP has also been demonstrated in different molluscan species, albeit in far less studies than what has been recorded for crustaceans, with these studies restricted to bacterial whole-cell priming against pathogenic Vibrio spp. [56,57,58] and Polyinosinic-polycytidylic acid (Poly(I:C)) against the devastating viral Ostreid herpesvirus (OsHV-1) [59,60] discussed in more detail later in this review in Section 4.2 and Section 4.5. Although TGIP has since been observed in various invertebrates, its molecular basis, ecological relevance, and consistency across species remain uncertain. Inconsistencies in TGIP success are likely limited within the literature due to selective publication biases in overreporting positive results that support TGIP. Additionally, host–pathogen interactions, degree of pathogenic exposure, and timing between parental priming, conception, and challenge of progeny affecting TGIP success.

4. Immunostimulants Utilised for Immune Priming and Trans-Generational Immune Priming Experiments

4.1. Protein

4.1.1. Flagellin

Flagellin is a major protein of the bacterial flagellum and acts as an immune stimulant in vertebrates and some invertebrates. For aquatic invertebrates, flagellin is utilised as a vaccine adjuvant or research tool for understanding host–pathogen interactions [61,62,63,64]. Flagellin A (FlaA) has demonstrated antiviral activity against highly pathogenic WSSV in shrimp species, Marsupenaeus japonicus [65]. Shrimps intramuscularly injected with 1 μg per 5 g shrimp, reduced WSSV viral load at 24 and 48 h post infection, and increased survival by 40% 96 h post-WSSV challenge [65] (

Table 1. Protein-based immunostimulants utilised in crustacean vaccination and immune priming experiments.

Immunostimulant	Source of Immunostimulant	Animal	Disease	Survival %	Administration	Source
Flagellin 2 fusion with VP28 and VP19	Salmonella typhimurium and WSSV	Macrobrachium nipponense and Palaemon paucidens	WSSV	84.2–89.5% at 15 days post challenge	Oral within feed	[71]
Flagellin A	Vibrio anguillarum	Marsupenaeus japonicus		40% at 4 days post challenge	Injection	[65]

). Prior work discovered that FlaA of bacterial species Vibrio anguillarum interacted with shrimp scaffold protein, Shoc2, involved in activating antibacterial immune responses. FlaA activated extracellular signal-regulated kinase (Erk) and signal transducer and activator of transcription (Stat) signalling pathways through Shoc2 [63]. Stat signalling regulates several antiviral effectors, demonstrating a critical role in viral clearance and inhibition of viral replication through the regulation of ficolin [66,67,68]. Ficolin recognises and attaches to carbohydrates on the virion surface, enhancing phagocytosis and complement activation [69,70]. Thus, the antiviral properties of flagellin may occur in shrimps through the interaction between flagellins, Shoc2 proteins, and subsequent Erk/Stat signalling activating immune effectors, such as ficolin, that are directly involved in innate immunity.

4.1.2. Structural Pathogen-Specific Components

Protein-based immunostimulants in crustacean immune priming experiments primarily target pathogen structural proteins, largely WSSV capsid proteins (VP28, VP19, VP26, VP24, and VP15), given their key roles in viral entry and assembly [72,73,74,75,76,77,78,79,80,81]. Numerous studies demonstrate these proteins enhance host protection across diverse crustaceans, including crayfish, freshwater prawns, and marine shrimp, although protection levels vary depending on the immunostimulant delivery method, concentration, and expression system employed [75,78,82,83,84]. Feed-based delivery of baculovirus-expressed VP28 produced slightly superior survival rates compared with immersion [85], while oral administration yielded protection levels marginally lower than intramuscular injection [77] (

Table 2. Viral capsid protein-based immunostimulants utilised in crustacean vaccination and immune priming experiments.

Immunostimulant	Source of Immunostimulant	Animal	Disease	Survival %	Administration	Source
VP28	WSSV	Penaeus monodon	WSSV	Immersion: 75% and 68.4% Feed: 81.7% and 76.7% 15 days post challenge	Immersion and feed (baculoviral) 3 and 15 days post vaccination	[85]
				86.3% and 73.5% at 3 and 15 days post treatment	Injection (baculoviral) 3 and 15 days post vaccination	[77]
		Marsupenaeus japonicus		Injection: 90% 25 days post challenge Feed: 85.7% 20 days post challenge	Injection and oral within feed	[86]
		Procambarus clarkii		VP28 protein: 43.5% VP28 baculovirus: 91.5% 30 days post challenge	Intramuscular injection	[88]
VP19 and VP466		Penaeus chinensis		Injection: VP19—49.8%, VP466—48.2% Feed: VP19—50.8%, VP466—10.8% 15 days post challenge	Injection and oral within feed	[87]
VP26 and VP28		Penaeus japonicus		VP26: 60% for 1 dose, 68% for 2 doses VP28: 24% for 1 dose, 96% for 2 doses 10 days post challenge	Injection—1 or 2 doses 30 days post vaccination	[75]
VP19 and VP28		Penaeus monodon		VP19: 55–70% 2 and 25 days post vaccination VP28: 55–45% 2 and 25 days post vaccination VP19 + VP28: 50–60% 2 and 25 days post vaccination 16 days post challenge	Intramuscular injection 2 and 25 days post vaccination	[78]
				VP19: 17% 0 days after vaccination VP28: 70% 0 and 3, 77% at 7, and 50% 21 days after vaccination VP19 + VP28: 50% 0 days post vaccination	Oral delivery	[83]
		Procambarus clarkii		Injection: VP28 at 60.4%, VP19 + VP28 at 63.5%, VP19 at 47.09% and VP28 at 60.17%, VP19 + VP28 at 53.5%, VP19 at 43.54% on 21 days post vaccination Feed: VP28 at 56.8%, VP19 + VP28 at 46.8%, VP19 at 24% 3 and VP28 at 51%, VP19 + VP28 at 43.5%, and VP19 at 18% on 21 days post vaccination Immersion: VP28 at 53.7%, VP19 + VP28 at 47%, VP19 at 17% on 3 and VP28 at 53.5%, VP19 + VP28 at 43%, VP19 at 13.7% on 21 days post vaccination 25 days post challenge	Intramuscular injection Oral Immersion	[84]
				3 days post injection: VP28: 91.2%, VP19: 49.1%, VP19 + VP28: 84.6% 15 days post challenge 21 days post injection: VP28: 78%, VP19: 17.9%, VP28 + VP19: 75.7% 15 days post challenge	Injection expressed in P. pastoris yeast	[82]
				VP19: 60% VP19 + VP29: 56.7% 10 days post challenge	Oral Synechococcus spp. PCC7942 with VP19, VP28 and VP19 + VP28 for 7 days, challenged 12 h later	[89]
				3 days post vaccination (i) VP28: 70%, VP19: 6%, VP19 + VP28: 42% (ii) VP28: 86%, VP19: 22%, VP19 + VP28: 65% (iii) VP28: 23%, VP19: 15%, VP19 + VP28: 27% (iv) VP28: 86%, VP19: 29%, VP19 + VP28: 72% 21 days post vaccination (i) VP28: 36%, VP19: 8%, VP19 + VP28: 22% (ii) VP28: 65%, VP19: 8%, VP19 + VP28: 44% (iii) VP28: 15%, VP19: 1%, VP19 + VP28: 8% (iv) VP28: 72%, VP19: 18%, VP19 + VP28: 65% 25 days post challenge	Oral delivery in feed for 25 days and expressed in P. pastoris yeast. (i) Whole culture inactivated (ii) Supernatants of inactivated cells (iii) Pelleted cells from whole culture resuspended in PBS (iv) Sonicated whole cells	[90]
				VP28: 94.7% VP19: 89.5% VP28 + VP19: 96.5%	Oral delivery within feed and expressed in baculoviral-infected silkworms, B. mori, expressing VP28 and VP19 proteins for 30 days	[91]
				VP28: 84.4% 20 days post challenge	Oral delivery within feed and expressed in baculoviral-infected silkworms, B. mori, expressing VP28 for 30 days	[92]
		Litopenaeus vannamei		VP28: 62.2% VP19: 57.8% VP19 + VP28: 71.1% 10 days post challenge	Oral Synechococcus spp. PCC7942 with VP19, VP28 and VP19 + VP28 for 10 days	[93]
				VP19: 33.3%, VP28: 58.3% 21 days post challenge	Oral within feed	[94]
		Penaeus japonicus		Feeding for 2 weeks: 3 days post feeding at 50 µg: 92.8%, 69.03%, and 80.3%; at 10 µg: 62.09%; and at 1 µg: 60.7% 7 days post feeding: 55.8% 14 days post feeding: 33.4% and 24.9% Sonicated, 3 days post feeding: 0%, 3.66% Sonicated, 7 days post feeding: 12.07% Sonicated, 14 days post feeding: 12.5%, 15.9% Feeding for 1 day: 50 µg 1 day post feeding: 72.5% Feeding for 3 days: 50 µg for 1 day post feeding: 73.7% Feeding for 1 week: 50 µg for 1 day post feeding: 76.9% 2 weeks following viral challenge	Oral feed for 14, 3, or 1 day	[95]
		Litopenaeus vannamei		100-fold LD50 WSSV: N-VP28: 23.6% (oral) NC-VP28: 2.4% (oral) 10-fold LD50 WSSV: N-VP28: 38.2% and 46.2% (oral) NC-VP28: 14.9% (oral) N-VP28: 59% (injected) 1-fold LD50 WSSV: N-VP28: 69.2% (oral) N-VP28: 73.1% (injected)	Truncated VP28 to remove N and N + C terminus. (N-VP28) and (NC-VP28) administered by intramuscular injection or oral feeding	[96]
				Homologous recombination VP28: 56.66% Transgenic VP28: 43.33% 10 days post challenge	Synechocystis spp. PCC6803 expressing VP28 either through homologous recombination or transgenic insertion orally given in feed and challenged 7 days later	[97]
				VP28: 80.2%	Oral Synechococcus spp. PCC6803 with VP19, VP28 and VP19 + VP28 for 10 days	[98]
				VP28: 87.10% 5 days post challenge	Oral within feed containing VP28 surface expression in S. cerevisiae	[99]
		Fenneropenaeus chinensis		B. subtilis spores and cells VP28: 3 days post vaccination: 72.7% 14 days post vaccination: 83.3% 28 days post vaccination: 71.7% 25 days post challenge	Oral with feed for 20 days	[100]
		Macrobrachium nipponense		3.33% Ana7120 + VP28: 45.6% 6.66% Ana 7120 + VP28: 62.2% 10 days post challenge	Oral-fed Anabaena spp. PCC7120 expressing VP28	[101]
		Litopenaeus vannamei		VP28: 68% 10 days post challenge	Oral-fed Anabaena spp. PCC7120 expressing VP28 for 10 days and challenged 10 days post feeding in post-larval shrimp	[102]
				Codon-optimised VP28: 87% Non-codon-optimised VP28: 0% 8 days post challenge	Transgenic microalgae C. reinhardtii expressing VP28 for oral delivery in feed	[103]
				VP28: 70% 14 days post challenge	Transgenic microalgae C. reinhardtii expressing VP28 within the nucleus for oral delivery in feed	[104]
		Procambarus clarkii		B. subtilis spores and cells VP28: 3 days post vaccination: 65.4% and 43.3% 14 days post vaccination: 78.3% and 51.7% 25 days post challenge	Oral with feed for 20 days	[105]
VP28 fused to CotB or CotC		Cambarus clarkii		B. subtilis spores 0 days post feeding: VP28-CotB: 37.9%, VP28-CotC: 44.8% 7 days post feeding: VP28-CotB: 46.4%, VP28-CotC: 50% and 21 days post feeding: VP28-CotB: 30%, VP28-CotC: 33.3% 15 days post challenge	Oral feeding for 7 days	[106]
VP28 fused to CotB		Litopenaeus vannamei		B. subtilis spores expressing VP28-CotB: 65% 14 days post challenge	Oral feeding	[107]
VP28 and VP26 fused to CotC				B. subtilis spores expressing VP28-CotC: 90% VP26-CotC: 100% 16 days post challenge		[108]
VP28, VP15, VP24, VP26, and VP19		Marsupenaeus japonicus		E. coli expressed: VP28: 50%, VP15: 78%, VP24: 56%, VP26: 44%, VP19: 27% B. mori expressed: VP15: 60%, VP19: 45%	Intramuscular injection	[72]
VP28 and VP36B		Litopenaeus vannamei		VP28: 100% VP36B: 0%	Intramuscular injection	[109]
VP28, VP26, and VP24		Cherax quadricarinatus		VP28: 58.33% and 66.67% VP26: 41.67% VP24: 33.33%	Intramuscular injection	[110]
VP28 fused VP24		Litopenaeus vannamei		VP28: 100% 14 days post challenge	Oral delivery of S. cerevisiae surface expressed VP28-VP24 fused proteins	[111]
VP15 truncated proteins		Marsupenaeus japonicus		VP15 (1–25): 31.6% VP15 (26–57): 57.9% VP15 (58–80): 47.6% VP15 (1–25, 58–80): 38.9% VP15: 42.1% 20 days post infection	Intramuscular injection	[73]
				VP15: 81% VP15 (26–57): 100% 20 days post challenge	Oral delivery within feed and expressed in baculoviral-infected silkworms, B. mori, expressing VP28 for 23 days and challenged 1 week after feeding	[74]

). Similar findings for M. japonicus, where intramuscular injection of recombinant VP28 (rVP28) conferred only a 4.3% higher survival than oral delivery 25 and 20 days post challenge, respectively [86]. Expression of VP19 and VP466 via baculoviral systems provided partial protection in Penaeus chinensis, with VP19-fed groups outperforming VP466, which exhibited approximately 40% lower efficacy [87].

In crayfish, intramuscular injection of VP28 achieved 91.2% and 78% survival at 3 and 21 days post vaccination, respectively, while VP19 elicited markedly lower protection (49.1% and 17.9%) [82]. A combination of both VP28 and VP19 demonstrated similar but lower protection at both timepoints (84.6% and 75.7%) compared to VP28 alone. Subsequent studies corroborated VP28’s superior efficacy across injection, immersion, and oral routes [83,94]. Injection remained the most effective route in P. monodon, Penaeus japonicus, and M. japonicus, consistent with crayfish studies, while baculoviral delivery of VP28 achieved comparable protection at 91.5% 30 days post challenge [88]. Similarly, in Cherax quadricarinatus, VP28 significantly outperformed VP26 and VP24 in conferring WSSV resistance [110], reinforcing VP28’s consistent immune-protection potential across crustaceans.

Recombinant expression of WSSV proteins in Escherichia coli and Bacillus subtilis has been the most prevalent in the literature [72,109]. Most notably, B. subtilis has emerged as a promising non-pathogenic, rapid, and cheap encapsulation method for oral delivery due to resilient spores [100,105,106,107]. B. subtilis spores expressing VP28 consistently provided higher efficacy (78.3%) compared to vegetative cells (5.17%) in Procambarus clarkii, with comparable results observed in Fenneropenaeus chinensis [100,105]. Comparable results occurred when VP28 fused with spore coat proteins (CotB/CotC) in Cambarus clarkii and Litopenaeus vannamei [106,107] and with VP26 fused to CotC in L. vannamei [108]. VP28 expression in Brevibacillus brevis and Brevibacillus chosshienensis also yielded high efficacy in P. japonicus, which reduced with time between priming and challenge, inactivation methods, and modifications in protein structure [95,96]. Algal (including Anabaena spp., Synechococcus spp., and Chlamydomonas reinhardtii) and yeast expression systems (including Pichia pastoris and Saccharomyces cerevisiae) are also cost-effective oral encapsulation methods and non-pathogenic, conferring survival rates between 62% and 100% post-WSSV challenge [82,89,93,97,98,99,101,102,103,104,111]. Silkworm expression platforms in B. mori have also facilitated oral immunization of VP28 and VP19, generating higher survival rates in crayfish (96.5%) and shrimp (VP19: 45%) compared to E. coli expression of VP19 (27%) in shrimp [72,91,92].

4.1.3. Host-Specific Proteins

Fortilin is a host-derived shrimp protein markedly upregulated during WSSV infections in P. monodon [112]. Initial priming studies employing fortilin explored both intramuscular and oral delivery to assess protection efficacy against WSSV in P. monodon shrimp [113]. Combined oral and injection administration conferred absolute protection, while single delivery methods conferred reduced efficacy, with minimal protection observed for oral delivery (10%) compared to injection (90%) (

Table 3. Host-protein-based immunostimulants in crustaceans in immune priming experiments.

Immunostimulant	Source of Immunostimulant	Animal	Disease	Survival %	Administration	Source
Fortilin	Penaeus monodon	Litopenaeus vannamei.	WSSV and YHV	WSSV 1% yeast fortilin: 66.7% 5% yeast fortilin: 91.7% 50 days post challenge Injection of purified fortillin: 100% 20 days post challenge YHV 5% yeast fortilin: >20%	Oral delivery with sonicated 1–5% yeast P. pastoris expressed fortilin protein 3 days prior to challenge	[114]
		Penaeus monodon	WSSV	Injection: 80% Injection + Feed: 100% and Feed: 10% 15 days post challenge	Intramuscular injection of fotilin protein and WSSV; injection and oral delivery in feed and feed alone prior to challenge	[113]
PmAV				purified rPmAV protein: 667% 15 days post challenge	Intramuscular injection of purified rPmAV protein, challenged with WSSV 24 h later	[115]
Rab7		Litopenaeus vannamei.		Rab7: 85% 13 days post challenge	Intra-muscular injection of purified P. monodon Rab7 protein + WSSV	[116]
				87% 5 days post challenge	Injection of Rab7 crude extract expressed in A. thaliana T87	[117]
				0.25 g Rab7/g: 26.7% 0.5 g Rab7/g: 46.7% 10 days post challenge	Oral delivery of Rab7 expressed in P. pastoris for yeast surface display in feed and fed for 7 days prior to challenge at two concentrations: 0.25 g and 0.5 g per g of feed	[118]

). Conversely, oral delivery of fortilin expressed in P. pastoris yeast (5% inclusion) achieved substantial protection in L. vannamei against WSSV (91%), though efficacy was markedly lower against yellow head virus (>20%) [114]. Similarly, an antiviral protein isolated from P. monodon also conferred moderate protection (66.7%) when administered by intramuscular injection [115].

Rab7 is another protein of eukaryotes, which has small GTPases involved in endosomal trafficking and phagocytosis [119,120,121] and is upregulated in WSSV-resistant P. japonicus or L. vannamei shrimp. Recombinant Rab7 isolated from P. monodon provided high survival (85%) when intramuscularly injected in L. vannamei challenged with WSSV [116]. Expression of Rab7 in Arabidopsis thaliana T87 cells, a cost-effective plant-based production platform, with crude extracts co-injected with WSSV, yielded 87% survival [117]. Oral delivery of Rab7 expressed and surface-displayed in P. pastoris for 7 days prior to WSSV challenge provided moderate protection, with survival rates of 26.7% and 46.7% at dosages of 0.25 g/g and 0.5 g/g feed, respectively [118]. To date, no studies have explored protein-based immunostimulants in molluscs, let alone those derived from pathogen structural proteins, with current efforts remaining nascent and primarily directed toward elucidating host–pathogen interactions rather than immune protection.

4.2. RNA

Nonspecific Double-Stranded RNA (dsRNA)

Double-stranded RNA (dsRNA) represents a well-established viral mimic capable of eliciting potent antiviral and inflammatory immune responses [122]. During viral infection, dsRNA accumulates and activates PRRs, such as retinoic acid-inducible gene I-like receptors (RIG-I) and TLR3, which induce type I interferons (IFNα, IFNβ, IFNω, IFNε, or IFNκ) and pro-inflammatory cytokines (IL-6, TNFα, and IL-12) to facilitate viral clearance [123,124,125]. In aquatic invertebrates, dsRNA application has predominantly centred on pathogen-specific RNA interference (RNAi) approaches to silence pathogen genes, thereby preventing infection [126]. Synthetic dsRNA analogues, including Poly(I:C), or non-target dsRNAs, also demonstrate significant immune-protective effects across various aquatic invertebrate species [127,128,129].

The use of nonspecific dsRNA as an antiviral immunostimulant in crustaceans was first demonstrated by Robalino et al. (2004), whereby various dsRNAs synthesised from immunoglobulins (duck and pig), noncoding genomic DNA (fish), bacterial vector (pBeloBAC11), and synthetic analogues such as Poly(C–G) and Poly(I:C) were evaluated against WSSV in L. vannamei [128]. Most nonspecific dsRNAs significantly reduced viral mortality except Poly(I:C), which failed to confer protection. Histopathological analysis confirmed the absence of viral inclusions and preservation of tissue integrity in dsRNA-primed shrimp 36–72 h post infection [128] (

Table 4. dsRNA immunostimulants utilised in crustacean and mollusc immune priming experiments.

Immunostimulant	Source of Immunostimulant	Animal	Disease	Survival %	Administration	Source
dsRNAs in vitro transcribed from duck, catfish, and pig antibodies; pBeloBAC11 and synthetic commercial Poly(C-G) and Poly(I:C)	Duck (Anas platyrhynchos), catfish (Ictalurus punctatus), pig (Sus scrofa), Escherichia coli plasmid, and synthetic dsRNA	Litopenaeus vannamei	WSSV	50–90% (IgH of duck, IgH catfish, IgG pig, and pBeloBAC11) 12 days post challenge with WSSV Poly(C-G): 90% Poly(I:C): <30%	Intramuscular injection of various nonspecific dsRNA, including duck IgH, catfish IgH, pig IgG, pBeloBAC11, Poly(C-G), and poly(I-C) 72 h prior to challenge	[128]
dsRNA in vitro transcribed from duck immunoglobulin	Duck (Anas platyrhynchos)			<60% across 50, 100, 150, and 200 bp of duck IgH dsRNA	Intramuscular injection of duck A. platyrhynchos IgH chain at different transcribed lengths (50, 100, 150, and 200 bp) and virally challenged 48 h	[130]
GFP dsRNA	Green fluorescent protein	Marsupenaeus japonicus		60% 14 days post challenge	Intramuscular injection with viral challenge 2 days post injection	[131]
Poly(I:C), imiquimod, and rVP28	Synthetic dsRNA, commercial compound, and WSSV			rVP28 + Poly(I:C): 52.1% rVP28 + imiquimod: 57.9% rVP28: 35% 11 days post challenge with WSSV Poly(I:C): 43.4% Imiquimod: 37.9% 12 days post challenge with WSSV	Intramuscular injection of 100 µg of Poly(I:C) or imiquimod or a combination of 2.5 µg VP28 protein and either 100 µg Poly(I:C) or imiquimod and challenged 7 days post injection with WSSV	[132]
HMW Poly(I:C), LMW Poly(I:C), OsHV-1 dsRNA and ssRNA, and GFP dsRNA	Synthetic dsRNA OsHV-1 Green fluorescent protein	Crassostrea gigas	OsHV-1 and Vibrio tasmaniensis LGP32	OsHV-1 challenge: HMW Poly(I:C): 100% LMW Poly(I:C): 97% OsHV-1 dsRNA: 83–90% OsHV-1 ssRNA: 90% GFP dsRNA: 83–90% 9 days post challenge V. tasmaniensis challenge: Poly(I:C) 13% 10 days post challenge OsHV-1 recurrent challenges: 1 day post Poly(I:C): 100% 14 days post Poly(I:C): 100% 28 days post Poly(I:C): 100% 56 days post Poly(I:C): 96% 126 days post Poly(I:C): 89% OsHV-1 Thau lagoon: Poly(I:C) 2015 1 day post injection: 80% 119 days post relocation Poly(I:C) 2016 1, 71, 99, and 126 days post injection: 83–92%	Intramuscular injection of low and high-molecular weight poly(I:C), ORF87 OsHV1-dsRNA and ssRNA, GFP dsRNA administered 1 day prior to OsHV-1 and V. tasmaniensis LGP32 challenge. Oysters were re-challenged with OsHV-1 at 1, 14, 28, 56, and 126 days after poly(I:C) treatment. Additionally, oysters taken from recurrent outbreak site: Thau lagoon were injected with Poly(I:C) and 1, 71, 99, and 126 days post injection were relocated back to Thau lagoon site.	[133]
Poly(I:C) and Vibrio splendidus	Synthetic dsRNA Vibrio splendidus		OsHV-1	Poly(I:C): 75–89% 48 h post challenge V. splendidus: 0% 48 h post challenge	Oysters intramuscularly injected with 240 µg of Poly(I:C) or heat-killed V. splendidus 1 day prior to OsHV-1 challenge	[127]
Poly(I:C)	Synthetic dsRNA		OsHV-1	Primed parents larvae 3 days prior: 85.6% 10 days prior: 62.5% Primed fathers larvae 79.4% Primed mother larvae 91.7% 48 h post OsHV-1 challenge	Oyster mothers or fathers were intramuscularly injected with Poly(I:C) at 3–10 days prior to spawning. Oyster larvae were trans-generationally immune primed and challenged with OsHV-1	[60]
				100% 10 days post challenge	Intramuscularly injected with Poly(I:C) and challenged with OsHV-1 10 days post priming	[50]
				Poly(I:C): 100% survival at 0 and 48 h post challenge, 59.7% at 96 h post challenge	Oyster parents were intramuscularly injected with Poly(I:C) 3 days prior to spawning. D. veliger (24 h post vaccination) larvae were immersion challenged with OsHV-1	[59]
			Vibrio harveyi	Poly(I:C): >50% 4 days post challenge Seawater control: >80% 4 days post challenge	Maternally primed oysters through intramuscular injection of Poly(I:C) 48 h prior to spawning. 4-days post-fertilisation larvae were challenged with V. harveyi by immersion	[134]
Poly(I:C) OsHV-1	Synthetic dsRNA OsHV-1		OsHV-1	Poly(I:C): 100% Heat-inactivated OsHV-1: 73.3% OsHV-1: 87.2% 14 days post challenge	Intramuscular injection of either Poly(I:C) or heat-inactvated or infectious OsHV-1 and challenged with OsHV-1	[135]
Poly(I:C)	Synthetic dsRNA	Haliotis laevigata × Haliotis rubra	HaHV-1	Intra-muscular injection 50% 12 days post challenge Intra-haemolymph injections 5 days prior to challenge: 100% 16 days prior to challenge: 75% 126 days prior to challenge: 75% 25 days post challenge	Intramuscular and intra-haemolymph injection of Poly(I:C) and 48 h or 5 days later, respectively, were immersion challenged with HaHV-1. Intra-haemolymph injections were administered 16 days and 126 days prior to challenge with HaHV-1	[136]

).

Building on this, Labreuche et al. (2010) investigated the role of dsRNA length in antiviral protection, using duck immunoglobulin dsRNAs of varying lengths [130]. Longer fragments (200 bp) significantly reduced mortality, while shorter fragments (150 and 100 bp) highly upregulated systemic interference defective 1 (Sid-1) and Argonaute 2 (Ago2) [130]. Sid-1 mediates dsRNA uptake and transport, whereas Ago2 functions in RNA interference and microRNA processing, implying a length-dependent modulation of antiviral pathways. In M. japonicus, exposure to GFP dsRNA enhanced survival following WSSV challenge and upregulated immune genes ((C-type lectin 2, hemocyte homeostasis-associated protein (HAAP), viral responsive protein 15 (VSP15), fibrinogen-related protein 1 (FREP1), and Dicer and Heat shock protein 90 (HSP90)) [131,137]. C-type lectin 2 and FREP1 bind to VP28, inhibiting infection and replication [138,139]. HAAP promotes hemocyte stability [140], and VSP15 is implicated in WSSV recognition and replication [141].

Dual immunostimulant strategies combining Poly(I:C) with rVP28 or the TLR7/8 agonist imiquimod [142] were tested in M. japonicus, yielding survival rates of 52% and 58%, respectively, compared to 43.3% and 37.9% for single-agent treatments [132]. Dual priming significantly upregulated immune effectors, including Rab7, toll, and TNF, with AMPs (lysozyme, penaeidin, and crustin). Similar upregulation of AMPs and Rab7 has been observed following oral rVP28 administration in intestinal and lymphoid organ tissues [86,116,143]. While in mammals, imiquimod is known to induce cytokines, such as IFNα, IL-12, and TNFα [144], consistent with the cytokine profile observed in shrimp treated with imiquimod.

In molluscs, Poly(I:C) has demonstrated potent antiviral activity against OsHV-1 in C. gigas exhibited by near-complete protection, with viral DNA detected in only one of nine oysters [127]. Poly(I:C) induced the upregulation of several antiviral genes MyD88 and IRF, alongside vertebrate interferon-stimulated homologs protein kinase R (PKR) and interferon-induced protein 44 (IFI44). In oysters, IFI44 is upregulated by viral infections, while PKR is an inhibitor of viral and cellular protein synthesis within vertebrate immunology [145,146]. Additional induction of Mpeg1 suggested enhanced cellular antiviral defence via lytic activity against infected cells [146]. Additionally, heat-inactivated Vibrio splendidus failed to protect oysters from OsHV-1, while Poly(I:C) also provided no protection from bacterial Vibrio tasmaniensis for trans-generationally primed oyster larvae, highlighting the specific antiviral nature of Poly(I:C)-induced responses [127,133,134].

Sublethal and inactivated OsHV-1 exposures offered partial protection (26.7% and 12.8% mortality), presumably through recognition of viral dsDNA, but still far below the 100% survival achieved with Poly(I:C) [135]. Long-term Poly(I:C)-induced protection persisting for up to 156 days post challenge has been reported in both laboratory and field conditions [133]. Poly(I:C)-treated oysters exhibited sustained upregulation of immune pathways, including IFN, TLR, NF-κB, Jak/Stat, and apoptosis signalling [50], indicating prolonged antiviral readiness.

Additionally, Poly(I:C) has demonstrated similar protection rates when administered by intra-haemolymph injection of Australian adult abalone hybrid species (Haliotis laevigata × Haliotis rubra) against Haliotid herpesvirus 1 (HaHV-1) [136]. This method of immune primer delivery through intramuscular injection was not as effective as immune primer delivery injected directly within the haemolymph. Similarly to oysters, this protection was long term, with abalone primed at both 16 and 126 days prior to viral challenge, exhibiting high survival rates. TGIP has also been shown to further extend Poly(I:C)’s protective effects in molluscs for antiviral defence, as offspring from maternally primed oysters exhibited markedly reduced OsHV-1 DNA loads and lower mortality compared to paternal priming [59,60]. The transfer of antiviral transcripts such as Viperin and IRF to eggs and larvae underscores the potential for Poly(I:C)-induced TGIP in oyster populations.

4.3. DNA

4.3.1. Flagellin

In Cho et al. (2017), the fusion of WSSV proteins, VP28 and VP19 with flagellin 2 derived from Salmonella typhimurium in an orally administered bivalent DNA vaccine enhanced efficacy against WSSV in Macrobrachium nipponense and Palaemon paucidens shrimp [71]. The inclusion of flagellin increased survival rates by >10% relative to monovalent formulations. Without flagellin, these bivalent DNA constructs demonstrated high efficacy (<80%), depending on the gene sequence order, where VP19 preceding VP28 produced somewhat lower mortality. When flagellin was incorporated, mortality decreased further to 15.8% (VP19 first) and 10.5% (VP28 first), indicating the host immune response was likely mediated through elevated NF-kB activation via several TLRs [71] (

Table 5. DNA-based immunostimulants utilised in crustacean immune priming experiments.

Immunostimulant	Source of Immunostimulant	Animal	Disease	Survival %	Administration	Source
Flagellin, VP28, and VP19	Salmonella typhimurium WSSV	Macrobrachium nipponense	WSSV	Ac-ie1VP28: 71.1% Ac-ie1VP19: 63.8% Ac-VP19-ie1VP28: 78.8% Ac-VP28-ie1VP19: 76.3% Ac-VP19-ie1VP28FL2: 84.2% Ac-VP28-ie1VP19FL2: 89.5%	Oral delivery of VP28 and VP19 fused with flagellin 2 bivalent baculoviral DNA vaccine. Shrimps fed for 7 days and then challenged with WSSV 7 days after feeding	[71]
CpG ODN plasmid (1681, 2216, 2006, 2395, and 2143)	Bacterial DNA	Litopenaeus vannamei	WSSV	<20% after 14 days post challenge and not statistically significant from untreated controls	Injected CpG ODN-rich plasmid (1681, 2216, 2006, 2395, and 2143) at 100 µg and then injection challenged with WSSV 12 h later	[147]
CpG ODN plasmid (1681, 2216, 2006, 2395, and 2143) and VP28 protein	Bacterial DNA WSSV			CpG ODN plasmid: 22.6% VP28: 28.8% 10 days post challenge	Oral delivery of VP28 expressed in yeast Y. lipolytica or CpG ODN plasmid (1681, 2216, 2006, 2395, and 2143), fed for 15 days, and then challenged with WSSV by injection	[148]
CpG ODN plasmid (1681, 2216, 2006, 2395, 1651)	Bacterial DNA	Eriocheir sinensis	Aeromonas hydrophila	C40: 33.8% mortality rate C100: 33.4% mortality rate Untreated controls: 44.2% mortality rate	Oral-fed two concentrations of CpG ODNs at 40 mg/kg (C40) and 100 mg/kg (C100) for 4 weeks	[149]
VP28, VP15, VP35, and VP281	WSSV	Penaeus monodon	WSSV	Experiment 1: Day 7: 73%, Day 14: 65%, Day 25: 17%, Day 50: 4% Experiment 2: VP28: 51%, VP281: 46%, VP35 and VP15: <10%, VP28 + VP281: 43% Experiment 3: VP28: 48%, VP281: 45%, VP28+ VP281: 41% Experiment 4: VP28: 30%, VP281: 34%, VP28+ VP281: 37% 20 days post challenge	Intramuscular injection of either (1) VP28 protein also injected at 2 doses on first and fifth day and challenged intramuscularly with WSSV on day 7, 14, 25, or 50 after second dose. (2) VP DNA in DNA vaccine vector pVAX1 with CMV promotor vaccinated once with single VPs and challenged 25 days later. (3) VP DNA administered in single treatments or combined and challenged on day 35 and (4) day 50	[150]
VP28				7 days post vaccination: 90% 14 days post vaccination: 76.66% 21 days post vaccination: 66.66% 30 days post vaccination: 56.66% 14 days post challenge	Intramuscular injection of VP28 in eukaryotic expression vector pcDNA3.1 and 7, 14, 21, and 30 days, later challenged with WSSV	[151]
				7 days post vaccination: 85% 15 days post vaccination: 65% 30 days post vaccination: 50% 14 days post challenge	Oral feed containing VP28 encapsulated in chitosan nanoparticles and 7, 15, and 30 days post feeding, challenged with WSSV	[152]
		Cambarus clarkii		7 days post vaccination: 83.3% 15 days post vaccination: 66.7% 25 days post vaccination: 56.7% 15 days post challenge	Oral feed of VP28 in attenuated S. typhimurium in pcDNA3.1 vector and 7, 15, and 25 days post vaccinated, challenged with WSSV	[153]
		Litopenaeus vannamei		7 days post vaccination: 52.5% 15 days post vaccination: 20% 5 days post challenge	Injected VP28 in eukaryotic expression vector pVAX1 and challenged with WSSV 7 and 14 days later	[154]
		Penaeus japonicus		VP28: 62.4% and PBS control: 70%, 12 days post-immersion challenge	Intramuscularly injected with VP28 in mammalian expression vector and challenged 7 days post vaccination with WSSV	[155]
		Marsupenaeus japonicus		7 days post vaccination: 78.5% at 12 days post challenge 30 days post vaccination: 26.2% 20 days post challenge	Intramuscularly injected with VP28 in pCMV eukaryotic expression vector and challenged 7 and 30 days post vaccination with WSSV	[143]
		Scylla serrate		Approx. 60% 10 days post challenge	Oral administration of VP28 in pcDNA 3.1 eukaryotic expression vector encapsulated in chitosan/tripolyphosphate nanoparticles and challenged with WSSV 5 days post feeding	[156]
VP28 and VP19		Procambarus clarkii		Injection: VP28: 86%, VP19: <50% and not statistically different from controls Oral: VP28: >80%, VP19: <40% and not statistically different from controls at 15 days post challenge	Injected with VP28 or VP19 DNA plasmids and challenged 5 days post injection with WSSV. Also orally given VP28 and VP19 DNA plasmids encapsulated in DH5α E. coli cells and challenged 4 days post feeding	[157]
PmAV	Penaeus monodon	Penaeus monodon		(i) 66.7% (ii) 27.8% (iii) 83.3% 15 days post challenge	Injected intramuscularly with PmAV in three different forms: (i) PmAV expressed protein, (ii) PmAV DNA plasmid, and (iii) PmAV DNA plasmid encapsulated in chitosan nanoparticles and challenged with WSSV 24 h later	[115]
XSVAS	XSV	Macrobrachium rosenbergii	WTD	Immersion: approx. 40% at 50 ng and 45% at 100 ng of XSVAS Feed: approx. 50% at 100 ng of XSVAS at 20 days post challenge	Immersed and orally delivered XSVAS gene of nodavirus encapsulated in chitosan nanoparticles and challenged with nodavirus 5 days post vaccination	[158]
Capsid protein	MrNV			20 days post feeding: 60% 40 days post feeding: 80% 10 days post challenge	Orally fed capsid protein of MrNV in DNA vector pVAX1 within feed for 40 days and challenged with MrNV 20- and 40-days post vaccination	[159]

).

4.3.2. CPG ODNS

CpG oligodeoxynucleotides (ODNs) represent unmethylated cytosine-guanine motifs derived from bacterial DNA that act as potent immunostimulants through TLR9 recognition [160,161]. In crustaceans, CpG administration has been shown to augment immune enzymatic activities, manifested through increased lysozyme, phenoloxidase (PO), and reactive oxygen species (ROS) activity within haemocytes [147,162,163,164,165], with comparable responses also reported in bivalves such as mussels [166].

Experimental investigations into the antiviral efficacy of CpG ODNs have revealed transient protection with injection of tandem CpG ODNs in L. vannamei delaying and only modestly reducing mortality 14 days post-WSSV challenge [147] (Table 5). In contrast, dietary supplementation further reduced viral burden by approximately 3.5-fold, accompanied by significant induction of PO, nitric oxide (NO), and key antiviral mediators such as Stat and Dicer [148]. Comparative analyses of structurally distinct CpG types (A, B, C, and P) identified type A as eliciting the strongest antiviral response, producing the lowest viral loads and the highest expression of Stat, IRF, Vago4, and IFN [167].

Beyond antiviral applications, CpG ODNs have demonstrated protection against bacterial pathogen Aeromonas hydrophila. In the Chinese mitten crab (Eriocheir sinensis), dietary inclusion of CpG conferred a 10% reduction in mortality upon challenge, while also promoting growth performance and a dose-dependent increase in lysozyme activity, indicative of enhanced antibacterial immunity [149]. Similarly, in freshwater crayfish (C. quadricarinatus), CpG administration elevated total haemocyte counts (THC), phagocytic index (PI), and lysozyme activity, with CpG ODN 2006 eliciting the most pronounced immunostimulatory effects [168].

4.3.3. Structural Pathogen-Specific DNA

Several WSSV structural proteins have been utilised in DNA vaccines, driven by eukaryotic promoters to facilitate in vivo expression within the host [154,155]. Among the earliest demonstrations of this concept, Rout et al. (2007) reported that intramuscular injection of P. monodon with DNA encoding VP15, VP28, VP35, and VP281 elicited varying degrees of protection following viral challenge [150] (Table 5). Survival rates were highest for VP28 (51%) and VP281 (46%), with combined vaccination of both modestly improving long-term protection at 35 and 50 days post priming. Similarly, Kumar et al. (2008) demonstrated enhanced survival in VP28 DNA-vaccinated shrimp, with elevated antiviral enzyme activities, including proPo and superoxide dismutase (SOD) across multiple tissues (head muscle, hepatopancreas, pleopods, and gut) and timepoints post vaccination (3, 15, and 30 days) [151]. Consistent with these observations, Kono et al. (2010) reported comparable survival rates in M. japonicus injected with VP28 DNA constructs under similar conditions [143].

Subsequent studies have investigated alternative delivery routes to improve vaccine practicality in aquaculture systems. For instance, Cherax clarkii crayfish vaccinated orally with S. typhimurium expressing VP28 DNA exhibited significant protection of 83.3%, 66.7%, and 56.7% at 7, 15, and 25 days post vaccination, respectively [153]. Comparable outcomes were reported when VP28 DNA was encapsulated in chitosan nanoparticles and administered orally to P. monodon [152]. Similar results were also documented in Scylla serrata crabs orally immunised with VP28 DNA encapsulated in chitosan/tripolyphosphate nanoparticles [156]. Moreover, Mu et al. (2012) evaluated both oral and injection delivery of VP28 DNA plasmid in P. clarkii, with both administration routes eliciting high protection [157]. DNA vaccine efficacy has also been demonstrated in the giant freshwater prawn Macrobrachium rosenbergii against white tail disease (WTD), where encapsulation of nodavirus small virus antisense (XSVAS) DNA within chitosan nanoparticles yielded moderate survival improvements via immersion and oral delivery [158]. A later study utilising the WTD capsid gene DNA achieved markedly enhanced protection, with survival peaking at 80% 40 days post vaccination [159]. However, if mortality was assessed for longer than 10 days, until the 20 days reported by [158] survival rates may appear more similar.

4.3.4. Host-Specific DNA

To date, only one study in P. monodon shrimp utilised host-specific DNA in vaccine work. Pathan et al. (2013) identified that the antiviral host protein (PmAV) derived from P. monodon when encapsulated in chitosan provided the highest protection (83.3%) compared to naked PmAV plasmid (27.8%) against WSSV [115] (Table 5). The authors concluded that the chitosan nanoparticles provided higher stability with naked DNA more prone to degradation by nucleases present in the host.

4.4. Carbohydrates

4.4.1. β-Glucan

β-glucans are poly-glucopyranoses featuring β-(1,3)-D linkages along their backbone, often with β-(1,6)-linked side branches, the degree and pattern of which determine their immunomodulatory properties [169]. As PAMPs, β-glucans engage PRRs to activate innate immune pathways, enhancing phagocytic activity (PA), lysozyme production, complement activation, and cytokine expression [169]. Early trials in P. monodon indicated variable protection against WSSV, with survival improving from 0–15% in juvenile shrimp and 18.3–31.6% in post-larval shrimp post-WSSV challenge following optimal β-1,3-glucan supplementation [170,171] (

Table 6. β-glucan-based immunostimulants utilised in crustacean and molluscan immune priming experiments.

Immunostimulant	Source of Immunostimulant	Animal	Disease	Survival %	Administration	Source
β-1.3-glucans	Schizophyllum commune	Penaeus monodon	WSSV	(i) 12.2% 6 days, 7.3% at 30 days, and 5.5% 120 days post challenge (ii) 10 days of feeding: 0% 6 days post challenge, 20 days feeding: >20% at 6 days, 15% at 30, and 13.3% 120 days post challenge.	Oral within feed and fed for 15 days to (i) post-larval shrimp, immersion challenged with WSSV and (ii) juvenile shrimp fed for either 10 or 20 days prior to post-injection challenge	[171]
				(i) 0% 12 days post challenge (ii) 30% 12 days and 20% 60 days post challenge (iii) 42.2% 12 days and 31.6% 60 days post challenge (iv) 24.4% 12 days and 18.3% 60 days post challenge	Oral within feed and fed for 20 days with β-1.3-glucan at different concentrations: (i) 1 kg−1, (ii) 2 kg−1, (iii) 10 kg−1, and (iv) 20 kg−1 and injection challenged with WSSV	[170]
	Debaryomyces hansenii S8, Debaryomyces hansenii S169, Candida tropicalis S186, and Saccharomyces cerevisiae S36			C. tropicalis (S186): 69% D. hansenii (S169): 27% D. hansenii (S8): 23% S. cerevisiae (S36): 4% 7 days post challenge	Oral inclusion of glucan in feed at 0.2% and fed for 21 days and fed twice daily and then orally challenged with WSSV	[174]
	Candida haemulonii S27 and Candida sake S165			C. haemulonii glucan: 42% C. sake glucan: 38% 7 days post challenge	Oral inclusion in feed at 0.2% and fed for 14 days and fed once every 7 days and then orally challenged with WSSV	[175]
	Laminaria digitata	Farfantepenaeus californiensis		β-glucan: 10% 6 days post challenge β-carotene: 0% 6 days post challenge Vitamin E: 0% 3 days post challenge	Oral within feed and fed β-glucan at 0.1%, β-carotene, and vitamin E at 0.01% for 23 days and then challenged with WSSV by intramuscular injection	[172]
	Candida sake	Fennerpenaeus indicus		Approx. values inferred from survival curve (i) 0.05% β-glucan: 17%, 0.1% β-glucan: 32%, 0.2% β-glucan: 54%, 0.3% β-glucan: 35%, 0.4% β-glucan: 27% 7 days post challenge (ii) Daily: 14%, once every 2 days: 21%, once every 5 days: 20%, once every 7 days: 35% once every 10 days: 16% 7 days post challenge (iii) Daily: 15%, once every 7 days: 40% once every 10 days: 20% 7 days post challenge	Orally administered in feed at (i) different concentrations (0.05, 0.1, 0.2, 0.3, and 0.4 g glucan/100 g feed) for 21 days and orally challenged with WSSV or (ii) at different feeding intervals at 0.2 g glucan/100 g feed (daily, once every 2 days, once every 5 days, once every 7 days, and once every 10 days) and orally challenged with WSSV after 40 days of feeding	[176]
β 1,3 1,6 glucan	Saccharomyces cerevisiae			(i) <40% (ii) 53.32% (iii) 48.32% 21 days post challenge	Oral within feed and fed β 1,3 1,6 glucan at different concentrations: (i) 2, (ii) 10, and (iii) 20 g kg−1 for 20 days and then challenged with WSSV by injection	[173]
β-1.3-glucans; CMG and SEG		Litopenaeus vannamei		Approx. values inferred from survival curve β -1.3-glucan: <50%, 0.2%: <80% CMG-A at 0.1%: <90%, 0.2%: >50% CMG-B at 0.1%: <80%, 0.2%: <60% CMG-C at 0.1%: <60%, 0.2%: <50% CMG-D at 0.1%: <60%, 0.2%: <40% SEG-A at 0.1%: <70%, 0.2%: < 60% SEG-B at 0.1%: <60%, 0.2%: <50% SEG-C at 0.1%: <80%, 0.2%: <40% SEG-D at 0.1%: <70%, 0.2%: <50% = 7 days post challenge	Oral within feed and fed for 35 days at two doses: 0.1% and 0.2% of CMG or SEG and their four derivatives representing different degrees of substation (A, B, C, and D) and then orally challenged with WSSV-infected shrimp tissue	[177]
β-1.3-glucans (water-soluble)		Penaeus monodon	WSSV and Vibrio parahaemolyticus	(i) V. parahaemolyticus challenge + 48 kDa: 0%, 35 kDa: 25%, 25 kDa: 35%, 15 kDa: 60%, 11 kDa: 30% WSSV challenge + 48 kDa: 0%, 35 kDa: 0%, 25 kDa: 16%, 15 kDa: 40%, 11 kDa: 32% (ii) V. parahaemolyticus challenge + 250 ppm: 18.75%, 500 ppm: 37.5%, 1000 ppm: 56.25%, 1500 ppm: 12.50%, 2000 ppm: 18.75%, 3000 ppm: 0% WSSV challenge + 250 ppm: 0%, 500 ppm: 0%, 1000 ppm: 38.1%, 1500 ppm: 0%, 2000 ppm: 0%, 3000 ppm: 0% 7 days post challenge	Oral administration of 15 kDa oligo β-glucan (γ-irradiated) in feed and (i) fed at different molecular weights: 48, 35, 25, 15, and 11 kDa at 1000 ppm for 90 days before bacterial or viral challenge. Additionally fed at (ii) different concentrations: 250, 500, 1000, 1500, 2000, and 3000 ppm for 90 days	[178]
β 1,3 1,6 glucan	Debaryomyces hansenii, Candida tropicalis, Candida humilis, Candida glabrata, Pichia kudriavzevyi, Wickerhamomyces anomalus, and Saccharomyces cerevisiae	Litopenaeus vannamei	WSSV	Approx. values inferred from survival curve D. hansenii β-glucan: 67% C. tropicalis β-glucan: 45% C. humilis β-glucan: 65% C. glabrata β-glucan: 30% P. kudriavzevyi β-glucan: 50% W. anomalus β-glucan: <40% S. cerevisiae β-glucan: 57.14% Control: 40% 5 days post challenge	Oral within feed of isolated purified β-glucans and fed for 75 days and then intramuscular injection challenged with WSSV	[179]
	Candida parapsilosis, Hortaea werneckii, Candida spencermartinsiae, Candida haemulonii, Candida oceani, Debaryomyces fabryi, Debaryomyces nepalensis, and Meyerozyma guilliermondii	Penaeus monodon		C. parapsilosis: 66.66% H. werneckii: 70.27% C. spencermartinsiae: 60.97% C. haemulonii: 48.64% C. oceani: 58.53% D. fabryi: 54.05% D. nepalensis: 45.7% M. guilliermondii: 43.24% 7 days post challenge	Orally fed and incorporated into diet at 0.2% and fed once every 7 days for a total of 45 days and then challenged with WSSV	[180]
β-1.3-glucans and whole yeast cells	Debaryomyces hansenii CBS8339, Yarrowia lipolytica, and Saccharomyces cerevisiae	Litopenaeus vannamei	Vibrio parahaemolyticus	Approx. values inferred from survival graph (i) (a) 76%, (b) 55%, (c) 67%, (d) 63%, (e) 78%, (f) 89%, (g) 93% (ii) (a) 87%, (b) 70%, (c) 78% (iii) (a) 91%, (b) 80%, (c) 77% 3 days post challenge	(i) Oral and immersion treatments administered twice daily with both oral, immersion, or both daily at different concentrations: (a) Glucan 2% feed, (b) D. hansenii 1% feed twice per day, (c) Y. lipolytica 1% feed twice per day, (d) D. hansenii 0.5% feed + immersion, (e) Y. lipolytica 0.5% feed + immersion, (f) D. hansenii + Y. lipolytica (1%, 1:1), (g) D. hansenii + Y. lipolytica (1%, 1:1) feed + D. hansenii + Y. lipolytica immersion (1:1) for 9 days (ii) fed at different frequencies (a) D. hansenii + Y. lipolytica (1%, 1:1) feed + D. hansenii + Y. lipolytica (1:1) immersion, added daily, (b) added every second day, and (c) added every third day for 9 days (iii) D. hansenii + Y. lipolytica (1%, 1:1) feed + D. hansenii + Y. lipolytica (1:1) immersion administered daily for 9 days and then challenged with V. parahaemolyticus at (a) day 1, (b) day 4, and (c) day 7	[181]
β-glucan	Chaetoceros muelleri and Thalassiosira weissflogii marine diatoms	Penaeus merguiensis		C. muelleri β-glucans: 82.2% T. weissflogii β-glucans: 77.8% Untreated control: 51.1% 10 days post challenge	Orally administered into feed different β-glucans derived from marine diatoms over 14 days and challenged with V. parahaemolyticus, with survival monitored over 10 days post infection	[182]
Glucan particles, WT yeast, Δmnn9 mutant yeast, and inactivated Aeromonas hydrophila	Saccharomyces cerevisiae and Aeromonas hydrophilia	Artemia	Vibrio campbellii, Vibrio proteolyticus	(i) (a) V. campbellii and V. proteolyticus: 0%, (b) V. campbellii: 0%, V. proteolyticus: 36%, (c) V. campbellii: 0%, V. proteolyticus: 1%, (d) V. campbellii: 1%, V. proteolyticus: 6%, (e) V. campbellii and V. proteolyticus: 0% (ii) (a) V. campbellii and V. proteolyticus: 50%, (b) V. campbellii and V. proteolyticus: 0%, (c) V. campbellii: 71%, V. proteolyticus: 86%, (d) V. campbellii: 0%, V. proteolyticus: 30% (iii) (a) V. campbellii and V. proteolyticus: 0%, (b) V. campbellii: 0% V. proteolyticus: 4%, (c) V. campbellii: 29% V. proteolyticus: 19%, (d) V. campbellii: 5% V. proteolyticus: 28%, (e) V. campbellii: 15% V. proteolyticus: 51%, (f) V. campbellii: 70% V. proteolyticus: 78%	Orally fed daily (i) with (a) inactivated A. hydrophila, (b) inactivated A. hydrophila + 10% WT yeast or (c) 10% WT yeast alone, (d) inactivated A. hydrophila + 1% WT yeast, and (e) 1% WT yeast alone, then challenged with either V. campbellii or V proteolyticus for 3 days (ii) Daily feeding of (a) S. cerevisiae Δmnn9 at 10% or (b) at 1% alone or (c) 10% Δmnn9 + inactivated A. hydrophila or (d) 1% Δmnn9 + A. hydrophila, then challenged with either V. campbellii or V proteolyticus for 3 days (iii) Daily feeding of (a) glucan particles, (b) WT yeast, (c) WT yeast + glucan particles, (d) inactivated A. hydrophila + glucan particles, (e) WT yeast + inactivated A. hydrophila, (f) WT yeast + inactivated A. hydrophila + glucan particles, then challenged with either V. campbellii or V proteolyticus for 3 days	[183]
Commercially available β-glucans: Biorigin, Sigma, Zymosan, MacroGard, Immunowall, Laminarin, and Chitin	Saccharomyces cerevisiae and Laminarin digitata		Vibrio campbellii	(a) 15%, (b) 61%, (c) 18%, (d) 9%, (e) 65%, (f) 66%, (g) 16%, (h) 46%, (i) 25% 3 days post challenge	Orally fed daily with (a) inactivated A. hydrophila alone or with (b) Zymosan, (c) Laminarin, (d) Chitin, (e) β-glucan (Sigma), (f) β-glucan (Sigma) + Chitin, (g) Biorigin, (h) MacroGard, and (i) Immunowall, then challenged with V. campbellii	[184]
β-glucan and glycyrrhizin	Saccharomyces cerevisiae and Glycyrrhiza glabra	Litopenaeus vannamei	Vibrio alginolyticus	Glycyrrhizin: 71.1% β-glucan: 65.6% 5 days post challenge	Orally fed 0.2% β-glucan or 0.06% glycyrrhizin diets four times a day for 18 days and challenged with V. alginolyticus by intramuscular injection	[185]
β-1,3-glucan and probiotics and organic acids	Saccharomyces cerevisiae, Bacillus velezensis, Bacillus amyloliquifaciens, Bacillus subtilis, Bacillus megaterium, and Brevibacillus parabrevis Formic acid, benzoic acid, and hydroxyl methylthio2-butanoic acid		Vibrio harveyi	Probiotic feed: 86% Organic acid feed: 81.33% β-1,3-glucan feed: 80.67% 4 days post challenge	Shrimp were orally fed four times daily for 60 days with either a probiotic feed (B. velezensis, B. amyloliquifaciens, B. subtilis, B. megaterium, and B. parabrevis), organic acid feed (formic, benzoic, and hydroxyl methylthio2-butanoic acid), or β-1,3-glucan feed and challenged with V. harveyi	[186]
β-1,3-glucan	Hordeum vulgare	Macrobrachium rosenbergii	Vibrio alginolyticus	Approx. values inferred from survival graph (a) 40%, (b) 40%, and (c) 15% 7 days post challenge with untreated controls: 40%	Immersed in four different concentrations of β-glucans: (a) 5 and (b) 10 and 15 mg L−1 for 1–3 h and immersion challenged with V. alginolyticus	[187]
	Saccharomyces cerevisiae		Aeromonas hydrophila	Approx. values inferred from survival graph 1.0 kg−1: 85% 2.0 kg−1: 50% 3.0 kg−1: 40% 10 days post challenge	Oral delivery of β-glucans at different concentrations (1.0, 2.0, and 3.0 kg−1) into feed and fed for 60 days, then challenged with A. hydrophila by intramuscular injection	[188]
				Approx. values inferred from survival graph 500 mg kg−1: 50% at 7 days and 10 days post challenge, 1000 mg kg−1: 65% at 7 days and 50% at 14 days post challenge, 1500 mg kg−1: 92% at 7 days and 70% at 14 days post challenge	Oral delivery of β-glucans at different concentrations (500, 1000, and 1500 mg kg−1) and fed twice a day and challenged with A. hydrophila by intramuscular injection	[189]
		Penaeus monodon	Vibrio vulnificus	(a) 10 days: 58.3%, 18 days: 50%, 43 days: 13.3%, (b) 10 days: 100%, 18 days: 80%, 43 days: 6.7%, (c) 10 days: 100%, 18 days: 70%, 43 days: 20%, (d) 10 days: 44.4%, 18 days: 50%, 43 days: 13.3%	Immersed at four different concentrations for 3 h at (a) 0.25, (b) 0.5, (c) 1, and (d) 2 mg/mL and challenged by immersion with V. vulnificus on day 10, 18, and 43 for 12 h	[190]
		Artemia franciscana	Vibrio harveyi	Approx. values inferred from survival graph WT β-glucan: (a) 42% (b) 40% Sigma β-glucan: (a) 40% (b) 42% Gas1 β-glucan: 50% 48 h post challenge	Orally fed different sizes of β-glucan (a) small and (b) large WT β-glucan and commercial β-glucan (Sigma). Gas1 small β-glucan was also utilised and challenged with V. harveyi	[191]
β-1,3-glucan and curdlan	Agrobacterium sp. ATCC 31749	Macrobrachium nipponense	Vibrio parahaemolyticus	Approx. values inferred from survival graph 0.1% β-glucan: 63% 0.2% β-glucan: 70% 1% β-glucan: 60% Curdlan: 50% 96 h post challenge	Fed 0.2% curdlan or different concentrations of β-1,3-glucan (0.1%, 0.2%, and 1.0%) for 6 weeks and fed four times a day, then challenged with V. parahaemolyticus by immersion for 96 h	[192]
WT yeast, Δmnn9 mutant yeast, and microalgae	Saccharomyces cerevisiae, Tisochrysis lutea (CCAP 927/14), and Chaetoceros muelleri (CCAP 1010/3)	Crassostrea gigas	Vibrio coralliilyticus	(i) Δmnn9 yeast: 66%, WT yeast: 48%, control: 53% 7 days post challenge (ii) Δmnn9 yeast at 5%: 3%, 10%: 7%, 25%: 14%, 50%: 23% 7 days post challenge	Oral delivered either (i) algae-based diet containing WT or Δmnn9 yeast for 24 h prior to challenge or (ii) different levels of Δmnn9 (0, 5, 10, 25, and 50%) into algae-based diet for 24 h prior to challenge	[193]
Yeast strains (gas1 and Δmnn9 and WT)	Saccharomyces cerevisiae	Artemia franciscana	Vibrio campbelii	(i) Δmnn9 yeast strain: 77.5%, gas1 yeast strain: 62.5%, WT yeast: 45.6% (ii) Δmnn9 yeast strain swap: 52.2%, gas1 yeast strain swap: 46.9%, WT yeast control: 40.8%	Heat-inactivated yeast strains were incorporated into feed and fed to shrimp for (i) 48 h prior to challenge with V. campbelii or (ii) fed WT for 72 h and then replaced with either Δmnn9 and gas1 strains for 8 h prior to challenge	[194]
β-glucan	Mushroom waste (King oyster mushroom and Maiitake)	Crassostrea gigas	Escherichia spp. and Vibrio spp.	β-glucan: 100% Control: 70% 48 h post challenge	Orally fed different feeds containing S. platensis and mushroom β-glucan extract for 48 h and immersed in combined Escherichia spp. and Vibrio spp.	[195]
β-1,3 1,6-glucan	Ganoderma lucidum	Haliotis diversicolor supertexta	Vibrio alginolyticus	40 µL/mL β-1,3 1,6-glucan: 33.33% 80 µL/mL β-1,3 1,6-glucan: 36.67% 7 days post challenge	β-1,3 1,6-glucan extracted from mushrooms injected at two different concentrations into abalone prior to challenge with V. alginolyticus	[196]

). Similar studies in brown (Farfantepenaeus californiensis) and Indian shrimp (Fennerpenaeus indicus) reported delayed or decreased mortality and elevated antioxidant enzyme activity and immunity with THC, total plasma protein (TPP), and PA following β-glucan administration [172,173].

β-glucan efficacy is strongly influenced by environmental conditions and glucan structure. Synergistic effects of higher temperature combined with β-glucan supplementation enhanced antiviral protection in L. vannamei against WSSV [197]. Modified water-soluble forms such as carboxymethyl-glucan (CMG), sulfoethyl-glucan (SEG), and γ-irradiated β-glucans induced stronger immune activation and reduced WSSV mortality compared to unmodified forms [177,178].

Marine yeast-derived glucans, notably from Candida sake and Debaryomyces hansenii, conferred survival benefits ranging from 37% to 70% following WSSV challenge in shrimp, accompanied by increased THC, respiratory burst, and AMP expression [176,179,180]. However, other glucans from Wickerhamomyces anomalus, Candida glabrata, Candida haemulonii, and C. sake have also reported increased viral susceptibility, highlighting the protective potential of β-glucans with molecular weight and branching [174,175,179]. Marine yeasts, Yarrowia lipolytica and D. hansenii, contain high concentrations of β-glucan and demonstrate bactericidal activity against Vibrio parahaemolyticus, significantly upregulating immune gene expression and enzymatic defences in L. vannamei [181,198].

Additional sources such as diatoms-derived glucans (Chaetoceros muelleri) and commercial preparations derived from S. cerevisiae with the addition of chitin also confer strong disease resistance, with survival rates exceeding 80% in P. merguiensis and Artemia spp. against V. campbelli and V. proteolyticus [182,183,184]. Additionally, other important Vibrio spp. have been utilised in β-glucan challenges, with enhanced immunity and survival against Vibrio spp. across multiple crustacean species when β-glucans were delivered either by immersion or feed prior to challenge [185,186,187,190,192]. β-glucan dietary supplementation has been shown to enhance M. rosenbergii immunity with elevated THC, PO, and proPo activity and survival following A. hydrophila [188,189] and WTD challenge [199]. Additionally, an early study demonstrated maternal priming with β-1,3-1,6-glucan derived from baker’s yeast (S. cerevisiae) conferred protection and overall reduction of clinical symptoms in P. monodon offspring following WSSV challenge [55].

In oysters, C. gigas fed algae diets supplemented with S. cerevisiae Δmnn9 mutant-improved survival post-Vibrio coralliilyticus challenge, an effect associated with heightened transcription of key immune effectors, including lysozyme, SOD, MyD88, NFκB, and β-glucan binding protein (βGBP) [193]. Many of these genes have been shown to be upregulated in response to β-glucan stimulation in other molluscs and crustaceans, such as Perna viridis [200], Artemia franciscana [191,194], F. chinensis [201], Ruditapes philippinarum [202], Chlamys farreri [203], and in previous work on C. gigas and Ostrea edulis [204,205,206].

Complementary findings were reported in oysters and fed diets composed of β-glucans extracted from mushrooms, whereby immunity was significantly enhanced and complete survival obtained against Escherichia spp. and Vibrio spp. 48 h post challenge [195]. In Taiwanese abalone (Haliotis diversicolor supertexta), in vivo administration of β-1,3/1,6-glucan significantly enhanced THC, respiratory burst, PO activity, PI, and phagocytic rate (PR), while also improving survival following Vibrio alginolyticus challenge [196]. This protection was attributed to β-glucan-induced activation of nonspecific immune mechanisms. Despite these encouraging findings, the application of β-glucans as immunostimulants in molluscan pathogen-challenge studies remains comparatively underdeveloped.

4.4.2. Peptidoglycan

Peptidoglycan (PGN), a fundamental structural component of bacterial cells, is readily perceived by PRRs, provoking a suite of innate immune responses, including macrophage activation and induction of cytokines, such as IL-1, IL-6, and TNF-α [207,208,209]. Within aquatic organisms, PGN has emerged as an immunomodulator, frequently utilised to delineate host–pathogen interactions and innate immune mechanisms [210,211,212,213,214]. Dietary PGN sourced from Bifidobacterium thermophilum (0.2–0.4 mg/kg/day) elicited dramatic improvements in survival for P. japonicus shrimp, with some feeding regimens achieving complete long-term protection [215] (

Table 7. Peptidoglycan-based immunostimulants utilised in crustacean immune priming experiments.

Immunostimulant	Source of Immunostimulant	Animal	Disease	Survival %	Administration	Source
PGN	Bifidobacterium thermophilum	Penaeus japonicus	WSSV Vibrio penaeicida	WSSV: (i) 100%, (ii) 100%, (iii) 95%, (iv) 85%, (v) 90% 40 days post challenge V. penaeicida: (i) 63.4% and (ii) 81.7% 10 days post challenge	Orally fed PGN at different concentrations and feeding schedules such as (i) 0.2 mg/kg/day for 7-day on/off cycles, (ii) 0.4 mg/kg/day with a 4-day on/off cycle, (iii) 0.2 mg/kg/day for 4-day on/3-day off, (iv) 2 day- on/2-day off, and (v) continuous feeding for 95 days, with shrimp challenged with V. penaeicida on day (i) 65 and (ii) 95 or WSSV	[215]
		Litopenaeus vannamei	Vibrio parahaemolyticus, WSSV	Approx. values inferred from survival graph Vibrio parahaemolyticus: 0.2 mg/kg: 60%, 1 mg/kg: 50%, 5 mg/kg: 80%, 25 mg/kg: 65% 125 mg/kg: 50% 3 days post challenge WSSV: 0.2 mg/kg: 50%, 1 mg/kg: 55%, 5 mg/kg: 65%, 25 mg/kg: 70% 125 mg/kg: 60% 5 days post challenge	Orally fed different concentrations of PGN (0.2, 1, 5, 25, and 125 mg/kg) for 8 weeks and injection challenged with either V. parahaemolyticus or WSSV	[216]
PGN + MOS		Penaeus monodon	WSSV	0.1% PGN + MOS: 40% 0.2% PGN + MOS: 80% 0.4% PGN + MOS: 25% 10 days post challenge	Orally fed different diets containing PGN and mannan oligosaccharide (MOS) at varying amounts (0.1, 0.2, and 0.4% PGN + MOS) for 8 weeks prior to challenge with WSSV via immersion challenge	[217]

). Similar dietary interventions in L. vannamei of PGN at higher concentrations (25 mg/kg) and over longer periods (8 weeks) increased survival and elevated pivotal immune enzyme activities, including SOD, PO, CAT, lysozyme, acid phosphatase (ACP), and alkaline phosphatase (AKP), while upregulating immune-related genes [216].

The integration of PGN with other functional feed additives, particularly mannan oligosaccharides (MOS), has revealed synergistic effects on growth performance, immune activation, and overall physiological homeostasis. Supplementation of P. monodon diets with combined PGN and MOS over an 8-week period notably enhanced weight gain and survival against WSSV, with the most pronounced effects at 0.2% inclusion accompanied by increased as THC and respiratory burst activity [217]. Comparable outcomes have been reported across diverse crustacean species, including shrimp, crabs, crayfish, and lobsters further substantiating the broad applicability of PGN-based immunostimulants in aquaculture [214,218,219,220,221,222,223].

Further pathogen challenge studies reinforce PGN’s immune-potentiating capacity with extended feeding of P. japonicus shrimp, with PGN demonstrating markedly increased survivorship following Vibrio penaeicida exposure [215]. Complementary studies indicate that dietary PGN at 5 mg/kg confers resistance to Vibrio parahaemolyticus, accompanied by coordinated upregulation of immune enzymes and signalling pathways consistent with earlier findings [216]. To date, no studies have directly tested PGN as an immunostimulant in molluscan pathogen-challenge experiments, with the explicit aim of enhancing host survival or resistance. Nevertheless, extensive evidence suggests that molluscs possess the cellular and molecular machinery to detect and respond to PGN, through peptidoglycan recognition protein (PGRP) family, which has been identified in scallops [224], clams [225,226,227,228,229], mussels [230,231], periwinkles [232], abalone [233], and oysters [234,235,236,237]. In these species, PGRPs are frequently localised within immunologically active tissue, such as the hepatopancreas [227,228,229,230,236], haemocytes [224,229,231,234,235,236], gill [226,228,230,231,234,236], and digestive gland [231,234], and are typically unregulated following bacterial challenge [224,225,226,227,229,230,231,233,235,236]. Furthermore, characterised PGRPs exhibit differential binding affinities for PGN and lipopolysaccharide (LPS) and yeast-derived components such as β-glucans [224,227,228,229,230,233,236], underscoring their functional versatility in recognising a spectrum of microbial elicitors.

Although no direct evidence demonstrates PGN-mediated protection against aquatic pathogens in molluscs, the widespread presence, inducibility, and ligand-binding competence of PGRPs strongly suggest that PGN-based immune-stimulation may confer at protective benefits in molluscs, providing a strong foundation for future investigations into PGN-driven immune priming in molluscs.

4.4.3. Lipopolysaccharide

Lipopolysaccharides (LPS) are major components of Gram-negative bacterial outer membranes and are recognised as PAMPs by TLR4, initiating a cascade of innate immune signalling events that culminate in inflammation-associated pathogen neutralisation [238,239,240]. Early foundational studies in horseshoe crab (Tachypleus tridentatus) identified LPS-binding proteins capable of detecting LPS and inducing robust innate immune activities [241,242] with comparable LPS-elicited responses characterised in abalone [243,244], oysters [245,246,247], clams [64], and shrimp [248,249]. However, LPS is not uniformly immunostimulatory; excessive or prolonged stimulation may suppress immune function through dose-dependent downregulation of key immune genes [250]. Although LPS research in aquatic invertebrates has largely focused on elucidating fundamental host–pathogen interactions, several studies demonstrate dietary or injected LPS can enhance antiviral and antibacterial resistance in penaeid shrimp. Dietary supplementation with Vibrio harveyi–derived LPS resulted in dose-dependent improvement in survival of post-larval (P. monodon) shrimp against WSSV; however, concentrations exceeding 50 mg kg⁻¹ or extending feeding prior to challenge reduced survival rates [251,252] (

Table 8. Lipopolysaccharide-based immunostimulants utilised in crustacean immune priming experiments.

Immunostimulant	Source of Immunostimulant	Animal	Disease	Survival %	Administration	Source
LPS	Vibrio harveyi	Penaeus monodon	WSSV	10 mg kg−1: 0%, 25 mg kg−1: 4%, 50 mg kg−1: 72%, 100 mg kg−1: 60% 12 days post challenge	Oral administration within diets at different concentrations (10, 25, 50, and 100 mg kg−1) for a month prior to challenge with WSSV by immersion	[251]
				Daily: 5%, 2 days: 53%, 5 days: 20%, 7 days: 15%	Oral administration within diets at 50 mg kg−1 with varying feeding frequencies (daily, every 2, 5, and 7 days) for 2 months and then challenged with WSSV by immersion	[252]
	Escherichia coli Vibrio harveyi		Vibrio harveyi	E. coli LPS 8 µg/body weight: 66.67%, 4 µg/body weight: 56.67%, untreated controls: 33.33% V. harveyi: 8 µg/body weight: 46.67%, 4 µg/body weight: 56.67% 10 days post challenge	Supplemented LPS from E. coli and V. harveyi into diet at two different concentrations (8 or 4 µg/body weight) and fed to juvenile shrimp for 4 weeks, then challenged with V. harveyi by immersion	[253]
LPS, fucoidan, carrageenan, inactivated Vibrio harveyi, and β-glucan	Vibrio harveyi Fucus vesiculasus Saccharomyces cerevisiae			Fucoidan: 82.2%, β-glucan: 84.4%, LPS: 84.4%, Carrageenan: 55.6%, heat-killed V. harveyi: 42.2%, and untreated controls: 51.1% 12 days post challenge	Orally fed diets containing 2.0 g kg−1 of LPS, fucoidan, carrageenan, inactivated V. harveyi, and β-glucan for 2 weeks and intramuscularly injection challenged with V. harveyi	[254]
LPS	Pantoea agglomerans	Penaeus japonicus	PAV	20 µg/kg shrimp−1/day: 75%, 40 µg/kg shrimp−1/day: 64.7%, 100 µg/kg shrimp−1/day: 52.9% 10 days post challenge	Oral administration of LPS within diets at varied concentrations (20, 40, and 100 µg/kg shrimp−1/day) and immersion challenged with PAV with feeding continued for 10 days after challenge	[255]
	Escherichia coli	Penaeus monodon	Vibrio parahaemolyticus	10 mg kg−1: 62.5%, 20 mg kg−1: 62.5%, 30 mg kg−1: 75%, 40 mg kg−1: 50%, and untreated controls: 50%	Dietary LPS administered at different concentrations (10, 20, 30, and 40 mg kg−1) over 10 days, then challenged with V. parahaemolyticus	[256]
		Litopenaeus vannamei	Vibrio parahaemolyticus WSSV	Approx. values inferred from survival graph V. parahaemolyticus: 0 mg kg−1: 45%, 0.2 mg kg−1: 50%, 1 mg kg−1: 78%, 5 mg kg−1: 65%, 25 mg kg−1: 70%, or 125 mg kg−1: 70% 3 days post challenge WSSV: 0 mg kg−1: 10%, 0.2 mg kg−1: 40%, 1 mg kg−1: 35%, 5 mg kg−1: 20%, 25 mg kg−1: 35%, or 125 mg kg−1: 30% 5 days post challenge	Shrimp-fed diets containing LPS at 0, 0.2, 1, 5, 25, or 125 mg kg−1 for 8 weeks and injection challenged with either V. parahaemolyticus or WSSV	[257]

).

Oral administration of LPS derived from Pantoea agglomerans enhanced immunity in P. japonicus, with lower doses providing better protection against penaeid acute viraemia (PAV), underscoring the dose-dependent balance between immune priming and immune exhaustion [255].

Beyond viral interactions, LPS has also been shown to improve bacterial resistance in juvenile P. monodon shrimp, with intermediate dietary levels enhancing survival following V. parahaemolyticus challenge and maximised PO activity [256]. In L. vannamei, low to moderate LPS inclusion (0.2–1 mg kg⁻¹) improved growth, immune enzyme activity, and survival against both WSSV and V. parahaemolyticus, whereas high concentrations suppressed ACP and AKP activity. Transcriptomic analyses indicate LPS modulates complex cellular processes and innate immunity pathways, including phosphatidylinositol, Wnt, and Jak-Stat signalling, suggesting broad immunomodulatory effects [257].

Dietary LPS also enhances protection against V. harveyi, with P. monodon shrimp fed V. harveyi-derived LPS displaying markedly increased survival, elevated PA, antibacterial activity, and respiratory burst, similar to results reported in other studies [250,254,258,259]. Consistent results were achieved for both injection vs. immersion-based challenge models, indicating that LPS-mediated immune stimulation is robust across different infection routes, regardless of feeding frequency [254,260]. LPS origin furthers influences immunostimulatory potency with E. coli-derived LPS inducing stronger immune activation and greater protection than V. harveyi-derived LPS against V. harveyi. Dose-dependent patterns were observed in high doses of V. harveyi-derived LPS, suggesting induced immune suppression, whereas higher doses of E. coli-derived LPS remained beneficial [253].

As previously mentioned, LPS stimulation has not been applied to pathogen-challenge immune priming experiments, despite haemocytes of mussels [261,262], abalone [243,244], and oysters [245,247] exhibit robust LPS-induced immune responses, including the upregulation of immune-related genes and increased effector activities. These findings demonstrate that molluscs possess the necessary cellular machinery for LPS recognition and sustained activation, providing a mechanistic basis for future LPS-focused immune priming studies.

4.5. Whole Cell

Whole-cell vaccines, derived from inactivated bacterial or viral pathogens, have long been central to studying invertebrate immunity and developing disease mitigation strategies. Their efficacy stems from the presentation of a highly complex assembly of PAMPs, including PGNs, LPS, flagellins, and nucleic acids that mimic natural infection, while regarded as safe for administration. Across crustaceans and molluscs, whole-cell preparations consistently enhance cellular immune functions and confer increased survival upon pathogen re-exposure.

In crustaceans, whole-cell vaccination represents one of the most extensively investigated immune priming approaches (

Table 9. Whole cell-based immunostimulants utilised in crustacean immune priming experiments.

Immunostimulant	Animal	Disease	Survival %	Administration	Source
Vibrio spp. Strain NU-1	Penaeus japonicus	Vibrio spp. Strain NU-1	Injection: 68.7% Immersion: 71.4% and spray: 63.2% 10 days post challenge with unvaccinated controls at 21.1%	Vibrio spp. Strain NU-1 was inactivated by formalin and either injected with 9.5 × 108 cells intramuscularly, immersed for 1 h in 1% bacteria solution or sprayed for 10 s and then challenged 30 days post vaccination by intramuscular injection of live Vibrio spp. Strain NU-1 at 1.9 × 103 or 2.6 × 103 cells per prawn	[265]
WSSV		WSSV	(i) Naturally exposed WSSV shrimp: 95%, Controls at 20% 16 days post challenge (ii) Housed together: 77% after second challenge, House individually: 64% after second challenge (iii) Naturally exposed WSSV: 15%, Non-primed: 35%, PBS: 30%, Experimentally challenged: 85% 23 days post inoculation (iv) Experimentally challenged: 77%, PBS: 21%, Non-centrifuged WSSV: 13%	Shrimps that were (i) natural survivors of WSSV or (ii) naïve shrimp that were primed with live WSSV and either reared together or in separate aquaria and intramuscularly challenged 32 days later. Shrimp were also challenged with (iii) the haemolymph serum from the natural survivors of WSSV, the serum of experimentally challenged shrimp, non-primed serum, or PBS and combined with WSSV and delivered intramuscularly into naïve shrimps. Additionally, (iv) centrifuged serum from experimentally challenged shrimp, PBS, or not centrifuged experimentally challenged shrimp was intramuscularly injected into smaller naïve shrimp	[269]
			(i) 1 week: 13%, 2 weeks: 9%, 3 weeks: 39%, 4 weeks: 58%, 1 month: 67%, 2 months: 54%, 3 months: 6% relative percent survival (ii) 1 week: -5%, 2 weeks: 14%, 3 weeks: 36%, 4 weeks: 50%, 1 month: 100%, 2 months: 38%, 3 months: 6% relative percental survival	Shrimps were intramuscularly injected with (i) WSSV at 1–4 weeks or 1–3 months post-initial exposure to virus or were injected with (ii) plasma treated with WSSV and then challenged with virus	[270]
WSSV, β-1,3-glucan, Vibrio penaeicida, and recombinant proteins of WSSV (rVP26, rVP28)			Formalin WSSV: 20%, heat-inactivated WSSV: 28% and controls: 24% to 40% formalin WSSV + β-1,3 glucan: 56%, formalin WSSV + V. penaeicida: 60%, formalin WSSV: 40% and PBS: 36% 14 days post challenge. rVP26: 60% at one and 68% at two vaccinations, rVP28: 24% at one and 96% at two vaccinations, E. coli protein: 8% at one and 20% with two vaccinations prior to challenge Formalin WSSV: 20%, formalin WSSV + V. penaeicida: 60% and PBS: 28% at 30 days post last vaccination with 3 vaccinations daily over 10 days	Intramuscular injection of either formalin or heat-inactivated WSSV alone or with immunostimulants; β-1,3-glucan, formalin-inactivated V. penaeicida. Recombinant proteins of WSSV (rVP26, rVP28) and control E. coli protein were also given at one or two dosages, followed by intramuscular challenge of WSSV at 10−4 dilution	[75]
WSSV	Fennerpenaeus indicus		First day: 0%, fifth day: 100%, tenth day: 100%, and fifteenth day post feeding: 0% at 10 days post challenge 0.025 g−1: 100% on the fifth and tenth day post feeding, 0.05 g−1: 100%, 0.075 g−1: 100%, 0.1 g−1: 100%	Formalin-inactivated WSSV was coated onto feed and fed to shrimps at 0.035 g−1 feed body weight d−1 for 7 consecutive days and challenged with WSSV orally on the first, fifth, tenth, and fifteenth day post feeding. Different dosages were trialed in feed (0.025 g−1, 0.05 g−1, 0.075 g−1, and 0.1 g−1) and challenged on the fifth and tenth day post feeding	[271]
	Procambarus clarkii		Relative survival rates: 2 mM BEI + WSSV: 77% on 7 days and 60% on 21 days 3 mM BEI + WSSV: 63% on 7 days and 30% on 21 days Heat-inactivated WSSV: 10% on 7 days and 3% on 21 days	Crayfish were intramuscularly injected with either BEI-inactivated WSSV or heat-inactivated WSSV, followed by WSSV challenge on the seventh and twenty-first day post vaccination	[272]
			Relative survival rates: Orally fed BEI-inactivated WSSV once: 60%, twice: 70%, three times: 75%, 17 days post challenge	Crayfish were orally fed BEI-inactivated WSSV for 1 day, 2 days, and 3 days prior to an oral challenge with WSSV	[273]
	Penaeus monodon		1 day post vaccination: 100% 5 days post vaccination: 50%, 8 days after challenge	Shrimp were orally vaccinated with formalin-inactivated WSSV feed at 1.75 × 106 DNA copies mL−1 per day for 7 days and challenged with 0.9 × 1013 viral DNA copies per gram tissue of WSSV at 1 and 5 days post vaccination	[274]
AquaVac™ Vibromax™ (MSD Animal Health, Boxmeer, NL)	Penaeus monodon and Litopenaeus vannamei	Vibrio parahaemolyticus	Relative survival rates: P. monodon: 50% (1/2 dose), 41% (1 dose), 59% (2 doses) at 1 day and 52% (1/2 dose), 72% (1 dose), and 48% (2 doses) 7 days post feeding L. vannamei: 50% (1/2 dose), 31% (1 dose), -4% (2 doses) at 1 day and 14% (1/2 dose), 36% (1 dose), and -9% (2 doses) 7 days post feeding	P. monodon and L. vannamei shrimp were fed Vibromax™ (formalin-inactivated V. anguillarum, V. ordalii, V. harveyi, V. parahaemolyticus, and V. vulnificus) enriched A. nauplii (1/2 dose, 1 dose, and 2 doses) and challenged via immersion with 1.0–1.4 × 105 CFU/mL of V. parahaemolyticus for 24 h at 1 day or 7 days post the 10-day feeding treatment.	[267]
Vibrio spp. Glucan	Penaeus monodon	Vibrio alginolyticus	Approx. values inferred from survival graph Indonesian shrimp Vibrio spp. + glucan: (i) 30%, (ii) 30%, (iii) 50%, Vibrio spp. alone: (i) 40%, (ii) 40%, (iii) 35%, glucan alone: (i) 15%, (ii) 30%, (iii) 20% Untreated controls: (i) 15%, (ii) 5%, (iii) 30% Thailand shrimp Vibrio spp. + glucan: (i) 55%, (ii) 55%, (iii) 95%, Vibrio spp. alone: (i) 65%, (ii) 60%, (iii) 80% Glucan alone: (i) 55%, (ii) 50%, (iii) 80% Untreated controls: (i) 35%, (ii) 55%, (iii) 70%	P. monodon sourced from Thailand and Indonesia and immersed for 5 h in either formalin-killed Vibrio species and glucan or inactivated Vibrio spp. or glucan alone. Shrimp were challenged by immersion at (i) 10, (ii) 20, and (iii) 30 days post vaccination with 107 V. alginolyticus bacteria per/mL. Survival was assessed over 10 days post challenge	[266]
Vibrio harveyi	Penaeus indicus	Vibrio harveyi	(i) Inactivated IN7 + IN7 challenge: 78%, Inactivated BP05 + BP05 challenge: 44.17%, non-primed controls + IN7: 63.80% or BP05 challenge: 6.50% (ii) Inactivated BP05 + IN7 challenge: 46.17%, BP04 challenge: 42.50%, BP05 challenge: 41%, and non-primed controls + IN7 challenge: 23.17%, BP04 challenge: 23.67%, BP05 challenge: 6.50% (iii) Live BP04 + BP04 challenge: 47.50%, IN7 challenge: 68.50%, BP03 challenge: 52.17%, BP05 challenge: 60.17%, non-primed controls + BP04: 23%, IN7 challenge: 35.50%, BP03 challenge: 21.50%, BP05 challenge: 31.33% Live IN7 + BP04 challenge: 37.33%, IN7 challenge: 50.50%, BP03 challenge: 43.7%, BP05 challenge: 38.17% (iv) Lyophilised BP04 + IN7 challenge: 39.82%, or BP04 challenge: 0.83%, non-primed controls + IN7 challenge: 51.59% or BP04 challenge: 0.33%	Shrimp were either primed by immersion with (i) formalin-inactivated V. harveyi strains (IN7 and BP05) at 2.07 × 107 cells mL−1 for 6 h (ii) lyophilised formalin-inactivated V. harveyi strain (BP05), (iii) and live strains (BP04 and IN7) following immersion challenge 48 h post vaccination at 1.24 × 106 cells mL−1 with different strains. Shrimp were also (iv) orally primmed (4 mg l−1 day−1), with BP04 strain and immersion challenged with IN7 and BP04 strains at 1.24 × 106 cells mL−1	[264]
Vibrio alginolyticus	Litopenaeus vannamei	Vibrio alginolyticus	(i) 70% (ii) 83.3% (iii) 40% at 7 days post challenge	Shrimp were vaccinated by injection with 3.8 × 105 cfu shrimp−1 of either (i) heat-killed or (ii) formalin-inactivated V. alginolyticus or (iii) marine saline and challenged by injection with live V. alginolyticus at 8.0 × 106 cfu shrimp−1 at 7 days post vaccination	[275]
			Formalin-killed V. alginolyticus: 60.71% Heat-killed Vibrio alginolyticus: 82.14% Untreated controls: 6.68% 10 days post challenge	Oral administration of heat-killed and formalin-inactivated V. alginolyticus for 7 days prior to injection challenge with 1.5 × 106 CFU mL−1 of live V. alginolyticus	[276]
Aeromonas hydrophila	Eriocheir sinensis	Aeromonas hydrophila	>60% 7 days post challenge	Formalin-inactivated A. hydrophila was injected into crabs at 1.2 × 105 cfu/g of crab and 7 days later injection challenged with live A. hydrophila at the same concentration	[277]
Vibrio parahaemolyticus	Scylla paramamosain	Vibrio parahaemolyticus	>50% 246 h post challenge	Crabs injected with heat-killed V. parahaemolyticus at 104 cfu/g of crab and were challenged 7 days later by injection with live V. parahaemolyticus at 5.0 × 103 cfu/g	[278]
			(i) 5 × 103 cells·g−1: 25%, 5 × 104 cells·g−1: 75.7%, 1 × 105 cells·g−1: 56.1%, 2 × 105 cells·g−1: 100%, 4 × 105 cells·g−1: 35% (ii) 12 h: 80%, 24 h: 75%, 72 h: 75%, 120 h: 70%, 168 h: 65% (iii) formalin-inactivated V. parahemolyticus: 73.5%, saline-injected: 32.1%, No injection: 28.4% 7 days post challenge	Crabs injected into the hemocoel with formalin-inactivated V. parahaemolyticus at (i) different dosages (5 × 103, 5 × 104, 1 × 105, 2 × 105, and 4 × 105 cells·g−1) and 72 h later challenged by injection with live V. parahemolyticus at a dose of 5.0 × 104 cells·g−1. Crabs were also challenged (ii) at different timepoints post vaccination (12–168 h) with 2.0 × 105 cells·g−1 inactivated V. parahemolyticus. Crabs were injected (iii) with 2.0 × 105 cells·g−1 of formalin-killed V. parahemolyticus prior to live challenge with V. parahemolyticus at 5.0 × 104 cells·g−1	[279]
Vibrio anguillarum	Fenneropenaeus merguiensis	Vibrio harveyi and Vibrio anguillarum	V. harveyi challenge: 106 CFU kg−1 feed: 40%, 108 CFU kg−1 feed: 63.43%, 1010 CFU kg−1 feed: 65%, 1012 CFU kg−1 feed: 63.43%, 8 days post challenge V. anguillarum challenge: 106 CFU kg−1 feed: 60%, 108 CFU kg−1 feed: 74%, 1010 CFU kg−1 feed: 72.77%, 1012 CFU kg−1 feed: 69.43% 8 days post challenge	Post-larval shrimp were orally vaccinated with different concentrations (106, 108, 1010, and 1012 CFU kg−1 feed) of formalin-inactivated V. anguillarum for 6 weeks and then challenged with live V. harveyi and V. anguillarum for 1 h at 106 CFU/mL−1 and 1010 CFU/mL−1, respectively	[280]
Vibrio alginolyticus and Vibrio harveyi	Litopenaeus vannamei		Heat-inactivated V. alginolyticus + formalin-inactivated V. alginolyticus + formalin-inactivated V. harveyi + formalin-inactivated V. harveyi + live V. harveyi challenge: 73% Heat-inactivated V. alginolyticus + formalin-inactivated V. alginolyticus + formalin-inactivated V. harveyi + formalin-inactivated V. harveyi + live V. alginolyticus challenge: 90% Untreated controls + live V. harveyi: 53% Untreated controls + live V. alginolyticus: 67% 7 days post challenge	Shrimp were either vaccinated by injection with heat-inactivated and formalin-inactivated V. alginolyticus and V. harveyi at 3.8 × 105 cfu shrimp−1 prior to challenge by injection with either live V. alginolyticus at 6.4 × 107 cfu/shrimp−1 or V. harveyi at 4.4 × 106 cfu/shrimp−1 5 weeks after vaccination	[281]

). Early investigations in penaeid shrimp employed inactivated Vibrio spp. to augment antibacterial immune responses [263] and improve survival following pathogenic challenge [264,265,266]. Building upon these early observations, commercial multivalent vaccine AquaVac™ Vibromax™, comprising of formalin-inactivated V. anguillarum, V. ordalii, V. harveyi, V. parahaemolyticus, and V. vulnificus, demonstrated substantial protective efficacy when administered orally via enriched A. nauplii in P. monodon and L. vannamei shrimp [267]. This multivalent vaccine reduced bacterial loads, increased survival against V. parahaemolyticus, and improved overall health. However, a follow-up study revealed variable efficacy across Vibrio species, with limited protection observed against V. harveyi, potentially due to confounding nutritional effects during challenge experiments [268].

Comparable broad-spectrum protection has also been reported in banana shrimp (F. merguiensis), where oral administration of inactivated V. anguillarum conferred cross-protection against both V. anguillarum and V. harveyi, yielding marginally higher protection (10%) against the homologous pathogen challenges [280]. Whole-cell vaccination did not impair growth, as vaccinated shrimp exhibited increased carcass lipid and protein content, reinforcing the suitability of such strategies for aquaculture applications [268,280].

Pathogen inactivation methods strongly influence vaccine efficacy. Formalin-inactivated V. alginolyticus provided longer-lasting protection due to improved preservation of antigenic epitopes, whereas heat inactivation may promote excessive LPS release and epitope denaturation, exhibiting a shorter immune response that resulted in higher mortalities [275]. Nevertheless, contradictory findings have reported heat-inactivated V. alginolyticus eliciting higher survival rates compared to formalin-inactivated preparations [276]. To address these inconsistencies, combined vaccines incorporating both heat- and formalin-inactivated V. harveyi and V. alginolyticus demonstrated significantly enhanced survival following live challenge, albeit with consistently higher protection against V. alginolyticus than V. harveyi [281].

In the Chinese mitten crab (E. sinensis), formalin-inactivated A. hydrophila significantly enhanced survival following homologous challenge, increasing cellular immunity [277]. Mud crab (Scylla paramamosain) primed with heat-killed V. parahaemolyticus also exhibited enhanced survival and upregulation of immune genes, along with strong down syndrome cell adhesion molecule (Dscam) expression upon V. parahaemolyticus challenge [278]. Dscam upregulation was also reported in A. hydrophila-primed crabs, supporting its proposed role in pathogen recognition and clearance within invertebrates [277,278]. Formalin-inactivated V. parahaemolyticus conferred marginally higher survival than heat-inactivated preparations, though immune responses (PO activity) were sometimes transient [277,279].

Early studies in whole-cell priming utilizing viral pathogens revealed increased WSSV resistance in shrimp following viral re-exposure was attributable to immune priming, rather than genetic selection with plasma from previously exposed individuals exhibiting viral neutralisation capacity [269,270,271]. Housing conditions influenced priming efficacy, with animals housed together conferring superior resistance than animals housed alone [269]. In crayfish (P. clarkii), binary ethylenimine (BEI)-inactivated WSSV significantly improved survival, whereas heat-inactivated virus failed to confer protection, likely due to capsid protein denaturation, essential for immune recognition [272]. Oral delivery trials confirmed that BEI and formalin-inactivation preserve viral structural integrity, eliciting robust immune priming responses [276,280]. Similar, albeit less pronounced, inactivation-dependent effects have been reported for bacterial vaccines in shrimp [272,275].

Delivery route of whole-cell vaccines has been shown to impact efficacy. Immersion priming, particularly using freeze-dried or freshly prepared V. harveyi, provided short-term protection against V. harveyi in P. indicus shrimp [264], while spray vaccination and injection yielded comparable survival outcomes in P. japonicus prawns against V. vulnificus [265]. Timing between priming and challenge, as well as co-administration with immunostimulants such as β-glucans, further enhanced long-term protection [75,266,274]. Collectively, vaccine efficacy depends greatly on the structural integrity maintained throughout inactivation processes [75,272,273].

Whole-cell vaccination strategies in molluscs remain comparatively underexplored relative to crustaceans; however, studies suggest that molluscs exhibit robust pathogen resistance (

Table 10. Whole cell-based immunostimulants utilised in molluscan immune priming experiments.

Immunostimulant	Animal	Disease	Survival %	Administration	Source
Listonella anguillarum	Chlamys farreri	Listonella anguillarum	(i) 100% (ii) 30% (iii) 30% 244 h (approx. >10 days) post challenge	Scallops were initially immersion challenged in 0.1 × 108 CFU mL of L. anguillarum for 8 h with no rechallenge for group (i) with immersion time increased to 100 h with no rechallenge (ii) or (iii) rechallenged with bacteria 6 days post-initial challenge for 100 h	[285]
Vibrio anguillarum	Chlamys farreri	Vibrio anguillarum and Micrococcus luteus	Heat-killed V. anguillarum + V. anguillarum challenge: 63.6% Heat-killed V. anguillarum + M. luteus: 74% and untreated controls + V. anguillarum: 41% Untreated controls + M. luteus: 79% after 24-h post challenge	Injected scallops with 1 × 108 CFU mL−1 of heat-killed V. anguillarum and challenged by injection with live V. anguillarum or M. luteus 7 days post-initial vaccination	[286]
	Ruditapes philippiinarum	Vibrio anguillarum	At 7 days post-initial challenge: 69% At 7 days post-second challenge: 82% and 60% in non-primed controls	Injected clams with V. anguillarum (5 × 107 CFU/mL) and injected again at 7 days post-initial challenge	[287]
Vibrio harveyi	Haliotis tuberculata	Vibrio harveyi	Single infection: 36% Second infection: 56% 15 days post challenge	Adult abalone sampled from local hatcheries in the Brittany region of France were immersed in 106 CFU mL−1 V. harveyi for 24 h and rechallenged with bacteria	[288]
			Saint-Malo: 95% Molene: 51% 28 days post challenge	Abalone at Saint-Malo, a site of recurrent V. harveyi infections, and Molene, a site not affected by V. harveyi in Brittany, France, were sampled and rechallenged by immersion of V. harveyi at 104 CFU mL−1 for 24 h. A second challenge was done with V. harveyi 28 days later	[289]
Bacterial: SY9, Yeast: SS1 and AY1	Haliotis midae	Vibrio anguillarum	Probiotic diet: 62% Commercial diet: 29% 7 days post challenge	Abalone were fed probiotic-supplemented diet, containing bacteria isolated from the gastrointestinal tract and digestive tract of abalone at 107 viable cells/g of dried feed for 2 weeks, then challenged with live V. anguillarum by injection at 1010 cfu/mL	[290]
Shewanella colwelliana WA64 and Shewanella olleyana WA65,	Haliotis discus hannai	Vibrio harveyi	Untreated controls: 80% at 1 week, 70% at 2 weeks, 75% at 3 weeks, 80% at 4 weeks: 20–23% S. colwelliana WA64: 73% at 1 week, 60% at 2 weeks, 53% at 3 weeks, 50% at 4 weeks S. olleyana WA65: 63% at 1 week, 70% at 2 weeks, 56% at 3 weeks, 60% at 4 weeks	Abalone were orally administered two probiotics isolated from the gastrointestinal tract of the abalone at 109 cell/g-1 of either probiotics for 1, 2, 3, and 4 weeks and challenged with V. harveyi by injection with 2 × 107	[291]
Agarivorans albus strain F1-UMA, Vibrio sp. Strain F15-UMA, and Vibrio sp. Strain C21-UMA	Haliotis rufescens	Vibrio parahaemolyticus	1 month post feeding Fed probiotics: 63.3% Untreated controls: 51.7% 7 months post feeding Fed probiotics: 51% Untreated controls: 72% 4 days post challenge	Juvenile abalone were fed macroalgae-supplemented with probiotics for 1 and 7 months and challenged with V. parahaemolyticus by injection at 1 × 106 CFU/g of abalone	[292]
Exiguobacterium JHEb1, Vibrio JH1 and Enterococcus JHLDc	Haliotis iris	Vibrio splendidus	Probiotic-fed abalone + V. splendidus: 78% Non-probiotic-fed abalone + V. splendidus: 66.7% at 48 h post challenge	Abalone were fed multi-strain probiotics incorporated into commercial feed at 3 × 109 CFU g−1 for 4 months and then challenged by injection with V. splendidus at 5 × 107 cfu/mL	[293]
Vibrio harveyi	Haliotis diversicolor	Vibrio harveyi	Approx. values inferred from survival graph 1.42 × 103 CFU mL−1: 72% 1.42 × 104 CFU mL−1: 86% 1.42 × 105 CFU mL−1: 79% 1.42 × 106 CFU mL−1: 82% 1.42 × 107 CFU mL−1: 78% Untreated control: 46% 9 days post challenge	Abalone were initially primed by injection with five different concentrations of V. harveyi (1.42 × 103 CFU mL−1, 1.42 × 104 CFU mL−1, 1.42 × 105 CFU mL−1, 1.42 × 106 CFU mL−1, and 1.42 × 107 CFU mL−1) and 15 days later challenged with V. harveyi at 1.58 × 106 CFU mL−1 by injection	[294]
Vibrio parahaemolyticus	Haliotis discus hannai	Vibrio parahaemolyticus	Approx. values inferred from survival graph V. parahaemolyticus prime abalone: 50% Untreated controls: 0% 10 days post challenge	Adult abalone were injected with 1 × 108 CFU/mL of live V. parahaemolyticus and 7 days later were injected with V. parahaemolyticus	[295]
Vibrio alginolyticus	Crassostrea gigas	Vibrio alginolyticus	Live V. alginolyticus 5 × 104 CFU/mL: 0% 5 × 105 CFU/mL: 0% 5 × 106 CFU/mL: 0% Heat-inactivated V. alginolyticus 5 × 104 CFU/mL: 100% 5 × 105 CFU/mL: 92.5% 5 × 106 CFU/mL: 92.5% 20 days post challenge	Formalin-inactivated and live V. alginolyticus were administered by immersion at different concentrations (5 × 104 CFU/mL, 5 × 105 CFU/mL, and 5 × 106 CFU/mL) and then challenged with live V. alginolyticus at 5 × 106 CFU/mL 7 days later	[57]
OsHV-1		OsHV-1	(i) 22 °C OsHV-1: 94.9%, 18 °C OsHV-1: 91.3%, (ii) 22 °C OsHV-1: 75.6% 18 °C unprimed control + 22 °C OsHV-1: 79.6% 22 °C unprimed control + 22 °C OsHV-1: 66.7% 18 °C unprimed control + 18 °C OsHV-1: 96.3% 10 days post challenge	Triploid oyster spat were intramuscularly injected at (i) 18 °C or (ii) 22 °C with OsHV-1 and then immersion challenged 35 days post-initial exposure for 6 h with 106 OsHV-1 DNA copies per oyster in 18 °C or 22 °C seawater	[296]
			Naturally exposed OsHV-1: 90.2% Naïve unprimed adult oysters: 8.3% Naïve unprimed oyster spat: 16.7% 10 days post challenge	Adult oysters that were previously naturally exposed to OsHV-1 were intramuscularly injected challenged with OsHV-1 at 5.07 × 104 OsHV-1 DNA copies/100 μL	[297]
OsHV-1 and Poly(I:C)			Heat-inactivated OsHV-1: 73.3% Live OsHV-1: 81.2% Poly(I:C): 100% Untreated controls: 47% OsHV-1 negative tissue homogenate: 45% 14 days post challenge	Heat-inactivated OsHV-1, live OsHV-1 (3 × 106 genome copies per oyster), and Poly(I:C) (250 μg per oyster) and were intra-muscularly injected into oysters at 18 °C. Temperature was increased 2 weeks later to 22 °C for 6 h and 1 week later were challenged with live OsHV-1 at 6 × 106 genome copies per oyster	[135]
			UV-inactivated OsHV-1: 97%, UV-treated virus-free solution: 23%, Poly(I:C): 100%, untreated controls: 13% 7 days post infection UV-inactivated OsHV-1: 100% at 8 days, 93% at 1 month, 87% at 2 months, and 63% at 3 months post priming for 7 days post challenge	UV-inactivated OsHV-1 (105 copies/μL of OsHV-1), UV-treated virus-free solution, and Poly(I:C) (100 μg per oyster) were injected into oysters and challenged by immersion to OsHV-1 24 h later. Oysters were rechallenged at 8 days, 1 month, 2 months, and 3 months by injection with 100 μL of 3.9 × 104 μg/μL of OsHV-1	[298]

). Heat-killed V. splendidus in oysters enhanced haemocyte activity, notably phagocytosis in a pathogen-specific manner [282]. Immersion of oysters in low concentrations of formalin-inactivated V. alginolyticus resulted in exceptionally high survival (100% and 92.5%), whereas priming with live V. alginolyticus at equivalent doses resulted in strong inflammatory responses and accelerated mortality [57]. These contrasting outcomes reflect differences in ROS-mediated production, whereby live bacteria induced excessive inflammatory responses and tissue damage and inactivated bacteria elicited a controlled protective oxidative response [57,283,284].

Whole-cell priming has been extensively investigated for OsHV-1 in C. gigas. Field observations demonstrated that adult oysters previously exposed to OsHV-1 exhibited markedly increased survival upon rechallenge (90.2%) compared to naïve spat or adults (16.7% and 8.3%, respectively) and carried reduced viral loads [297]. Experimental work confirms non-lethal OsHV-1 exposure at 18 °C enhances resistance upon viral challenge at 22 °C, highlighting the synergistic relationship between temperature and viral susceptibility for OsHV-1 [296]. Live and heat-inactivated OsHV-1 conferred substantial (81.2% and 73.3%) protection, though Poly(I:C) consistently provides the highest survival (100%) [283]. Ultraviolet (UV)-inactivated OsHV-1 achieved protection levels comparable to Poly(I:C) (97–100%), with long-lasting immunity persisting over 3 months post priming, supported by upregulation of interferon-like and NF-κB-associated antiviral pathways, including RLRs, MyD88, IRFs, and viperin [284].

In scallops (C. farreri), both live and inactivated bacteria induce strong innate immune activation exhibited through phagocytosis, enzymatic activity, and PRRs activation with markedly improved survival upon re-exposure [285,286]. Manila clams (R. philippinarum) also exhibit enhanced AMP expression, immune enzyme activity, PRR signalling, and overall survival following secondary live or inactivated V. anguillarum challenge [290]. These findings closely parallel the protective effects observed in oysters and reinforce the efficacy of whole-cell-induced protection across bivalves [282,286].

In gastropods, particularly abalone, whole-cell priming and natural re-exposure studies provide compelling evidence for long-term immune enhancement. European abalone (Haliotis tuberculata) from the Saint-Malo region of Brittany, which experiences recurrent V. harveyi outbreaks, exhibit elevated survival and enhanced phagocytic capacity 2 months post challenge, along with significantly reduced bacterial loads in the gills, implying coordinated mucosal and systemic protection [289]. This resistance has been attributed to haemolymph AMPs and hemocyanins, which inhibit systemic bacterial growth and transmission [299,300], with reduced bacterial loads associated with localised mechanisms that impair bacterial attachment and lysozyme-mediated pathogen clearance [301,302].

Additional studies report reduced mortality and strong transcriptional responses (e.g., ferritin, heat shock protein HSP84, and fatty acid binding protein) upon secondary V. harveyi exposure [288]. Dose-dependent priming effects have been demonstrated in small abalone (H. diversicolor), with intermediate doses of live V. harveyi providing optimal protection upon secondary challenge [294]. Transcriptomic analyses revealed upregulation of PRRs, immune effector pathways associated with phagocytosis, calcium signalling, and metabolic regulation involved in pathogen defence and clearance. Similar immune responses were observed in Japanese disc abalone (Haliotis discus hannai) following priming with live V. parahaemolyticus, with activation of PRRs (TLRs and RIG-I), NF-κB, TNF, and MAPK signalling pathways within haemocytes [289,294,295,299,300].

Probiotic-based whole-cell exposure also enhances growth and disease resistance in abalone. Probiotics isolated from the gastrointestinal tract of African abalone (Haliotis midae) significantly enhanced growth and survival with elevated haemocyte THC and PA during V. anguillarum challenge [290]. Non-virulent Shewanella species isolated from H. discus hannai gastrointestinal tract improved survival and both cellular and humoral immunity when incorporated into feed [291], likely via mechanisms, such as quorum sensing, melanin secretion, and other antibacterial characteristics [303,304,305]. Prolonged probiotic feeding has similarly reduced pathogen loads and conferred sustained protection against Vibrio species in Haliotis iris and Haliotis rufescens [292,293,306]. Collectively, these findings support persistent, nonspecific immune stimulation through whole-cell or probiotic exposure, consistent with immune priming theory in invertebrates [32].

TGIP extends invertebrate immune priming by enabling parental immune experience to enhance offspring disease resistance. Evidence across crustaceans and molluscs demonstrates that exposure to whole-cell bacterial or viral pathogens can enhance disease resistance in offspring, primarily through maternal transfer of immune effectors and epigenetic modifications. TGIP was first reported in Daphnia magna, where maternal exposure to Pasteuria ramosa resulted in strain-specific enhancement of offspring fitness and survival [52]. Artemia species have since become key TGIP models due to their short generation times. In A. nauplii, maternal priming with V. campbellii significantly improved survival across F1–F3 generations following homologous challenge, although this protection declined with successive generations from 40% improvement in F1, 70% in F2, and to ~20% in F3, indicating progressive dilution of inherited immunity [307] (

Table 11. Whole cell-based immunostimulants utilised in crustacean and molluscan transgenerational immune priming experiments.

Immunostimulant	Animal	Disease	Survival %	Administration	Source
Vibrio anguillarum	Chlamys farreri	Vibrio anguillarum	Primed trochophore (22 h post fertilisation): 78%, non-primed trochophore: 48% Primed D-shaped larvae (2 days post fertilisation): 40%, non-primed D-shaped larvae: 24% Primed gastrula (18 h post fertilisation): 80%, non-primed gastrula: 65%	Scallop mothers were intramuscularly injected with heat-killed V. anguillarum (1 × 108 cells/mL) at 18 °C, and then offspring were challenged with live V. anguillarum (5 × 108 cells/L) by immersion at 20 °C	[56]
Vibrio campbellii	Artermia nauplii	Vibrio campbellii	Approx. values inferred from survival graph Primed F1 offspring: 85%, non-primed F1 offspring: 50%, primed F2 offspring: 90%, non-primed F2 offspring: 20%, primed F3 offspring: 50%, non-primed F3 offspring: 30% 36 h post challenge.	A single A. nauplii female (F0) was grown in V. campbelli (107 cells mL−1) culture conditions and then reproduced with male grown in normal culture conditions. The offspring F1, F2, and F3 larval generations were challenged with live V. campbelli (107 cells mL−1) in the culture media.	[307]
	Artemia franciscana	Vibrio campbellii and Vibrio harveyi	Approx. values inferred from survival graph Homologous challenge + primed F1 larvae: 80%, F1 cysts: 60%, non-primed F1 larvae: 60%, non-primed cysts: 40%, primed F2 larvae: 70%, primed cysts: 60%, non-primed F2 larvae: 55%, non-primed cysts: 30%, primed F3 larvae: 40%, primed cysts: 60%, non-primed F3 larvae: 30%, non-primed cysts: 45% Heterologous challenge + primed F1 larvae: 70%, primed cysts: 60%, non-primed F1 larvae: 65%, non-primed cysts: 65%, primed F2 larvae: 58%, primed cysts: 58%, non-primed F2 larvae: 63%, non-primed cysts: 60%, primed F3 larvae: 62%, primed cysts: 42%, non-primed F3 larvae: 50%, non-primed cysts: 39%:	Instar larvae (2 days post hatching) (F0) were immersion challenged in V. campbelli (107 cells mL−1) repeatedly prior to reproductive maturity. Then females reproduced F1, F2, and F3 larval offspring, and within these generations, their cysts as well were challenged with live V. campbelli (107 cells mL−1) or V. harveyi (107 cells mL−1) by immersion	[308]
Vibrio campbellii and Vibrio parahaemolyticus		Vibrio campbellii, Vibrio harveyi, and Vibrio parahemolyticus	Approx. values inferred from survival graph Homologous challenge + live V. parahaemolyticus primed F1 larvae: 40%, F2 larvae: 30%, F3 larvae: 30%, inactivated V. parahaemolyticus primed F1 larvae: 50%, F2 larvae: 40%, F3 larvae: 45%, non-primed F1 larvae: 20%, F2 larvae: 20%, F3 larvae: 30%, live V. campbellii primed F1 larvae: 50%, F2 larvae: 50%, F3 larvae: 40%, Inactivated V. campbellii primed F1 larvae: 40%, F2 larvae: 55%, F3 larvae: 45%, non-primed F1 larvae: 20%, F2 larvae: 25%, F3 larvae: 10% Live V. parahaemolyticus primed + V. campbellii challenge F1 larvae: 20%, F2 larvae: 40%, F3 larvae: 35%, inactivated V. parahaemolyticus primed + V. campbellii challenge F1 larvae: 35%, F2 larvae: 40%, F3 larvae: 30%, non-primed F1 larvae: 25%, F2 larvae: 20%, F3 larvae: 10% Live V. campbellii primed + V. parahaemolyticus challenge F1 larvae: 25%, F2 larvae: 45%, F3 larvae: 42%, inactivated V. campbellii primed + V. parahaemolyticus challenge F1 larvae: 30%, F2 larvae: 50%, F3 larvae: 30%, non-primed F1 larvae: 20%, F2 larvae: 30%, F3 larvae: 30% 2 days post challenge	Parental generation (2 days post hatching) was primed with live or inactivated V. campbelli (107 cells mL−1) or V. parahaemolyticus (107 cells mL−1) by immersion prior to reproductive maturity and offspring (1 day old) were homologous or heterologous challenged with live campbelli (107 cells mL−1) or V. parahaemolyticus (107 cells mL−1) or V. harveyi (107 cells mL−1) by immersion	[310]
Vibrio splendidus	Crassostrea gigas	Vibrio splendidus	Naïve-only parents + V. splendidus: 84.7% Naïve-only parents + no challenge: 96.4% Primed female and naïve male + V. splendidus: 96.75% Primed female and naïve male + no challenge: 94.9% Primed male and naïve female + V. splendidus: 95.9% Primed male and naïve female + no challenge: 93.7%	Oyster parents were injected with V. splendidus (2 × 108 CFU/mL) and spawned 7 days post priming in the following conditions (naïve-only parents, primed female + naïve male, naïve female + primed male). Offspring at the blastula stage (4.5 h post fertilisation) were challenged by immersion with V. splendidus (1 × 107 CFU/mL) until offspring reached D-veliger developmental stage (24 h post fertilisation)	[58]

). Similar multigenerational species-specific protection was observed in A. franciscana offspring following whole-cell priming with Vibrio species, whereas heterologous challenges yielded substantially reduced protection [308]. Enhanced resistance in F1 and F2 generations was associated with elevated expression of heat shock protein 70 (HSP70) and high mobility group box 1 (HMGB1) [309]. Importantly, this protection was accompanied by persistent epigenetic signatures, including altered H3 and H4 acetylation and enrichment of H3K4me3 across both parental and offspring generations, suggesting an epigenetic inheritance of immune memory [308].

Consistent evidence demonstrates parental priming with live or inactivated V. campbellii or V. parahaemolyticus enhances homologous protection across F1–F3 generations, with markedly lower protection during heterologous challenge [310]. This inherited resistance correlates with elevated expression of immune-related genes, including peroxinectin (pxn), proPO, LPS-binding protein, βGBP, hmgb1, and HSP90, alongside epigenetic modifications, including altered H3/H4 acetylation and m6A RNA methylation, reinforcing an epigenetic basis for TGIP [308,310].

TGIP is also well documented in molluscs. In C. farreri, maternal priming with heat-killed V. anguillarum during oocyte maturation increased offspring levels of immune effector proteins, Cu/Zn-SOD activity, and antibacterial capacity, accompanied by significantly reduced mortality following bacterial challenge [56]. These findings were consistent with earlier work in insects, demonstrating that offspring immune competence is strongly influenced by maternal immunological history [53].

In oysters, maternal priming with formalin-inactivated V. alginolyticus significantly enhanced offspring resistance to multiple Vibrio species [57], while V. splendidus exposure during spawning increased egg-associated lysozyme, SOD activity, immune gene expression (TLR2, MACPF, and FBG), and antibacterial activity [58]. Following fertilisation, maternally primed larvae exhibited significantly elevated CAT, lysozyme, and SOD activity, whereas paternally priming only resulted in increased expression of immune genes (galectin, MyD88, and LBP) in offspring, which was also demonstrated in maternally primed offspring. Both parental treatments, however, significantly reduced larval mortality upon V. splendidus challenge [58].

Collectively, these studies demonstrate that whole-cell TGIP operates across multiple molluscan and crustacean species, conferring pathogen and strain-specific protection via maternally transferred immune effectors and heritable epigenetic modifications. Although protection can persist across multiple generations, particularly in rapidly reproducing species such as Artemia, the strength of protection typically declines over time, underscoring the need for further research into mechanisms that support durable, long-term inherited immunity.

5. Challenges to Immune Priming

5.1. Synthesis of Immune Primers

There are several practical, technical, and regulatory constraints associated with immunostimulant synthesis, all of which can influence their efficacy, safety, and reproducibility in invertebrate aquaculture. Many immunostimulants are biologically derived (β-glucan, LPS, PGN, nucleic acids, and whole cell), and differences in organism source, strain, culture conditions, and purification methods can result in substantial batch-to-batch variation in molecular composition and immunostimulatory potency. β-glucan provides a well-studied example of how physicochemical properties governing immunological outcomes, such as solubility, molecular weight, and branching patterns, strongly influence immune activation and protection capacity. Given the limited solubility of β-glucans and associated suboptimal immunoenhancement, chemical and physical modifications (carboxymethylation, sulfoethylation, and γ-irradiation) have been employed to improve solubility and efficacy. Although these modified β-glucans have enhanced antiviral efficacy [177,178], these approaches are costly, require specialised infrastructure, and rely on hazardous reagents, limiting their scalability in aquaculture [311].

Moreover, chemical modification can alter β-glucan structure with unpredictable consequences for immunogenicity [312]. Some modified marine yeast-derived β-glucans enhance WSSV protection, whereas others increase susceptibility [175,176,179], suggesting that immunogenicity depends on molecular weight and structure. Conflicting results across studies highlight the complexity of β-glucan structure and function in immune priming. Recombinant protein vaccines present additional challenges as they require genetically modified production systems, are labour-intensive to manufacture [313], and often elicit weaker immunogenic response than whole-cell formulations, which possess a more complex array of PAMPs. Many immunostimulants, particularly nucleic acids, exhibit chemical or biological instability and degrade rapidly due to fluctuations in pH and nucleases [314]. To address this, encapsulation techniques, including nanoparticle-based delivery systems (chitosan, liposomes, and virus-like particles), are increasingly employed to enhance the stability and delivery. However, these encapsulation methods present their own challenges in scalability, short half-lives, high cost, and impracticability in oral delivery [314].

5.2. Administration

Immune priming strategies in molluscs and crustaceans are constrained by both biological and logistical challenges in administration. Injection remains the most experimentally robust delivery method, offering precise control over dosage, reduced variability in uptake, and direct delivery to target tissues, enabling rapid and strong immune responses [315]. It is also the most effective route for delivering immunostimulants such as poly(I:C) in oysters [127,133,135] and baculovirus-expressed VP28 in crayfish [88]. Despite its experimental advantages, injections are inherently labour-intensive, are stressful, require skilled personnel, and are fundamentally impractical for large-scale aquaculture.

Immersion offers a more scalable alternative, enabling high-throughput exposure with reduced handling; however, uptake through epithelial surfaces such as gills is highly variable across developmental stages and species, resulting in inconsistent immune activation and efficacy [49]. Oral administration via feed represents the most operationally feasible strategy for large-scale aquaculture, but faces substantial challenges, including degradation of immunostimulants within the digestive tract, variable ingestion rates, and limited bioavailability.

Selecting an administration route should therefore consider logistics and the natural transmission of the target pathogen and the subsequent host–pathogen interactions [316]. Immune priming strategies that mimic natural infection routes may improve immunological relevance and protective outcomes. This principle is exemplified in TGIP studies in Caenorhabditis elegans, where oral exposure to Orsay virus, reflecting the natural route of viral transmission produced heritable antiviral protection in progeny, while injection failed to confer transgenerational protection [317,318]. Similarly, WSSV spreads orally in shrimp, often through cannibalism [319], yet oral or immersion delivery of VP28 generally provides weaker protection than injection of the same antigen. Despite these limitations, immersion and oral-based approaches remain the only practical method for aquaculture, offering scalability and reduced animal handling, and can stimulate both systemic and mucosal immunity.

Recent advances in encapsulation technologies, including algae and nanoparticle formulations, have improved immunostimulant stability, uptake, and efficacy for both immersion and oral delivery routes, offering promising solutions to current delivery constraints [103,152,271,273].

5.3. Toxicity

Optimising dosage and dosing regimes is critical for effective immune priming in molluscs and crustaceans, as both insufficient and excessive doses can paradoxically exacerbate susceptibility to infection. Many immunostimulants have narrow therapeutic windows, with dose-dependent adverse effects reported across multiple immune priming strategies. For instance, recombinant FlaA becomes toxic when administered at doses above 20 µg in shrimp, reducing survival even without WSSV challenge and causing near-total mortality following challenge, highlighting the consequences of excessive immune activation [65].

Comparable dose-dependent effects have been documented for β-glucans, where overdosing or repeated administration can lead to immunosuppression, immune exhaustion, and immune fatigue, where the energetic costs of sustained immune activation outweigh the benefits and ultimately reduce disease resistance [176]. These effects vary with glucan source and structure, as some marine yeast-derived β-glucans have maintained elevated THC and survival rates even after prolonged use [174,179].

Poly(I:C) further illustrates the trade-off between potency and toxicity, inducing strong antiviral responses and high survival in oysters [59,127,133,282,320]. Yet its documented toxicity at high concentrations in vertebrates, such as that observed in rhesus monkeys (30 mg kg⁻¹) and beagles (5 mg kg⁻¹) [321,322,323], raises safety and regulatory concerns for aquaculture use. In contrast, inactivated OsHV-1 provides lower protection but is considered more acceptable by regulatory authorities.

Collectively, these findings emphasise the necessity for rigorous optimisation of dosage and frequency to balance efficacy while avoiding immunopathology, metabolic burden, and fitness costs.

5.4. Fitness Costs

Immune priming in invertebrates can enhance pathogen resistance; however, the initiation and maintenance are inherently metabolically costly, often imposing significant fitness costs to maintain [44,134,324]. The mechanisms underpinning the persistence of long-term immune enhancement remain not fully elucidated across and between species despite increasing empirical evidence for both immune priming and TGIP [325]. A clear example of these trade-offs occurs in the Peruvian calico scallop Argopecten purpuratus, where V. splendidus challenge revealed a pronounced trade-off between reproduction and immune defense [320]. Following spawning, scallops exhibited marked reductions in haemocyte abundance, protein content, metabolic rate, respiratory activity, and immunological activity, reflecting that the energetic costs of reproduction constrain immune competence.

Fitness costs have also been associated with TGIP, where sustained immune activation or parental investment in immune defence can negatively impact fecundity, growth rates, lifespan, and reproductive ability [326,327,328,329,330,331,332]. Such trade-offs suggest that enhanced offspring immunity may come at the expense of parental condition, influencing the evolutionary stability of TGIP. Inconsistencies reported in TGIP outcomes may therefore reflect differences in experimental design, biological context, or publication bias favouring positive findings [333]. In molluscs, these trade-offs are particularly evident. In oysters, maternal poly(I:C) priming increased larval survival against OsHV-1; however, simultaneously reduced larval growth altered the microbiome and increased opportunistic Vibrio species abundance [142]. These findings highlight that while immune priming and TGIP can provide short-term protection, their metabolic and fitness costs constrain long-term application, particularly in aquaculture, where rapid growth and reproductive output are critical.

6. Conclusions

In summary, immunostimulants represent a promising and environmentally sustainable approach for enhancing disease resistance in molluscan and crustacean aquaculture, owing to their capacity to activate innate immune defences in a broad or pathogen-specific manner while remaining biodegradable and generally safe for human consumption. Experimental evidence demonstrates that immunostimulant-mediated immune priming can reduce pathogen burden, enhance immune activation, and improve survival following challenge; however, significant barriers to commercial translation remain. These include constraints surrounding synthesis, administration, dose-dependent toxicity, and fitness costs linked to sustained immune activation. Although advances such as chemical modification, encapsulation technologies, and dosage optimisation have improved efficacy and delivery, further research is required to elucidate the mechanistic basis of immune priming in non-model aquaculture species and to evaluate the durability and long-term consequences of protection. Addressing these knowledge gaps will be critical for enabling the safe, scalable, and effective integration of immunostimulant-based immune priming strategies into intensive aquaculture production systems.