Autophagy and white spot syndrome virus infection in crustaceans

Highlights

• •Recent findings on the autophagy-related molecules involved in anti-WSSV immunity are summarized.
• •Different functions of autophagy in the process of WSSV infection are discussed and prospected.
• •The regulatory mechanisms of autophagy on WSSV infection are pointed out.

Abstract

Autophagy is an evolutionarily conserved process of degradation in eukaryotes, which can form double-membrane vesicles for delivering the trapped cargo to lysosome for degradation, also facilitate host cells against the invasion of foreign pathogens. Recently, autophagy was reported to participate in viral infection in crustaceans. White spot syndrome virus (WSSV) is the most severely viral pathogen for farmed crustaceans, particularly in crayfish and shrimp. In this review, we summarized and discussed the current findings of autophagy involved in WSSV infection in crustaceans, particularly focusing on the identified autophagy-related molecules and their effects on viral infection. We hope this summary will provide us a better understanding of autophagy and its contribution to antiviral immunity in crustaceans.

1. Introduction

According to the statistics of the United Nations Food and Agriculture Organization (FAO), the global aquaculture production of crustaceans was 9.4 million tons accounting for 11.4% of the world aquaculture production in 2018, among which widely farmed shrimp, crayfish, prawns, and river crabs are of high nutritional value as high-quality protein sources making great economic benefits in aquaculture [1]. However, aquatic crustaceans are often threatened by a variety of exotic pathogens, of which the virus has caused great harm to farmed crustaceans. As one of the most severe viral pathogens, white spot syndrome virus (WSSV), a member of the family Nimaviridae consisting of only one single genus Whispovirus, is an enveloped and rod-shaped double-strands DNA virus that has a wide host range, including crayfish, shrimp, crab and lobster, which has been causing huge economic losses [2]. Unfortunately,the research on the pathogenic mechanism of WSSV has not been deep enough, making the prevention and control of white spot disease not available so far. Due to the lack of adaptive immunity unique to vertebrates, crustaceans mainly rely on the innate immune system, mainly composed of cellular immunity and humoral immunity, to resist the invasion of pathogens including viruses, while autophagy has been recently shown to play a vital role as an important cellular immune defense against invading virus [3], which might be helpful for anti-WSSV designing in crustacean farming.

Autophagy is the process of delivering cytoplasmic materials to lysosomes where the cargos are further degraded [4], which involves many cellular processes, including the responses to starvation, cell death, and microorganism elimination. The concept of autophagy was first proposed in 1962, and the autophagy-related (ATG) genes ATG1 and microtubule-associated protein 1 light chain 3 (LC3) were cloned firstly in the 1990s, which greatly promoted the research on autophagy in diseases and immunity [5]. There are three different autophagy types, including macroautophagy, chaperone-mediated autophagy, and microautophagy according to the different transport modes of intracellular substrates. Besides, autophagy (herein referred to as macroautophagy) is the most studied cellular process that cytoplasmic materials are wrapped by bilayer membrane to form autophagosomes and then delivered to lysosomes for degradation [4]. Autophagy can remove damaged organelles, protein aggregates and excess cytoplasmic materials to effectively maintain the cellular homeostasis. Equally important, the host also uses the intracellular degradation pathway of autophagy to resist the infection of extracellular pathogens, that is, the direct elimination of intracellular pathogenic microorganisms through autophagy [6]. Currently, an increasing number of studies have shown autophagy is closely related to innate immunity after the invasion of pathogens, especially innate immune response to virus. Among arthropods, the model organism such as Drosophila [7] and mosquitoes [8] have been studied more and more deeply in terms of autophagy, and autophagy has been proven to play a fundamental role in growth, metamorphosis, hunger and immunity. In crustaceans, so far, the effect of autophagy on viruses has also made preliminary research advances, mainly focusing on the phenomena and regulatory machinery of proviral and antiviral autophagy, but there are still many research blind spots that need further experimental evidence. In this review, we mainly summarized and discussed the recent progress on the role of autophagy in anti-WSSV response in crustaceans, which might help us have a better insight of autophagy and provide new strategies and viewpoints for the research on the role of autophagy in antiviral immunity in crustaceans.

2. Autophagy involved in WSSV infection

Autophagy is an evolutionarily highly conserved degradation process that maintains the health of the host and the homeostasis of the cellular environment and helps the immune system to eliminate invading pathogens [9]. Generally, for viral infection, previous reports found that the viral protein or any single step in the viral life cycle, including viral adsorption, invasion, membrane fusion, replication and transcription could all trigger autophagy, which has important effects on viral infection [10]. In mammals, studies have shown that autophagy played a dual role in the process of viral infection, on the one hand, viruses have developed reverse mechanisms that evading or inhibiting autophagy even exploiting autophagy to promote viral entry and replication for achieving the successful infection [11]. On the other hand, autophagy played an antiviral role by directly degrading virus particles or regulating the inflammation and interferon-related innate immune responses; what's more, autophagy could also degrade the components of the viral particles and then present antigens to T lymphocytes to activate adaptive immunity in mammals [11, 12]. As one of the important aquatic economic species, crustaceans are facing with a variety of threats from viruses during the breeding process [13], especially WSSV. Similarly, in crustaceans, autophagy has also been found to play a dual role in the process of WSSV infection. In the following, we discussed in detail the different roles of autophagy in WSSV infection and the relevant molecular mechanisms according to the recently reported studies (Fig. 1).

Fig. 1.

Fig.1 Schematic representation of the different roles of autophagy and its associated molecules in WSSV infection. GB: γ-aminobutyric acid receptor-associated protein (GABARAP); Ub: ubiquitin; VCP: valosin-containing protein; CME: clathrin-mediated endocytosis; TRIM: tripartite motif.

2.1. Autophagy promoted WSSV invasion

Generally, the first step for the virus to effective infection is to enter the cells. The virus could be recognized by specific receptors or receptor-like molecules on the cell membrane surface to trigger the subsequent viral internalization process [14]. In the meantime, there is an important association between viral invasion and autophagy activation in host cells, and many viruses such as herpesviruses have been confirmed to induce autophagy during their cellular entry [9]. Further studies also have shown that ATG proteins might play a positive role in the process of virus entry. For example, in mammals, ATG protein ATG16L1 could promote echovirus 7 to enter polarized Caco-2 intestinal epithelial cells [15]. In addition, the foot-and-mouth disease virus (FMDV) could induce autophagosomes in the early stage of viral infection, and autophagosomes also could be activated by UV-inactivated virus or empty virus capsids, which suggested that the induction of autophagy might occur during the virus bind to cell surface receptors, and FMDV could trigger the formation of autophagosomes during cell entry to help the early replication of virus [16].

In crustaceans, previous studies have reported that WSSV mainly depends on three endocytic pathways to enter host cells, including clathrin-mediated endocytosis (CME) pathway, macropinocytosis pathway and caveolae-mediated endocytosis pathway, among them, the CME pathway has been proven to be one of the most important ways for WSSV entry into host cells. Therefore, WSSV could bind to cell surface molecules and enter the cell through different endocytosis pathways, in which many specific molecules are not only recruited by the virus particles to help the virus enter and internalize, but also could trigger innate immunity through cellular receptors to play an antiviral response [14]. Interestingly, autophagy was also found to be positively correlated with WSSV entry into haematopoietic tissue (Hpt) cells in red claw crayfish Cherax quadricarinatus [17]. Specifically, the cellular entry of WSSV was significantly promoted at 1 hpi in Hpt cells after induction of autophagy by rapamycin (autophagy inducer); while inhibition of autophagy via autophagy antagonist L-asparagine (L-Asn) could reduce the cellular entry of WSSV. These findings indicated that WSSV could utilize autophagy to promote viral entry into host cells [17]. Moreover, the study also identified a γ-aminobutyric acid receptor-associated protein (CqGABARAP) from red claw crayfish, which is an autophagy-associated protein involved in WSSV entry into Hpt cells. WSSV entry was strongly inhibited at 1 hpi in Hpt cells after gene silencing of CqGABARAP [17]. On the contrary, when Hpt cells were pretreated with recombinant CqGABARAP protein (rCqGABARAP) before WSSV infection, WSSV entry was enhanced at 1 hpi in Hpt cells. What's more, rCqGABARAP-mediated enhancement of WSSV entry was inhibited after treatment of CPZ (a specific inhibitor of the CME pathway), implying that CqGABARAP could participate in the WSSV entry into Hpt cells in the CME-dependent manner [17] (Fig. 1). In addition, it has been also reported that the expression level of miR-52, predicted to target ATG protein 8b, was up-regulated after WSSV infection in crayfish Hpt cells at 1 hpi, which also demonstrated that autophagy might be involved in WSSV invasion in crayfish [18].

In order to complete a successful infection, WSSV could not only use autophagy to enter cells but also utilize the process of autophagy to promote its own transcription and replication. Research has proven that the copy number of viral particles was increased at 36 h post-WSSV infection after autophagy was activated by rapamycin in shrimp hemocytes. Reversely, the copy number of WSSV particles was decreased when autophagy was inhibited by chloroquine (autophagy inhibitor), which indicated that autophagy could promote the transcription and replication of WSSV [19]. And in this process, miR-13b and miR-71, as an important regulator of gene transcription, were both up-regulated in shrimp hemocytes after the activation of autophagy by rapamycin. Additionally, the overexpression of miR-13b by injecting miR-13b mimic increased the copy number of WSSV and at the same time enhanced the autophagy level in shrimp hemocytes, suggesting that miR-13b could increase WSSV replication via promoting autophagy activity. After that, it was also found that miR-13b negatively regulated the target gene knickkopf gene to enhance the autophagic activity and further to promote viral infection [19]. Similarly, miR-71 could also negatively regulate its target gene cap-1 to bridge autophagy and WSSV infection [19]. These findings indicated that miR-13b/miR-71, as an important regulator targeting knickkopf genes/cap-1 gene, could connect WSSV with autophagy, in a positive correlation, to achieve the viral replication (Fig. 1). So far, there are still relatively few studies on the involvement of autophagy in pro-WSSV infection, and many key molecules need to be explained urgently. For example, how does WSSV induce autophagy when it invades cells? Which receptors, ATG proteins and signaling pathways are involved in WSSV-induced autophagy? These are still necessary to be further explored.

2.2. Autophagy mediated the degradation of WSSV

As an important pathway to eliminate the invading pathogens in host cells, autophagy can be induced to degrade the virus or viral components. For example, autophagy could reduce the number of progeny viruses after herpes simplex virus-1 (HSV-1) infection, indicating that autophagy is capable of inhibiting virus replication in mammals [20]. Similarly, in Drosophila, it was also found that vesicular stomatitis virus (VSV) infection could induce autophagy, and gene silencing of the ATG genes ATG18, ATG12 or ATG7 made Drosophila more susceptible to VSV, indicating that autophagy has an antiviral effect in Drosophila [8]. In crustaceans, autophagy could also be triggered and had an antiviral role during viral infection. For instance, the tripartite motif (TRIM) family proteins have been reported to induce autophagy to inhibit WSSV replication in crustaceans [21]. TRIM proteins were a family of proteins that are highly conserved in evolution, most of which contain the RING-finger domain and can be regarded as an E3 ubiquitin ligase. TRIM proteins have been reported to participate in many cellular activities such as cell cycle, immune regulation, autophagy, apoptosis and cancer [22]. In mammals, TRIM proteins have been proven to play an important role in regulating autophagy to resist viral infection. For example, TRIM23 could regulate TANK Binding Kinase 1 (TBK1) mediated autophagy to limit virus infection by ubiquitin modification. Likewise, some progress has been made on TRIM proteins in the antiviral autophagy of crustaceans. It's reported that a TRIM50-like gene and TRIM32-like gene identified from Penaeus monodon were both up-regulated after WSSV infection or after the induction of autophagy by rapamycin in P. monodon; while the autophagy activity were both reduced after gene silencing of PmTRIM50-like gene or PmTRIM32-like gene. Thus, the PmTRIM50-like gene and PmTRIM32-like gene were both involved in the regulation of antiviral autophagy in P. monodon. Additionally, the WSSV replication was strongly inhibited in both hemocytes and intestines in shrimp. Furthermore, the PmTRIM50-like protein exhibited binding affinity to WSSV envelope proteins, such as VP24, VP26 and VP28, to ubiquitinate these envelope proteins for the subsequent clearance via autophagic degradation [21] (Fig. 1). Meanwhile, PmTRIM32-like protein was also necessary for the induction of autophagy, which played an antiviral role by binding to WSSV envelope protein VP28 for its ubiquitination to induce the autophagic degradation [23] (Fig. 1). Hence, TRIM proteins are essential for the autophagy-mediated antiviral pathway. However, some key questions are remained to be further addressed. For instance, what signaling pathway does TRIM use to target WSSV for autophagic degradation? Which ATG autophagy-related molecules are involved in the process of TRIM-mediated autophagy?

Usually, double-stranded DNA viruses have to reach the perinuclear area to release the viral genome into the host cell nucleus for transcription and replication, during which the viral nucleocapsid containing the viral genome has to evade the immune effects of the host cell like the autophagic degradation [24]. Recently, WSSV was found to evade the host autophagic degradation by using the endosomal transport system, and the key gene-AAA⁺ ATPase valosin-containing protein (CqVCP) was positively recruited during the endosomal transport of WSSV in red claw crayfish [25]. As a member of the AAA⁺ ATPase protein family, VCP/p97 was widely expressed that could work with its cofactors, as a molecular chaperone, to transport cargo to lysosomes. It has been reported that VCP participated in various cellular activities such as cell cycle regulation and endolysosomal-degradation [26]. Importantly, VCP could induce autophagy by facilitating the de-ubiquitinase activity of ataxin-3 towards Beclin-1 or regulating the assembly and activity of the Beclin-1-containing phosphatidylinositol-3-kinase (PI3K) complex [27]. Intriguingly, the endosomal transport of WSSV was substantially limited when CqVCP ATPase activity was inhibited in crayfish Hpt cells, in which WSSV was gathered in the damaged endosomal system [25]. Furthermore, blocking of CqVCP activity resulted in the significantly increased ratio of CqGABARAP-II/Cqβ-actin, an indication for induced autophagic activity, in Hpt cells followed by a decreased WSSV replication, in which the dysfunctional endosomal transporting system mediated by blocking of CqVCP could recruit autophagosomes to fuse with the endosomes, containing aggregated WSSV virions, for the subsequent clearance of WSSV via the autophagic degradation, thus autophagy could be induced to clean the aggregated WSSV in endosomes in case of CqVCP dysfunction [25] (Fig. 1). However, the mechanism by which VCP regulates autophagy in crustaceans is still unclear, which needs further investigations.

Collectively, these studies show that the enhanced autophagy activity can directly promote the degradation of WSSV. Besides, autophagy can also be involved in the regulation of interferon-related innate immunity and inflammation against viruses in mammals. For example, the unique pattern recognition receptors (PRR) of autophagy pathway–selective autophagy receptorssequestosome1, could identify pathogen-associated molecular patterns (PAMPs) of exotic pathogens, and directly target autophagy to eliminate the invading viruses, but it could also act as a signal enhancer to promote the signal transduction between pathogen-related molecular patterns and PRRs, thus promoting the enhancement of IFN response [12]. Considering that currently in crustaceans, the first crustacean IFN regulatory factor (IRF)-like gene, identified from P. vannamei, could regulate the transcription of Vago4 and Vago5 genes in the same manner to IFN, supporting the view that Vago might play the role of IFN-like [28]. However, whether crustaceans have the antiviral mechanism of autophagy connecting the IFN system similar to that of vertebrates is still controversial, and there is no direct evidence that autophagy functioning as the regulator and enhancer in the interferon-like pathway against viral infection. Therefore, whether autophagy can induce interferon-like signaling pathways and participate in the antiviral response also needs further investigations.

3. Molecular regulation of autophagy in crustaceans

As described above, autophagy indeed affects viral infection. However, the molecular regulation of autophagy on WSSV infection has not been fully addressed in crustaceans. Up to now, more than 40 ATG genes have been identified as essential molecules for autophagy regulation, which are highly conserved in yeast and mammals that are involved in the formation, regulation, elongation and maturation of autophagy [29]. In addition to ATG genes, the process of autophagy is also regulated by other factors, such as the classic autophagy regulatory factors mammalian target of rapamycin (mTOR) and adenosine 5‘-monophosphate (AMP)-activated protein kinase (AMPK), both of which could catalyze the phosphorylation of unc-51 like autophagy activating kinase 1 (ULK1) to regulate autophagy activity [30]. At present, although a lot of ATG molecules have been identified in mammals and yeast, relatively little research has been carried out in crustaceans in terms of the autophagy regulation associated factors. Yet an increasing number of studies have indicated that autophagy plays a vital role in the innate immunity of crustaceans, in the following, the ATG molecules involved in innate immunity, especially some ATG molecules related to antiviral immunity in crustaceans are summarized and discussed according to the recent reports in crustaceans (Table 1), which provides us new insights and better understanding about the molecular mechanism of autophagy in crustaceans.

Table 1.

Autophagy related molecules in anti-WSSV immunity in crustaceans.

Autophagy related molecules	Homologue	Effect	Species	References
ATG8	CqGABARAP	Promoting WSSV entry	Cherax quadricarinatus	[17, 40]
Rab GTPase	PmRab7 LvRab7	Benefiting viral infection by binding to VP28	Penaeus monodon Litopenaeus vannamei	[46], [47], [48]
Valosin-containing protein (VCP/p97)	CqVCP	Facilitating endosomal transport of WSSV to avoid autophagic elimination	Cherax quadricarinatus	[25]
MicroRNA (miRNA)	miR-71 and miR-13b	Induction of antiviral autophagy	Marsupenaeus. japonicus	[19]
Tripartite motif family proteins	PmTRIM50-like, PmTRIM32-like	Ubiquitinating WSSV envelope proteins for autophagic degradation	Penaeus monodon	[21, 23]

3.1. Autophagy-related genes in crustaceans

Currently, there are few studies on the regulation of autophagy in aquatic animals, especially in crustaceans, which mainly focused on the comparative analysis of differential ATG genes and some major regulators of autophagy pathway for various cellular stress responses [31]. Among them, ATG6 /Beclin1 and ATG8, as the core genes of the autophagy pathway, have been verified in participating in the process of autophagy and regulating the innate immunity in crustaceans.

3.1.1. Beclin1

Beclin1, the homologous gene of yeast ATG6/vacuolar protein sorting 30 (VPS30) in mammals, was firstly identified in mice with lethal Sindbis virus infection, which is involved in cancer, aging, apoptosis, autophagy and other cellular processes [32, 33]. Moreover, Beclin1 could participate in different stages of autophagy from the formation of autophagosomes to the maturation of autophagosomes. Importantly, at the beginning of autophagy, the formation of the VPS34-VPS15-Beclin1 core complex occurred to induce autophagy by promoting the recruitment of ATG genes into autophagic vesicles. In this process, Beclin1 could be phosphorylated by ULK1 and then act as an integral scaffold of the phosphoinositide-3-kinase (PI3K) complex to promote the localization of autophagic proteins to autophagic vesicles [32].

In mammals and Drosophila, the reports about Beclin1 in autophagy are more in-depth, while the study in crustaceans, especially about function-related research, is very few. Recently, EsBeclin1 from Eriocheir sinensis was identified as a homologue of invertebrate Beclin1, in which the mRNA transcripts of EsBeclin1 in hemocytes was up-regulated after immune stimulations with Lipopolysaccharide (LPS) or post challenge of Aeromonas hydrophila from 3 to 24 h, suggesting that EsBeclin1 might be involved in the antibacterial activity of E. sinensis. Importantly, the mRNA expression of antimicrobial peptides (AMPs), such as EsALF2 (Anti-lipopolysaccharide factor2), EsLYZ (Lysozyme), EsCrus (Crustin) and EsCrus2, were significantly down-regulated at 6 h after LPS stimulation post gene silencing of EsBeclin1, indicating that EsBeclin1 could participate in the innate immune response against bacterial infection in E. sinensis by regulating the expressions of AMPs [34]. However, the association between the antibacterial mechanism of EsBeclin1 and autophagy needs further experimental evidence. Additionally, Beclin1 homologue was also identified in P. vannamei using the next-generation DNA sequencing technique [35], but the function-related research of Beclin1 has been not yet reported, particularly, the role of Beclin1 involved in autophagy is still unclear in crustaceans. Hence, further research is needed to explore how Beclin1, as an ATG gene, participates in the antibacterial response in crustaceans.

3.1.2. ATG8 family proteins

ATG8 family proteins were first identified in yeast as the ATG ubiquitin-like protein, involved in multiple cellular processes such as intracellular membrane trafficking and the formation of autophagosomes [36]. ATG8 had three subfamilies in humans, including LC3, GABARAP and Golgi-associated ATPase enhancer of 16 kDa (GATE-16) [36]. Generally, LC3 includes two mutually convertible forms namely LC3-I and LC3-II. After autophagy is induced, cytoplasmic LC3-I is catalyzed by E1-like enzyme ATG7 and E2-like enzyme ATG3, coupled with the substrate PE on the surface of the autophagosome membrane to form LC3-II. Hence, LC3-II is an important symbol of autophagosome formation, in which the conversion of LC3-I to LC3-II is increased, so the ratio of LC3-II/LC3-I is mostly used to indicate the degree of autophagy formation [37]. Furthermore, GABARAP is a ubiquitin-like modifier, also as a member of the ATG8 protein family, participates in the fusion of autophagosome and lysosome as scaffold proteins by recruiting ULK1 and beclin1 complex to the nucleation site and participates in different forms of autophagy [38]. Similarly, the ratio of LC3-II/LC3-I can also be used as a marker for autophagy activation in crustaceans. For example, the ratio of LC3-II/LC3-I was significantly increased in hemocytes after stimulation with CpG oligonucleotide in E. sinensis, indicating that CpG could induce autophagy in E. sinensis to activate the immune response [39]. Besides, GABARAP, as a homologue of LC3, has also been identified and proved to be involved in autophagy in crustaceans. In red claw crayfish, CqGABARAP expression was up-regulated after WSSV infection in Hpt cells, indicating autophagy was involved in WSSV infection [40]. Intriguingly, autophagic activity was positively correlated with WSSV entry and CqGABARAP could help WSSV locate on the cell membrane then promote WSSV enter the host cells by binding to VP28 and cytoskeleton components [17]. Furthermore, the GABARAP gene was also identified in E. sinensis, in which EsGABARAP was highly expressed in the hemocyte and hepatopancreas, and the expression of EsGABARAP in hemocyte was up-regulated after bacterial challenge with Listonella anguillarum, suggesting that EsGABARAP might mediate autophagy or apoptosis of E. sinensis hemocyte to play an antibacterial effect, but the specific signaling pathways and regulatory mechanisms have not yet been explored [41].

3.2. Other factors involved in autophagy in crustaceans

In addition to ATG genes, there are many other regulatory factors involved in the immune response of autophagy in crustaceans. As explained in the previous section, both the VCP and TRIM proteins, which have been introduced in detail, could participate in the regulation of autophagy in crustaceans. In addition to VCP and TRIM proteins, there are also some autophagy-related molecules such as Rab GTPase and microRNAs, which help us a better understanding of the autophagy-related immune response in crustaceans, providing new ideas for studying the efficient antiviral strategies related to autophagy.

3.2.1. Rab GTPase

Rab GTPase, a kind of small GTPase, belongs to the Ras-like GTPase superfamily, which can regulate vesicle transport, endosomal trafficking pathway and membrane fusion process [42]. Rab proteins have been shown to be involved in different stages of autophagy. For example, Rab1, Rab5, Rab11, Rab32 and Rab33B were involved in the formation of autophagosomes, while Rab7, Rab8B and Rab24 worked on the maturation of autophagosomes in mammals [43]. In addition, Rab7 also could participate in the fusion of autophagosomes and lysosomes to promote autophagy degradation. For example, Hepatitis C virus (HCV) could inhibit the expression of Rab7 to inhibit the autophagic degradation of the invading virus [44]. In crustaceans, Rab proteins are gradually being studied, which have been proven to participate in antiviral immunity. Rab5B, Rab6A and Rab7 were firstly cloned in Litopenaeus vannamei, and the mRNA expression of LvRab5B, LvRab6A and LvRab7 in the hepatopancreas was significantly up-regulated after infection with infectious hypodermal and hematopoietic necrosis virus or WSSV, implying that LvRab5B, LvRab6A and LvRab7 might be involved in immune response towards viral infection in crustacean [45]. Furthermore, Rab7 has been reported to bind to the WSSV envelope protein VP28 in a dose-dependent manner, and vivo neutralization assay showed that compared with the control group injected with WSSV, the shrimp mortality of the groups injected with WSSV plus the recombinant PmRab7 (15%) or WSSV plus anti-Rab7 antibody were both significantly decreased, which suggested that the binding of endogenous PmRab7 with VP28 was necessary for WSSV infection in P. monodon, and PmRab7 might help WSSV infection by avoiding the degradation of lysosome [46]. Besides, the gene silencing of Rab7 by dsRNA-Rab7 could inhibit the replication of Laem-Singh virus in P. monodon [47] and Taura syndrome virus in L. vannamei, hence, Rab7 could help the viral replication [48]. Taken together, Rab7 played a vital role in the endosomal trafficking pathway of WSSV infection in crustaceans, but the antiviral mechanism of dsRNA-Rab7 has not been fully understood. When Rab7 was silenced by dsRNA-Rab7, how to block the endosomal transport process of WSSV, how to mediate WSSV degradation, and whether WSSV could be degraded through the autophagy-lysosome pathway, whether Rab proteins could regulate the activity of autophagy to achieve the antiviral function in crustaceans, these questions remain unclear, thus the in-depth research is still needed in the future.

3.2.2. MicroRNAs

MicroRNA (miRNA) is a class of evolutionarily conserved non-coding small RNA, with a length of about 21–23 nucleotides in length, which was firstly identified in nematodes, regulating gene expression in the way of post-transcriptional regulation, changing the protein levels to affect many biological processes. So far, more and more studies have proved that miRNA has been recruited in autophagy regulation to resist virus invasion [49]. In crustaceans, miRNA, as a key post-transcriptional regulatory factor, can simultaneously regulate the expression of multiple target genes. Therefore, miRNA might also play an important role in the regulation of virus infection and autophagy. The researches about miRNA-mediated virus infection and host autophagy provided us with new insights. It has been directly proved that miR-13b and miR-71 could serve as a bridge between host autophagy and WSSV infection in Marsupenaeus. japonicus hemocytes, and autophagy could promote WSSV infection in the regulation of miRNA. Further research showed that miR-13b and miR-71 targeted knickkopf gene and cap-1 gene, respectively, to regulate the WSSV infection and host autophagy, suggesting that miRNA was necessary for host autophagy and viral infection [19]. So far, although there were few studies on ATG molecules, they all provided us with some new ideas to better understand the mechanism of autophagy in crustaceans, in terms of miRNA regulation.

4. Conclusion and future perspective

Collectively, autophagy is an important component in the innate immunity of crustaceans, mainly focusing on the antiviral effect. Studies have found that autophagy plays a positive role in WSSV entry and replication, on the contrary, autophagy could effectively block WSSV transcription and replication by autophagic degradation. An increasing number of ATG molecules involved in innate immunity are summarized and discussed in crustaceans, thus, autophagy has a dual effect during WSSV infection, but there are now too few studies on the anti-viral and pro-viral mechanism of autophagy in crustaceans. Furthermore, the mechanisms of the organism that sense the virus and induce autophagy, as well as the pathway of autophagy signal have not been specifically investigated. In particular, the mechanism of how to balance the interaction between autophagy's pro-viral and anti-viral effects is still unclear. All in all, the solution of these questions will help to further explore the autophagy mechanism of crustaceans and even invertebrates, which could provide new ideas for the antiviral studies in crustaceans.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.