Immune System Of Invertebrates Research Articles

Generating prophylactic immunity against arboviruses in vertebrates and invertebrates.

The World Health Organization recently declared a global initiative to control arboviral diseases. These are mainly caused by pathogenic flaviviruses (such as dengue, yellow fever and Zika viruses) and alphaviruses (such as chikungunya and Venezuelan equine encephalitis viruses). Vaccines represent key interventions for these viruses, with licensed human and/or veterinary vaccines being available for several members of both genera. However, a hurdle for the licensing of new vaccines is the epidemic nature of many arboviruses, which presents logistical challenges for phase III efficacy trials. Furthermore, our ability to predict or measure the post-vaccination immune responses that are sufficient for subclinical outcomes post-infection is limited. Given that arboviruses are also subject to control by the immune system of their insect vectors, several approaches are now emerging that aim to augment antiviral immunity in mosquitoes, including Wolbachia infection, transgenic mosquitoes, insect-specific viruses and paratransgenesis. In this Review, we discuss recent advances, current challenges and future prospects in exploiting both vertebrate and invertebrate immune systems for the control of flaviviral and alphaviral diseases.

Invertebrate Immunity, Natural Transplantation Immunity, Somatic and Germ Cell Parasitism, and Transposon Defense.

While the vertebrate immune system consists of innate and adaptive branches, invertebrates only have innate immunity. This feature makes them an ideal model system for studying the cellular and molecular mechanisms of innate immunity sensu stricto without reciprocal interferences from adaptive immunity. Although invertebrate immunity is evolutionarily older and a precursor of vertebrate immunity, it is far from simple. Despite lacking lymphocytes and functional immunoglobulin, the invertebrate immune system has many sophisticated mechanisms and features, such as long-term immune memory, which, for decades, have been exclusively attributed to adaptive immunity. In this review, we describe the cellular and molecular aspects of invertebrate immunity, including the epigenetic foundation of innate memory, the transgenerational inheritance of immunity, genetic immunity against invading transposons, the mechanisms of self-recognition, natural transplantation, and germ/somatic cell parasitism.

White spot syndrome virus impact on the expression of immune genes and gut microbiome of black tiger shrimp Penaeus monodon

The gut microbiome plays an essential role in the immune system of invertebrates and vertebrates. Pre and pro-biotics could enhance the shrimp immune system by increasing the phenoloxidase (PO), prophenoloxidase (ProPO), and superoxide dismutase activities. During viral infection, the host immune system alteration could influence the gut microbiome composition and probably lead to other pathogenic infections. Since the JAK/STAT pathway is involved in white spot syndrome virus (WSSV) infection, we investigated the intestine immune genes of STAT-silenced shrimp. During WSSV infection, expression levels of PmVago1, PmDoral, and PmSpätzle in PmSTAT-silenced shrimp were higher than normal. In addition, the transcription levels of antimicrobial peptides, including crustinPm1, crustinPm7, and PmPEN3, were higher in WSSV-challenged PmSTAT-silenced shrimp than the WSSV-infected normal shrimp. Meanwhile, PmSTAT silencing suppressed PmProPO1, PmProPO2, and PmPPAE1 expressions during WSSV infection. The microbiota from four shrimp tested groups (control group, WSSV-infected, PmSTAT-silenced, and PmSTAT-silenced infected by WSSV) was significantly different, with decreasing richness and diversity due to WSSV infection. The relative abundance of Bacteroidetes, Actinobacteria, and Planctomycetes was reduced in WSSV-challenged shrimp. However, at the species level, P. damselae, a pathogen to human and marine animals, significantly increased in WSSV-challenged shrimp. In constrast, Shewanella algae, a shrimp probiotic, was decreased in WSSV groups. In addition, the microbiota structure between control and PmSTAT-silenced shrimp was significantly different, suggesting the importance of STAT to maintain the homeostasis interaction with the microbiota.

Functions of hemocytes in immune reactions of annelida

This article discusses the role of hemocytes in the immune defense of Annelid. Annelida are bilaterally symmetric, three-layered animals with a secondary body cavity. The immune system of invertebrates does not contain immunoglobulins or interacting subpopulations of lymphocytes as components. Hemocytes occupy one of the main roles in this system.

Hemocytes: A Useful Tool for Assessing the Toxicity of Microplastics, Heavy Metals, and Pesticides on Aquatic Invertebrates.

Invertebrates have long been an important tool for assessing water pollution due to their characteristics as intermediate consumers in aquatic ecosystem food chains. Most of the time, the effects of contaminants are measured by their effect on oxidative status or by mortality, although there already exists an easier tool-hemocytes. Hemocytes are circulating cells with a very important role in the immune system of invertebrates, which can be found within the hemolymph, analogous to the blood in vertebrates. The collection of hemolymph samples is easy, fast, minimally invasive, and poses no danger to the life of invertebrates. The purpose of this review was to highlight the advantages of using hemolymph for toxicity assays of various substances, including heavy metals, micro- and nano-plastics, pesticides, hydrocarbons, and oil spills. A literature search was conducted for this purpose using the most common and most often used databases, with a focus on the most recent and relevant studies. Bivalve mollusks, crustaceans, and gastropods were chosen for this investigation. This review found a growing number of studies choosing to use hemolymph as the standard methodology for toxicology assays, confirming their qualities as reliable tools.

Establishment of the immunological self in juvenile Patiria pectinifera post-metamorphosis.

Ontogeny of the immune system is a fundamental immunology issue. One indicator of immune system maturation is the establishment of the immunological self, which describes the ability of the immune system to distinguish allogeneic individuals (allorecognition ability). However, the timing of immune system maturation during invertebrate ontogeny is poorly understood. In the sea star Patiria pectinifera, cells that have dissociated from the embryos and larvae are able to reconstruct larvae. This reconstruction phenomenon is possible because of a lack of allorecognition capability in the larval immune system, which facilitates the formation of an allogeneic chimera. In this study, we revealed that the adult immune cells of P. pectinifera (coelomocytes) have allorecognition ability. Based on a hypothesis that allorecognition ability is acquired before and after metamorphosis, we conducted detailed morphological observations and survival time analysis of metamorphosis-induced chimeric larvae. The results showed that all allogeneic chimeras died within approximately two weeks to one month of reaching the juvenile stage. In these chimeras, the majority of the epidermal cell layer was lost and the mesenchymal region expanded, but cell death appeared enhanced in the digestive tract. These results indicate that the immunological self of P. pectinifera is established post-metamorphosis during the juvenile stage. This is the first study to identify the timing of immune system maturation during echinodermal ontogenesis. As well as establishing P. pectinifera as an excellent model for studies on self- and non-self-recognition, this study enhances our understanding of the ontogeny of the immune system in invertebrates.

Do heritable immune responses extend physiological individuality?

Immunology and its philosophy are a primary source for thinking about biological individuality. Through its discriminatory function, the immune system is believed to delineate organism and environment within one generation, thus defining the physiological individual. Based on the paradigmatic instantiations of immune systems, immune interactions and, thus, the physiological individual are believed to last only for one generation. However, in recent years, transgenerationally persisting immune responses have been reported in several phyla, but the consequences for physiological individuality have not yet been explored. In this article, I will introduce an invertebrate immune system that is RNA-based and operates through a heritable silencing/licensing paradigm. I will discuss how such a perspective on immune systems can illuminate our conceptions of individuality. I will particularly introduce an account of immunological individuality that is not restricted to one generation.

Study of the haemocytic parameters of cockroaches (Periplaneta americana) post stimulation with bacterial spores

Haemocytes and haemolymph are the major mediators of immunity in cockroaches (Periplaneta americana). The six different types of haemocytes reported are prohaemocytes, plasmatocytes, granular haemocytes, oenocytoids, spherules and adipohaemocytes. These cells have their own structural and functional uniqueness. In this study, we injected cockroaches with bacterial spores and compared the number and size of the haemocytes in control and inoculated populations. The number of viable haemocytes in the inoculated population was less than that in control. There was a significant increase in the number and size of plasmatocytes in the immunized cockroaches. The adipohaemocytes also showed increase in size in the inoculated population. Both plasmatocytes and adipohaemocytes exhibit phagocytic activity. Additionally, more production of superoxide anions in the haemocytes of inoculated cockroaches are indicative of increased phagocytosis. We also report a change in protein profile of haemolymph of the injected cockroaches. The heightened activity of few haemocytes and bio-molecules in the immunized cockroaches, hint towards an activated cellular and humoral immune response due to inoculation. Together, these findings re-establish the fact that the invertebrate immune system responds variably to antigenic challenge.

Identification of a Novel Antimicrobial Peptide From the Ancient Marine Arthropod Chinese Horseshoe Crab, Tachypleus tridentatus.

Humoral immunity is the first line of defense in the invertebrate immune system, and antimicrobial peptides play an important role in this biological process. A novel antimicrobial peptide, termed Tatritin, was identified and characterized in hemolymph of Chinese horseshoe crab, Tachypleus tridentatus, infected with Gram-negative bacteria via transcriptome analysis. Tatritin was significantly induced by bacterial infection in hemolymph and gill. The preprotein of Tatritin consists of a signal peptide (21 aa) and a mature peptide (47 aa) enriched by cysteine. The putative mature peptide was 5.6 kDa with a theoretical isoelectric point (pI) of 9.99 and showed a α-helix structure in the N-terminal and an anti-parallel β-sheet structure in the cysteine-stabilized C-terminal region. The chemically synthesized peptide of Tatritin exhibited a broad spectrum of antimicrobial activity against Gram-negative and Gram-positive bacteria and fungi. Furthermore, Tatritin may recognize and inhibit pathogenic microorganisms by directly binding to LPS, DNA, and chitin. In addition, administration of Tatritin reduced the mortality of zebrafish after bacterial infection. Due to its broad-spectrum antimicrobial activity in vivo and in vitro and the sensitivity to drug-resistant bacterial strains, Tatritin peptide can be used as a new type of drug for infection treatment or as an immune enhancer in animals.

CircRNA75 and CircRNA72 Function as the Sponge of MicroRNA-200 to Suppress Coelomocyte Apoptosis Via Targeting Tollip in Apostichopus japonicus

Circular RNAs (circRNAs) act as essential regulators in many biological processes, especially in mammalian immune response. Nonetheless, the functions and mechanisms of circRNAs in the invertebrate immune system are largely unclarified. In our previous work, 261 differentially expressed circRNAs potentially related to the development of Apostichopus japonicus skin ulceration syndrome (SUS), which is a major problem restricting the sea cucumber breeding industry, were identified by genome-wide screening. In this study, via miRanda analysis, both circRNA75 and circrRNA72 were shown to share the miR-200 binding site, a key microRNA in the SUS. The two circRNAs were verified to be increased significantly in LPS-exposed primary coelomocytes, similar to the results of circRNA-seq in sea cucumber under Vibrio splendidus-challenged conditions. A dual-luciferase assay indicated that both circRNA75 and circRNA72 could bind miR-200 in vivo, in which circRNA75 had four binding sites of miR-200 and only one for circRNA72. Furthermore, we found that miR-200 could bind the 3’-UTR of Toll interacting protein (Tollip) to negatively mediate the expression of Tollip. Silencing Tollip increased primary coelomocyte apoptosis. Consistently, inference of circRNA75 and circRNA72 could also downregulate Tollip expression, thereby increasing the apoptosis of primary coelomocytes, which could be blocked by miR-200 inhibitor treatment. Moreover, the rate of si-circRNA75-downregulated Tollip expression was higher than that of si-circRNA72 under an equivalent amount. CircRNA75 and circRNA72 suppressed coelomocyte apoptosis by sponging miR-200 to promote Tollip expression. The ability of circRNA to adsorb miRNA might be positively related to the number of binding sites for miRNA.

Characterization of a thioester-containing protein from the pearl oyster Pinctada fucata and its role in phagocytosis

Thioester-containing proteins (TEPs) superfamily is known to play important innate immune functions in vertebrates and invertebrates, and involved in recognition, and killing of invading pathogens. Here, a TEP gene (designated as Pf-TEP) was obtained from the pearl oyster Pinctada fucata by RT-PCR and rapid amplification of cDNA ends (RACE). The Pf-TEP cDNA consists of 4335 bp with an open reading frame (ORF) of 3639 bp encoding a protein of 1212 amino acids. The deduced TEP protein possessed the characteristic structure features present in its homologs and contained the A2M_N_2, A2M, A2M_comp, A2M_recep, and thioester domain, as well as the conserved Cys, His, and Glu residues. Phylogenetic analysis revealed that Pf-TEP is closely related to the TEPs from other invertebrates. Pf-TEP mRNA was expressed in all examined tissues including gill, digestive gland, adductor muscle, mantle and hemocyte, while the highest expression was found in gill. Following challenge with Vibrio alginolyticus, Pf-TEP expression was significantly induced in hemocytes. Further knockdown of Pf‐TEP by specific siRNA could significantly reduce the phagocytosis of V. alginolyticus by hemocytes in vitro. These results would help increase understanding of TEP existed as an ancient complement-like molecular and therefore provide new insights into the roles of TEP in the invertebrate immune system.

Molecular Characterization of Complement Component 3 (C3) in the Pearl Oyster Pinctada fucata Improves Our Understanding of the Primitive Complement System in Bivalve.

As the central component in the complement system, complement component 3 (C3) plays essential roles in both the innate and adaptive immune responses. Here, a C3 gene (designated as pf-C3) was obtained from the pearl oyster Pinctada fucata by RT-PCR and rapid amplification of cDNA ends (RACE). The pf-C3 cDNA consists of 5,634 bp with an open reading frame (ORF) of 5,193 bp encoding a protein of 1,730 amino acids with a 19 residue signal peptide. The deduced pf-C3 protein possessed the characteristic structural features present in its homologs and contained the A2M_N_2, ANATO, A2M, A2M_comp, A2M_recep, and C345C domains, as well as the C3 convertase cleavage site, thioester motif, and conserved Cys, His, and Glu residues. Phylogenetic analysis revealed that pf-C3 is closely related to the C3s from other mollusks. Pf-C3 mRNA was expressed in all examined tissues including gill, digestive gland, adductor muscle, mantle and foot, while the highest expression was found in the digestive gland. Following the challenge with Vibrio alginolyticus, pf-C3 expression was significantly induced in hemocytes. Luciferase reporter assays indicated that pf-C3a could activate the NF-κB signal pathway in HEK293T cells. Further knockdown of pf-C3 by specific siRNA could significantly reduce the phagocytosis of V. alginolyticus by hemocytes in vitro. These results would help increase understanding of the function of C3 in the invertebrate immune system and therefore provide new insights into the roles of the primitive complement system in invertebrates.

Molecular characterization, redox regulation, and immune responses of monothiol and dithiol glutaredoxins from disk abalone (Haliotis discus discus)

Glutaredoxins (Grxs) are well-known oxidoreductases involved in a wide range of redox activities in organisms. In this study, two invertebrate Grxs (AbGrx1-like and AbGrx2) from disk abalone were identified and characterized in an effort to gain a deeper understanding into their immune and redox regulatory roles. Both AbGrxs share typical thioredoxin/Grx structures. AbGrx1-like and AbGrx2 were identified as monothiol and diothiol Grxs, respectively. AbGrxs were significantly expressed at the egg and 16-cell stage of early abalone development. Although the expression of both AbGrxs demonstrated similar patterns, the expression of AbGrx1-like was higher than AbGrx2 during development stages. In contrast, AbGrx2 expression was significantly higher than that of AbGrx1-like in adult tissues. Highest AbGrx1-like expression was observed in the hepatopancreas and digestive tract, while highest AbGrx2 expression was found in the gills, followed by the mantle, in healthy adult abalone tissues. The highest expression of AbGrx1-like was observed in the gills at 12 h and 6 h post injection (p.i) of Vibrio parahemolyticus and other stimulants, respectively. The highest expression of AbGrx2 in the gills were observed at 120 h, 6 h, 24 h, and 12 h post injection of V. parahaemolyticus, Listeria monocytogenes, Viral hemorrhagic septicemia virus, and Polyinosinic:polycytidylic acid, respectively. AbGrxs possessed significant 2-hydroxyethyl disulfide (HED) and dehydroascorbate (DHA) reduction activity, but AbGrx2 exhibited higher redox activity than AbGrx1-like. Altogether, our results suggest an important role of AbGrx1-like and AbGrx2 in redox homeostasis, as well as in the invertebrate immune defense system. Our findings will aid the development of new disease management strategies for this economically valuable species.

Enhanced Immune Protection of Mud Crab Scylla paramamosain in Response to the Secondary Challenge by Vibrio parahaemolyticus.

“Immune priming” plays a vital part in the immune system of invertebrates, protecting against recurrent infections by pathogens, and can provide some ideas for the prevention and treatment of invertebrate diseases. Many invertebrates have been demonstrated recently to have immune priming, but the relevant mechanisms are not known. Expression of immune system–related genes in the hemocytes and hepatopancreas of the mud crab (Scylla paramamosain) before and after repeated stimulation with Vibrio parahaemolyticus were analyzed by real-time fluorescence quantitative polymerase chain reaction. Some molecules that may participate in the immune priming of S. paramamosain were screened out, and their possible roles in immune priming were interpreted. Crabs injected first with heat-killed V. parahaemolyticus (HkVp group) or physiologic (0.9%) saline (PS group) were rechallenged at 168 h with live V. parahaemolyticus (HkVp+Vp group and PS+Vp group, respectively). The log-rank test shows a significant difference in survival rate between the HkVp+Vp group and the other groups after the ICH (p < 0.05). Expression of genes involved in the toll-like receptor (TLR) signaling pathway and some antimicrobial peptide genes were detected. By, respectively, comparing gene quantification at different time points in hemocytes and the hepatopancreas, the molecules that may play a part in the early stage of the immune priming of S. paramamosain in the hemocytes are found to be down syndrome cell adhesion molecule (Dscam), Hyastatin, Cactus, Arasin, antilipopolysaccharide factor 3 (ALF3), ALF4, ALF5, and ALF6 as well as later acting molecules, such as Crustin, Dorsal, Pelle, and myeloid differentiation factor 88 (MyD88). The molecules that functioned throughout the entire period are TLR and Spaetzle. In the hepatopancreas, the molecules that may play a part in the early stages of immune priming are Dscam, Hyastatin, Arasin, ALF6, Pelle, Spaetzle, Dorsal and, in the later stage, ALF4. The molecules that functioned throughout the entire period are TLR, Crustin, Cactus, MyD88, ALF3, and ALF5. In summary, the immune function of S. paramamosain is enhanced after it receives the same repetitive stimulation by V. parahaemolyticus, indicating immune priming in S. paramamosain. Our study enriches research on immune priming in invertebrates and lays the foundation for further studies revealing the molecular mechanism of immune priming in crabs.

Accumulation and trafficking of zinc oxide nanoparticles in an invertebrate model, Bombyx mori, with insights on their effects on immuno-competent cells

Zinc oxide nanoparticles (ZnO NPs) are used in many applications; however, their interactions with cells, immune cells in particular, and potential health risk(s) are not fully known. In this manuscript, we have demonstrated the potential of ZnO NPs to cross the gut barrier in an invertebrate model, Bombyx mori, and that they can reach the hemolymph where they interact with and/or are taken up by immune-competent cells resulting in various toxic responses like decline in hemocyte viability, ROS generation, morphological alterations, apoptotic cell death, etc. Exposure to these NPs also resulted in alteration of hemocyte dynamics including an immediate increase in THC, possibly due to the release of these hemocytes either from enhanced rate of cell divisions or from attached hemocyte populations, and decline in percentage of prohemocytes and increase in percentage of two professional phagocytes, i.e., granulocytes and plasmatocytes, possibly due to the differentiation of prohemocytes into phagocytes in response to a perceived immune challenge posed by these NPs. Taken together, our data suggest that ZnO NPs have the potential to cross gut barrier and cause various toxic effects that could reverse and the insects could return to normal physiological states as there is restoration and repair of various systems and their affected pathways following the clearance of these NPs from the insect body. Our study also indicates that B. mori has the potential to serve as an effective alternate animal model for biosafety, environmental monitoring and screening of NPs, particularly to evaluate their interactions with invertebrate immune system.

Is pallial mucus involved in Ostrea edulis defenses against the parasite Bonamia ostreae?

Bonamia ostreae is an intrahemocytic parasite that has been responsible for severe mortalities in the flat oyster Ostrea edulis since the 1970́s. The Pacific oyster Crassostrea gigas is considered to be resistant to the disease and appears to have mechanisms to avoid infection. Most studies carried out on the invertebrate immune system focus on the role of hemolymph, although mucus, which covers the body surface of molluscs, could also act as a barrier against pathogens. In this study, the in vitro effect of mucus from the oyster species Ostrea edulis and C. gigas on B. ostreae was investigated using flow cytometry. Results showed an increase in esterase activities and mortality rate of parasites exposed to mucus from both oyster species. In order to better understand the potential role of mucus in the defense of the oyster against parasites such as B. ostreae, liquid chromatography and tandem mass spectrometry were used to describe and compare mucus protein composition from both species. In all oyster species, pallial mucus contains a high level of proteins; however, O. edulis mucus produced a variety of proteins that could be involved in the immune response against the parasite, including Cu/Zn extracellular superoxide dismutase, thioxiredoxin, peroxiredon VI, heat shock protein 90 as well as several hydrolases. Conversely, a different set of antioxidant proteins, hydrolases and stress related proteins were identified in mucus from C. gigas. Our results suggest an innate immunity adaptation of oysters to develop a specific response against their respective pathogens. The mucosal protein composition also provides new insights for further investigations into the immune response in oysters.

An improved method for testing invertebrate encapsulation response as shown in the honey bee

In 1882, Metchnikoff documented the encapsulation response (ER) of the invertebrate immune system. Since then, researchers have used Metchnikoff’s method to quantify immune function—and examine its relationship with ecological and behavioral factors—across various insect taxa. While scientists continue to uncover information regarding invertebrate immunity, behavioral ecology, and ecological immunology, the basics of Metchnikoff’s method have remained unchanged. All but two previous studies investigating insect immunity have used sterile or PBS-coated inducers, although we know that the immune system recognizes specific pathogens. To account for the specificity of the immune system, we modified Metchnikoff’s method and coated nylon monofilaments with pathogen-associated molecular patterns (PAMPs). Using honey bees (Apis mellifera), we examined ER using implants coated with PAMPs (“PAMPlants”) found on known honey bee parasites and pathogens. Lipopolysaccharide (LPS), peptidoglycan (PGN), and β-1, 3-glucan (B13G) PAMPlants mimicked an infection with Gram-negative bacteria, Gram-positive bacteria, and fungi, respectively. Our PAMPlants induced stronger responses than the control implants in both singly- (one PBS-coated or PAMP-coated implant) and doubly- (internal control; one PBS-coated and one PAMP-coated implant) implanted animals. In doubly-implanted individuals, there was a significant increase in response to B13G and LPS when compared with internal controls. The PGN and BSA did not differ from the internal controls in the doubly implanted individuals. These methods provide an improvement when exploring responses to specific pathogens and exploring topics within the field of invertebrate ecological immunity. When applied to social systems, these methods can be used to examine the evolution of disease resistance in societies.

Expression of Macrobrachium rosenbergii lipopolysaccharide- and β-1,3-glucan-binding protein (LGBP) in Saccharomyces cerevisiae and evaluation of its immune function

Pattern recognition proteins (PRPs) activate the innate immune system in invertebrates, and lipopolysaccharide- and β-1,3-glucan-binding protein (LGBP) is an important PRP with various biological functions. Here, the open reading frame (ORF) of Macrobrachium rosenbergii LGBP (MrLGBP) was cloned into plasmid vector pHAC181, then integrated into downstream of the GAL1 promoter of Saccharomyces cerevisiae strain GAL1-ScRCH1 via homologous recombination, followed by its expression in the yeast eukaryotic system. The resulting recombinant LGBP contained a 3 × HA-tag at its C terminus and had a molecular weight of about 45 kDa, as evaluated by western blot analysis. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were ranged from 0.340 to 0.802 and 1.189–1.810 μM, respectively. The recombinant MrLGBP protein agglutinated almost all tested bacteria except Bacillus thuringiensis and Staphylococcus aureus. These results revealed that this recombinant protein exhibited antimicrobial activity against some Gram-positive and Gram-negative bacteria. M. rosenbergii prawns were fed with the recombinant yeast strain MrLGBP for 1 month and challenged with the most common crustacean pathogen, Vibrio parahaemolyticus. These prawns showed lower mortality and higher enzymatic activity and expression levels of immunity genes than did the control groups. All these results suggest that MrLGBP may play important roles in the innate immunity of crustaceans, and recombinant strain S. cerevisiae MrLGBP may be useful for the development of an effective immune feed additive in the future.

Immunotoxicity in green mussels under perfluoroalkyl substance (PFAS) exposure: Reversible response and response model development.

The immunotoxicity of 4 commonly detected perfluoroalkyl substances (PFASs), namely, perfluorooctanesulfonate (PFOS), perfluorooctanoic acid (PFOA), perfluorononanoic acid (PFNA), and perfluorodecanoic acid (PFDA) was investigated by measuring biomarkers of the immune profile of green mussels, Perna viridis. The biomarkers included neutral red retention, phagocytosis, and spontaneous cytotoxicity, all of which were tested on mussel hemocytes. Hemocytes are an important component of the invertebrate immune system. We found that exposure to PFASs could lead to reduced hemocyte cell viability and suppress immune function by up to 50% of normal performance within the experimental exposure range. The results indicate that PFASs have an immunotoxic potential and thus could pose severe health risks to aquatic organisms. The reported immunotoxicity is likely to result from the compounds' direct and indirect interactions with the hemocyte membrane, and therefore likely to affect the functionality of these cells. The immunotoxic response was found to be related to the organism's burden of PFASs, and was reversible when the compounds were removed from the test organisms. Based on this relationship, models using an organism's PFAS concentration and bioaccumulation factor (BAF) as the independent variables were established to quantify PFAS-induced immunotoxicity. The models help us to gain a better understanding of the toxic mechanism of PFASs, and provide a tool to evaluate adverse effects for the whole group of compounds with one mathematical equation. Environ Toxicol Chem 2018;37:1138-1145. © 2018 SETAC.

Role of Antheraea pernyi serpin 12 in prophenoloxidase activation and immune responses.

Serine protease inhibitors play a key role in the immune system of invertebrates by controlling proteolytic cascades. Besides its importance, the knowledge on immune functions of serpins in most of insects is fragmentary. In the present study, we identified serpin-12 from Antheraea pernyi encoding a predicted 402 amino acid residue protein (Apserpin-12). We expressed the recombinant protein in Escherichia coli and the purified protein was used for the synthesis of rabbit anti-Apserpin-12 polyclonal antibodies and functional studies. Quantitative real-time ploymerase chain reaction (qRT-PCR) analysis revealed that the knock-down of Apserpin-12 enhanced the prophenoloxidase (PPO) cascade stimulated by Micrococcus luteus in hemolymph, whereas addition of recombinant Apserpin-12 protein along with same elicitor led to down-regulate PPO activation. Following different microbial challenge (E. coli, Beauveria bassiana, M. Luteus, and nuclear polyhedrosis virus), the expression of Apserpin-12 mRNA was induced significantly. Furthermore, the Apserpin-12 double-stranded RNA administration elicited the expression of antimicrobial peptides, while the treatment with recombinant protein suppressed their expression. Tissue profile of Apserpin-12 indicated that it is expressed in all examined tissues, that is, hemolymph, malpighian tubules, midgut, silk gland, integument, and fat body with variation in their transcript levels. We concluded that Apserpin-12 may regulate PPO activation and inhibit the production of antimicrobial peptides in A. pernyi, suggesting important role in its immune system.