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After publication of the article, a reader raised concerns about two figures: In Figure 5A, lane 1 looks very similar to the top band in lane 7 and lane 2 looks very similar to the top band in lane 5. In Figure 7B, in the P/Dscam panel, lanes 1 through 6 all look very similar to each other; in the WWSV VP28 panel, lanes 2 and 3 look very similar to each other; and in the WSSV VP28 panel, lanes 5 through 9 all look very similar to each other. These concerns were brought to the attention of the Corresponding Author, Kenneth Soderhall, along with a request to provide the raw blots for each of the two figures to allow for full examination. In response, Dr. Soderhall informed the journal that the authors were unable to provide the raw blots, stating that the first author, Apiruck Watthanasurorot, who conducted the experiments and who handled and assembled these figures, has been out of contact and he has the original files. A researcher in Dr. Soderhall’s group, who was not involved in the original study, has repeated the experiments and confirmed the results in Figures 5A and 7B. In addition, other published findings support the conclusions of this paper. However, the authors are retracting this publication due to the possibility of misrepresentation of the data. An investigation of Figure 5A and Figure 7B in this article by Uppsala University, conducted by an Expert Group for Scientific Misconduct at the Central Ethical Review Board, led to the recommendation to retract the article on the grounds of image manipulation by Apiruck Watthansurorot, and the unavailability of the original images. All of the authors, except Apiruck Watthansurorot who could not be contacted, have agreed to this retraction. The authors apologize to the readers and editors of PLOS Pathogens for these errors.

The gC1qR/p32 protein is a multiple receptor for several proteins and pathogens. We cloned a gC1qR homologue in a crustacean, Pacifastacus leniusculus, and analyzed the expression of P. leniusculus C1qR (PlgC1qR) in various tissues. The gC1qR/p32 transcript was significantly enhanced by white spot syndrome virus (WSSV) infection 6 h after viral infection both in vitro in a hematopoietic tissue cell culture (Hpt) and in vivo compared to appropriate controls. Moreover, PlgC1qR silencing in both the Hpt cell culture and live crayfish enhanced the WSSV replication. In addition, by making a recombinant PlgC1qR protein we could show that if this recombinant protein was injected in a crayfish, Pacifastacus leniusculus, followed by injection of WSSV, this significantly reduced viral replication in vivo. Furthermore, if the recombinant PlgC1qR was incubated with Hpt cells and then WSSV was added, this also reduced viral replication. These experiments clearly demonstrate that recombinant PlgC1qR reduce WSSV replication both in vivo and in vitro. The results from a far-Western overlay and glutathione S-transferase pull-down assays showed that PlgC1qR could bind to VP15, VP26, and VP28. Altogether, these results demonstrate a role for PlgC1qR in antiviral activity against WSSV.

Invertebrates rely on innate immunity to respond to the entry of foreign microorganisms. One of the important innate immune responses in arthropods is the activation of prophenoloxidase (proPO) by a proteolytic cascade finalized by the proPO-activating enzyme (ppA), which leads to melanization and the elimination of pathogens. Proteolytic cascades play a crucial role in innate immune reactions because they can be triggered more quickly than immune responses that require altered gene expression. Caspases are intracellular proteases involved in tightly regulated limited proteolysis of downstream processes and are also involved in inflammatory responses to infections for example by activation of interleukin 1ß. Here we show for the first time a link between caspase cleavage of proPO and release of this protein and the biological function of these fragments in response to bacterial infection in crayfish. Different fragments from the cleavage of proPO were studied to determine their roles in bacterial clearance and antimicrobial activity. These fragments include proPO-ppA, the N-terminal part of proPO cleaved by ppA, and proPO-casp1 and proPO-casp2, the fragments from the N-terminus after cleavage by caspase-1. The recombinant proteins corresponding to all three of these peptide fragments exhibited bacterial clearance activity in vivo, and proPO-ppA had antimicrobial activity, as evidenced by a drastic decrease in the number of Escherichia coli in vitro. The bacteria incubated with the proPO-ppA fragment were agglutinated and their cell morphology was altered. Our findings show an evolutionary conserved role for caspase cleavage in inflammation, and for the first time show a link between caspase induced inflammation and melanization. Further we give a more detailed understanding of how the proPO system is regulated in time and place and a role for the peptide generated by activation of proPO as well as for the peptides resulting from Caspase 1 proteolysis., Author Summary Melanization is an important reaction in most multicellular organisms, both animals and plants. The initiation steps of this reaction in invertebrates are catalyzed by the prophenoloxidase (proPO) activating system a proteolytic enzyme cascade, which primary function is to recognize cell wall products from microorganisms and respond by activation of the system and generation of immune effector molecules. This cascade requires careful regulation to achieve spatial and temporal control to avoid dangerous side effects. We here show that a Caspase1-like enzyme can inactivate proPO when ppA is not activating the proPO to avoid deleterious effects and further we show for the first time that the N-terminal peptide from ppA cleavage of proPO (activation of proPO) has an important biological function as also the Caspase1 cleaved fragments. Our results also show that Caspase 1-induced inflammatory response is evolutionarily conserved and is linked to melanization.

Daily, circadian rhythms influence essentially all living organisms and affect many physiological processes from sleep and nutrition to immunity. This ability to respond to environmental daily rhythms has been conserved along evolution, and it is found among species from bacteria to mammals. The hematopoietic process of the crayfish Pacifastacus leniusculus is under circadian control and is tightly regulated by astakines, a new family of cytokines sharing a prokineticin (PROK) domain. The expression of AST1 and AST2 are light-dependent, and this suggests an evolutionarily conserved function for PROK domain proteins in mediating circadian rhythms. Vertebrate PROKs are transmitters of circadian rhythms of the suprachiasmatic nucleus (SCN) in the brain of mammals, but the mechanism by which they function is unknown. Here we demonstrate that high AST2 expression is induced by melatonin in the brain. We identify RACK1 as a binding protein of AST2 and further provide evidence that a complex between AST2 and RACK1 functions as a negative-feedback regulator of the circadian clock. By DNA mobility shift assay, we showed that the AST2-RACK1 complex will interfere with the binding between BMAL1 and CLK and inhibit the E-box binding activity of the complex BMAL1-CLK. Finally, we demonstrate by gene knockdown that AST2 is necessary for melatonin-induced inhibition of the complex formation between BMAL1 and CLK during the dark period. In summary, we provide evidence that melatonin regulates AST2 expression and thereby affects the core clock of the crustacean brain. This process may be very important in all animals that have AST2 molecules, i.e. spiders, ticks, crustaceans, scorpions, several insect groups such as Hymenoptera, Hemiptera, and Blattodea, but not Diptera and Coleoptera. Our findings further reveal an ancient evolutionary role for the prokineticin superfamily protein that links melatonin to direct regulation of the core clock gene feedback loops., Author Summary Most living organisms are able to sense the time and in particular time of day by their internal clocks. So-called clock proteins control these internal clockworks. BMAL1 and CLK are two important clock proteins, which together form a complex that serves as a transcription factor and controls the production of diurnal proteins. These diurnal proteins, in turn, inhibit the formation of clock proteins so that the concentration of the different proteins in the cell oscillates back and forth throughout the day. External factors may affect the balance of clock proteins, and one such important factor is light. Melatonin is a darkness hormone produced in the brain of most animals during the night, and here we show that melatonin controls the formation of a protein named AST2 in crayfish. AST2 belongs to a group of proteins found in many arthropods, such as spiders, scorpions, crustaceans, and some insects, whose function has been unknown until now. Now we demonstrate that AST2 is induced by melatonin at night and then functions in the internal biological clock by preventing BMAL1 and CLK to form a complex. In this way, AST2 acts as a link between melatonin and the internal biological clock.

During evolution, the innate and adaptive immune systems were developed to protect organisms from non-self substances. The innate immune system is phylogenetically more ancient and is present in most multicellular organisms, whereas adaptive responses are restricted to vertebrates. Arthropods lack the blood cells of the lymphoid lineage and oxygen-carrying erythrocytes, making them suitable model animals for studying the regulation of the blood cells of the innate immune system. Many crustaceans have a long life span and need to continuously synthesize blood cells, in contrast to many insects. The hematopoietic tissue (HPT) of Pacifastacus leniusculus provides a simple model for studying hematopoiesis, because the tissue can be isolated, and the proliferation of stem cells and their differentiation can be studied both in vivo and in vitro. Here, we demonstrate new findings of a physical link between the HPT and the brain. Actively proliferating cells were localized to an anterior proliferation center (APC) in the anterior part of the tissue near the area linking the HPT to the brain, whereas more differentiated cells were detected in the posterior part. The central areas of HPT expand in response to lipopolysaccharide-induced blood loss. Cells isolated from the APC divide rapidly and form cell clusters in vitro; conversely, the cells from the remaining HPT form monolayers, and they can be induced to differentiate in vitro. Our findings offer an opportunity to learn more about invertebrate hematopoiesis and its connection to the central nervous system, thereby obtaining new information about the evolution of different blood and nerve cell lineages.

The Down syndrome cell adhesion molecule, also known as Dscam, is a member of the immunoglobulin super family. Dscam plays an essential function in neuronal wiring and appears to be involved in innate immune reactions in insects. The deduced amino acid sequence of Dscam in the crustacean Pacifastacus leniusculus (PlDscam), encodes 9(Ig)-4(FNIII)-(Ig)-2(FNIII)-TM and it has variable regions in the N-terminal half of Ig2 and Ig3 and the complete Ig7 and in the transmembrane domain. The cytoplasmic tail can generate multiple isoforms. PlDscam can generate more than 22,000 different unique isoforms. Bacteria and LPS injection enhanced the expression of PlDscam, but no response in expression occurred after a white spot syndrome virus (WSSV) infection or injection with peptidoglycans. Furthermore, PlDscam silencing did not have any effect on the replication of the WSSV. Bacterial specific isoforms of PlDscam were shown to have a specific binding property to each tested bacteria, E. coli or S. aureus. The bacteria specific isoforms of PlDscam were shown to be associated with bacterial clearance and phagocytosis in crayfish., Author Summary Invertebrate animals lack an adaptive immune system and have no antibodies. Vertebrate antibodies belong to the immunoglobulin super family of proteins, and one other member of this large family is the Down syndrome cell adhesion molecule or Dscam. Of specific interest is that Dscam proteins in invertebrates show a great diversity of isoforms, and its gene structure in Drosophila melanogaster and other insect species allow for more than 30,000 different isoforms. Dscam proteins are important for the interaction between neurons in insects, but recently a role for this hypervariable protein in immune defense has been shown. Here, we show that Dscam proteins with similar highly variable structures are present in a crustacean, the freshwater crayfish Pacifastacus leniusculus. We also found that specific isoforms could be induced in the animal after injection of different bacteria. The Dscam isoforms induced by Escherichia coli were found to cluster together in a phylogenetic analysis. Furthermore we produced recombinant proteins of the different isoforms that were induced by E. coli and Staphylococcus aureus and we could demonstrate that these proteins can bind specifically to their corresponding bacteria. The bacteria specific isoforms of Dscam were also shown to be associated with bacterial clearance and phagocytosis in crayfish. Our study therefore provides new insights into the function of invertebrate Dscams in immunity.

The gC1qR/p32 protein is a multiple receptor for several proteins and pathogens. We cloned a gC1qR homologue in a crustacean, Pacifastacus leniusculus , and analyzed the expression of P. leniusculus C1qR ( Pl gC1qR) in various tissues. The gC1qR/p32 transcript was significantly enhanced by white spot syndrome virus (WSSV) infection 6 h after viral infection both in vitro in a hematopoietic tissue cell culture (Hpt) and in vivo compared to appropriate controls. Moreover, PlgC1qR silencing in both the Hpt cell culture and live crayfish enhanced the WSSV replication. In addition, by making a recombinant Pl gC1qR protein we could show that if this recombinant protein was injected in a crayfish, Pacifastacus leniusculus , followed by injection of WSSV, this significantly reduced viral replication in vivo . Furthermore, if the recombinant Pl gC1qR was incubated with Hpt cells and then WSSV was added, this also reduced viral replication. These experiments clearly demonstrate that recombinant Pl gC1qR reduce WSSV replication both in vivo and in vitro . The results from a far-Western overlay and glutathione S -transferase pull-down assays showed that PlgC1qR could bind to VP15, VP26, and VP28. Altogether, these results demonstrate a role for Pl gC1qR in antiviral activity against WSSV.

Aeromonas hydrophila is the most common Aeromonas species causing infections in human and other animals such as amphibians, reptiles, fish and crustaceans. Pathogenesis of Aeromonas species have been reported to be associated with virulence factors such as lipopolysaccharides (LPS), bacterial toxins, bacterial secretion systems, flagella, and other surface molecules. Several mutant strains of A. hydrophila AH-3 were initially used to study their virulence in two animal species, Pacifastacus leniusculus (crayfish) and Tenebrio molitor larvae (mealworm). The AH-3 strains used in this study have mutations in genes involving the synthesis of flagella, LPS structures, secretion systems, and some other factors, which have been reported to be involved in A. hydrophila pathogenicity. Our study shows that the LPS (O-antigen and external core) is the most determinant A. hydrophila AH-3 virulence factor in both animals. Furthermore, we studied the immune responses of these hosts to infection of virulent or non-virulent strains of A. hydrophila AH-3. The AH-3 wild type (WT) containing the complete LPS core is highly virulent and this bacterium strongly stimulated the prophenoloxidase activating system resulting in melanization in both crayfish and mealworm. In contrast, the ΔwaaE mutant which has LPS without O-antigen and external core was non-virulent and lost ability to stimulate this system and melanization in these two animals. The high phenoloxidase activity found in WT infected crayfish appears to result from a low expression of pacifastin, a prophenoloxidase activating enzyme inhibitor, and this gene expression was not changed in the ΔwaaE mutant infected animal and consequently phenoloxidase activity was not altered as compared to non-infected animals. Therefore we show that the virulence factors of A. hydrophila are the same regardless whether an insect or a crustacean is infected and the O-antigen and external core is essential for activation of the proPO system and as virulence factors for this bacterium.