A putative G protein-coupled receptor involved in innate immune defense of Procambarus clarkii against bacterial infection

The immune functions of G protein-coupled receptor (GPCR) were widely investigated in mammals. However, limited researches on immune function of GPCRs were reported in invertebrates. In the present study, the immune functions of HP1R gene, a putative GPCR identified from red swamp crayfish Procambarus clarkii were reported. Expression of HP1R gene was significant up-regulated in response to heat-killed Aeromonas hydrophila challenge. HP1R gene silencing mediated by RNA interference significantly enhanced the susceptibility of red swamp crayfish to A. hydrophila and Vibrio alginolyticus, indicating that HP1R was required for red swamp crayfish to defend against bacterial challenge. In HP1R-silenced crayfish, increased bacterial burden and decreased THC in response to bacterial challenge were observed when compared with control crayfish. No significant difference of proPO gene expression was observed between HP1R-silenced and control crayfish after challenge with heat-killed A. hydrophila. However, PO activity in response to bacterial challenge was significantly reduced in HP1R-silenced crayfish. The results collectively indicated that HP1R was an important immune molecule which was required for red swamp crayfish to defend against bacterial infection.     

A highly virulent pathogen, Aeromonas hydrophila, from the freshwater crayfish Pacifastacus leniusculus

Aeromonas spp. are characteristic bacteria of freshwaters and many of them can be components of the bacterial flora of aquatic animals and may become pathogens on animals including humans. In this study Aeromonas hydrophila was isolated from the freshwater crayfish, Pacifastacus leniusculus, and was found to be a highly pathogenic bacterium among many isolated bacteria. Mortality reached 100% within 6 h when 200 μl of 1.24 × 10⁷ CFU/ml was applied by injection. Histopathological studies of moribund crayfish showed that extensive necrotic nuclei and clump-infiltrated hemocytes were found in observed tissues including gill, heart, hepatopancreas and the circulatory system. To verify how crayfish are susceptible to this bacterium, crude extracellular products (ECPs) obtained from culture supernatant of A. hydrophila was studied either in vivo or in vitro. ECPs (200 μl) were able to kill crayfish by injection. In an in vitro study, ECPs induced cytotoxicity of hemocytes as well as hematopoietic cells in a dose- and time-dependent manner after 30 min post inoculation. Two genes coding for endotoxins were also found in this isolate of A. hydrophila. This indicates that the bacterial endotoxins are the causative agents of crayfish mortality. Moreover, the effect of temperature on the infectivity of A. hydrophila to crayfish was also studied. At 4 °C, all crayfish survived, whereas at 20 °C the animals died rapidly after bacterial challenge. At this low temperature A. hydrophila did not replicate or replicated at a very low degree and hence crayfish could probably mount effective cellular reactions towards A. hydrophila.     

Involvement of peroxinectin in the defence of red swamp crayfish Procambarus clarkii against pathogenic Aeromonas hydrophila

Cell adhesion factors are important immune components for invertebrate to immobilize, phagocytose or encapsulate invasive microorganisms and foreign particles. In this study, a new cell adhesion factor, peroxinectin (refered as Pcpxin) was isolated from hemocytes of red swamp crayfish (Procambarus clarkii). The full-length cDNA of Pcpxin was 3014 bp encoding a protein of 819 amino acid residues with a predicted molecular weight of 89.0 kDa and a calculational isoelectric point of 6.93. The putative amino acid sequence contained a peroxidase domain and a signal peptide of 21 amino acid residues, and exhibited high identity to peroxinectin from Pacifastacus leniusculus (85%), Fenneropenaeus chinensis (62%) and Scylla serrata (58%), as well as peroxidase from Camponotus floridanus (40%), Pediculus humanus corporis (39%), and Culex quinquefasciatus (38%). Quantitative real time PCR revealed that mRNA expression of Pcpxin in hemocytes could be inhibited by challenge with heat-killed Aeromonas hydrophila, suggesting that Pcpxin was involved in immune responses to A. hydrophila. RNA interference (RNAi) experiment demonstrated that silencing Pcpxin significantly reduced the survival rate of red swamp crayfishes after challenge with A. hydrophila, which indicated that Pcpxin was important for P. clarkii to survive A. hydrophila infection. Moreover, silencing Pcpxin inhibited the up-regulation of crustin1 and lysozyme expression in response to challenge with heat-killed A. hydrophila. This result suggested that Pcpxin might participate in antibacterial peptide gene expression and thereby might be involved in signal transduction pathway regulating the expression of antibacterial peptide gene.     

Expression of Developmental-Stage-Specific Genes in the Gilthead Sea Bream Sparus aurata L.

The mechanism of early fish development as well as the control of egg quality is of great importance for the ability of the oocyte to develop after fertilization. Embryonic development is initially regulated by maternally provided mRNAs and later by the zygotic genome. Maternal mRNAs have an important role in initiating processes crucial to patterning the developing fish embryo. Furthermore, it has been shown that maternal RNA plays an important role in egg quality. The identification and characterization of candidate maternal genes in non-model fish species with important aquaculture interest like the gilthead sea bream Sparus aurata L. is of importance for future studies related to egg quality. The broodstock of the gilthead sea bream produces large quantities of eggs with a high and non-controllable quality variation. In the present study, we have studied the gene expression of 16 genes (gapdh 1 and 2, cathepsin D, L, S and Z, erk1, jnk1, p38 alpha and p38 delta, ppar alpha, beta and gamma, tubulin beta, ferritin M, cyclinA2) of different functional categories in seven developmental stages. The 16 genes were chosen based on their putative involvement in egg quality and regulation of early development. In total, 11 showed a characteristic gene expression pattern pinpointing to the possible function as maternal genes and thus may function as molecular biomarker for egg quality.     

The raspberry Gene Is Involved in the Regulation of the Cellular Immune Response in Drosophila melanogaster

Drosophila is an extremely useful model organism for understanding how innate immune mechanisms defend against microbes and parasitoids. Large foreign objects trigger a potent cellular immune response in Drosophila larva. In the case of endoparasitoid wasp eggs, this response includes hemocyte proliferation, lamellocyte differentiation and eventual encapsulation of the egg. The encapsulation reaction involves the attachment and spreading of hemocytes around the egg, which requires cytoskeletal rearrangements, changes in adhesion properties and cell shape, as well as melanization of the capsule. Guanine nucleotide metabolism has an essential role in the regulation of pathways necessary for this encapsulation response. Here, we show that the Drosophila inosine 5''-monophosphate dehydrogenase (IMPDH), encoded by raspberry (ras), is centrally important for a proper cellular immune response against eggs from the parasitoid wasp Leptopilina boulardi. Notably, hemocyte attachment to the egg and subsequent melanization of the capsule are deficient in hypomorphic ras mutant larvae, which results in a compromised cellular immune response and increased survival of the parasitoid.     

An insect TEP in a crustacean is specific for cuticular tissues and involved in intestinal defense

In an attempt to identify genes encoding thioester-containing proteins in the freshwater crayfish, Pacifastacus leniusculus, three different cDNAs were found. A phylogenetic analysis of these proteins indicates that they can be classified into two subfamilies: two alpha-2-macroglobulins (Pl-A2M1, Pl-A2M2) showing a close similarity to shrimp A2M, and one insect TEP-like protein (Pl-TEP). This is the first report of an insect TEP-like protein in a crustacean. Crayfish Pl-A2M1, Pl-A2M2 and Pl-TEP cDNAs encode proteins with 1480, 1586 or 1507 amino acids, respectively. Pl-A2M1, Pl-A2M2 and Pl-TEP have the basic domain structure and functionally important residues for each molecule, and their mRNA was detected in different parts of the body, suggesting that they may have different functions. Pl-A2M1 was mainly expressed in hemocytes and Pl-A2M2 was highly expressed in heart and nerve, while Pl-TEP was exclusively expressed in cuticular tissues such as gill and intestine. RNA interference of Pl-TEP in vivo resulted in that these animals were slightly less resistant when fed with the bacterium, Pseudomonas libanensis/gessardii. Furthermore, when TEP activity was blocked using methylamine followed by bacterial feeding, the animals were killed to a higher extent compared to a control group. Taken together, this indicates that Pl-TEP and/or Pl-A2M1, Pl-A2M2 may be important for the immune defense in crayfish intestine and function as a pattern recognition protein in crayfish cuticular tissues.     

Damselfly eggs alter their development rate in the presence of an invasive alien cue but not a native predator cue

Biological invasions are a serious problem in natural ecosystems. Local species that are potential prey of invasive alien predators can be threatened by their inability to recognize invasive predator cues. Such an inability of prey to recognize the presence of the predator supports the naïve prey hypothesis. We exposed eggs of a damselfly, Ischnura elegans, to four treatments: water with no predator cue (control), water with a native predator cue (perch), water with an invasive alien predator cue (spinycheek crayfish) that is present in the damselfly sampling site, and water with an invasive alien predator cue (signal crayfish) that is absent in the damselfly sampling site but is expected to invade it. We measured egg development time, mortality between ovipositing and hatching, and hatching synchrony. Eggs took longer to develop in the signal crayfish group (however, in this group, we also observed high green algae growth), and there was a trend of shorter egg development time in the spinycheek crayfish group than in the control group. There was no difference in egg development time between the perch and the control group. Neither egg mortality nor hatching synchrony differed between groups. We suggest that egg response to signal crayfish could be a general stress reaction to an unfamiliar cue or an artifact due to algae development in this group. The egg response to the spinycheek crayfish cue could be caused by the predation of crayfish on damselfly eggs in nature. The lack of egg response to the perch cue could be caused by perch predation on damselfly larvae rather than on eggs. Such differences in egg responses to alternative predator cues can have important implications for understanding how this group of insects responds to biological invasions, starting from the egg stage.     

Phenoloxidase activation in the embryo of the common cuttlefish Sepia officinalis and responses to the Ag and Cu exposure

The prophenoloxidase (proPO) system catalyzing the melanin production is considered as implicated in the innate immune system in invertebrates. The phenoloxidase (PO)-like activity was detected in the cuttlefish embryo sampled at the end of the organogenesis and few hours before hatching. Various modulators of the PO activity were used to assess the triggering of the proPO activating system. The results demonstrated the evidence of a true PO activity in the cuttlefish embryo. However, SDS and LPS granted contrasting effects on the PO-like activity between the developmental stages suggesting a progressive maturation of the proPO system from the embryonic to the juvenile stages. In eggs exposed to dissolved trace metals all along the embryonic development, Ag (1.2 μg L⁻¹) inhibited the PO-like activity in the cuttlefish embryo except at hatching time, suggesting the synthesis of a new “juvenile” form of the PO enzyme. In similar conditions as for Ag, Cu (230 μg L⁻¹) stimulated and then inhibited the PO-like activity according to a progressive metal accumulation within the egg and suggesting the occurrence of a threshold, above which the toxicity of the essential metal reduced the PO activity.     

The Involvement of Hemocyte Prophenoloxidase in the Shell-Hardening Process of the Blue Crab,Callinectes sapidus

Cuticular structures of arthropods undergo dramatic molt-related changes from being soft to becoming hard. The shell-hardening process of decapod crustaceans includes sclerotization and mineralization. Hemocyte PPO plays a central role in melanization and sclerotization particularly in wound healing in crustaceans. However, little is known about its role in the crustacean initial shell-hardening process. The earlier findings of the aggregation of heavily granulated hemocytes beneath the hypodermis during ecdysis imply that the hemocytes may be involved in the shell-hardening process. In order to determine if hemocytes and hemocyte PPO have a role in the shell-hardening of crustaceans, a knockdown study using specific CasPPO-hemo-dsRNA was carried out with juvenile blue crabs, Callinectes sapidus. Multiple injections of CasPPO-hemo-dsRNA reduce specifically the levels of CasPPO-hemo expression by 57% and PO activity by 54% in hemocyte lysate at the postmolt, while they have no effect on the total hemocyte numbers. Immunocytochemistry and flow cytometry analysis using a specific antiserum generated against CasPPO show granulocytes, semigranulocytes and hyaline cells as the cellular sources for PPO at the postmolt. Interestingly, the type of hemocytes, as the cellular sources of PPO, varies by molt stage. The granulocytes always contain PPO throughout the molt cycle. However, semigranulocytes and hyaline cells become CasPPO immune-positive only at early premolt and postmolt, indicating that PPO expression in these cells may be involved in the shell-hardening process of C. sapidus.     

A cell adhesion factor from crayfish haemocytes has degranulating activity towards crayfish granular cells

A factor able to mediate cell adhesion of semigranular and granular haemocytes of the crayfish Pacifastacus leniusculus was recently purified from crayfish haemocyte lysate (Johansson and Söderhäll, J. Cell Biol.106, 1795–1803, 1988). It is a protein with a mass of 76 kDa, and its activity seems to be generated concomitantly with the activation of the prophenoloxidase (proPO) activating system. In this paper, we present evidence that this same protein is also responsible for the previously reported degranulating activity of a crayfish haemocyte lysate, in which the proPO system has been activated. First, the 76 kDa band in SDS-polyacrylamide electrophoresis seems to be a single protein, since in isoelectric focusing the purified cell adhesion factor fraction migrated as one band with an isoelectric point of 7.2. Second, this fraction was also able to degranulate crayfish granular cells in vitro, and third, antibodies to this 76 kDa protein, which are known to block cell adhesion, could also inhibit degranulation in vitro.     

A study of uptake of radiolabeled host proteins and protein synthesis during development of eggs of the endoparasitoid,Microplitis croceipes (Cresson) (Braconidae)

The uptake of radiolabeled haemolymph and fat body proteins from fourth instar larvae of Heliothis zea (Boddie) by eggs of Microplitis croceipes (Cresson) was examined by SDS-polyacrylamide gel electrophoresis and by autoradiography. None of the ¹²⁵I-labeled haemolymph proteins was detected in eggs exposed to the proteins in vivo. Although several of the proteins were observed in eggs incubated with the labeled proteins in vitro, none of these proteins was degraded or resynthesized into new structural proteins during development of the embryo. Similarly, no significant uptake of labeled fat body proteins by the eggs could be detected in vitro. On the other hand, protein synthesis measured by incorporation of [³⁵S]methionine occurred throughout egg development. Proteins were synthesized at least 1 hr after the egg was deposited into the host. The protein patterns of eggs on one-dimensional SDS gels were complex and ranged in size from less than 18,500 to more than 330,000 mol. wt. The protein band patterns of the newly synthesized proteins showed some qualitative differences at 1–8, 16–32 and 40 hr after egg deposition. We conclude that eggs do not absorb or utilize the host apoproteins (or degradation products) but instead synthesize proteins de novo from free amino acids in the host haemolymph.     

Caspase-1-Like Regulation of the proPO-System and Role of ppA and Caspase-1-Like Cleaved Peptides from proPO in Innate Immunity

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.     

Repair of a genetically-caused defect in oogenesis in Drosophila melanogaster by transplantation of cytoplasm from wild-type eggs and by injection of pyrimidine nucleosides

Eggs produced by homozygous mutant rudimentary (r⁹, 154.5) females of Drosophila melanogaster die during embryogenesis, apparently because the mutant female fails to incorporate certain substances into the egg during oogenesis. These eggs can be rescued by injecting them at the preblastoderm stage with wild-type nuclei and cytoplasm or wild-type cytoplasm alone from unfertilized eggs. Some preblastoderm eggs injected with 1.5% of egg volume of cytoplasm from unfertilized wild-type eggs were able to complete both embryonic and postembryonic development and emerged as adults, whereas not a single uninjected control egg was able to complete embryonic development. The eggs of rudimentary mothers can also be rescued by injecting each egg at the blastoderm stage with 0.01 μg of pyrimidine nucleosides. The results demonstrate that a pyrimidine deficiency is the cause of abortive embryogenesis, and confirm the previous finding that the rudimentary mutation leads to pyrimidine auxotrophy.     

Salmonella Typhimurium infection causes defects and fastening of Caenorhabditis elegans developmental stages

Salmonella infection is known to cause a 50% reduction in the lifespan of Caenorhabditis elegans. But the mechanism behind this reduction is not reported. The current study deals with the Salmonella infection mediated egg retention in the worm leading to various developmental and morphological defects and disruption of temporal regulation of developmental timing in C. elegans. Worm’s delayed egg-laying response to Salmonella infection causes several defects in eggs, including over-folding of developing embryo, increased egg size, and losing the osmotic stress resistance. Also, the infected eggs show delayed and reduced hatching. With significantly downregulated lin-28a, col-72 and col-87, we observed a disrupted L2, but L3, L4, and adult developmental stages reach faster during infection. The precocious development of L3, L4, and the adult stage is further indicated by upregulation of stage-specific genes viz. rnh-1.3, col-158 and col-176 (L3), col-17, col-38 and col-49 (L4), and col-19, col-7 (adult). The significant upregulation of the flp-1 gene indicates reduced egglaying, and the flp-1(ok2811) null mutant further supported the Salmonella infectionmediated phenotype. Similar phenotypes are primarily evident in multiple generations up to F5 and F6. Salmonella infection causes a range of developmental anomalies and shortening of worm life span through various regulatory pathways.