The legacy of larval infection on immunological dynamics over metamorphosis.

Insect metamorphosis promotes the exploration of different ecological niches, as well as exposure to different parasites, across life stages. Adaptation should favour immune responses that are tailored to specific microbial threats, with the potential for metamorphosis to decouple the underlying genetic or physiological basis of immune responses in each stage. However, we do not have a good understanding of how early-life exposure to parasites influences immune responses in subsequent life stages. Is there a developmental legacy of larval infection in holometabolous insect hosts? To address this question, we exposed flour beetle (Tribolium castaneum) larvae to a protozoan parasite that inhabits the midgut of larvae and adults despite clearance during metamorphosis. We quantified the expression of relevant immune genes in the gut and whole body of exposed and unexposed individuals during the larval, pupal and adult stages. Our results suggest that parasite exposure induces the differential expression of several immune genes in the larval stage that persist into subsequent stages. We also demonstrate that immune gene expression covariance is partially decoupled among tissues and life stages. These results suggest that larval infection can leave a lasting imprint on immune phenotypes, with implications for the evolution of metamorphosis and immune systems. This article is part of the theme issue 'The evolution of complete metamorphosis'.

Immune gene regulation in the gut during metamorphosis in a holo- versus a hemimetabolous insect.

During metamorphosis, holometabolous insects completely replace the larval gut and must control the microbiota to avoid septicaemia. Rapid induction of bactericidal activity in the insect gut at the onset of pupation has been described in numerous orders of the Holometabola and is best-studied in the Lepidoptera where it is under control of the 20-hydroxyecdysone (20E) moulting pathway. Here, using RNAseq, we compare the expression of immune effector genes in the gut during metamorphosis in a holometabolous (Galleria mellonella) and a hemimetabolous insect (Gryllus bimaculatus). We find that in G. mellonella, the expression of numerous immune effectors and the transcription factor GmEts are upregulated, with peak expression of three antimicrobial peptides (AMPs) and a lysozyme coinciding with delamination of the larval gut. By contrast, no such upregulation was detectable in the hemimetabolous Gr. bimaculatus. These findings support the idea that the upregulation of immune effectors at the onset of complete metamorphosis is an adaptive response, which controls the microbiota during gut replacement. This article is part of the theme issue 'The evolution of complete metamorphosis'.

The microbiota profile and transcriptome analysis of immune response during metamorphosis stages in orange spotted grouper (Epinephelus coioides).

Metamorphosis is a transformation process in larval development associated with changes in morphological and physiological features, including the immune system. The gastrointestinal tract harbors a plethora of bacteria, which might affect the digestion and absorption of nutrients, immunity, and gut-brain crosstalk in the host. In this study, we have performed metagenomic and transcriptomic analyses on the intestines of grouper at the pre-, mid- and post-metamorphosis stages. The sequencing data of 16S rRNA gene showed drastic changes in the microbial communities at different developmental stages. The transcriptomic data revealed that the leukocyte transendothelial migration and the phagosome pathways might play important roles in mediating immunity in grouper at the three developmental stages. This information will increase our understanding of the metamorphosis process in grouper larvae, and shed light on the development of antimicrobial strategy during larval development.

Mechanisms of tail resorption during anuran metamorphosis.

Amphibian metamorphosis has historically attracted a good deal of scientific attention owing to its dramatic nature and easy observability. However, the genetic mechanisms of amphibian metamorphosis have not been thoroughly examined using modern techniques such as gene cloning, DNA sequencing, polymerase chain reaction or genomic editing. Here, we review the current state of knowledge regarding molecular mechanisms underlying tadpole tail resorption.

Long term effects of carbaryl exposure on antiviral immune responses in Xenopus laevis.

Water pollutants associated with agriculture may contribute to the increased prevalence of infectious diseases caused by ranaviruses. We have established the amphibian Xenopus laevis and the ranavirus Frog Virus 3 (FV3) as a reliable experimental platform for evaluating the effects of common waterborne pollutants, such as the insecticide carbaryl. Following 3 weeks of exposure to 10 ppb carbaryl, X. laevis tadpoles exhibited a marked increase in mortality and accelerated development. Exposure at lower concentrations (0.1 and 1.0 ppb) was not toxic, but it impaired tadpole innate antiviral immune responses, as evidenced by significantly decreased TNF-α, IL-1ß, IFN-I, and IFN-III gene expression. The defect in IFN-I and IL-1ß gene expression levels persisted after metamorphosis in froglets, whereas only IFN-I gene expression in response to FV3 was attenuated when carbaryl exposure was performed at the adult stage. These findings suggest that the agriculture-associated carbaryl exposure at low but ecologically-relevant concentrations has the potential to induce long term alterations in host-pathogen interactions and antiviral immunity.

Stepwise metamorphosis of the tubeworm Hydroides elegans is mediated by a bacterial inducer and MAPK signaling.

Diverse animal taxa metamorphose between larval and juvenile phases in response to bacteria. Although bacteria-induced metamorphosis is widespread among metazoans, little is known about the molecular changes that occur in the animal upon stimulation by bacteria. Larvae of the tubeworm Hydroides elegans metamorphose in response to surface-bound Pseudoalteromonas luteoviolacea bacteria, producing ordered arrays of phage tail-like metamorphosis-associated contractile structures (MACs). Sequencing the Hydroides genome and transcripts during five developmental stages revealed that MACs induce the regulation of groups of genes important for tissue remodeling, innate immunity, and mitogen-activated protein kinase (MAPK) signaling. Using two MAC mutations that block P. luteoviolacea from inducing settlement or metamorphosis and three MAPK inhibitors, we established a sequence of bacteria-induced metamorphic events: MACs induce larval settlement; then, particular properties of MACs encoded by a specific locus in P. luteoviolacea initiate cilia loss and activate metamorphosis-associated transcription; finally, signaling through p38 and c-Jun N-terminal kinase (JNK) MAPK pathways alters gene expression and leads to morphological changes upon initiation of metamorphosis. Our results reveal that the intricate interaction between Hydroides and P. luteoviolacea can be dissected using genomic, genetic, and pharmacological tools. Hydroides' dependency on bacteria for metamorphosis highlights the importance of external stimuli to orchestrate animal development. The conservation of Hydroides genome content with distantly related deuterostomes (urchins, sea squirts, and humans) suggests that mechanisms of bacteria-induced metamorphosis in Hydroides may have conserved features in diverse animals. As a major biofouling agent, insight into the triggers of Hydroides metamorphosis might lead to practical strategies for fouling control.

Comparative proteomics analysis of silkworm hemolymph during the stages of metamorphosis via liquid chromatography and mass spectrometry.

The silkworm is a lepidopteran insect that has an open circulatory system with hemolymph consisting of blood and lymph fluid. Hemolymph is not only considered as a depository of nutrients and energy, but it also plays a key role in substance transportation, immunity response, and proteolysis. In this study, we used LC-MS/MS to analyze the hemolymph proteins of four developmental stages during metamorphosis. A total of 728 proteins were identified from the hemolymph of the second day of wandering stage, first day of pupation, ninth day of pupation, and first day as an adult moth. GO annotations and categories showed that silkworm hemolymph proteins were enriched in carbohydrate metabolism, proteolysis, protein binding, and antibacterial humoral response. The levels of nutrient, immunity-related, and structural proteins changed significantly during development and metamorphosis. Some, such as cuticle, odorant-binding, and chemosensory proteins, showed stage-specific expression in the hemolymph. In addition, the expression of several antimicrobial peptides exhibited their highest level of abundance in the hemolymph of the early pupal stage. These findings provide a comprehensive proteomic insight of the silkworm hemolymph and suggest additional molecular targets for studying insect metamorphosis.

Molecular cloning, characterization and expression analysis of cathepsin O in silkworm Bombyx mori related to bacterial response.

Cathepsins are the main members of the cysteine family and play important roles in immune response in vertebrates. The Cathepsin O of Bombyx mori (BmCathepsin O) was cloned from the hemocytes by the rapid amplification of cDNA ends (RACE). The genomic DNA was 6131bp long with a total of six exons and five introns. Its pre-mRNA was spliced to generate two spliceosomes. By comparisons with other reported cathepsins O, it was concluded that the identity between them ranged from 29 to 39%. Expression analysis indicated that BmCathepsin O was specific-expressed in hemocytes, and highly expressed at the 4th molting and metamorphosis stages. Immunofluorescence assay and qRT-PCR showed that BmCathepsin O was expressed in granulocytes and plasmatocytes. Interestingly, BmCathepsin O was significantly up-regulated after stimulated by 20-hydroxyecdysone (20-E) in vivo, which suggested that BmCathepsin O may be regulated by 20E. Moreover, activation of BmCathepsin O was also observed in hemocytes challenged by Escherichia coli, indicating its potential involvement in the innate immune system of silkworm, B. mori. In summary, our studies provide a new insight into the functional features of Cathepsin O.

MANF silencing, immunity induction or autophagy trigger an unusual cell type in metamorphosing Drosophila brain.

Glia are abundant cells in the brain of animals ranging from flies to humans. They perform conserved functions not only in neural development and wiring, but also in brain homeostasis. Here we show that by manipulating gene expression in glia, a previously unidentified cell type appears in the Drosophila brain during metamorphosis. More specifically, this cell type appears in three contexts: (1) after the induction of either immunity, or (2) autophagy, or (3) by silencing of neurotrophic factor DmMANF in glial cells. We call these cells MANF immunoreactive Cells (MiCs). MiCs are migratory based on their shape, appearance in brain areas where no cell bodies exist and the nuclear localization of dSTAT. They are labeled with a unique set of molecular markers including the conserved neurotrophic factor DmMANF and the transcription factor Zfh1. They possess the nuclearly localized protein Relish, which is the hallmark of immune response activation. They also express the conserved engulfment receptor Draper, therefore indicating that they are potentially phagocytic. Surprisingly, they do not express any of the common glial and neuronal markers. In addition, ultrastructural studies show that MiCs are extremely rich in lysosomes. Our findings reveal critical molecular and functional components of an unusual cell type in the Drosophila brain. We suggest that MiCs resemble macrophages/hemocytes and vertebrate microglia based on their appearance in the brain upon genetically challenged conditions and the expression of molecular markers. Interestingly, macrophages/hemocytes or microglia-like cells have not been reported in the fly nervous system before.

Expression of immune genes during metamorphosis of Xenopus: a survey.

This review focuses on what is known about the immune transcriptome during metamorphosis of Xenopus laevis and Silurana (Xenopus) tropicalis. This subject is of importance to obtain a global understanding of the physiological changes operating during metamorphosis. In turn, a good knowledge of the physiology of amphibian metamorphosis may contribute to the fight against amphibian decline and help the development of alternative toxicologic assays. By examining what is known on the expression of innate and adaptive immune genes during metamorphosis, it becomes clear that our knowledge of the anatomy of the tadpole "immunome" is fragmentary. Since a wealth of data sits in cDNA sequences, I am making a first attempt to enrich our knowledge on this subject. I exemplify that mining EST data can rapidly provide us with the necessary tools to unravel the cross-talk between thyroid hormone signalling during metamorphosis and larval immune system changes.

Comparative and developmental study of the immune system in Xenopus.

Xenopus laevis is the model of choice for evolutionary, comparative, and developmental studies of immunity, and invaluable research tools including MHC-defined clones, inbred strains, cell lines, and monoclonal antibodies are available for these studies. Recent efforts to use Silurana (Xenopus) tropicalis for genetic analyses have led to the sequencing of the whole genome. Ongoing genome mapping and mutagenesis studies will provide a new dimension to the study of immunity. Here we review what is known about the immune system of X. laevis integrated with available genomic information from S. tropicalis. This review provides compelling evidence for the high degree of similarity and evolutionary conservation between Xenopus and mammalian immune systems. We propose to build a powerful and innovative comparative biomedical model based on modern genetic technologies that takes take advantage of X. laevis and S. tropicalis, as well as the whole Xenopus genus. Developmental Dynamics 238:1249-1270, 2009. (c) 2009 Wiley-Liss, Inc.

The immune system is involved in Xenopus metamorphosis.

Amphibian metamorphosis provides a model to elucidate the mechanisms underlying how vertebrates reconstitute a body plan and how the immune system develops during ontogeny. In Xenopus, T cells are expanded from the early developmental stages just after hatching. These T cells switch from larval-type in an easily tolerizable state into an adult-type having a potent immune responsiveness comparable to that of mammals. During metamorphosis, tadpoles exhibit morphological changes in skin that completely transforms from larval-type to adult-type. Only tail tissue behaves differently; it remains a larval-type tissue until it disappears at the end of metamorphosis. Thus, at metamorphic climax, four different types of cells co-exist in a tadpole body: larval tissue cells; adult tissue cells; larval immune cells; and adult immune cells. Based on the results showing that tadpole tail skin is rejected by syngeneic adult, it is proposed that the elimination of the larval tissue cells by the adult T cells that occurs during metamorphosis is immunologically mediated. Recent results indicate that the antigenic proteins expressed in the metamorphosing skin cells participate in the process of tail regression. This chapter describes how animals adjust and survive through such crises associated with large scale replacement of entire body cells.

A new isotype of immunoglobulin heavy chain in the urodele amphibian Pleurodeles waltl predominantly expressed in larvae.

Up to now, it was thought that urodele amphibians possessed only two IgH isotypes, IgM (mu) and IgY (upsilon). By screening a Pleurodeles waltl Ig cDNA mini-library, we identified three isotypes: IgM, IgY and a previously unknown class. IgM are multimeric molecules and represent the most abundant isotype throughout the life of P. waltl. IgY are likely the counterpart of mammalian IgA. The new isotype has typical Ig H-chain characteristics and is expressed as both secretory and membrane forms. Our analyses indicate that this isotype is restricted to Pleurodeles. Consequently, we named it "IgP" (pi) for Pleurodeles. This isotype is mainly expressed after hatching. Its expression decreases after metamorphosis. Our data indicate that IgP-expressing B cells present some similarities with mammalian B1-cells.

Larval Pacific herring, Clupea pallasii (Valenciennes), are highly susceptible to viral haemorrhagic septicaemia and survivors are partially protected after their metamorphosis to juveniles.

Pacific herring were susceptible to waterborne challenge with viral haemorrhagic septicaemia virus (VHSV) throughout their early life history stages, with significantly greater cumulative mortalities occurring among VHSV-exposed groups of 9-, 44-, 54- and 76-day-old larvae than among respective control groups. Similarly, among 89-day-1-year-old and 1+year old post-metamorphosed juveniles, cumulative mortality was significantly greater in VHSV-challenged groups than in respective control groups. Larval exposure to VHSV conferred partial protection to the survivors after their metamorphosis to juveniles as shown by significantly less cumulative mortalities among juvenile groups that survived a VHS epidemic as larvae than among groups that were previously naïve to VHSV. Magnitude of the protection, measured as relative per cent survival, was a direct function of larval age at first exposure and was probably a reflection of gradual developmental onset of immunocompetence. These results indicate the potential for easily overlooked VHS epizootics among wild larvae in regions where the virus is endemic and emphasize the importance of early life history stages of marine fish in influencing the ecological disease processes.

Dynamic changes of free nucleotides in the silkworm undergoing embryonic diapause induced by environmental factors.

Free nucleotide contents in newly hatched larvae and final instar larvae were examined in a bivoltine race (Daizo) of the silkworm, Bombyx mori, by high pressure liquid chromatography (HPLC). ATP and UTP were higher in whole bodies especially in newly hatched larvae experienced the regimen of continuous light and warm temperature (25 degrees C)(LW-condition) throughout the embyrogenesis than in those experienced the regimen of continuous darkness and low temperature (15 degrees C) (DL-condition). ATP and UTP increased rapidly during the wandering stage and their difference was significant between LW-condition and DL-condition. These results indicated that the triphosphate nucleotide contents induced by environmental factors might involve in diapause destination.

Rearing density and susceptibility of Rana pipiens metamorphs to cercariae of a digenetic trematode.

Cercariae of many digenean trematodes target particular developmental stages of their hosts. For some digeneans that are parasites of amphibians, infection appears timed to host metamorphosis. The success and timing of metamorphosis is itself affected by a number of factors, including host density. We investigated the degree to which rearing density of Rana pipiens larvae influenced time to metamorphosis and snout-to-vent length and mass at metamorphosis, as well as establishment of cercariae of the trematode Manodistomum syntomentera Stafford, 1905. As expected, individuals metamorphosed later, were smaller, and weighed less at metamorphosis if they were reared under intermediate to high densities compared with low densities, in experimental outdoor mesocosms. Cercariae establishment was higher in smaller metamorphs that took longer to metamorphose within the low-density treatment. Additionally, cercariae establishment was lower in larvae from the low-density tanks compared with larvae from the intermediate- to high-density tanks. However, more tadpoles had failed to metamorphose in the intermediate to high rearing densities by the time cercariae were no longer available from natural collections of first intermediate hosts, Physa spp. Larval amphibians under crowded conditions should experience increased susceptibility to trematode establishment in nature, but only if they metamorphose within the time period when cercariae are still available.

Persistence of host response against glochidia larvae in Micropterus salmoides.

Host fish acquire resistance to the parasitic larvae (glochidia) of freshwater mussels (Unionidae). Glochidia metamorphose into juvenile mussels while encysted on host fish. We investigated the duration of acquired resistance of largemouth bass, Micropterus salmoides (Lacepède, 1802) to glochidia of the broken rays mussel, Lampsilis reeveiana (Call, 1887). Fish received three successive priming infections with glochidia to induce an immune response. Primed fish were held at 22-23 degrees C and were challenged (re-infected) at intervals after priming. Metamorphosis success was quantified as the percent of attached glochidia that metamorphosed to the juvenile stage and were recovered alive. Metamorphosis success at 3, 7, and 12 months after priming was significantly lower on primed fish (26%, 40%, and 68% respectively) than on control fish (85%, 93%, and 92% respectively). A second group of largemouth bass was similarly primed and blood was extracted. Immunoblotting was used to detect host serum antibodies to L. reeveiana glochidia proteins. Serum antibodies were evident in primed fish, but not in naive control fish. Acquired resistance of host fish potentially affects natural reproduction and artificial propagation of unionids, many of which are of conservation concern.

Metamorphosis and collagen-IV-fragments stimulate innate immune response in the greater wax moth, Galleria mellonella.

A novel link between development and immunity in insects is introduced. Transiently enhanced expression of lysozyme, gallerimycin and the insect metalloproteinase inhibitor was discovered at the onset of metamorphosis of the greater wax moth, Galleria mellonella. Relative quantification of mRNAs encoding for these antimicrobial peptides using real-time PCR documents their induced expression during transformation of last instar larvae into prepupae and upon injection of either recombinant interstitial collagenase (MMP-1) or small-sized fragments of collagen type IV. The latter were also found to stimulate both nuclear import of c-Rel-proteins in the fat body, implicating activation of Toll or Imd-related signaling pathways, and subsequent synthesis of antimicrobial peptides. Obtained results implicate that degradation of collagen-IV by either microbial metalloproteinases associated with invading pathogens or endogenous matrix metalloproteinases contributing to degradation of extracellular matrix during metamorphosis stimulate innate immune responses.

Analyses of immune responses to ontogeny-specific antigens using an inbred strain of Xenopus laevis (J strain).

In this chapter, the procedures for specific detection of ontogenic emerging antigens during animal development are described. Anuran metamorphosis has provided us with a good experimental model for investigation of the mechanisms of tissue remodeling. The establishment of a syngeneic strain of Xenopus laevis described in this chapter has enabled us to perform a unique experiment to develop antibodies that specifically react to ontogenic antigens by immunizing syngeneic animals. This strategy was successful because the antibody repertoires produced in the adult frog serum were well subtracted by a number of common antigens expressed in syngeneic larvae. Here we show, using results of immunohistochemical and T-cell proliferation analyses that adult frogs exhibit humoral and cell-mediated immune responses to larva- or metamorphosis-specific antigen molecules in epidermal cells.

Cytotoxic activity of blowfly Calliphora vicina hemocytes.

The cytotoxic activity of hemocytes isolated from larvae of the blowfly Calliphora vicina was tested using human myelogenous leukemia K562 cells as target. Both single cell and cytotoxicity assays demonstrated that the hemocytes recognize the K562 cells as nonself, firmly attach to their surface and induce target destruction in a manner resembling the effect of mammalian cytotoxic lymphocytes. The cytotoxic activity increased dramatically in the course of larval metamorphosis and was considerably higher shortly before the onset of pupariation, compared to the activity of human peripheral blood or mouse spleen lymphocytes. In insects, the cytotoxic hemocytes may take part in defense against eukaryotic parasites and in the elimination of aberrant self cells, as well as in developmental processes such as metamorphosis.