The physiological effect of polystyrene nanoplastic particles on fish and human fibroblasts.

Numerous studies have identified the detrimental effects for the biosphere of large plastic debris, the effect of microplastics (MPs) and nanoplastics (NPs) is less clear. The skin is the first point of contact with NPs, and skin fibroblasts have a vital role in maintaining skin structure and function. Here, a comparative approach is taken using three fibroblast cell lines from the zebrafish (SJD.1), human male newborn (BJ-5ta) and female adult (HDF/TERT164) and their response to polystyrene NP (PS-NPs) exposure is characterized. Cells were exposed to environmentally relevant PS-NP sizes (50, 500 and 1000 nm) and concentrations (0.001 to 10 µg/ml) and their uptake (1000 nm), and effect on cell viability, proliferation, migration, reactive oxygen species (ROS) production, apoptosis, alkaline phosphatase (ALP) and acid phosphatase (AP) determined. All fibroblasts took up PS-NPs, and a relationship between PS-NP particle size and concentration and the inhibition of proliferation and cell migration was identified. The inhibitory effect of PS-NPs on proliferation was more pronounced for human skin fibroblasts. The presence of PS-NPs negatively affected fibroblast migration in a time-, size- and concentration-dependent manner with larger PS-NPs at higher concentrations causing a more significant inhibition of cell migration, with human fibroblasts being the most affected. No major changes were detected in ROS production or apoptosis in NP challenged fibroblasts. While the ALP activity was increased in all fibroblast cell lines, only fish fibroblasts showed a significant increase in AP activity. The heterogeneous response of fibroblasts induced by PS-NPs was clearly revealed by the segregation of HDF, BJ.5ta and SJD.1 fibroblasts in principal component analysis. Our results demonstrate that PS-NP exposure adversely affected cellular processes in a cell-type and dose-specific manner in distinct fibroblast cell lines, emphasizing the need for further exploration of NP interactions with different cell types to better understand potential implications for human health.

Fish lysozyme gene family evolution and divergent function in early development.

Lysozymes are an ancient group of antimicrobial enzymes of the innate immune system. Here we provide a comparative analysis of the evolution and function of lysozymes during early development in fish, the most speciose vertebrate group. In fishes, lineage and species-specific evolution of both C-type (chicken or conventional) and G-type (goose type) genes occurred. Phylogenetic analysis revealed that the teleost lysozyme G-type members group with the tetrapod homologues but the teleost C-type form three different clusters with the tetrapods. Most of the teleost C-type cluster with tetrapod Lyz but there are some that group with the mammalian Lyzl1/2 and LALBA. This suggests that early in gnathostome evolution these genes already existed and that lyzl1/2 and lalba genes are present in fish and tetrapods. Gene synteny analysis to confirm sequence orthologies failed to identify conserved genome regions between teleosts and other vertebrates lysozyme gene regions suggesting that in the ancestral bony fish genome lyz, lyzl1/2, lalba and lyg precursor genes were transposed to different chromosome regions. The homologue of the mammalian lactalbumin (LALBA) gene was identified for the first time in teleosts and was expressed in skin and during egg and larval development. Lysozyme activity was detected in teleost eggs and varied between species and in the gilthead sea bream lyg and lalba transcript abundance differed in eggs and larvae from different brood stock suggesting differences exist in maternal innate immune protection.

The calcitonin-like system is an ancient regulatory system of biomineralization.

Biomineralization is the process by which living organisms acquired the capacity to accumulate minerals in tissues. Shells are the biomineralized exoskeleton of marine molluscs produced by the mantle but factors that regulate mantle shell building are still enigmatic. This study sought to identify candidate regulatory factors of molluscan shell mineralization and targeted family B G-protein coupled receptors (GPCRs) and ligands that include calcium regulatory factors in vertebrates, such as calcitonin (CALC). In molluscs, CALC receptor (CALCR) number was variable and arose through lineage and species-specific duplications. The Mediterranean mussel (Mytilus galloprovincialis) mantle transcriptome expresses six CALCR-like and two CALC-precursors encoding four putative mature peptides. Mussel CALCR-like are activated in vitro by vertebrate CALC but only receptor CALCRIIc is activated by the mussel CALCIIa peptide (EC50 = 2.6 ×10-5 M). Ex-vivo incubations of mantle edge tissue and mantle cells with CALCIIa revealed they accumulated significantly more calcium than untreated tissue and cells. Mussel CALCIIa also significantly decreased mantle acid phosphatase activity, which is associated with shell remodelling. Our data indicate the CALC-like system as candidate regulatory factors of shell mineralization. The identification of the CALC system from molluscs to vertebrates suggests it is an ancient and conserved calcium regulatory system of mineralization.

A Blood-Free Diet to Rear Anopheline Mosquitoes.

Malaria research requires large-scale breeding and production conditions for mosquitoes (Anopheles spp.) in captivity. The sustainable and reliable production of mosquitoes is currently inhibited by the supply of fresh vertebrate blood. Alternatives to blood are required to promote efficient control strategies for malaria and other vector borne diseases that are transmitted by blood feeding insects. With this in mind, artificial liquid diets were formulated as substitutes for fresh vertebrate blood. Herein we report a blood-free artificial liquid diet that delivers feeding rates similar to blood and mimics the physiological effects of a fresh vertebrate blood meal. The diet induces ovarian and egg maturation of Anopheles mosquitoes and also produces good larval survival and development of functional adults. The formulated blood-free liquid diet is an important advance towards sustainable mosquito breeding in captivity and will reduce the maintenance costs of mosquito colonies and eliminate the need for fresh vertebrate blood.

Cartilage acidic protein 1 promotes increased cell viability, cell proliferation and energy metabolism in primary human dermal fibroblasts.

Cartilage acidic protein 1 (CRTAC1) is an extracellular matrix protein of human chondrogenic tissue that is also present in other vertebrates, non-vertebrate eukaryotes and in some prokaryotes. The function of CRTAC1 remains unknown but the protein's structure indicates a role in cell-cell or cell-matrix interactions and calcium-binding. The aim of the present study was to evaluate the in vitro effects of hCRTAC1-A on normal human dermal fibroblasts (NHDF). A battery of in vitro assays (biochemical and PCR), immunofluorescence and a biosensor approach were used to characterize the protein's biological activities on NHDF cells in a scratch assay. Gene expression analysis revealed that hCRTAC1-A protein is associated with altered levels of expression for genes involved in the processes of cell proliferation (CXCL12 and NOS2), cell migration (AQP3 and TNC), and extracellular matrix-ECM regeneration and remodeling (FMOD, TIMP1, FN1) indicating a role for hCRTAC1-A in promoting these activities in a scratch assay. In parallel, the candidate processes identified by differential gene transcription were substantiated and extended using Electric cell-substrate impedance sensing (ECIS) technology, immunofluorescence and cell viability assays. Our findings indicate that hCRTAC1-A stimulated cell proliferation, migration and ECM production in primary human fibroblasts in vitro.

Extracellular electrophysiological based sensor to monitor cancer cells cooperative migration and cell-cell connections.

Herein, we describe an electrophysiological based sensor that reproducibly monitors and quantifies in real-time collective migration and the formation of cell-cell junctions by C6 glioma cells seeded on top of electrodes. The signal amplitude and frequency generated by the migrating cells changed over time and these parameters were used to accurately calculate the migration speed. Electrophysiological measurements could also distinguish individual from collective cell migration. The migration of densely packed cells generated strong signals, while dispersed cells showed weak bioelectrical activity. We propose this electrophysiological technique as a cell-based biosensor to gain insight into the mechanisms of cooperative migration of cancer cells. Possible applications include screening for anti-migratory compounds, which may lead to the development of novel strategies for antineoplastic chemotherapy.

Evolution and diversity of alpha-carbonic anhydrases in the mantle of the Mediterranean mussel (Mytilus galloprovincialis).

The α-carbonic anhydrases (α-CAs) are a large and ancient group of metazoan-specific enzymes. They generate bicarbonate from metabolic carbon dioxide and through calcium carbonate crystal formation play a key role in the regulation of mineralized structures. To better understand how α-CAs contribute to shell mineralization in the marine Mediterranean mussel (Mytilus galloprovincialis) we characterized them in the mantle. Phylogenetic analysis revealed that mollusc α-CA evolution was affected by lineage and species-specific events. Ten α-CAs were found in the Mediterranean mussel mantle and the most abundant form was named, MgNACR, as it grouped with oyster nacreins (NACR). Exposure of the Mediterranean mussel to reduced water salinity (18 vs 37 ppt), caused a significant reduction (p < 0.05) in mantle esterase activity and MgNACR transcript abundance (p < 0.05). Protonograms revealed multiple proteins in the mantle with α-CA hydratase activity and mapped to a protein with a similar size to that deduced for monomeric MgNACR. Our data indicate that MgNACR is a major α-CA enzyme in mantle and that by homology with oyster nacreins likely regulates mussel shell production. We propose that species-dependent α-CA evolution may contribute to explain the diversity of bivalve shell structures and their vulnerability to environmental changes.

Fresh-blood-free diet for rearing malaria mosquito vectors.

Mosquito breeding depends on the supply of fresh vertebrate blood, a major bottleneck for large-scale production of Anopheles spp. Feeding alternatives to fresh blood are thus a priority for research, outdoor large-cage trials and control interventions. Several artificial meal compositions were tested and Anopheles oogenesis, egg laying and development into the next generation of adult mosquitoes were followed. We identified blood-substitute-diets that supported ovarian development, egg maturation and fertility as well as, low progeny larval mortality, and normal development of offspring into adult mosquitoes. The formulated diet is an effective artificial meal, free of fresh blood that mimics a vertebrate blood meal and represents an important advance for the sustainability of Anopheles mosquito rearing in captivity.

Evolution of the glucagon-like system across fish.

In fishes, including the jawless lampreys, the most ancient lineage of extant vertebrates, plasma glucose levels are highly variable and regulation is more relaxed than in mammals. The regulation of glucose and lipid in fishes in common with mammals involves members of the glucagon (GCG)-like family of gastrointestinal peptides. In mammals, four peptides GCG, glucagon-like peptide 1 and 2 (GLP1 and GLP2) and glucose-dependent insulinotropic peptide (GIP) that activate four specific receptors exist. However, in lamprey and other fishes the glucagon-like family evolved differently and they retained additional gene family members (glucagon-related peptide, gcrp and its receptor, gcrpr) that are absent from mammals. In the present study, we analysed the evolution of the glucagon-like system in fish and characterized gene expression of the family members in the European sea bass (Dicentrarchus labrax) a teleost fish. Phylogenetic analysis revealed that multiple receptors and peptides of the glucagon-like family emerged early during the vertebrate radiation and evolved via lineage specific events. Synteny analysis suggested that family member gene loss is likely to be the result of a single gene deletion event. Lamprey was the only fish where a putative glp1r persisted and the presence of the receptor gene in the genomes of the elephant shark and coelacanth remains unresolved. In the coelacanth and elephant shark, unique proglucagon genes were acquired which in the former only encoded Gcg and Glp2 and in the latter, shared a similar structure to the teleost proglucagon gene but possessed an extra exon coding for Glp-like peptide that was most similar to Glp2. The variable tissue distribution of the gene transcripts encoding the ligands and receptors of the glucagon-like system in an advanced teleost, the European sea bass, suggested that, as occurs in mammals, they have acquired distinct functions. Statistically significant (p < .05) down-regulation of teleost proglucagon a in sea bass with modified plasma glucose levels confirmed the link between these peptides and metabolism. The tissue distribution of members of the glucagon-like system in sea bass and human suggests that evolution of the brain-gut-peptide regulatory loop diverged between teleosts and mammals despite the overall conservation and similarity of glucagon-like family members.

Corticotropin-releasing hormone family evolution: five ancestral genes remain in some lineages.

The evolution of the peptide family consisting of corticotropin-releasing hormone (CRH) and the three urocortins (UCN1-3) has been puzzling due to uneven evolutionary rates. Distinct gene duplication scenarios have been proposed in relation to the two basal rounds of vertebrate genome doubling (2R) and the teleost fish-specific genome doubling (3R). By analyses of sequences and chromosomal regions, including many neighboring gene families, we show here that the vertebrate progenitor had two peptide genes that served as the founders of separate subfamilies. Then, 2R resulted in a total of five members: one subfamily consists of CRH1, CRH2, and UCN1. The other subfamily contains UCN2 and UCN3. All five peptide genes are present in the slowly evolving genomes of the coelacanth Latimeria chalumnae (a lobe-finned fish), the spotted gar Lepisosteus oculatus (a basal ray-finned fish), and the elephant shark Callorhinchus milii (a cartilaginous fish). The CRH2 gene has been lost independently in placental mammals and in teleost fish, but is present in birds (except chicken), anole lizard, and the nonplacental mammals platypus and opossum. Teleost 3R resulted in an additional surviving duplicate only for crh1 in some teleosts including zebrafish (crh1a and crh1b). We have previously reported that the two vertebrate CRH/UCN receptors arose in 2R and that CRHR1 was duplicated in 3R. Thus, we can now conclude that this peptide-receptor system was quite complex in the ancestor of the jawed vertebrates with five CRH/UCN peptides and two receptors, and that crh and crhr1 were duplicated in the teleost fish tetraploidization.

Allatostatin-type A, kisspeptin and galanin GPCRs and putative ligands as candidate regulatory factors of mantle function.

Allatostatin-type A (AST-A), kisspeptin (KISS) and galanin (GAL) G-protein coupled receptor (GPCR) systems share a common ancestral origin in arthropods and the vertebrates where they regulate metabolism and reproduction. The molluscs are the second most diverse phylum in the animal kingdom, they occupy an important phylogenetic position, and their genome is more similar to deuterostomes than the arthropods and nematodes and thus they are good models for studies of gene family evolution and function. This mini-review intends to extend the current knowledge about AST-A, KISS and GAL GPCR system evolution and their putative function in the mollusc mantle. Comparative evolutionary analysis of the target GPCR systems was established by identifying homologues in genomes and tissue transcriptome datasets available for molluscs and comparing them to those of other metazoan systems. Studies in arthropods have revealed the existence of the AST-A system but the loss of homologues of the KISS and GAL systems. Homologues of the insect AST-AR and vertebrate KISSR genes were found in molluscs but putative GALR genes were absent. Receptor gene number suggested that members of this family have suffered lineage specific evolution during the molluscan radiation. In molluscs, orthologues of the insect AST-A peptides were not identified but buccalin peptides that are structurally related were identified and are putative receptor agonists. The identification of AST-AR and KISSR genes in molluscs strengthens the hypotheses that in metazoans members of the AST-AR subfamily share evolutionary proximity with KISSRs. The variable number of receptors and large repertoire of buccalin peptides may be indicative of the functional diversity of the AST-AR/KISSR systems in molluscs. The identification of AST-A and KISS receptors and ligands in the mantle transcriptome indicates that in molluscs they may have acquired a novel function and may play a role in shell development or sensory detection in the mantle.

Unravelling the Evolution of the Allatostatin-Type A, KISS and Galanin Peptide-Receptor Gene Families in Bilaterians: Insights from Anopheles Mosquitoes.

UNLABELLED: Allatostatin type A receptors (AST-ARs) are a group of G-protein coupled receptors activated by members of the FGL-amide (AST-A) peptide family that inhibit food intake and development in arthropods. Despite their physiological importance the evolution of the AST-A system is poorly described and relatively few receptors have been isolated and functionally characterised in insects. The present study provides a comprehensive analysis of the origin and comparative evolution of the AST-A system. To determine how evolution and feeding modified the function of AST-AR the duplicate receptors in Anopheles mosquitoes, were characterised. Phylogeny and gene synteny suggested that invertebrate AST-A receptors and peptide genes shared a common evolutionary origin with KISS/GAL receptors and ligands. AST-ARs and KISSR emerged from a common gene ancestor after the divergence of GALRs in the bilaterian genome. In arthropods, the AST-A system evolved through lineage-specific events and the maintenance of two receptors in the flies and mosquitoes (Diptera) was the result of a gene duplication event. Speciation of Anopheles mosquitoes affected receptor gene organisation and characterisation of AST-AR duplicates (GPRALS1 and 2) revealed that in common with other insects, the mosquito receptors were activated by insect AST-A peptides and the iCa2+-signalling pathway was stimulated. GPRALS1 and 2 were expressed mainly in mosquito midgut and ovaries and transcript abundance of both receptors was modified by feeding. A blood meal strongly up-regulated expression of both GPRALS in the midgut (p < 0.05) compared to glucose fed females. Based on the results we hypothesise that the AST-A system in insects shared a common origin with the vertebrate KISS system and may also share a common function as an integrator of metabolism and reproduction. HIGHLIGHTS: AST-A and KISS/GAL receptors and ligands shared common ancestry prior to the protostome-deuterostome divergence. Phylogeny and gene synteny revealed that AST-AR and KISSR emerged after GALR gene divergence. AST-AR genes were present in the hemichordates but were lost from the chordates. In protostomes, AST-ARs persisted and evolved through lineage-specific events and duplicated in the arthropod radiation. Diptera acquired and maintained functionally divergent duplicate AST-AR genes.

PACAP system evolution and its role in melanophore function in teleost fish skin.

Pituitary adenylate cyclase-activating polypeptide (PACAP) administered to tilapia melanophores ex-vivo causes significant pigment aggregation and this is a newly identified function for this peptide in fish. The G-protein coupled receptors (GPCRs), adcyap1r1a (encoding Pac1a) and vipr2a (encoding Vpac2a), are the only receptors in melanophores with appreciable levels of expression and are significantly (p < 0.05) down-regulated in the absence of light. Vpac2a is activated exclusively by peptide histidine isoleucine (PHI), which suggests that Pac1a mediates the melanin aggregating effect of PACAP on melanophores. Paradoxically activation of Pac1a with PACAP caused a rise in cAMP, which in fish melanophores is associated with melanin dispersion. We hypothesise that the duplicate adcyap1ra and vipr2a genes in teleosts have acquired a specific role in skin and that the melanin aggregating effect of PACAP results from the interaction of Pac1a with Ramp that attenuates cAMP-dependent PKA activity and favours the Ca(2+)/Calmodulin dependent pathway.

STC1 interference on calcitonin family of receptors signaling during osteoblastogenesis via adenylate cyclase inhibition.

Stanniocalcin 1 (STC1) and calcitonin gene-related peptide (CGRP) are involved in bone formation/remodeling. Here we investigate the effects of STC1 on functional heterodimer complex CALCRL/RAMP1, expression and activity during osteoblastogenesis. STC1 did not modify CALCRL and ramp1 gene expression during osteoblastogenesis when compared to controls. However, plasma membrane spatial distribution of CALCRL/RAMP1 was modified in 7-day pre-osteoblasts exposed to either CGRP or STC1, and both peptides induced CALCRL and RAMP1 assembly. CGRP, but not STC1 stimulated cAMP accumulation in 7-day osteoblasts and in CALCRL/RAMP1 transfected HEK293 cells. Furthermore, STC1 inhibited forskolin stimulated cAMP accumulation of HEK293 cells, but not in CALCRL/RAMP1 transfected HEK293 cells. However, STC1 inhibited cAMP accumulation in calcitonin receptor (CTR) HEK293 transfected cells stimulated by calcitonin. In conclusion, STC1 signals through inhibitory G-protein modulates CGRP receptor spatial localization during osteoblastogenesis and may function as a regulatory factor interacting with calcitonin peptide members during bone formation.

New insights into the evolution of vertebrate CRH (corticotropin-releasing hormone) and invertebrate DH44 (diuretic hormone 44) receptors in metazoans.

The corticotropin releasing hormone receptors (CRHR) and the arthropod diuretic hormone 44 receptors (DH44R) are structurally and functionally related members of the G protein-coupled receptors (GPCR) of the secretin-like receptor superfamily. We show here that they derive from a bilaterian predecessor. In protostomes, the receptor became DH44R that has been identified and functionally characterised in several arthropods but the gene seems to be absent from nematode genomes. Duplicate DH44R genes (DH44 R1 and DH44R2) have been described in some arthropods resulting from lineage-specific duplications. Recently, CRHR-DH44R-like receptors have been identified in the genomes of some lophotrochozoans (molluscs, which have a lineage-specific gene duplication, and annelids) as well as representatives of early diverging deuterostomes. Vertebrates have previously been reported to have two CRHR receptors that were named CRHR1 and CRHR2. To resolve their origin we have analysed recently assembled genomes from representatives of early vertebrate divergencies including elephant shark, spotted gar and coelacanth. We show here by analysis of synteny conservation that the two CRHR genes arose from a common ancestral gene in the early vertebrate tetraploidizations (2R) approximately 500 million years ago. Subsequently, the teleost-specific tetraploidization (3R) resulted in a duplicate of CRHR1 that has been lost in some teleost lineages. These results help distinguish orthology and paralogy relationships and will allow studies of functional conservation and changes during evolution of the individual members of the receptor family and their multiple native peptide agonists.

Fish genomes provide novel insights into the evolution of vertebrate secretin receptors and their ligand.

The secretin receptor (SCTR) is a member of Class 2 subfamily B1 GPCRs and part of the PAC1/VPAC receptor subfamily. This receptor has long been known in mammals but has only recently been identified in other vertebrates including teleosts, from which it was previously considered to be absent. The ligand for SCTR in mammals is secretin (SCT), an important gastrointestinal peptide, which in teleosts has not yet been isolated, or the gene identified. This study revises the evolutionary model previously proposed for the secretin-GPCRs in metazoan by analysing in detail the fishes, the most successful of the extant vertebrates. All the Actinopterygii genomes analysed and the Chondrichthyes and Sarcopterygii fish possess a SCTR gene that shares conserved sequence, structure and synteny with the tetrapod homologue. Phylogenetic clustering and gene environment comparisons revealed that fish and tetrapod SCTR shared a common origin and diverged early from the PAC1/VPAC subfamily group. In teleosts SCTR duplicated as a result of the fish specific whole genome duplication but in all the teleost genomes analysed, with the exception of tilapia (Oreochromis niloticus), one of the duplicates was lost. The function of SCTR in teleosts is unknown but quantitative PCR revealed that in both sea bass (Dicentrarchus labrax) and tilapia (Oreochromis mossambicus) transcript abundance is high in the gastrointestinal tract suggesting it may intervene in similar processes to those in mammals. In contrast, no gene encoding the ligand SCT was identified in the ray-finned fishes (Actinopterygii) although it was present in the coelacanth (lobe finned fish, Sarcopterygii) and in the elephant shark (holocephalian). The genes in linkage with SCT in tetrapods and coelacanth were also identified in ray-finned fishes supporting the idea that it was lost from their genome. At present SCTR remains an orphan receptor in ray-finned fishes and it will be of interest in the future to establish why SCT was lost and which ligand substitutes for it so that full characterization of the receptor can occur.

Nematode and arthropod genomes provide new insights into the evolution of class 2 B1 GPCRs.

Nematodes and arthropods are the most speciose animal groups and possess Class 2 B1 G-protein coupled receptors (GPCRs). Existing models of invertebrate Class 2 B1 GPCR evolution are mainly centered on Caenorhabditis elegans and Drosophila melanogaster and a few other nematode and arthropod representatives. The present study reevaluates the evolution of metazoan Class 2 B1 GPCRs and orthologues by exploring the receptors in several nematode and arthropod genomes and comparing them to the human receptors. Three novel receptor phylogenetic clusters were identified and designated cluster A, cluster B and PDF-R-related cluster. Clusters A and B were identified in several nematode and arthropod genomes but were absent from D. melanogaster and Culicidae genomes, whereas the majority of the members of the PDF-R-related cluster were from nematodes. Cluster A receptors were nematode and arthropod-specific but shared a conserved gene environment with human receptor loci. Cluster B members were orthologous to human GCGR, PTHR and Secretin members with which they probably shared a common origin. PDF-R and PDF-R related clusters were present in representatives of both nematodes and arthropods. The results of comparative analysis of GPCR evolution and diversity in protostomes confirm previous notions that C. elegans and D. melanogaster genomes are not good representatives of nematode and arthropod phyla. We hypothesize that at least four ancestral Class 2 B1 genes emerged early in the metazoan radiation, which after the protostome-deuterostome split underwent distinct selective pressures that resulted in duplication and deletion events that originated the current Class 2 B1 GPCRs in nematode and arthropod genomes.

Feeding and the rhodopsin family g-protein coupled receptors in nematodes and arthropods.

In vertebrates, receptors of the rhodopsin G-protein coupled superfamily (GPCRs) play an important role in the regulation of feeding and energy homeostasis and are activated by peptide hormones produced in the brain-gut axis. These peptides regulate appetite and energy expenditure by promoting or inhibiting food intake. Sequence and function homologs of human GPCRs involved in feeding exist in the nematode roundworm, Caenorhabditis elegans (C. elegans), and the arthropod fruit fly, Drosophila melanogaster (D. melanogaster), suggesting that the mechanisms that regulate food intake emerged early and have been conserved during metazoan radiation. Nematodes and arthropods are the most diverse and successful animal phyla on Earth. They can survive in a vast diversity of environments and have acquired distinct life styles and feeding strategies. The aim of the present review is to investigate if this diversity has affected the evolution of invertebrate GPCRs. Homologs of the C. elegans and D. melanogaster rhodopsin receptors were characterized in the genome of other nematodes and arthropods and receptor evolution compared. With the exception of bombesin receptors (BBR) that are absent from nematodes, a similar gene complement was found. In arthropods, rhodopsin GPCR evolution is characterized by species-specific gene duplications and deletions and in nematodes by gene expansions in species with a free-living stage and gene deletions in representatives of obligate parasitic taxa. Based upon variation in GPCR gene number and potentially divergent functions within phyla we hypothesize that life style and feeding diversity practiced by nematodes and arthropods was one factor that contributed to rhodopsin GPCR gene evolution. Understanding how the regulation of food intake has evolved in invertebrates will contribute to the development of novel drugs to control nematodes and arthropods and the pests and diseases that use them as vectors.

The interplay between tubulins and P450 cytochromes during Plasmodium berghei invasion of Anopheles gambiae midgut.

BACKGROUND: Plasmodium infection increases the oxidative stress inside the mosquito, leading to a significant alteration on transcription of Anopheles gambiae detoxification genes. Among these detoxification genes several P450 cytochromes and tubulins were differently expressed, suggesting their involvement in the mosquito's response to parasite invasion. P450 cytochromes are usually involved in the metabolism and detoxification of several compounds, but are also regulated by several pathogens, including malaria parasite. Tubulins are extremely important as components of the cytoskeleton, which rearrangement functions as a response to malaria parasite invasion. METHODOLOGY/PRINCIPAL FINDINGS: Gene silencing methods were used to uncover the effects of cytochrome P450 reductase, tubulinA and tubulinB silencing on the A. gambiae response to Plasmodium berghei invasion. The role of tubulins in counter infection processes was also investigated by inhibiting their effect. Colchicine, vinblastine and paclitaxel, three different tubulin inhibitors were injected into A. gambiae mosquitoes. Twenty-four hours post injection these mosquitoes were infected with P. berghei through a blood meal from infected CD1 mice. Cytochrome P450 gene expression was measured using RT-qPCR to detect differences in cytochrome expression between silenced, inhibited and control mosquitoes. Results showed that cytochrome P450 reductase silencing, as well as tubulin (A and B) silencing and inhibition affected the efficiency of Plasmodium infection. Silencing and inhibition also affected the expression levels of cytochromes P450. CONCLUSIONS: Our results suggest the existence of a relationship between tubulins and P450 cytochromes during A. gambiae immune response to P. berghei invasion. One of the P450 cytochromes in this study, CYP6Z2, stands out as the potential link in this association. Further work is needed to fully understand the role of tubulin genes in the response to Plasmodium infection.

Plasmodium infection alters Anopheles gambiae detoxification gene expression.

BACKGROUND: Anopheles gambiae has been shown to change its global gene expression patterns upon Plasmodium infection. While many alterations are directly related to the mosquito's innate immune response, parasite invasion is also expected to generate toxic by-products such as free radicals. The current study aimed at identifying which loci coding for detoxification enzymes are differentially expressed as a function of Plasmodium berghei infection in midgut and fat body tissues. RESULTS: Using a custom-made DNA microarray, transcript levels of 254 loci primarily belonging to three major detoxification enzyme families (glutathione S-transferases, cytochrome P450 monooxygenases and esterases) were compared in infected and uninfected mosquitoes both during ookinete invasion and the release of sporozoites into the hemocoel. The greatest changes in gene expression were observed in the midgut in response to ookinete invasion. Interestingly, many detoxification genes including a large number of P450s were down-regulated at this stage. In the fat body, while less dramatic, gene expression alterations were also observed and occurred during the ookinete invasion and during the release of sporozoites into the hemocoel. While most gene expression changes were tissue-related, CYP6M2, a CYP previously associated with insecticide resistance, was over-expressed both in the midgut and fat body during ookinete invasion. CONCLUSIONS: Most toxicity-related reactions occur in the midgut shortly after the ingestion of an infected blood meal. Strong up-regulation of CYP6M2 in the midgut and the fat body as well as its previous association with insecticide resistance shows its broad role in metabolic detoxification.