The impact of low erythrocyte density in human blood on the fitness and energetic reserves of the African malaria vector Anopheles gambiae

Background Anaemia is a common health problem in the developing world. This condition is characterized by a reduction in erythrocyte density, primarily from malnutrition and/or infectious diseases such as malaria. As red blood cells are the primary source of protein for haematophagous mosquitoes, any reduction could impede the ability of mosquito vectors to transmit malaria by influencing their fitness or that of the parasites they transmit. The aim of this study was to determine the impact of differences in the density of red blood cells in human blood on malaria vector (Anopheles gambiae sensu stricto) fitness. The hypotheses tested are that mosquito vector energetic reserves and fitness are negatively influenced by reductions in the red cell density of host human blood meals commensurate with those expected from severe anaemia. Methods Mosquitoes (An. gambiae s.s.) were offered blood meals of different packed cell volume(PCV) of human blood consistent with those arising from severe anaemia (15%) and normalPCV (50%). Associations between mosquito energetic reserves (lipid, glucose and glycogen)and fitness measures (reproduction and survival) and blood meal PCV were investigated. Results The amount of protein that malaria vectors acquired from blood feeding (indexed by haematin excretion) was significantly reduced at low blood PCV. However, mosquitoes feeding on blood of low PCV had the same oviposition rates as those feeding on blood of normal PCV, and showed an increase in egg production of around 15%. The long-term survival of An. gambiae s.s was reduced after feeding on low PCV blood, but PCV had no significant impact on the proportion of mosquitoes surviving through the minimal period required to develop and transmit malaria parasites (estimated as 14 days post-blood feeding). The impact of blood PCV on the energetic reserves of mosquitoes was relatively minor. Conclusions These results suggest that feeding on human hosts whose PCV has been depleted due to severe anaemia does not significantly reduce the fitness or transmission potential of malaria vectors, and indicates that mosquitoes may be able exploit resources for reproduction more efficiently from blood of low rather than normal PCV

Pathogen Entrapment by Transglutaminase—A Conserved Early Innate Immune Mechanism

Clotting systems are required in almost all animals to prevent loss of body fluids after injury. Here, we show that despite the risks associated with its systemic activation, clotting is a hitherto little appreciated branch of the immune system. We compared clotting of human blood and insect hemolymph to study the best-conserved component of clotting systems, namely the Drosophila enzyme transglutaminase and its vertebrate homologue Factor XIIIa. Using labelled artificial substrates we observe that transglutaminase activity from both Drosophila hemolymph and human blood accumulates on microbial surfaces, leading to their sequestration into the clot. Using both a human and a natural insect pathogen we provide functional proof for an immune function for transglutaminase (TG). Drosophila larvae with reduced TG levels show increased mortality after septic injury. The same larvae are also more susceptible to a natural infection involving entomopathogenic nematodes and their symbiotic bacteria while neither phagocytosis, phenoloxidase or—as previously shown—the Toll or imd pathway contribute to immunity. These results firmly establish the hemolymph/blood clot as an important effector of early innate immunity, which helps to prevent septic infections. These findings will help to guide further strategies to reduce the damaging effects of clotting and enhance its beneficial contribution to immune reactions

Assessment and validation of a suite of reverse transcription-quantitative PCR reference genes for analyses of density-dependent behavioural plasticity in the Australian plague locust

<p>Abstract</p> <p>Background</p> <p>The Australian plague locust, <it>Chortoicetes terminifera</it>, is among the most promising species to unravel the suites of genes underling the density-dependent shift from shy and cryptic solitarious behaviour to the highly active and aggregating gregarious behaviour that is characteristic of locusts. This is because it lacks many of the major phenotypic changes in colour and morphology that accompany phase change in other locust species. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is the most sensitive method available for determining changes in gene expression. However, to accurately monitor the expression of target genes, it is essential to select an appropriate normalization strategy to control for non-specific variation between samples. Here we identify eight potential reference genes and examine their expression stability at different rearing density treatments in neural tissue of the Australian plague locust.</p> <p>Results</p> <p>Taking advantage of the new orthologous DNA sequences available in locusts, we developed primers for genes encoding 18SrRNA, ribosomal protein L32 (RpL32), armadillo (Arm), actin 5C (Actin), succinate dehydrogenase (SDHa), glyceraldehyde-3P-dehydrogenase (GAPDH), elongation factor 1 alpha (EF1a) and annexin IX (AnnIX). The relative transcription levels of these eight genes were then analyzed in three treatment groups differing in rearing density (isolated, short- and long-term crowded), each made up of five pools of four neural tissue samples from 5^thinstar nymphs. SDHa and GAPDH, which are both involved in metabolic pathways, were identified as the least stable in expression levels, challenging their usefulness in normalization. Based on calculations performed with the geNorm and NormFinder programs, the best combination of two genes for normalization of gene expression data following crowding in the Australian plague locust was EF1a and Arm. We applied their use to studying a target gene that encodes a Ca²⁺binding glycoprotein, <it>SPARC</it>, which was previously found to be up-regulated in brains of gregarious desert locusts, <it>Schistocerca gregaria</it>. Interestingly, expression of this gene did not vary with rearing density in the same way in brains of the two locust species. Unlike <it>S. gregaria</it>, there was no effect of any crowding treatment in the Australian plague locust.</p> <p>Conclusion</p> <p>Arm and EF1a is the most stably expressed combination of two reference genes of the eight examined for reliable normalization of RT-qPCR assays studying density-dependent behavioural change in the Australian plague locust. Such normalization allowed us to show that <it>C. terminifera </it>crowding did not change the neuronal expression of the <it>SPARC </it>gene, a gregarious phase-specific gene identified in brains of the desert locust, <it>S. gregaria</it>. Such comparative results on density-dependent gene regulation provide insights into the evolution of gregarious behaviour and mass migration of locusts. The eight identified genes we evaluated are also candidates as normalization genes for use in experiments involving other Oedipodinae species, but the rank order of gene stability must necessarily be determined on a case-by-case basis.</p

The main actors involved in parasitization of Heliothis virescens larva

At the moment of parasitization by another insect, the host Heliothis larva is able to defend itself by the activation of humoral and cellular defenses characterized by unusual reactions of hemocytes in response to external stimuli. Here, we have combined light and electron microscopy, staining reactions, and immunocytochemical characterization to analyze the activation and deactivation of one of the most important immune responses involved in invertebrates defense, i.e., melanin production and deposition. The insect host/parasitoid system is a good model to study these events. The activated granulocytes of the host insect are a major repository of amyloid fibrils forming a lattice in the cell. Subsequently, the exocytosed amyloid lattice constitutes the template for melanin deposition in the hemocel. Furthermore, cross-talk between immune and neuroendocrine systems mediated by hormones, cytokines, and neuromodulators with the activation of stress-sensoring circuits to produce and release molecules such as adrenocorticotropin hormone, alpha melanocyte-stimulating hormone, and neutral endopeptidase occurs. Thus, parasitization promotes massive morphological and physiological modifications in the host insect hemocytes and mimics general stress conditions in which phenomena such as amyloid fibril formation, melanin polymerization, pro-inflammatory cytokine production, and activation of the adrenocorticotropin hormone system occur. These events observed in invertebrates are also reported in the literature for vertebrates, suggesting that this network of mechanisms and responses is maintained throughout evolution

Neuropeptide Receptor Transcriptome Reveals Unidentified Neuroendocrine Pathways

Neuropeptides are an important class of molecules involved in diverse aspects of metazoan development and homeostasis. Insects are ideal model systems to investigate neuropeptide functions, and the major focus of insect neuropeptide research in the last decade has been on the identification of their receptors. Despite these vigorous efforts, receptors for some key neuropeptides in insect development such as prothoracicotropic hormone, eclosion hormone and allatotropin (AT), remain undefined. In this paper, we report the comprehensive cloning of neuropeptide G protein-coupled receptors from the silkworm, Bombyx mori, and systematic analyses of their expression. Based on the expression patterns of orphan receptors, we identified the long-sought receptor for AT, which is thought to stimulate juvenile hormone biosynthesis in the corpora allata (CA). Surprisingly, however, the AT receptor was not highly expressed in the CA, but instead was predominantly transcribed in the corpora cardiaca (CC), an organ adjacent to the CA. Indeed, by using a reverse-physiological approach, we purified and characterized novel allatoregulatory peptides produced in AT receptor-expressing CC cells, which may indirectly mediate AT activity on the CA. All of the above findings confirm the effectiveness of a systematic analysis of the receptor transcriptome, not only in characterizing orphan receptors, but also in identifying novel players and hidden mechanisms in important biological processes. This work illustrates how using a combinatorial approach employing bioinformatic, molecular, biochemical and physiological methods can help solve recalcitrant problems in neuropeptide research

Transcriptome Analysis of the Desert Locust Central Nervous System: Production and Annotation of a Schistocerca gregaria EST Database

) displays a fascinating type of phenotypic plasticity, designated as ‘phase polyphenism’. Depending on environmental conditions, one genome can be translated into two highly divergent phenotypes, termed the solitarious and gregarious (swarming) phase. Although many of the underlying molecular events remain elusive, the central nervous system (CNS) is expected to play a crucial role in the phase transition process. Locusts have also proven to be interesting model organisms in a physiological and neurobiological research context. However, molecular studies in locusts are hampered by the fact that genome/transcriptome sequence information available for this branch of insects is still limited. EST information is highly complementary to the existing orthopteran transcriptomic data. Since many novel transcripts encode neuronal signaling and signal transduction components, this paper includes an overview of these sequences. Furthermore, several transcripts being differentially represented in solitarious and gregarious locusts were retrieved from this EST database. The findings highlight the involvement of the CNS in the phase transition process and indicate that this novel annotated database may also add to the emerging knowledge of concomitant neuronal signaling and neuroplasticity events. EST data constitute an important new source of information that will be instrumental in further unraveling the molecular principles of phase polyphenism, in further establishing locusts as valuable research model organisms and in molecular evolutionary and comparative entomology

Primary biliary cirrhosis

Primary biliary cirrhosis (PBC) is an immune-mediated chronic cholestatic liver disease with a slowly progressive course. Without treatment, most patients eventually develop fibrosis and cirrhosis of the liver and may need liver transplantation in the late stage of disease. PBC primarily affects women (female preponderance 9–10:1) with a prevalence of up to 1 in 1,000 women over 40 years of age. Common symptoms of the disease are fatigue and pruritus, but most patients are asymptomatic at first presentation. The diagnosis is based on sustained elevation of serum markers of cholestasis, i.e., alkaline phosphatase and gamma-glutamyl transferase, and the presence of serum antimitochondrial antibodies directed against the E2 subunit of the pyruvate dehydrogenase complex. Histologically, PBC is characterized by florid bile duct lesions with damage to biliary epithelial cells, an often dense portal inflammatory infiltrate and progressive loss of small intrahepatic bile ducts. Although the insight into pathogenetic aspects of PBC has grown enormously during the recent decade and numerous genetic, environmental, and infectious factors have been disclosed which may contribute to the development of PBC, the precise pathogenesis remains enigmatic. Ursodeoxycholic acid (UDCA) is currently the only FDA-approved medical treatment for PBC. When administered at adequate doses of 13–15 mg/kg/day, up to two out of three patients with PBC may have a normal life expectancy without additional therapeutic measures. The mode of action of UDCA is still under discussion, but stimulation of impaired hepatocellular and cholangiocellular secretion, detoxification of bile, and antiapoptotic effects may represent key mechanisms. One out of three patients does not adequately respond to UDCA therapy and may need additional medical therapy and/or liver transplantation. This review summarizes current knowledge on the clinical, diagnostic, pathogenetic, and therapeutic aspects of PBC