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1.  Variation in spatial and temporal incidence of the crustacean pathogen Hematodinium perezi in environmental samples from Atlantic Coastal Bays 
Aquatic Biosystems  2013;9:11.
Hematodinium perezi, a parasitic dinoflagellate, infects and kills blue crabs, Callinectes sapidus, along the Atlantic and Gulf coasts of the United States. The parasite proliferates within host hemolymph and tissues, and also produces free-swimming biflagellated dinospores that emerge from infected crabs. Infections in C. sapidus recur annually, and it is not known if biotic or environmental reservoirs contribute to reinfection and outbreaks. To address this data gap, a quantitative PCR assay based on the internal transcribed spacer 2 (ITS2) region of H. perezi rRNA genes was developed to asses the temporal and spatial incidence of the parasite in Delaware and Maryland coastal bays.
A previously-used PCR assay for H. perezi, based on the small subunit rRNA gene sequence, was found to lack adequate species specificity to discriminate non-Hematodinium sp. dinoflagellate species in environmental samples. A new ITS2-targeted assay was developed and validated to detect H. perezi DNA in sediment and water samples using E. coli carrying the H. perezi rDNA genes. Application of the method to environmental samples identified potential hotspots in sediment in Indian River Inlet, DE and Chincoteague Bay, MD and VA. H. perezi DNA was not detected in co-occurring shrimp or snails, even during an outbreak of the parasite in C. sapidus.
H. perezi is present in water and sediment samples in Maryland and Delaware coastal bays from April through November with a wide spatial and temporal variability in incidence. Sampling sites with high levels of H. perezi DNA in both bays share characteristics of silty, organic sediments and low tidal currents. The environmental detection of H. perezi in spring, ahead of peak prevalence in crabs, points to gaps in our understanding of the parasite’s life history prior to infection in crabs as well as the mode of environmental transmission. To better understand the H. perezi life cycle will require further monitoring of the parasite in habitats as well as hosts. Improved understanding of potential environmental transmission to crabs will facilitate the development of disease forecasting.
PMCID: PMC3651331  PMID: 23641869
Blue crab; Hematodinium; Parasite; Disease reservoir; Fishery
2.  Temporal distribution of genetically homogenous ‘free-living’ Hematodinium sp. in a Delmarva coastal ecosystem 
Aquatic Biosystems  2012;8:16.
Significant damage to crustacean fisheries worldwide has been associated with Hematodinium sp. It has been postulated that Hematodinium sp. requires passage through the water column and/or intermediate hosts to complete its life cycle. Thus, an understanding of the prevalence and seasonality of Hematodinium sp. within environmentally-derived samples should yield insight into potential modes of disease transmission, and how these relate to infection cycles in hosts.
We conducted a two year survey, from 2010–2011, in which 48 of 546 (8.8%) of environmental samples from the Maryland and Virginia coastal bays were positive for Hematodinium sp. between April and November, as based upon endpoint PCR analysis specific to blue crab isolates. Detection in both water and sediment was roughly equivalent, and there were no obvious seasonal patterns. However, there was a high detection in April water samples, which was unanticipated owing to the fact that crabs infected with Hematodinium sp. have not been observed in this early month of the seasonal disease cycle. Focusing on three sites of high prevalence (Sinnickson, VA; Tom’s Cove, VA; and Newport Bay, MD) Hematodinium sp. population diversity was analyzed using standard cloning methods. Of 131 clones, 109 (83.2%) were identical, 19 displayed a single nucleotide substitution, and 4 contain two nucleotide substitutions.
Our data suggests a continuous presence of Hematodinium sp. in both water and sediment of a combined Maryland and Virginia coastal bay ecosystem. The detection of Hematodinium sp. in the water column in April is an earlier manifestation of the parasite than predicted, pointing to an as yet unknown stage in its development prior to infection. That the population is relatively homogenous ranging from April to November, at three distinct sites, supports a hypothesis that one species of Hematodinium is responsible for infections within the ecosystem.
PMCID: PMC3413547  PMID: 22828185
Hematodinium; Life cycle; Environment; Population
3.  New Host Range for Hematodinium in Southern Australia and Novel Tools for Sensitive Detection of Parasitic Dinoflagellates 
PLoS ONE  2013;8(12):e82774.
Hematodinium is a parasitic dinoflagellate and emerging pathogen of crustaceans. It preferably manifests in haemolymph of marine decapod crustaceans, killing a large variety of genera with significant impacts on fisheries worldwide. There is, however, evidence that some crustacean stocks harbor high prevalence, low intensity infections that may not result in widespread host mortality and are therefore hard to detect. The most widely used methods for detection of Hematodinium are conventional blood smears and polymerase chain reaction (PCR) against ribosomal RNAs. Blood smears demand a trained investigator, are labor intensive and not readily scalable for high-throughput sampling. PCRs only detect parasite DNA and can also suffer from false negatives and positives. In order to develop alternative detection tools for Hematodinium cells in decapod crustaceans we employed an immunological approach against a newly identified, abundant dinoflagellate-specific nuclear protein—Dinoflagellate/Viral NucleoProtein (DVNP). Both immunofluorescence assay (IFA) and Western blot methods against DVNP showed high sensitivity of detection. The Western blot detects Hematodinium parasites to levels of 25 parasites per milliliter of crustacean haemolymph, with the potential for sample pooling and screening of large samples. Using both PCR and these new tools, we have identified Hematodinium cells present in three new host crab taxa, at high prevalence but with no sign of pathogenesis. This extends the known range of Hematodinium to southern Australia.
PMCID: PMC3855790  PMID: 24324829
4.  Characterization and Molecular Epidemiology of a Fungal Infection of Edible Crabs (Cancer pagurus) and Interaction of the Fungus with the Dinoflagellate Parasite Hematodinium 
This study reports on an emerging fungal disease of the edible crab, Cancer pagurus. Juvenile (prerecruit) crabs were found to be subject to this disease condition during the months of May to September at two intertidal sites in South Wales, United Kingdom. Histopathology revealed that the fungi overwhelm the host response in the tissues, leading to progressive septicemia. The causative agent of this infection was isolated and grown in pure culture and was identified as a member of the Ophiocordyceps clade by sequencing of the small subunit of the fungal ribosomal DNA (rDNA). Of the crabs naturally infected with the fungus, 94% had a coinfection with the parasitic dinoflagellate Hematodinium species. To determine if there was any interaction between the two disease-causing agents, apparently fungus-free crabs, both with and without natural Hematodinium infections, were challenged with the fungal isolate. The presence of Hematodinium caused a significant reduction in fungal multiplication in the hemocoel of the crabs in comparison to that in Hematodinium-free individuals. Histopathology of coinfected crabs showed a systemic multiplication of Hematodinium within host tissues, leading to a rapid death, while Hematodinium-free crabs experimentally infected with the fungal isolate died due to fungal sepsis (septicemia) with the same characteristic pathology as seen in natural infections.
PMCID: PMC3568540  PMID: 23160130
5.  Absence of Ca2+-Induced Mitochondrial Permeability Transition but Presence of Bongkrekate-Sensitive Nucleotide Exchange in C. crangon and P. serratus 
PLoS ONE  2012;7(6):e39839.
Mitochondria from the embryos of brine shrimp (Artemia franciscana) do not undergo Ca2+-induced permeability transition in the presence of a profound Ca2+ uptake capacity. Furthermore, this crustacean is the only organism known to exhibit bongkrekate-insensitive mitochondrial adenine nucleotide exchange, prompting the conjecture that refractoriness to bongkrekate and absence of Ca2+-induced permeability transition are somehow related phenomena. Here we report that mitochondria isolated from two other crustaceans, brown shrimp (Crangon crangon) and common prawn (Palaemon serratus) exhibited bongkrekate-sensitive mitochondrial adenine nucleotide transport, but lacked a Ca2+-induced permeability transition. Ca2+ uptake capacity was robust in the absence of adenine nucleotides in both crustaceans, unaffected by either bongkrekate or cyclosporin A. Transmission electron microscopy images of Ca2+-loaded mitochondria showed needle-like formations of electron-dense material strikingly similar to those observed in mitochondria from the hepatopancreas of blue crab (Callinectes sapidus) and the embryos of Artemia franciscana. Alignment analysis of the partial coding sequences of the adenine nucleotide translocase (ANT) expressed in Crangon crangon and Palaemon serratus versus the complete sequence expressed in Artemia franciscana reappraised the possibility of the 208-214 amino acid region for conferring sensitivity to bongkrekate. However, our findings suggest that the ability to undergo Ca2+-induced mitochondrial permeability transition and the sensitivity of adenine nucleotide translocase to bongkrekate are not necessarily related phenomena.
PMCID: PMC3387235  PMID: 22768139
6.  A Quantitative Assessment of the Role of the Parasite Amoebophrya in the Termination of Alexandrium fundyense Blooms within a Small Coastal Embayment 
PLoS ONE  2013;8(12):e81150.
Parasitic dinoflagellates of the genus Amoebophrya infect free-living dinoflagellates, some of which can cause harmful algal blooms (HABs). High prevalence of Amoebophrya spp. has been linked to the decline of some HABs in marine systems. The objective of this study was to evaluate the impact of Amoebophrya spp. on the dynamics of dinoflagellate blooms in Salt Pond (MA, USA), particularly the harmful species Alexandrium fundyense. The abundance of Amoebophrya life stages was estimated 3–7 days per week through the full duration of an annual A. fundyense bloom using fluorescence in situ hybridization coupled with tyramide signal amplification (FISH- TSA). More than 20 potential hosts were recorded including Dinophysis spp., Protoperidinium spp. and Gonyaulax spp., but the only dinoflagellate cells infected by Amoebophrya spp. during the sampling period were A. fundyense. Maximum A. fundyense concentration co-occurred with an increase of infected hosts, followed by a massive release of Amoebophrya dinospores in the water column. On average, Amoebophrya spp. infected and killed ∼30% of the A. fundyense population per day in the end phase of the bloom. The decline of the host A. fundyense population coincided with a dramatic life-cycle transition from vegetative division to sexual fusion. This transition occurred after maximum infected host concentrations and before peak infection percentages were observed, suggesting that most A. fundyense escaped parasite infection through sexual fusion. The results of this work highlight the importance of high frequency sampling of both parasite and host populations to accurately assess the impact of parasites on natural plankton assemblages.
PMCID: PMC3852033  PMID: 24324668
7.  Detection and Discovery of Crustacean Parasites in Blue Crabs (Callinectes sapidus) by Using 18S rRNA Gene-Targeted Denaturing High-Performance Liquid Chromatography▿ †  
Applied and Environmental Microbiology  2008;74(14):4346-4353.
Recently, we described a novel denaturing high-performance liquid chromatography (DHPLC) approach useful for initial detection and identification of crustacean parasites. Because this approach utilizes general primers targeted to conserved regions of the 18S rRNA gene, a priori genetic sequence information on eukaryotic parasites is not required. This distinction provides a significant advantage over specifically targeted PCR assays that do not allow for the detection of unknown or unsuspected parasites. However, initial field evaluations of the DHPLC assay suggested that because of PCR-biased amplification of dominant host genes it was not possible to detect relatively rare parasite genes in infected crab tissue. Here, we describe the use of a peptide nucleic acid (PNA) PCR hybridization blocking probe in association with DHPLC (PNA-PCR DHPLC) to overcome inherent PCR bias associated with amplification of rare target genes by use of generic primers. This approach was utilized to detect infection of blue crabs (Callinectes sapidus) by the parasitic dinoflagellate Hematodinium sp. Evaluation of 76 crabs caught in Wassaw Sound, GA, indicated a 97% correspondence between detection of the parasite by use of a specific PCR diagnostic assay and that by use of PNA-PCR DHPLC. During these studies, we discovered one crab with an association with a previously undescribed protist symbiont. Phylogenetic analysis of the amplified symbiont 18S rRNA gene indicated that it is most closely related to the free-living kinetoplastid parasite Procryptobia sorokini. To our knowledge, this is the first report of this parasite group in a decapod crab and of this organism exhibiting a presumably parasitic life history.
PMCID: PMC2493178  PMID: 18502913
8.  First record of a parasitic septate gregarines (Apicomplexa: Sporozoea) in the shrimp Peneaus monodon in Sundarbans of West Bengal 
Investigations on the incidence of septate gregarines in shrimp have immense importance because of severe pathogenicity of the parasite. The septate gregarines infect the midgut of shrimp Peneaus monodon and severe infection disturbs the intestinal tissues. Mostly gregarines of the genus Nematopsis have been identified from cultured peneaid shrimp. It has worldwide in distribution. In India, gregarine parasites have so far been reported from penaeid shrimps of Bombay and Kerala. The species which was isolated from the midgut of shrimp Peneaus monodon collected from Kharibari area of Sunderbans. 9 out of 20 i.e. 45% of the randomly sampled hosts were found to be infected with a species of the genus Nematopsis. Different developmental stages including trophozoites, sporadins, and gametocysts of the Nematopsis sp. infecting the shrimp have been isolated. No correlations have been established between incidence of infection and environmental parameters.
PMCID: PMC3081698  PMID: 21526032
Nematopsis sundarbanensis; Peneaus monodon; Septate gregarines; Gut parasite; Sundarbans
9.  Density dynamics of diverse Spiroplasma strains naturally infecting different species of Drosophila 
Fly  2013;7(3):204-210.
Facultative heritable bacterial endosymbionts can have dramatic effects on their hosts, ranging from mutualistic to parasitic. Within-host bacterial endosymbiont density plays a critical role in maintenance of a symbiotic relationship, as it can affect levels of vertical transmission and expression of phenotypic effects, both of which influence the infection prevalence in host populations. Species of genus Drosophila are infected with Spiroplasma, whose characterized phenotypic effects range from that of a male-killing reproductive parasite to beneficial defensive endosymbiont. For many strains of Spiroplasma infecting at least 17 species of Drosophila, however, the phenotypic effects are obscure. The infection prevalence of these Spiroplasma vary within and among Drosophila species, and little is known about the within-host density dynamics of these diverse strains. To characterize the patterns of Spiroplasma density variation among Drosophila we used quantitative PCR to assess bacterial titer at various life stages of three species of Drosophila naturally-infected with two different types of Spiroplasma. For naturally infected Drosophila species we found that non-male-killing infections had consistently lower densities than the male-killing infection. The patterns of Spiroplasma titer change during aging varied among Drosophila species infected with different Spiroplasma strains. Bacterial density varied within and among populations of Drosophila, with individuals from the population with the highest prevalence of infection having the highest density. This density variation underscores the complex interaction of Spiroplasma strain and host genetic background in determining endosymbiont density.
PMCID: PMC4049854  PMID: 23846301
Spiroplasma; Drosophila; vertically-transmitted endosymbiont; endosymbiont density
10.  Dinoflagellate phylogeny revisited: Using ribosomal proteins to resolve deep branching dinoflagellate clades 
The alveolates are composed of three major lineages, the ciliates, dinoflagellates, and apicomplexans. Together these ‘protist’ taxa play key roles in primary production and ecology, as well as in illness of humans and other animals. The interface between the dinoflagellate and apicomplexan clades has been an area of recent discovery, blurring the distinction between these two clades. Moreover, phylogenetic analysis has yet to determine the position of basal dinoflagellate clades hence the deepest branches of the dinoflagellate tree currently remain unresolved. Large-scale mRNA sequencing was applied to 11 species of dinoflagellates, including strains of the syndinean genera Hematodinium and Amoebophrya, parasites of crustaceans and dinoflagellates, respectively, to optimize and update the dinoflagellate tree. From the transcriptome-scale data a total of 73 ribosomal protein-coding genes were selected for phylogeny. After individual gene orthology assessment, the genes were concatenated into a >15,000 amino acid alignment with 76 taxa from dinoflagellates, apicomplexans, ciliates, and the outgroup heterokonts. Overall the tree was well resolved and supported, when the data was subsampled with gblocks or constraint trees were tested with the approximately unbiased test. The deepest branches of the dinoflagellate tree can now be resolved with strong support, and provides a clearer view of the evolution of the distinctive traits of dinoflagellates.
PMCID: PMC4144664  PMID: 24135237
Dinoflagellate; Alveolate; Heterokont; Apicomplexan; Ribosomal protein
11.  Progression of Plasmodium berghei through Anopheles stephensi Is Density-Dependent 
PLoS Pathogens  2007;3(12):e195.
It is well documented that the density of Plasmodium in its vertebrate host modulates the physiological response induced; this in turn regulates parasite survival and transmission. It is less clear that parasite density in the mosquito regulates survival and transmission of this important pathogen. Numerous studies have described conversion rates of Plasmodium from one life stage to the next within the mosquito, yet few have considered that these rates might vary with parasite density. Here we establish infections with defined numbers of the rodent malaria parasite Plasmodium berghei to examine how parasite density at each stage of development (gametocytes; ookinetes; oocysts and sporozoites) influences development to the ensuing stage in Anopheles stephensi, and thus the delivery of infectious sporozoites to the vertebrate host. We show that every developmental transition exhibits strong density dependence, with numbers of the ensuing stages saturating at high density. We further show that when fed ookinetes at very low densities, oocyst development is facilitated by increasing ookinete number (i.e., the efficiency of ookinete–oocyst transformation follows a sigmoid relationship). We discuss how observations on this model system generate important hypotheses for the understanding of malaria biology, and how these might guide the rational analysis of interventions against the transmission of the malaria parasites of humans by their diverse vector species.
Author Summary
Malaria, one of the world's most devastating parasitic diseases, is caused by protozoan parasites of the genus Plasmodium and is transmitted between mammalian hosts by Anopheles mosquitoes. Within the mosquito, the parasite undergoes four sequential developmental transformations as it passes from the bloodmeal through the mosquito's midgut epithelium to the salivary glands, from where the parasite is inoculated when the mosquito bites the vertebrate host. This study demonstrates, in a laboratory model, that parasite input density at every developmental stage in the mosquito regulates output to the ensuing form. Statistical models were fitted to experimental data to identify and describe the most appropriate functional relationships. In all cases, the relationships between two consecutive parasite stages can saturate at high parasite densities, suggesting that at high parasite densities parasite numbers may have to be reduced substantially to effect an appreciable decrease in parasite transmission. These results may help establish a rational basis for new studies on species of medical importance and further our understanding of how interventions designed to reduce parasite survival within the mosquito might be expected to impact upon transmission.
PMCID: PMC2156095  PMID: 18166078
12.  Two Bacterial Genera, Sodalis and Rickettsia, Associated with the Seal Louse Proechinophthirus fluctus (Phthiraptera: Anoplura) 
Applied and Environmental Microbiology  2016;82(11):3185-3197.
Roughly 10% to 15% of insect species host heritable symbiotic bacteria known as endosymbionts. The lice parasitizing mammals rely on endosymbionts to provide essential vitamins absent in their blood meals. Here, we describe two bacterial associates from a louse, Proechinophthirus fluctus, which is an obligate ectoparasite of a marine mammal. One of these is a heritable endosymbiont that is not closely related to endosymbionts of other mammalian lice. Rather, it is more closely related to endosymbionts of the genus Sodalis associated with spittlebugs and feather-chewing bird lice. Localization and vertical transmission of this endosymbiont are also more similar to those of bird lice than to those of other mammalian lice. The endosymbiont genome appears to be degrading in symbiosis; however, it is considerably larger than the genomes of other mammalian louse endosymbionts. These patterns suggest the possibility that this Sodalis endosymbiont might be recently acquired, replacing a now-extinct, ancient endosymbiont. From the same lice, we also identified an abundant bacterium belonging to the genus Rickettsia that is closely related to Rickettsia ricketsii, a human pathogen vectored by ticks. No obvious masses of the Rickettsia bacterium were observed in louse tissues, nor did we find any evidence of vertical transmission, so the nature of its association remains unclear.
IMPORTANCE Many insects are host to heritable symbiotic bacteria. These heritable bacteria have been identified from numerous species of parasitic lice. It appears that novel symbioses have formed between lice and bacteria many times, with new bacterial symbionts potentially replacing existing ones. However, little was known about the symbionts of lice parasitizing marine mammals. Here, we identified a heritable bacterial symbiont in lice parasitizing northern fur seals. This bacterial symbiont appears to have been recently acquired by the lice. The findings reported here provide insights into how new symbioses form and how this lifestyle is shaping the symbiont genome.
PMCID: PMC4959230  PMID: 26994086
13.  Mutualism Breakdown by Amplification of Wolbachia Genes 
PLoS Biology  2015;13(2):e1002065.
Most insect species are associated with vertically transmitted endosymbionts. Because of the mode of transmission, the fitness of these symbionts is dependent on the fitness of the hosts. Therefore, these endosymbionts need to control their proliferation in order to minimize their cost for the host. The genetic bases and mechanisms of this regulation remain largely undetermined. The maternally inherited bacteria of the genus Wolbachia are the most common endosymbionts of insects, providing some of them with fitness benefits. In Drosophila melanogaster, Wolbachia wMelPop is a unique virulent variant that proliferates massively in the hosts and shortens their lifespan. The genetic bases of wMelPop virulence are unknown, and their identification would allow a better understanding of how Wolbachia levels are regulated. Here we show that amplification of a region containing eight Wolbachia genes, called Octomom, is responsible for wMelPop virulence. Using Drosophila lines selected for carrying Wolbachia with different Octomom copy numbers, we demonstrate that the number of Octomom copies determines Wolbachia titers and the strength of the lethal phenotype. Octomom amplification is unstable, and reversion of copy number to one reverts all the phenotypes. Our results provide a link between genotype and phenotype in Wolbachia and identify a genomic region regulating Wolbachia proliferation. We also prove that these bacteria can evolve rapidly. Rapid evolution by changes in gene copy number may be common in endosymbionts with a high number of mobile elements and other repeated regions. Understanding wMelPop pathogenicity and variability also allows researchers to better control and predict the outcome of releasing mosquitoes transinfected with this variant to block human vector-borne diseases. Our results show that transition from a mutualist to a pathogen may occur because of a single genomic change in the endosymbiont. This implies that there must be constant selection on endosymbionts to control their densities.
An elegant experimental evolution approach reveals that a strain of the symbiotic bacterium Wolbachia that over-replicates and shortens the life of its fruit fly host owes this property to the amplification of a small region of its genome. Read the accompanying Primer.
Author Summary
Insects frequently carry intracellular bacteria that are passed from generation to generation through their eggs. These intracellular symbionts can be beneficial or parasitic, but because of their mode of transmission, they are always dependent on the reproduction of their carriers. They therefore have to control their own growth in order to minimize deleterious effects on the host. Bacteria of the genus Wolbachia are the most common maternally transmitted intracellular bacteria in insects. Most Wolbachia variants that are naturally associated with the fruit fly Drosophila melanogaster are benign to their hosts and provide them with protection against viruses. However, wMelPop is a virulent Wolbachia variant that over-replicates massively and shortens the lifespan of its fruit fly host. Here we show that amplification of a Wolbachia genomic region containing eight genes—called Octomom—is responsible for the pathogenic effects of wMelPop. Our results provide a link between genotype and phenotype in Wolbachia and show that virulence in symbionts can be simply caused by increases in gene copy number. These results also indicate that gene copy number variation may be a common mechanism underlying rapid evolution of intracellular symbionts.
PMCID: PMC4323108  PMID: 25668031
14.  These Squatters Are Not Innocent: The Evidence of Parasitism in Sponge-Inhabiting Shrimps 
PLoS ONE  2011;6(7):e21987.
Marine sponges are frequently inhabited by a wide range of associated invertebrates, including caridean shrimps. Symbiotic shrimps are often considered to be commensals; however, in most cases, the relationship with sponge hosts remains unclear. Here we demonstrate that sponge-inhabiting shrimps are often parasites adapted to consumption of sponge tissues. First, we provide detailed examination of morphology and stomach contents of Typton carneus (Decapoda: Palaemonidae: Pontoniinae), a West Atlantic tropical shrimp living in fire sponges of the genus Tedania. Remarkable shear-like claws of T. carneus show evidence of intensive shearing, likely the result of crushing siliceous sponge spicules. Examination of stomach contents revealed that the host sponge tissue is a major source of food for T. carneus. A parasitic mode of life is also reflected in adaptations of mouth appendages, in the reproduction strategy, and in apparent sequestration of host pigments by shrimp. Consistent results were obtained also for congeneric species T. distinctus (Western Atlantic) and T. spongicola (Mediterranean). The distribution of shrimps among sponge hosts (mostly solitary individuals or heterosexual pairs) suggests that Typton shrimps actively prevent colonisation of their sponge by additional conspecifics, thus protecting their resource and reducing the damage to the hosts. We also demonstrate feeding on host tissues by sponge-associated shrimps of the genera Onycocaris, Periclimenaeus, and Thaumastocaris (Pontoniinae) and Synalpheus (Alpheidae). The parasitic mode of life appears to be widely distributed among sponge-inhabiting shrimps. However, it is possible that under some circumstances, the shrimps provide a service to the host sponge by preventing a penetration by potentially more damaging associated animals. The overall nature of interspecific shrimp-sponge relationships thus warrants further investigation.
PMCID: PMC3140983  PMID: 21814564
15.  Evolutionary Relationships among Primary Endosymbionts of the Mealybug Subfamily Phenacoccinae (Hemiptera: Coccoidea: Pseudococcidae) ▿  
Applied and Environmental Microbiology  2010;76(22):7521-7525.
Mealybugs (Coccoidea: Pseudococcidae) are sap-sucking plant parasites that harbor bacterial endosymbionts within specialized organs. Previous studies have identified two subfamilies, Pseudococcinae and Phenacoccinae, within mealybugs and determined the primary endosymbionts (P-endosymbionts) of the Pseudococcinae to be Betaproteobacteria (“Candidatus Tremblaya princeps”) containing Gammaproteobacteria secondary symbionts. Here, the P-endosymbionts of phenacoccine mealybugs are characterized based on 16S rRNA from the bacteria of 20 species of phenacoccine mealybugs and four outgroup Puto species (Coccoidea: Putoidae) and aligned to more than 100 published 16S rRNA sequences from symbiotic and free-living bacteria. Phylogenetic analyses recovered three separate lineages of bacteria from the Phenacoccinae, and these are considered to be the P-endosymbionts of their respective mealybug hosts, with those from (i) the mealybug genus Rastrococcus belonging to the Bacteroidetes, (ii) the subterranean mealybugs, tribe Rhizoecini, also within Bacteroidetes, in a clade sister to cockroach endosymbionts (Blattabacterium), and (iii) the remaining Phenacoccinae within the Betaproteobacteria, forming a well-supported sister group to “Candidatus Tremblaya princeps.” Names are proposed for two strongly supported lineages: “Candidatus Brownia rhizoecola” for P-endosymbionts of Rhizoecini and “Candidatus Tremblaya phenacola” for P-endosymbionts of Phenacoccinae excluding Rastrococcus and Rhizoecini. Rates of nucleotide substitution among lineages of Tremblaya were inferred to be significantly faster than those of free-living Betaproteobacteria. Analyses also recovered a clade of Gammaproteobacteria, sister to the P-endosymbiont lineage of aphids (“Candidatus Buchnera aphidicola”), containing the endosymbionts of Putoidae, the secondary endosymbionts of pseudococcine mealybugs, and the endosymbionts of several other insect groups.
PMCID: PMC2976180  PMID: 20851962
16.  Genomic Insights into Processes Driving the Infection of Alexandrium tamarense by the Parasitoid Amoebophrya sp. 
Eukaryotic Cell  2014;13(11):1439-1449.
The regulatory circuits during infection of dinoflagellates by their parasites are largely unknown on the molecular level. Here we provide molecular insights into these infection dynamics. Alexandrium tamarense is one of the most prominent harmful algal bloom dinoflagellates. Its pathogen, the dinoflagellate parasitoid Amoebophrya sp., has been observed to infect and control the blooms of this species. We generated a data set of transcripts from three time points (0, 6, and 96 h) during the infection of this parasite-host system. Assembly of all transcript data from the parasitoid (>900,000 reads/313 Mbp with 454/Roche next-generation sequencing [NGS]) yielded 14,455 contigs, to which we mapped the raw transcript reads of each time point of the infection cycle. We show that particular surface lectins are expressed at the beginning of the infection cycle which likely mediate the attachment to the host cell. In a later phase, signal transduction-related genes together with transmembrane transport and cytoskeleton proteins point to a high integration of processes involved in host recognition, adhesion, and invasion. At the final maturation stage, cell division- and proliferation-related genes were highly expressed, reflecting the fast cell growth and nuclear division of the parasitoid. Our molecular insights into dinoflagellate parasitoid interactions point to general mechanisms also known from other eukaryotic parasites, especially from the Alveolata. These similarities indicate the presence of fundamental processes of parasitoid infection that have remained stable throughout evolution within different phyla.
PMCID: PMC4248694  PMID: 25239978
17.  Marine dinoflagellates show induced life-history shifts to escape parasite infection in response to water-borne signals. 
Many dinoflagellate species form dormant resting cysts as a part of their life cycle, and in some freshwater species, hatching of these cysts can be delayed by the presence of water-borne signals from grazing zooplankton. Some marine dinoflagellates can form temporary cysts, which may function to resist unfavourable short-term environmental conditions. We investigated whether the marine dinoflagellate Alexandrium ostenfeldii is able to induce an increased resistance to the parasitic flagellate Parvilucifera infectans by forming temporary cysts. We performed several laboratory experiments where dinoflagellates were exposed either to direct contact with parasites or to filtered water from cultures of parasite-infected conspecifics (parasite-derived signals). Infection by P. infectans is lethal to motile A. ostenfeldii cells, but temporary cysts were more resistant to parasite infection. Furthermore, A. ostenfeldii induced a shift in life-history stage (from motile cells to temporary cysts) when exposed to parasite-derived water-borne signals. The response was relaxed within a couple of hours, indicating that A. ostenfeldii may use this behaviour as a short-term escape mechanism to avoid parasite infection. The results suggest that intraspecies chemical communication evoked by biotic interactions can be an important mechanism controlling life-history shifts in marine dinoflagellates, which may have implications for the development of toxic algal blooms.
PMCID: PMC1691648  PMID: 15209107
18.  Definitive, Intermediate, Paratenic, and Accidental Hosts of Angiostrongylus cantonensis and its Molluscan Intermediate Hosts in Hawai‘i 
Eosinophilic meningitis caused by infection with Angiostrongylus cantonensis, a parasitic nematode, is an emerging infectious disease of humans and other animals, known as angiostrongyliasis or rat lungworm disease. Symptoms range from headache and muscle spasms in mild cases to coma and even death. Many human cases have been recorded around the world, with the majority in tropical and subtropical locations. The increase in numbers of human cases and the expansion of the geographic distribution of cases make this parasite and its hosts important research foci. Definitive hosts include various rat species such as Rattus norvegicus, R. rattus, and R. exulans, and a number of land and freshwater snails and slugs have previously been identified as intermediate hosts.1 Both definitive and intermediate hosts are obligate to the life cycle of A. cantonensis. Paratenic hosts span a wide range of fauna and are not needed in the nematode's life cycle, but act as reservoirs in which different larval stages of the parasite can persist but not develop further; they include freshwater shrimp, flatworms, and frogs.2–4 Accidental hosts, including humans and other mammals, as well as birds, permit development from the third larval stage to the subadult (fifth) stage but are then dead ends for the parasite.5,6 These hosts are infected primarily through consumption of raw or undercooked intermediate or paratenic hosts, either intentionally or accidentally via contaminated produce.7
In Hawai‘i, there have been recent outbreaks with cases of infection on four of the main islands. Since there is currently a limited consensus on appropriate therapy, steps to prevent infection should be taken. The first step to facilitate this and to lay the groundwork for future management of the hosts is to identify the intermediate hosts of A. cantonensis and to determine its geographic distribution within the Hawaiian Islands. To do this over 1000 specimens of 37 terrestrial and freshwater snail and slug species (30 introduced, 7 native) from the six largest Hawaiian Islands were screened using a molecular approach.8 Total DNA was extracted from foot tissue of each specimen and was amplified using Angiostrongylus-specific primers.8 Amplicons were visualized on agarose gels to determine if specimens were positive or negative for A. cantonensis. All of the positive specimens and a random sample of all other specimens tested were also reamplified using species-specific primers.9 All positive samples were still positive with the newer primers. The parasite was present in 16 (14 alien, 2 native) of these species, from five of the six largest Hawaiian Islands. These species represent 10 phylogenetically diverse terrestrial pulmonate families and 2 more distantly related caenogastropod families (one terrestrial and one freshwater). This broad phylogenetic representation demonstrates that this parasite is not host specific, to the extent that perhaps even any snail or slug species could act as an intermediate host.
PMCID: PMC3689485
Angiostrongyliasis; Eosinophilic meningitis; Hawai‘i; Nematodes; Rat lungworm disease; Slugs; Snails
19.  Erythrocyte G Protein as a Novel Target for Malarial Chemotherapy 
PLoS Medicine  2006;3(12):e528.
Malaria remains a serious health problem because resistance develops to all currently used drugs when their parasite targets mutate. Novel antimalarial drug targets are urgently needed to reduce global morbidity and mortality. Our prior results suggested that inhibiting erythrocyte Gs signaling blocked invasion by the human malaria parasite Plasmodium falciparum.
Methods and Findings
We investigated the erythrocyte guanine nucleotide regulatory protein Gs as a novel antimalarial target. Erythrocyte “ghosts” loaded with a Gs peptide designed to block Gs interaction with its receptors, were blocked in β-adrenergic agonist-induced signaling. This finding directly demonstrates that erythrocyte Gs is functional and that propranolol, an antagonist of G protein–coupled β-adrenergic receptors, dampens Gs activity in erythrocytes. We subsequently used the ghost system to directly link inhibition of host Gs to parasite entry. In addition, we discovered that ghosts loaded with the peptide were inhibited in intracellular parasite maturation. Propranolol also inhibited blood-stage parasite growth, as did other β2-antagonists. β-blocker growth inhibition appeared to be due to delay in the terminal schizont stage. When used in combination with existing antimalarials in cell culture, propranolol reduced the 50% and 90% inhibitory concentrations for existing drugs against P. falciparum by 5- to 10-fold and was also effective in reducing drug dose in animal models of infection.
Together these data establish that, in addition to invasion, erythrocyte G protein signaling is needed for intracellular parasite proliferation and thus may present a novel antimalarial target. The results provide proof of the concept that erythrocyte Gs antagonism offers a novel strategy to fight infection and that it has potential to be used to develop combination therapies with existing antimalarials.
Erythrocyte G protein signaling is needed for intracellular malarial parasite proliferation and thus may present a novel antimalarial target.
Editors' Summary
New drugs for treatment of malaria are urgently needed, because the malaria parasite has evolved resistance against virtually all types of commonly used drugs. When a person is bitten by a malaria-infected mosquito, the parasite first infects the person's liver cells before going on to infect red blood cells, where the parasites multiply and develop into a parasite stage called a schizont. The red blood cells then burst and release more schizonts into the bloodstream; it is this “blood stage” of infection in humans that causes the symptoms of disease. Therefore efforts to develop new drugs against malaria often focus on this “blood stage” of infection. One strategy for developing new drugs is termed the “host-targeted” approach. This means that rather than trying to block processes occurring within the parasite itself, a drug can be developed which blocks processes within the person's red blood cells, and which would otherwise be needed for the parasite to complete its life cycle. It will be difficult for malaria parasites to evolve resistance to such a drug, because changes in a person's red blood cells occur much more slowly than in the parasites themselves.
Why Was This Study Done?
This research group has been studying a set of molecular processes within human red blood cells which seemed to be required for entry of malaria parasites into the cells. They wanted to get a better understanding of those processes and, specifically, to find out whether it would be possible to use particular molecules to block those processes, and by doing so to prevent malaria parasites from entering and multiplying within red blood cells. In particular, when the malaria parasites invade the red blood cell, they form membranes around the red blood cell, containing lipids and proteins “hijacked” from the red blood cell membrane. These researchers already knew that two particular proteins were hijacked in this way; the β2-adrenergic receptor (β2-AR) and heterotrimeric G protein (Gs). These two proteins act together to pass messages across the surface of the membrane to inside the cell. Small molecules could be used to block signaling through β2-AR and Gs, and therefore potentially to provide a new way of preventing malaria parasites from entering red blood cells and multiplying within them.
What Did the Researchers Do and Find?
Firstly, the researchers made red blood cell “ghosts” in which to study these molecular processes. This meant that they took fresh red blood cells from healthy human volunteers, burst them to remove half the contents and loaded them with markers and other cargoes before resealing the membranes of the cell. These resealed markers and cargoes allowed them to see what was happening inside the cells. Malaria parasites were able to invade these ghosts normally and multiply within them. When the researchers introduced a specific peptide (a molecule consisting of a short series of amino acids), they found that it blocked Gs signaling within the ghosts. This peptide also prevented malaria parasites from developing inside the ghosts. Therefore, they concluded that Gs signaling inside the red blood cell was important for the parasite life cycle. The researchers then examined a drug called propranolol which is already known to act on Gs signaling and which is commonly prescribed for high blood pressure. This drug also blocked development of malaria parasites inside the ghosts when used at a particular concentration. Finally, the researchers studied the effect of giving propranolol, along with other antimalarial drugs, to human malaria parasites in a culture dish and to mice injected with a malaria parasite that infects rodents. In these experiments, adding propranolol reduced the amount of other “parasite-targeted” drugs that were needed to effectively treat malarial infection in tissue culture and in mice.
What Do These Findings Mean?
Showing that the Gs signaling pathway is important for the malaria parasite's life cycle opens up new possibilities for drug development. Specifically, propranolol (which is already approved for treatment of high blood pressure and other conditions) might itself provide a new candidate therapy, either alone or in combination with existing drugs. These combinations would first, however, need to be tested in human clinical trials, perhaps by seeing whether they have antimalarial activity in people who have not responded to existing antimalarial drugs. Since it acts to lower blood pressure, which can already be low in some people with malaria, there are some concerns that propranolol might not be a suitable drug candidate for use, especially with existing antimalarial drugs that also reduce blood pressure. However, other molecules which block Gs signaling could be tested for activity against malaria should propranolol prove not to be an ideal drug candidate.
Additional Information.
Please access these Web sites via the online version of this summary at
The World Health Organization publishes a minisite containing links to information about all aspects of malaria worldwide, including treatment, prevention, and current programs for malaria control
Medicines for Malaria Venture is a collaboration between public and private organizations (including the pharmaceutical industry) that aims to fund and manage the development of new drugs for treatment and prevention of malaria
Wikipedia entries for drug discovery and drug development (Wikipedia is an internet encyclopedia that anyone can edit)
PMCID: PMC1716186  PMID: 17194200
20.  Global Gene Expression Analysis of the Zoonotic Parasite Trichinella spiralis Revealed Novel Genes in Host Parasite Interaction 
Trichinellosis is a typical food-borne zoonotic disease which is epidemic worldwide and the nematode Trichinella spiralis is the main pathogen. The life cycle of T. spiralis contains three developmental stages, i.e. adult worms, new borne larva (new borne L1 larva) and muscular larva (infective L1 larva). Stage-specific gene expression in the parasites has been investigated with various immunological and cDNA cloning approaches, whereas the genome-wide transcriptome and expression features of the parasite have been largely unknown. The availability of the genome sequence information of T. spiralis has made it possible to deeply dissect parasite biology in association with global gene expression and pathogenesis.
Methodology and Principal Findings
In this study, we analyzed the global gene expression patterns in the three developmental stages of T. spiralis using digital gene expression (DGE) analysis. Almost 15 million sequence tags were generated with the Illumina RNA-seq technology, producing expression data for more than 9,000 genes, covering 65% of the genome. The transcriptome analysis revealed thousands of differentially expressed genes within the genome, and importantly, a panel of genes encoding functional proteins associated with parasite invasion and immuno-modulation were identified. More than 45% of the genes were found to be transcribed from both strands, indicating the importance of RNA-mediated gene regulation in the development of the parasite. Further, based on gene ontological analysis, over 3000 genes were functionally categorized and biological pathways in the three life cycle stage were elucidated.
Conclusions and Significance
The global transcriptome of T. spiralis in three developmental stages has been profiled, and most gene activity in the genome was found to be developmentally regulated. Many metabolic and biological pathways have been revealed. The findings of the differential expression of several protein families facilitate understanding of the molecular mechanisms of parasite biology and the pathological aspects of trichinellosis.
Author Summary
Trichinellosis of human and other mammals was caused through the ingestion of the parasite Trichinella sparilis in contaminated meat. It is a typical zoonotic disease that affects more than 10 million people world-wide. Parasites of the genus Trichinella are unique intracellular pathogens. Adult Trichinella parasites directly release newborn larvae which invade striated muscle cells and causes diseases. In this study, we profiled the global transcriptome in the three developmental stages of T. spiralis. The transcriptomic analysis revealed the global gene expression patterns from newborn larval stage through muscle larval stage to adults. Thousands of genes with stage-specific transcriptional patterns were described and novel genes involving host-parasite interaction were identified. More than 45% of the protein-coding genes showed evidence of transcription from both sense and antisense strands which suggests the importance of RNA-mediated gene regulation in the parasite. This study presents a first deep analysis of the transcriptome of T. spiralis, providing insight information of the parasite biology.
PMCID: PMC3429391  PMID: 22953016
21.  Fusion between Leishmania amazonensis and Leishmania major Parasitophorous Vacuoles: Live Imaging of Coinfected Macrophages 
Protozoan parasites of the genus Leishmania alternate between flagellated, elongated extracellular promastigotes found in insect vectors, and round-shaped amastigotes enclosed in phagolysosome-like Parasitophorous Vacuoles (PVs) of infected mammalian host cells. Leishmania amazonensis amastigotes occupy large PVs which may contain many parasites; in contrast, single amastigotes of Leishmania major lodge in small, tight PVs, which undergo fission as parasites divide. To determine if PVs of these Leishmania species can fuse with each other, mouse macrophages in culture were infected with non-fluorescent L. amazonensis amastigotes and, 48 h later, superinfected with fluorescent L. major amastigotes or promastigotes. Fusion was investigated by time-lapse image acquisition of living cells and inferred from the colocalization of parasites of the two species in the same PVs. Survival, multiplication and differentiation of parasites that did or did not share the same vacuoles were also investigated. Fusion of PVs containing L. amazonensis and L. major amastigotes was not found. However, PVs containing L. major promastigotes did fuse with pre-established L. amazonensis PVs. In these chimeric vacuoles, L. major promastigotes remained motile and multiplied, but did not differentiate into amastigotes. In contrast, in doubly infected cells, within their own, unfused PVs metacyclic-enriched L. major promastigotes, but not log phase promastigotes - which were destroyed - differentiated into proliferating amastigotes. The results indicate that PVs, presumably customized by L. major amastigotes or promastigotes, differ in their ability to fuse with L. amazonensis PVs. Additionally, a species-specific PV was required for L. major destruction or differentiation – a requirement for which mechanisms remain unknown. The observations reported in this paper should be useful in further studies of the interactions between PVs to different species of Leishmania parasites, and of the mechanisms involved in the recognition and fusion of PVs.
Author Summary
Many non-viral intracellular pathogens lodge within cell vesicles known as “parasitophorous vacuoles” (PVs), which exhibit a variety of pathogen-dependent functional and compositional phenotypes. PVs of the protozoan Leishmania are similar to the digestive organelles known as phagolysosomes. We asked if, in phagocytes infected with two different Leishmania species, would the two parasites be found in the same or in separate vacuoles? Of the species chosen, Leishmania amazonensis develops within large vacuoles which shelter many parasites; in contrast, Leishmania major lodges in small PVs containing one or two parasites. In the present experiments, the species and their life-cycle stages (extracellular promastigotes, and intracellular amastigotes) were distinguished by means of fluorescent markers, and the intracellular localization of the parasites was examined in living cells. We report here that, whereas L. major amastigotes remained within their individual vacuoles, L. major promastigotes were delivered to L. amazonensis vacuoles, in which they survived and multiplied but were unable to differentiate into amastigotes. A species-specific vacuole was thus required for L. major differentiation. The model should be useful in cellular and molecular studies of the biology of these parasites and of their parasitophorous vacuoles.
PMCID: PMC2998430  PMID: 21151877
22.  Beyond Symbiosis: Cleaner Shrimp Clean Up in Culture 
PLoS ONE  2015;10(2):e0117723.
Cleaner organisms exhibit a remarkable natural behaviour where they consume ectoparasites attached to “client” organisms. While this behaviour can be utilized as a natural method of parasitic disease control (or biocontrol), it is not known whether cleaner organisms can also limit reinfection from parasite eggs and larvae within the environment. Here we show that cleaner shrimp, Lysmata amboinensis, consume eggs and larvae of a harmful monogenean parasite, Neobenedenia sp., in aquaculture. Shrimp consumed parasite eggs under diurnal (63%) and nocturnal (14%) conditions as well as infectious larvae (oncomiracidia) diurnally (26%). Furthermore, we trialled the inclusion of cleaner shrimp for preventative parasite management of ornamental fish, Pseudanthias squamipinnis, and found shrimp reduced oncomiracidia infection success of host fish by half compared to controls (held without shrimp). Fish held without cleaner shrimp exhibited pigmentation changes as a result of infection, possibly indicative of a stress response. These results provide the first empirical evidence that cleaner organisms reduce parasite loads in the environment through non-symbiotic cleaning activities. Our research findings have relevance to aquaculture and the marine ornamental trade, where cleaner shrimp could be applied for prophylaxis and control of ectoparasite infections.
PMCID: PMC4338302  PMID: 25706952
23.  A Plasmodium Phospholipase Is Involved in Disruption of the Liver Stage Parasitophorous Vacuole Membrane 
PLoS Pathogens  2015;11(3):e1004760.
The coordinated exit of intracellular pathogens from host cells is a process critical to the success and spread of an infection. While phospholipases have been shown to play important roles in bacteria host cell egress and virulence, their role in the release of intracellular eukaryotic parasites is largely unknown. We examined a malaria parasite protein with phospholipase activity and found it to be involved in hepatocyte egress. In hepatocytes, Plasmodium parasites are surrounded by a parasitophorous vacuole membrane (PVM), which must be disrupted before parasites are released into the blood. However, on a molecular basis, little is known about how the PVM is ruptured. We show that Plasmodium berghei phospholipase, PbPL, localizes to the PVM in infected hepatocytes. We provide evidence that parasites lacking PbPL undergo completely normal liver stage development until merozoites are produced but have a defect in egress from host hepatocytes. To investigate this further, we established a live-cell imaging-based assay, which enabled us to study the temporal dynamics of PVM rupture on a quantitative basis. Using this assay we could show that PbPL-deficient parasites exhibit impaired PVM rupture, resulting in delayed parasite egress. A wild-type phenotype could be re-established by gene complementation, demonstrating the specificity of the PbPL deletion phenotype. In conclusion, we have identified for the first time a Plasmodium phospholipase that is important for PVM rupture and in turn for parasite exit from the infected hepatocyte and therefore established a key role of a parasite phospholipase in egress.
Author Summary
Leaving their host cell is a crucial process for intracellular pathogens, allowing successful infection of other cells and thereby spreading of infection. Plasmodium parasites infect hepatocytes and red blood cells, and inside these cells they are contained within a vacuole like many other intracellular pathogens. Before parasites can infect other cells, the surrounding parasitophorous vacuole membrane (PVM) needs to be ruptured. However, little is known about this process on a molecular level and Plasmodium proteins mediating lysis of the PVM during parasite egress have not so far been identified. In this study, we characterize a Plasmodium phospholipase and show that it localizes to the PVM of parasites within hepatocytes. We demonstrate that parasites lacking this protein have a defect in rupture of the PVM and thereby in host cell egress. In conclusion, our study shows for the first time that a phospholipase plays a role in PVM disruption of an intracellular eukaryotic parasite.
PMCID: PMC4364735  PMID: 25786000
24.  Classification and identification of Pfiesteria and Pfiesteria-like species. 
Environmental Health Perspectives  2001;109(Suppl 5):661-665.
Dinoflagellates can be classified both botanically and zoologically; however, they are typically put in the botanical division Pyrrhophyta. As a group they appear most related to the protistan ciliates and apicomplexans at the ultrastructure level. Within the Pyrrhophyta are both unarmored and armored forms of the dominant, motile flagellated stage. Unarmored dinoflagellates do not have thecal or wall plates arranged in specific series, whereas armored species have plates that vary in thickness but are specific in number and arrangement. In armored dinoflagellates, the plate pattern and tabulation is a diagnostic character at the family, subfamily, and even genus levels. In most cases, the molecular characterization of dinoflagellates confirms the taxonomy on the basis of external morphology; this has been demonstrated for several groups. Together, both genetic and morphological criteria are becoming increasingly important for the characterization, separation, and identification of dinoflagellates species. Pfiesteria and Pfiesteria-like species are thinly armored forms with motile dinospore stages characterized by their distinct plate formulae. Pfiesteria piscicida is the best-known member of the genus; however, there is at least one other species. Other genetically and morphologically related genera, now grouped under the common names of "Lucy," "Shepherd's crook," and cryptoperidiniopsoid, are being studied and described in separate works. All these other heterotrophic dinoflagellate groups, many of which are thought to be benign, co-occur in estuarine waters where Pfiesteria has been found.
PMCID: PMC1240595  PMID: 11677173
25.  Simultaneous transcriptional profiling of Leishmania major and its murine macrophage host cell reveals insights into host-pathogen interactions 
BMC Genomics  2015;16:1108.
Parasites of the genus Leishmania are the causative agents of leishmaniasis, a group of diseases that range in manifestations from skin lesions to fatal visceral disease. The life cycle of Leishmania parasites is split between its insect vector and its mammalian host, where it resides primarily inside of macrophages. Once intracellular, Leishmania parasites must evade or deactivate the host's innate and adaptive immune responses in order to survive and replicate.
We performed transcriptome profiling using RNA-seq to simultaneously identify global changes in murine macrophage and L. major gene expression as the parasite entered and persisted within murine macrophages during the first 72 h of an infection. Differential gene expression, pathway, and gene ontology analyses enabled us to identify modulations in host and parasite responses during an infection. The most substantial and dynamic gene expression responses by both macrophage and parasite were observed during early infection. Murine genes related to both pro- and anti-inflammatory immune responses and glycolysis were substantially upregulated and genes related to lipid metabolism, biogenesis, and Fc gamma receptor-mediated phagocytosis were downregulated. Upregulated parasite genes included those aimed at mitigating the effects of an oxidative response by the host immune system while downregulated genes were related to translation, cell signaling, fatty acid biosynthesis, and flagellum structure.
The gene expression patterns identified in this work yield signatures that characterize multiple developmental stages of L. major parasites and the coordinated response of Leishmania-infected macrophages in the real-time setting of a dual biological system. This comprehensive dataset offers a clearer and more sensitive picture of the interplay between host and parasite during intracellular infection, providing additional insights into how pathogens are able to evade host defenses and modulate the biological functions of the cell in order to survive in the mammalian environment.
Electronic supplementary material
The online version of this article (doi:10.1186/s12864-015-2237-2) contains supplementary material, which is available to authorized users.
PMCID: PMC4696162  PMID: 26715493
Leishmania; Transcriptome; RNA-seq; Differentiation; Host-pathogen interactions; Macrophage; Mouse

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