Factor Affecting Sex Ratios of a Mermithid Parasite of Mosquitoes

The ratio of male to female Reesimermis nielseni Tsai and Grundmann, a nematode parasite of mosquito larvae, increased as the number of parasites per host increased. Hosts with a single nematode produced 9% males compared with essentially 100% males in hosts with more than 7 parasites; hosts with 3 nematodes produced about equal numbers of males and females. Males of R. nielseni generally emerged before females because of the earlier death of multiple-infected mosquitoes. The species of the host mosquito influenced the sex ratio, but the size of a specific host at the time of invasion did not. Host diet also had a noticeable influence on the sex ratio of the nematode: singly infected hosts from a starved population produced 92% males compared with 13% in the normally fed group. The importance of these factors in the mass rearing of R. nielseni is discussed.     

Effects of Host Diet on Romanomermis culicivorax,a Mermithid Parasite of Mosquitoes

When larval mosquitoes (Aedes aegypti) infected with the mermithid nematode Romanomermis culicivorax were fed on a diet low in quantity or protein content or both, the number of postparasites which emerged from the hosts decreased and host mortality increased marginally. Parasitic development was prolonged and became asynchronous in nutritionally deprived hosts. Nematodes emerged from insects infected by more than one nematode before the remaining juveniles comprising such infections had completed parasitic development; this resulted in substantial reductions in postparasite nttmbers. Host development was retarded by low protein and/or reduced diets. Postparasites emerging from second and third instars were reduced in size and in the amount of stored nutriment compared to those recovered from hosts fed on a high protein diet ad libitum. A greater proportion of the rnermithids developed into males in hosts fed on reduced diets but not in hosts fed on low protein diets.     

Penetration and Development of the Mermithid Nematode Reesimermis nielseni in Eighteen Species of Mosquitoes

The susceptibility of 18 species of mosquitoes to the infective stage of the mermithid nematode Reesimermis nielseni was compared to that of Culex pipiens quinquefasciatus. Thirteen species were more susceptible, with three Anopheles species and Culiseta inornata the most susceptible. Aedes triseriatus, Culex territans, and Psorophora ferox were highly resistant. Resistance to R. nielseni appeared to be behavioral, physical, or physiological, and some host species exhibited one or more types of defense mechanisms. No noticeable differences were apparent in the degree of susceptibility of a native-and a laboratory strain of C. p. quinquqfasciatus to R. nielseni.     

Biochemical Characterization of Anopheles gambiae SRPN6, a Malaria Parasite Invasion Marker in Mosquitoes

Serine proteinase inhibitors of the serpin family are well known as negative regulators of hemostasis, thrombolysis and innate immune responses. Additionally, non-inhibitory serpins serve functions as chaperones, hormone transporters, or anti-angiogenic factors. In the African malaria mosquito, Anopheles gambiae s.s., at least three serpins (SRPNs) are implicated in the innate immune response against malaria parasites. Based on reverse genetic and cell biological analyses, AgSRPN6 limits parasite numbers and transmission and has been postulated to control melanization and complement function in mosquitoes. This study aimed to characterize AgSRPN6 biophysically and determine its biochemical mode of action. The structure model of AgSRPN6, as predicted by I-Tasser showed the protein in the native serpin fold, with three central β-sheets, nine surrounding α-helices, and a protruding reactive center loop. This structure is in agreement with biophysical and functional data obtained from recombinant (r) AgSRPN6, produced in Escherichia coli. The physical properties of purified rAgSRPN6 were investigated by means of analytical ultracentrifugation, circular dichroism, and differential scanning calorimetry tools. The recombinant protein exists predominantly as a monomer in solution, is composed of a mixture of α-helices and β-sheets, and has a mid-point unfolding temperature of 56°C. Recombinant AgSRPN6 strongly inhibited porcine pancreatic kallikrein and to a lesser extent bovine pancreatic trypsin in vitro. Furthermore, rAgSRPN6 formed inhibitory, SDS-stable, higher molecular weight complexes with prophenoloxidase-activating proteinase (PAP)1, PAP3, and Hemolymph protein (HP)6, which are required for melanization in the lepidopteran model organism, Manduca sexta. Taken together, our results strongly suggest that AgSRPN6 takes on a native serpin fold and is an inhibitor of trypsin-like serine proteinases.     

Development and Fecundity of Reesimermis nielseni,a Nematode Parasite of Mosquitoes

Maturation of the mermithid nematode Reesimermis nielseni to the adult stage began by the tenth day after emergence of the nematodes from their hosts at ambient temperatures (24-27 C). Most postparasitic males and females reached the adult stage after 50 and 70 days, respectively. The first females exhibiting egg development and oviposition were observed 25-30 days after emergence, but some oviposition was still taking place 150 days later. Reesimermis nielseni laid an average 2,480 eggs per female over an 18-day oviposition period. A majority of the mature eggs hatched within 7 h after the cultures were flooded. The preparasites are short-lived, and only a few were able to infect exposed hosts after 72 h.     

Biological Control of Mosquitoes with Mermithids

Mermithid nematodes parasitizing mosquitoes have substantial potential for vector control. Studies on the physiological ecology of Romanomermis culicivorax have defined some of the general requirements of mermithid nematodes and produced general guidelines for the experimental release of mermithids in biological control. Experimental field studies have established the biological control potential of R. culicivorax, but further development and ulilization of this parasite will require a substantial commitlnent of scientific man-years and ftmds.     

Rickettsia Species in African Anopheles Mosquitoes

Background

There is higher rate of R. felis infection among febrile patients than in healthy people in Sub-Saharan Africa, predominantly in the rainy season. Mosquitoes possess a high vectorial capacity and, because of their abundance and aggressiveness, likely play a role in rickettsial epidemiology.

Methodology/Principal Findings

Quantitative and traditional PCR assays specific for Rickettsia genes detected rickettsial DNA in 13 of 848 (1.5%) Anopheles mosquitoes collected from Côte d’Ivoire, Gabon, and Senegal. R. felis was detected in one An. gambiae molecular form S mosquito collected from Kahin, Côte d’Ivoire (1/77, 1.3%). Additionally, a new Rickettsia genotype was detected in five An. gambiae molecular form S mosquitoes collected from Côte d’Ivoire (5/77, 6.5%) and one mosquito from Libreville, Gabon (1/88, 1.1%), as well as six An. melas (6/67, 9%) mosquitoes collected from Port Gentil, Gabon. A sequence analysis of the gltA, ompB, ompA and sca4 genes indicated that this new Rickettsia sp. is closely related to R. felis. No rickettsial DNA was detected from An. funestus, An. arabiensis, or An. gambiae molecular form M mosquitoes. Additionally, a BLAST analysis of the gltA sequence from the new Rickettsia sp. resulted in a 99.71% sequence similarity to a species ({"type":"entrez-nucleotide","attrs":{"text":"JQ674485","term_id":"386870496","term_text":"JQ674485"}}JQ674485) previously detected in a blood sample of a Senegalese patient with a fever from the Bandafassi village, Kedougou region.

Conclusion

R. felis was detected for the first time in An. gambiae molecular form S, which represents the major African malaria vector. The discovery of R. felis, as well as a new Rickettsia species, in mosquitoes raises new issues with respect to African rickettsial epidemiology that need to be investigated, such as bacterial isolation, the degree of the vectorial capacity of mosquitoes, the animal reservoirs, and human pathogenicity.     

Mermithid Parasitism of Black Flies (Diptera: Simuliidae)

Mermithid nematodes are common parasites of black flies and play a significant role in the natural regulation of these medically important insects. Infection levels tend to he moderate and perennial, with epizootics rare and highly localized. Mermithid parasitism almost invariably results in the death of the black fly, and thus considerable attention has focused on the potential of these nematodes as biocontrol agents. Early instar black fly larvae appear most susceptible to infection, and integumental penetration hy mermithid preparasites is the only known mode of entry. Postparasitic nematodes typically emerge before host pupation. However, carryover of parasitism into adult simuliids is an important mechanism for local dispersal and recolonization of upstream areas. Following emergence, the mermithids molt to the adult stage. Copulation ensues, the females then laying eggs which eventually give rise to the next generation of infective preparasites. The number of described species is conservatively estimated at 35-40, with most species within the genera Mesomermis, Gastromermis, and Isomermis. The taxonomy of this group of mermithids is a challenging and little explored area. Host-specificity statements, therefore, must be made cautiously because of these systematic problems and others within the Simuliidae. In most instances, temporal and spatial factors limit the host range of these mermithids among simuliid species. Differential susceptibilities anmng larvae concurrently present within the same microhabitat probably reflect varying degrees of host attractiveness and behavioral-physiological resistance. Effects of parasitism on the host may include prevention of metamorphosis, sterility, intersexual development, and behavior modification. Evaluation of the technical feasibility of mermithid control of black flies has been stymied by the limitations of current inoculum-production technology. Continued advances in in vivo and in vitro culture methods are required to accelerate the research process.     

In Vitro Replication of Nosema algerae (Microsporidia), a Parasite of Anopheline Mosquitoes, in Human Cells above 36° C

Microsporidia form a large and ubiquitous group of obligately intracellular parasitic eukaryotes, increasingly recognized as pathogens in humans. Transmission of invertebrate microsporidia to mammals has been considered impossible because temperature seemed to be a limiting factor for development. Nosema algerae, a microsporidian of anopheline mosquitoes, was cultured in human muscle fibroblasts at temperatures of 31 degrees C and 38 degrees C. This is the first record of an invertebrate microsporidian developing in human cells at a temperature above 36 degrees C. The ultrastructure of N. algerae growing in human muscle fibroblasts is similar to that of Brachiola vesicularum, a microsporidian species previously described in the muscle of an AIDS patient.     

Isolation,Characterization and Synthesis of DNA from a Malaria Parasite*

SYNOPSIS. Methods for the isolation and purification of DNA from the intraerythrocytic stages of the avian malaria parasite Plasmodium lophurae are described. The DNA of P. lophurae was found to have a 19 mole-percent guanine plus cytosine (G+C) composition as determined by melting temperature and density measurements in CsCl gradients, whereas that of the host cell nucleus was 35 molepercent G+C. Synthesis of DNA by P. lophurae was studied by following the incorporation of P³² during the in vitro growth of plasmodia in infected cell suspension cultures. DNA synthesis was linear during the multiple nuclear divisions of a single growth cycle in highly synchronous cultures. Separation of P³² labeled parasite DNA from the DNA of the duck erythrocyte on CsCl gradients showed that only parasite DNA was synthesized.     

Diversity and Expression of MicroRNAs in the Filarial Parasite,Brugia malayi

Human filarial parasites infect an estimated 120 million people in 80 countries worldwide causing blindness and the gross disfigurement of limbs and genitals. An understanding of RNA-mediated regulatory pathways in these parasites may open new avenues for treatment. Toward this goal, small RNAs from Brugia malayi adult females, males and microfilariae were cloned for deep-sequencing. From ∼30 million sequencing reads, 145 miRNAs were identified in the B. malayi genome. Some microRNAs were validated using the p19 RNA binding protein and qPCR. B. malayi miRNAs segregate into 99 families each defined by a unique seed sequence. Sixty-one of the miRNA families are highly conserved with homologues in arthropods, vertebrates and helminths. Of those miRNAs not highly conserved, homologues of 20 B. malayi miRNA families were found in vertebrates. Nine B. malayi miRNA families appear to be filarial-specific as orthologues were not found in other organisms. The miR-2 family is the largest in B. malayi with 11 members. Analysis of the sequences shows that six members result from a recent expansion of the family. Library comparisons found that 1/3 of the B. malayi miRNAs are differentially expressed. For example, miR-71 is 5–7X more highly expressed in microfilariae than adults. Studies suggest that in C.elegans, miR-71 may enhance longevity by targeting the DAF-2 pathway. Characterization of B. malayi miRNAs and their targets will enhance our understanding of their regulatory pathways in filariads and aid in the search for novel therapeutics.     

辽宁发现库蚊黄病毒

2011年从辽宁省丹东地区蚊虫样品中分离到6株病毒,采用逆转录-聚合酶链式扩增(RT-PCR)检测方法,结果黄病毒属通用引物和库蚊黄病毒特异性引物均为阳性。6株病毒核苷酸序列经基因库(GenBank)比对后,证实6株病毒为库蚊黄病毒,为我国首次报道。将病毒NS5基因和E基因核苷酸序列与GenBank中10株库蚊黄病毒参考毒株核苷酸序列进行比较,并构建遗传进化树,结果显示本次分离的6株病毒与美国和日本的毒株亲缘关系较近。     

Isolation of Mitochondria from Orobanche, a "Total" Root Angiospermic Parasite

Mitochondria were isolated from the scapes of Orobanche cernua. On the basis of comparative studies with four media, a mannitol grinding medium was selected at a slightly alkaline pH and supplemented with EDTA, metabisulphite, BSA and soluble PVP. Various intermediates of the Krebs cycle, pyruvate, glutamate and NADH were oxidized by the isolated particles, though with different efficiencies. The rate of oxidation was steady for every intermediate tested over a 60 min period, except for NADH. In the majority of cases, the oxidation was accompanied by efficient coupling with phosphorylation. Antimycin A led to significant reduction in the oxygen uptake in the presence of succinate and complete suppression of phosphate-esterification. The amount of protein recovered in the mitochondrial fraction was comparable with the reported recoveries from a number of plant tissues. The biochemical integrity of mitochondria in the particulate fraction appeared possible from: (a) the absence of respiratory response to added cytochrome c during the oxidation of succinate, (b) insignificant oxidation of exogenously added pyruvate in the absence of sparker and (c) the ability to satisfactorily couple phosphorylation to the oxidation of a number of substrates.     

Alternative Protein Secretion in the Malaria Parasite Plasmodium falciparum

Plasmodium falciparum invades human red blood cells, residing in a parasitophorous vacuole (PV), with a parasitophorous vacuole membrane (PVM) separating the PV from the host cell cytoplasm. Here we have investigated the role of N-myristoylation and two other N-terminal motifs, a cysteine potential S-palmitoylation site and a stretch of basic residues, as the driving force for protein targeting to the parasite plasma membrane (PPM) and subsequent translocation across this membrane. Plasmodium falciparum adenylate kinase 2 (Pf AK2) contains these three motifs, and was previously proposed to be targeted beyond the parasite to the PVM, despite the absence of a signal peptide for entry into the classical secretory pathway. Biochemical and microscopy analyses of PfAK2 variants tagged with green fluorescent protein (GFP) showed that these three motifs are involved in targeting the protein to the PPM and translocation across the PPM to the PV. It was shown that the N-terminal 37 amino acids of PfAK2 alone are sufficient to target and translocate GFP across the PPM. As a control we examined the N-myristoylated P. falciparum ADP-ribosylation factor 1 (PfARF1). PfARF1 was found to co-localise with a Golgi marker. To determine whether or not the putative palmitoylation and the cluster of lysine residues from the N-terminus of PfAK2 would modulate the subcellular localization of PfARF1, a chimeric fusion protein containing the N-terminus of PfARF1 and the two additional PfAK2 motifs was analysed. This chimeric protein was targeted to the PPM, but not translocated across the membrane into the PV, indicating that other features of the N-terminus of PfAK2 also play a role in the secretion process.