No Association Between MTHFR A1298C and MTRR A66G Polymorphisms, and MS in an Australian Cohort

Multiple sclerosis (MS) is a complex neurological disease that affects the central nervous system (CNS) resulting in debilitating neuropathology. Pathogenesis is primarily defined by CNS inflammation and demyelination of nerve axons. Methionine synthase reductase (MTRR) is an enzyme that catalyzes the remethylation of homocysteine (Hcy) to methionine via cobalamin and folate dependant reactions. Cobalamin acts as an intermediate methyl carrier between methylenetetrahydrofolate reductase (MTHFR) and Hcy. MTRR plays a critical role in maintaining cobalamin in an active form and is consequently an important determinant of total plasma Hcy (pHcy) concentrations. Elevated intracellular pHcy levels have been suggested to play a role in CNS dysfunction, neurodegenerative, and cerebrovascular diseases. Our investigation entailed the genotyping of a cohort of 140 cases and matched controls for MTRR and MTHFR, by restriction length polymorphism (RFLP) techniques. Two polymorphisms: MTRR A66G and MTHFR A1298C were investigated in an Australian age and gender matched case-control study. No significant allelic frequency difference was observed between cases and controls at the α = 0.05 level (MTRR χ^2 = 0.005, P = 0.95, MTHFR χ^2 = 1.15, P = 0.28). Our preliminary findings suggest no association between the MTRR A66G and MTHFR A1298C polymorphisms and MS.

Changes in neutrophil surface receptor expression, degranulation, and respiratory burst activity following moderate and high intensity exercise

Intense exercise stimulates the systemic release of a variety of factors that alter neutrophil surface receptor expression and functional activity. These alterations may influence resistance to infection after intense exercise. The aim of this study was to examine the influence of exercise intensity on neutrophil receptor expression, degranulation (measured by plasma and intracellular myeloperoxidase concentrations), and respiratory burst activity. Ten well-trained male runners ran on a treadmill for 60 min at 60% [moderate-intensity exercise (MI)] and 85% maximal oxygen consumption [high-intensity exercise (HI)]. Blood was drawn immediately before and after exercise and at 1 h postexercise. Immediately after HI, the expression of the neutrophil receptor CD16 was significantly below preexercise values (P < 0.01), whereas MI significantly reduced CD35 expression below preexercise values (P < 0.05). One hour after exercise at both intensities, there was a significant decline in CD11b expression (P < 0.05) and a further decrease in CD16 expression compared with preexercise values (P < 0.01). CD16 expression was lower 1 h after HI than 1 h after MI (P < 0.01). Immediately after HI, intracellular myeloperoxidase concentration was less than preexercise values (P < 0.01), whereas plasma myeloperoxidase concentration was greater (P < 0.01), indicating that HI stimulated neutrophil degranulation. Plasma myeloperoxidase concentration was higher immediately after HI than after MI (P < 0.01). Neutrophil respiratory burst activity increased after HI (P < 0.01). In summary, both MI and HI reduced neutrophil surface receptor expression. Although CD16 expression was reduced to a greater extent after HI, this reduction did not impair neutrophil degranulation and respiratory burst activity.

Brick detail, Agriculture and Entomology Building, Brisbane

Detailed view of venting in brick work. Bricks used were commons, with prevalent dark blue iron staining.

The Genus Wolbachia

Numerous invertebrate species form long lasting symbioses with bacteria (Buchner, 1949; Buchner, 1965). One of the most common of these bacterial symbionts is Wolbachia pipientis, which has been estimated to infect anywhere from 15–75% of all insect species (Werren et al., 1995a; West et al., 1998; Jeyaprakash and Hoy, 2000; Werren and Windsor, 2000) as well as many species of arachnids, terrestrial crustaceans and filarial nematodes (O’Neill et al., 1997a; Bandi et al., 1998). In most arthropod associations, Wolbachia act as reproductive parasites manipulating the reproduction of their hosts to enhance their own vertical transmission. There appears to be little direct fitness cost to the infected host besides the costs arising from the reproductive manipulations. However instances have been reported where Wolbachia can be either deleterious (Min and Benzer, 1997; Bouchon et al., 1998) or beneficial (Girin and Boultreau, 1995; Stolk and Stouthamer, 1995; Wade and Chang, 1995; Vavre et al., 1999b; Dedeine et al., 2001) to their hosts. Wolbachia were first described as intracellular Rickettsia-like organisms (RLOs), infecting the gonad cells of the mosquito, Culex pipiens (Hertig and Wolbach, 1924), and were later named 'Wolbachia pipientis' (Hertig, 1936). It was not until the work of Yen and Barr (Yen and Barr, 1971; Yen and Barr, 1973) that Wolbachia were implicated in causing crossing incompatibilities between different mosquito populations (Laven, 1951; Ghelelovitch, 1952). When polymerase chain reaction (PCR) diagnostics for Wolbachia became available, it became clear that this agent was both extremely widespread and also responsible for a range of different reproductive phenotypes in the different hosts it infected (O’Neill et al., 1992; Rousset et al., 1992; Stouthamer et al., 1993). The most common of these are cytoplasmic incompatibility, inducing parthenogenesis, overriding host sex-determination, and male-killing (O’Neill et al., 1997a). As of the time of this writing, more than 450 different Wolbachia strains with unique gene sequences, different phenotypes, and infecting different hosts have been deposited in GenBank and the Wolbachia host database (http://www.wolbachia.sols. uq.edu.au).

Evidence for a global Wolbachia replacement in Drosophila melanogaster

Wolbachia are maternally inherited intracellular α-Proteobacteria found in numerous arthropod and filarial nematode species [1, 2 and 3]. They influence the biology of their hosts in many ways. In some cases, they act as obligate mutualists and are required for the normal development and reproduction of the host [4 and 5]. They are best known, however, for the various reproductive parasitism traits that they can generate in infected hosts. These include cytoplasmic incompatibility (CI) between individuals of different infection status, the parthenogenetic production of females, the selective killing of male embryos, and the feminization of genetic males [1 and 2]. Wolbachia infections of Drosophila melanogaster are extremely common in both wild populations and long-term laboratory stocks [6, 7 and 8]. Utilizing the newly completed genome sequence of Wolbachia pipientis wMel [9], we have identified a number of polymorphic markers that can be used to discriminate among five different Wolbachia variants within what was previously thought to be the single clonal infection of D. melanogaster. Analysis of long-term lab stocks together with wild-caught flies indicates that one of these variants has replaced the others globally within the last century. This is the first report of a global replacement of a Wolbachia strain in an insect host species. The sweep is at odds with current theory that cannot explain how Wolbachia can invade this host species given the observed cytoplasmic incompatibility characteristics of Wolbachia infections in D. melanogaster in the field [6].

Wolbachia replication and host cell division in Aedes albopictus

Wolbachia pipientis is an obligate intracellular endosymbiont of a range of arthropod species. The microbe is best known for its manipulations of host reproduction that include inducing cytoplasmic incompatibility, parthenogenesis, feminization, and male-killing. Like other vertically transmitted intracellular symbionts, Wolbachiarsquos replication rate must not outpace that of its host cells if it is to remain benign. The mosquito Aedes albopictus is naturally infected both singly and doubly with different strains of Wolbachia pipientis. During diapause in mosquito eggs, no host cell division is believed to occur. Further development is triggered only by subsequent exposure of the egg to water. This study uses diapause in Wolbachia-infected Aedes albopictus eggs to determine whether symbiont replication slows or stops when host cell division ceases or whether it continues at a low but constant rate. We have shown that Wolbachia densities in eggs are greatest during embryonation and then decline throughout diapause, suggesting that Wolbachia replication is dependent on host cell replication.

Molecular phylogeny of Wolbachia endosymbionts in southeast asian mosquitoes (Diptera: Culicidae) based on wsp gene sequences

Wolbachia are maternally inherited intracellular bacteria that infect a wide range of arthropods and nematodes and are associated with various reproductive abnormalities in their hosts. Insect-associated Wolbachia form a monophyletic clade in the α-Proteobacteria and recently have been separated into two supergroups (A and B) and 19 groups. Our recent polymerase chain reaction (PCR) survey using wsp specific primers indicated that various strains of Wolbachia were present in mosquitoes collected from Southeast Asia. Here, we report the phylogenetic relationship of the Wolbachia strains found in these mosquitoes using wsp gene sequences. Our phylogenetic analysis revealed eight new Wolbachia strains, five in the A supergroup and three in the B supergroup. Most of the Wolbachia strains present in Southeast Asian mosquitoes belong to the established Mors, Con, and Pip groups.

Determination of Wolbachia genome size by Pulsed-Field Gel Electrophoresis

Genome sizes of six different Wolbachia strains from insect and nematode hosts have been determined by pulsed-field gel electrophoresis of purified DNA both before and after digestion with rare-cutting restriction endonucleases. Enzymes SmaI, ApaI, AscI, and FseI cleaved the studied Wolbachia strains at a small number of sites and were used for the determination of the genome sizes of wMelPop, wMel, and wMelCS (each 1.36 Mb), wRi (1.66 Mb), wBma (1.1 Mb), and wDim (0.95 Mb). The Wolbachia genomes studied were all much smaller than the genomes of free-living bacteria such as Escherichia coli (4.7 Mb), as is typical for obligate intracellular bacteria. There was considerable genome size variability among Wolbachia strains, especially between the more parasitic A group Wolbachia infections of insects and the mutualistic C and D group infections of nematodes. The studies described here found no evidence for extrachromosomal plasmid DNA in any of the strains examined. They also indicated that the Wolbachia genome is circular.

Wolbachia infections of phlebotomine sand flies (Diptera: Psychodidae)

Old and New World phlebotomine sand fly species were screened for infection with Wolbachia, intracellular bacterial endosymbionts found in many arthropods and filarial nematodes. Of 53 samples representing 15 species, nine samples of four species were found positive for Wolbachia by polymerase chain reaction amplification using primers for the Wolbachia surface protein (wsp) gene. Five of the wsp gene fragments from four species were cloned, sequenced, and used for phylogenetic analysis. These wsp sequences were placed in three different clades within the arthropod associated Wolbachia (groups A and B), suggesting that Wolbachia has infected sand flies on more than one occasion. Two distantly related sand fly species, Lutzomyia (Psanthyromyia) shannoni (Dyar) and Lutzomyia (Nyssomyia) whitmani (Antunes & Coutinho), infected with an identical Wolbachia strain suggest a very recent horizontal transmission.

Wolbachia infection and expression of cytoplasmic incompatibility in Armigeres subalbatus (Diptera: Culicidae)

Polymerase chain reaction screening revealed that Armigeres subalbatus (Coquillett), a vector of filariasis, was infected with the intracellular bacteria Wolbachia. Laboratory crosses between infected males and uninfected females resulted in less than half the number of offspring than control crosses between uninfected individuals when young (2- to 3-d-old) males were used in the cross. However, incompatibility was lost when old (14- to 17-d-old) males were used. Field-collected females did not show detectable cytoplasmic incompatibility, and this may be because of the age at which males mate in the field. We used head pigment fluorescence levels to age field males collected from mating swarms, and found that 25-63% of swarming males were older than 13 d. Male age may be one factor influencing the observed low levels of cytoplasmic incompatibility detected in the field.

Wolbachia neither induces nor suppresses transcripts encoding antimicrobial peptides

Wolbachia are intracellular maternally inherited microorganisms that are associated with reproductive abnormalities such as cytoplasmic incompatibility (CI), feminization and parthenogenesis in the various arthropod species they infect. Surveys indicate that these bacteria infect more than 16% of all insect species as well as isopods, mites and nematodes, making Wolbachia one of the most ubiquitous parasites yet described. However, nothing is known about the interactions of this bacterium with the host's immune system. We studied the expression of inducible antimicrobial markers in the adults of two Wolbachia infected insect species, Drosophila simulans and Aedes albopictus. The lack of available immune markers in the mosquito species led us to clone part of the defensin gene from this species, which was found to be very similar to the other mosquito defensins cloned from Anopheles gambiae and Aedes aegypti. Comparisons of the expression pattern of the antibacterial markers between Wolbachia-infected and cured lines, and also between bacteria-challenged and unchallenged adults indicated that Wolbachia does not either constitutively induce or suppress the transcription of these antibacterial genes. In addition, no difference in the transcription of these genes was found between double and single Wolbachia-infected strains or between strains in which Wolbachia has different tissue tropisms.

Wolbachia: why these bacteria are important to genome research

Intracellular bacteria of the genus Wolbachia were first discovered in mosquitoes in the 1920s. Their superficial similarity to pathogenic rickettsia initially raised interest in them as potential human pathogens. However, injection experiments with mice showed that they were non-pathogenic, and they were subsequently classified as symbionts of insects. Until the 1970s, Wolbachia was considered to infect a limited number of species of mosquitoes. It is now clear that Wolbachia is an extremely common intracellular agent of invertebrates, infecting nearly all the major groups of arthropods and other terrestrial invertebrates. Its wide host range and abundance can be attributed partly to the unusual phenotypes it exerts on the host it infects. These include the induction of parthenogenesis (the production of female offspring from unmated mothers) in certain insects, the feminization of genetic male crustaceans to functional phenotypic females, and the failure of fertilization in hosts when males and females have a different infection status (cytoplasmic incompatibility). All of these phenotypes favor maternal transmission of the intracellular Wolbachia. In the last year, Wolbachia has also been shown to be a widespread symbiont of filarial nematodes. It appears that Wolbachia is needed by the adult worm for normal fertility, indicating that Wolbachia is behaving like a classic mutualist in this case. This discovery exemplifies that the extent of the host range of Wolbachia and its associated phenotypes is still far from fully understood.

Gene organization of the dnaA region of Wolbachia

The dnaA region of Wolbachia, an intracellular bacterial parasite of insects, is unique. A glnA cognate was found upstream of the dnaA gene, while neither of the two open reading frames detected downstream of dnaA has any homologue in the database. This unusual gene arrangement may reflect requirements associated with the unique ecological niche this agent occupies.

Evolution of Wolbachia pipientis transmission dynamics in insects

Wolbachia pipientis is an intracellular bacterial parasite of arthropods that enhances its transmission by manipulating host reproduction, most commonly by inducing cytoplasmic incompatibility. The discovery of isolates with modified cytoplasmic incompatibility phenotypes and others with novel virulence properties is an indication of the potential breadth of evolutionary strategies employed by Wolbachia.