The endosymbiont Wolbachia rebounds following antibiotic treatment.

Antibiotic treatment has emerged as a promising strategy to sterilize and kill filarial nematodes due to their dependence on their endosymbiotic bacteria, Wolbachia. Several studies have shown that novel and FDA-approved antibiotics are efficacious at depleting the filarial nematodes of their endosymbiont, thus reducing female fecundity. However, it remains unclear if antibiotics can permanently deplete Wolbachia and cause sterility for the lifespan of the adult worms. Concerns about resistance arising from mass drug administration necessitate a careful exploration of potential Wolbachia recrudescence. In the present study, we investigated the long-term effects of the FDA-approved antibiotic, rifampicin, in the Brugia pahangi jird model of infection. Initially, rifampicin treatment depleted Wolbachia in adult worms and simultaneously impaired female worm fecundity. However, during an 8-month washout period, Wolbachia titers rebounded and embryogenesis returned to normal. Genome sequence analyses of Wolbachia revealed that despite the population bottleneck and recovery, no genetic changes occurred that could account for the rebound. Clusters of densely packed Wolbachia within the worm's ovarian tissues were observed by confocal microscopy and remained in worms treated with rifampicin, suggesting that they may serve as privileged sites that allow Wolbachia to persist in worms while treated with antibiotic. To our knowledge, these clusters have not been previously described and may be the source of the Wolbachia rebound.

Pyruvate produced by Brugia spp. via glycolysis is essential for maintaining the mutualistic association between the parasite and its endosymbiont, Wolbachia.

Human parasitic nematodes are the causative agents of lymphatic filariasis (elephantiasis) and onchocerciasis (river blindness), diseases that are endemic to more than 80 countries and that consistently rank in the top ten for the highest number of years lived with disability. These filarial nematodes have evolved an obligate mutualistic association with an intracellular bacterium, Wolbachia, a symbiont that is essential for the successful development, reproduction, and survival of adult filarial worms. Elimination of the bacteria causes adult worms to die, making Wolbachia a primary target for developing new interventional tools to combat filariases. To further explore Wolbachia as a promising indirect macrofilaricidal drug target, the essential cellular processes that define the symbiotic Wolbachia-host interactions need to be identified. Genomic analyses revealed that while filarial nematodes encode all the enzymes necessary for glycolysis, Wolbachia does not encode the genes for three glycolytic enzymes: hexokinase, 6-phosphofructokinase, and pyruvate kinase. These enzymes are necessary for converting glucose into pyruvate. Wolbachia, however, has the full complement of genes required for gluconeogenesis starting with pyruvate, and for energy metabolism via the tricarboxylic acid cycle. Therefore, we hypothesized that Wolbachia might depend on host glycolysis to maintain a mutualistic association with their parasitic host. We did conditional experiments in vitro that confirmed that glycolysis and its end-product, pyruvate, sustain this symbiotic relationship. Analysis of alternative sources of pyruvate within the worm indicated that the filarial lactate dehydrogenase could also regulate the local intracellular concentration of pyruvate in proximity to Wolbachia and thus help control bacterial growth via molecular interactions with the bacteria. Lastly, we have shown that the parasite's pyruvate kinase, the enzyme that performs the last step in glycolysis, could be a potential novel anti-filarial drug target. Establishing that glycolysis is an essential component of symbiosis in filarial worms could have a broader impact on research focused on other intracellular bacteria-host interactions where the role of glycolysis in supporting intracellular survival of bacteria has been reported.

Chemotactic responses of Brugia pahangi infective third-stage larvae to tris(hydroxymethyl)aminomethane-related compounds and amino acids.

Since the exogenous compound tris(hydroxymethyl)aminomethane (Tris) showed a potent chemoattractant activity for Brugia pahangi infective third-stage larvae (L3), it was assumed that, in natural infection to a host, filarial L3 can be expected to recognize an endogenous Tris-related compound. In addition, a few amino acids have been identified as water-soluble attractants for second-stage juveniles of Meloidogyne incognita, a plant parasitic nematode. Therefore, the present study assesses the in vitro chemotactic responses of B. pahangi L3 to Tris-related compounds and amino acids using an agar-plate assay. Among Tris-related compounds, 2-amino-1,3-propanediol (APD) and 2-amino-2-methyl-1,3-propanediol (AMPD) exhibited a potent chemoattractant activity for filarial L3 at a level similar to Tris. Furthermore, arginine (Arg) was identified as a potent attractant for filarial L3 among amino acids. In addition, filarial L3 were attracted to Arg, APD and AMPD in mild alkaline conditions rather than acidic conditions. The chemoattractant activity of the three compounds for filarial L3 was observed in concentrations between 6.3 and 200 mm. This is the first report to demonstrate that Arg, APD and AMPD are potent chemoattractants for B. pahangi L3. Endogenous Arg and APD, in particular, may be involved in the regulation of the chemotactic behaviour of filarial L3 in the infection to a host. The present results will help to elucidate the mechanism of filarial skin-penetrating invasion of a host.

Effect of Brugia pahangi co-infection with Plasmodium berghei ANKA in gerbils (Meriones unguiculatus).

Malaria and lymphatic filariasis (LF) are two leading and common mosquito-borne parasitic diseases worldwide. These two diseases are co-endemic in many tropical and sub-tropical regions and are known to share vectors. The interactions between malaria and filarial parasites are poorly understood. Thus, this study aimed at establishing the interactions that occur between Brugia pahangi and Plasmodium berghei ANKA (PbA) co-infection in gerbils. Briefly, the gerbils were matched according to age, sex, and weight and grouped into filarial-only infection, PbA-only infection, co-infection, and control group. The parasitemia, survival and clinical assessment of the gerbils were monitored for a period of 30 days post Plasmodium infection. The immune responses of gerbils to both mono and co-infection were monitored. Findings show that co-infected gerbils have higher survival rate than PbA-infected gerbils. Food and water consumption were significantly reduced in both PbA-infected and co-infected gerbils, although loss of body weight, hypothermia, and anemia were less severe in co-infected gerbils. Plasmodium-infected gerbils also suffered hypoglycemia, which was not observed in co-infected gerbils. Furthermore, gerbil cytokine responses to co-infection were significantly higher than PbA-only-infected gerbils, which is being suggested as a factor for their increased longevity. Co-infected gerbils had significantly elicited interleukin-4, interferon-gamma, and tumor necrotic factor at early stage of infection than PbA-infected gerbils. Findings from this study suggest that B. pahangi infection protect against severe anemia and hypoglycemia, which are manifestations of PbA infection.

First study of topical selamectin efficacy for treating cats naturally infected with Brugia malayi and Brugia pahangi under field conditions.

Lymphatic filariae are important human and animal parasites. Infection by these parasites could lead to severe morbidity and has significant socioeconomic impacts. Topical selamectin is a semi-synthetic macrocyclic lactone that is widely used to prevent heartworm infection. Up until now, there were no studies that investigated the efficacy of selamectin in lymphatic filariae. Therefore, we aimed to study the chemotherapeutic and chemoprophylactic efficacies of selamectin use for cats in brugian filariasis-endemic areas in Southern Thailand. To assess chemotherapeutic efficacy of topical selamectin, eight Brugia malayi and six Brugia pahangi microfilaremic cats were treated with a single administration of topical selamectin. For chemoprophylactic efficacy assessment, a single application of topical selamectin was administrated to 9 healthy, uninfected cats. The cats in both groups were subjected to a monthly blood testing for microfilariae and filarial DNA for 1 year. Topical selamectin treatment in B. malayi and B. pahangi microfilaremic cats showed 100% effectivity in eradicating microfilaremia but only 78.5% effectivity in eliminating filarial DNA. In the chemoprophylactic group, selamectin demonstrated 66.7% efficacy in preventing B. malayi infection. Our findings suggest that a single administration of 6 mg/kg topical selamectin given every two months could effectively prevent B. malayi infection. Application of topical selamectin twice a year could block circulating microfilariae. Since there are no treatment guidelines currently available for lymphatic filarial infection in cats, the data obtained from this study could be used to guide the management of brugian lymphatic filarial infection in reservoir cats.

Chemoattractant activity of tris-(hydroxymethyl)aminomethane for Brugia pahangi infective third-stage larvae.

Urocanic acid (UCA) is known as a major chemoattractant for Strongyloides stercoralis infective third-stage larvae (L3). Since Brugia pahangi is a skin-penetrating parasitic nematode similar to S. stercoralis, UCA was expected to be a chemoattractant for B. pahangi L3. Thus, the chemoattractant activity of UCA for B. pahangi L3 was assessed. The chemotactic responses of B. pahangi L3 to UCA or acetic acid (CH3COOH) dissolved in amine solutions were assessed using an agar-plate assay. A test solution of 200 mm UCA dissolved in aqueous 270 mm tris(hydroxymethyl)aminomethane (Tris) significantly attracted B. pahangi L3 compared with deionized water (DW), while neither a solution of 200 mm UCA dissolved in aqueous 230 mm ammonia (NH3) nor 290 mm triethylamine (TEA) significantly attracted L3. Similarly, a test solution of 200 mm CH3COOH dissolved with 200 mm Tris significantly attracted L3, but neither a test solution of 200 mm CH3COOH plus 200 mm NH3 nor 200 mm TEA attracted L3. Furthermore, L3 were significantly attracted to 200 mm Tris alone, compared with DW, but avoided 200 mm NH3 and 200 mm TEA. Moreover, the chemoattractant activity of Tris for L3 was observed even at a low concentration of 25 mm, and it was observed in a mild alkaline condition but not in an acidic condition. The present study reveals that Tris is a potential chemoattractant for B. pahangi L3 while UCA is not. This finding will contribute to an understanding of the mechanisms of skin-penetrating infection of filarial L3.

Antifilarial and Antibiotic Activities of Methanolic Extracts of Melaleuca cajuputi Flowers.

We evaluated the activity of methanolic extracts of Melaleuca cajuputi flowers against the filarial worm Brugia pahangi and its bacterial endosymbiont Wolbachia. Anti-Wolbachia activity was measured in worms and in Aedes albopictus Aa23 cells by PCR, electron microscopy, and other biological assays. In particular, microfilarial release, worm motility, and viability were determined. M. cajuputi flower extracts were found to significantly reduce Wolbachia endosymbionts in Aa23 cells, Wolbachia surface protein, and microfilarial release, as well as the viability and motility of adult worms. Anti-Wolbachia activity was further confirmed by observation of degraded and phagocytized Wolbachia in worms treated with the flower extracts. The data provided in vitro and in vivo evidence that M. cajuputi flower extracts inhibit Wolbachia, an activity that may be exploited as an alternative strategy to treat human lymphatic filariasis.

A novel member of the let-7 microRNA family is associated with developmental transitions in filarial nematode parasites.

BACKGROUND: Filarial nematodes are important pathogens in the tropics transmitted to humans via the bite of blood sucking arthropod vectors. The molecular mechanisms underpinning survival and differentiation of these parasites following transmission are poorly understood. microRNAs are small non-coding RNA molecules that regulate target mRNAs and we set out to investigate whether they play a role in the infection event. RESULTS: microRNAs differentially expressed during the early post-infective stages of Brugia pahangi L3 were identified by microarray analysis. One of these, bpa-miR-5364, was selected for further study as it is upregulated ~12-fold at 24 hours post-infection, is specific to clade III nematodes, and is a novel member of the let-7 family, which are known to have key developmental functions in the free-living nematode Caenorhabditis elegans. Predicted mRNA targets of bpa-miR-5364 were identified using bioinformatics and comparative genomics approaches that relied on the conservation of miR-5364 binding sites in the orthologous mRNAs of other filarial nematodes. Finally, we confirmed the interaction between bpa-miR-5364 and three of its predicted targets using a dual luciferase assay. CONCLUSIONS: These data provide new insight into the molecular mechanisms underpinning the transmission of third stage larvae of filarial nematodes from vector to mammal. This study is the first to identify parasitic nematode mRNAs that are verified targets of specific microRNAs and demonstrates that post-transcriptional control of gene expression via stage-specific expression of microRNAs may be important in the success of filarial infection.

Filarial antigens mediate apoptosis of human monocytes through Toll-like receptor 4.

BACKGROUND: Apoptosis of several host cells induced by parasites/parasite products has been investigated in human filariasis to understand immune hyporesponsiveness. However, apoptosis of monocytes-one of the major antigen presenting cells in peripheral circulation, which are chronically exposed to filarial antigens in infected subjects-is yet to be understood. METHODS: Apoptosis of human monocytes with Brugia pahangi antigen (BpA) was demonstrated by scoring several apoptotic markers using flow cytometry. Ability of BpA and plasma of infected subjects to suppress lymphocyte proliferation was demonstrated by (3)H thymidine incorporation assay and carboxyfluorescein succinimidyl ester dilution assay. RESULTS: BpA induced significant apoptosis of normal human monocytes, primarily through Toll-like receptor 4 (TLR4), and suppressed phytohemagglutinin (PHA)-mediated proliferation of normal human T lymphocytes. However, monocytes of Wuchereria bancrofti-infected subjects were resistant to BpA-induced apoptosis. Plasma of infected subjects also mediated apoptosis of normal monocytes, presumably due to circulating filarial antigens, and resulted in inhibition of PHA-induced proliferation. CONCLUSION: Normal human monocytes were found to be qualitatively different from those of filariasis-infected subjects; whereas filarial antigens mediate apoptosis of normal human monocytes through TLR4, those of infected subjects were found to be resistant.

Reactive oxygen species production and Brugia pahangi survivorship in Aedes polynesiensis with artificial Wolbachia infection types.

Heterologous transinfection with the endosymbiotic bacterium Wolbachia has been shown previously to induce pathogen interference phenotypes in mosquito hosts. Here we examine an artificially infected strain of Aedes polynesiensis, the primary vector of Wuchereria bancrofti, which is the causative agent of Lymphatic filariasis (LF) throughout much of the South Pacific. Embryonic microinjection was used to transfer the wAlbB infection from Aedes albopictus into an aposymbiotic strain of Ae. polynesiensis. The resulting strain (designated "MTB") experiences a stable artificial infection with high maternal inheritance. Reciprocal crosses of MTB with naturally infected wild-type Ae. polynesiensis demonstrate strong bidirectional incompatibility. Levels of reactive oxygen species (ROS) in the MTB strain differ significantly relative to that of the wild-type, indicating an impaired ability to regulate oxidative stress. Following a challenge with Brugia pahangi, the number of filarial worms achieving the infective stage is significantly reduced in MTB as compared to the naturally infected and aposymbiotic strains. Survivorship of MTB differed significantly from that of the wild-type, with an interactive effect between survivorship and blood feeding. The results demonstrate a direct correlation between decreased ROS levels and decreased survival of adult female Aedes polynesiensis. The results are discussed in relation to the interaction of Wolbachia with ROS production and antioxidant expression, iron homeostasis and the insect immune system. We discuss the potential applied use of the MTB strain for impacting Ae. polynesiensis populations and strategies for reducing LF incidence in the South Pacific.

Nematode Hsp90: highly conserved but functionally diverse.

Nematodes are amongst the most successful and abundant organisms on the planet with approximately 30 000 species described, although the actual number of species is estimated to be one million or more. Despite sharing a relatively simple and invariant body plan, there is considerable diversity within the phylum. Nematodes have evolved to colonize most ecological niches, and can be free-living or can parasitize plants or animals to the detriment of the host organism. In this review we consider the role of heat shock protein 90 (Hsp90) in the nematode life cycle. We describe studies on Hsp90 in the free-living nematode Caenorhabditis elegans and comparative work on the parasitic species Brugia pahangi, and consider whether a dependence upon Hsp90 can be exploited for the control of parasitic species.

Intraperitoneal development of the filarial nematode Brugia malayi in the Mongolian jird (Meriones unguiculatus).

In the present study, we describe intraperitoneal development of the FR3 strain of Brugia malayi in Mongolian jirds (Meriones unguiculatus). The third molt for male worms occurred between 4 and 7 days postinfection (dpi) and between 4 and 8 dpi for females. The fourth and final molt occurred between days 21 and 29 for males and 25 and 34 for females, considerably earlier than the times reported for subcutaneous infection models using cats and jirds. The timing of the third molt coincided largely with reports for subcutaneous Brugia pahangi infections of cats and jirds, but the final molt occurred considerably later and lasted longer than those reported for subcutaneous B. pahangi models. Spermatogenesis occurred by at least 50 dpi in adult males, and insemination of females likely occurred between 50 and 60 dpi. Microfilariae were observed in the uteri and ovejectors of adult females at 65 dpi.

Integrated Histological and Molecular Analysis of Filarial Species and Associated Wolbachia Endosymbionts in Human Filariasis Cases Presenting Atypically in Thailand.

Atypical presentations of filariasis have posed diagnostic challenges due to the complexity of identifying the causative species and the difficulties in both diagnosis and treatment. In this study, we present the integrative histological and molecular analysis of seven atypical filariasis cases observed in regions of nonendemicity of Thailand. All filariasis cases were initially diagnosed based on histological findings. To confirm the causative species, molecular characterization based on both filarial mitochondrial (mt 12S rRNA and COI genes) and nuclear ITS1 markers was performed, together with the identification of associated Wolbachia bacterial endosymbionts. Among the cases studied, Brugia pahangi (N = 3), Brugia malayi (N = 1), Dirofilaria sp. "hongkongensis" (N = 2), and a suspected novel filarial species genetically related to Pelecitus copsychi (N = 1) were identified. By targeting the 16S rRNA gene, Wolbachia was also molecularly amplified in two cases of infection with Dirofilaria sp. "hongkongensis." Phylogenetic analysis further revealed that the detected Wolbachia could be classified into supergroups C and F, indicating the high genetic diversity of this endosymbiont in Dirofilaria sp. "hongkongensis." Furthermore, this study demonstrates the consistency between histological findings and species identification based on mitochondrial loci rather than on the nuclear ITS1. This suggests the utility of mitochondrial markers, particularly COI, as a highly sensitive and reliable diagnostic tool for the detection and differentiation of filarial species in clinical specimens. Precise identification of the causative species will facilitate accurate diagnosis and treatment and is also essential for the development of epidemiological and preventive strategies for filariasis.

Ocular Brugia pahangi Filariasis Complicated by Severe Macular Damage in Thailand: Case Report and Literature Review.

Extralymphatic filariasis caused by filaria of zoonotic origins has been frequently reported in Thailand over recent years. Here, we report the first case of ocular filariasis in a 7.5-year-old Thai boy who initially presented with progressive conjunctival redness and blurred vision in his right eye. A small, slender, coiled worm was found and surgically removed from the right anterior chamber. Histopathological examination illustrated predominant eosinophilic inflammation surrounding the parasite, which showed smooth and thin cuticle, prominent lateral chords, flat and broad muscle cells, one intestine, and two reproductive tubes with unsegmented ova, typically characteristic of a female adult Brugia filarial nematode. The parasite was also molecularly identified as B. pahangi, based on mitochondrial cytochrome c oxidase subunit I sequence analysis. The patient was then empirically prescribed albendazole, systemic prednisolone, and topical methylprednisolone. Unfortunately, his vision did not recover after 2 months due to severe maculopathy, most likely resulting from parasitic infestation and subsequent vitreous inflammation. To the best of our knowledge, this is the first case of ocular infestation by B. pahangi with visual complications that occurred outside a filariasis-endemic area of Thailand. Furthermore, this report provides clinical data on preceding cases of B. pahangi filariasis formally reported in southeast Asian countries, including Thailand and Malaysia, which facilitate a better understanding of the epidemiology of this sporadic zoonotic infection for effective disease elimination.

Brugia pahangi: immunization with early L3 ES alters parasite migration, and reduces microfilaremia and lymphatic lesion formation in gerbils (Meriones unguiculatus).

Previous studies have shown that intradermally (ID) injected Brugia pahangi L3 s migrate through various tissues and into the lymphatics of gerbils in a distinct pattern. Excretory/secretory products (ES) produced at the time of invasion of B. pahangi are likely to be important in this early migration phase of the parasite life cycle in their rodent host. Hence, early L3 ES was collected from 24h in vitro cultures of B. pahangi L3 larvae and used in immunization experiments to investigate the effect of immunity to early L3 ES on worm migration, survival and development of B. pahangi. Immunization of gerbils with ES in RIBI adjuvant produced antibodies to numerous ES proteins eliciting a strong humoral response to ES and indirect fluorescent antibody (IFA) assay using anti-ES serum recognized the ES proteins on the surface of B. pahangi L3 larvae. Following ES immunization, gerbils were challenged either ID or intraperitoneally (IP) with 100 L3 s of B. pahangi and euthanized at 3 or 106 days post inoculation (DPI). Immunization with early ES slowed the migration of ID inoculated L3 at 3 DPI and significantly altered the locations of adult worms at 106 DPI. Immunization did not induce protection in any treatment group. However, immunized animals had significantly fewer microfilariae per female worm suggesting the antigens in ES are important in microfilariae development or survival in the host. The number of lymphatic granulomas was also significantly reduced in ES immunized animals. It is important to note that microfilariae serve as a nidus in these granulomas. Our results shows immunization with early Brugia malayi L3 ES alters the worm migration, affects circulating microfilarial numbers and reduces lymphatic granulomas associated with B. pahangi infection in gerbils.

Successful treatment of Brugia pahangi in naturally infected cats with ivermectin.

Lymphatic filariasis is a common parasitic disease of cats in tropical regions including Thailand. The objective of this study was to determine the efficacy of ivermectin against microfilariae of Brugia pahangi in naturally infected cats. Eight cats naturally infected with B. pahangi were divided into control (untreated) and treated groups. Cats in the latter group were given ivermectin injection at 400 µg/kg weekly for 2 months. Microfilariae were counted every week until 48 weeks. Microfilaremia was significantly decreased in the treated group 4 weeks after starting the treatment and become zero at week 9 and afterwards. On the other hand, cats in the control group had high microfilaremia throughout the study. It was successful to treat and control B. pahangi infection in naturally infected cats using ivermectin.

A case report of Brugian filariasis outside an endemic area in Thailand.

A 2-year-old boy living outside the endemic area of lymphatic filariasis in Surat Thani Province, Thailand, developed a high fever. To investigate the cause of his presenting symptoms, blood was collected and microfilariae were detected and identified as Brugia malayi using thick blood smear staining. The sources of the infection were investigated. Microfilariae from two domestic cats residing in the boy's village were detected and identified as B. pahangi using a high-resolution melting real-time polymerase chain reaction analysis. The possible sources of this cryptic infection are discussed.

A high-throughput sensory assay for parasitic and free-living nematodes.

Sensory pathways first elucidated in Caenorhabditis elegans are conserved across free-living and parasitic nematodes, even though each species responds to a diverse array of compounds. Most nematode sensory assays are performed by tallying observations of worm behavior on two-dimensional planes using agarose plates. These assays have been successful in the study of volatile sensation but are poorly suited for investigation of water-soluble gustation or parasitic nematodes without a free-living stage. In contrast, gustatory assays tend to be tedious, often limited to the manipulation of a single individual at a time. We have designed a nematode sensory assay using a microfluidics device that allows for the study of gustation in a 96-well, three-dimensional environment. This device is suited for free-living worms and parasitic worms that spend their lives in an aqueous environment, and we have used it to show that ivermectin inhibits the gustatory ability of vector-borne parasitic nematodes. Insight box Nematodes are powerful model organisms for understanding the sensory biology of multicellular eukaryotes, and many parasitic species cause disease in humans. Simple sensory assays performed on agarose plates have been the bedrock for establishing the neuronal, genetic, and developmental foundations for many sensory modalities in nematodes. However, these classical assays are poorly suited for translational movement of many parasitic nematodes and the sensation of water-soluble molecules (gustation). We have designed a device for high-throughput nematode sensory assays in a gel matrix. This 'gustatory microplate' is amenable to several species and reveals novel responses by free-living and parasitic nematodes to cues and drugs.

Diversity in parasitic nematode genomes: the microRNAs of Brugia pahangi and Haemonchus contortus are largely novel.

BACKGROUND: MicroRNAs (miRNAs) play key roles in regulating post-transcriptional gene expression and are essential for development in the free-living nematode Caenorhabditis elegans and in higher organisms. Whether microRNAs are involved in regulating developmental programs of parasitic nematodes is currently unknown. Here we describe the the miRNA repertoire of two important parasitic nematodes as an essential first step in addressing this question. RESULTS: The small RNAs from larval and adult stages of two parasitic species, Brugia pahangi and Haemonchus contortus, were identified using deep-sequencing and bioinformatic approaches. Comparative analysis to known miRNA sequences reveals that the majority of these miRNAs are novel. Some novel miRNAs are abundantly expressed and display developmental regulation, suggesting important functional roles. Despite the lack of conservation in the miRNA repertoire, genomic positioning of certain miRNAs within or close to specific coding genes is remarkably conserved across diverse species, indicating selection for these associations. Endogenous small-interfering RNAs and Piwi-interacting (pi)RNAs, which regulate gene and transposon expression, were also identified. piRNAs are expressed in adult stage H. contortus, supporting a conserved role in germline maintenance in some parasitic nematodes. CONCLUSIONS: This in-depth comparative analysis of nematode miRNAs reveals the high level of divergence across species and identifies novel sequences potentially involved in development. Expression of novel miRNAs may reflect adaptations to different environments and lifestyles. Our findings provide a detailed foundation for further study of the evolution and function of miRNAs within nematodes and for identifying potential targets for intervention.

In vitro chemotactic responses of Brugia pahangi infective larvae to sodium ions.

In vitro chemotactic responses of infective third-stage larvae (L3) of Brugia pahangi to NaCl, Na2HPO4, KCl, K2HPO4, MgCl2 and CaCl2 were assessed. Compared to deionized water as a control, 200 mm NaCl and 100 mm Na2HPO4 significantly attracted L3 (P < 0.01 and P < 0.01), whereas L3 were likely to avoid 200 mm KCl and 100 mm K2HPO4 (P < 0.05 and P < 0.05). L3 showed no significant tendency to avoid or to be attracted to 200 mm CaCl2 and 200 mm MgCl2. Furthermore, NaCl exhibited a significant chemoattractant activity for L3 at a low concentration of 100 mm.