Diverse host-seeking behaviors of skin-penetrating nematodes.

Skin-penetrating parasitic nematodes infect approximately one billion people worldwide and are responsible for some of the most common neglected tropical diseases. The infective larvae of skin-penetrating nematodes are thought to search for hosts using sensory cues, yet their host-seeking behavior is poorly understood. We conducted an in-depth analysis of host seeking in the skin-penetrating human parasite Strongyloides stercoralis, and compared its behavior to that of other parasitic nematodes. We found that Str. stercoralis is highly mobile relative to other parasitic nematodes and uses a cruising strategy for finding hosts. Str. stercoralis shows robust attraction to a diverse array of human skin and sweat odorants, most of which are known mosquito attractants. Olfactory preferences of Str. stercoralis vary across life stages, suggesting a mechanism by which host seeking is limited to infective larvae. A comparison of odor-driven behavior in Str. stercoralis and six other nematode species revealed that parasite olfactory preferences reflect host specificity rather than phylogeny, suggesting an important role for olfaction in host selection. Our results may enable the development of new strategies for combating harmful nematode infections.

Immune response to a Trichinella spiralis infection in house mice from lines selectively bred for high voluntary wheel running.

Four lines of mice bred for high voluntary wheel running (HR lines) have high baseline circulating corticosterone levels and increased daily energy expenditure as compared with four non-selected control (C) lines. High corticosterone may suppress immune function and competing energy demands may limit ability to mount an immune response. We hypothesized that HR mice have a reduced immune response and therefore a decreased ability to fight an infection by Trichinella spiralis, an ecologically relevant nematode common in mammals. Infections have an acute, intestinal phase while the nematode is migrating, reproducing and traveling throughout the bloodstream, followed by a chronic phase with larvae encysted in muscles. Adult males (generation 55 of the selection experiment) were sham-infected or infected by oral gavage with ~300 J1 T. spiralis larvae. During the chronic phase of infection, mice were given wheel access for 6 days, followed by 2 days of maximum aerobic performance trials. Two weeks post-infection, infected HR had significantly lower circulating immunoglobulin E levels compared with infected C mice. However, we found no statistical difference between infected HR and C mice in numbers of encysted larvae within the diaphragm. As expected, both voluntary running and maximum aerobic performance were significantly higher in HR mice and lower in infected mice, with no line type-by-infection interactions. Results complement those of previous studies suggesting decreased locomotor abilities during the chronic phase of T. spiralis infection. However, despite reduced antibody production, breeding for high voluntary wheel exercise does not appear to have a substantial negative impact on general humoral function.

Discovery of a highly synergistic anthelmintic combination that shows mutual hypersusceptibility.

The soil-transmitted helminths or nematodes (hookworms, whipworms, and Ascaris) are roundworms that infect more than 1 billion of the poorest peoples and are leading causes of morbidity worldwide. Few anthelmintics are available for treatment, and only one is commonly used in mass drug administrations. New anthelmintics are urgently needed, and crystal (Cry) proteins made by Bacillus thuringiensis are promising new candidates. Combination drug therapies are considered the ideal treatment for infectious diseases. Surprisingly, little work has been done to define the characteristics of anthelmintic combinations. Here, by means of quantitative assays with wild-type and mutants of the roundworm Caenorhabditis elegans, we establish a paradigm for studying anthelmintic combinations using Cry proteins and nicotinic acetylcholine receptor (nAChR) agonists, e.g., tribendimidine and levamisole. We find that nAChR agonists and Cry proteins, like Cry5B and Cry21A, mutually display what is known in the HIV field as hypersusceptibility--when the nematodes become resistant to either class, they become hypersensitive to the other class. Furthermore, we find that when Cry5B and nAChR agonists are combined, their activities are strongly synergistic, producing combination index values as good or better than seen with antitumor, anti-HIV, and insecticide combinations. Our study provides a powerful means by which anthelmintic combination therapies can be examined and demonstrate that the combination of nAChR agonists and Cry proteins has excellent properties and is predicted to give improved cure rates while being recalcitrant to the development of parasite resistance.

Efficacy of the mermithid nematode, Romanomermis iyengari, for the biocontrol of Anopheles gambiae, the major malaria vector in sub-Saharan Africa.

BACKGROUND: The intensive use of chemical insecticides against mosquitoes has led to the development of widespread insecticide resistance. Control of Anopheles mosquitoes in malaria endemic areas of sub-Saharan Africa has become increasingly difficult. There is an urgent need for malaria control programmes to adopt more integrated mosquito management approaches that include sustainable, nonchemical solutions. The mermithid nematode Romanomermis iyengari is one of several natural control alternatives to synthetic pesticides for mosquito suppression. This study evaluated the effectiveness of the nematode R. iyengari for control of Anopheles gambiae. METHODS: The nematode R. iyengari was mass-produced, and pre-parasitic stage (J2) were used for laboratory and field experiments. In laboratory experiments, two concentrations of pre-parasitics (5 and 10 J2 per larva) were tested against first- (L1), second- (L2) and third-instar (L3) larvae of An. gambiae. Infected larvae were observed daily to determine their mortality rate and the number of post-parasitic nematodes emerging from dead larvae. In field experiments, 3500, 4000 and 5000 J2/m2 were sprayed in separate natural Anopheles breeding sites. After treatment, the larval mosquito density in the breeding sites was assessed every 5-7 days. RESULTS: Laboratory results showed that larval An. gambiae is susceptible to nematode infection: 100% L1 larvae died within 24 hours post-treatment, and 100% of both L2 and L3 larvae died within 7 days, regardless of nematode concentrations. The average number of post-parasitic nematodes emerging per larva increased with increasing nematode concentration. In field experiments, the monthly applications of 3500 to 5000 pre-parasitic nematodes per m2 eliminated larval mosquito development in Anopheles- and mixed breeding sites. Larval mosquito density dramatically decreased five days after the first treatment in all treated sites and was maintained at a very low level during the whole experimental period. Basically, only early instar larva were detected in treated sites throughout the test period. The average number of post-parasitic nematodes emerging per larva collected in treated sites was 1.45, 2, and 5.7 respectively for sites treated with 3500, 4000, and 5000 J2/m2. CONCLUSIONS: Malaria mosquito larvae is susceptible to R. iyengari infection in West Africa. Parasitism intensity depends on tested nematode concentrations. Monthly application of 3500 J2/ m2 was enough to control effectively larval An. gambiae in wetlands and floodable locations in West Africa.

Large-scale production of the malaria vector biocontrol agent Romanomermis iyengari (Nematoda: Mermithidae) in Benin, West Africa.

Background: The mermithid nematode Romanomermis iyengari is one of several natural control alternatives to synthetic pesticides for mosquito suppression. The commonly used mass rearing procedure of R. iyengari involves the use of coarse sand as a substrate for nematode maturation and oviposition. The coarse sand technique gives excellent nematode productivity in North America. However, under West African climatic conditions, this technique generates relatively lesser amounts of infectious worms. We evaluated coconut coir fibres as a replacement for coarse sand to improve yields in large-scale production of R. iyengari in Benin, West Africa. Materials and Methods: Culex quinquefasciatus was the host for the nematodes, and mosquitoes were blood-fed on chickens. Four days after blood feeding, egg rafts were collected and transferred into trays, each containing 2 l of water. The mosquito larvae were fed with fish food. When the mosquito larvae reached the second instar, preparasites (J2) were added (3 J2/larva) to the incubation trays. Eight days after infection, post-parasitic juveniles were separated from the water containing dead mosquito larvae and other debris using sieves and needles; 2 g of them were deposited in containers with coarse sand or coconut coir fibres and water. Three hours later, the water was drained, the jars covered and stored for eight weeks, after which J2 abundance was determined, using a total of 320 containers for each substrate. The abundance of J2 preparasites was also assessed 3-5 months after storage to determine the impact of long-term storage on the J2 yield. Results: After 2 months storage, 2 g of post-parasites (~457 females and 583 males) yielded an average of 559,300±6094 J2 and 155,818±4427 J2 per container for coconut fibres and for coarse sand, respectively. During long-term storage, yields of J2 on coconut fibres substrate slowly decreased from 442,180±9322 J2 (3 months storage) to 163,632±12,416 J2 per container (5 months storage). On coarse sand substrate, the yield was relatively low and decreased from 49,812±1200 J2 at 3 months storage to 3046±229 J2 at 5 months storage. Conclusion: Under West African climatic conditions, coconut coir fibres gave significantly higher preparasitic nematode yields than the coarse sand technique.

Immunoglobulin responses of northern elephant and Pacific harbor seals naturally infected with Otostrongylus circumlitus.

Immunoglobulin (Ig) binding patterns of Pacific harbor seals (PHS, Phoca vitulina richardsi) and northern elephant seals (NES, Mirounga angustirostris) to tissues of adult Otostrongylus circumlitus were examined by immunoblotting to investigate the role of age in the unusual response of juvenile NES to infection with O. circumlitus. Serum was taken from NES between March 1997 and March 2001 and from PHS between May 1996 and August 1999. The serum of seals infected with O. circumlitus contained antibodies that bound to all nematode tissues examined. Intensity of band staining on Western blots suggested that there were higher levels of antibody recognizing the excretory-secretory (ES) glands in the serum of NES that were 1 yr and older and in the majority of PHS compared with that in 2- to 9-mo-old NES. All juvenile NES infected with O. circumlitus and a proportion of the PHS and older NES infected with O. circumlitus contained Ig specific to a 28 kDa protein band that was dominant in the female reproductive tract of the nematode. The Ig binding patterns of NES and PHS to adult Parafilaroides sp., larval Pseudoterranova sp., and larval and adult Anisakis sp. differed sufficiently from that of O. circumlitus that immunoblotting for the 28 kDa protein could be useful for diagnosis of this parasite in juvenile NES. The banding patterns suggest that O. circumlitus nematodes die and disintegrate in PHS and NES and that NES of 1 yr and older and most PHS respond differently to the ES glands than 2- to 9-mo-old NES.

Biological evidence for the world's smallest tRNAs.

Due to their function as adapters in translation, tRNA molecules share a common structural organization in all kingdoms and organelles with ribosomal protein biosynthesis. A typical tRNA has a cloverleaf-like secondary structure, consisting of acceptor stem, D-arm, anticodon arm, a variable region, and T-arm, with an average length of 73 nucleotides. In several mitochondrial genomes, however, tRNA genes encode transcripts that show a considerable deviation of this standard, having reduced D- or T-arms or even completely lack one of these elements, resulting in tRNAs as small as 66 nts. An extreme case of such truncations is found in the mitochondria of Enoplea. Here, several tRNA genes are annotated that lack both the D- and the T-arm, suggesting even shorter transcripts with a length of only 42 nts. However, direct evidence for these exceptional tRNAs, which were predicted by purely computational means, has been lacking so far. Here, we demonstrate that several of these miniaturized armless tRNAs consisting only of acceptor- and anticodon-arms are indeed transcribed and correctly processed by non-encoded CCA addition in the mermithid Romanomermis culicivorax. This is the first direct evidence for the existence and functionality of the smallest tRNAs ever identified so far. It opens new possibilities towards exploration/assessment of minimal structural motifs defining a functional tRNA and their evolution.

Ascaroside signaling is widely conserved among nematodes.

BACKGROUND: Nematodes are among the most successful animals on earth and include important human pathogens, yet little is known about nematode pheromone systems. A group of small molecules called ascarosides has been found to mediate mate finding, aggregation, and developmental diapause in Caenorhabditis elegans, but it is unknown whether ascaroside signaling exists outside of the genus Caenorhabditis. RESULTS: To determine whether ascarosides are used as signaling molecules by other nematode species, we performed a mass spectrometry-based screen for ascarosides in secretions from a variety of both free-living and parasitic (plant, insect, and animal) nematodes. We found that most of the species analyzed, including nematodes from several different clades, produce species-specific ascaroside mixtures. In some cases, ascaroside biosynthesis patterns appear to correlate with phylogeny, whereas in other cases, biosynthesis seems to correlate with lifestyle and ecological niche. We further show that ascarosides mediate distinct nematode behaviors, such as retention, avoidance, and long-range attraction, and that different nematode species respond to distinct, but overlapping, sets of ascarosides. CONCLUSIONS: Our findings indicate that nematodes utilize a conserved family of signaling molecules despite having evolved to occupy diverse ecologies. Their structural features and level of conservation are evocative of bacterial quorum sensing, where acyl homoserine lactones (AHLs) are both produced and sensed by many species of gram-negative bacteria. The identification of species-specific ascaroside profiles may enable pheromone-based approaches to interfere with reproduction and survival of parasitic nematodes, which are responsible for significant agricultural losses and many human diseases worldwide.

Lines of mice with chronically elevated baseline corticosterone levels are more susceptible to a parasitic nematode infection.

Chronically elevated circulating plasma glucocorticoid concentrations can have suppressive effects on immune function in mammals. House mice (Mus domesticus) that have been selectively bred for high voluntary wheel running exhibit chronically elevated (two-fold, on average) plasma corticosterone (CORT) levels and hence are an interesting model to study possible glucocorticoid-induced immune suppression. As an initial test of their immunocompetence, we compared the four replicate high runner (HR) lines with their four non-selected control (C) lines by subjecting them to infection by a parasitic nematode, Nippostrongylus brasiliensis. At generation 36 of the selection experiment, 10 adult males from each of the eight lines were inoculated subcutaneously with approximately 600 third-stage larval N. brasiliensis, and then sacrificed 12 days after injection. Neither spleen mass nor number of adult nematodes in the small intestine differed significantly between HR and C lines. However, the eight lines differed significantly in nematode counts, and the line means for nematode infestation were significantly positively related to baseline circulating CORT concentration measured in males from generations 34 and 39. Therefore, although selective breeding for high locomotor activity may not have resulted in a generally compromised immune response, results of this study are consistent with the hypothesis that glucocorticoids can have immunosuppressive effects.