Severe Infection of Nippostrongylus brasiliensis in Bandicota bengalensis Inhabiting Commensal Areas of Punjab, India: Prevalence, Risk Factor Analysis, Molecular Identification and Phylogenesis.

PURPOSE: The present study aimed to record the prevalence, risk factors, molecular identification, and phylogeny of Nippostrongylus brasiliensis found in the small intestine of the lesser bandicoot rat, Bandicota bengalensis, a wild rodent species. METHODS: A total of 100 bandicoot rats live trapped at two commensal urban locations (50 each), i.e., a fish market and railway station, in Ludhiana, Punjab State (India), from November 2020 to October 2021, were analysed for the presence of N. brasiliensis, a nematode parasite of zoonotic importance. RESULT: Overall, the small intestine of 43.00% of the rats was found severely infected with bright red coloured adult N. brasiliensis of both sexes (total of 1439 specimens). Faecal samples contained ellipsoidal and thin-shelled eggs measuring 62.25-74.70 m in length and 33.20-37.35 m in breadth. No significant (P > 0.05) effect of host age, sex, or season was observed on the rate of infection. The parasite intensity and mean abundance ranged from 27.68-38.04 and 10.52-18.26, respectively, indicating a high risk of disease transmission. Based on the morphology, the nematode parasite was identified as Nippostrongylus sp. Molecular identification was confirmed through PCR amplification of the mitochondrial cytochrome oxidase I gene, which showed a single band of approximately 355 bp. A comparison of the present isolate with the available sequences of Nippostrongylus species across the globe showed 100% nucleotide homology with N. brasiliensis sequences available in GenBank from Japan (AP017690), the USA (U57035), and New Zealand (NC033886). CONCLUSION: The study indicates that B. bengalensis inhabiting commensal urban areas is a reservoir host for N. brasiliensis, which if transmitted to humans and animals visiting the area may pose a potential health risk. The study thus suggests proper rodent population management close to human habitations to avoid the transmission of disease-causing agents.

Lentiviral transduction facilitates RNA interference in the nematode parasite Nippostrongylus brasiliensis.

Animal-parasitic nematodes have thus far been largely refractory to genetic manipulation, and methods employed to effect RNA interference (RNAi) have been ineffective or inconsistent in most cases. We describe here a new approach for genetic manipulation of Nippostrongylus brasiliensis, a widely used laboratory model of gastrointestinal nematode infection. N. brasiliensis was successfully transduced with Vesicular Stomatitis Virus glycoprotein G (VSV-G)-pseudotyped lentivirus. The virus was taken up via the nematode intestine, RNA reverse transcribed into proviral DNA, and transgene transcripts produced stably in infective larvae, which resulted in expression of the reporter protein mCherry. Improved transgene expression was achieved by incorporating the C. elegans hlh11 promoter and the tbb2 3´-UTR into viral constructs. MicroRNA-adapted short hairpin RNAs delivered in this manner were processed correctly and resulted in partial knockdown of ß-tubulin isotype-1 (tbb-iso-1) and secreted acetylcholinesterase B (ache-B). The system was further refined by lentiviral delivery of double stranded RNAs, which acted as a trigger for RNAi following processing and generation of 22G-RNAs. Virus-encoded sequences were detectable in F1 eggs and third stage larvae, demonstrating that proviral DNA entered the germline and was heritable. Lentiviral transduction thus provides a new means for genetic manipulation of parasitic nematodes, including gene silencing and expression of exogenous genes.

Dafachronic acid and temperature regulate canonical dauer pathways during Nippostrongylus brasiliensis infectious larvae activation.

BACKGROUND: While immune responses to the murine hookworm Nippostrongylus brasiliensis have been investigated, signaling pathways regulating development of infectious larvae (iL3) are not well understood. We hypothesized that N. brasiliensis would use pathways similar to those controlling dauer development in the free-living nematode Caenorhabditis elegans, which is formally known as the "dauer hypothesis." METHODS: To investigate whether dafachronic acid activates the N. brasiliensis DAF-12 homolog, we utilized an in vitro reporter assay. We then utilized RNA-Seq and subsequent bioinformatic analyses to identify N. brasiliensis dauer pathway homologs and examine regulation of these genes during iL3 activation. RESULTS: In this study, we demonstrated that dafachronic acid activates the N. brasiliensis DAF-12 homolog. We then identified N. brasiliensis homologs for members in each of the four canonical dauer pathways and examined their regulation during iL3 activation by either temperature or dafachronic acid. Similar to C. elegans, we found that transcripts encoding antagonistic insulin-like peptides were significantly downregulated during iL3 activation, and that a transcript encoding a phylogenetic homolog of DAF-9 increased during iL3 activation, suggesting that both increased insulin-like and DAF-12 nuclear hormone receptor signaling accompanies iL3 activation. In contrast to C. elegans, we observed a significant decrease in transcripts encoding the dauer transforming growth factor beta ligand DAF-7 during iL3 activation, suggesting a different role for this pathway in parasitic nematode development. CONCLUSIONS: Our data suggest that canonical dauer pathways indeed regulate iL3 activation in the hookworm N. brasiliensis and that DAF-12 may be a therapeutic target in hookworm infections.

Molecular characterization of cosmopolitan and potentially co-invasive helminths of commensal, murid rodents in Gauteng Province, South Africa.

Concurrent studies of helminth parasites of introduced and native rodent species are few and miss the opportunity to identify potential co-invasive parasite species. This study employed molecular tools to infer the phylogeny and elucidate the origin of potentially co-invasive parasites of commensal, murid rodents by assessing introduced Rattus norvegicus, Rattus rattus, Rattus tanezumi, and native Mastomys coucha in Gauteng Province, South Africa. Genotypes of Nippostrongylus brasiliensis recovered from R. norvegicus are nearly identical to those recovered from elsewhere in the world. The pinworms, Aspiculurus tetraptera, recovered from introduced R. tanezumi and R. rattus, Syphacia muris recovered from R. tanezumi, and Syphacia obvelata recovered from indigenous M. coucha have affiliations to those recovered of laboratory rodents from the USA and China. Syphacia obvelata was previously only known as a commensal endoparasite of laboratory rodents, and the S. muris genotype recovered from R. tanezumi in this study shows an affiliation to a genotype recovered from the same host species in Indonesia which is part of the native range. The study emphasizes the need for surveillance of potential co-invasive species and contributes in documenting genetic diversity of endoparasites of well-known hosts.

De novo assembly of the complex genome of Nippostrongylus brasiliensis using MinION long reads.

BACKGROUND: Eukaryotic genome assembly remains a challenge in part due to the prevalence of complex DNA repeats. This is a particularly acute problem for holocentric nematodes because of the large number of satellite DNA sequences found throughout their genomes. These have been recalcitrant to most genome sequencing methods. At the same time, many nematodes are parasites and some represent a serious threat to human health. There is a pressing need for better molecular characterization of animal and plant parasitic nematodes. The advent of long-read DNA sequencing methods offers the promise of resolving complex genomes. RESULTS: Using Nippostrongylus brasiliensis as a test case, applying improved base-calling algorithms and assembly methods, we demonstrate the feasibility of de novo genome assembly matching current community standards using only MinION long reads. In doing so, we uncovered an unexpected diversity of very long and complex DNA sequences repeated throughout the N. brasiliensis genome, including massive tandem repeats of tRNA genes. CONCLUSION: Base-calling and assembly methods have improved sufficiently that de novo genome assembly of large complex genomes is possible using only long reads. The method has the added advantage of preserving haplotypic variants and so has the potential to be used in population analyses.

Molecular Characterization of Nippostrongylus brasiliensis (Nematoda: Heligmosomatidae) from Mus musculus in India.

Mus musculus (Rodentia: Muridae) has generally been infected with a rodent hookworm Nippostrongylus brasiliensis. In this report, we present morphological and molecular identification of N. brasiliensis by light and scanning electron microscopy and PCR amplification of mitochondrial cytochrome c oxidase subunit 1 (cox1) gene and the protein sequences encoded by cox1 gene, respectively. Despite the use of N. brasiliensis in many biochemistry studies from India, their taxonomic identification was not fully understood, especially at the species level, and no molecular data is available in GenBank from India. Sequence analysis of cox1 gene in this study revealed that the present specimen showed close identity with the same species available in GenBank, confirming that the species is N. brasiliensis. This study represents the first record of molecular identification of N. brasiliensis from India and the protein structure to better understand the comparative phylogenetic characteristics.

Mucosal trapping and degradation of Nippostrongylus brasiliensis occurs in the absence of STAT6.

Hookworms represent a major infectious burden globally, especially in developing countries. The murine hookworm Nippostrongylus brasiliensis is normally cleared in a manner dependent on IL-13, IL4-R and STAT6 signalling. Here we have used STAT6-deficient animals to model a non-resistant population and describe 2 novel STAT6-independent processes for the clearance of N. brasiliensis. During primary infection STAT6-/- animals are able to clear gut-dwelling N. brasiliensis by a mechanism involving the trapping and degradation of worms in the gut mucosa. Here, a previously undescribed STAT6-independent up-regulation of Relm-ß was observed which correlated with the mucosal trapping and degradation of worms. Previous studies have indicated that during secondary infection STAT6 deficient animals fail to expel adult worms and remain susceptible to re-infection and long-term colonization of the gut. We report here that an initial partially protective response occurs early upon re-infection in the absence of STAT6, and that a late-phase protective secondary response arises in the gut of STAT6-deficient mice leading to the clearance of the majority of N. brasiliensis, through their trapping and death in the mucosal layer of the lower region of the small intestine. These findings show that there are a number of redundant effector pathways which act to reduce worm burden in the gut which can be activated by mechanisms that do not work through the dominant STAT6 signalling pathway and may be useful as targets for future vaccination strategies against resistant hookworm strains.

Hsp12.6 expression is inducible by host immunity in adult worms of the parasitic nematode Nippostrongylus brasiliensis.

Heat shock proteins (Hsp) are a family of stress-inducible molecular chaperones that play multiple roles in a wide variety of animals. However, the roles of Hsps in parasitic nematodes remain largely unknown. To elucidate the roles of Hsps in the survival and longevity of nematodes, particularly at the 2 most critical stages in their lifecycle, the infective-L3 stage and adult stage, which is subjected to host-derived immunological pressure, we examined the temporal gene transcription patterns of Hsp12.6, Hsp20, Hsp70, and Hsp90 throughout the developmental course of the nematode Nippostrongylus brasiliensis by reverse transcriptase real-time PCR. Nb-Hsp70 and Nb-Hsp90 expression were observed throughout the nematode's lifecycle, while the expression of Nb-Hsp20 was restricted to adults. Interestingly, Nb-Hsp12.6 showed a biphasic temporal expression pattern; i.e., it was expressed in infective-L3 larvae and in adults during worm expulsion from immunocompetent rats. However, the activation of Nb-Hsp12.6 in adult worms was aborted when they infected permissive athymic-rnu/rnu rats and was only marginal when they infected mast-cell-deficient Ws/Ws rats, which exhibited a low response of rat mast cell protease (RMCP) II and resistin-like molecule (Relm)-ß expression compared to those observed in immunocompetent rats. Moreover, the activation of Nb-Hsp12.6 was reversed when adult worms were transplanted into the naive rat intestine. These features of Nb-Hsp12.6, the expression of which is not only stage-specific in infective-L3, but is also inducible by mucosal immunity in adults, have implications for the survival strategies of parasitic nematodes in deleterious environmental conditions both outside and inside the host.

Activation of Nippostrongylus brasiliensis infective larvae is regulated by a pathway distinct from the hookworm Ancylostoma caninum.

Developmentally arrested infective larvae of strongylid nematodes are activated to resume growth by host-derived cues encountered during invasion of the mammalian host. Exposure of Nippostrongylus brasiliensis infective larvae to elevated temperature (37°C) is sufficient to activate signalling pathways which result in resumption of feeding and protein secretion. This occurs independently of exposure to serum or glutathione, in contrast to the hookworm Ancylostoma caninum, and is not initiated by chemical exsheathment. No qualitative differences in protein secretion were induced by host serum as visualised by two-dimensional SDS-PAGE, although exposure of larvae to an aqueous extract of rat skin did stimulate secretion of a small pre-synthesised bolus of proteins. Infective larvae began feeding after a lag period of 3-4 h at 37°C, reaching a maximum of 90% of the population feeding by 48 h. Neither a membrane permeant analogue of cyclic GMP nor muscarinic acetylcholine receptor agonists stimulated feeding at 20°C, and high concentrations of both compounds inhibited temperature-induced activation. LY294002, an inhibitor of phosphatidylinositol 3-kinase, Akt inhibitor IV, an inhibitor of Akt protein kinase, and ketoconazole, an inhibitor of cytochrome P450, all blocked resumption of feeding and protein secretion at 37°C. Serotonin increased the rate of feeding assessed by uptake of radiolabelled BSA, but could not initiate feeding independently of elevated temperature. Collectively, the data suggest that the early signalling events for larval activation in N. brasiliensis differ substantially from A. caninum, but that they may converge at pathways downstream of phosphatidylinositol 3-kinase involving steroid hormone synthesis.

C-type lectins from the nematode parasites Heligmosomoides polygyrus and Nippostrongylus brasiliensis.

The C-type lectin superfamily is highly represented in all metazoan phyla so far studied. Many members of this superfamily are important in innate immune defences against infection, while others serve key developmental and structural roles. Within the superfamily, many proteins contain multiple canonical carbohydrate-recognition domains (CRDs), together with additional non-lectin domains. In this report, we have studied two gastrointestinal nematode parasites which are widely used in experimental rodent systems, Heligmosomoides polygyrus and Nippostrongylus brasiliensis. From cDNA libraries, we have isolated 3 new C-type lectins from these species; all are single-CRD proteins with short additional N-terminal domains. The predicted Hp-CTL-1 protein contains 156 aa, Nb-CTL-1 191 aa and Nb-CTL-2 183 aa; all encode predicted signal peptides, as well as key conserved sequence motifs characteristic of the CTL superfamily. These lectins are most similar to C. elegans CLEC-48, 49 and 50, as well as to the lectin domains of mammalian immune system proteins CD23 and CD206. RT-PCR showed that these H. polygyrus and N. brasiliensis genes are primarily expressed in the gut-dwelling adult stages, although Nb-CTL-2 transcripts are also prominent in the free-living infective larval (L3) stage. Polyclonal antibodies raised to Hp-CTL-1 and Nb-CTL-1 reacted to both proteins by ELISA, and in Western blot analysis recognised a 15-kDa band in secreted proteins of adult N. brasiliensis (NES) and a 19-kDa band in H. polygyrus ES (HES). Anti-CTL-1 antibody also bound strongly to the cuticle of adult H. polygyrus. Hence, live parasites release C-type lectins homologous to some key receptors of the mammalian host immune system, raising the possibility that these products interfere in some manner with immunological recognition or effector function.

Impaired resistance in early secondary Nippostrongylus brasiliensis infections in mice with defective eosinophilopoeisis.

Eosinophils are an important feature of immune responses to infections with many of the tissue-invasive helminth parasites. The cytokine IL-5 and a high-affinity double GATA-binding site within the GATA-1 promoter are critical for eosinophilopoiesis. In this study, we believe we demonstrate for the first time that defects in eosinophilopoiesis are associated with impaired resistance to Nippostrongylus brasiliensis. Primary and secondary infections were established in wildtype (WT), IL-5(-/-) and DeltadblGATA mice. Resistance to secondary infections was impaired in IL-5(-/-) and DeltadblGATA mice, with significantly more larvae able to reach the lungs 2 days p.i. Pulmonary inflammation was minimal in all strains in the first 2 days of both primary and secondary infections, suggesting that eosinophil-dependent resistance occurred before larvae reached this site. Intestinal worm burdens and/or parasite egg production in primary infections were greater in animals with defective eosinophilopoiesis. While larvae did reach the gut by day 3 of secondary infections of WT and IL-5(-/-) mice, worms were expelled by day 7, even in the complete absence of eosinophils in tissues of the small intestine. This and our previous studies indicate that N. brasiliensis are likely to be exquisitely sensitive to attack by eosinophils soon after entry into the skin. Eosinophils in the gut may make a modest contribution to resistance on first exposure to the parasite, but are not required for expulsion in either primary or secondary infections. In order to mount an effective immune response it may be vital for the host to identify and attack the parasite before it implements immune evasion strategies and migrates to other anatomical sites. These observations may be of particular significance for the development of successful vaccines against hookworms and other nematodes.

Nippostrongylus brasiliensis: identification of intelectin-1 and -2 as Stat6-dependent genes expressed in lung and intestine during infection.

Elimination of the helminth parasite Nippostrongylus brasiliensis from infected mice is mediated by IL-4 or IL-13 and dependent on the IL-4Ralpha chain and the transcription factor Stat6 in non-hematopoietic cells. However, it is not clear which Stat6-dependent effector molecules mediate worm expulsion. We identified intelectin-1 and -2 as Stat6-dependent genes that are induced during infection. Intelectins can bind galactofuranose, a sugar present only in microorganisms and might therefore serve as microbial pattern element. To analyze whether constitutive expression of intelectin-1 or -2 leads to accelerated pathogen clearance, transgenic mice were generated which express high levels of these genes selectively in the lung. Infection with N. brasiliensis or Mycobacterium tuberculosis did not result in accelerated pathogen clearance in transgenic as compared to wild-type mice. Further, no significant modulation of the immune response in lung or lymph nodes was observed. Thus, under these conditions, intelectins did not enhance pathogen clearance.

Identification and characterization of a novel antigen from the nematode Nippostrongylus brasiliensis recognized by specific IgE.

Identification and characterization of IgE-inducing antigens are important for elucidating the mechanisms involved in IgE-mediated immune responses in allergic diseases and parasite infections. While many allergens have been characterized, little is known about parasite antigens inducing specific IgE following infection. In order to identify antigens from the nematode Nippostrongylus brasiliensis, we generated an IgE-producing B cell hybridoma from N. brasiliensis-infected C57BL/6 mice and constructed a cDNA phage display library from N. brasiliensis. We successfully cloned and expressed an N. brasiliensis antigen (Nb-Ag1) that showed specific binding to anti-N. brasiliensis IgE. Nb-Ag1 localized to the pharynx of adult N. brasiliensis, suggesting that Nb-Ag1 is a potential pharyngeal gland antigen. Nb-Ag1-specific IgE could be detected in the serum of N. brasiliensis-infected mice, but only for a short time and only following a challenge infection. In contrast, local administration of Nb-Ag1 during primary, secondary and tertiary infections induced Nb-Ag1-specific IgE-mediated active cutaneous anaphylaxis. Therefore, amongst the high amounts of polyclonal total IgE, low levels of parasite-specific IgE responses are induced during primary helminth infections. Here, we show that even such low levels of parasite-specific IgE are sufficient to prime mast cells in vivo and mediate degranulation.

[Separation and purification of recombinant Nippostrongylus brasiliensis acetylcholinesterase from culture medium of genetic engineering Pichia pastoris].

To develop a simple, fast and highly efficient method for the separation and purification of recombinant Nippostrongylus brasiliensis acetylcholinesterase (NbAChE) from culture medium of genetic engineering Pichia pastoris, Q-Sepharose Fast Flow chromatographic medium was used. The chromatographic column was 20 cm x 3.5 cm i. d. The elution buffer A was 20 mmol/L NaH2PO4-Na2HPO4 (pH 8) and buffer B was 1 mol/L NaCl + 20 mmol/L NaH2PO4- Na2HPO4(pH 8). The elution gradient was nonlinear. It was firstly eluted with 10% B for 300 min, then with 30% B for 300 min, finally with 100% B for 300 min. The flow rate of mobile phase was 6 mL/min. The obtained recombinant NbAChE was proved to be homogeneous on sodium-dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and its relative molecular mass was estimated to be approximately 66 000, which was consistent with that reported in literature. The total activity recovery of this purification method was 52.6% and the purification factor was 3.87. The final specific activity of recombinant NbAChE was 2 837 U/mg. This chromatographic process is simple and highly efficient. It can be used to separate and purify recombinant NbAChE from culture medium of Pichia pastoris harboring NbAChE gene.

Insecticide detection through protein engineering of Nippostrongylus brasiliensis acetylcholinesterase B.

The sensitivity of acetylcholinesterase (AChE) biosensors for insecticide detection could be increased substantially by engineering AChE B of Nippostrongylus brasiliensis. The introduction of 10 single and 4 double mutations into the AChE peptide chain led to an increase in sensitivity to 10 of the 11 insecticides tested. The combination of three mutants with the wild-type enzyme in a multienzyme biosensor array enabled the detection of 11 out of the 14 most important organophosphates and carbamates at concentrations below 10 microg/kg, the maximum residue limit of infant food. The detection limit for pirimiphos methyl could be reduced from 10 microg/L to a value as low as 1 ng/L (3.5 x 10(-)(12) mol/L). The newly created biosensors exhibited an extraordinary high storage stability. There was no loss of sensitivity of N. brasiliensis AChE B, immobilized on screen-printed, disposable electrodes, even after 17-month storage at room temperature.

Signal sequence analysis of expressed sequence tags from the nematode Nippostrongylus brasiliensis and the evolution of secreted proteins in parasites.

BACKGROUND: Parasitism is a highly successful mode of life and one that requires suites of gene adaptations to permit survival within a potentially hostile host. Among such adaptations is the secretion of proteins capable of modifying or manipulating the host environment. Nippostrongylus brasiliensis is a well-studied model nematode parasite of rodents, which secretes products known to modulate host immunity. RESULTS: Taking a genomic approach to characterize potential secreted products, we analyzed expressed sequence tag (EST) sequences for putative amino-terminal secretory signals. We sequenced ESTs from a cDNA library constructed by oligo-capping to select full-length cDNAs, as well as from conventional cDNA libraries. SignalP analysis was applied to predicted open reading frames, to identify potential signal peptides and anchors. Among 1,234 ESTs, 197 (~16%) contain predicted 5' signal sequences, with 176 classified as conventional signal peptides and 21 as signal anchors. ESTs cluster into 742 distinct genes, of which 135 (18%) bear predicted signal-sequence coding regions. Comparisons of clusters with homologs from Caenorhabditis elegans and more distantly related organisms reveal that the majority (65% at P < e-10) of signal peptide-bearing sequences from N. brasiliensis show no similarity to previously reported genes, and less than 10% align to conserved genes recorded outside the phylum Nematoda. Of all novel sequences identified, 32% contained predicted signal peptides, whereas this was the case for only 3.4% of conserved genes with sequence homologies beyond the Nematoda. CONCLUSIONS: These results indicate that secreted proteins may be undergoing accelerated evolution, either because of relaxed functional constraints, or in response to stronger selective pressure from host immunity.

A long insertion reverts the functional effect of a substitution in acetylcholinesterase.

Proteins are thought to undertake single substitutions, deletions and insertions to explore the fitness landscape. Nevertheless, the ways in which these different kind of mutations act together to alter a protein phenotype remain poorly described. We introduced incrementally the single substitution W290A and a 26 amino acid long insertion at the 297 location in the Nippostrongylus brasiliensis acetylcholinesterase B sequence and analysed in vitro the induced changes in the hydrolysis rate of three hemi-substrates: pirimicarb, paraoxon methyl and omethoate. The substitution decreased the hydrolysis rate of the three hemi-substrates. The insertion did not influence this kinetic alteration induced by the substitution for the former hemi-substrate, but reverted it for the two others. These results show that two different kinds of mutations can interact together to influence the direction of a protein's adaptative walk on the fitness landscape.

Lactic dehydrogenase virus infection reduces the expulsion of the nematode Nippostrongylus brasiliensis.

The interleukin (IL)-13-mediated goblet cell response is the major host effector system involved in the expulsion of Nippostrongylus brasiliensis. Lactic dehydrogenase virus (LDV) induced higher levels of N. brasiliensis egg production compared with controls, but the effect of LDV infection on worm expulsion of, and goblet cell and IL-13 responses to, N. brasiliensis have not been studied. In this study, the effects of LDV infection on these host responses against N. brasiliensis were examined. Mice with chronic LDV infection showed significantly lower worm expulsion rates than non-LDV-infected mice after N. brasiliensis infection, and there were no significant differences in the ratio of female versus male adult worms between control and LDV-infected mice. The number of goblet cells in LDV-infected mice was significantly lower than that in controls. In addition, the levels of IL-13 gene expression in lymph nodes were significantly lower in LDV-infected mice compared with controls. These results suggest that LDV infection reduces the protective immune responses against N. brasiliensis infection by the suppression of IL-13 production.

A distinct family of acetylcholinesterases is secreted by Nippostrongylus brasiliensis.

A third variant of acetylcholinesterase (AChE A) secreted by the parasitic nematode Nippostrongylus brasiliensis has been isolated which shows 63-64% identity to AChE B and AChE C, with a truncated carboxyl terminus and a short internal insertion relative to AChEs from other species. Three of the fourteen aromatic residues which line the active site gorge in Torpedo AChE are substituted by non-aromatic residues (Y70T, W279D and F288M). All three enzymes have 8 cysteine residues in conserved positions, including 6 which have been implicated in disulphide bonds in other AChEs. Phylogenetic analysis suggests that these enzymes form a distinct group which evolved after speciation and are most closely related to ACE-2 of Caenorhabditis elegans. Recombinant AChE A secreted by Pichia pastoris was monomeric and hydrophilic, with a substrate preference for acetylthiocholine and negligible activity against butyrylthiocholine. A model structure of AChE A built from the coordinates of the Torpedo californica AChE suggests that W345 (F331 in Torpedo) limits the docking of butyrylcholine. This model is consistent with mutational analysis of the nematode enzymes. Expression of AChE A is regulated at the transcriptional level independently of the other 2 secreted variants, with maximal expression by fourth stage larvae and young adult worms. These enzymes thus appear to represent an unusual family of AChEs with conserved structural features which operate outside the normal boundaries of known functions in regulation of endogenous neurotransmitter activity.