The Trypanosoma brucei AIR9-like protein is cytoskeleton-associated and is required for nucleus positioning and accurate cleavage furrow placement.

AIR9 is a cytoskeleton-associated protein in Arabidopsis thaliana with roles in cytokinesis and cross wall maturation, and reported homologues in land plants and excavate protists, including trypanosomatids. We show that the Trypanosoma brucei AIR9-like protein, TbAIR9, is also cytoskeleton-associated and colocalizes with the subpellicular microtubules. We find it to be expressed in all life cycle stages and show that it is essential for normal proliferation of trypanosomes in vitro. Depletion of TbAIR9 from procyclic trypanosomes resulted in increased cell length due to increased microtubule extension at the cell posterior. Additionally, the nucleus was re-positioned to a location posterior to the kinetoplast, leading to defects in cytokinesis and the generation of aberrant progeny. In contrast, in bloodstream trypanosomes, depletion of TbAIR9 had little effect on nucleus positioning, but resulted in aberrant cleavage furrow placement and the generation of non-equivalent daughter cells following cytokinesis. Our data provide insight into the control of nucleus positioning in this important pathogen and emphasize differences in the cytoskeleton and cell cycle control between two life cycle stages of the T. brucei parasite.

Ecotin-like serine peptidase inhibitor ISP1 of Leishmania major plays a role in flagellar pocket dynamics and promastigote differentiation.

Leishmania ISPs are ecotin-like natural peptide inhibitors of trypsin-family serine peptidases, enzymes that are absent from the Leishmania genome. This led to the proposal that ISPs inhibit host serine peptidases and we have recently shown that ISP2 inhibits neutrophil elastase, thereby enhancing parasite survival in murine macrophages. In this study we show that ISP1 has less serine peptidase inhibitory activity than ISP2, and in promastigotes both are generally located in the cytosol and along the flagellum. However, in haptomonad promastigotes there is a prominent accumulation of ISP1 and ISP2 in the hemidesmosome and for ISP2 on the cell surface. An L. major mutant deficient in all three ISP genes (Δisp1/2/3) was generated and compared with Δisp2/3 mutants to elucidate the physiological role of ISP1. In in vitro cultures, the Δisp1/2/3 mutant contained more haptomonad, nectomonad and leptomonad promastigotes with elongated flagella and reduced motility compared with Δisp2/3 populations, moreover it was characterized by very high levels of release of exosome-like vesicles from the flagellar pocket. These data suggest that ISP1 has a primary role in flagellar homeostasis, disruption of which affects differentiation and flagellar pocket dynamics.

Analysis of intracellular enzyme activity by surface enhanced Raman scattering.

Dysfunctional intracellular enzymatic activity is believed to be an underlying cause of a myriad of diseases. We present the first use of surface enhanced Raman scattering (SERS) as a detection technique capable of reporting intracellular activity of a specific enzyme. Careful choice of reagents allowed the preparation of high resolution cellular activity maps highlighting the specific conversion of the commonly used ELISA reagent 5-bromo-4-chloro-3-indolyl ß-D-galactopyranoside (X-Gal), by wild type ß-galactosidase enzymes. Further, through co-addition of X-Gal substrate and inhibitors we were able to demonstrate that intracellular substrate conversion occurred predominantly through an enzymatically specific pathway. The data presented therefore supports the application of SERS probes as sensitive, specific sensors of biochemical activity and demonstrates the use of SERS probes for the first time as beacons capable of high resolution subcellular localisation of native enzymes.

Trypanosoma brucei metacaspase 4 is a pseudopeptidase and a virulence factor.

Metacaspases are caspase family cysteine peptidases found in plants, fungi, and protozoa but not mammals. Trypanosoma brucei is unusual in having five metacaspases (MCA1-MCA5), of which MCA1 and MCA4 have active site substitutions, making them possible non-enzymatic homologues. Here we demonstrate that recombinant MCA4 lacks detectable peptidase activity despite maintaining a functional peptidase structure. MCA4 is expressed primarily in the bloodstream form of the parasite and associates with the flagellar membrane via dual myristoylation/palmitoylation. Loss of function phenotyping revealed critical roles for MCA4; rapid depletion by RNAi caused lethal disruption to the parasite's cell cycle, yet the generation of MCA4 null mutant parasites (Δmca4) was possible. Δmca4 had normal growth in axenic culture but markedly reduced virulence in mice. Further analysis revealed that MCA4 is released from the parasite and is specifically processed by MCA3, the only metacaspase that is both palmitoylated and enzymatically active. Accordingly, we have identified that the multiple metacaspases in T. brucei form a membrane-associated proteolytic cascade to generate a pseudopeptidase virulence factor.

The use of nano polymeric self-assemblies based on novel amphiphilic polymers for oral hydrophobic drug delivery.

PURPOSE: To investigate the use of nano self-assemblies formed by polyallylamine (PAA) modified with 5 or 10% mole fluorenylmethoxy carbonyl (Fmoc(5)/(10)), dimethylamino-1-naphthalenesulfonyl (Dansyl(5)/(10)) and 5% mole cholesteryl group (Ch(5)) for oral hydrophobic drug delivery. METHODS: Propofol, griseofulvin and prednisolone were loaded into amphiphilic PAAs. Particle size and morphology of drug-loaded self-assemblies were determined using photon correlation spectroscopy and transmission electron microscopy. Solubilising capacity, in vitro drug release and formulation stability were analysed by HPLC, and in vitro biocompatibility studies (haemolysis and cytotoxicity) were carried out on bovine erythrocytes and Caco-2 cells, respectively. Dansyl(10) and Ch(5) griseofulvin formulations were administered intra-gastrically to rats, and drug plasma levels were analysed by HPLC. RESULTS: Drug-encapsulated self-assemblies typically have hydrodynamic size of 300-400 nm. Dansyl(10) exhibited universal drug solubiliser property and had significantly improved prednisolone, griseofulvin and propofol solubility by 145, 557 and 224-fold, respectively. Fmoc polymers resulted in modest drug solubility improvement. These polymers were non-haemolytic, did not enhance cytotoxicity compared to unmodified PAA, and demonstrated significant increase in griseofulvin plasma concentration compared to griseofulvin in water after oral administration. CONCLUSIONS: Ch(5) and Dansyl(10) showed promising potential as nano-carriers for oral hydrophobic drug delivery.

Enhanced gene expression in tumors after intravenous administration of arginine-, lysine- and leucine-bearing polyethylenimine polyplex.

The potential of gene therapy to treat cancer is currently limited by the low expression of therapeutic genes in the tumors. Because amino acids are known to have excellent properties in cell penetration and gene expression regulation, we investigated if the conjugation of arginine (Arg), lysine (Lys) and leucine (Leu) onto the surface of the gene delivery system polyethylenimine (PEI) could lead to an improved gene expression in tumors. The intravenous administration of Arg-, Lys- and Leu-bearing PEI polyplexes led to a significant increase of gene expression in the tumor, with a ß-galactosidase expression amount at least threefold higher than that obtained after treatment with unmodified PEI polyplex. The three amino acid-bearing PEI polyplexes led to similar levels of gene expression in the tumor. The treatments were well tolerated by the mice. Arg-, Lys- and Leu-bearing PEI polyplexes are therefore highly promising gene delivery systems for cancer therapy.

Campylobacter jejuni 81-176 forms distinct microcolonies on in vitro-infected human small intestinal tissue prior to biofilm formation.

Human small and large intestinal tissue was used to study the interaction of Campylobacter jejuni with its target tissue. The strain used for the study was 81-176 (+pVir). Tissue was processed for scanning and transmission electron microscopy, and by immunohistochemistry for light microscopy. Organisms adhered to the apical surface of ileal tissues at all time points in large numbers, in areas where mucus was present and in distinct groups. Microcolony formation was evident at 1-2 h, with bacteria adhering to mucus on the tissue surface and to each other by flagellar interaction. At later time points (3-4 h), biofilm formation on ileal tissue was evident. Flagellar mutants did not form microcolonies or biofilms in tissue. Few organisms were observed in colonic tissue, with organisms present but not as abundant as in the ileal tissue. This study shows that C. jejuni 81-176 can form microcolonies and biofilms on human intestinal tissue and that this may be an essential step in its ability to cause diarrhoea in man.

The molecular shape of poly(propylenimine) dendrimer amphiphiles has a profound effect on their self assembly.

The shape of dendrimer amphiphiles has an unexpected effect on their self-assembly. A series of diaminobutane poly(propylenimine) generation 3 dendrimer (DAB-dendr-(NH(2))(16)) amphiphiles has been synthesized, bearing an average of five (PD5), three (PD3) and one (PD1) palmitoyl group(s) per dendrimer molecule. Additionally DAB-dendr-(NH(2))(16) was derivatized with a layer of poly(ethylene glycol) (PEG, degree of polymerization = 12) groups and conjugated to an average of 1 palmitoyl group at the PEG end (PPD1). A final amphiphile resulted from the conjugation of DAB-dendr-(NH(2))(16) with 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-succinimidylpropionate (DSPE-PEG(3400)-SPA), i.e.: DPD5 (with 4 DSPE-PEG arms). The critical micellar concentration in aqueous media followed the trend: DPD5 < PD5 = PD3 < PD1 < PPD1 and amphiphiles eventually formed 10-20 nm monomolecular or multimolecular micelles and/or 200 nm spheres or tubules. Aggregation was entropy driven, as expected, for DPD5, PD5 and PD1 and enthalpy driven with the most hydrophilic compound PPD1, but was unexpectedly enthalpy driven for PD3. PD3 aggregates formed low capacity hydrophobic domains with a limited capacity for encapsulation of cyclosporine A; encapsulation levels (mole drug per mole polymer) were 0.099, 0.014, 0.099, and 0.735 for PD1, PD3, PD5, and DPD5 and, respectively. We conclude that star shaped amphiphiles such as PD3 are sterically hindered from self-assembling into high capacity hydrophobic domains in aqueous media. Amphiphile-membrane interactions were promoted by hydrophobic groups, but diminished by PEG moieties. DPD5 is the most suitable amphiphile for biomedical applications.

Rapid cell mapping using nanoparticles and SERRS.

Bone marrow-derived immune cells (macrophages) treated with gold and silver nanoparticles before fixation and dye staining have been analysed by multiple wavelength line scanning surface enhanced resonance Raman scattering (SERRS) mapping. The method yields high selectivity and sensitivity within short analysis times, identifying nanoparticle aggregates in secondary lysosomes. Using routine cell stains, the output from fluorescence, Raman and SERRS is quantified at four wavelengths of excitation, demonstrating the potential at longer biologically compatible wavelengths of using nanoparticles with cell stains for superior cell mapping.

GPI-anchored proteins and free GPI glycolipids of procyclic form Trypanosoma brucei are nonessential for growth, are required for colonization of the tsetse fly, and are not the only components of the surface coat.

The procyclic form of Trypanosoma brucei exists in the midgut of the tsetse fly. The current model of its surface glycocalyx is an array of rod-like procyclin glycoproteins with glycosylphosphatidylinositol (GPI) anchors carrying sialylated poly-N-acetyllactosamine side chains interspersed with smaller sialylated poly-N-acetyllactosamine-containing free GPI glycolipids. Mutants for TbGPI12, deficient in the second step of GPI biosynthesis, were devoid of cell surface procyclins and poly-N-acetyllactosamine-containing free GPI glycolipids. This major disruption to their surface architecture severely impaired their ability to colonize tsetse fly midguts but, surprisingly, had no effect on their morphology and growth characteristics in vitro. Transmission electron microscopy showed that the mutants retained a cell surface glycocalyx. This structure, and the viability of the mutants in vitro, prompted us to look for non-GPI-anchored parasite molecules and/or the adsorption of serum components. Neither were apparent from cell surface biotinylation experiments but [3H]glucosamine biosynthetic labeling revealed a group of previously unidentified high apparent molecular weight glycoconjugates that might contribute to the surface coat. While characterizing GlcNAc-PI that accumulates in the TbGPI12 mutant, we observed inositolphosphoceramides for the first time in this organism.

McArthur revisited: fluorescence microscopes for field diagnostics.

Few scientific instruments become eponymous with their inventors. Among those that have is the 'McArthur'. As a student in the 1930s, John Norris McArthur wanted a portable microscope to take on field trips. His rugged pocket field microscope [Mcarthur, J. (1958) A new concept in microscope design for tropical medicine. Am. J. Trop. Med. Hyg. 7, 382-385] remains a classic of compact design and performance, and has been used for malaria diagnosis over several decades. The 'McArthur' has dimensions of 102x63x51mm (McArthur folded the 160mm path length with a prism) and uses phase-contrast and specialised oil immersion objective lenses. Later, a plastic version was developed and further adapted for the Open University by Kirk & Sons, UK [McArthur J. (1971) The McArthur microscope--open university model. Trans. R. Soc. Trop. Med. Hyg. 65, 438].

Tumour gene expression from C12 spermine amphiphile gene delivery systems.

Gene therapy requires safe and efficient gene delivery systems. Towards this aim both the gene formulation and tumour transfection ability of C12 spermine amphiphiles were tested. Five amphiphiles were synthesised and characterised: 1-[N,N-bis(3-aminopropyl)-1,4-butane diamine] dodecane (12G0--a C12 spermine amphiphile), a poly(ethylene glycol) (PEG, MW = 2 kDa) derivative of 12G0, 1,12-[N,N-bis(3-aminopropyl)-1,4-butane diamine] dodecane (12G1--a C12 spermine bolaamphiphile) and N-methyl quaternary ammonium derivatives of both 12G0 (12QG0) and 12G1 (12QG1). All amphiphiles except 12G0, which precipitates, yield nanoparticles in aqueous media with and without DNA. Thus when 12G0 is substituted with either quaternary ammonium or PEG groups it forms nanoparticles both with and without DNA. The minimum nitrogen, phosphate ratio required to completely condense DNA (NP) was inversely proportional to the particles' zeta potential (zeta), NP = 1626/zeta(0.98). Biological testing showed that both PEG and quaternary ammonium groups diminished the membrane lytic ability of these C12 amphiphiles. On intratumoural injection, while PEG groups hamper gene transfer, the quaternary ammonium amphiphile (12QG0) produces tumour confined gene expression that is 80% of that produced by linear poly(ethylenimine) (LPEI, MW = 22 kDa); while the intratumoural injection of LPEI produced significant gene expression in the liver and lung, making 12QG0 suitable for the administration of cytotoxic tumouricidal genes.

The effects of a mutant connexin 26 on epidermal differentiation.

To elucidate the mode of action of dominant mutant connexins in causing inherited skin diseases, transgenic mice were produced that express the true Vohwinkel syndrome-associated mutant Cx26 (D66H), from a keratin 10 promoter, specifically in the suprabasal epidermal keratinocytes. Following birth, the transgenic mice developed keratoderma similar to that of human carriers of Cx26 (D66H). Expression of the transgene resulted in a loss of Cx26 and Cx30 at intercellular junctions of epidermal keratinocytes and accumulation of these connexins in the cytoplasm. Injection of primary mouse keratinocytes with Lucifer Yellow showed no difference in terms of dye spreading between transgenic and non transgenic keratinocytes in vitro. Expression of the mutant Cx26 (D66H) did not interfere with the formation of the epidermal water barrier during late embryonic development. Attempts to produce transgenic mice expressing the wild type form of Cx26 from the K10 promoter failed to produce viable animals although transgenic embryos were recovered at days 9 and 12 of gestation, suggesting that the transgene might be embryonic lethal.

TsetseEP, a gut protein from the tsetse Glossina morsitans, is related to a major surface glycoprotein of trypanosomes transmitted by the fly and to the products of a Drosophila gene family.

African trypanosomes live in the lumen of the gut of tsetse (Glossina) and may have to face an immune response. As yet, it is unclear whether they are sensitive to antimicrobial peptides in vivo, but for some years there has been indirect evidence that one or more lectins can influence the infection. We have purified a protein complex from midgut extracts that, by SDS-PAGE, is a doublet of 37 and 38 kDa in a ratio of 3:1. Through prediction from corresponding cDNA clones, the full-length protein (tsetseEP) contains 320 amino acids, including a signal peptide. There is apparently only one gene encoding this protein. Towards the C terminus, the protein contains a run of 59 (EP) repeats, which surprisingly is what comprises almost the entire mature EP procyclin molecule present on the surface of trypanosomes in the tsetse gut. Drosophila contains a number of genes encoding proteins, of unknown function, with the same cysteine pattern as tsetseEP; this pattern is not reported for any other protein. Immunoblotting with a monoclonal antibody against (EP) repeats reveals expression in the gut, but not salivary glands, of female and male flies, whether or not fed. Immunoelectron microscopy shows the presence in vesicles in midgut cells and in the lumen of the gut. Attempts to demonstrate lectin activity were thwarted by limited availability of the protein complex.

In vitro and in vivo gene transfer with poly(amino acid) vesicles.

Non-viral gene delivery systems utilise either amine lipids or polyamines and although non-viral gene delivery systems are said to have a superior safety profile to viruses, the polyamines such as poly(L-lysine) are toxic when used without derivatisation and usually require specific receptor mediated uptake and/or endosomolytic agents to be effective. However, the conversion of poly(L-lysine) and poly(L-ornithine) polyamino acids into amphiphilic vesicle forming polymers reduces the toxicity of the polyamino acids and enables the resulting polyamino acid vesicles to deliver genes both in vitro and in vivo in the absence of receptor specific ligands and endosomolytic agents. The incorporation of a distearoylphosphatidylethanolamine poly(ethylene glycol)-galactosamine conjugate (with the galactosamine unit at the distal end of the poly(ethylene glycol) moiety) into the polyamino acid formulations improved in vitro gene transfer in the case of the amphiphilic poly(L-ornithine) (POP) although no in vivo targeting was detected with the galactosamine formulations. We conclude that the conversion of poly(L-lysine) and poly(L-ornithine) into amphiphilic colloid forming molecules reduces their toxicity, thus allowing these systems to be used for gene transfer in vivo. It is possible that this approach may be extended to other polyamines.

The release of model macromolecules may be controlled by the hydrophobicity of palmitoyl glycol chitosan hydrogels.

A non-covalently cross-linked palmitoyl glycol chitosan (GCP) hydrogel has been evaluated as an erodible controlled release system for the delivery of hydrophilic macromolecules. Samples of GCP with hydrophobicity decreasing in the order GCP12>GCP11>GCP21 were synthesised and characterised by 1H NMR. Hydrogels were prepared by freeze-drying an aqueous dispersion of the polymer in the presence or absence of either a model macromolecule fluorescein isothiocyanate-dextran (FITC-dextran, MW 4400), and/or amphiphilic derivatives Gelucire 50/13 or vitamin E d-alpha-tocopherol polyethylene glycol succinate. Gels were analysed for aqueous hydration, FITC-dextran release, and bioadhesion, and imaged by scanning electron microscopy. The gels were highly porous and could be hydrated to up to 95x their original weight without an appreciable volume change and most gels eventually eroded. Hydration and erosion were governed by the hydrophobicity of the gel and the presence of the amphiphilic additives. GCP gels could be loaded with up to 27.5% (w/w) of FITC-dextran by freeze-drying a dispersion of GCP in a solution of FITC-dextran. The controlled release of FITC-dextran was governed by the hydrophobicity of the gel following the trend GCP21>GCP11>GCP12. GCP gels were bioadhesive but less so than hydroxypropylmethylcellulose, Carbopol 974NF (7:3) tablets.

Gene transfer with three amphiphilic glycol chitosans--the degree of polymerisation is the main controller of transfection efficiency.

A number of studies have examined the possibility of delivering genes for the treatment of genetic diseases using various polymers and lipids. We have previously demonstrated the gene transfer ability of amphiphilic polymers (a soluble amine polymer covalently bound to lipid pendant groups). In the current communication we explore the gene transfer activity of amphiphilic glycol chitosans. Glycol chitosan was acid depolymerised to give polymers of various molecular weights. Palmitoyl or hexadecyl and in some cases additional N-methyl quaternary ammonium groups were attached to the polymers. DNA binding was studied by measuring the reduced fluorescence of ethidium bromide and the polyplex particle size and zeta potential. Biological characterisation of the polyplexes involved haemolysis, cytotoxicity and gene transfer assays. For the 22 polymers tested, DNA binding was optimum at a nitrogen to phosphate ratio of 2:1 and above. Polyplexes were 200-500 nm in diameter with a neutral or positive zeta potential. The haemolytic activity of the N-methyl polymers was studied and no haemolysis was detected up to a concentration of 10 mg ml-1. Cytotoxicity studies showed that the biocompatibility of glycol chitosan was adversely affected by a combination of a palmitoyl group and depolymerisation and that biocompatibility was subsequently restored with the introduction of N-methyl groups. In vitro transfection efficiency superior to the cationic lipid formulation N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methyl sulphate (DOTAP) was seen with depolymerised glycol chitosan in the A431 cell line only and with the depolymerised N-methyl quaternary ammonium amphiphilic derivatives in both the A431 and A549 cell lines. Degree of polymerisation (DP) was the most important controller of transfection efficiency and transfection resided within polymers with a DP of 73-171. High DP polymers diminished DNA-cell association, the first step in the cellular gene transfer process, thus apparently diminishing cell uptake. In vivo transfection with the N-methyl quaternary ammonium amphiphile was best at a DP of 86 and this glycol chitosan amphiphile gave superior liver and heart gene expression levels when compared to both Exgen 500 (linear polyethylenimine) and Superfect (a polyamidoamine dendrimer).

Antitumor activity of the tea polyphenol epigallocatechin-3-gallate encapsulated in targeted vesicles after intravenous administration.

AIM: The therapeutic potential of epigallocatechin-3-gallate (EGCG), a green tea polyphenol with anticancer properties, is limited by its inability to specifically reach tumors following intravenous administration. The purpose of this study was to determine whether a tumor-targeted vesicular formulation of EGCG would suppress the growth of A431 epidermoid carcinoma and B16-F10 melanoma in vitro and in vivo. MATERIALS & METHODS: Transferrin-bearing vesicles encapsulating EGCG were administered intravenously to mice bearing subcutaneous A431 and B16-F10 tumors. RESULTS: The intravenous administration of EGCG encapsulated in transferrin-bearing vesicles resulted in tumor suppression in 40% of A431 and B16-F10 tumors. Animal survival was improved by more than 20 days compared with controls. CONCLUSION: Encapsulation of EGCG in transferrin-bearing vesicles is a promising therapeutic strategy.

Identification and Functional Characterisation of CRK12:CYC9, a Novel Cyclin-Dependent Kinase (CDK)-Cyclin Complex in Trypanosoma brucei.

The protozoan parasite, Trypanosoma brucei, is spread by the tsetse fly and causes trypanosomiasis in humans and animals. Both the life cycle and cell cycle of the parasite are complex. Trypanosomes have eleven cdc2-related kinases (CRKs) and ten cyclins, an unusually large number for a single celled organism. To date, relatively little is known about the function of many of the CRKs and cyclins, and only CRK3 has previously been shown to be cyclin-dependent in vivo. Here we report the identification of a previously uncharacterised CRK:cyclin complex between CRK12 and the putative transcriptional cyclin, CYC9. CRK12:CYC9 interact to form an active protein kinase complex in procyclic and bloodstream T. brucei. Both CRK12 and CYC9 are essential for the proliferation of bloodstream trypanosomes in vitro, and we show that CRK12 is also essential for survival of T. brucei in a mouse model, providing genetic validation of CRK12:CYC9 as a novel drug target for trypanosomiasis. Further, functional characterisation of CRK12 and CYC9 using RNA interference reveals roles for these proteins in endocytosis and cytokinesis, respectively.

A unique Kelch domain phosphatase in Plasmodium regulates ookinete morphology, motility and invasion.

Signalling through post-translational modification (PTM) of proteins is a process central to cell homeostasis, development and responses to external stimuli. The best characterised PTM is protein phosphorylation which is reversibly catalysed at specific residues through the action of protein kinases (addition) and phosphatases (removal). Here, we report characterisation of an orphan protein phosphatase that possesses a domain architecture previously only described in Plantae. Through gene disruption and the production of active site mutants, the enzymatically active Protein Phosphatase containing Kelch-Like domains (PPKL, PBANKA_132950) is shown to play an essential role in the development of an infectious ookinete. PPKL is produced in schizonts and female gametocytes, is maternally inherited where its absence leads to the development of a malformed, immotile, non-infectious ookinete with an extended apical protrusion. The distribution of PPKL includes focussed localization at the ookinete apical tip implying a link between its activity and the correct deployment of the apical complex and microtubule cytoskeleton. Unlike wild type parasites, ppkl(-) ookinetes do not have a pronounced apical distribution of their micronemes yet secretion of microneme cargo is unaffected in the mutant implying that release of microneme cargo is either highly efficient at the malformed apical prominence or secretion may also occur from other points of the parasite, possibly the pellicular pores.