The proton-translocating inorganic pyrophosphatases (H+-PPases) are primary electrogenic H+ pumps that derive energy from the hydrolysis of inorganic pyrophosphate (PPi). They are widely distributed among most land plants and have also been found in several species of protozoan parasites. Here we describe, for the first time, the molecular cloning and functional characterization of a gene encoding an H+-pyrophosphatase in the protozoan scuticociliate parasite Philasterides dicentrarchi, which infects turbot. The predicted P. dicentrarchi PPase (PdPPase) consists of 587 amino acids of molecular mass 61·7 kDa and an isoelectric point of 5·0. Several motifs characteristic of plant vacuolar H+-PPases (V–H+-PPases) were also found in the PdPPase, which contains all the sequence motifs of the prototypical type I V–H+-PPase from Arabidopsis thaliana vacuolar pyrophosphatase type I (AVP1) plant. The PdPPase has a characteristic residue that determines strict K+-dependence, but unlike AVP1, PdPPase contains an N-terminal signal peptide (SP) sequence. Antibodies generated by vaccination of mice with a genetic or recombinant protein containing a partial sequence of the PdPPase and a common motif with the polyclonal antibody PABHK specific to AVP1 recognized a single band of about 62 kDa in western blots. These antibodies specifically stained both vacuole and the alveolar membranes of trophozoites of P. dicentrarchi. H+ transport was partially inhibited by the bisphosphonate pamidronate (PAM) and completely inhibited by NaF. The bisphosphonate PAM inhibited both H+-translocation and gene expression. PdPPase and PAM also inhibited in vitro growth of the ciliates. The apparent lack of V–H+-PPases in vertebrates and the parasite sensitivity to PPI analogues may provide a molecular target for developing new drugs to control scuticociliatosis.

Gyrodactylid parasites were observed on non-native populations of North-American freshwater catfishes, Ameiurus nebulosus and Ameiurus melas (Siluriformes: Ictaluridae), at several sites in the Elbe River basin, Czech Republic, Europe. Using a combination of morphological and genetic analyses, the parasites infecting A. nebulosus were determined to be Gyrodactylus nebulosus, a North American parasite co-introduced to Europe along with its Ameiurus fish hosts. Subtle morphometrical differences, as well as seasonal variations, were observed among parasites collected from A. nebulosus and A. melas. The host-related variation was further supported through genetic analysis of the partial 18S rDNA, ITS1-5.8S-ITS2 and COI, showing 0.2, 3.0 and 4.8% divergence, respectively. Consistent genetic differences indicated there were two distinct genotypes. Subtle morphological differences associated with the shape of sickle toe, anchor root and ventral bar membrane, according to host species, also supported the description of a new cryptic species, Gyrodactylus melas n. sp., infecting A. melas. Multivariate morphometrical analysis of haptoral hard parts showed significant differences between the anchor lengths of G. nebulosus and G. melas n. sp. However, the measurements of the haptoral hard structures partially overlapped between species, limiting the usage of these parameters for species delineation.

With rapid developments in DNA and protein sequencing technologies, combined with powerful bioinformatics tools, a continued acceleration of gene identification in parasitic helminths is predicted, potentially leading to discovery of new drug and vaccine targets, enhanced diagnostics and insights into the complex biology underlying host-parasite interactions. For the schistosome blood flukes, with the recent completion of genome sequencing and comprehensive transcriptomic datasets, there has accumulated massive amounts of gene sequence data, for which, in the vast majority of cases, little is known about actual functions within the intact organism. In this review we attempt to bring together traditional in vitro cultivation approaches and recent emergent technologies of molecular genomics, transcriptomics and genetic manipulation to illustrate the considerable progress made in our understanding of trematode gene expression and function during development of the intramolluscan larval stages. Using several prominent trematode families (Schistosomatidae, Fasciolidae, Echinostomatidae), we have focused on the current status of in vitro larval isolation/cultivation as a source of valuable raw material supporting gene discovery efforts in model digeneans that include whole genome sequencing, transcript and protein expression profiling during larval development, and progress made in the in vitro manipulation of genes and their expression in larval trematodes using transgenic and RNA interference (RNAi) approaches.

Distribution of 9-O-acetylated sialic acids (9-O-AcSA) on Leishmania donovani has been previously reported. Considering their role in recognition, the differential distribution of sialic acids especially 9-O-acetylated sialic acids in avirulent (UR6) versus virulent (AG83 and GE1) promastigotes of Leishmania donovani and its role in entry into macrophages was explored. Fluorimetric-HPLC, fluorimetric determination and ELISA revealed 14-, 8- and 5-fold lower sialic acids in UR6 as compared to AG83. Interestingly, on UR6, flow cytometry indicated lower (α2→6)-linked sialoglycoproteins along with minimal 9-O-acetylated sialoglycoproteins by Scatchard analysis. Further, UR6 demonstrated a 9- and 14·5-fold lower infectivity and phagocytic index than AG83. Additionally, de-O-acetylation and de-sialylation of AG83 demonstrated a 3- and 1·5-fold reduced phagocytic index. The role of 9-O-AcSA in entry was further confirmed by pre-blocking the macrophage surface with a cocktail of sugars followed by microscopic quantification. The phagocytic index of AG83 exclusively through 9-O-AcSA was significantly high. Interestingly, AG83 produced higher metacyclic promastigotes containing increased 9-O-AcSA as compared to avirulent UR6 supporting its virulent nature. Taken together; our results conclusively demonstrate the increased presence of 9-O-acetylated sialic acid on promastigotes of virulent Leishmania donovani as compared to avirulent UR6 and their subsequent role in entry within macrophages.

Animals frequently host organisms on their surface which can be beneficial, have no effect or a negative effect on their host. Ectoparasites, by definition, are those which incur costs to their host, but these costs may vary. Examples of avian ectoparasites are chewing lice which feed exclusively on dead feather or skin material; therefore, costs to their bird hosts are generally considered small. Theoretically, many possible proximate effects exist, like loss of tissue or food, infected bites, transmission of microparasitic diseases or reduced body insulation due to loss of feathers, which may ultimately also have fitness consequences. Here, we experimentally examined a possible negative impact of 2 feather-eating louse species (Meropoecus meropis and Brueelia apiastri) on male and female European bee-eaters (Merops apiaster) by removing or increasing louse loads and comparing their impact to a control group (lice removed and immediately returned) after 1 month. A negative effect of chewing lice was found on body mass and sedimentation rate and to a lesser extent on haematocrit levels. Males and females lost more weight when bearing heavy louse loads, and were more susceptible to infestations as indicated by the higher sedimentation rate. Our results further suggest differences in sex-specific susceptibility.

The specimens of this new species of mite were collected by Mr Gordon Thompson, formerly curator of the Science Museum of the Institute of Jamaica, from the legs of a specimen of the golden thrush, Turdus aurantiacus Gmelin, taken at Morant Bay Road, 14½ miles east of Kingston, 14 January 1947. The bird was a male, and in a letter dated 11 March 1947 he says the mites were ‘extracted from a “growth” after macerating it for a while in caustic potash. This is the only “growth” of its kind I have found among approximately two hundred birds examined in Jamaica. The bird seemed quite healthy.’ I have to thank Mr Thompson for his kindness in handing me this, together with other acarine material, for study and determination.

In a previous paper (1935) an account was given of the prostigmatic mites parasitic on South African lizards, belonging to the two endemic families, the Zonuridae and Gerrhosauridae. The present paper deals with the fauna of the two remaining families of lizards, the Geckonidae and Agamidae, parasitised exclusively by prostigmatic mites. Trägardh (1905) and Hirst (1926) have made the most considerable contributions to the knowledge of the parasites of Geckonid and Agamid lizards; the latter placed the group on a sound systematic basis, the former, in addition to his descriptions of new forms, contributed valuable notes on the development and morphology of the species studied. With regard to mite parasites, however, up to the present, no systematic investigation has been attempted of all the species belonging to any one family of lizards from a given zoo-geographical region. In the present paper this has been attempted; the great majority of Geckonid and all the species of Agamid lizards from the South African subregion, either as living or spirit specimens, have been examined; from most of them mite parasites have been obtained. Both families of lizards are, genealogically speaking, derived from a long-established, Saurian stock; both are, in South Africa, extremely well demarcated, homogeneous families, with their main structural characteristics varying within narrow limits. In neither of them does loss or degeneration of the limbs occur. Each family is parasitised by a single genus of prostigmatic mites, the Geckonidae by Geckobia, the Agamidae by Pterygosoma; while Geckobia is found on the Geckonidae and on no other family, Pterygosoma is also a parasite of Gerrhosaurid lizards on which three species have been found. In a previous paper by the writer (1935, p. 3) Pimeliaphilus was cited as occurring on Geckonidae. Trägardh, who erected the genus (1905, p. 31), actually found his species P. podapolipophagus living under the elytra of a beetle, Pimelia, but the other two species of the genus, P. insignis Berl. and P. tenuipes Hirst, both occur on Geckonid lizards. Neither of these two species have been found in South Africa and it is extremely unlikely that they occur in this region.

The almost universally-occurring aggregated distributions of helminth burdens in host populations have major significance for parasite population ecology and evolutionary biology, but the mechanisms generating heterogeneity remain poorly understood. For the direct life cycle monogenean Discocotyle sagittata infecting rainbow trout, Oncorhynchus mykiss, variables potentially influencing aggregation can be analysed individually. This study was based at a fish farm where every host individual becomes infected by D. sagittata during each annual transmission period. Worm burdens were examined in one trout population maintained in isolation for 9 years, exposed to self-contained transmission. After this year-on-year recruitment, prevalence was 100% with intensities 10–2628, mean 576, worms per host. Parasite distribution, amongst hosts with the same age and environmental experience, was highly aggregated with variance to mean ratio 834 and negative binomial parameter, k, 0.64. The most heavily infected 20% of fish carried around 80% of the total adult parasite population. Aggregation develops within the first weeks post-infection; hosts typically carried intensities of successive age-specific cohorts that were consistent for that individual, such that heavily-infected individuals carried high numbers of all parasite age classes. Results suggest that host factors alone, operating post-infection, are sufficient to generate strongly overdispersed parasite distributions, rather than heterogeneity in exposure and initial invasion.

Within the genus Cryptosporidium 2 lineages have evolved, one adapted to the acid environment of the stomach and abomasum, the other comprising parasites that multiply in the small intestine. We tested whether the release of sporozoites from oocysts, a process known as excystation, is triggered by conditions which mimic the site of infection. Specifically, we exposed oocysts from gastric and intestinal Cryptosporidium species to acid conditions or to a neutral solution of taurocholic acid, at 37°C. We found that oocysts from the gastric species C. muris and C. andersoni excysted in both conditions, whereas the intestinal species C. parvum and C. hominis did not respond to acid. When the effect of temperature alone was tested on C. muris and C. parvum, only oocysts from the former species excysted in significant numbers. Oocysts from intestinal species did not respond to temperature alone, nor to acidity. These observations are consistent with the need of gastric species to rapidly excyst and release the sporozoites upon ingestion, and indicate that Cryptosporidium oocysts have evolved to maximize delivery of sporozoites to the region of the gastro-intestinal tract where the parasite multiplies.

We cloned and expressed a novel gene encoding a 32-kDa merozoite protein of Babesia gibsoni (BgP32). The length of nucleotide sequence of the cDNA was 1464 bp with an open reading frame of 969 bp. The truncated recombinant BgP32 (rBgP32) without a signal peptide and C-terminal hydrophobic sequence was expressed in Escherichia coli as a soluble glutathione-S-transferase (GST) fusion protein. Western blotting demonstrated that the native protein was 32-kDa, consistent with molecular weight of the predicted mature polypeptide. Enzyme-linked immunosorbent assay (ELISA) using rBgP32 detected specific antibodies from 8 days to 541 days post-infection in the sequential sera from a dog experimentally infected with B. gibsoni. Moreover, the antigen did not cross-react with B. canis subspecies and closely related protozoan parasites, indicating that rBgP32 is a specific diagnostic antigen. Analysis of 47 sera taken from dogs with anaemic signs revealed that rBgP32 detected a higher proportion of B. gibsoni seropositive samples (77%) than its previously identified rBgP50 (68%) homologue. These results indicate that the BgP32 is a novel immunodominant antigen of B. gibsoni, and rBgP32 might be useful for diagnosis of B. gibsoni infection.

The following is a continuation of the foregoing Report (Parasitology, xv, pp. 1–13). As in the first part, the numbers are those given to the original tubes at Cambridge.

The isolation of 2 genomic clones has allowed us to further characterize a Schistosoma mansoni serine protease designated SmSP1. The deduced amino acid sequence (248aa) considered as a ‘light chain’ encoding the active site, presents significant homologies with mouse plasma kallikrein and human factor I light chain. The secondary structure of SmSP1 ‘light chain’ is correctly predicted and may be sufficient by itself to constitute an active enzyme. The biological function of SmSP1 is unknown, however, the homology with 2 serine proteases suggests that SmSP1 may play a role in the evasion of the host immune response. This is supported by the presence of the native protein corresponding to SmSP1 particularly in schistosomula released products (SRP) and in male dorsal spines. The expression of this enzyme is differentially regulated throughout the parasite life-cycle. However, infected animals with S. mansoni did not produce specific antibodies to recombinant SmSP1. The lack of such response could be advantageous to the parasite by protecting itself from host effector mechanisms.

Two strains of Schistosoma mansoni were used to investigate the hereditary basis of species-specific host recognition by analysing behavioural responses of miracidia to snail-conditioned water. An Egyptian strain of S. mansoni, capable of distinguishing its host snail Biomphalaria alexandrina from other snails was cycled repeatedly through Biomphalaria glabrata, the intermediate host of a Brazilian strain known to respond even to non-susceptible snails with high intensity. After 5 cycles in the non-natural host, miracidia of the Egyptian strain still retained their preference for the original host snail. In a second experiment, host-finding behaviour of hybrids between these two parasite strains was studied. In the F1 generation, hybrids of both parental combinations showed the same low degree of specificity as the pure-bred Brazilian strain. Approximately one quarter of F2 hybrids proved to be as discriminatory as the Egyptian strain, confirming dominant Mendelian inheritance of non-specificity in schistosome miracidial host-finding behaviour. Moreover, hybrids seem to have lost the ability to develop in B. alexandrina, possibly suggesting a link between host recognition and host compatibility. The heredity of this behavioural trait is of evolutionary and epidemiological significance, since a shift to low host-finding specificity might have been a prerequisite for S. mansoni to acquire new host snails after being introduced to South America by the slave trade.

Very little work has been done on the systematic study of entozoic trematodes of Australian fishes. Possibly the earliest contribution to the subject was a work by Kreft (1871), “On Australian Entozoa.” Other contributors to this branch of the subject are Wm Nicoll, S. J. Johnston, and T. Harvey Johnston, their studies being chiefly based on parasites of fishes from Queensland and New South Wales waters. So far as can be ascertained there has been as yet only one contribution to the literature on trematode parasites of fishes of Southern Australian waters, a paper by T. Harvey Johnston recently published.

Throughout the world, infection with parasites is extremely common. Prevalence is highest in the warm countries of the tropics and subtropics, but infection occurs in all climatic zones. The figures usually quoted for the prevalence of specific parasites attain values akin to those used to describe astronomical distances. The World Health Organization (1985) estimated that some 2582 million of the earth's inhabitants live in areas where they risk contracting malaria and that many – in Africa south of the Sahara alone probably 200 million – remain chronically and persistently infected. Estimates for other parasites are similarly impressive; 600 million at risk of infection with schistosomiasis, with some 200 million actually infected; 900 million exposed to lymphatic filarial parasites, with some 90 million currently infected; for onchocerciasis, probably some 40 million cases spread throughout Africa, Central and South America and the Eastern mediterranean. Yet, impressive as these estimates may be, they are dwarfed by those for some intestinal helminths. Recent assessments suggest that 1 person in 4 of the world's population carries Ascaris lumbricoides (Schultz, 1982), while the same proportion is believed to be infected with one or other of the hookworms (Gilman, 1982).

The monogenean Diplozoon paradoxum occurs on Abramis brama, Gobio gobio, Phoxinus phoxinus and Rutilus rutilus in northern England. In any one area, the infection is present in some waters but absent in others and predominates in rivers rather than in ponds or reservoirs.

D. paradoxum is randomly distributed on the gills, sides of the gill apparatus, hemibranchs and surfaces of primary lamellae of G. gobio, P. phoxinus and R. rutilus (except for P. phoxinus where inner hemibranchs carry more worms), but prevails on median sectors of the gills of these hosts. In A. brama the parasite is more numerous on certain gills, on the right side of the gill apparatus, and on the inner hemibranchs, but is randomly disposed on primary lamellar surfaces and along the gills. Except on G. gobio attachment is predominantly to two primary lamellae rather than one. This may reduce dislodging effects of the gill ventilating current by leaving portions of the lamellar surfaces unoccupied and allowing unimpeded flow in these regions.

This work was conducted during tenure of a Science Research Council Research Studentship. Research facilities in the Department of Zoology, University of Leeds, were kindly provided by Professor J. M. Dodd. Other facilities in the Biological Station, St John's, Newfoundland, were provided by the Director, Dr W. Templeman. Statistical advice offered by Dr E. Broadhead, Department of Zoology, and Professor C. L. Oakley, Department of Bacteriology, both of the University of Leeds, is gratefully acknowledged. I am indebted to Dr R. Wynne Owen, Department of Zoology, for constant advice and encouragement and for critical reading of the manuscript.

Didymobothrium rudolphii (Cestoda: Spathebothriidea) was collected seasonally from the sand sole, Solea lascaris, off the northern, central and southern areas of the Portuguese coast. Morphological and molecular analyses were conducted in order to examine the possible existence of cryptic species and to facilitate the circumscription of their morphological boundaries. Data were compared between D. rudolphii specimens from each of the 3 geographical areas and 4 seasons, and principal components analysis of 18 morphological characters was used to detect differences. Two distinct genotypes were present with sequence divergences of 1·9% and 2·1% in the large subunit (lsrDNA) and second internal transcribed spacer (ITS-2) of ribosomal DNA (rDNA), respectively. The less common ‘central’ genotype was present only off the central area from summer to winter, whereas the ‘common’ genotype was present throughout the year off the northern and southern areas, but only during spring in the central area. No sequence variation was found within each genotype. The presence of 2 distinct genetic entities was supported by morphological analyses, which showed the ‘central’ genotype specimens to be more slender and elongate, although morphometric ranges overlapped considerably for most characters. Molecular phylogenetic analysis of 4 of the 5 known genera of the Spathebothriidea showed Spathebothrium to be the earliest branching lineage and the 2 genotypes of Didymobothrium formed a sister group to Cyathocephalus. The concordance of genetic differences with variation in host diet according to season and locality could account for sympatric speciation occurring in the central region of the Portuguese coast.

Montgomery (1917) showed that Nairobi sheep disease was commonly transmitted by Rhipicephalus appendiculatus. In 1931 we confirmed Montgomery's findings and extended them considerably in respect of transmission by this species of tick. It was shown that infection of any instar would result in transmission by the next succeeding stage, which would then in the normal course lose its infection during the next moult. A female tick infected in this manner as an adult would pass infection through the eggs to the larvae of the next generation. Further it was demonstrated that the transmitting stage could be reinfected if the reaction of the host animal commenced before the infecting meal was completed, thus ensuring the carrying on of infetion for a further stage.

This ultrastructural study has shown that there is a layer of dense flocculent material on the surface of juvenile Fasciola hepatica incubated in vitro with specific bovine antiserum. This material corresponds to the complexes of secreted glycocalyx and bovine antibody previously characterized by fluorescence microscopy. Bovine eosinophils attach closely to those regions of the parasite's surface that are free of flocculent precipitates. This close attachment is followed by degranulation of the eosinophils into the narrow zone between the cells and the parasite. Only in these regions is damage, in the form of vacuolation of the tegument, seen within the juvenile F. hepatica. It is concluded that the inability of bovine eosinophils to kill juvenile F. hepatica in the presence of specific antiserum results from the presence of a protective layer, consisting of antigen/antibody complexes, on the parasite's surface.

Previous work on the effect of temperature and humidity upon the survival of unfed fleas is criticised in respect of experimental technique. In the present investigation measures have been taken to eliminate as many variables as possible and the methods employed to this end are described.

In the experiments, 2027 Xenopsylla cheopis, 999 X. astia and 646 Ceratophyllus fasciatus were employed, all unfed and less than 24 hours old at the commencement of the different experiments. Records were kept individually so that a statistical measure of consistency could be made.

The following conclusions have been reached:

(1) Duration of life of unfed fleas is not influenced by sex.

(2) High temperatures and low humidities tend to shorten life; conversely, low temperatures and high humidities, with a possible optimum of about 90 per cent., tend to produce longer lives.

(3) Humidity is at its maximum efficiency in this respect at about 18° C.

(4) At 37° C. humidity ceases to have any effect upon the duration of life of unfed fleas, and the evidence suggests that there is also a low temperature at which humidity has no influence upon survival.

(5) There is no direct proportion between survival of unfed fleas and saturation deficiency of the atmosphere at any temperature.

Comparing the species, it was found that:

(1) Ceratophyllus fasciatus was the longest lived, at similar atmospheric conditions.

(2) Xenopsylla astia lived longer than X. cheopis at all humidities at 23° C.