The material which forms the subject of this communication was derived from Dr W. A. Lamborn's collection, and was placed at my disposal through the kindness of Dr Guy A. K. Marshall, Director of the Imperial Bureau of Entomology, London. It comprised eight larvae of Stegomyia scutellaris Walker, which had been preserved in formaldehyde solution (10 per cent.), and the specimens were mounted individually on microscope slides in the same medium.

There is a general lack of basic information concerning one class of glycoconjugate, the glycolipids, from parasitic nematodes. As the prototype, the neutral glycolipid fraction derived from adult males of Ascaris suum was investigated as to its chromatographic, differential chemical staining, antigenic and chemical properties. The thin-layer chromato-graphy-resolved neutral fraction glycolipids could be classified into components of fast and slow migrating band groups. Immunoreactivity was restricted to the latter as detected by IgG and IgM anti-neutral fraction glycolipid antibody levels in serial infection sera of mice. Similarities of chromatography, antigenicity and serological cross-reactivity have been extended to the neutral glycolipid fractions of other parasitic nematodes: Litomosoides carinii and Nippostrongylus brasiliensis. Chemical, differential chemical staining and enzymatic analyses identified the Ascaris suum antigenic, slow migrating band group of components as amphoteric glycosphingolipids, and not the originally hypothesized glyco-glycerolipids or glycosylphosphatidylinositols, that contained typical neutral monosaccharide constituents and a zwitter-ionic phosphodiester linkage, most probably phosphocholine. Glycosphingolipid-immunoreactivity is eliminated on cleavage of the zwitterionic phosphodiester linkage by hydrofluoric acid treatment.

The intergenic spacer of the ribosomal RNA gene array from the monogenean Gyrodactylus salaris was isolated using PCR amplification. PCR products were cloned and sequenced. Three different fragments of 0·63, 1·0 and 2·62 kb, were consistently obtained. These showed homology at the 5′ and 3′ termini but differed in their overall size and intervening sequence. The 5′ end showed homology to various 28S ribosomal RNA gene sequences, suggesting that this represented the 3′ terminus of the G. salaris 28S ribosomal RNA gene. A number of features common to other eukaryotic intergenic spacers were found in the longest sequence, including A + T rich sequences, palindromic sequences and tandemly repeated elements. Two regions of 23 bp sequences arranged in non-identical tandem repeats were identified. There were 9 repeats in both regions, separated by 81 bp of non-repetitive sequence. The repeat units from the two regions shared some similarity at their 3′ ends. The G. salaris intergenic spacer sequence was examined for sequence motifs involved in the transcription of the ribosomal RNA genes in other species. Several regions with homology to transcription start sites were identified.

The various suggestions concerning the mode of spreading of Monocystid Gregarines parasitic in earthworms have been already summarised by Minchin (1903), Hesse (1909) and more recently by Bhatia (1924). The last named author seemed to find that the spores from the vesicula seminalis may escape during copulation with the seminal fluid, may be transferred to the receptacula seminis to appear finally inside the cocoon of the earthworm. This observation has led him to suppose that the earthworm may become infected before it emerges from the cocoon. He does not, however, consider this mode of infection as the principal one, but accepts the general view that the majority of spores are set free only after the death of the worm and the complete disintegration of its body.

Bonamia ostreae is a protozoan parasite that infects the European flat oyster Ostrea edulis, causing systemic infections and resulting in massive mortalities in populations of this valuable bivalve species. In this work, we have characterized B. ostreae actin genes and used their sequences for a phylogenetic analysis. Design of different primer sets was necessary to amplify the central coding region of actin genes of B. ostreae. Characterization of the sequences and their amplification in different samples demonstrated the presence of 2 intragenomic actin genes in B. ostreae, without any intron. The phylogenetic analysis placed B. ostreae in a clade with Minchinia tapetis, Minchinia teredinis and Haplosporidium costale as its closest relatives, and demonstrated that the paralogous actin genes found in Bonamia resulted from a duplication of the original actin gene after the Bonamia origin.

1. The spermatophore of the argasid tick Argas persicus, is a sac enclosing a mass of sperm and a number of other structures which are rapidly extruded into the vagina of the female tick during copulation.

2. During copulation the first part of the contents of the spermatophore to be extruded consists of a clear tip with two lateral semi-opaque areas surrounded by white borders and a mass of opaque white material followed by the sperm.

3. The male accessory reproductive gland is redescribed. It consists of a stem portion which gives off: dorsally, the unpaired anterior dorsal granular lobe and the paired posterior dorsal and third lateral granular lobes; the small paired second lateral granular lobes from the mid-region; ventrally, the small unpaired median granular lobe and the large paired first lateral granular lobes and, opening into the ejaculatory duct, the small, paired anterior and posterior spongy lobes.

4. The anterior and posterior spongy lobes secrete an acid mucopolysaccharide which thinly coats the spermatophore.

5. The median granular lobe secretes a phospholipoprotein rich in phenolic groups and a polyphenol oxidase.

6. The first lateral granular lobes secrete a basic protein which interacts with the secretions of the median granular lobe to form the main wall of the spermatophore.

7. The second lateral granular lobes secrete the tenuous mucoprotein membrane which surrounds the sperm mass within the spermatophore and also the large protein granules which adhere to the inner surface of this membrane.

8. The third lateral granular lobe secretes the mass of opaque, white material in the spermatophore; possibly this is pharmacologically active and analogous to the secretion of the opaque glands of Rhodnius.

9. The posterior dorsal granular lobes secrete a non-specific esterase at their tip and a protein in the mid-region; these form, respectively, the lateral semi-opaque areas and their white borders in the spermatophore.

10. The anterior dorsal granular lobe secretes an acid mucopolysaccharideprotein complex which forms the clear tip of the extruded contents of the spermatophore.

1. Previous work by the writer drew attention to the fact that the pH index of natural waters was in some manner correlated with the presence or absence of particular mosquito larvae.

2. The observation led other investigators to study this hypothesis, and a short summary of their published records is given.

3. The actual significance of the pH index was, however, obscure, and the present work has been undertaken to attempt to demonstrate the actual significance of the pH in mosquito breeding-places.

4. By gradually eliminating first one and then another of the interacting factors, the writer has been able to demonstrate (a) that if the pH of the normal environment is changed the development of the larvae is adversely affected; (b) that under bacteriologically sterile conditions the foregoing statement is no longer true; and (c) that consequently the acid or alkaline reaction of the medium, within ordinary limits, has no direct effect upon the development of the larvae.

5. Nevertheless, it is shown that the influence of the pH. is of unquestionable importance and that its significance lies in the fact that, under natural conditions, it indicates the favourable or unfavourable association of chemical and biological factors in the breeding-places upon which the successful or unsuccessful development of the larvae depends.

6. It may therefore be stated that although the pH index is not an infallible guide, it does in the majority of instances provide a reliable index as to whether the necessary combination of chemical and biological factors in any given water are such that they will permit the development of the larvae of particular species. Some species find their optimum conditions for development in waters which are acid, others in waters which are alkaline, and it can be shown that to alter the pH of such waters is often to bring about a complete change in the biological group associations, which frequently means the acquired abnormal dominance of factors unfavourable to a particular species of larvae. Clearly, the system is to some extent elastic, and in rare instances what are in general acidophile larvae may be found in waters showing alkalinity.

7. The chief conclusion is, however, that in most instances in various localities the larvae of certain species do actually show a restriction to waters exhibiting a pH index within a definite short range, and that the pH index is consequently often a reliable index also as to whether the chemical and biological group associations will favour or preclude the successful development of such larvae.

8. A new technique is described for the production of a high percentage of successful cultures of Aëdes argenteus larvae and pupae under bacteriologically sterile conditions.

9. The phase of “suspended development” has been investigated and is found to have its probable explanation in the temporary or complete disappearance of micro-organisms on which the conversion of the organic materials of the environment, to a suitable larval diet, depends.

The localization and distribution of the serotoninergic components of the nervous system in the hydatid organism, Echinococcus granulosus, were determined by immunocytochemical techniques in conjunction with confocal scanning laser microscopy (CSLM). The distribution of serotonin immunoreactivity (IR) paralleled that previously described for cholinesterase activity, although it was more widespread. Nerve cell bodies and nerve fibres immunoreactive for 5-HT were present throughout the central nervous system (CNS), occurring in the paired lateral, posterior lateral and rostellar ganglia, their connecting commissures and nerve rings in the scolex and in the ten longitudinal nerve cords that run posteriorly throughout the body of the worm. A large population of nerve cell bodies was associated with the lateral nerve cords. In the peripheral nervous system (PNS), immunoreactive nerve fibres occurred in well-developed nerve plexuses innervating the somatic musculature and the musculature of the rostellum and suckers. The genital atrium and associated reproductive ducts were richly innervated with serotoninergic nerve cell bodies and nerve fibres.

Antibody-resistant strains are less sensitive to suramin and antrycide than antibody-sensitive strains. When living trypanosomes were exposed to suramin and antrycide in vitro, antibody-resistant strains needed 50 times more drugs than antibody-sensitive trypanosomes in order to make them non-infectious to mice. In therapeutic experiments in mice the minimal therapeutic dose of drugs for antibody-sensitive strains was 0·1 mg. but for resistant strains it was 0·3 mg./20 g. mice. Rabbits treated prophylactically with suramin resisted infection with the antibody-sensitive strain for a period of 4 months, but failed to resist infection with the antibody-resistant strain after 2 months.

Rabbits treated prophylactically with antrycide pro-salt, resisted infection with antibody-sensitive strains for a period of 2 months, but failed to resist infection with the antibody-resistant strain even 1 month after injection with the drug. Although trypanosomes can become drug resistant without being antibody resistant it is suggested that, under natural conditions, drug-resistant strains in animals and man develop from antibody-resistant strains, particularly when trypanostatic drugs are used. It is suggested in conclusion from these experiments that strains of trypanosomes which are exposed for some time to antibodies and become antibody resistant after passage through animals like rabbits, as well as those strains frequently passaged through mice, should be used in all tests for the efficiency of chemotherapeutic drugs.

Serine protease inhibitors (serpin) play essential roles in many organisms. Mammalian serpins regulate the blood coagulation, fibrinolysis, inflammation and complement activation pathways. In parasitic helminths, serpins are less well characterized, but may also be involved in evasion of the host immune response. In this study, a Schistosoma japonicum serpin (SjB10), containing a 1212 bp open reading frame (ORF), was cloned, expressed and functionally characterized. Sequence analysis, comparative modelling and structural-based alignment revealed that SjB10 contains the essential structural motifs and consensus secondary structures of inhibitory serpins. Transcriptional profiling demonstrated that SjB10 is expressed in adult males, schistosomula and eggs but particularly in the cercariae, suggesting a possible role in cercarial penetration of mammalian host skin. Recombinant SjB10 (rSjB10) inhibited pancreatic elastase (PE) in a dose-dependent manner. rSjB10 was recognized strongly by experimentally infected rat sera indicating that native SjB10 is released into host tissue and induces an immune response. By immunochemistry, SjB10 localized in the S. japonicum adult foregut and extra-embryonic layer of the egg. This study provides a comprehensive demonstration of sequence and structural-based analysis of a functional S. japonicum serpin. Furthermore, our findings suggest that SjB10 may be associated with important functional roles in S. japonicum particularly in host-parasite interactions.

With the aim of evaluating the utility of marine parasites as indicators of zoogeographical regions in the South West Atlantic, we analyzed data on assemblages of long-lived larval parasites of 488 specimens of Percophis brasiliensis distributed in 11 samples from nine localities covering the entire distribution of the species in the Argentine biogeographical Province. Near half a million long-lived parasite individuals belonging to 17 species present in the whole sample displayed clear latitudinal patterns. Data for parasite assemblages at infracommunity and component community levels were analysed in relation to the geographical distance. Significant similarity decay of parasite assemblages over distance was observed, with those based on abundances and mean abundances showing departures from predicted values of regressions. These departures were represented by higher dissimilarities between samples coming from different zoogeographical regions than between those caught within the same region, independently of the distance separating them. Consequently, zoogeographical regions were identified in a distance-decay context. Multivariate analyses corroborated a close fit of similarity between assemblages to existing zoogeographical classifications. Regressions representing distance decay of similarity, and the identification of their outliers, can therefore shed light on the existence of discontinuities or uniformities in the geographic distribution of parasite assemblages and, in turn, in the zoogeography of their fish hosts.

This study examines the effects of Taenia crassiceps cysticerci on the viability of a human T lymphocyte cell line (Jurkat). Both budding and non-budding T. crassiceps metacestodes were cultured over 24 and 48 h in the presence of Jurkat cells. Cell viability decreased with increasing numbers of cysticerci, particularly budding cysticerci. Single cell gel electrophoresis (comet) analysis, which grades DNA damage, showed a significant increase in apoptosis at 24 and 48 h. The morphology of treated cells was determined using acridine orange with the classical morphology of apoptotic bodies seen to increase with increasing cysticerci numbers over time. These results indicate that parasite-induced apoptosis occurs during murine cysticercosis. Such a mechanism may be important in survival of other metacestode infections of medical or veterinary importance.

To recognize the stage conversion of Toxoplasma gondii between tachyzoite and bradyzoite in live host cells, a transgenic T. gondii line, which expressed stage-specific red and green fluorescence, was constructed. T. gondii PLK strain tachyzoites were stably transformed with genes encoding red fluorescent protein (DsRed Express) and green fluorescent protein (GFP) under the control of tachyzoite-specific SAG1 and bradyzoite-specific BAG1 promoters, respectively. The resulting transgenic parasite was designated PLK/DUAL. When PLK/DUAL was cultured in pH 7·0 medium, the PLK/DUAL zoites expressed red fluorescence, but no detectable levels of green fluorescence were observed. The PLK/DUAL zoites reacted with anti-SAG1 antibody, but not anti-BAG1 antiserum. When PLK/DUAL was cultured under high pH conditions, or in the presence of the p38 MAPK inhibitor SB202190, a small number of zoites expressed green fluorescence and were BAG1 positive. C57BL/6J mice were infected with PLK/DUAL tachyzoites. During the acute and reactivating phase, zoites expressed red fluorescence. However, green fluorescence was not detectable. By contrast, latent cysts expressed green fluorescence. The stage-specific dual fluorescence of PLK/DUAL facilitates identification of the parasitic stage in live cells, with the advantage that fixation or immunostaining is not required.

To evaluate the distribution of asymptomatic infection by Leishmania infantum in a metropolis in Brazil with different relative risks (RRs) for disease and risk factors associated with the infection, an ecological study was conducted using a Bayesian approach to estimate the RR of human visceral leishmaniasis (HVL) based on cases between 2008 and 2011. The areas were categorized and selected according to disease incidence: low (area-1), medium (area-2) and high (area-3). Cross-sectional study enrolling 935 children was used to estimate the prevalence of infection by L. infantum. Volunteers from these three areas were tested for L. infantum infection by ELISA (rK39 and soluble antigens). Infection prevalence rates were estimated and compared with the RR of disease. Multilevel logistic regression model evaluated the relationship between infection and the analysed variables. The RR of HVL was distributed heterogeneously in the municipality. The infection prevalence rates were: 34·9% in area-1; 29·3% in area-2; and 33·6% in area-3, with no significant differences between these areas. The variables ‘Presence of backyards in the neighbourhood’ and ‘Younger children’ were associated with L. infantum infection. We conclude that infection by L. infantum affects a significant proportion of the infant population regardless of the RR of disease.

A cDNA encoding Fg14-3-3 protein 1 was cloned by immunoscreening of an adult-stage Fasciola gigantica cDNA library using a rabbit antiserum against tegumental antigens of the parasite. The protein has a deduced amino acid sequence of 252 residues and a calculated molecular weight of 28·7 kDa. It shows sequence identity values between 57·6 and 58·1% to the human 14-3-3 beta, zeta, theta, and eta proteins and is in a phylogenetic cluster with the 14-3-3 protein 1 of Schistosoma spp. Nucleic acid analyses indicate that the Fg14-3-3 protein 1 is encoded by a single copy gene and that this gene is expressed as a transcript of 1250 nucleotides. In adult and 4-week-old parasites the gene's transcriptional and translational products were localized in the gut epithelium, parenchyma, tegument cells, and in the reproductive organs. An antiserum against recombinant Fg14-3-3 protein 1 detected a slightly smaller 14-3-3 protein in the parasite's excretion/secretion material and showed cross-reactivity with 14-3-3 proteins in extracts of other trematodes and mouse. Antibodies against Fg14-3-3 protein were detected in the sera of rabbits as early as 2 weeks after infection with metacercariae of F. gigantica and the antibody titre increased continuously over a 10-week observation period.

Changes in the specialization of parasite–host interactions will be influenced by variations in host species composition. We evaluated this hypothesis by comparing the composition of bats and bat flies within a roost cave over one annual. Five bat and five bat fly species occupied the cave over the course of the study. Bat species composition was 40% different in the rainy season compared with the dry–cold and dry–warm seasons. Despite the incorporation of three new bat species into the cave during the rainy season, bat fly species composition was not affected by seasonality, since the bats that arrived in the rainy season only contributed one new bat fly species at a low prevalence. Bat–bat fly ecological networks were less specialized in the rainy season compared with the dry–cold and dry–warm seasons because of the increase of host overlap among bat fly species during this season. This study suggests that seasonality promote: (1) differences in host species composition, and (2) a reduction in the specialization of host–parasite ecological networks.

Studies of blood parasite infection in nestling birds rarely find a high prevalence of infection. This is likely due to a combination of short nestling periods (limiting the age at which nestlings can be sampled) and long parasite prepatent periods before gametocytes can be detected in peripheral blood. Here we examine rates of blood parasite infection in nestlings from three Columbid species in the UK. We use this system to address two key hypotheses in the epidemiology of avian haemoparasites: first, that nestlings in open nests have a higher prevalence of infection; and second, that nestlings sampled at 14 days old have a higher apparent infection rate than those sampled at 7 days old. Open-nesting individuals had a 54% infection rate compared with 25% for box-nesters, probably due to an increased exposure of open-nesting species to dipteran vectors. Nestlings sampled at 14 days had a 68% infection rate compared with 32% in nestlings sampled at 7 days, suggesting that rates of infection in the nest are high. Further work should examine nestlings post-fledging to identify rates of successful parasite infection (as opposed to abortive development within a dead-end host) as well as impacts on host post-fledging survival and behaviour.

Schistosoma kisumuensis n. sp. is described based on 6 adult males and 2 adult females collected from the circulatory system of 3 murid rodent species, Pelomys isseli, Mastomys natalensis, and Dasymys incomtus. Specimens were collected from a single location, Nyabera Swamp, in Kisumu, Kenya in the Lake Victoria Basin. This new species is morphologically similar to members of the S. haematobium group, currently represented by 8 species parasitizing artiodactyls and primates, including humans. Schistosoma kisumuensis differs from these species by producing relatively small Schistosoma intercalatum-like eggs (135·2×52·9 μm) with a relatively small length to width ratio (2·55). Comparison of approximately 3000-base-pair sequences of nuclear rDNA (partial 28S) and mtDNA (partial cox1, nad6, 12S) strongly supports the status of S. kisumuensis as a new species and as a sister species of S. intercalatum. The cox1 genetic distance between these two species (6·3%) is comparable to other pairwise comparisons within the S. haematobium group. Separation of the Congo River and Lake Victoria drainage basins is discussed as a possible factor favoring the origin of this species.

Haemonchosis is an acute problem in India. Three species of Haemonchus occur. These are H. contortus (Rudolphi, 1803) which occurs in sheep, goat, cattle, buffalo and other ruminants; H. longistipes Railliet && Henry, 1909 occurring in camels and H. similis Travassos, 1941 in cattle. Seasonal fluctuation of H. contortus occurs, with infection being more frequent in autumn ≻ summer ≻ winter ≻ spring, and infection in sheep and goats has been reported to be 100 %. Various aspects of haemonchosis have been investigated. However, the fundamental and the most challenging areas in Haemonchus research remain almost untouched. In order to forecast the development of haemonchosis, epidemiological studies should be undertaken on the prevalence, distribution and seasonal incidence of the worm to understand the effects of climatological factors like rainfall, humidity and temperature. The sparse and inadequate studies of metabolism need to be extended to other areas of Haemonchus biochemistry to provide additional possibilities for exploration of hostparasite differences. Clinical haemonchosis in sheep, goats and other ruminants should be studied in order to calculate losses caused by mortality and the cost of anthelmintic medication. Most anthelmintics are expensive, and are not easily available to our farming community, therefore a search for many more indigenous anthelmintics (such as Wopell, Krimos, Sonex etc) must be made. Detailed knowledge of the mode of action and route of entry of anthelmintics at the molecular level could also be beneficial, particularly when the strains of H, contortus have been identified as resistant to some anthelmintics. The prospects of producing a vaccine against H. contortus should be explored. Attempts in this field have already been made by overseas workers (see Clegg && Smith, 1978) and in India too, work has been undertaken at Kerala (Sathianesan, personal communication). Therefore, because of its economic importance and wide distribution, H. contortus provides fascinating research material particularly in the warmer regions of the world.