Mating preference inSchistosoma mansoni

We investigated the suitability of an in vitro culture system for measurement of mating behavior ofSchistosoma mansoni. The criteria used to evaluate this system were the level of phosphorylated nucleotides, egg production, and mating status of parasites. The level of ATP, ADP, AMP, and G6-P was measured at different time intervals during cultivation of worm pairs and remained essentially the same as that of control worms for up to 6 days. Egg production was observed in this system during 19 days of cultivation. Peak egg production occurred on day 4 with 72% of the total eggs being laid during the first week of cultivation. The variability in the number of eggs produced by different pairs ofS. mansoni necessitated the selection and matching of tubes with the same number of eggs after 48 hr. This permitted the detection of small changes in egg production by decreasing intertube variation. Mating recognition between male and femaleS. mansoni was evaluated by culturing separated adult worms with their original partner or with a different partner. During the first 24 hr, mating occurred among a greater percentage of worm pairs comprised of their original partner than among worm pairs comprised of different partners (P < 0.001). After 48 and 72 hr of cultivation, these differences were not statistically significant. Similar results were obtained with a culture of mixed males and females. Two drugs were studied for their effects on the mating ofS. mansoni in vitro. Aminoglutethimide (AG) at a concentration of 1 × 10^–4 had no effect on the frequency of mating whereas diethyldithiocarbamate (DDC) completely inhibited mating at a concentration of 3 × 10^–6 M and reduced the level of ATP in these worms.     

Elemental changes during sexual maturation inSchistosoma mansoni

Changes in the elemental composition ofSchistosoma mansoni in relation to age and sexual status are described, and studies on the structural localization of calcium in adult worms and the elemental composition of the tegument-subtegumental tissues are reviewed. Some preliminary results on the distribution of Ca²⁺-dependent ATPase activity in the tegument of worms from both mixed and single-sex infections are also presented.     

Portal serum constituents possible determinants for anatomical localization ofSchistosoma mansoni during maturation and reproduction

Coupled adult pairs ofSchistosoma mansoni were incubated in medium containing either peripheral or portal serum from rat, rabbit, hamster, or man, and egg production was measured daily. In all cases egg production was significantly increased for pairs in the presence of portal sera compared with that in the presence of peripheral sera. Fractionation of rabbit portal serum according to molecular weight demonstrated that the most active component(s) were in the range of 2000 to 50,000. Similarly, a rat portal serum fraction in the range of 2000 to 30,000 molecular weight was most stimulatory. These fractions were as effective in stimulating oviposition as whole portal serum. Conclusions: (1) portal serum factor(s) exist that stimulateS. mansoni oviposition in vitro; (2) they are present in susceptible and nonsusceptible hosts; and (3) the molecular weight range for the active components is larger than would be expected for simple carbohydrates, amino acids, or free fatty acids absorbed from the gastrointestinal tract.     

Limitations to schistosome growth and maturation in nonpermissive hosts

The life cycle of schistosomes is reviewed in its various steps, both in permissive hosts (in which the cycle is completed) and in nonpermissive hosts (which excrete no viable eggs as a result of the infection). A large worm loss occurs at (or after) the lung stage in both types of hosts (normal attrition) and some nonpermissive hosts (like the rat) have an additional elimination of worms from the portal circulation. Worm growth and reproductive maturation are also impaired in several nonpermissive hosts and the possible host-parasite interactions leading to such limitations are discussed, with special reference to hormonal influences. Attention is also given to peculiar phenomena occurring in some hosts, like the late portal worm elimination in rhesus monkeys, the migration from mesenteric veins to lungs in rats, and the block to egg excretion in guinea pigs. The steps of the schistosome life cycle which appear vulnerable in several hosts are contrasted with the steps which are carried out successfully in the majority of hosts studied.     

Biology of tegument associated IgG-Fc and C3 receptors inSchistosoma mansoni

Mechanisms by which schistosomes escape host immune responses are reviewed, with particular emphasis regarding the possible contributions of host or host-like Fc and C3 receptors on adult parasites.     

Extraction of intersexual chemoattractants fromSchistosoma mansoni

FemaleSchistosoma mansoni and their excretory-secretory (ES) products were extracted with a series of solvents to provide fractions of varying polarity. These fractions were assayed for chemoattractivity to males in vitro. One major component of these mixtures was found to be nonattractive and was identified as cholesterol.n-Pentane- and ether-soluble fractions derived from ES products exhibited chemoattractive activity comparable to that possessed by whole-worm extracts, but appeared to be simpler mixtures.     

In vitro orientation of maleSchistosoma mansoni to extracts derived from female schistosomes

Chemical orientation of adult maleSchistosoma mansoni was studied during cultivation in vitro. Directional preference was assessed in culture vessels marked with compass coordinates by the application of circular statistics for determining clustering and orientation to a predicted direction. Organic solvent extracts of fresh female schistosomes and supernatant fluids from 72-hr cultures of female parasites were tested for potential chemotactic activity. Analysis showed significant (P < 0.05) directional preference and clustering of male worms towards test compounds at all time periods (24, 48, and 72 hr) with one mixture of female extracts and at one of three time periods with a second female extract. Male worms did not respond to ecdysone, cholesterol, or solvent controls by orienting in predicted direction, although clustering was observed on two of 12 occasions. These results indicate the presence of a chemoattractant compound(s) in female extracts.     

Internal chemical communication within flatworms

An understanding of reproductive function is important for control of parasitic helminths. In cestodes and trematodes virtually nothing is known about regulatory and coordinating mechanisms that control maturation, gamete formation, egg production, and related processes. Neurosecretory neurons have been reported in various species but specific modes of action of neurohormones have yet to be demonstrated. The role of ecdysone is being investigated.     

Time-lapse video tape documentation of chemical orientation bySchistosoma mansoni in vitro

In vitro, the opposite sexes ofS. mansoni are attracted to each other by some means of premating communication which culminates in aggregation and copulation of sexual pairs within 24 hr. The system used for time-lapse video tape documentation of worm sexual behavior in vitro is described and evidence of sexual chemoattraction is presented.     

Effects of steroids and steroid synthesis inhibitors on fecundity ofSchistosoma mansoni in vitro

In vitro egg production bySchistosoma mansoni was examined following a 72-hr incubation period in the presence of various steroids, steroid precursors, or steroid synthesis inhibitors. Mevinolin, an inhibitor of cholesterol biosynthesis, significantly decreased egg production at 10^–6 M. However, neither cholesterol (at 10^–5 M) nor any of its hydroxylated derivatives (at 10^–4 M) affectedS. mansoni fecundity. Dianisidine, an inhibitor of cholesterol metabolism to hormonal products, was also ineffective in influencing egg production. The steroid hormones progesterone, 17-hydroxyprogesterone and medroxyprogesterone acetate, all significantly decreased egg production; however, parasite muscle tension was also significantly reduced by the concentrations of these steroids needed to produce an effect on egg laying. Various androgenic hormones (androsterone, androstenedione, testosterone) and estrogenic hormones (17-estradiol and estrone) demonstrated no effect on in vitro egg production. Significant elevation in the rate of parasite oviposition was seen in the presence of the corticosteroid prednisolone (at 10^–5 M), but not by dexamethasone, a corticosteroid analog.     

Developmental changes in energy metabolism ofSchistosoma mansoni and physiological role of oxygen in maintaining parasite function

The time course of the conversion from a cyanide-sensitive to a cyanide-insensitive energy metabolism in immatureSchistosoma mansoni was followed by correlating transitions in CO₂ and lactate formation with physiological properties of the parasite. Volume conducted electrical potentials and measurement of CO₂ evolution indicate that 3-hr posttransformational schistosomula are highly sensitive to 1 mM cyanide. By 24 hr after transformation, evolution of CO₂ under control conditions is reduced by 77% from 3-hr levels, while lactate excretion rises by 84%. At the 24-hr stage, neither cyanide nor rotenone affects the frequency or magnitude of endogenous electrical transients, but cyanide does eliminate 83 % of the already reduced levels of CO₂ evolved in 24-hr schistosomula. The adult parasite evolves a low level of CO₂ which is reduced by 88% in the presence of 1 mM cyanide. No significant Pasteur effect is detected, however, and endogenous electrical activity as well as mechanical responses of the adult musculature are unaffected by cyanide exposure. Furthermore, adult schistosomes were not adversely affected in terms of the physiological parameters measured by 24-hr incubations in oxygen-free medium. Adults were only marginally affected by 24-hr exposure to several respiratory inhibitors, but responded rapidly to some uncouplers of oxidative phosphorylation, including 2,4-DNP, carbonyl cyanide 3-chlorphenylhydrazone, and closantel. Our results indicate that schistosomula continue to rely on cyanide-sensitive respiratory components for at least 3 hr after transformation; by 24 hr, however, the parasites are metabolically similar to the adult stage, i.e, they depend on lactate fermentation for most of their energy requirements.     

Influence of mating on surface nutrient exchange in schistosomes

In schistosomes, the mating process influences male-female transfer and gender-specific exchange of nutrients. The paired male schistosome provides glucose to the female partner. Male-to-female intertegumental transfer of¹⁴C-labeled glucose,¹⁴C-labeled 3-O-methylglucose, [¹⁴C]2-deoxyglucose and 2-fluorodeoxyglucose has been demonstrated in schistosomes. This phenomenon has been studied extensively inSchistosoma mansoni, and confirmed inSchistosoma japonicum, as well asS. haematobium, using radioactive pulsing methods. Male schistosomes contain significantly greater quantities (nmol/mg worm water) of glucose than do females. The transfer of glucose is apparently not an energy-dependent process, but occurs along this concentration gradient. Most, if not all, of the glucose utilized by the female is transferred from the male partner via tegumentary-facilitated diffusion mechanisms, free diffusion, or some combination of these two components. Unpaired male schistosomes contain greater quantities of glycogen than do comparable paired schistosomes, indicating that the presence of a female in the gynecophoral canal depletes the reserves of the male partner; this is additional indirect evidence for male-to-female transfer of glucose. Tegumentary surface uptake of acidic amino acids has been compared in paired and separated male and female schistosomes. InS. mansoni, a saturable carrier-mediated mechanism has been defined which operates only in unpaired male and unpaired female teguments. In contrast, the uptake of aspartate and glutamate is not seen in paired worms of this species. Tegumental uptake of acidic amino acids is not observed in paired or unpaired male or femaleS. japonicum. However, inS. haematobium, significant quantities of aspartate are taken up by both paired and unpaired male schistosomes. Measurable aspartate uptake is seen in paired femaleS. haematobium, but in the separated female, there is minimal uptake of this acidic amino acid. Thus the permeability of the teguments of human schistosome species to acidic amino acids is modified in response to the paired state inS. mansoni andS. haematobium, but these characteristics are not shared byS. japonicum.     

Lectin studies of surface carbohydrates and induction of gland secretion in the free-living stages ofSchistosoma mansoni

Secretion from glands was observed when cercariae ofSchistosoma mansoni were exposed to certain lectins. Lectins fromMaclura pomifera, Pisum sativum, andTriticum vulgaris, were effective at 7.5 /ml. The effects of cercarial gland secretion caused byT. vulgaris agglutinin andArachis hypogaea agglutinin were blocked by pretreatment with the inhibiting glycan. Discharge of glands was not visualized after exposure of miracidia to lectins. The distribution of five labeled lectins was determined on live miracidia and cercariae. OnlyT. vulgaris agglutinin generally labeled the cercarial and miracidial bodies. Specific labeling occurred with the other lectins on the anterior, in glands or on their secretions, in flame cells in both stages, and on an unidentified ring of cells in miracidia. The possible mechanisms involved in changes caused by the lectins are discussed.     

Kernel fatty acid and triacylglycerol composition for three almond cultivars during maturation

The kernel oil content, kernel FA and TAG composition, kernel moisture content, and kernel weight as well as fruit weight of three almond cultivars (Achaak, Mazetto, and Perlees) were monitored during the maturation of kernels. Lipid fractions of all almond samples were extracted using a mixture of chloroform and methanol. FAMF and TAG contained in these fractions were analyzed by GC and HPLC, respectively. The ratio of kernel to fruit weight appears to be a good indicator of almond kernel development. The total lipid content of developing almond kernels exhibited a sigmoidal pattern with time, similar to seeds and kernels of other higher plants; the cultivar Achaak showed a higher rate of lipid accumulation. The proportion of eleic acid (0) dominated at the later stage of maturation for all three almond cultivars. Although there was no significant difference in the FA composition for the three cultivars studied, marked differences were observed in their TAG profiles. Ten TAG species identified were LLL, LLO, LnOO, LOO, LOP, PLP, OOO, POO, POP, and SOO, where L represents linoleic acid; Ln, linolenic acid; P, palmitic acid; and S, stearic acid. The difference in the TAG profile can be useful for distinguishing various cultivars. The oil of Mazetto cultivar kernes exhibited a TAG composition comparable to that of olive oil.     

Chemical communication in adult schistosomes

Lipids released bySchistosoma mansoni adult males attract females in vitro. Lipid release is modulated by the presence of other worms. AlthoughS. mansoni males release lipid when paired with females, the release is enhanced when they are separated.S. japonicum adults release more free sterols when incubated individually than when incubated together. Similarly, individually incubatedS. haematobium males release more free sterols than when incubated in groups. However,S. haematobium females incubated in groups release more free fatty acids than do equal numbers of males or pairs incubated in groups. There is evidence thatS. mansoni adult females concomitantly accumulate and release cholesterol in the absence of an exogenous supply, although de novo synthesis of cholesterol in schistosomes has not yet been demonstrated. Schistosomula and adult schistosomes incorporate exogenous lipids. Lipids are incorporated chiefly through the tegument. Cholesterol is transferred between males and females.     

Structural and metabolic changes in femaleSchistosoma mansoni following male stimulation

Pairing of males and females from single-sex infections results in the multiplication and differentiation of undifferentiated cells of the vitelline lobule culminating in the production of mature vitelline cells involved in egg shell formation. These changes are accompanied by increases in the rate of uptake of tyrosine, thymidine, and an increased accumulation of calcium.     

Identification of sex-linked antigens ofSchistosoma mansoni by immunoelectrophoresis and immunoblotting

Immunoelectrophoresis and immunoblotting techniques, using sera from hyperimmune rabbits and infected mice, were used to analyze antigens ofSchistosoma mansoni. A comparison was made between the antigenic composition of adult male and female worms, isolated from monosexually and bisexually infected hamsters. A large number of female-specific and a few male-specific antigens were detected. The antigenic composition of females isolated from monosexually infected hamsters was shown to be very different from that of paired females. The presence of a major female-specific antigen with an apparent molecular weight of 32 kd in paired females only, suggests that synthesis of this antigen by female worms is initiated by the male partner. Analysis of the humoral immune response of mice receiving bisexual or monosexual infections showed that both mono- and bisexually infected mice develop a major humoral immune response to a 36-kd polypeptide antigen. A response to a major polypeptide antigen with an apparent molecular weight of 60 kd was observed in bisexually infected mice only. No antibodies reactive with this 60-kd polypeptide are formed during mon-osexual infections with either male or female worms. The 60-kd polypeptide was, however, present in monosexually reared females. This presence, and the presence in bisexually reared males, suggests a transfer of this antigen from females to males.     

Isolation,identification, and synthesis of sex pheromone components of female tea cluster caterpillar,Andraca bipunctata walker (Lepidoptera: Bombycidae) in Taiwan

Octadecanal (18:Ald), (E)-11-octadecenal (E11–18:Ald), (E)-14-octadecenal (E14–18:Ald) and (E,E)-11,14-octadecadienal (E11,E14–18:Ald) were isolated and identified as major components from the pheromone glands of the tea cluster caterpillar,Andraca bipunctata, in Taiwan by analyzing the mass spectra of gland components and their DMDS adducts. GC retention times and mass spectra of the components were in agreement with those of authentic synthetic compounds. The average amount of 18:Ald,E11–18:Ald,E14–18:Ald andE11,E14–18:Ald per female gland (1 to 3 days old) was 121±76, 50±20, 187±75, and 237±110 ng, respectively, in a ratio of 20:8:31:41. SyntheticE11,E14–18:Ald caught more males than each of the other three components or blank control in field trapping tests.E11,E14–18:Ald is reported as an insect sex pheromone for the first time. Male antenna responded toE11,E14–18:Ald strongly in an EAG analysis. Furthermore, 4 hr after the injection of PBAN (pheromone biosynthetic activating neuropeptide) into decapitated female moths (2 days old), the percentage of theE11,E14–C18 Ald in the gland extract increased from 0% to 75.5%, which was also significantly more than that of unligated and uninjected control at 55.1%. All these data indicated thatE11,E14–18:Ald is the sex pheromone of theAndraca bipunctata in Taiwan.     

Influence of lectins on female sex pheromone reception byHeterodera schachtii (Nematoda: Heteroderidae) males

A bioassay was developed in which the chemotactic behavior of males of the sugarbeet cyst nematodeHeterodera schachtii towards the female sex pheromone can be observed and documented at any time. The standardized test, performed under sterile conditions, gives reproducible results within 1–2 hr. Incubation of males with the lectins fromCanavalia ensiformis, Triticum vulgare, Arachis hypogaea, Helix pomatia, andLimax flavus did not affect chemotactic attraction to the sex pheromone. Supplementing the bioassay medium with the lectin fromC. ensiformis also had no effect.C. ensiformis lectin binding to the surface of the exudate produced by the secretory gland cell of the amphids, the main chemoreceptors, did not inhibit the passage of fluorochrome fluorescein isothiocyanate into the amphidial canals, where the receptor dendrites are localized. Amphidial exudate synthesis was enhanced during pheromone reception.     

A material isolated from human hands that attracts female mosquitoes

The residue left on glass surfaces by human hands was found to be attractive to femaleAedes aegypti (L.) andAnopheles quadrimaculatus Say mosquitoes. The material lost half of its activity in 1 hr. A solvent wash technique was developed to recover and concentrate the residuum from handled glass beads. The residuum could be recovered effectively with absolute ethanol and less effectively with several other solvents. More mosquitoes were attracted to heated than to unheated residuum, an indication of its volatility. Also, attraction of the residuum decreased with decreasing concentration or dose. Concentrated residuum collections, stored under refrigeration and tested for longevity, showed no appreciable loss of attractiveness up to 60 days of storage.Diptera: Culicidae.This research was supported partly by the Medical Research and Development Command, Office of the Surgeon General, U.S. Army. Mention of a commercial or proprietary product does not constitute an endorsement by the USDA.