Schistosoma: cross-reactivity and antigenic community among different species

It is not unusual to find common molecules among different species of the genus Schistosoma. When those molecules are antigenic, they may be used in immunodiagnosis and vaccines, but they could also be applied to taxonomic and evolutionary studies. To study cross-reactivity and antigenic community among different species of schistosomes, plasmas from laboratory animals infected with Schistosoma bovis, S. guineensis, S. rodhaini, S. haematobium, and four strains of S. mansoni were evaluated with a crude extract of adult worms of S. mansoni by Western blot. Using the multiple antigen blot assay, plasmas from these infected animals were exposed to a selected group of synthetic peptides from Sm28GST, Sm28TPI, Sm elastase, Sm97, Sm32, Sm31, and Sm Cathepsin L. The results presented herein demonstrate differential cross-reactivity and antigenic community among the Mansoni and Haematobium groups of schistosomes, which is of relevance as an additional new tool for phylogenetic studies of schistosomes as well as for diagnosis and vaccine purposes.     

Vitellogenin and vitellin from the blowfly Calliphora vicina: Occurrence,purification and antigenic characterization

The occurrence and purification of vitellogenin and vitellin from Calliphora vicina Rob.-Dev. (= C. erythrocephala (Meig.)) are described together with the preparation of specific anti-vitellogenin antibodies. C. vicina vitellogenin and vitellin were purified from ovaries and eggs respectively; both proteins contain two polypeptide subunits identical to the dominating polypeptides in the growing oocytes. The polypeptides show molecular weights of 52,000 and 48,500 respectively, and are associated with carbohydrate and lipid. Polypeptides of similar size could be identified in haemolymph from yolk-depositing females, but were absent in ovariectomized females. The anti-bodies specifically precipitated the vitellogenin polypeptides from fat body homogenates of females depositing yolk or from the purified vitellogenin. Therefore, these antibodies were judged suitable for use in a study on the ultrastructural localization of vitellogenin in fat body cells (Thomsenet al., 1980).     

Trigeminal and facial innervation of cirri in three teleost species

Summary Innervation of the cirri in three teleost species (Hypsoblennius gilberti, Hypsoblennius gentilis, Oxylebius pictus) was investigated with the use of HRP- and cobalttracing techniques. All projections were found to be ipsilateral. Labeled cells were demonstrated in both portions of the trigeminal ganglion and in the facial ganglion. Cirrus nerve fibers running in the trigeminal nerve project to terminal fields in an isthmic sensory trigeminal nucleus, to areas adjacent to the descending trigeminal root in the brainstem, and to the medial funicular nucleus in the medulla. Distribution of labeled cells in the trigeminal ganglion complex suggests a functional distinction of the two ganglion portions. Cirrus nerve fibers belonging to the facial nerve terminate in a circumscribed part of of the facial lobe, indicating a somatotopic projection. Pathways were principally the same in all three species investigated. Findings of facial innervation of teleost cirri suggest a suspected gustatory function of teleost head appendages.     

Intracellular ion levels in erythrocytes and hepatocytes isolated from three teleost species

Intracellular concentrations of Na⁺ and K⁺ were similar (∼75 mmol l⁻¹) in rainbow trout Oncorhynchus mykiss hepatocytes directly following isolation by collagenase digestion, but partial recovery occurred over 6 h with K⁺ levels increasing to 110 mmol l⁻¹ and Na⁺ levels decreasing to 42 mmol l⁻¹. Black bullhead Ameiurus melas hepatocytes exhibited higher intracellular concentrations of K⁺ (90 mmol l⁻¹) than Na⁺ (55 mmol l⁻¹) with no recovery occurring over 6 h following cell isolation. Concentrations of Na⁺, K⁺ and Cl⁻ in eel Anguilla rostrata hepatocytes were similar (∼ 55 mmol l⁻¹) following isolation, with no recovery occurring over time. Erythrocytes from all species apparently did not experience an intracellular ion imbalance following isolation as indicated by high K⁺ levels (<140 mmol l⁻¹) and low Na⁺ levels (<40 mmol l⁻¹) during the entire 24-h monitoring period. Although hepatocytes from all species exhibited an ion imbalance post-isolation, comparison of their in vitro intracellular Na⁺ and K⁺ concentrations with those in plasma demonstrated that directionally correct ion gradients still exist across the cell membrane, albeit differing from those that would be found in the tissue in vivo .     

Intracellular pH regulation in hepatocytes isolated from three teleost species.

The mechanisms of intracellular pH (pH(i)) regulation were studied in hepatocytes isolated from three species of teleost: rainbow trout (Oncorhynchus mykiss), black bullhead (Ameiurus melas) and American eel (Anguilla rostrata). Intracellular pH was monitored over time using the pH-sensitive fluorescent dye BCECF in response to acid loading under control conditions and in different experimental media containing either low Na(+) or Cl(-) concentrations, the Na(+)-H(+) exchanger blocker amiloride or the blocker of the V-type H(+)-ATPase, bafilomycin A(1). In trout and bullhead hepatocytes, recovery to an intracellular acid load occurred principally by way of a Na(+)-dependent amiloride-sensitive Na(+)-H(+) exchanger. In eel hepatocytes, the Na(+)-H(+) exchanger did not contribute to recovery to an acid load though evidence suggests that it is present on the cell membrane and participates in the maintenance of steady-state pH(i). The V-type H(+)-ATPase did not participate in recovery to an acid load in any species. A Cl(-)-HCO(3)(-) exchanger may play a role in recovery to an acid load in eel hepatocytes by switching off and retaining base that would normally be tonically extruded. Thus, it is clear that hepatocytes isolated from the three species are capable of regulating pH(i), principally by way of a Na(+)-H(+) exchanger and a Cl(-)-HCO(3)(-) exchanger, but do not exploit identical mechanisms for pH(i) recovery. J. Exp. Zool. 284:361-367, 1999.     

The isolation, purification and amino-acid sequence of insulin from the teleost fish Cottus scorpius (daddy sculpin)

Insulin from the principal islets of the teleost fish, Cottus scorpius (daddy sculpin), has been isolated and sequenced. Purification involved acid/alcohol extraction, gel filtration, and reverse-phase high-performance liquid chromatography to yield nearly 1 mg pure insulin/g wet weight islet tissue. Biological potency was estimated as 40% compared to porcine insulin. The sculpin insulin crystallised in the absence of zinc ions although zinc is known to be present in the islets in significant amounts. Two other hormones, glucagon and pancreatic polypeptide, were copurified with the insulin, and an N-terminal sequence for pancreatic polypeptide was determined. The primary structure of sculpin insulin shows a number of sequence changes unique so far amongst teleost fish. These changes occur at A14 (Arg), A15 (Val), and B2 (Asp). The B chain contains 29 amino acids and there is no N-terminal extension as seen with several other fish. Presumably as a result of the amino acid substitutions, sculpin insulin does not readily form crystals containing zinc-insulin hexamers, despite the presence of the coordinating B10 His.     

Surface folds in superficial epidermal cells of three species of teleost fish

Scanning electron microscope studies of fish epidermis demonstrate whorled arrangements of microfolds on the surface of superficial epidermal cells in the following species of teleost fish: the guppyfish Poecilia reticulata (Peters), the goldfish Carassius auratus auratus (L.), and the clown anemone fish Amphiprion percula (Lacépède). The pattern is most pronounced in epidermal cells covering the scales, but is also seen in the epidermal cells of the tail and body fins. The whorled pattern was not present on the gill epithelium or the surface of the eyes. These observations differ from transmission electron microscope studies which have described the skin surface as being composed of microvilli.     

Mycorrhizal associations and reproductive isolation in three closely related Orchis species

Background and Aims

The maintenance of species boundaries in sympatric populations of closely related species requires some kind of reproductive isolation that limits gene flow among species and/or prevents the production of viable progeny. Because in orchids mycorrhizal fungi are needed for seed germination and subsequent seedling establishment, orchid–mycorrhizal associations may be involved in acting as a post-mating barrier.

Methods

We investigated the strength of post-mating barriers up to the seed germination stage acting between three closely related Orchis species (Orchis anthropophora, O. militaris and O. purpurea) and studied the role of mycorrhizal fungi in hybridization by burying seed packets of pure and hybrid seeds. After retrieval and assessment of seed germination, the fungi associating with protocorms originating from hybrid and pure seeds were determined and compared with those associating with adult individuals using DNA array technology.

Results

Whereas pre-zygotic post-mating barriers were rather weak in most crosses, post-zygotic post-mating barriers were stronger, particularly when O. purpurea was crossed with O. anthropophora. Germination trials in the field showed that seed germination percentages of hybrid seeds were in most cases lower than those originating from pure crosses. In all species pair combinations, total post-mating reproductive isolation was asymmetric. Protocorms associated with a smaller range of fungal symbionts than adult plants, but there was considerable overlap in mycorrhizal associations between protocorms and their respective parents.

Conclusions

Our results suggest that mycorrhizal associations contribute little to reproductive isolation. Pre-mating barriers are probably the main factors determining hybridization rates between the investigated species.     

Partial purification, stereospecificity and stoichiometry of three dehalogenases from a Rhizobium species

Abstract A fast-growing species of Rhizobium that utilized 2,2-dichloropropionate (2,2DCP) and d,l -2-chloropropionate ( dl -2CP) as sole sources of carbon and energy was shown to contain three inducible dehalogenases. These enzymes differed in their substrate specificities: dehalogenase II degraded 2,2DCP, d - and l -2CP, monochloroacetate (MCA) and dichloroacetate (DCA) whilst dehalogenase I showed activity only towards l -2CP and DCA. Dehalogenase III liberated halide from d -2CP and MCA. This is the first report of a dehalogenase acting solely on the d -isomer of a haloalkanoate. All three dehalogenases inverted the isomeric configuration during dehalogenation, forming d (−) and l (+) lactate from l - and d -2CP, respectively.     

Floral traits and isolation of three sympatric Aquilegia species in the Qinling Mountains, China

Floral isolation has been considered to be an important reproductive mechanism governing the species diversification in many genera. In a classic example Aquilegia, sympatric species from North America with diverse floral traits are generally associated with specialized pollinators that prohibit interspecific hybridization. It remains unclear whether species diversification in the genus from Eurasia is also maintained by floral isolation. We investigated floral phenology, floral characteristics and pollinators in three sympatric Aquilegia species (A. ecalcarata, A. incurvata and A. yabeana) in the Qinling Mountains, Shanxi Province, China from 2001 to 2005. The spurless A. ecalcarata flowers earlier than the other two species with nectar spurs but their floral phenology overlaps. Major pollinators of A. ecalcarata are syrphid flies while bumblebees are major for A. incurvata and A. yabeana. Therefore our observations confirm that mechanical isolation through differential pollinators could contribute reproductive isolation between spurless and spurred species, as demonstrated by studies from North America. Whether floral isolation plays a major role in the reproductive isolation between two spurred species (A. incurvata and A. yabeana), however, remains to be seen. Further studies are required to quantify the potential role of geographical isolation because they occupy different habitats.     

Sex-pairing pheromones and reproductive isolation in three sympatric Cornitermes species (Isoptera, Termitidae, Syntermitinae)

The species-specificity of pairing has been studied in three sympatric Neotropical termites: Cornitermes bequaerti, Cornitermes cumulans and Cornitermes silvestrii (Termitidae, Syntermitinae). Bioassays showed that sex attraction was highly species-specific between C. bequaerti and C. cumulans but not between C. cumulans and C. silvestrii. The sex-pairing pheromone of the three species is secreted by the tergal glands of female alates. It consists of a common compound (3Z,6Z,8E)-dodeca-3,6,8-trien-1-ol. In C. bequaerti, this polyunsaturated alcohol is the only compound of the sex-pairing pheromone, whereas it is associated with the oxygenated sesquiterpene (E)-nerolidol in C. cumulans, and with (E)-nerolidol and (Z)-dodec-3-en-1-ol in C. silvestrii. (3Z,6Z,8E)-Dodeca-3,6,8-trien-1-ol is responsible for sexual attraction, whereas (E)-nerolidol, which is inactive in eliciting attraction of male alates, is responsible for the species-specificity of the attraction. This is the first time that a multicomponent sex-pairing pheromone has been identified in termites. The role of (Z)-dodec-3-en-1-ol present on the surface of the tergal glands of the female alates of C. silvestrii could not be definitively determined, but it is suggested that this compound could be involved in the species-specificity of sex attraction with other sympatric species of Cornitermes. Our study shows that the reproductive isolation in termites is due to a succession of factors, as the chronology of dispersal flights, the species-specificity of sex-pairing pheromones and the species-specific recognition.     

Improved method for purification of Xenopus laevis vitellogenin and demonstration of cross-reactivity among wide-range species by radioimmunoassay

This study was performed to improve the purification of Xenopus vitellogenin and establish the radioimmunoassay. The procedure of purification consisted of ammonium precipitation, DEAE-Sephadex chromatography and Sephadex G-200 gel chromatography. Using this procedure, 934 mg vitellogenin was purified from 49 ml of estradiol treated female Xenopus plasma (about 19 mg/ml). Vitellogenins purified from male and female plasma after a single injection of estradiol showed good correspondence in electrophoretic patterns and amino acid compositions, indicating that vitellogenin synthesis in the male occurs in four different genes as in the female. The radioimmunoassay for vitellogenin was established using an antibody in the plasma obtained from rabbits injected with purified Xenopus female vitellogenin. The titer was 20,000 times dilution of the plasma, and the minimum detectable amount of vitellogenin was 0.1 microgram. The cross-reactivity of this antibody with newt vitellogenin was about 65% and that of chick 6%. The cross-reaction was also observed in female bullfrog plasma. Vitellogenin content was increased gradually during the first 6 days after injection of estradiol in female and the elevated level of vitellogenin dropped afterward.     

Genetic differentiation, behavioural reproductive isolation and mixis cues in three sibling species of monogonont rotifers

Many aquatic species usually considered to be 'cosmopolitan' have been identified as cryptic species complexes, based on deep genetic differentiation. However, reproductive isolation among sibling cryptic species has rarely been studied, and interspecific hybridization is common in some taxa.We investigated isolation mechanisms and possible introgression among three cyclical parthenogenetic rotifer species in the Epiphanes senta complex that are found in very different freshwater habitats: temperate floodplains, subtropical desert rock pools and a tropical alpine lake. Whereas Epiphanes ukera is reproductively isolated from E. chihuahuaensis and E. hawaiiensis, the latter hybridize under laboratory conditions.While reproductive isolation is incomplete, RAPD profiles indicated unique genetic signatures and showed no evidence for introgression, indicating that these three species are diverging and have independent evolutionary trajectories.Testing cues for sexual reproduction in these cyclic parthenogens demonstrated that mixis in E. chihuahuaensis and E. ukera is influenced by population density, whereas E. hawaiiensis females rarely produce mictic offspring regardless of density. Different mixis cues are likely to separate sexual periods and effectively cause reproductive isolation between the species. Epiphanes ukera and E. chihuahuaensis males display mate guarding behaviour, and E. ukera males distinguish between conspecific and heterospecific females in mate choice experiments. Geographic isolation, along with different cues for mixis induction and mate recognition, act as reproductive barriers among these sibling species.     

Vitellogenin and vitellin of a millipede Spirostreptus asthenes: Occurrence,isolation and partial characterization

Vitellogenin (Vg), the yolk precursor protein and its product vitellin (Vn) have been identified in hemolymph and fat body of females and mature oocytes in the millipede Spirostreptus asthenes employing double immunodiffusion technique. These two proteins are absent in males indicating that they are female specific. Immunoelectrophoresis has shown that there is only one vitellogenic protein present in S. asthenes. Vg and Vn were isolated by gel filtration. Polyacrylamide gel electrophoresis (PAGE) analysis revealed that Vg and Vn are glycolipoproteins. Vg contains 48.8% protein, 2.2% carbohydrate and 48.9% lipid. Vn is comprised of 52% protein, 2.3% carbohydrate and 45.4% lipid. The lipid components of Vg and Vn include mainly phospholipids such as phosphatidic acid, sphingomyelin, phosphatidyl choline, phosphatidyl ethanolamine and cholesterol. On SDS-PAGE analysis both Vg and Vn yielded five sub units each. The molecular weight of the sub units of Vg was found to be 135, 115, 105, 73 and 56 kDa and those of Vn were 125, 110, 100, 68, and 53 kDa. The vitellogenic system of S. asthenes resembles that of insects. The phylogenetic relationship of the vitellogenic system of this millipede with other arthropod groups is discussed.     

Mating behaviour and reproductive isolation of three species of reptile tick

The mating behaviour of three species of reptile tick, Aponomma hydrosauri, Amblyomma albolimbatum and Amb. limbatum is similar and involves a female sex pheromone which activates males to detach from their hosts and search. After contacting females, a stereotyped six-phase courtship sequence occurs. There are qualitative differences between the species in courtship behaviour at phase 3 (reversal of position by dorsally mounted males) and phase 6 (the copulation position). On-host observations of non-conspecific mating show that females of the three species have species-specific activation pheromones, which is contrary to reports in other species of tick. Such specificity should result in reproductive isolation of the three species; however, under certain circumstances it may not prevent non-conspecific contacts between the sexes. Off-host observations of courtship behaviour show that once males of the three species contact non-conspecific females, they attempt courtship and are persistent with their courtship. Rarely did non-conspecific courtships proceed beyond phase 4 of the courtship sequence, as non-conspecific females did not lift their bodies to allow males venter contact. Differences between the species in leg orientation in the copulation position, together with body size differences, are responsible for a complete barrier to successful non-conspecific copulation. The observations illustrate the role that behavioural mechanisms play in reproductively isolating these three species of tick.     

Production,partial purification and bioassay of toxic metabolites of three plant pathogenic species ofColletotrichum in Nigeria

Colletotrichum lindemuthianum, C. truncatum andC. graminicola, produced metabolites in culture which induced necrotic lesions on susceptible hosts. The highest production was obtained from Richard's medium under shake incubation. The toxic metabolites of these pathogens fluoresce at 254 nm and 366 nm under ultraviolet light. The crude metabolites of theseColletotrichum species inhibited seed germination at the concentration of 100 µg/ml while the potency of the metabolites decreases with increase in dilution to 0.1 µg/ml. Similarly, the metabolites also inhibited growth of seedlings of hosts to the pathogen at 100 µg/ml and the potency again reduced with increasing dilution to 0.10 µg/ml.