An Example of Circular Statistics in Chronobiological Studies: Analysis of Polymorphism in the Emergence Rhythms of Schistosoma Mansoni Cercariae

Chronobiological data concerning the emergence rhythms of Schistosoma mansoni cercariae were transformed into circular variables and analysed using circular statistics. The method proposed allowed three chronobiological patterns to be distinguished for the parasite populations studied, and statistical tests for the homogeneity of samples in each group and the significant differences between groups were also carried out.     

The Difference Between Activity When in Bed and Out of Bed. II. Subjects on 27-Hour “Days”

Nine healthy subjects have been studied while exposed to the normal alternation of light and dark, but with their sleep and activity pattern adjusted to a 27-h “day” for 17 imposed “days.” Rectal temperature showed clearly the competing influences of 27-h and 24-h components, and these were separated by the method of “purification.” The method indicated that the endogenous component had a constant amplitude throughout the experiment and remained entrained to solar (24-h) time; by contrast, the exogenous component followed the imposed 27-h “day” and increased rectal temperature in proportion to the amount of subjects' activity. Wrist movement was used to assess activity while in bed (attempting sleep) and out of bed (when naps were forbidden). While these results confirmed adherence of the subjects to the imposed 27-h “days,” they also showed that the dichotomy between “out of bed” activity and “in bed” inactivity depended on the phase relationship between endogenous (24h) and exogenous (27h) components. Thus, the dichotomy was highest and was equal to that during control days (with a conventional 24-h life-style) when the two components were in phase and lowest when the solar and imposed day were in antiphase. This was due to changes in activity, both during time spent in bed and out of bed.

We confirm that this protocol can produce valuable information about the properties of the circadian system in humans and the value of the process of purification of temperature data. We have established also that the very simple and noninvasive measurement of wrist movement, coupled with its use to calculate dichotomy indices, provides valuable information that both confirms and extends the results obtained from the more conventional (butalso more invasive) measurement of rectal temperature.     

Comparative Morphometrics and Associated Growth Trends of Two Benthophagous Cichlid Species from Lake Malawi (Pisces, Perciformes)

Body shape differences between two benthophagous, sympatric cichlid species, Ctenopharynx pictus and Otopharynx sp. “heterodon nankhumba” were examined for 14 morphometric characters using measurements obtained from 11 morphological landmarks and additional 6 characters from the head region using principal component and regression analyses. The two species, though not closely related, superficially resemble each other and, being benthic feeders, directly compete for food resources unless morphological differences lead to trophic specialization. Hence, the present study explores the presence of such morphological variations. Principal component analysis (PCA), revealed that only four characters loaded highly on PC II and, therefore, account for most of the variations in the external morphology between the two species. C. pictus has longer lower jaws reflecting longer gape, and a shorter abdominal cavity than O. sp. “heterodon nakhumba”. The regression analyses showed significant differences in the allometric coefficients of the lower jaw and abdominal cavity, which might have contributed to differences in overall shape detected between the two species. The difference in gape size between the two species may be related to different feeding habits employed by each species, and such specializations are suggested to reduce interspecific competition for food resources. We suggest that the ensuing ecological separation promotes sympatry of these two species.     

cis-Platinum(II)diaminodichloride-induce mutagenesis in E. coli K12: crowding depression of mutagenesis

cis-Platinum(II)diamminodichloride (cis-PDD)-induced mutations to prototrophy were studied in Escherichia coli. Mutagenesis was not detected in a recA nor in a lexA mutant, but as greater in uvrA strain than in a repair-proficient strain, at a given treatment of cis-PDD. Increasing the plating density above 10⁵ cells per plate did not give an equivalent increase in revertants per plate [crowding depression of mutagenesis (Bockrath et al., 1980)]. Growth rates were similar at different plating densities and crowdign depression of mutagenesis was observed in both excision-proficient and excision-deficient strains.

A filtrate of a plate wash from crowded plates, of either treated or untreated cultuers, further reduced the mutation frequenciews over that due to crowding depression of mutagenesis.     

Rasnitsynala sigambrorum gen. et sp. n., a small odonatopterid ("Eomeganisoptera", "Erasipteridae") from the early Late Carboniferous of Hagen-Vorhalle (Germany)

Besides Erasipteroides valentini (Brauckmann in Brauckmann, Koch & Kemper, 1985), Zessinella siope Brauckmann, 1988, and Namurotypus sippeli Brauckmann & Zessin, 1989, Rasnitsynala sigambrorum gen. et sp. n. is the fourth species of the Odonatoptera from the early Late Carboniferous (Early Pennsylvanian: Namurian B, Marsdenian) deposits of the important Hagen-Vorhalle Konservat-Lagerstätte in Germany. With its wing-span of about 55 mm it is unusually small even for the “Eomeganisoptera”. Its venation resembles other small “Eomeganisoptera”, in particular Zessinella siope. This is why it is here assigned to the probably paraphyletic “Erasipteridae” Carpenter, 1939.     

A biochemical approach toward the systematics of the Leptothorax “muscorum”Group in North America (Hymenoptera: Formicidae)

Enzyme patterns have been used to distinguish among a number of morphologically very similar ant species belonging to the Leptothorax “muscoru” group in North America. Not counting the already described species L. retractus, L. sphagnicolus and L. crassipilis, the complex apparently consists of at least three or four more different taxa.     

Phylogeny of "Oxycanus" lineages of hepialid moths from New Zealand inferred from sequence variation in the mtDNA COI and II gene regions

The phylogeny of the New Zealand hepialid moths was estimated from a 527-bp nucleotide sequence from the mitochondrial DNA cytochrome oxidase subunit I and II gene regions. New haplotypes were identified for Wiseana cervinata, W. copularis, and W. signata. Phylogenetic reconstructions using maximum parsimony and maximum likelihood methods indicated that the four hepialid lineages Aenetus, Aoraia, "Oxycanus" Cladoxycanus, and "Oxycanus" s. str. hypothesized by Dugdale (1994) based on a morphological taxonomic revision were monophyletic within New Zealand. Addition of exemplars from the Australian genera Fraus, Jeana, Oxycanus, and Trictena to the data set tentatively support the monophyly of the New Zealand "Oxycanus" lineages. Estimated times of divergence for the genus Wiseana taxa fitted well with known geological events and suggest that the genus may have diverged 1-1.5 mya.     

Making the most of mitochondrial genomes--markers for phylogeny, molecular ecology and barcodes in Schistosoma (Platyhelminthes: Digenea)

An increasing number of complete sequences of mitochondrial (mt) genomes provides the opportunity to optimise the choice of molecular markers for phylogenetic and ecological studies. This is particularly the case where mt genomes from closely related taxa have been sequenced; e.g., within Schistosoma. These blood flukes include species that are the causative agents of schistosomiasis, where there has been a need to optimise markers for species and strain recognition. For many phylogenetic and population genetic studies, the choice of nucleotide sequences depends primarily on suitable PCR primers. Complete mt genomes allow individual gene or other mt markers to be assessed relative to one another for potential information content, prior to broad-scale sampling. We assess the phylogenetic utility of individual genes and identify regions that contain the greatest interspecific variation for molecular ecological and diagnostic markers. We show that variable characters are not randomly distributed along the genome and there is a positive correlation between polymorphism and divergence. The mt genomes of African and Asian schistosomes were compared with the available intraspecific dataset of Schistosoma mansoni through sliding window analyses, in order to assess whether the observed polymorphism was at a level predicted from interspecific comparisons. We found a positive correlation except for the two genes (cox1 and nad1) adjoining the putative control region in S. mansoni. The genes nad1, nad4, nad5, cox1 and cox3 resolved phylogenies that were consistent with a benchmark phylogeny and in general, longer genes performed better in phylogenetic reconstruction. Considering the information content of entire mt genome sequences, partial cox1 would not be the ideal marker for either species identification (barcoding) or population studies with Schistosoma species. Instead, we suggest the use of cox3 and nad5 for both phylogenetic and population studies. Five primer pairs designed against Schistosoma mekongi and Schistosoma malayensis were tested successfully against Schistosoma japonicum. In combination, these fragments encompass 20-27% of the variation amongst the genomes (average total length approximately 14,000bp), thus providing an efficient means of encapsulating the greatest amount of variation within the shortest sequence. Comparative mitogenomics provides the basis of a rational approach to molecular marker selection and optimisation.     

Systematic implications of the sesquiterpene lactones in the “strumarium” morphological complex (Xanthium strumarium, Asteraceae) of Europe, Asia and Africa

In most populations of the “strumarium” morphological complex of Xanthium strumarium L. (sensu lato) in northern Europe and in India, a new compound, xanthinosin, is the only detectable sesquiterpene lactone. In populations of this morphological complex in Portugal and Egypt as well as in eastern Asia, USSR, Korea, Hong Kong and Taiwan, xanthinin and xanthatin occur as major constituents along with xanthinosin. Experimental F, hybrids between pistillate Indian plants which contained only xanthinosin and staminate plants from Hong Kong which contained a mixture of xanthinin, xanthatin and xanthinosin produced a mixture of compounds in which the percentage of xanthinin increased relative to its percentage in the Hong Kong parent. The sesquiterpenoid data suggest that the various taxa in the “strumarium” morphological complex can be divided into three groups: (a) X. strumarium (sensu stricto) and X. indicum König, containing primarily or exclusively xanthinosin; (b) X. sibiricum Patrin and X. brasilicum Vell., with xanthinin and xanthinosin predominating; and (c) X. inaequilaterum DC., with almost equal proportions of xanthinin, xanthatin and xanthinosin. Two other taxa of the complex. X. japonicum Widd. and X. abyssinicum Wallr., were not available for inclusion in the present study.     

Production of bifunctional proteins by Aspergillus awamori: llama variable heavy chain antibody fragment (V(HH)) R9 coupled to Arthromyces ramosus peroxidase (ARP)

The Arthromyces ramosus peroxidase gene (arp) was genetically fused to either the 5′- or 3′-terminal ends of the gene encoding llama variable heavy chain antibody fragment V_HH R9, resulting in the fusion expression cassettes ARP-R9 or R9-ARP. Aspergillus awamori transformants were obtained which produced up to 30 mg l⁻¹ fusion protein in the culture medium. Both fusion proteins showed peroxidase activity in an ABTS activity test. Considerable amounts of fusion protein were detected intracellularly, suggesting that the fungus encounters problems in secreting these kind of proteins. ELISA experiments showed that ARP-R9 was less able to bind its antigen, the azo-dye RR6, as compared to R9-ARP. Furthermore, in contrast to R9-ARP, ARP-R9 bound to RR6 did not show peroxidase activity anymore. These results indicate that fusion of ARP to the C-terminus of the antibody fragment V_HH R9 (R9-ARP) is the preferred orientation.     

Discovery of Maritrema obstipum (Digenea: Microphallidae) from migratory birds in Korea

Adults of Maritrema obstipum (Digenea: Microphallidae) were found in the intestines of 4 species of migratory birds, including the sanderling (Crocethia alba), Kentish plover (Charadrius alexandrines), Mongolian plover (Charadrius mongolus), and red-necked stint (Calidris ruficollis), collected from Yubu Island, Chungcheongnam-do, Korea. The worms of were 451 × 265 μm in size, and were easily identifiable as Maritrema species by the presence of the cirrus sac, and the ring-like distribution of the vitellaria. More specifically, the ejaculatory duct curved posteromedially, and the 2 parts of vitelline follicles were found to be distinct at the posterior end. The eggs were brown-colored, and 19.8 × 12.3 μm in size. All these findings implicated M. obstipum as the pertinent species of the worms. Beside these, adult worms of Gynaecotyla squatarolae, Parvatrema duboisi, and Acanthoparyphium sp. were also discovered. This is the first report establishing migratory birds as the natural definitive hosts for M. obstipum.     

An example of molecular co-evolution: reactive oxygen species (ROS) and ROS scavenger levels in Schistosoma mansoni/Biomphalaria glabrata interactions

The co-evolution between hosts and parasites involves huge reciprocal selective pressures on both protagonists. However, relatively few reports have evaluated the impact of these reciprocal pressures on the molecular determinants at the core of the relevant interaction, such as the factors influencing parasitic virulence and host resistance. Here, we address this question in a host-parasite model that allows co-evolution to be monitored in the field: the interaction between the mollusc, Biomphalaria glabrata, and its trematode parasite, Schistosoma mansoni. Reactive oxygen species (ROS) produced by the haemocytes of B. glabrata are known to play a crucial role in killing S. mansoni. Therefore, the parasite must defend itself against oxidative damage caused by ROS using ROS scavengers in order to survive. In this context, ROS and ROS scavengers are involved in a co-evolutionary arms race, and their respective production levels by sympatric host and parasite could be expected to be closely related. Here, we test this hypothesis by comparing host oxidant and parasite antioxidant capabilities between two S. mansoni/B. glabrata populations that have co-evolved independently. As expected, our findings show a clear link between the oxidant and antioxidant levels, presumably resulting from sympatric co-evolution. We believe this work provides the first supporting evidence of the Red Queen Hypothesis of reciprocal evolution for functional traits at the field-level in a model involving a host and a eukaryotic parasite.     

Parachorius semsanganus sp. n. (Coleoptera, Scarabaeidae, Scarabaeinae) from Laos and its significance in the phylogeny of Oriental Deltochilini

The new species Parachorius semsanganussp. n. is described from Laos. This enigmatic Oriental deltochiline represents a "morphological link" between Parachorius and Cassolus by sharing characters of the two genera. The fact that Parachorius semsanganus cannot be unequivocally placed in either of these two genera stresses some more general problems of the current classification of Parachorius and Cassolus. Such problems can be solved only in the course of phylogenetic analysis, the need of which is briefly outlined.     

Discovery of Parvatrema duboisi and Parvatrema homoeotecnum (Digenea: Gymnophallidae) from Migratory Birds in Korea

Adult worms of Parvatrema spp. (Digenea: Gymnophallidae) were found in the intestines of 2 species of migratory birds, i.e., a great knot, Calidris tenuirostris, and 2 Mongolian plovers, Charadrius mongolus, in the coastal area of Gunsan-si, Jeollabuk-do in October 2009. The recovered Parvatrema worms were 79 in total number and composed of 2 species. The worms from a great knot were 289 µm in length with the oral and ventral sucker ratio of 2 : 1. They had a single vitellarium, and their intrauterine eggs were 25.0 × 17.5 µm in size. These findings were compatible with P. duboisi (Dollfus, 1923) Bartoli, 1974 (syn. P. timondavidi Bartoli, 1963). The worms recovered from the Mongolian plovers were smaller in length than P. duboisi and had 2 vitellaria. The oral and ventral sucker ratio was 2.5 : 1, and the eggs were 17.5 × 8.8 µm in size. These worms were assigned to be P. homoeotecnum James, 1964. This is the first report on the natural final hosts of Parvatrema spp. in Korea.     

Preliminary observation on the role of conditioning and mechanical Interference with egg masses and Juveniles in the competitive relationships Between Helisoma Durvi (Wetherby) and the Intermediate host of Schistosoma Mansoni Sambon: Biomphalaria Camerunensis (Boettger).

Biological control of the intermediate hosts of schistosomiasis by means of the introduction oa a competitor snail Helisoma duryi has been suggested. The effect of conditioning by Biomphalaria camerunensis and H. duryi respectively on growth and egg laying capacity of B. camerunensis and on the hatchability of B. camerunensis egg masses was compared. No differences were found besides an indication of a growth promoting effect of B. camerunensis conditioning.B. camerunensis egg masses were found to be much more sensitive to mechanical interference from snails than were H. duryi egg masses. Both juvenile and adult H. duryi caused a great reduction in the number of B. camerunensis egg masses and adult B. camerunensis showed a great tendency to destroy its own egg masses. Newly hatched B. camerunensis appeared to be destroyed to a greater extent by adult H. duryi than by adult B. camerunensis.     

Morphological and Ecological Characterization of Steinernema feltiae (Rhabditida: Steinernematidae) Rioja Strain Isolated from Bibio hortulanus (Diptera: Bibionidae) in Spain

A new strain of Steinernema feltiae (Rhabditida: Steinernematidae) was isolated in La Rioja (Spain) from larvae of Bibio hortulanus (Diptera: Bibionidae). A comparative morphometric analysis of this new strain and four additional S. feltiae isolates was performed. Although significant differences in morphometric measurements were observed, PCR-RFLP profiles and sequence analysis of the ITS region of rDNA confirmed the identity of the new strain as A2 RFLP type of S. feltiae. A comparative morphometric study among nematodes from three hosts, Galleria mellonella (Lepidoptera: Pyralidae), Spodoptera littoralis (Lepidoptera: Noctuidae) and B. hortulanus, was conducted. Ecological characterization of the Rioja isolate was performed in G. mellonella larvae. Larval mortality was 75.3 and 78.12% in penetration and sand column assays, respectively, and the percentage of penetrating infective juveniles was 12.0 and 2.8% in these assays. Larval mortality in the one-on-one bioassay was 4.2%, and in exposure-time bioassays, it was 50% at 11.25 hours. Relationships between morphometric characteristics and host mortality are discussed for this new strain of entomopathogenic nematode.     

Discovery of Maritrema jebuensis n. sp. (Digenea: Microphallidae) from the Asian Shore Crab, Hemigrapsus sanguineus, in Korea

Maritrema spp. (Digenea: Microphallidae) are parasites of birds, but have not been found in the Republic of Korea. In this study, metacercariae of Maritrema sp. were discovered in the Asian shore crab, Hemigrapsus sanguineus, caught in the mud-flats of Jebu-do, Hwasung-gun, Gyeonggi-do, and the adult flukes were confirmed by experimental infection into mice. Based on the symmetric ribbon-like vitellarium, adult flukes of Maritrema sp. were identified, but did not belong to previously described species in terms of the following morphologic characteristics: ceca reaching to the lateral wall at the anterior border of the ovary; ventral sucker larger than oral sucker; a prominent metraterm; and vitellarium forming a complete ring. Hence, we named this microphallid M. jebuensis n. sp. after the island where the second intermediate hosts were collected. From this study, it has been shown that Maritrema sp. is distributed in Korea and transmitted by the Asian shore crab, H. sanguineus.     

Fasciola gigantica and Schistosoma mansoni: vaccine potential of recombinant glutathione S-transferase (rFgGST26) against infections in mice

Recombinant Fasciola gigantica glutathione S-transferase (rFgGST26) was expressed in Escherichia coli. This protein had 86% and 56% sequence identity with 26 kDa GST from Fasciolahepatica and Schistosoma mansoni, respectively. Polyclonal antibody raised in ICR mice against rFgGST26 recognized immunoblotted 26 kDa native GSTs from F. gigantica and S. mansoni. rFgGST26 was used as a vaccine in combination with Freund’s adjuvant to evaluate the induction of immune responses and protection against F. gigantica and S. mansoni infection in mice. Mice were immunized via subcutaneous (s.c.), intramuscular (i.m.) or intradermal (i.d.) routes. Strong protection (77-84%) against F. gigantica was observed in all routes. Immunization via s.c. route induced immune response with IgG1 isotype predominating, while i.m. and i.d. routes resulted in mixed IgG1/IgG2a immune responses. Passive intraperitoneal transfer of IgG1 predominating antisera from s.c. rFgGST26-immunized donors to naïve recipient mice resulted in 47% protection against F. gigantica infection. This suggests that the mechanism of resistance depends on the presence of specific antibody against rFgGST26. Immunization with rFgGST26 via i.m. and i.d. routes resulted in significant cross protection (55%) against S. mansoni infection in the i.d. route with mixed IgG1/IgG2a response with IgG1 isotype predominating. This indicated that rFgGST26 is a good vaccine candidate against F. gigantica in mice and could also provide cross protection against S. mansoni.     

Holostephanus metorchis (Digenea: Cyathocotylidae) from chicks experimentally infected with metacercariae from a fish, Pseudorasbora parva, in the Republic of Korea

Holostephanus metorchis (Digenea: Cyathocotylidae) is a parasite of birds, transmitted by freshwater fishes. H. metorchis adults were recovered from chicks experimentally infected with metacercariae collected from freshwater fishes, Pseudorasbora parva. The metacercariae were oval, surrounded with thick fibrous capsules. In adult flukes, the holdfast organ occupied the ventral concavity, and the anterior testis did not reach the level of the ventral sucker. Based on these morphological characteristics, these flukes were identified as H. metorchis.     

Molecular Characterization of Gastrothylax crumenifer (Platyhelminthes: Gastrothylacidae) from Goats in the Western Part of India by LSU of Nuclear Ribosomal DNA

The rumen parasite, Gastrothylax crumenifer (Platyhelminthes: Gastrothylacidae), is a highly pathogenic trematode parasite of goat (Capra hircus). It sucks blood that causes acute disease like anemia, and severe economic losses occur due to morbidity and mortality of the ruminant infected by these worms. The study of these rumen paramphistomes, their infection, and public health importance remains unclear in India especially in the western part of state Uttar Pradesh (U.P.), Meerut, India, where the goat meat consumption is very high. This paper provides the molecular characterization of G. crumenifer recovered from the rumen of Capra hircus from Meerut, U.P., India by the partial sequence of 28S rDNA. Nucleotide sequence similarity searching on BLAST of 28S rDNA from parasites showed the highest identity with those of G. crumenifer from the same host Capra hircus. This is the first report of molecular identification of G. crumenifer from this part of India.