Ultrastructure of Babesia WA1 (Apicomplexa: Piroplasma) during infection of erythrocytes in a hamster model

Babesia Washington-1 (WA1) is a newly identified intraerythrocyte infectious agent of human babesiosis in the western United States. The purpose of the present study is to describe the ultrastructural changes in affected erythrocytes during the infectious process in a susceptible animal model, the golden Syrian hamster. Two, 1-mo-old female hamsters were inoculated intraperitoneally (i.p.) with 1.8 x 10(9) Babesia WA1-infected erythrocytes originally isolated from a human case and serially passaged in hamsters. Saphenous vein blood samples (20 microl) were collected at 0, 24, 36, 48, 60, 72, 84, and 96 hr postinoculation (PI). Parasitemia was determined at each time interval by quick staining of blood smears showing 0, 2.5, 5, 10, 12.5, 22.5, 70, and almost 100% parasitemic erythrocytes at the corresponding PI time interval, respectively. Animals showed weakness and dehydration 72 hr PI inoculation, and were killed by 96 hr PI. Selected blood samples from 0, 24, 48, 72, and 96 hr were fixed in cacodylate buffer, dehydrated in ethanol gradients, resin embedded, and then thin sectioned and stained with uranyl acetate and lead citrate for transmission electron microscopy or gold-coated for scanning electron microscopy (SEM). Shape and surface membrane changes in erythrocytes were demonstrated by SEM and were more evident at 72 and 96 hr PI. Infected erythrocytes underwent changes in shape 24 hr PI, from few protrusions to several perforations, some of them resembling a "swiss cheese" appearance 96 hr PI. Several erythrocytes had irregular surface membranes and Babesia WA1 organisms were seen at different stages of development within erythrocytes, from single trophozoites to several merozoites (young trophozoites), some of them dividing to form typical tetrads. In general, Babesia WAI induced severe morphological changes in the erythrocytes, and these changes were more evident in almost all infected cells 96 hr PI.     

狭义五福花科的分子系统学和物种分化

狭义五福花科(Adoxaceae.s.s)仅含3属4种,但该科的物种分化、系统发育和分类一直存在争议。本文通过测定东方五福花和血满草的ITS(核糖体DNA内转录间隔区)序列,构建了包括狭义五福花科(4种)、广义忍冬科接骨木属、荚属以及其余4属植物在内的系统发育树。研究结果不支持狭义五福花科内根据形态学证据做出的系统发育假设四福花不是该科中最早分化出来的种;该物种与五福花属的两个物种形成一个单系群,与另一分支华福花属相对应。该科中3个物种,四福花、五福花和华福花之间的分化主要是在二倍体水平上的异域分化,而东方五福花则是通过多倍化形成的。粗略的时间估算表明这些物种之间的分化较晚,可能在第三纪末至第四纪早中期,与青池高原近期强烈隆升以及冰期气候反复变化形成的环境变迁密切相关。     

Molecular Phylogeny and Species Speciation of Adoxaceae. s. s

It remains unclear about the speciation and phylogeny of Adoxaceae s. s ., a small family with 3 genera and 4 species . In this paper , ITS ( nuclear DNA internal transcribed spacer) regions of Adoxa orientalis and Sambucus adnata were firstly sequenced . Phylogenetic trees were constructed for all species of Adoxaceae (four species ) , Sambucus, Viburnum and four genera of Caprifoliaceae . The divergences among four species of this family were further calculated based on the calibration of the fossil records of the Caprifoliaceae and the general evolutionary rate of herbs for ITS . The phylogenetic analyses did not support the previous assumptions on the phylogeny and species divergence of Adoxaceae s. s . based on the morphological evidence : Tetradoxa is not the firstly diverged and it clustered with two species of Adoxa as a monophylogenetic group , paralleling to the other lineage comprising of monotypic Sinadoxa . The allopatric speciation at the diploid level might have contributed to the differentiation among Sinadoxa corydalifolia, Tetradoxa omeiensis and Adoxa moschatellina and the polyploidy to the origin of A. orientalis . The crude timing based on ITS sequence differentiation suggested a recent divergence among all four species probably between the late Miocene and the Tertiary and this speciation process might be closely correlated with habitat fragmentation and change due to the extensive uplifts of the Qinghai-Tibetan Plateau and climatic oscillation during the glacial and interglacial ages occurred at this stage .     

Cytogenetics of Alismatales s.s.: chromosomal evolution and C-banding

Ten species of Alismatales, five of Alismataceae, four of Limnocharitaceae and one of Hydrocharitaceae were studied with regard to chromosome number, chromosome morphology, and pattern of Giemsa C-bands. The genus Echinodorus had a diploid chromosome number of 22 for all species that were analyzed and a karyotypic formula of 2m + 20a. For the family Limnocharitaceae, Hydrocleys nymphoides had a diploid chromosome number of 16, Hydrocleys martii (4m + 2sm + 10a) had a diploid chromosome number of 16, Limnocharis flava had a diploid chromosome number of 20 and L. laforestii (4m + 16a) had a diploid chromosome number of 20. The only species of Hydrocharitaceae that was studied exhibited a karyotype that consisted of a diploid chromosome number of 28 and a karyotypic formula of 4m + 6sm + 4a. The distribution pattern of the C-banded karyotype in Echinodorus showed four blocks of constitutive heterochromatin in two smaller acrocentric pairs that corresponded to the heterochromatic NORs. In E. lanceolatus, 14 bands in the termini of the arms beyond the heterochromatic NORs of seven acrocentric pairs were also observed. Idiograms are presented and the karyotypic evolution patterns for the studied groups are discussed.     

Isolation of a field strain of Babesia bigemina (Piroplasma: Babesiidae) and establishment of in vitro culture for antigen production

Isolation of a field strain of Babesia bigemina (Piroplasma: Babesiidae) and establishment of in vitro culture for antigen production. Bovine b abesiosis, caused by Babesia bigemina, is a barrier for livestock development; it results in high economic loss to Mexican livestock. Control requires adequate antigens for diagnosis and vaccination programs. However, because of antigenic variation among Babesia strains, it is necessary to use antigens prepared from local strains. The purpose of the present study was to isolate a local field strain and to establish the in vitro culture of B. bigemina by the evaluation of the constituent's concentration of culture media. Thirty engorged female Boophilus microplus were collected from cattle suffering clinical babesiosis (B. bigemina) in Yucatan state, Mexico. These ticks were sent to the laboratory for detection of Babesia sp. vermicules. Eggs were kept at 83-85 % humidity and 27 degrees C until hatching. Larvae were transferred to an esplenectomized calf (B-1). The resulting nymphs were transferred to an esplenectomized calf (B-2). Twelve days later, B. bigemina (local strain) was detected in calf B-2 and its infected blood was frozen in liquid nitrogen to initiate the in vitro culture. The Microaerophilus Stationary Phase (MASP) in vitro culture method was used to reactivate the parasite. Three different concentrations of culture media (70, 60 and 50%), serum (30, 40 and 50%) and uninfected red blood cells (5, 10 and 15 %) were used in order to know the convenient concentrations to obtain the highest percentage of infected red blood cells (PEI). The cultured strain was used to prepare antigens for the Immunofluorescence Antibody Test (IFAT) and several concentrations of serum and conjugate were tested. Strain isolation was successful; 30 days were needed to obtain a PEI of 1.5%. The isolated strain was frozen in liquid nitrogen and the parasites were reactivated with the in vitro culture MASP method. The concentration of culture media that produced the highest PEI (14%) (p < 0.05) was 30% serum, 70% M199 and 5%. Uninfected Red Blood cells antigens were successfully used in the IFAT and the best dilutions to differentiate between positive and negative controls were serum 1:80 and conjugate 1:80. The isolated B. bigemina local strain requires particular conditions of in vitro culture by the MASP method to reach high numbers of infected red blood cells, needed to prepare and provide high quality antigens for serological diagnosis of B. bigemina.     

Fruit structure and systematics of Monimiaceae s.s. (Laurales)

Fruit structure (anatomy) was studied in 27 species of 15 genera of Monimiaceae s.s. Almost all have apocarpous gynoecia, with the carpels more or less surrounded by a floral cup. The fruitlets are presented on the opened floral cup, which, depending on its pre‐ and post‐floral development, differentially contributes to the attractive part of the mature fruit. Morphologically similar fruits may differ conspicuously in anatomical structure. Based on anatomical characters two different fruit forms were found: drupe(let)s (with compact sclerenchymatic endocarp forming a stone: putamen) and berry(let)s (with parenchymatic endocarp, and mesocarp parenchyma containing isolated sclereid nests). Four types of drupelets differing by the endocarp structure were tentatively distinguished: (1) the Monimia‐type has a many‐cell‐layered putamen of large isodiametric sclereids, interrupted on the ventral side by few radial rows of small sclereids; (2) the Hortonia‐type has a few‐cell‐layered putamen of isodiametric, especially thick‐walled sclereids – it may be composed of two lateral halves, i.e. with the sclerenchyma partially interrupted on the ventral and dorsal sides (but without rows of small sclereids); (3) the Mollinedia‐type has a few‐cell‐layered putamen, with more or less radially elongate sclereids with wavy cell walls; and (4) the Hedycarya‐type has a one‐cell‐layered putamen of pronouncedly radially elongate sclereids with wavy cell walls. Drupelets of some taxa with a single‐cell‐layered endocarp with only weakly thickened cell walls may represent a transition from drupelets to berrylets. The fruit structure supports three major clades recognized earlier by morphological studies and by molecular phylogenetic analyses: (1) Monimioideae (Monimia‐type drupelets), (2) Hortonieae of Mollinedioideae (Hortonia‐type drupelets), and (3) the remainder of Mollinedioideae (Hedycarya‐ and Mollinedia‐types) and berrylets. Fruit structure also supports the close relationship of Monimiaceae and Lauraceae. © 2007 The Linnean Society of London, Botanical Journal of the Linnean Society, 2007, 153 , 265–285.     

Multimodal pyrethroid resistance in malaria vectors, Anopheles gambiae s.s., Anopheles arabiensis, and Anopheles funestus s.s. in western Kenya

Anopheles gambiae s.s., Anopheles arabiensis, and Anopheles funestus s.s. are the most important species for malaria transmission. Pyrethroid resistance of these vector mosquitoes is one of the main obstacles against effective vector control. The objective of the present study was to monitor the pyrethroid susceptibility in the 3 major malaria vectors in a highly malaria endemic area in western Kenya and to elucidate the mechanisms of pyrethroid resistance in these species. Gembe East and West, Mbita Division, and 4 main western islands in the Suba district of the Nyanza province in western Kenya were used as the study area. Larval and adult collection and bioassay were conducted, as well as the detection of point mutation in the voltage-gated sodium channel (1014L) by using direct DNA sequencing. A high level of pyrethroid resistance caused by the high frequency of point mutations (L1014S) was detected in An. gambiae s.s. In contrast, P450-related pyrethroid resistance seemed to be widespread in both An. arabiensis and An. funestus s.s. Not a single L1014S mutation was detected in these 2 species. A lack of cross-resistance between DDT and permethrin was also found in An. arabiensis and An. funestus s.s., while An. gambiae s.s. was resistant to both insecticides. It is noteworthy that the above species in the same area are found to be resistant to pyrethroids by their unique resistance mechanisms. Furthermore, it is interesting that 2 different resistance mechanisms have developed in the 2 sibling species in the same area individually. The cross resistance between permethrin and DDT in An. gambiae s.s. may be attributed to the high frequency of kdr mutation, which might be selected by the frequent exposure to ITNs. Similarly, the metabolic pyrethroid resistance in An. arabiensis and An. funestus s.s. is thought to develop without strong selection by DDT.     

Genetic differentiation of Anopheles claviger s.s. in France and neighbouring countries

An investigation of polymorphism of 11 autosomal and one sex-linked allozyme loci was made on 18 samples of Anopheles claviger Meigen (Diptera: Culicidae) from localities across France and neighbouring sites in Germany and Switzerland, plus one sample of Anopheles petragnani Del Vecchio from the French Pyrénées. Genetic differentiation between these two sibling species was confirmed (Nei genetic distance 0.33-0.44) and two genetically distinct groups of populations were identified within An. claviger. These two forms of An. claviger showed contiguous geographical distributions, Group I found across western and Central France, Group II in eastern France and nearby parts of Germany and Switzerland. The two groups were in contact in a region near the Rhone Valley where two intermediate samples were found. The taxonomic significance of this finding is discussed in the context of the recent climatic history of Europe and in relation to the vector potential of each member of the An. claviger complex.     

Comparison of reproductive success in two orchids: the nectarless Dactylorhiza majalis s.s. and the nectar-producing Gymnadenia conopsea s.l.

Differences in reproductive biology between two orchid species were examined: Eight Danish populations of the nectarless Dactylorhiza majalis ssp. majalis and six Swedish populations of the nectar-producing Gymnadenia conopsea ssp. densiflora . Population size varied from 24 to 1700 flowering individuals in D. majalis and from 100 to 1200 in G. conopsea. Dactylorhiza majalis had only half as many flowers per spike as G. conopsea . Fruit-set of D. majalis ranged from 16 to 39%, much lower than the fruit-set of G. conopsea (78–91%). Thus in the species with a few flowers and no nectar a lower fruit-set was observed than in the species with many flowers and presence of nectar. The low fruit-set of D. majalis was pollen-limited and fruit-set was positively correlated with pollinia removal and the latter was negatively correlated with population size. In both species, fruit-set and population size were uncorrelated.     

A new taxonomic system of the Campanulaceae s.s.

The Campanulaceae s.s. have been proved to be monophyletic, but the subdivision of the family is still controversial among authors. To investigate the intrafamilial structure of the Campanulaceae s.s., four chloroplast DNA fragments, atpB, matK, rbcL, and petD with the petB-petD spacer region, were chosen for molecular phylogenetic analysis, and 90 taxa representing 36 genera of this family were sampled. The result shows that the Campanulaceae s.s. consist of three strongly supported monophyletic clades. This result is highly correlated with the data from palynology and external morphology. Therefore, we propose to establish a new three tribal classification system of Campanulaceae s.s. The Cyanantheae is characterized by colpate or colporate pollen with elongate apertures and a loculicidal capsule which is dehiscent by apical valves, or a berry. The other two tribes have porate pollen with poroid apertures, but the Campanuleae possesses a poricidal capsule which is dehiscent by lateral pores or valves, or a dry indehiscent fruit, whereas the Wahlenbergieae possesses a loculicidal capsule which is dehiscent by apical valves, pores, or opercula. In the Cyanantheae, we recognize 6 subtribes and 10 genera. In addition, keys to tribes of Campanulaceae s.s. and to subtribes and genera of the Cyanantheae are presented.     

Phylogeny and classification of the Elachistidae s.s. (Lepidoptera: Gelechioidea)

Abstract. The classification of the gelechioid family Elachistidae (Lepidoptera) is revised on the basis of a phylogenetic analysis. Pee-Wee analysis of 131 characters of adult and pupal morphology and larval mode of life, coded for seventy elachistid species, results in a classification with three recognized genera: Perittia, Stephensia and Elachista. Elachista is further divided into four subgenera. The phylogenetic relationships of the genera and subgenera are (Perittia (Stephensia ((E. sg. Dibrachia (E. sg. Hemiprosopa, E. sg. Aphelosetia)) (E. sg. Elachista)))). Twenty-four new generic synonyms and thirty-eight new generic combinations of species are proposed. A checklist is given for the species of Elachistidae in their revised generic combinations, including nine new synonymies. Due to secondary homonymy, Elachista dasycara nom. n. is proposed as a new name for Eurynome albella Chambers.     

On the distribution and genetic differentiation of Anopheles gambiae s.s. molecular forms

This paper summarises published and unpublished data on the spatial and temporal distribution, and on the genetic characterisation of molecular forms M and S of Anopheles gambiae s.s. The two forms are characterised by a high level of gene-flow restriction, by a largely overlapping geographical and temporal distribution, and by a low degree of genetic differentiation. Floating paracentric inversions on chromosome-2 are shown to be shared by the two forms, although with very different frequencies of alternative arrangements, confirming that these inversions are most probably involved in ecotypic adaptation, rather than in the building of reproductive barriers. Further studies and tools are needed to throw light on the genetic and biological differentiation of M and S to improve the knowledge of the real composition of the vector system, of its demography, population genetics and dynamics, also in view of the possible consequences on the transmission of human pathogens in sub-Saharan Africa. Preliminary results and perspectives of the use of transposable element insertion sites as markers of genetic differentiation and tools for population genetic studies are discussed.     

Scanning electron microscopic studies of Centaurea L. s.s. pollen

Abstract

The purpose of this study is to analyse, by means of Scanning Electron Microscopy (SEM), the validity of the typification of the pollen of Centaurea L. s.s., carried out by Wagenitz (1955) with light microscopy and based on the exine structure and sculpturing. The pollen of six species have been analyzed: one species for every type of pollen present in Italy: C. sempervirens L., C. alpina L., C. scabiosa L., C. alba L., C. montana L., C. cyanus L.

The validity of the pollen typification suggested by Wagenitz has been confirmed also at the ultramicroscopic level. The ultrastructure and the sculturing of the sporoderm are described in detail and some discrepancies, mainly due to the different potentialities offered by the two methods, are pointed out.