Leishmania (Viannia) panamensis: An in vitro assay using the expression of GFP for screening of antileishmanial drug

Promastigotes of Leishmania (Viannia) panamensis were successfully transfected with p6.5-egfp to express green fluorescent protein. The transfectants remained infective to macrophages, providing an in vitro model for screening antileishmanial drugs. This was demonstrated by flow cytometry of macrophage-associated GFP after exposure of infected cultures to known antileishmanial drugs, i.e. amphotericin B and glucantime^®. Fluorescence of GFP diminished progressively from infected cells with increasing drug concentrations used in both cases. The availability of this fluorescent assay for infection of macrophages by L. (V.) panamensis facilitates drug discovery program for the Viannia species, which differ significantly from those of the Leishmania subgenus.     

Morphological and genetic differentiation of two loliginid squids, Uroteuthis (Photololigo) chinensis and Uroteuthis (Photololigo) edulis (Cephalopoda: Loliginidae), in Asia

The squids Uroteuthis (Photololigo) edulis and Uroteuthis (Photololigo) chinensis (family Loliginidae) are commercially important fishery species in many coastal regions of Asia. The morphologies of these two squids are very similar, and identification based on morphology has been inadequate. The occurrence of cryptic species in the family Loliginidae has been reported. The widely distributed U. (P.) chinensis and U. (P.) edulis are believed to comprise several cryptic species. In this study, the taxonomic status of the two species in East Asia was elucidated by morphological and genetic analyses. Analysis of U. (P.) chinensis from Hong Kong and Xiamen (China) and U. (P.) edulis from Yamaguchi (Japan) and Shanghai (China) was performed in order to determine the effectiveness of different morphometric variables in discriminating between the two species. Multivariate analysis of 27 morphometric indices revealed no new morphological characters for the taxonomic identification of the two taxa, which can be distinguished by the teeth shape and number on arm sucker rings, and the percentage of hectocotylized part of left arm IV of males. The morphometric differences between U. (P.) edulis individuals from the two localities is most probably due to differences in the maturity stages of the sampled individuals between the two localities. Genetic analysis based on the mitochondrial COI and 16S rRNA genes revealed a high divergence of 15.5% and 7.5% respectively, indicating that U. (P.) edulis and U. (P.) chinensis are distinct species.     

Seasonal growth dynamics of Laurencia papillosa and Gracilaria coronopifolia from a highly eutrophic reef in southern Taiwan: temperature limitation and nutrient availability

Both field and laboratory studies were used to investigate the effects of temperature limitation and nutrient availability on seasonal growth dynamics of Laurencia papillosa and Gracilaria coronopifolia from a nearshore coral reef in the southern tip of Taiwan during 1999-2000. L. papillosa was a summer blooming alga abundant in August-November and G. coronopifolia was abundant year round except April-May. L. papillosa blooms in the summer were attributed to its preference for high temperatures and highly sensitivity to low temperatures. A wider temperature range and a significant stimulation of growth by high N inputs can explain the appearance of G. coronopifolia year round and also its maximum growth in November-March. Levels of dissolved inorganic nitrogen (DIN) and soluble reactive phosphorus (SRP) in water column were extremely high, but the growth of these two rhodophytes still suffered nutrient limitation that the type and severity of nutrient limitation were variable over time and also between two species. The growth of L. papillosa was limited by P in the early growth stage (August-September) as indicated by decreased tissue P contents, increased C/P and N/P molar ratios and increased alkaline phosphatase activity (APA) and in the later growth stage, it was subjected to N-limitation, evidenced by decreased tissue N contents and C/P and N/P molar ratios and increased tissue P contents. The growth of G. coronopifolia was also P-limited as indicated by increased tissue N contents and concomitantly decreased tissue P contents, while marked drops in tissue P contents below the subsistence level in mid September and December 1999 reveal severe P limitation, which was supported by increased alkaline phosphatase activity. Higher critical nutrient contents and nutrient thresholds for maximum growth of G. coronopifolia suggest that G. coronopifolia faced more frequent nutrient limitation compared to L. papillosa. In conclusion, the results from these laboratory and field studies provide evidence that the seasonal abundance of L. papillosa and G. coronopifolia from southern Taiwan was determined by seasonal variations in seawater temperatures and nutrient concentrations as well as different physiological growth strategies. Seawater temperature and nutrient availability were important determinants of seasonal abundance of L. papillosa while the seasonal abundance of G. coronopifolia was influenced by nutrient availability.     

Presence of Nosema ceranae in honeybees (Apis mellifera) in Uruguay

The microsporidium Nosema ceranae is an emergent pathogen of European honeybees Apis mellifera. Using a PCR-RFLP diagnosis, 29 samples of infected honeybees obtained in 2007-2008 (N = 26), 2004 (N = 2) and before 1990 (N = 1) were analyzed for the presence of Nosema apis and N. ceranae. Only N. ceranae was found in all samples, indicating that this species dispersed to Uruguay (and likely the region) at some time before 1990. The presence of N. ceranae in Uruguay is not associated with an increase of Nosemosis, and its role in colony loss seems to be irrelevant.     

Pollen morphology of Chinese Curcuma L. and Boesenbergia Kuntz (Zingiberaceae): Taxonomic implications

In order to find new non-molecular evidence to support the phylogenetic and taxonomic position, pollen grains of 20 populations of 16 species of Chinese Curcuma L. and Boesenbergia Kuntz (Zingiberaceae) were investigated under SEM and TEM. The pollen grains are spherical and ovoid, nonaperturate. The pollen wall is composed of a very thin exine and a thick intine. The exine is psilate or echinate. The intine consists of two layers, i.e., a thick, channeled layer (exintine) and an inner homogenous layer (endintine). The results reveal morphological congruence between the pollen grains of species of Curcuma, which according to DNA sequence data appears to be a polyphyletic genus. However the uniform pollen morphology in Curcuma provides no evidence to divide this genus into separate taxonomic entities. Our results on pollen morphology also do not provide any additional evidence to either unite or segregate Boesenbergia albomaculata and Curcumorpha longiflora in the same genus and demonstrate that more taxonomic data on the genus Boesenbergia and its relatives are needed before a final decision can be made.     

Host specificity of microsporidia pathogenic to the gypsy moth, Lymantria dispar (L.): Field studies in Slovakia

Several species of microsporidia are important chronic pathogens of Lymantria dispar in Europe but have never been recovered from North American gypsy moth populations. The major issue for their introduction into North American L. dispar populations is concern about their safety to native non-target insects. In this study, we evaluated the susceptibility of sympatric non-target Lepidoptera to two species of microsporidia, Nosema lymantriae and Vairimorpha disparis, isolated from European populations of L. dispar and applied in field plots in Slovakia. Application of ultra low volume sprays of the microsporidia maximized coverage of infective spores in a complex natural environment and, thus, exposure of non-target species to the pathogens. Of 653 non-target larvae collected from plots treated with V. disparis in 2002, 18 individual larvae representing nine species in four families were infected. These plots were monitored for two subsequent seasons and V. disparis was not recovered from non-target species. Of 2571 non-target larvae collected in N. lymantriae-treated sites, one larva was found to be infected. Both species of microsporidia, particularly N. lymantriae, appear to have a very narrow host range in the field, even when an inundative technique is used for their introduction. V. disparis infections in L. dispar exceeded 40% of recovered larvae in the treated study sites; infection rates were lower in sites sprayed with N. lymantriae. Several naturally-occurring pathogens were recorded from the non-target species. The most common pathogen, isolated from 21 species in eight families, was a microsporidium in the genus Cystosporogenes.     

In vivo study of human Plasmodium knowlesi inMacaca fascicularis

Plasmodium knowlesi is a malaria parasite of Old World monkeys and is infectious to humans. In this study Macaca fascicularis was used as a model to understand the host response to P. knowlesi using parasitological and haematological parameters. Three M. fascicularis of either sex were experimentally infected with P. knowlesi erythrocytic parasites from humans. The pre-patent period for P. knowlesi infection in M. fascicularis ranged from seven to 14 days. The parasitemia observed was 13,686-24,202 parasites per μL of blood for asexual stage and 88-264 parasites per μL of blood for sexual stage. Periodicity analysis adopted from microfilaria periodicity technique of asexual stage showed that the parasitemia peak at 17:39 h while the sexual stage peaked at 02:36 h. Mathematical analysis of the data indicates that P. knowlesi gametocytes tend to display periodicity with a peak (24:00-06:00) that coincides with the peak biting activity (19:00-06:00) of the local vector, Anopheles latens. The morphology of P. knowlesi resembled P. falciparum in early trophozoite and P. malariae in late trophozoite. However, it may be distinguishable by observing the appliqué appearance of the cytoplasm and the chromatin lying inside the ring. Haematological analysis on macaques with knowlesi malaria showed clinical manifestations of hypoglycaemia, anaemia and hyperbilirubinemia. Gross examination of spleen and liver showed malaria pigments deposition in both organs.     

Atypical foliar anatomy related to Kranz syndrome in Paspalum inaequivalve and Paspalum microstachyum (Poaceae: Panicoideae: Paniceae)

Paspalum L. is a large and complex genus, enclosing more than 300 species, whose boundaries and infrageneric classification are still being studied. Recent phylogenetic analyses suggest that Paspalum inaequivalve Raddi and Paspalum microstachyum J. Presl, from the Inaequivalvia informal group, should be excluded from Paspalum. Focused on the unclear taxonomic position of P. inaequivalve and P. microstachyum, their leaf anatomy was studied, and some atypical features related to C₄ photosynthesis were found. This atypical Kranz syndrome is the aim of this research. Transverse leaf blade sections from fresh and herbarium material of P. inaequivalve were studied by light, fluorescence, and transmission microscopy. Additionally, sheaths and culms of P. inaequivalve and leaf blades of P. microstachyum were observed by light microscopy. δ¹³C isotope discrimination was determinated for P. inaequivalve. We compared our results with available anatomical data from related taxa. As well as typical mesophyll cells (PCA) and mestome sheath cells (PCR), a third type of cells, here called ‘globose parenchymatous cells’, was observed in leaf blades of P. inaequivalve and P. microstachyum. These cells are placed externally to the mestome sheaths of the first and second vascular bundles, they have thin walls, with no developed suberine lamella, few chloroplasts with 1–2 starch grains, thylakoids not organized in grana, and a large central vacuole. The globose parenchymatous cells represent a novel trait in P. inaequivalve and P. microstachyum, further supporting the close relation between both species and their exclusion from the genus Paspalum. This atypical Kranz syndrome has not been described in Paspalum before, but the globose parenchymatous cells here described resemble the distinct cells considered as remnants of the outer parenchymatous sheath described for Anthaenantiopsis, some sections of Panicum L., and Chaetium Nees, providing possible taxonomic significance.     

Mycobiota associated with the ambrosia beetle Scolytodes unipunctatus (Coleoptera: Curculionidae, Scolytinae)

Ambrosia beetles (Coleoptera: Scolytinae) are associated with strictly entomochoric and mutualistic fungi. We studied the mycobiota associated with Scolytodes unipunctatus, ambrosia beetles that infest Cecropia trees in Central America. Isolates were characterized using morphology and rDNA sequences (ITS region, LSU, and SSU rDNA). Four species are described here: Raffaelea scolytodis sp. nov. (Ophiostomatales), Gondwanamyces scolytodis sp. nov., Custingophora cecropiae sp. nov., and Graphium sp. (Microascales). The genus Custingophora is emended to include Knoxdaviesia anamorphs of Gondwanamyces based on uniformity of DNA sequences and phenotype.     

不同群落中米氏冰草和羊草的年龄结构动态

米氏冰草和羊草都是根茎型禾草,是典型的无性系植物。采取单位面积挖掘取样法,对呼伦贝尔沙地植被次生演替过程中的米氏冰草和羊草种群分株年龄结构进行调查。结果表明,在单生和混合群落中,米氏冰草分株分别由3和4个年龄级组成,羊草均由2个年龄级组成;米氏冰草分株数量和生物量年龄结构变化基本一致,即随着生长年限的延长,呈减少低龄级和增加高龄级比例的趋势,使单生群落中始终为增长型,但接近于稳定型,混生群落中由增长型变为稳定型。羊草分株数量和生物量年龄结构在连续2a均为明显的增长型。羊草各龄级平均单株生产力均高于米氏冰草,平均是米氏冰草的5.2倍。米氏冰草和羊草种群分株年龄结构的变化,蕴含着米氏冰草种群的优势地位将被羊草种群取代的趋势。     

Diversimorbus metrosiderotis gen. et sp. nov. and three new species of Holocryphia (Cryphonectriaceae) associated with cankers on native Metrosideros angustifolia trees in South Africa

The Cryphonectriaceae includes important tree pathogens, especially on the Myrtales. During a routine disease survey in the Western Cape Province of South Africa, a fungus resembling the Eucalyptus pathogen Holocryphia eucalypti was observed on native Metrosideros angustifolia (Myrtales). The aims of this study were to identify the fungus and to expand surveys for fungi in the Cryphonectriaceae on M. angustifolia. Fungi were identified based on DNA sequence comparisons and morphological features, and their pathogenicity was tested on M. angustifolia under field conditions. Based on morphology and multigene phylogenetic analyses of DNA sequence data from six gene regions, we describe a new genus including a single species and three new species of Holocryphia (Cryphonectriaceae) from M. angustifolia. These fungi are provided with the names Diversimorbus metrosiderotis gen. et sp. nov., Holocryphia capensis sp. nov., Holocryphia gleniana sp. nov., and Holocryphia mzansi sp. nov. We also revise H. eucalypti, the type of the genus, to include only isolates from Eucalyptus in South Africa. Research results indicated that H. mzansi may undergo host shifts between different tree genera in the Myrtaceae. Inoculation tests showed that isolates of all the newly described species can cause lesions on the branches of M. angustifolia, indicating that they are all pathogens of this tree.     

New case of long-term persistence of Paranosema locustae (Microsporidia) in melanopline grasshoppers (Orthoptera: Acrididae: Melanoplinae) of Argentina

We report an additional case of long-term persistence of Paranosema locustae in grasshoppers of Argentina. The pathogen was introduced from North America on rangeland at Loncopué, Neuquén province. Microsporidia were not detected in pre-introduction samples whereas infected grasshoppers were found 11 years after introduction. Affected grasshoppers were the melanoplines Dichroplus elongatus, Dichroplus maculipennis, and Scotussa lemniscata, some of them with high spore loads. The case highlights the ability of P. locustae to recycle in local grasshopper communities by parasitizing susceptible species other than the natural hosts.     

Fruit anatomy of the genus Pimpinella L. (Apiaceae) in Iran

The genus Pimpinella L., with about 170–180 species in the world, is one of the largest genera of the family Apiaceae (Umbelliferae). Based on the Flora Iranica treatment, this genus has 25 species in the Iranian plateau, including 19 species in Iran, and six of those (P. tragioides, P. deverroides, P. pastinacifolia, P. anisactis, P. khorasanica and P. khayyamii) are endemic.     

Use of spent mushroom substrates from Agaricus subrufescens (syn. A. blazei, A. brasiliensis) and Lentinula edodes productions in the enrichment of a soil-based potting media for lettuce (Lactuca sativa) cultivation: Growth promotion and soil bioremediation

This study aimed to assess physicochemical and microbiological properties of fresh spent mushroom substrates (SMSs) – without post-crop heat treatment – from Agaricus subrufescens and Lentinula edodes production to optimize the use of these residues in the soil enrichment for lettuce growth promotion and soil remediation. Organic matter and C content of both SMSs were high. Fresh A. subrufescens SMS was a good source of N, P and K. On the other hand, L. edodes SMS presented a lower concentration of these nutrients and a high level of immaturity. Both SMSs presented high electric conductivity values (2.5–3.4 mS/cm). Microbiological analysis, based upon enumeration of culturable bacteria (thermophilic and mesophilic) and fungi, and also evolution of CO₂, showed that SMSs played higher microbial diversity than soil control. Laccase activity from A. subrufescens SMS tended to remain constant during a 2-month period, while L. edodes SMS presented low laccase activity throughout the same period. Agaricus subrufescens and L. edodes were able to grow on a PDA (Potato Dextrose Agar) media supplemented with different concentrations of atrazine (1–50 μg/ml), degraded the herbicide, attaining rates of 35% and 26%, respectively. On experiments of lettuce growth promotion using a soil-based potting media with different SMS rates, 5% and 10% (dw) rates of A. subrufescens SMS resulted in higher lettuce aerial dry weights than the rates of 25% and 40%, the chemical fertilization (NPK) and the control (soil). At 10% supplementation, lettuce aerial dry weight increased 2.2 and 1.3 times compared to the control and the NPK treatment, respectively. Protein content increased along with SMS rates. Fresh A. subrufescens SMS was an excellent supplement for lettuce growth promotion and showed potential for remediation of biocides possibly due to improved microbial diversity and enzymatic activity. Fresh L. edodes SMS was not a good fertilizer, at least under the conditions tested. However, microbiological analysis showed that promising results may be achieved when using fresh L. edodes SMS for soil remediation.     

Lavandula angustifolia essential oil as a novel and promising natural candidate for tick (Rhipicephalus (Boophilus) annulatus) control

Lavandula angustifolia is a well known herbal medicine with a variety of useful properties, including its acaricidal effect. This experiment was carried out to study the bioacaricidal activity of L. angustifolia essential oil (EO) against engorged Rhipicephalus (Boophilus) annulatus (Acari; Ixodidae) females. For this purpose six serial concentrations (0.5, 1.0, 2.0, 4.0, 6.0 and 8.0% w/v) of L. angustifolia EO were used. There was considerable mortality in concentrations more than 4.0% although there were no differences between 6.0 and 8.0% in all measured criteria. The mortality rate 24 h after inoculation was 73.26 and 100% in groups treated with 4.0 and 8.0% EO, respectively. Lavender EO also reduced tick egg weight in a concentration-dependent manner. The amount of eggs produced varied from 0.12 g (at 0.5% EO) to 0.00 g (at 8.0% EO) but did not differ statistically from the control. L. angustifolia EO caused 100% failure in egg laying at 6.0 and 8.0% whereas this value in the control group was zero. A positive correlation between L. angustifolia EO concentration and tick control, assessed by relative mortality rate and egg-laying weight, was observed by the EO LC/EC₅₀, which, when calculated using the Probit test, was 2.76-fold higher than the control. Lavender is a promising acaricidal against R. (B.) annulatusin vitro.     

Notes on the reproductive biology of Phrynus exsul Harvey, 2002 (Arachnida: Amblypygi: Phrynidae)

Mating behaviour and spermatophore morphology of the only Old World species of the Phrynidae, Phrynus exsul Harvey, 2002, are described and compared to the reproductive biology of other whip spiders. Mating behaviour and spermatophore morphology clearly show that P. exsul is correctly included in the Phrynidae. But some details and other characters suggest that P.exsul has no close relation with any of the Neotropical species of Phrynus Lamarck, 1793, probably having been evolutionary distinct for a long time from the Neotropical species of Phrynus. Its occurrence on the Indonesian Island Flores thus remains an enigma; is P. exsul a relic from a more global distribution of the genus Phrynus in the past or the result of prehistoric rafting across the ocean?     

Phylogenic analysis of the genus Leishmania by cytochrome b gene sequencing

In a previous report (Luyo-Acero et al., 2004), we demonstrated that cytochrome b (Cyt b) gene analysis is an effective method for classifying several isolates of the genus Leishmania; hence, we have further applied this method to other Leishmania species in an effort to enhance the accuracy of the procedure and to construct a new phylogenic tree. In this study, a total of 30 Leishmania and Endotrypanum WHO reference strains, clinical isolates from our patients assigned to 28 strains (human and non-human pathogenic species) and two species of the genus Endotrypanum were analyzed. The Cyt b gene in each sample was amplified by PCR, and was then sequenced by several primers, as reported previously. The phylogenic tree was constructed based on the results obtained by the computer software MEGA v3.1 and PAUP* v4.0 Beta. The present phylogenic tree was almost identical to the traditional method of classification proposed by Lainson and Shaw (1987). However, it produces the following suggestions: (1) exclusion of L. (Leishmania) major from the L. (L.) tropica complex; (2) placement of L.tarentolae in the genus Sauroleishmania; (3) L. (L.) hertigi complex and L. (V.) equatorensis close to the genus Endotrypanum; (4) L. (L.) enrietti, defined as L. (L.) mexicana complex, placed in another position; and (5) L. (L.) turanica and L. (L.) arabica are located in an area far from human pathogenic Leishmania strains. Cyt b gene analysis is thus applicable to the analyzing phylogeny of the genus Leishmania and may be useful for separating non-human pathogenic species from human pathogenic species.     

A new ovine Babesia species transmitted by Hyalomma anatolicum anatolicum

The pathogenicity and morphology of a large Babesia species, Babesia sp. Xinjiang, are described here. The parasite has very low virulence for sheep, and caused no detectable clinical symptoms. Splenectomized sheep infected with the parasite showed mild fever and low parasitemia and would recover gradually. If splenectomized sheep were immuno-suppressed with dexamethasone, the parasitemia could reach 8.5%, and death occurred. A splenectomized calf could not be infected with the Babesia species. Paired parasites were the typical form of the Babesia species in erythrocytes and the average size of a pair of parasites was 2.42 (±0.35) μm × 1.06 (±0.22) μm. Merozoites were found in the gut, salivary gland, haemolymph, ovary and eggs of female Hyalomma anatolicum anatolicum engorged on sheep infected with the parasites. The results of experimental transmission showed that the larval, nymph and adult stages of H. a. anatolicum could transmit the Babesia species to sheep.     

A common pathway for metabolism of 4-hydroxybenzylglucosinolate in Pieris and Anthocaris (Lepidoptera: Pieridae)

Caterpillars of Pieris rapae L. (Lepidoptera: Pieridae) convert 4-hydroxybenzylglucosinolate (sinalbin) in brassicaceous plants into 4-hydroxybenzylcyanide sulfate (HBC sulfate), with 4-hydroxybenzylcyanide (HBC) as intermediate. This apparently serves as a detoxification, because alternative formation of a mustard oil is avoided. We confirmed the capacity of P. rapae to convert the intermediate HBC into HBC sulfate. Four additional Pieridae – Anthocaris cardamines L., Pieris virginiensis Harris, Pieris napi oleracea Edwards and Pieris brassicae L., likewise excreted HBC sulfate after ingesting leaves with topically added HBC or leaves naturally containing sinalbin and myrosinase, but not after ingesting control leaves devoid of HBC and sinalbin. We confirmed the capacity of the most distantly related pierid species (A. cardamines) for converting ingested (topically added) sinalbin into HBC sulfate. Larvae of two non-pierid Brassicaceae-feeding insects, the oligophagous sawfly Athalia rosae L. (Hymenoptera: Tenthrenidae) and the polyphagous moth Spodoptera frugiperda (Smith) (Lepidoptera: Noctuidae), did not excrete HBC sulfate after ingesting sinalbin-containing leaves or topically added HBC.