Chinese phlebotomine sandflies of subgenus Adlerius nitzulescu, 1931 (Diptera: Psychodidae) and the identity of Phlebotomus sichuanensis Leng & Yin, 1983. Part I--Taxonomical study and geographical distribution

Four species of Adlerius phlebotomine sandflies have been recorded in China, namely: P. chinensis Newstead, 1916 (Pc), P. fengi Leng & Zhang, 1994; P. longiductus Parrot, 1928 and P. sichuanensis Leng & Yin, 1983 (Ps). Adlerius phlebotomies are the main vectors of visceral leishmaniasis (VL) in China; three of them are acknowledged as VL vectors and P. fengi is considered a potential VL vector for southwestern mountainous region. Different opinion has been raised to the validity of identity of Ps by some investigators from Shanghai and Shanxi who consider Ps to be a large type of Pc instead of an isolate species. The center of controversy is whether Ps is an isolate taxon or a large type of Pc. The present authors have carried out a series of comparative studies for these two flies on: 1 quantitative and qualitative morphological characters of four Chinese Adlerius phlebotomies; and 2. differences in geographical distribution. All specimens of Pc and Ps used in the present study are collected where their holotypes-paratypes were produced--West Mountain, West Suburb, Beijing and Lixian County, Sichuan Province. The results have forcefully proved that Ps is an isolate species instead of a so-called large type Pc according to the concept of species. The clarification of their taxonomical identities is meaningful because both of them are VL vectors in different epidemic areas in China; especially Ps is an important VL vector in high mountainous regions of southwestern China and some extend to the Loess Plateau of northwestern China, where VL still exists and it is also the first Phlebotomine sandfly discovered in Tibet, the locality being near Assam in India (Leng et al. 1990).     

Phlebotomus (Adlerius) halepensis vector competence for Leishmania major and Le. tropica

In Eurasia, phlebotomine sandflies of the subgenus Adlerius (Diptera: Psychodidae) comprise about 20 known species. Some are suspected vectors of visceral leishmaniasis (VL) and at least one species has been implicated as a vector of cutaneous leishmaniasis (CL). We tested Phlebotomus (Adlerius) halepensis Theodor (Jordan strain) for CL vector competence, compared with three standard vectors: Phlebotomus (Phlebotomus) duboscqi N-L. from Senegal, Phlebotomus (Paraphlebotomus) sergenti Parrot from Turkey and the Neotropical Lutzomyia longipalpis (L. & N) (Jacobina strain). Sandfly females were membrane-fed on amastigote suspensions of Leishmania major Y. & S. and Le. tropica (Wright) (Kinetoplastida: Trypanosomatidae) and examined for parasite development 3, 6 and 10 days post-infection. Phlebotomus halepensis showed high susceptibility to both leishmanias, supporting typical suprapylarian parasite development similar to the other vectors. Phlebotomus halepensis infection rates were approximately 90% for Le. major and approximately 80% for Le. tropica, with high parasite densities. Development of infections was relatively fast, colonizing the thoracic midgut by 6 days post-bloodmeal in every case and reaching the stomodeal valve in >80% of flies. In late-stage infections, 10 days post-bloodmeal, nearly all P. halepensis females had cardia and stomodeal valve filled with very high numbers of parasites and some Le. tropica-infected females had promastigotes in the pharynx and proboscis. Host choice experiments in the laboratory showed that P. halepensis females fed readily on rat or rabbit and preferred the human forearm. In view of its vector competence and partial anthropophily, we infer that P. halepensis is a potential vector of cutaneous as well as visceral leishmaniases.     

Phylogenetic analysis of Phlebotomus species belonging to the subgenus Larroussius (Diptera, psychodidae) by ITS2 rDNA sequences

In the genealogy of Phlebotomus (Diptera: Psychodidae), morphological analyses have indicated that the subgenus Larroussius is a monophyletic group which is most closely related to the subgenera Transphlebotomus and Adlerius. We conducted a phylogenetic analysis of the relationships among six representative species of the subgenus Larroussius and one species representatitive of the Phlebotomus subgenus, assessing sequences of the Second Internal Transcribed Spacer (ITS2) of the ribosomal RNA (rRNA). Three of the species (P. perniciosus, P. ariasi and P. perfiliewi perfiliewi) were collected in different parts of the Mediterranean area. The trees estimated from parsimony and neighbour-joining analyses supported the monophyly of the Larroussius subgenus inferred from the morphological analysis. According to our data, P. ariasi may be a sister group to the rest of the Larroussius subgenus, although additional sequence data are needed to confirm this observation. Our results suggest that P. perniciosus and P. longicuspis are distinct species, in spite of the fact that there are only slight morphological differences. The strict congruence between the phylogeny of the Larroussius subgenus inferred from the ITS2 sequences and that based on morphological studies further confirmed the ability of the spacer sequence to identify recently-derived affiliations.     

Bionomics of phlebotomine sandflies in the Galilee focus of cutaneous leishmaniasis in northern Israel

The bionomics of phlebotomine sandflies (Diptera: Psychodidae) were studied for three years (2001-2003) in the Galilee focus of cutaneous leishmaniasis in northern Israel, where the causative Leishmania tropica (Kinetoplastida: Trypanosomatidae) is transmitted by Phlebotomus (Adlerius) arabicus Theodor and Phlebotomus (Paraphlebotomus) sergenti Parrot, comprising 22% and 8%, respectively, of the local sandfly fauna sampled by light traps. The predominant species overall was Phlebotomus (Larroussius) tobbi Adler & Theodor (51%) with lesser numbers of Phlebotomus (Adlerius) simici Theodor (11%), Phlebotomus (Larroussius) syriacus Adler & Theodor (5%), Phlebotomus (Larroussius) perfiliewi Perfil'ev (3%) and Phlebotomus (Phlebotomus) papatasi Scopoli (0.05%). Sandfly adult populations were prevalent from April to November and peaked between June and August, being more abundant through the summer in irrigated habitats, such as gardens and orchards, than in open grassland. Of the two cutaneous leishmaniasis vectors, P. sergenti preferred boulder mounds located at the outskirts of settlements, whereas P. arabicus was more abundant overall and near houses in particular. Females of all these sandfly species displayed a peak of activity after sunset (20.00-22.00 hours), whereas activity of males persisted longer through the night. Another slight increase in activity was noted before dawn (02.00-04.00 hours). Phlebotomus arabicus appears to be the main vector of L. tropica in the Galilee focus, due to its denser populations, more endophily and preference for peridomestic habitats than shown by P. sergenti in northern Israel.     

Incrimination of Phlebotomus kandelakii and Phlebotomus balcanicus as vectors of Leishmania infantum in Tbilisi, Georgia

A survey of potential vector sand flies was conducted in the neighboring suburban communities of Vake and Mtatsminda districts in an active focus of visceral Leishmaniasis (VL) in Tbilisi, Georgia. Using light and sticky-paper traps, 1,266 male and 1,179 female sand flies were collected during 2006-2008. Five Phlebotomus species of three subgenera were collected: Phlebotomus balcanicus Theodor and Phlebotomus halepensis Theodor of the subgenus Adlerius; Phlebotomus kandelakii Shchurenkova and Phlebotomus wenyoni Adler and Theodor of the subgenus Larroussius; Phlebotomus sergenti Perfil'ev of the subgenus Paraphlebotomus. Phlebotomus sergenti (35.1%) predominated in Vake, followed by P. kandelakii (33.5%), P. balcanicus (18.9%), P. halepensis (12.2%), and P. wenyoni (0.3%). In Mtatsminda, P. kandelakii (76.8%) comprised over three fourths of collected sand flies, followed by P. sergenti (12.6%), P. balcanicus (5.8%), P. halepensis (3.7%), and P. wenyoni (1.1%). The sand fly season in Georgia is exceptionally short beginning in early June, peaking in July and August, then declining to zero in early September. Of 659 female sand flies examined for Leishmania, 12 (1.8%) specimens without traces of blood were infected including 10 of 535 P. kandelakii (1.9%) and two of 40 P. balcanicus (5.0%). Six isolates were successfully cultured and characterized as Leishmania by PCR. Three isolates from P. kandelakii (2) and P. balcanicus (1) were further identified as L. infantum using sequence alignment of the 70 kDa heat-shock protein gene. Importantly, the sand fly isolates showed a high percent identity (99.8%-99.9%) to human and dog isolates from the same focus, incriminating the two sand fly species as vectors. Blood meal analysis showed that P. kandelakii preferentially feeds on dogs (76%) but also feeds on humans. The abundance, infection rate and feeding behavior of P. kandelakii and the infection rate in P. balcanicus establish these species as vectors in the Tbilisi VL focus.     

Experimental transmission of Leishmania tropica to hyraxes (Procavia capensis) by the bite of Phlebotomus arabicus

The ability of the sand fly Phlebotomus (Adlerius) arabicus to transmit Leishmania tropica was studied experimentally using hyraxes (Procavia capensis), natural reservoir hosts of the parasite. Sand flies became infected with L. tropica after feeding on a lesion of needle-inoculated hyrax. Moreover, P. arabicus fed with L. tropica promastigotes transmitted the parasite to hyraxes by bite during a second bloodmeal. Although the animals remained asymptomatic after infective sand fly bite, they were PCR positive and infectious for naive sand flies. We have thus demonstrated cyclical transmission of L. tropica by P. arabicus in hyraxes. This confirms experimentally the vectorial competence of P. (Adlerius) arabicus, and demonstrates that asymptomatic reservoir hosts are infectious to appropriate vectors.     

Distribution of phlebotomine sandflies, of the subgenus Larroussius, in Morocco

Adult sandflies (Diptera: Psychodidae) of the genus Phlebotomus and of the subgenus Larroussius were collected with sticky traps from thirty-four localities in central and south-western Morocco. Four Larroussius species were present in this area, Phlebotomus perniciosus Newstead, being the most widespread and abundant species particularly in the mountainous area, was sympatric with the closely related species Phlebotomus longicuspis Nitzulescu. Only one male of P. perniciosus displayed the bifid copulatory valves characteristic of the species. All specimens showed copulatory valves curved at their apex. The number of coxite hairs is a taxonomical tool that differentiates this morph of P. perniciosus from P. longicuspis. Phlebotomus ariasi Tonnoir was collected from six mountainous stations. Phlebotomus mariae Rioux et al. was scarce; only five males were collected and were often associated with P. ariasi.     

Sandflies (Diptera: Psychodidae) in a focus of visceral leishmaniasis in White Nile, Sudan

Visceral leishmaniasis (VL) has been known to occur since the 1980s on the western bank of the White Nile River (Central Sudan), 150 km south of Khartoum, and has resulted in high mortality. The most recent outbreak of the disease in this area began in 2006. Entomological surveys were carried out during May 2008, June 2010 and May and July 2011 in the White Nile area. Sandflies were collected using Centers for Disease Control light traps and sticky oil traps in the village of Kadaba and the nearby woodland. Phlebotomus females were dissected for the presence of Leishmania promastigotes. A total of 17,387 sandflies, including six species of Phlebotomus and 10 species of Sergentomyia, were identified. The Phlebotomus species recorded were Phlebotomus orientalis, Phlebotomus papatasi, Phlebotomus bergeroti, Phlebotomus duboscqi, Phlebotomus rodhaini and Phlebotomus saevus. P. orientalis was collected in both habitats. The relative abundance of P. orientalis in the woodland habitat was higher than that recorded in the village habitat. In the woodland habitat, there was a notable increase in the relative abundance of P. orientalis during the surveys conducted in 2008 and 2010 compared to 2011. None of the 311 P. orientalis females dissected were infected with Leishmania promastigotes, although relatively high parous rates were recorded in both habitats. Based on the distribution of P. orientalis recorded in this study, this species is the most likely vector of VL in the endemic focus in the White Nile area. Further investigation is required to elucidate the seasonal abundance and distribution of the vector, as well as the transmission season of VL in both habitats so that appropriate control strategies for the vector can be designed.     

Phlebotomines of the Isle of Cyprus. III. Species inventory

Three surveys on canine leishmaniasis were carried out in Cyprus (1993, 1998 and 1999) emphasise the presence of eleven species of phlebotomine sandflies: Phlebotomus (Phlebotomus) papatasi, P. (Paraphlebotomus) alexandri, P. (Pa.) jacusieli (first mention in Cyprus), P. (Pa.) sergenti, P. (Larroussius) galilaeus, P. (L.) tobbi, P. (Transphlebotomus) economidesi, P. (T.) mascittii, Sergentomyia (Sergentomyia) azizi (its specific statute is validated by the authors), S. (S.) fallax et S. (S.) minuta. P. (Adlerius) kyreniae was not caught during the surveys. The authors propose hypothesis of settlement of the island by phlebotomine sandflies according this species inventory. A first migration period took probably place during the Miocene time and a second one during the Pleistocene time.     

Morphometric and molecular differentiation of Phlebotomus (Phlebotomus) sandflies

The closely related sandfly species of the subgenus Phlebotomus namely, Phlebotomus papatasi (Scopoli, 1786), Phlebotomus duboscqi Neveu‐Lemair, 1906 and Phlebotomus bergeroti Parrot, 1934 (Diptera: Psychodidae), are major vectors of Leishmania major (Kinetoplastida: Trypanosomatidae), the causative agent of cutaneous leishmaniasis in the Old World. Although allopatric in most of their distribution, the three species exist sympatrically in many places in central and eastern Sudan. Males of the three species can be distinguished using morphological characters; however, females are much harder to identify, thus complicating epidemiological studies. We carried out a morphometric and a molecular study to determine reliable morphological features and develop a polymerase chain reaction (PCR) assay for distinguishing females of these species. Males and females from each species were collected from sites in Sudan, East Africa and from one site in Mali, West Africa. Males were analysed morphologically and 20 characters and 10 character ratios were used in a stepwise discriminant analysis. This led to the identification of four characters with high discriminant loading scores sufficient for accurate male species identification. Male DNA was then used for the development of a PCR‐based species diagnostic based on the second internal transcribed spacer (ITS2) of the ribosomal DNA. A set of four primers was developed to generate fragment sizes that are specific to each species and can reliably identify females as well as hybrid DNA. Both the morphometric and the molecular findings of this study have important applications for studies of the epidemiology of cutaneous leishmaniasis.     

Fellomyces mexicanus sp. nov., a new member of the yeast genus Fellomyces isolated from lichen Cryptothecia rubrocincta collected in Mexico

Two strains isolated from the lichen Cryptothecia rubrocincta collected in Mexico are described as new members of the sterigmatoconidia-producing genus Fellomyces. Based on the identical sequences of the D1/D2 regions of 26S rDNA, as well as the results of the cluster analysis of the AFLP fingerprints, the strains have been shown to be conspecific. The 26S rDNA-based phylogeny has indicated that Fellomyces mexicanus clusters within a clade including species such as four other Fellomyces isolates from lichens: F. chinensis, F. lichenicola, F. sichuanensis and F. thailandicus. F. mexicanus is characterised by the presence of xylose in the cell walls and CoQ10 in the mitochondrial membrane. The type culture is strain HB25=CBS8279.     

Genetic polymorphism in sympatric species of the genus Phlebotomus, with special reference to Phlebotomus perniciosus and Phlebotomus longicuspis (Diptera, Phlebotomidae)

The Random Amplified Polymorphic DNA assay was used to study genetic variation within and between five Phlebotomus species belonging to three subgenera: P. (Larroussius) ariasi, P. (L.) longicuspis, P. (L.) perniciosus, P.(Paraphlebotomus) sergenti and P. (Phlebotomus) papatasi sympatric in southern Spain and proven vector of leishmaniasis. Two cluster analysis were proposed: one according to sandfly species and populations, the second according individual specimens of Phlebotomus perniciosus, Phlebotomus longicuspis s.l. and intermediate morphological specimens between these species. The results obtained are closely correlated with the taxonomy classically accepted for the subgenera and with the automatic classifications made by other authors which use morphological and isoenzymatic data. The validity of the species Phlebotomus longicuspis is also discussed.     

Distribution and altitudinal structuring of phlebotomine sand flies (Diptera: Psychodidae) in southern Anatolia, Turkey: their relation to human cutaneous leishmaniasis.

The two Old World genera, Phlebotomus and Sergentomyia, were both recorded in southern Anatolia in Turkey. Phlebotomus species predominated and comprised about 93% of the entire collection (3,172 specimens). Out of the sixteen species identified, two belonged to the genus Sergentomyia: S. dentata and S. theodori. The remaining fourteen species in the genus Phlebotomus were grouped under four subgenera including some species that are elsewhere known to act as vectors of human cutaneous leishmaniasis. Most of the Phlebotomus were P. tobbi (32.5%), but P. papatasi, P. transcaucasicus, P. halepensis, P. galilaeus, P. sergenti, P. syriacus, P. neglectus, P. simici, P. alexandri, P. similis, P jacusieli, P. perfiliewi, and P. brevis were also identified. There were two associations of sand fly fauna with altitudinal gradient; the first one at relatively higher altitudes and the second one at lower altitudes. The transition between these two assemblages was within the range of 800-1,000 m. It is likely that Adana and Hatay provinces are transitional areas between western and eastern Anatolia. Mountains do not appear to be important geographical barriers for sand fly distribution. We also found that the proven vector P. sergenti is a widely distributed species throughout southern Anatolia and this species, together with its closely related species P. similis, shows sympatry in Konya Province.     

Mass spectrometry-based chemotaxonomic classification of Penicillium species (P. echinulatum, P. expansum, P. solitum, and P. oxalicum) and its correlation with antioxidant activity

In this study, 4 Penicillium species (17 strains) were classified on the basis of metabolite profile (chemotaxonomy) by using liquid chromatography-electrospray ionization ion trap-mass spectrometry (LC-ESI-MS), gas chromatography-ion trap-mass spectrometry (GC-IT-MS) and multivariate statistical analysis. The LC-ESI-MS-based dendrogram was similar to the internal transcribed spacer (ITS)-based dendrogram, in that Penicillium oxalicum was separated from the other 3 species. Moreover, vermiculidiol, meleagrin, oxaline, glandicolin A and B, and secalonic acid D were identified as metabolites that enable discrimination of Penicillium species by partial least squares discriminant analysis (PLS-DA). Evaluation of the species-specific metabolites produced by P. expansum, P. echinulatum, and P. solitum revealed that the 3 species differed from each other. On the other hand, GC-IT-MS-based dendrogram revealed that P. expansum was clearly classified separately from the other 3 species, and this result correlated with the antioxidant activity of the 4 species: P. expansum had a higher radical scavenging activity than the other 3 species. The metabolites produced in higher amounts in P. expansum were gluconic acid (12, 29, 33); andrastin A (16), B (15), and C (17); chaetoglobosin C (14), a class of sugar (31, 32); and salicylic acid (28). The results of this study demonstrated that metabolite-based chemotaxonomy could be used not only as a classification method but also as a tool for evaluation of species-specific activities.     

多叶重楼遗传多样性的RAPD分析

应用RAPD技术检测了多叶重楼(Paris polyphyfzo)2个变种4个居群的遗传多样性,并与1个凌云重楼(P．cronquistii)居群进行了比较。选择的16个随机引物在5个居群中共检测到246个多态位点。在居群水平上,滇重楼2个居群的多态位点百分比(PP鳓分别为57．43％和54．67%,Shannon指数分别为0．3080和0．2830;七叶一枝花2个居群的PPB分别为56．33％和57．75%,Shannon指数分别为0、3080和0．3293。在变种水平上,滇重楼的PPB为75．14％,Shannon指数为0．3922,遗传分化系数(Gst)为0．3085;七叶一枝花的PPB为80．31％,Shannon指数为0．3992,遗传分化系数(Gst)为0．3726;在种的水平PPB达92．05%,遗传分化系数Gst达0．5151。聚类分析显示滇重楼和七叶一枝花有较近的亲缘关系,而与凌云重楼遗传距离较远。此结果从分子水平上支持了过去将滇重楼和七叶一枝花划分为1个种下2个变种的形态分类观点。     

About the presence of Phlebotomus sergenti Parrot, 1917 (Diptera: Psychodidae) in Eastern Sicily, Italy

The note reports the data of a three-year sand fly investigation (1997-99) carried out in Eastern Sicily (Italy) with the aim to study the distribution of Phlebotomus sergenti. The survey involved a densely inhabited area at the foot of Mount Etna and the area of Iblei mounts. A total of 9,095 sand flies, of which 63.4% males, were captured. Five species belonging to the genus Phlebotomus (P. perniciosus, P. perfiliewi, P. neglectus, P. sergenti and P. papatasi) and one to the genus Sergentomyia (S. minuta) were identified. Both the prevalence and distribution of the species were different within the two areas studied. In Mount Etna area, P. perniciosus (77.7%) was the prevalent species followed by S. minuta (19.8%), P. sergenti (2.0%), P. neglectus (0.3%) and P. papatasi (0.2%). While in Iblei mounts region S. minuta (84.5%) showed the highest prevalence, followed by P. perniciosus (14.4%), P. perfiliewi (0.9%) and P. neglectus (0.1%). Here, P. sergenti was a very rare species (< 0.02). P. sergenti was mostly associated to domestic habitats of peri-urban and urban zones located between two and 750 m a.s.l. The density values of P. sergenti, expressed as number of specimens/m2 of sticky trap, were between 0.3 and 5.5 with the highest value in the hilly collecting sites. The low observed abundance of P. sergenti does not allow to draw any prediction on the role that the species could play in the transmission of leishmaniasis in Sicily.     

Molecular phylogeny of mangroves. VI. Intraspecific genetic variation in mangrove species Excoecaria agallocha L. (Euphorbiaceae).

Genomic DNA from 84 individuals of Excoecaria agallocha from seven mangrove populations were analysed for random amplified polymorphic DNAs (RAPDs) using 16 random 10-mer primers. Polymorphism within populations varied from 20% to 31%. At the interpopulation level, 111/149 (74%) of RAPDs were polymorphic. Restriction fragment length polymorphism (RFLP) analysis of 21 individuals (3 individuals randomly selected from the 7 populations) using 30 probe-enzyme combinations revealed a high level of interpopulation polymorphism (62.2%) indicating interpopulation genetic divergence. The polymorphic RAPDs and RFLPs were pooled, and clustering was carried out based on mean similarity for individual populations. The dendrogram showed groupings of populations from the West and East Coasts of India into separate clusters, at 60% similarity level. Further, RAPD and RFLP analysis of male and female plants showed approximately the same level of variation in both sexes, and no sex-linked markers were found. These results demonstrate that considerable intrapopulation and interpopulation genetic variations exist in E. agallocha, and that lack of genetic variation is not the reason for the morphological uniformity observed across the range of the species.     

Absence of outer caudal setae on all larval instars of Phlebotomus tobbi from the Ionian Greek islands

Larval instars 2, 3 and 4 of Phlebotomus tobbi Adler, Theodor & Lourie from the Greek islands of Corfu and Zakynthos were found to have two caudal setae instead of the four usually present on these instars of Phlebotomus larvae. In a scanning electron microscope comparison with larvae of P. papatasi (Scopoli), a sensillum was seen in place of each outer seta of P. tobbi larvae, suggesting secondary loss of the setae. As the larvae of less than a tenth of the species and subspecies of the genus Phlebotomus have been seen and described, it cannot be assumed that those of P. tobbi are unique in having only two caudal setae. However, four caudal setae in late instars can no longer be considered as a constant character of the genus Phlebotomus. Observations on the larvae of P. tobbi also raise the question of the unknown function of the caudal setae.     

四种竹子的花器官形态描述

近几年来南京林业大学竹种园中4种竹子出现开花现象.首次描述了鹅毛竹(Shibaoea chinensis)和异叶苦竹(Arundinaria simonii f.heterophylla)的花部形态特征,补充描述了月月竹(Chimonobambusa sichuanensis)和福建茶秆竹(Arundinaria amabilis var.convexa)的花部形态特征.