Trypanosoma theileri: In vitro cultivation in tsetse fly and vertebrate cell culture systems

Epimastigote forms of Trypanosoma theileri were grown at 25°C in insect cell culture media and in Glossina tissue cultures for more than 6 months. Doubling times of 10–14 h during exponential growth were observed. In cell cultures which had been derived from pupal tsetse flies growth rates were higher than in cell free media; in a larval cell line, however, growth of T. theileri was inhibited. Ecdysteroids and juvenile hormone I reduced multiplication of T. theileri in cell free media. When T. theileri was incubated in different sera only fetal calf serum (FCS) supported growth. Epimastigote forms transformed into trypomastigote bloodforms when cultured at 37°C in FCS, vertebrate cell cultures, and Eagle's medium, but not in insect media or Glossina cell cultures. Oxygen uptake of epimastigotes could be inhibited by rotenone antimycin A and cyanide; trypomastigotes were not affected by these inhibitors.     

DNA fingerprinting analysis in the solitary bee Megachile rotundata: variability and nest mate genetic relationships.

The most conventional approach for evaluating genetic variability in an insect population involves assessing the degree of enzyme polymorphism. Hymenoptera display a relatively low level of genetic variability compared with most insect species. DNA probes consisting of tandemly repeated sequences are powerful tools for detecting polymorphisms when employed to develop DNA fingerprinting (DNAfp) profiles in a wide range of organisms. This report describes genetic variability in the solitary bee species Megachile rotundata as assessed by DNAfp using the M13 sequence and a synthetic oligonucleotide sequence homologous to a hypervariable region of the alpha-globin gene. DNAfp comparisons among offspring were used to analyze genealogical structure in M. rotundata nests. The results indicate that polyandry, by a large number of males, is not a common phenomenon in M. rotundata bee species. In the present analysis, it is likely that the broods raised in single nests are mostly the offspring of one singly mated female. However, the data does not preclude that for certain nests two males could have been involved in the mating process.     

中国甘草属植物的核型分析

本文对国产甘草属11种,1变种进行了染色体核型分析,2n=2x=16,并对其种间关系进行了讨论。     

Application of kDNA as a molecular marker to analyse Leishmania infantum diversity in Portugal

Around the Mediterranean basin Leishmania infantum is an important parasite causing canine leishmaniasis and visceral and cutaneous clinical forms in both immunocompetent and immunocompromised humans. Efficient monitoring and evaluation of epidemiology with discriminatory molecular markers are required. We investigated the genetic diversity of L. infantum in Portugal by polymerase chain amplification and restriction fragment length polymorphism analysis of kinetoplastid DNA, as molecular marker. We analysed 120 Portuguese isolates of L. infantum plus 16 other non-Portuguese isolates (as a reference group) from humans, dogs and sand flies. The Portuguese population showed a high degree of polymorphism with a total of 13 profiles identified. The predominant profile was A, which was only detected in the Portuguese samples.

The kinetoplastid DNA PCR-RFLP assay described here was suitable for use directly with biological samples and the profiles obtained were stable during long-term growth in vitro and in laboratory animals.     

The antiviral drug valacyclovir successfully suppresses salivary gland hypertrophy virus (SGHV) in laboratory colonies of Glossina pallidipes

Many species of tsetse flies are infected with a virus that causes salivary gland hypertrophy (SGH) symptoms associated with a reduced fecundity and fertility. A high prevalence of SGH has been correlated with the collapse of two laboratory colonies of Glossina pallidipes and colony maintenance problems in a mass rearing facility in Ethiopia. Mass-production of G. pallidipes is crucial for programs of tsetse control including the sterile insect technique (SIT), and therefore requires a management strategy for this virus. Based on the homology of DNA polymerase between salivary gland hypertrophy virus and herpes viruses at the amino acid level, two antiviral drugs, valacyclovir and acyclovir, classically used against herpes viruses were selected and tested for their toxicity on tsetse flies and their impact on virus replication. While long term per os administration of acyclovir resulted in a significant reduction of productivity of the colonies, no negative effect was observed in colonies fed with valacyclovir-treated blood. Furthermore, treatment of a tsetse colony with valacyclovir for 83 weeks resulted in a significant reduction of viral loads and consequently suppression of SGH symptoms. The combination of initial selection of SGHV-negative flies by non-destructive PCR, a clean feeding system, and valacyclovir treatment resulted in a colony that was free of SGH syndromes in 33 weeks. This is the first report of the use of a drug to control a viral infection in an insect and of the demonstration that valacyclovir can be used to suppress SGH in colonies of G. pallidipes.     

Analysis of DNA polymorphism in populations of the Volga-Ural region using genome fingerprinting with phage M13 DNA]

The hyperpolymorphism of minisatellite DNA hybridizing with DNA of bacteriophage M13 was analyzed in seven Turkic and Finno-Ugric populations from the Volga-Urals region. In total, hybridization revealed 80 BspRI genomic DNA fragments ranging in size from 1.7 to 10 kb; the average frequency of an individual fragment was 0.299 +/- 0.020. The average number of hybridization fragments per pattern (varying from 14 to 20 in different populations) and frequencies of individual fragments showed significant interpopulation differences. Parameters of this polymorphic system were assumed to reflect phenotypic diversity of populations. Genome fingerprinting with the use of phage M13 can be employed in the studies of population genetic structure and differentiation and in forensic medicine, for more accurate personal identification.     

毒鹿花菌——一个发现于中国的毒蘑菇新种

基于形态学和系统发育研究结果,本研究报道在中国华中和西南地区发现的一个隶属于鹿花菌属鹿花菌亚属的有毒新种——毒鹿花菌。该种在形态上与鹿花菌Gyromitra esculenta十分相似,但毒鹿花菌的囊盘被分化较为明显,外表面几乎由一层栅状细胞排列而成,而鹿花菌的囊盘被基本没有分化。此外,毒鹿花菌通常具有更长的菌柄并生于阔叶林地上。为了准确理解鹿花菌的物种概念,本研究指定了一份采自法国的标本（即HMAS 21460）作为该种的附加模式。     

微地形对优势种群点格局和关联性的影响

不同的空间格局类型和关联性可以反映出不同生境下植物种群的资源环境适应策略.老鹳草与米口袋是甘南亚高寒草甸的主要物种,通过野外群落调查和室内试验,运用点格局分析法中的Ripley K函数,对不同坡向的米口袋和老鹳草种群的空间分布格局及种间关联性进行分析.结果表明:从阳坡-西坡-阴坡,土壤含水量和养分含量逐渐增加,土壤温度和光照度逐渐降低.米口袋和老鹳草在3个坡向(阴坡、西坡、阳坡)上的分布呈现出明显差异,随着坡向由阴坡向阳坡的转变,米口袋种群数量呈递增趋势,老鹳草种群数量则表现出递减趋势.在阴坡,米口袋种群在0~1.8 m尺度上呈聚集分布,在1.8~5.0 m尺度上聚集强度减弱并趋向于随机分布,老鹳草种群在0~5.0 m尺度上均以聚集分布为主;在西坡,米口袋种群在0~1.2 m尺度上呈聚集分布,在1.2~5.0 m研究尺度上趋向于随机分布,老鹳草种群在0~2.2 m尺度上呈聚集分布,在2.2~5.0 m研究尺度上趋向于随机分布;在阳坡,米口袋与老鹳草种群在0~5.0 m尺度内均表现出随机分布.阴坡上这两种物种在0~1.0 m尺度上表现为正相关,在1.0~5.0 m尺度不相关;西坡上两物种在0~2.1 m尺度上呈负关联,在2.1~5.0 m尺度上两个物种间关联度均趋向于不相关;阳坡上这两个物种在0~5.0 m尺度上表现出不相关.     

中国近海甲藻环沟藻属2个新记录种:纺锤环沟藻和莫氏环沟藻

甲藻环沟藻属于一类无色素体、表面有脊的裸甲藻, 因可捕食一些重要的赤潮生物而在海洋生态系统中扮演着重要的角色。有关中国近海环沟藻属的物种多样性信息非常有限。本文报道了2个新记录种——纺锤环沟藻(Gyrodinium fusiforme)和莫氏环沟藻(G. moestrupii)。纺锤环沟藻细胞呈纺锤形, 长48.0-58.0 μm, 宽18.0-23.0 μm, 长宽比为2.4-3.0, 和模式种相比体型和长宽比都较小。莫氏环沟藻细胞也呈纺锤形, 长约30 μm, 宽约15 μm。我们测定了纺锤环沟藻和莫氏环沟藻大亚基的部分序列, 并根据大亚基序列利用最大似然法和贝叶斯法建立了系统发育树。结果显示环沟藻属是单源的, 纺锤环沟藻和裂缝环沟藻(G. fissum)聚合在一起, 但是与螺旋环沟藻(G. spirale)分离。纺锤环沟藻和莫氏环沟藻分别可以摄食米氏凯伦藻(Karenia mikimotoi)和具齿原甲藻(Prorocentrum dentatum), 前者在米氏凯伦藻赤潮中的大量出现显示它可以促进赤潮的消退。     

Genetic relationships between subspecies of the tsetse fly Glossina morsitans inferred from variation in mitochondrial DNA sequences

A 750 base pair segment of DNA from the tsetse fly Glossina morsitans morsitans was isolated by means of molecular cloning. It was shown by DNA hybridization to have substantial sequence homology with a defined region of the mitochondrial genomes of several Drosophila species. When used as a probe against DNA prepared from single tsetse flies, the cloned sequence revealed local restriction site variation between members of the G. morsitans subspecies complex. This feature was used to demonstrate maternal inheritance of the sequence in progeny of hybrid crosses and to assemble comparative restriction maps for a 3-kilobase segment of each mitochondrial genome. The data obtained from these exercises point to a higher genetic identity between G. m. morsitans and G. m. centralis than between either form and G. m. submorsitans.     

中国地星属新记录种

在对采自内蒙古自治区和吉林省的地星标本进行研究中,发现3个中国新记录种：草原地星Geastrum campestre、匈牙利地星Geastrum hungaricum和施魏地星Geastrum schweinitzii,本文提供3个种的形态描述、原生态图、线条图、担孢子和孢丝的扫描电镜照片以及ITS序列的GenBank号。     

Prospects of a fungus-contamination device for the control of tsetse fly Glossina fuscipes fuscipes

The prospects of the fungus Metarhizium anisopliae (Metsch.) Sorok. applied in contamination devices (Cds) to control tsetse fly Glossina fuscipes fuscipes Newstead was tested in a field experiment in Lake Victoria from 2 March 1999 to 31 August 2000. One hundred and sixty pyramidal traps mounted with Cds were deployed along the lakeshore and rivers on Mfangano Island. Contamination devices were loaded with 1.5-2.0 g of dry conidia/Cd. On the second island, Nzenze Island, four pyramidal traps fitted with plastic bags were deployed and served as the conventional 'trap and kill' population suppression method. A third island, Ngodhe Island, remained untreated and served as a control. Cds were recharged monthly with fresh conidia; plastic bags were also changed monthly. The apparent changes in population density were monitored weekly using biconical traps set at random on the three islands. To assess the incidence of M. anisopliae in tsetse flies on Mfangano Island, flies captured during monitoring were maintained in the laboratory and their mortality recorded. Fly population was reduced to 82.4 and 95.8% relative to untreated control on Mfangano and Nzenze islands, respectively, during the experimental period. Compared to the fungus-treated island, the number of flies caught in monitoring traps increased considerably in 'trap kill' treatment at 5 months after the treatments were removed. The incidence of M. anisopliae in fly populations was low during the 12 weeks following the initiation of the experiment but increased afterward until termination of the treatment. M. anisopliae could still be recovered from fly populations at 3 months after termination of the treatment, although the incidence was low. The results of this study have shown that application of M. anisopliae in a contamination device can suppress the population of G. fuscipes fuscipes comparable to the 'trap and kill' technology.     

Immunogenicity and Serological Cross-Reactivity of Saliva Proteins among Different Tsetse Species

Tsetse are vectors of pathogenic trypanosomes, agents of human and animal trypanosomiasis in Africa. Components of tsetse saliva (sialome) are introduced into the mammalian host bite site during the blood feeding process and are important for tsetse’s ability to feed efficiently, but can also influence disease transmission and serve as biomarkers for host exposure. We compared the sialome components from four tsetse species in two subgenera: subgenus Morsitans: Glossina morsitans morsitans (Gmm) and Glossina pallidipes (Gpd), and subgenus Palpalis: Glossina palpalis gambiensis (Gpg) and Glossina fuscipes fuscipes (Gff), and evaluated their immunogenicity and serological cross reactivity by an immunoblot approach utilizing antibodies from experimental mice challenged with uninfected flies. The protein and immune profiles of sialome components varied with fly species in the same subgenus displaying greater similarity and cross reactivity. Sera obtained from cattle from disease endemic areas of Africa displayed an immunogenicity profile reflective of tsetse species distribution. We analyzed the sialome fractions of Gmm by LC-MS/MS, and identified TAg5, Tsal1/Tsal2, and Sgp3 as major immunogenic proteins, and the 5''-nucleotidase family as well as four members of the Adenosine Deaminase Growth Factor (ADGF) family as the major non-immunogenic proteins. Within the ADGF family, we identified four closely related proteins (TSGF-1, TSGF-2, ADGF-3 and ADGF-4), all of which are expressed in tsetse salivary glands. We describe the tsetse species-specific expression profiles and genomic localization of these proteins. Using a passive-immunity approach, we evaluated the effects of rec-TSGF (TSGF-1 and TSGF-2) polyclonal antibodies on tsetse fitness parameters. Limited exposure of tsetse to mice with circulating anti-TSGF antibodies resulted in a slight detriment to their blood feeding ability as reflected by compromised digestion, lower weight gain and less total lipid reserves although these results were not statistically significant. Long-term exposure studies of tsetse flies to antibodies corresponding to the ADGF family of proteins are warranted to evaluate the role of this conserved family in fly biology.     

Comparison of the susceptibility of different Glossina species to simple and mixed infections with Trypanosoma (Nannomonas) congolense savannah and riverine forest types

Abstract Teneral Glossina morsitans mositans, G.m.submorsitans, G.palpalis gambiensis and G.tachinoides were allowed to feed on rabbits infected with Trypanosoma congolense savannah type or on mice infected with T.congolense riverine-forest type. The four tsetse species and subspecies were also infected simultaneously in vitro on the blood of mice infected with the two clones of T.congolense via a silicone membrane. The infected tsetse were maintained on rabbits and from the day 25 after the infective feed, the surviving tsetse were dissected in order to determine the infection rates.
Results showed higher mature infection rates in morsitans-gwup tsetse flies than in palpalis-group tsetse flies when infected with the savannah type of T.congolense. In contrast, infection rates with the riverine-forest type of T.congolense were lower, and fewer flies showed full development cycle. The intrinsec vectorial capacity of G.m.submorsitans for the two T.congolense types was the highest, whereas the intrinsic vectorial capacity of G.p.gambiensis for the Savannah type and G.m.morsitans for the riverine-forest type were the lowest. Among all tsetse which were infected simultaneously with the two types of T.congolense , the polymerase chain reaction detected only five flies which had both trypanosome taxa in the midgut and the proboscis. All the other infections were attributable to the savannah type.
The differences in the gut of different Glossina species and subspecies allowing these two sub-groups of T.congolense to survive better and undergo the complete developmental cycle more readily in some species than other are discussed.     

Utility of the polymerase chain reaction-restriction fragment length polymorphisms of the intergenic spacer region of the rDNA for characterizing Gibberella fujikuroi isolates

In the present report, a total of thirty-one isolates of Gibberella fujikuroi (Sawada) Wollenw. species complex of Fusarium (section Liseola) morphologically classified as F. moniliforme according to the taxonomy of Nelson, Toussoun and Marasas (1983) were analyzed for their ability to produce fumonisin B₁ and fumonisin B₂ by an optimized liquid chromatographic method. They were isolated from three hosts (Zea mays, Musa sapientum and Pinus pinea). The results indicate that M. sapientum is a preferential host for G. fujikuroi isolates with low or null capacity for producing fumonisins, while isolates from Z. mays and P. pinea are generally high fumonisin producers.

The molecular characterization of isolates was carried out in parallel using an optimized, simple and low-cost method for isolating DNA from filamentous fungi and polymerase chain reaction-restriction fragment length polymorphisms (PCR-RFLP) of the rDNA intergenic spacer (IGS) region. The haplotypes obtained with Hha I enzyme and combinations of Hha I, EcoR I, Alu I, Pst I and Xho I enzymes provided very characteristic groupings of G. fujikuroi isolates as a function of host type and fumonisin B₁ and B₂ producing capacity. IGS region restriction patterns showed no relationship to isolate geographical origin. This is the first report on this method's capacity to detect polymorphism permitting discrimination between G. fujikuroi isolates from different hosts and with different toxigenic profiles.     

Seasonal distribution and abundance of tsetse flies (Glossina spp.) in the Faro and Deo Division of the Adamaoua Plateau in Cameroon

Ten years after the large-scale tsetse control campaigns in the important cattle rearing areas of the Faro and Deo Division of the Adamaoua Plateau in Cameroon, the seasonal distribution and abundance of tsetse flies (Glossina spp.) were determined. During a period of 12 consecutive months (January-December 2005), the tsetse population was monitored along four trap transects consisting of a total of 32 traps and two flyround transects traversing the study area, which comprised the tsetse-infested valley, a buffer zone and the supposedly tsetse-free plateau. Throughout the study period, a total of 2195 Glossina morsitans submorsitans and 23 Glossina tachinoides were captured in the traps and 1007 G. m. submorsitans (78.8% male flies) were captured along the flyround transects. All G. tachinoides and almost all G. m. submorsitans were captured in the valley. Five G. m. submorsitans were captured in traps located in the buffer zone, whereas no flies were captured in traps located on the plateau. The index of apparent abundance (IAA) of G. m. submorsitans was substantially higher in the areas close to game reserves. In the remaining part of the valley, where wildlife is scarce and cattle are present during transhumance (dry season), the IAA of tsetse was substantially lower. In this part of the valley, the abundance of tsetse seemed to be associated with the presence of cattle, with the highest IAA during transhumance when cattle are present and the lowest apparent abundance during the rainy season when cattle have moved to the plateau. It is concluded that the distribution of tsetse in a large part of the valley undergoes substantial seasonal changes depending on the presence or absence of cattle. The repercussions of those findings for the control of tsetse in the valley and the probability of reinvasion of the plateau are discussed.     

Distribution and abundance of trypanosome (subgenus Nannomonas) infections of the tsetse fly Glossina pallidipes in southern Africa

Over 10000 Glossina pallidipes tsetse flies were collected during two field studies in the Zambezi Valley, Zimbabwe and one in the Luangwa Valley, Zambia. These were screened for mature trypanosome infections and 234 dot-blot preparations were made of infected midguts, which were screened using DNA probes or PCR with primers specific to different species or types of the trypanosome subgenus Nannomonas. Over 70% of midgut infections were successfully identified as either Trypanosoma godfreyi, T. simiae or three types of T. congolense, savannah, riverine-forest and Kilifi. The relative abundance of species and types did not vary significantly between study locations, habitat, season or tsetse age or sex, although there were differences between DNA probe and PCR results. Mixed species and/or mixed type infections were common and were more often detected using PCR. The distribution of infections among flies was highly aggregated, but there was no tendency for multiple infections to accumulate in older flies, implying that sequential superinfection may be uncommon. Possible explanations for these patterns are discussed.     

AM真菌控制蚕豆枯萎病发生的根际微生物效应

在温室盆栽条件下,研究了接种尖孢镰刀菌蚕豆专化型和丛枝菌根(AM)真菌(摩西球囊霉Gm、扭形球囊霉Gt、根内球囊霉Gi及幼套球囊霉Ge)对灭菌连作土壤中蚕豆幼苗生长、枯萎病发生、根际镰刀菌数量和微生物代谢功能多样性的影响.结果表明: 接种AM真菌能显著增加蚕豆幼苗地上部和地下部鲜质量;接种Gm、Gt、Gi和Ge真菌使蚕豆枯萎病病情指数分别显著降低94.0%、60.0%、64.0%和94.0%,使蚕豆根际镰刀菌数量分别显著降低98.6%、74.3%、77.8%和90.4%,以Gm和Ge真菌对蚕豆枯萎病的抑制效应最好.接种Gm、Gt和Ge显著提高了根际微生物对糖类(CH)、氨基酸类(AA)、羧酸类(CA)和酚酸类(PA)碳源的利用,使蚕豆根际微生物的AWCD值分别显著提高34.4%、31.5%和50.8%;而接种Gi对AA、CA、PA类碳源利用和AWCD值均无显著影响,使微生物对CH类碳源的利用显著降低59.3%.主成分和相关分析表明, 接种AM真菌明显改变了蚕豆根际微生物的群落结构.接种Gt真菌提高了蚕豆根际微生物对AA类碳源(β-甲基-D-葡萄糖苷、D-半乳糖酸-γ-内酯、D-甘露醇、N-乙酰基-D-葡萄糖胺、D-纤维二糖)和CA类碳源(D-半乳糖醛酸)的利用,而接种Gi真菌降低了微生物对以上碳源的利用;接种Gm和Ge真菌提高了根际微生物对L-精氨酸和4-羟基苯甲酸的利用.表明糖类和羧酸类是接种Gt真菌后蚕豆根际土壤微生物利用的主要碳源, 氨基酸和酚酸类是接种Gm和Ge后蚕豆根际土壤微生物利用的主要碳源.接种AM真菌显著提高了蚕豆根际微生物的活性,改变了微生物群落结构并抑制病原菌的增殖,是AM真菌减轻了蚕豆枯萎病的发生的重要原因.不同AM真菌减轻了蚕豆枯萎病的发生与其改变微生物的碳源利用密切相关.     

A contribution to Gymnosphaera (Cyatheaceae) in mainland Asia: Two new species,reinstatement of Cyathea bonii,and their phylogenetic positions

Gymnosphaera represents a minor lineage within the scaly tree-fern family Cyatheaceae. Tropical and subtropical mainland Asia is a main distribution area of Gymnosphaera. However, the species diversity of Gymnosphaera is currently incompletely known in mainland Asia due to lacking critical revision. Here we present new findings of species diversity and their relationships to mainland Asian Gymnosphaera based on field surveys, the examination of herbarium collections, and phylogenetic analyses of sequences of multiple chloroplast and nuclear regions. Two new species, G. saxicola from southwestern Yunnan and G. bachmaensis from central Vietnam, are established. Traditionally recognized G. podophylla is revealed to be a complex, from which G. bonii is reinstated as a distinct species. Our phylogenetic analyses identified four clades within Gymnosphaera in mainland Asia: G. denticulata clade, G. gigantea clade, G. podophylla clade, and G. salletii clade. The new species G. saxicola, which is special for its saxicolous habitat, was resolved as sister to G. austroyunnanensis in the G. salletii clade. The newly discovered G. bachmaensis, which is characterized specially by the spathulate frond, was positioned in the G. podophylla clade, being sister to G. bonii. The mountainous region from south-central Vietnam northwards to western Yunnan is a diverse center of Gymnosphaera and more species of this group are probably to be discovered there.     

西藏朗错秋季裸鲤属营养生态位及种间食物关系

西藏朗错海拔4300 m,是典型的高原湖泊之一。湖内具有兰格湖裸鲤和拉孜裸鲤共存的现象。本研究采用碳、氮稳定同位素技术分析了2018年秋季兰格湖裸鲤和拉孜裸鲤的食物组成和营养生态位,探讨了两种裸鲤的种间食物关系,包括饵料相似性和重叠系数。结果表明: 兰格湖裸鲤主要摄食蜉蝣类幼虫、摇蚊类幼虫、着生藻类、钩虾类和浮游生物;拉孜裸鲤主要摄食蜉蝣类幼虫、着生藻类、摇蚊类幼虫、钩虾类和水生维管束植物。两种裸鲤的营养生态位存在重叠,兰格湖裸鲤的δ¹³C值变幅(CR=1.60)、δ¹⁵N值变幅(NR=1.89)和凸多边形,总面积(TA=1.80)均小于拉孜裸鲤,差值分别为0.09、0.57和1.29。两种裸鲤的饵料相似性系数(94.2％)和重叠系数(91.5%)均较高,表明秋季西藏朗错两种裸鲤可能存在激烈的种间食物竞争关系。