Cytogenetic studies of some species complexes of Anopheles in Thailand and Southeast Asia

Recent studies on cytogenetics, behavioral, geographical and distinct morphological characters on adult, pupal and larval stages have revealed that "balabacensis" is a species complex. Anopheles dirus the mainland species, is distributed widely in Thailand and is renowned for its role as primary vector of human malarial parasites. Further, evidence from cytogenetic and taxonomic studies suggests that "An. dirus" is a species complex comprising at least four distinct species provisionally designated: dirus A, B, C and D. These cryptic species are distinguishable only partially morphologically, but can be separated on the basis of metaphase chromosomes using the Giemsa and Hoechst 33258 staining techniques. Apparently, these siblings show distinct patterns of geographic distribution in Thailand and Peninsular Malaysia. The recognition of dirus as a complex of species in Thailand and Peninsular Malaysia requires a re-evaluation of the role that the individual members of this complex have in the transmission of malaria parasites in this region. Cytological analysis of gene rearrangements in ovarian polytene chromosomes has shown that An. maculatus is a sibling-species complex consisting of at least four species in Thailand provisionally designated: maculatus A, B, C and G. These siblings are sympatric in some populations. Furthermore, species B is so highly polymorphic for chromosome rearrangements that four geographic forms can be recognized. It is not known whether these four forms are subspecies or yet further species within the species B complex. These sibling-species must be differentiated in order to understand any differential capabilities in their transmission of human malaria parasites. Anopheles nivipes was elevated from synonymy under An. philippinensis to full species status by Reid, a decision recently confirmed by cross mating experiments. The Thailand Malaria Division does not differentiate these two species and only identifies An. philippinensis, yet, An. nivipes is by far the most common of the two species in Thailand. Furthermore, preliminary surveys of the ovarian polytene chromosomes of several widely separated populations of An. nivipes in Thailand have revealed at least two distinct chromosomal types of nivipes based on fixed inversions on the X chromosomes.     

Population cytogenetics of the malaria vector Anopheles leucosphyrus group

Until recently, very little was known of Anopheles species complexes and their relationships to epidemiology and malaria transmission in Southeast Asia. During the past eight years, extensive studies on the genetics of natural populations of anopheline mosquitoes in this region, involving the interdisciplinary efforts of taxonomists, operational entomologists and biologists, have revealed groups of cryptic species of Anopheles vectors, particularly the An. leucos phyrus group. This species group comprise seventeen species and two subspecies widely distributed in the forested areas of Southeast Asia. Among these species. An. dirus Peyton and Harrison, has been shown by cytogenetic and morphological studies to be a complex of at least seven isomorphic species, provisionally designated species A, B, C, D, E, F and takasagoensis, on the Southeast Asian mainland. Cytological identification of these species is based on distinct banding patterns of salivary gland polytene chromosomes as well as heterochromatin differences in mitotic karyotypes. The five species found in Thailand (A-D, F) exhibit distinct geographic distributions. Species A is widespread throughout Thailand except in the south. Species B had been found in sympatry with species C in southern Thailand and both seem to show north-south clinal geographic variation. Species D is common on the west side of southern Thailand and along the Thai-Burmese border in sympatry with species A. Species F, An. nemophilous Peyton and Ramalingam, has been found in a population at the Thai-Malaysian border in this study although it was known to be common in southern and western Thailand and Peninsular Malaysia. Species E is known only from western India. The five species found in Thailand also exhibit seasonal variation in relative abundance and different nocturnal biting cycles. Chromosomal polymorphisms have been observed in mitotic and polytene chromosomes of An. dirus A and D. Species B and C also show heterochromatin variation in the sex chromosomes, but are monomorphic for the standard sequence in polytene chromosomes. These biological characteristics of the An. dirus complex may have implications for understanding the epidemiology of malaria in Southeast Asia. Recent cytogenetic studies of wild-caught samples of An. leucosphyrus from Sumatra, Kalimantan and southern Thailand have revealed the presence of two distinct species within this taxon. Species A is widely distributed in southern Thailand, East Malaysia and Kalimantan, while species B is confined to Sumatra. The two isomorphic species are vectors of human malaria within their range of distribution.(ABSTRACT TRUNCATED AT 400 WORDS)     

Microsatellite markers for population genetic studies in Aedes aegypti (Diptera: Culicidae) from Côte d'Ivoire: evidence for a microgeographic genetic differentiation of mosquitoes from Bouaké

In West Africa, Aedes aegypti (Diptera: Culicidae) (Linnaeus, C., 1762. Zweyter Theil, enhalt Beschreibungen veschiedener wichtiger Naturalien. In: Hasselquist, F. (Ed.), Reise nach Palastina in den Jahren von 1749 bis 1752, Rostock, Germany, pp. 267-606) represents the principal vector of yellow fever. This study reports the use of microsatellite markers to characterise various A. aegypti populations from C?te d'Ivoire according to a north-south transect, and to perform a temporal genetic survey of the mosquitoes. Three microsatellite loci were used to analyse individuals from four different places: Kabolo, Bouaké, and two different districts of Abidjan. We found that the four populations are genetically distinct except the two Abidjan populations. In the Bouaké population, the coexistence of two cryptic species, not morphologically distinguishable, seems to account for the extensive heterozygote deficiency observed. Comparison of mosquitoes from Bouaké 1 year apart indicated that a dramatic change occurred in the structuring of this population over time. Taken together these results indicate that microsatellite markers could be useful for identifying various populations of A. aegypti on a microgeographic scale and to assess for temporal variation within mosquito populations.     

用聚合酶链反应区分我国大劣按蚊复合体A和D种

目的：建立一种鉴别大劣按蚊复合体Ａ和Ｄ种的ＰＣＲ检测技术。方法：根据大劣按蚊Ａ和Ｄ种核糖体ＤＮＡ第二内转录间隔区的序列差异，设计种特异引物，通过ＰＣＲ扩增出种特异长度（Ａ种为３７４ｂｐ，Ｄ种为６６３ｂｐ）的片段区分蚊种。结果：该法敏感性达１／１６００单蚊抽提ＤＮＡ或１／５单条蚊腿水匀浆ＤＮＡ。应用该法检测大劣按蚊ＡＦＲＩＭＳ实验室品系１０例，ＨＮ实验室品系２０例，以及采自海南省白沙、和平、罗葵、毛阳等地野外样本１４８例，均显示Ａ种特异带；云南省勐腊地区野外样本３０例，全部为Ｄ种特异带。结论：提供一种简便可靠的蚊种鉴别ＰＣＲ法，确认我国海南和云南的大劣按蚊分别为Ａ种和Ｄ种，为深入研究我国大劣按蚊复合体不同成员种的地理分布、生态习性和传疟作用创造了条件。     

Molecular variation and phylogeny of the Anopheles minimus complex (Diptera: Culicidae) inhabiting Southeast Asian countries, based on ribosomal DNA internal transcribed spacers, ITS1 and 2, and the 28S D3 sequences

Anopheles minimus (Theobald) is one of the most important vectors of human malaria in Southeast Asia. Morphological studies now have revealed five sibling species as its complex, designated as species A to E. The present study investigated the genetic divergence among An. minimus populations from four countries (Japan, China, Thailand and Indonesia), based on the DNA sequences data of the D3 (the third domain of the 28S ribosomal gene) and ITS2 (the second internal transcribed spacer of the ribosomal gene) is reported. The D3 and ITS2 phylogenetic trees, and the electrophoretic profile of ITS1 (the first internal transcribed spacer of the ribosomal gene) indicated that our An. minimus populations are comprised of three groups: the Japanese population as group I, the population from Guangxi Province of China (GX population) as group II, and others, as group III. The results showed the morphological similarity of group III and GX with the species complex A and B, respectively. It is interesting that both two species A (YN population) and species B (GX) occur in China, and that both species, An. minimus species A (LB-95 population) and the closer population An. flavirostris (Ludlow) (LB-00 population) appeared to be present on the Lombok Island of Indonesia, although in far separated localities. Moreover, this molecular evidence confirms the previous suggestion that the population from the Ishigaki Island of Japan should be classified as a new genetic status species E.     

Co-occurrence of parasites sensu lato in alimentary tract of patients hospitalised in lower Silesia

Up to now, the co-occurrence of parasites sensu stricto and fungi were confirmed mostly in the oral cavity. The aim of the present parallel parasitological and mycological investigations was to determine co-occurrence of protozoa, helminths and fungi (parasites sensu lato) both in smears from cavity and in stool samples of 93 patients undergoing the non-parasitological treatment in two hospitals of Lower Silesia (Poland). Among the examined patients parasites sensu lato (13 species) were found in 62 cases (63%). Trofozoites of Trichomonas tenax and/or Entamoeba gingivalis as well as fungi (Candida albicans, C. krusei, C. tropicalis and C. pseudotropicalis) occurred in 10% of the examined patients. Almost all infected cases (90%) were joined with infection of common invasion of protozoa and fungi. In stool common invasion of parasites sensu lato (Giardia intestinalis, Entamoeba coli, Ascaris lumbricoides, Taenia sp., Enterobius vermicularis as well as Candida albicans, C. krusei, C. pseudotropicalis, C. guilliermondii and Rhodotorula sp.) were found in 12% of the patiens. The co-occurrence of parasites and fungi was stated in the half of invasions. C. albicans was the most often co-occurring species in alimentary tract.     

Allozymes as Diagnostic Characters of Sibling Species of Drosophila

Considerable enzyme variation that is genetically controlled exists within and between natural populations of Drosophila. Our studies of two groups of sibling species show that allozyme differences can be used as species-diagnostic characters. A locus is defined as diagnostic if an individual can be correctly assigned to one of two species with a probability of 99% or higher. Between 15 and 32% of the loci studied are diagnostic for any two of the sibling species. If several diagnostic loci are used, the species of any individual can be diagnosed with virtual certainty.     

Occurrence of a sibling species complex within neotropical lymnaeids,snail intermediate hosts of fascioliasis

The delimitation of cryptic species within the genus Lymnaea, which are the main vectors of fascioliasis, remains a topic of controversy. An analysis of genetic variability based on 12 enzyme loci revealed different fixed alleles at 9 loci between two sympatric samples of Lymnaea viatrix at the type locality in Lima, Peru. The absence of heterozygotes within this locality indicates the presence of isolated populations or cryptic species within L. viatrix. Significant genetic differences were also found between these two L. viatrix samples from Lima and other populations of L. viatrix in South America and in addition to species such as L. truncatula, L. cubensis and L. columella. Moreover, the lack of variability within each Lymnaea samples studied indicates the existence of a high selfing rate in each species.     

Mitochondrial cytochrome b variation in populations of the visceral leishmaniasis vector Lutzomyia longipalpis across eastern Brazil

A population analysis of peridomestic, light-trapped, field specimens of the phlebotomine sand fly Lutzomyia longipalpis was targeted to six locations representing a geographic transect across eastern Brazil. Mitochondrial cytochrome b gene sequences established the pattern of genetic variation among the populations. Alignment of a 261-basepair region at the 3' end of cytochrome b identified 30 haplotypes and 21 segregating sites from 78 sand flies. Pairwise comparisons indicated statistically significant population structuring between northern and southern populations, as well as structuring among the southern populations. Prominent spatial clustering was evident for two of the populations in a minimum spanning network of the haplotypes, but sequence divergence was not sufficient to indicate cryptic species.     

Occurrence of sibling species of Lutzomyia longipalpis (Diptera: Psychodidae) in Venezuela: first evidence from reproductively isolated sympatric populations

The delimitation of cryptic species within the main vector of the American visceral leishmaniasis, Lutzomyia longipalpis, remains a topic of controversy. An analysis of genetic variability based on 8 enzymatic loci revealed fixed differences in 2 diagnostic loci, adenylate kinase (Ak) and hexokinase (Hk), between sympatric and allopatric populations at 4 localities in Venezuela. The absence of heterozygotes for these 2 loci within 1 locality indicates, for the first time, the presence of 2 sympatric reproductively isolated populations or cryptic species within L. longipalpis. Significant differences were also detected between these cryptic species in the allele frequencies of glucose-6-phosphate isomerase (Gpi) and malate dehydrogenase, decarboxylating (Me). One species showed mean heterozygosities that ranged between 6.6% and 6.7%, with 1.6-1.9 alleles detected per locus, while the other had mean heterozygosities that ranged from 4.3% to 6.3%, with 1.3-1.6 alleles per locus. Comparisons of isozyme profiles with published data suggests that 1 species is similar to the L. longipalpis described in Colombian and Brazilian populations, whereas the other has not been previously reported.     

Microsporidial AIDS cholangiopathy due to Encephalitozoon intestinalis: case report and review

Microsporidia are increasingly recognized as opportunistic infections in immunodeficient patients, predominantly patients with AIDS. The two microsporidia most commonly associated with disease in AIDS patients are Enterocytozoon bieneusi and Encephalitozoon intestinalis (previously known as Septata intestinalis). The most common clinical presentation of microsporidiosis in AIDS patients is diarrhea, most commonly caused by the Enterocytozoon bieneusi species. Encephalitozoon intestinalis is a recently described species that has been reported to cause disseminated human infection including cholangitis. We report a case of AIDS cholangiopathy that presented with abdominal pain and cholestatic liver tests. Ultrasound examination and ERCP revealed a picture of sclerosing cholangitis. Bile samples obtained at ERCP were negative for microsporidia; stool studies for microsporidia and cryptosporidia were also negative. No organisms were identified on routine light microscopy of the biopsy specimens from the duodenum, ampulla, and bile duct. E. intestinalis spores were demonstrated in the bile duct biopsies, by methylene blue and azure 11 staining and confirmed by electron microscopy. Albendazole therapy was successful in eradicating E. intestinalis with clinical improvement and improvement in CD4 count. However, the cholangiographic picture did not improve and repeat cholangiography revealed progressive bile duct injury. Albendazole therapy was delayed and may have been too late to prevent bile duct damage; the drug had to be approved by the US Food and Drug Administration for compassionate use. This is an unusual case of sclerosing cholangitis caused by an unusual organism and requiring biliary sphincterotomy and stent placement for progressive stricturing despite eradication of the infection.     

Detection of Babesia and Anaplasma species in rabbits from Texas and Georgia, USA

Rabbits have been shown to harbor a suite of zoonotic organisms, including a Babesia species, Borrelia burgdorferi, and Anaplasma phagocytophilum. In this study, we conducted a molecular survey for various tick-borne pathogens in three species of rabbits from Texas and Georgia. Of 18 black-tailed jackrabbits (Lepus californicus) tested from Texas, six (28%) were polymerase chain reaction (PCR) positive for Babesia, and nucleotide sequencing revealed two distinct species or strains. Two jackrabbits were infected with a Babesia species or strain (Babesia sp. A) that was nearly identical (99.9%) to a piroplasm previously detected in humans from Washington state, and the remaining four jackrabbits were infected with a Babesia species (Babesia sp. B) that was most similar (99.7%) to a Babesia species detected in cottontail rabbits from Massachusetts and humans from Kentucky and Missouri. Eleven (61%) black-tailed jackrabbits were positive for A. bovis, and one was positive for A. phagocytophilum. Two of four desert cottontails (Sylvilagus audubonii) from Texas were positive for the Babesia sp. B, and one desert cottontail each was positive for A. bovis and A. phagocytophilum. One of these desert cottontails was coinfected with the Babesia sp. B and A. phagocytophilum, and five jackrabbits were coinfected with Babesia species and A. bovis. Of 19 eastern cottontails (S. floridanus) from Georgia, only one (5.3%) was positive for A. phagocytophilum, and three (15.8%) were positive for A. bovis. No rabbits from Texas or Georgia were positive for Borrelia species. The only tick species detected on the Texas and Georgia rabbits was the rabbit tick, Haemaphysalis leporispalustris. These data extend the geographic and host range of these pathogens, and because both the Babesia species and A. phagocytophilum are potential zoonotic pathogens, it is important to be aware that these organisms are enzootic in parts of the southern United States.     

Geometric Morphological Differences Distinguish Populations of Scup in the Northwestern Atlantic Ocean

Abstract

Scup Stenotomus chrysops, a commercially important marine species, are distributed from Nova Scotia to south Florida, and may represent morphologically distinct populations across their range. It was determined whether there were morphological differences between a North Atlantic Bight (NAB) population (41°N) and two South Atlantic Bight (SAB) populations (30°N and 32°N) of this species from April 2005 to July 2005, when the populations had formed spawning groups. Morphology was compared among populations by means of a geometric, landmark-based analysis of morphological and meristic traits for 180 individuals that were sexed and staged to maturity. A backward, stepwise discriminant functions analysis (DFA) produced a model that generated DFA scores that differed significantly between the NAB and SAB populations. Forehead and body depth dimensions differed among populations but not sexes. The DFA model predicted similar scores for 32 new NAB individuals and 31 new SAB individuals; however, scores were consistently underpredicted for the NAB individuals and overpredicted for the SAB individuals. We concluded that distinct northern and southern populations support a two-stock or two-species hypothesis; however, it is unclear whether this stock structure is related to reproductive or other life history traits. Either phenotypic plasticity or divergent selection may explain the morphological dissimilarities among these populations, but their influence on individual fitness remains unknown.     

Is human giardiasis caused by two different Giardia species?

We have recently sequenced the genome of the human Giardia intestinalis assemblage B isolate GS.1 comparisons to the earlier sequenced genome of the human assemblage A isolate WB showed that the average amino acid identity in 4,300 orthologous proteins was only 78%. Here we discuss these results in the light of new genome sequencing data from the hoofed-animal assemblage E (isolate P15, isolated from a pig) and further characterization of assemblage A and B isolates from humans. There is a highly conserved set of core genes (4,557 genes, 91% of genome) common to all isolates. The largest genomic differences are found in variable, Giardia-specific gene families and a large number of chromosomal rearrangements were detected, even between different chromosomes. Surprisingly, the assemblage E and A isolates are more similar at the amino-acid level than the two human isolates are to each other. This strengthens our earlier data suggesting that humans are infected by two different species of Giardia.     

Some applications of bloodmeal identifications in relation to the epidemiology of vector-borne tropical diseases.

No attempt has been made to give a complete review of the value of bloodmeal identifications of haematophagous arthropods in relation to the epidemiology of disease but rather to choose certain topics of current interest. A brief review of the current techniques for bloodmeal identification is given with particular reference to recent studies on mosquitoes and sandflies using the haemagglutination inhibition test. New data on mixed feeding by Anopheles gambiae (a mixture of species B and C) collected in Ethiopia confirm the earlier work with A. sacharovi that interrupted feeding is important. Details of current attempts to measure the cryptic portion of multiple meals using blood-typing techniques are given. Information collected on the feeding patterns of Culex univittatus group and Mansonia uniformis from Kenya demonstrates that caution must be shown in interpreting blood meal analyses since sampling techniques as well as several other factors can affect the results. A recent survey of sandflies in West Bengal suggest that a dangerous situation now exists and an outbreak of visceral leishmaniasis could occur.     

Determination of discriminatory power of genetic markers used for genotyping Giardia duodenalis

Small subunit ribosomal DNA (SSU-rDNA), glutamate dehydrogenase (gdh), beta-giardin, triosephosphate isomerase (tpi), and elongation factor 1-alpha (ef1-alpha) genes are useful genetic markers for genotypic analysis of the intestinal protozoan, Giardia duodenalis (syn. G. lamblia, G. intestinalis), the cause of enteric disease in humans. To quantitatively compare the discriminatory power of these loci, 43 fecal samples were collected from central, northern and eastern Thailand and G. duodenalis specimens were analyzed using PCR-based genotyping and subcloning methods. Approximately equal prevalence of assemblage A (21) and B (22) were present among these populations. Analysis of Simpson's index and Wallace coefficient values from assemblage B isolates together with the data obtained from GenBank showed that the combination of two loci provides a higher discrimination power for subgenotyping G. duodenalis than using any single locus.     

Prevalence of Giardia intestinalis in domestic dogs in Warsaw

INTRODUCTION: Giardia intestinalis is the most common intestinal protozoan parasite, which infects humans, dogs and other mammals throughout the world. So far eight genotypes of the parasite have been described of which four were found in dogs. Assemblages A-I and B infect either dogs or humans. Assemblages C and D occur only in dogs. The aim of the study was to determine the prevalence and genotypes of G. intestinalis in domestic dogs of Warsaw area. MATERIAL AND METHODS: From October 2005 to March 2006 fecal samples were collected from 350 dogs and examined using light microscopy and PCR techniques. RESULTS: 5.14% of dogs was found to be positive for G. intestinalis by microscopy and prevalence of 9.14% was found by PCR. The PCR amplicons were sequenced and the DNA sequences were compared with Giardia sequences in GeneBank database. The analysis revealed assemblage A-I in 1.71% of dogs, assemblage C in 1.14% and assemblage D in 6.28% of dogs in Warsaw. According to literature, the genotype A can infect humans however a role of dogs as a reservoir of human giardiosis in Poland is not known.     

Cryptic species within the cosmopolitan desiccation-tolerant moss Grimmia laevigata

The common cushion moss Grimmia laevigata (Bridel) Bridel grows on bare rock in a broad range of environments on every continent except Antarctica. As such, it must harbor adaptations to a remarkably broad set of environmental stresses, the extremes of which can include very high temperatures, prolonged nearly complete desiccation, and high ultraviolet B (UVB) exposure. Yet, like many mosses, G. laevigata shows very little morphological variability across its cosmopolitan range. This presents an evolutionary puzzle, the solution to which lies in understanding the phylogeographic structure of this morphologically simple organism. Here we report the results of an analysis of amplified fragment length polymorphisms (AFLPs) in G. laevigata, focusing on individuals from the California Floristic Province. We found evidence that populations within California constitute two distinct geographically overlapping cryptic species. Each clade harbors multiple private alleles, indicating they have been genetically isolated for some time. We suggest that the existence of cryptic species within G. laevigata, in combination with its life history, growth habits, and extreme desiccation tolerance, makes this moss an ideal research tool and a candidate for a biological indicator of climate change and pollution.     

Ectoparasites and associated pathogens of free-roaming and captive animals in zoos of South Carolina

A survey of ectoparasites and their associated pathogens was conducted in two South Carolina zoos, from 2004 to 2007. Dead, wild birds and mammals, as well as captive animals examined during routine veterinary checks constituted the study populations. Ectoparasites were tested for species of Anaplasma, Bartonella, Coxiella burnetii, Ehrlichia, Rickettsia, and Trypanosoma. Forty-six species of ectoparasites were collected from 133 free-roaming and captive hosts and their associated nesting and bedding materials. Six vector-borne pathogens were detected molecularly in the ectoparasites, including Anaplasma phagocytophilum in the tick Ixodes dentatus Marx from an eastern cottontail rabbit, Bartonella clarridgeiae in the cat flea Ctenocephalides felis (Bouché) from a Virginia opossum, Bartonella sp. Oh6 in the squirrel flea Orchopeas howardi (Baker) from an eastern grey squirrel, Bartonella sp. T7498 in the sucking louse Neohaematopinus sciuri Jancke from a squirrel, Rickettsia sp. Rf2125 in C. felis from a zookeeper and a grizzly bear, and Rickettsiales sp. Ib 2006 in Ixodes brunneus Koch from an American crow. While the pathology of some of these pathogens is poorly known, Anaplasma phagocytophilum (causative agent of human granulocytic anaplasmosis) and Bartonella clarridgeiae (causative agent of a disease similar to cat-scratch disease) can infect humans. Ectoparasites and their pathogens, especially those originating from free-roaming animals, present a potential threat to captive animals and humans.