Concordant Evolution of a Symbiont with Its Host Insect Species: Molecular Phylogeny of Genus Glossina and Its Bacteriome-Associated Endosymbiont, Wigglesworthia glossinidia

Many arthropods with restricted diets rely on symbiotic associations for full nutrition and fecundity. Tsetse flies (Diptera: Glossinidae) harbor three symbiotic organisms in addition to the parasitic African trypanosomes they transmit. Two of these microorganisms reside in different gut cells, while the third organism is harbored in reproductive tissues and belongs to the genus Wolbachia. The primary symbiont (genus Wigglesworthia glossinidia) lives in differentiated epithelial cells (bacteriocytes) which form an organ (bacteriome) in the anterior gut, while the secondary (S) symbionts are present in midgut cells. Here we have characterized the phylogeny of Wigglesworthia based on their 16S rDNA sequence analysis from eight species representing the three subgenera of Glossina: Austenina (=fusca group), Nemorhina (=palpalis group), and Glossina (=morsitans group). Independently, the ribosomal DNA internal transcribed spacer-2 (ITS-2) regions from these species were analyzed. The analysis of Wigglesworthia indicated that they form a distinct lineage in the γ subdivision of Proteobacteria and display concordance with their host insect species. The trees generated by parsimony confirmed the monophyletic taxonomic placement of Glossina, where fusca group species formed the deepest branch followed by morsitans and palpalis groups, respectively. The placement of the species Glossina austeni by both the traditional morphological and biochemical criteria has been controversial. Results presented here, based on both the ITS-2 and the symbiont 16S rDNA sequence analysis, suggest that Glossina austeni should be placed into a separate fourth subgenus, Machadomyia, which forms a sister-group relationship with the morsitans group species. Received: 17 March 1998 / Accepted: 1 May 1998     

Phytogeny of genusGlossina (Diptera:Glossinidae) according to ITS2 sequences

The flies of genusGlossina (Diptera: Glossinidae) are an important vector of African trypanosomiases which cause diseases in humans and animals. The ribosomal DNA Internal Transcribed Spacer-2 (ITS-2) region sequences from differentGlossina species were PCR-amplified and analyzed in order to construct a molecular phylogeny for genusGlossina. Trees generated by parsimony confirmed the monophyletic taxonomic placement of genusGlossina wherefusca group species formed the deepest branch followed bymorsitans andpalpalis groups, respectively. The placement ofGlossina austeni by both the traditional morphological and biochemical criteria has been controversial. Results presented here, based on ITS-2 locus sequence analysis, suggest thatGlossina austeni can be placed into a separate subgenerus which forms a sister-group relationship with themorsitans group species.     

Functional Morphology of the Phallosome in Glossina (Diptera, Glossinidae) and Its Evolutionary Implications

Current views on the phylo-genetjc relations of the three species groups within the genus Glossina are critically reviewed. In respect of the male genitalia, the fusca group is here regarded as more specialized than the morsitans group. The detailed phallosome morphology of G. austeni is compared with that of G. fuscipes , on the basis of their mode of action in living flies. The comparison is extended to fusca group species, using dead material only. The presumed phylogeny of Glossina is presented in outline; the evolution of the genus has involved a progressive invasion of riverine and later of forest habitats, from an original semi-arid grassy woodland habitat.     

Tissue distribution and prevalence of Wolbachia infections in tsetse flies, Glossina spp

Tsetse flies Glossina spp. (Diptera: Glossinidae) harbor three different symbiotic microorganisms, one being an intracellular Rickettsia of the genus Wolbachia. This bacterium infects a wide range of arthropods, where it causes a variety of reproductive abnormalities, one of which is termed cytoplasmic incompatibility (CI) that, when expressed, results in embryonic death due to disruptions in fertilization events. We report here that in colonized flies, Wolbachia infections can be detected in 100% of sampled individuals, while infections vary significantly in field populations. Based on Wolbachia Surface Protein (wsp) gene sequence analysis, the infections associated with different fly species are all unique within the A group of the Wolbachia pipientis clade. In addition to being present in germ-line tissues, Wolbachia infections have been found in somatic tissues of several insects. Using a Wolbachia-specific PCR-based assay, the tissue tropism of infections in Glossina morsitans morsitans Westwood, Glossina brevipalpis Newstead and Glossina austeni Newstead were analysed. While infections in G. m. morsitans and G. brevipalpis were limited to reproductive tissues, in G. austeni, Wolbachia could be detected in various somatic tissues.     

Detection and characterization of <Emphasis Type="Italic">Wolbachia</Emphasis> infections in laboratory and natural populations of different species of tsetse flies (genus <Emphasis Type="Italic">Glossina</Emphasis>)

BACKGROUND: Wolbachia is a genus of endosymbiotic α-Proteobacteria infecting a wide range of arthropods and filarial nematodes. Wolbachia is able to induce reproductive abnormalities such as cytoplasmic incompatibility (CI), thelytokous parthenogenesis, feminization and male killing, thus affecting biology, ecology and evolution of its hosts. The bacterial group has prompted research regarding its potential for the control of agricultural and medical disease vectors, including Glossina spp., which transmits African trypanosomes, the causative agents of sleeping sickness in humans and nagana in animals. RESULTS: In the present study, we employed a Wolbachia specific 16S rRNA PCR assay to investigate the presence of Wolbachia in six different laboratory stocks as well as in natural populations of nine different Glossina species originating from 10 African countries. Wolbachia was prevalent in Glossina morsitans morsitans, G. morsitans centralis and G. austeni populations. It was also detected in G. brevipalpis, and, for the first time, in G. pallidipes and G. palpalis gambiensis. On the other hand, Wolbachia was not found in G. p. palpalis, G. fuscipes fuscipes and G. tachinoides. Wolbachia infections of different laboratory and natural populations of Glossina species were characterized using 16S rRNA, the wsp (Wolbachia Surface Protein) gene and MLST (Multi Locus Sequence Typing) gene markers. This analysis led to the detection of horizontal gene transfer events, in which Wobachia genes were inserted into the tsetse flies fly nuclear genome. CONCLUSIONS: Wolbachia infections were detected in both laboratory and natural populations of several different Glossina species. The characterization of these Wolbachia strains promises to lead to a deeper insight in tsetse flies-Wolbachia interactions, which is essential for the development and use of Wolbachia-based biological control methods.     

Sex pheromone of the tsetse species, Glossina austeni: isolation and identification of natural hydrocarbons, and bioassay of synthesized compounds

Copulatory responses of male Glossina austeni (Newstead) (Diptera: Glossinidae), that were elicited after contact with frozen female tsetse, were not observed after solvent washing of cuticular lipids. Chromatographic analysis of extracts from laboratory-reared and field-collected G. austeni females yielded natural hydrocarbons that were highly stimulatory to males. Most of this activity was produced by compounds in the alkene fraction. Gas chromatograms (GC) contained five natural alkenes; these were separated by preparative GC for bioassays conducted in Tanzania. The two major alkenes were identified using gas chromatography-mass spectrometry (GC-MS) to be 13,17-dimethyltritriacont-1-ene and 13,17-dimethylpentatriacont-1-ene, after the samples had undergone derivatization using dimethyl disulphide and saturation with deuterium. These alkenes and natural alkanes were quantified from G. austeni of both sexes from laboratory and field samples to confirm that their presence was consistent in this species. Trials of synthetic samples resulted in the order of biological activity for the stereoisomers of 13,17-dimethyltritriacont-1-ene as follows: S,R-33:1 > R,S- 33:1 > S,S-33:1 > R,R-33:1. Dose-response data showed an ED(50) at 5 microg per treated, solvent-washed male decoy. Of the four stereoisomers of 13,17-dimethylpentatriacont-1-ene, R,R-35:1 showed the most activity. This is the first report of alkene-induced sexual activity in males of the genus Glossina.     

Abundance and distribution of the tsetse flies, Glossina austeni and G. brevipalpis, in different habitats in South Africa

The distribution and abundance of Glossina austeni Newstead and Glossina brevipalpis Newstead (Diptera: Glossinidae) were studied in the three main vegetation types in Zululand, KwaZulu-Natal, South Africa. During a period of 12 months, a trap transect consisting of 38 H-traps traversing the three vegetation types was monitored. The Index of Apparent Abundance (IAA) for G. brevipalpis was high in indigenous forest and open grassland but lower in exotic plantations. Glossina austeni, on the other hand, was captured mainly in or adjacent to indigenous forest. The seasonal trend in the IAA did not differ between vegetation types. The findings on the distribution of G. brevipalpis are in contrast with the historic records. Historically, this species was considered to be restricted to areas with a dense overhead canopy and high relative humidity. The repercussions of these findings for the epidemiology of livestock trypanosomiasis and the control of tsetse in Zululand are discussed.     

Comparative study on the infection rates of different laboratory strains of Glossina species by Trypanosoma congolense

Teneral Glossina morsitans centralis Machado, G.austeni Newstead, G.palpalis palpalis Robineau-Desvoidy, G.p.gambiensis Vanderplank, G.fuscipes fuscipes Newstead, G.tachinoides Westwood and G.brevipalpis Newstead, from laboratory-bred colonies, were fed at the same time on the flanks of ten goats infected with Trypanosoma congolense Broden isolated in Tanzania or in Nigeria. The seven tsetse species were infected over the range 0.3-49.2%. Survival of both T.congolense isolates was best in G.m.centralis, poorest in G.austeni and the four palpalis group tsetse, with G.brevipalpis intermediate. It is suggested that there are differences in the gut of different laboratory-bred cultures of Glossina Westwood species and subspecies such that T.congolense parasites can survive better in the gut of some than in others and undergo cyclical development to metacyclics in the hypopharynx.     

Evaluation and improvement of sticky traps as monitoring tools for Glossina austeni and G. brevipalpis (Diptera: Glossinidae) in north-eastern KwaZulu-Natal, South Africa

The attractiveness of various colours, colour combinations and sizes of sticky traps of the 3-dimensional trap (3DT), cross-shaped target (XT), rectangular screen (RT) and monopanels were evaluated for their efficacy to capture Glossina austeni Newstead and G. brevipalpis Newstead in north-eastern KwaZulu-Natal, South Africa. The 3-dimensional shapes of the XT and 3DT in light blue (l.blue) and white were significantly (ca. 3.1-6.9 times) better than the RT for G. austeni. On bicoloured XTs, G. austeni landed preferentially on electric blue (e.blue) (58%) and black (63%) surfaces when used with white; while for G. brevipalpis, significantly more landed on e.blue (60-66%) surfaces when used with l.blue, black or white surfaces. Increased trap size increased the catches of G. brevipalpis females and both sexes of G. austeni significantly. Temoocid and polybutene sticky materials were equally effective and remained durable for 2-3 weeks. The glossy shine of trap surfaces did not have any significant effect on the attraction and landing responses of the two species. The overall trap efficiency of the e.blue/l.blue XT was 23% for G. brevipalpis and 28% for G. austeni, and that of the e.blue/black XT was 16% for G. brevipalpis and 51% for G. austeni. Larger monopanels, painted e.blue/black on both sides, increased the catches of G. austeni females significantly by up to four times compared to the standard e.blue/black XT. This monopanel would be recommended for use as a simple and cost effective survey tool for both species in South Africa.     

Comparison between pars intercerebralis neurosecretory cells of Glossina and those of other higher Diptera

Three types of the A-neurosecretory cells (NSCs) were found in the pars intercerebralis (PI) of the adult brain of four species of tsetse flies (Glossina palpalis, G. pallidipes, G. austeni and G. morsitans). The typical formula of their composition is: 8 A1-, 14 A2- and 4 A3-cells. After permanganate oxidation, the neurosecretory materials (NSMs) of Al- and A3-cells are rich in strong acid groups. The A2-NSM contains both strong and weak acid groups after this treatment; besides, it retains a weak affinity to acid dyes. In addition to the A-cells, the PI of Glossina species seems to include a number of NSCs of the type B.
The data obtained indicate a great similarity of the NSC composition in the PI of Glossina and other related higher dipterans.
In addition to the NSCs, giant neurons and particular cells with large cytoplasmic inclusions were also found in the PI of Glossina. The cells with large cytoplasmic inclusions correspond apparently to vacuolated cells previously observed in the brain of some flies. It is suggested that the considerable complication in reproductive biology which took place in tsetse flies had no effect on the composition of the NSCs in their PI when compared to that in related species with more ordinary cycles of reproduction.     

Efficiency of DNA barcodes for species delimitation: A case in Pterygiella Oliv. (Orobanchaceae)

DNA barcoding is becoming an increasingly popular means to identify species. The obscure discrimination in the genus Pterygiella calls into question the re-assessment of the criterion for species delimitation. We collected 20 individuals, representing all five described species of this genus in its distributional range. The aim was to use three proposed barcode DNA regions (rbcL, matK, and ITS) to diagnose Pterygiella species, and examine which barcode is more suitable for discerning the congeneric and related species. The results showed that the core barcodes matK and rbcL were comparatively less effective. However, the ITS region, especially ITS-1 and ITS-2, successfully identified all species in the genus. Furthermore, the secondary structure of ITS-2 RNA, especially compensatory base changes, appears complementary to classical primary sequence analysis for DNA barcoding.     

Efficiency of DNA barcodes for species delimitation: A case in Pterygiella Oliv.(Orobanchaceae)

DNA barcoding is becoming an increasingly popular means to identify species. The obscure discrimination in the genus Pterygiella calls into question the re-assessment of the criterion for species delimitation. We collected 20 individuals, representing all five described species of this genus in its distributional range. The aim was to use three proposed barcode DNA regions (rbcL, matK, and ITS) to diagnose Pterygiella species, and examine which barcode is more suitable for discerning the congeneric and related species. The results showed that the core barcodes matK and rbcL were comparatively less effective. However, the ITS region, especially ITS-1and ITS-2, successfully identified all species in the genus. Furthermore, the secondary structure of ITS-2 RNA, especially compensatory base changes, appears complementary to classical primary sequence analysis for DNA barcoding.     

Phylogenetic inferences in Antennaria (Asteraceae: Gnaphalieae: Cassiniinae) based on sequences from Nuclear Ribosomal DNA internal transcribed spacers (ITS)

The phylogenetic relationships among sexually reproducing species of Antennaria (Asteraceae) are poorly understood. An earlier cladistic analysis based on morphology did not fully resolve the phylogeny of these taxa and therefore a different approach using molecular data was explored. The internal transcribed spacer regions (ITS-1 and ITS-2) of nuclear ribosomal DNA were sequenced for 30 species of Antennaria and one species from each of the outgroup genera Anaphalis, Ewartia, Leontopodium, and Pseudognaphalium. The ITS-1 sequence in Antennaria ranged from 253 to 260 base pairs (bp) in length, and the proportion of nucleotide differences between pairs of species of Antennaria ranged from 1 to 14%. For ITS-2, the divergence between pairs of species of Antennaria ranged from 0 to 8%. ITS-2 is shorter than ITS-1, ranging from 213 to 219 bp. Phylogenetic analysis indicates that, relative to the outgroups included, Antennaria is a well-supported monophyletic group. Based on the genera surveyed, Leontopodium appears to be the sister genus of Antennaria. The general topology of the molecular trees agrees with that based on previous morphological analyses and indicates that Antennaria is composed of six clades of equal rank, corresponding to the traditionally recognized informal groups, the Geyeriae, Argenteae, Arcuatae, Dimorphae, Pulcherrimae, and Catipes. Sequence and morphological data indicate that the Alpinae and Dioicae are unnatural, polyphyletic units that should be abandoned and redefined as the monophyletic Catipes group. Phylogenetic analysis of ITS sequences also suggests the dissociation of A. stenophylla from the Dimorphae, where it is traditionally placed, and its affiliation with the Argenteae, as well as the placement of A. arcuata in its own group.     

Host preferences of tsetse (Diptera: Glossinidae) based on bloodmeal identifications

An enzyme-linked immunosorbent assay (ELISA) was developed to identify the origin of vertebrate blood in the guts of 29 245 wild-caught flies of eleven Glossina species from various ecological zones of Africa. Depending on the quality of the bloodmeal samples, 62.8% of the samples were identified and could be assigned to a host-group (e.g. ruminant), family (e.g. Bovidae) or species (e.g. Bos spp.). A total of 13 145 samples (44.9%) was identifiable up to the species level. With a few exceptions, the present results are in agreement with earlier published reports. Glossina austeni and G. fuscipleuris seemed to have a distinct feeding preference for Suidae (mainly bushpig). Glossina morsitans ssp. fed mainly on Suidae (mainly warthog), although local variations were observed and in some areas hippopotamus or ruminants replaced the warthog as the main host. Bushbuck seemed to be the principal food source for G. longipalpis and G. fusca . Glossina pallidipes fed mainly on ruminants (buffalo, bushbuck and cattle) but, depending on host availability and location, Suidae were also important hosts. Hippopotamus was identified as the main source of blood-meals for G. brevipalpis . The main hosts for G. longipennis were Suidae (mainly bushpig) and not rhinoceros as had been reported 40 years earlier. The opportunistic feeding behaviour of the palpalis tsetse group was confirmed. The results showed that changes in environment, fauna and host availability may result in modification of tsetse feeding patterns.     

Molecular phylogeny ofCheirolophus (Asteraceae:Cardueae-Centaureinae) based on ITS sequences of nuclear ribosomal DNA

The genusCheirolophus has an interesting western Mediterranean and Macaronesian distribution. Here we investigate the delimitation of the genus and its exclusion from the large genusCentaurea, the systematic position of the related genusPaleocyanus, the delimitation of some species and the phylogeny of the group. We have carried out a phylogenetic analysis of the PCR-generated sequences of the internal transcribed spacers (ITS-1 and ITS-2) of the nuclear ribosomal DNA. The results suggest that the genus, includingPaleocyanus crassifolius is monophyletic; thus, a new combination of this species underCheirolophus is proposed. The Macaronesian group of species is also monophyletic, indicating a single colonization of the archipelago. The poor resolution of microspecies in the Macaronesian group reinforces the hypothesis of a very recent differentiation of the group.     

Activation of three species of tsetse (Glossina spp.) in response to host derived stimuli

Recordings were made of the activation of hungry Glossina morsitans morsitans Westwood, G. pallidipes Austen, and G. austeni Newstead in response to odours from ox breath and ox urine, and a moving visual stimulus, in a wind tunnel. The spontaneous activity of G.m.morsitans was very low (less than 4% of males and 2% of females active per min during control periods). That of G.austeni and G.pallidipes was in the region of 20% except for G.pallidipes females when in excess of 40% were active during control periods. Addition of ox urine odours to the airstream had no effect on activity in any of the species investigated but addition of ox breath odours to the airstream significantly increased activity of G.pallidipes and of G.m.morsitans, although for the latter only approximately 12% of flies were active. For G.austeni the addition of ox breath odours resulted in a significant increase in activity of females but not of males. The moving visual stimulus resulted in a significant increase in the activity of both sexes of G.austeni and G.m.morsitans but no change in the activity of G.pallidipes. The low level of spontaneous activity and the low response to ox breath odours in a strain of G.m.morsitans maintained in the laboratory since 1969 was compared with a new colony of this species which originated from puparia collected in Zimbabwe in 1991. No differences in either spontaneous activity or the response to ox breath odour was recorded, but females from the new colony were significantly more responsive to a moving visual stimulus.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparative study on the susceptibility of different laboratory strains of Glossina species to Trypanosoma simiae

Abstract. Teneral tsetse of four Glossina species from laboratory-reared colonies were fed on four Large White pigs infected with three different stocks of Trypanosoma simiae isolated in Coast Province, Kenya. Thereafter the tsetse were maintained on goats and dissected on day 28 to determine the trypanosome infection rates. Glossina brevipalpis was as susceptible as G.pallidipes whilst G.palpalis gambiensis was not susceptible to T.simiae CP 11 a stock causing acute infection, which was isolated from a wild G.austeni. Glossina brevipalpis was as susceptible as G.pallidipes to another stock causing acute infection, T.simiae CP 813 isolated from a wild G.pallidipes. Glossina morsitans centralis was also as susceptible as G.brevipalpis and G.pallidipes whilst G.p.gambiensis was not susceptible to this T.simiae stock. Glossina m.centralis showed very low susceptibility to a stock causing chronic infection, T.simiae CP 1896 isolated from a bushpig, whilst G.brevipalpis, G.p.gambiensis and G.pallidipes could not be infected by this T.simiae stock. Male Glossina were generally more susceptible than females to the three T.simiae stocks.     

Generic and species concepts in Microglena (previously the Chlamydomonas monadina group) revised using an integrative approach

Traditionally the genus Microglena Ehrenberg has been used to contain species that belong to the Chrysophyceae; however, the type species of Microglena, M. monadina, represents a green alga, which was later transferred to the genus Chlamydomonas. The taxonomic status of the genus has therefore remained unclear. We investigated 15 strains previously assigned to C. monadina and two marine species (C. reginae and C. uva-maris) using an integrative approach. Phylogenetic analyses of SSU and ITS rDNA sequences revealed that all strains form a monophyletic lineage within the Chlorophyceae containing species from different habitats. The strains studied showed similar morphology with respect to cell shape and size, but showed differences in chloroplast and pyrenoid structures. Some representatives of this group have the same type of sexual reproduction (homothallic advanced anisogamy). Three different morphotypes could be recognized. Strains belonging to type I have a cup-shaped chloroplast with a massive basal part, in which a large, single, ellipsoidal pyrenoid is located. The members of type II also have a cup-shaped chloroplast, which is partly lobed and has a thinner basal part than type I; here the pyrenoid is half-ring or horseshoe-shaped and occupies different positions in the chloroplast depending on the strain. The strains of type III have multiple pyrenoids, which appear to have developed from the subdivision of a single ring-shaped pyrenoid into several parts. We compared the results of our morphological investigations with the literature and found that 15 strains could be identified with existing species. Two strains did not fit with any described species. As a result of our study, we transfer all strains to the genus Microglena, propose 11 new combinations, and describe two new species. Comparison of the ITS-1 and ITS-2 secondary structures confirmed the species delineations. All species have characteristic compensatory base changes in their ITS secondary structures and are supported by ITS-2 DNA barcodes.     

Copulatory behaviour of the tsetse flies Glossina morsitans and G.austeni

ABSTRACT. The copulatory behaviour of Glossina morsitans morsitans West. and G.austeni Newst. was analysed by filming. After a male and female engaged genitalia, the male performed a repertoire of five actions for 3–4 min: (1) 'rubbing' his metathoracic, tibiotarsal joint against the region of genital contact; (2) 'stroking' or hitting the female's head and thorax with his meso- and metathoracic legs; (3) 'wing flick' by moving his mesothoracic legs in a rowing motion whilst at the same time vibrating the wings as in normal flight; (4) 'wing vibration' with the wings vibrated in the closed position; (5) 'wings out' in which the wings are moved out to the flying position without any observable vibration. Each action was repeated many times, to give variable individual sequences, but declined in frequency exponentially over the first 3–4 min in copulo. The two species differed in the frequency of acts. Shortly before separation, a few hours later, actions 1 and 2 reappeared. Receptive females exhibited little overt behaviour except in the maintenance of a passive stance. Refractory females rejected a mating attempt by flexing the abdomen ventrally, vibrating their wings in the closed position, and pushing the male with meso- and metathoracic legs. The significance and possible functions of male behaviour are discussed in relation to mating in Glossina and other Diptera.     

Phylogenetic Relationship ofPopulusSections by ITS Sequence Analysis

ITS sequences of 15 representative species of five sections in the genus Populus L. were determined. By using direct sequencing of PCR product, it was found that the fragments of internal transcribed spacers (ITS) are about 594 bp in length. The length of ITS-1 and ITS-2 is about 220 bp and 210 bp, respectively, while that of 5.8s is 164 bp. Its G+C content is about 69.0%. The number of phylogenetically informative loci is higher in ITS-2 than in ITS-1. Transversion and transition are two main factors that drive the ITS evolution, and more insertions and deletions occurred in ITS-2. Taking Salix matsudana Koidz. and Salix suchowensis Cheng as outgroups, phylogenetic analysis of ITS sequences using PAUP 4.0 software indicated that Populus is monophyletic group and can be divided into two main clades. One is the section Leuce , and the other is the remaining sections.