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.     

“Wigglesworthia morsitans” Folate (Vitamin B9) Biosynthesis Contributes to Tsetse Host Fitness

Closely related ancient endosymbionts may retain minor genomic distinctions through evolutionary time, yet the biological relevance of these small pockets of unique loci remains unknown. The tsetse fly (Diptera: Glossinidae), the sole vector of lethal African trypanosomes (Trypanosoma spp.), maintains an ancient and obligate mutualism with species belonging to the gammaproteobacterium Wigglesworthia. Extensive concordant evolution with associated Wigglesworthia species has occurred through tsetse species radiation. Accordingly, the retention of unique symbiont loci between Wigglesworthia genomes may prove instrumental toward host species-specific biological traits. Genome distinctions between “Wigglesworthia morsitans” (harbored within Glossina morsitans bacteriomes) and the basal species Wigglesworthia glossinidia (harbored within Glossina brevipalpis bacteriomes) include the retention of chorismate and downstream folate (vitamin B₉) biosynthesis capabilities, contributing to distinct symbiont metabolomes. Here, we demonstrate that these W. morsitans pathways remain functionally intact, with folate likely being systemically disseminated through a synchronously expressed tsetse folate transporter within bacteriomes. The folate produced by W. morsitans is demonstrated to be pivotal for G. morsitans sexual maturation and reproduction. Modest differences between ancient symbiont genomes may still play key roles in the evolution of their host species, particularly if loci are involved in shaping host physiology and ecology. Enhanced knowledge of the Wigglesworthia-tsetse mutualism may also provide novel and specific avenues for vector control.     

Glossina morsitans morsitansandGlossina palpalis palpalis:Dosage Compensation Raises Questions about the Milligan Model for Control of Trypanosome Development

Gooding, R. H., and McIntyre, G. S. 1998.Glossina morsitans morsitansandGlossina palpalis palpalis: Dosage compensation raises questions about the Milligan model for control of trypanosome development.Experimental Parasitology90, 244–249. Evidence that dosage compensation occurs in tsetse flies was obtained by comparing the activities of X chromosome-linked enzymes, arginine phosphokinase and glucose-6-phosphate dehydrogenase inGlossina m. morsitansand hexokinase and phosphoglucomutase inGlossina p. palpalis, with the activity of an autosome-linked enzyme, malate dehydrogenase, in each species. The shortcomings of the X chromosome model for the control ofTrypanozoonmaturation in tsetse are discussed in light of these findings and previously published reports on the lack of fitness effects of matureTrypanozooninfections in tsetse and on published results on antitrypanosomal factors in male and female tsetse flies.     

Origin and Evolution of Paralogous rRNA Gene Clusters Within the Flatworm Family Dugesiidae (Platyhelminthes, Tricladida)

Analysis of the 18S rDNA sequences of five species of the family Dugesiidae (phylum Platyhelminthes, suborder Tricladida, infraorder Paludicola) and eight species belonging to families Dendrocoelidae and Planaridae and to the infraorder Maricola showed that members of the family Dugesiidae have two types of 18S rDNA genes, while the rest of the species have only one. The duplication event also affected the ITS-1, 5.8S, ITS-2 region and probably the 28S gene. The mean divergence value between the type I and the type II sequences is 9% and type II 18S rDNA genes are evolving 2.3 times more rapidly than type I. The evolutionary rates of type I and type II genes were calibrated from biogeographical data, and an approximate date for the duplication event of 80–120 million years ago was calculated. The type II gene was shown, by RT-PCR, to be transcribed in adult individuals of Schmidtea polychroa, though at very low levels. This result, together with the fact that most of the functionally important positions for small-subunit rRNA in prokaryotes have been conserved, indicates that the type II gene is probably functional. Received: 24 March 1998 / Accepted: 17 March 1999     

A cytogenetical study of Glossina fuscipes fuscipes including a comparison of the polytene chromosome maps with those of Glossina austeni (Diptera,Glossinidae)

The male meiotic sequence is described for the tsetse fly Glossina fuscipes fuscipes together with the polytene chromosome maps and all principal cytological markers. The diploid chromosome number is 2n=6 and includes a pair of large submetacentric autosomes (L₁), a shorter pair of metacentric autosomes (L₂), and an X and Y which constitute a heteromorphic pair. Male meiosis is normally achiasmate although evidence is presented which suggests that chiasmata do form in about 1% of males. A detailed comparison between the polytene chromosomes of this species and Glossina austeni indicates that although they must have had a common ancestor, G. austeni is genetically more closely related to morsitans group tsetses.     

Functional morphology and anatomy of the polypneustic lobes of the last larval instar of tsetse flies,Glossina spp. (Diptera: Glossinidae)

This study examines the external and internal anatomy of the polypneustic (respiratory) lobes in 8 species of tsetse larvae (Diptera: Glossinidae). In the more primitive fusca group, the respiratory lobes are either ring-like (Glossina longipennis) or partially divided into 2 lobes (G. brevipalpis). Two distinctly separated lobes are present in the palpalis group (G. palpalis, G. tachinoides, G. fuscipes) and in the morsitans group (G. morsitans, G. pallidipes, G. austeni). Air enters the polypneustic lobes through narrow slits (stigmata) on the tips of numerous small spiracular papillae that are arranged in rows on both the outer and inner surfaces of the polypneustic lobes. The openings on the spiracular papillae connect to an air tube that is sculptured with septa and pegs. The air tubes connect to an outer air chamber that is likewise replete with a network of pegged septa. The outer air chamber is connected to a felt chamber containing a dense network of filamentous septa (spicules) that appear to function as an air filter. The felt chamber opens into a large, sculptured inner air chamber that connects directly to the regular tracheal trunk. The polypneustic lobes are unusually hard and brittle due to strong sclerotization of the cuticle and are permeated with numerous cuticular pores. There is no evidence that trichomes or other structures present on the respiratory lobes are innervated.     

Analysis of Multiple Tsetse Fly Populations in Uganda Reveals Limited Diversity and Species-Specific Gut Microbiota

The invertebrate microbiome contributes to multiple aspects of host physiology, including nutrient supplementation and immune maturation processes. We identified and compared gut microbial abundance and diversity in natural tsetse flies from Uganda using five genetically distinct populations of Glossina fuscipes fuscipes and multiple tsetse species (Glossina morsitans morsitans, G. f. fuscipes, and Glossina pallidipes) that occur in sympatry in one location. We used multiple approaches, including deep sequencing of the V4 hypervariable region of the 16S rRNA gene, 16S rRNA gene clone libraries, and bacterium-specific quantitative PCR (qPCR), to investigate the levels and patterns of gut microbial diversity from a total of 151 individuals. Our results show extremely limited diversity in field flies of different tsetse species. The obligate endosymbiont Wigglesworthia dominated all samples (>99%), but we also observed wide prevalence of low-density Sodalis (tsetse''s commensal endosymbiont) infections (<0.05%). There were also several individuals (22%) with high Sodalis density, which also carried coinfections with Serratia. Albeit in low density, we noted differences in microbiota composition among the genetically distinct G. f. fuscipes flies and between different sympatric species. Interestingly, Wigglesworthia density varied in different species (10⁴ to 10⁶ normalized genomes), with G. f. fuscipes having the highest levels. We describe the factors that may be responsible for the reduced diversity of tsetse''s gut microbiota compared to those of other insects. Additionally, we discuss the implications of Wigglesworthia and Sodalis density variations as they relate to trypanosome transmission dynamics and vector competence variations associated with different tsetse species.     

Phylogeny of genus Glossina (Diptera: Glossinidae) according to ITS2 sequences

The flies of genus Glossina (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 different Glossina species were PCR-amplified and analyzed in order to construct a molecular phylogeny for genus Glossina. Trees generated by parsimony confirmed the monophyletic taxonomic placement of genus Glossina where fusca group species formed the deepest branch followed by morsitans and palpalis groups, respectively. The placement of Glossina austeni by both the traditional morphological and biochemical criteria has been controversial. Results presented here, based on ITS-2 locus sequence analysis, suggest that Glossina austeni can be placed into a separate subgenerus which forms a sister-group relationship with the morsitans group species.     

Sodalis glossinidius (Enterobacteriaceae) and Vectorial Competence of Glossina palpalis gambiensis and Glossina morsitans morsitans for Trypanosoma congolense Savannah Type

Sodalis glossinidius is an endosymbiont of Glossina palpalis gambiensis and Glossina morsitans morsitans, the vectors of Trypanosoma congolense. The presence of the symbiont was investigated by PCR in Trypanosoma congolense savannah type-infected and noninfected midguts of both fly species, and into the probosces of flies displaying either mature or immature infection, to investigate possible correlation with the vectorial competence of tsetse flies. Sodalis glossinidius was detected in all midguts, infected or not, from both Glossina species. It was also detected in probosces from Glossina palpalis gambiensis flies displaying mature or immature infection, but never in probosces from Glossina morsitans morsitans. These results suggest that, a) there might be no direct correlation between the presence of Sodalis glossinidius and the vectorial competence of Glossina, and b) the symbiont is probably not involved in Trypanosoma congolense savannah type maturation. It could however participate in the establishment process of the parasite.     

Group I Introns from Zygomycota: Evolutionary Implications for the Fungal IC1 Intron Subgroup

The origins of fungal group I introns within nuclear small-subunit (nSSU) rDNA are enigmatic. This is partly because they have never been reported in basal fungal phyla (Zygomycota and Chytridiomycota), which are hypothesized to be ancestral to derived phyla (Ascomycota and Basidiomycota). Here we report group I introns from the nSSU rDNA of two zygomycete fungi, Zoophagus insidians (Zoopagales) and Coemansia mojavensis (Kickxellales). Secondary structure analyses predicted that both introns belong to the IC1 subgroup and that they are distantly related to each other, which is also suggested by different insertion sites. Molecular phylogenetic analyses indicated that the IC1 intron of Z. insidians is closely related to the IC1 intron inserted in the LSU rDNA of the basidiomycete fungus Clavicorona taxophila, which strongly suggests interphylum horizontal transfer. The IC1 intron of C. mojavensis has a low phylogenetic affinity to other fungal IC1 introns inserted into site 943 of nSSU rDNA (relative to E. coli 16S rDNA). It is noteworthy that this intron contains a putative ORF containing a His–Cys box motif in the antisense strand, a hallmark for nuclear-encoded homing endonucleases. Overall, molecular phylogenetic analyses do not support the placement of these two introns in basal fungal IC1 intron lineages. This result leads to the suggestion that fungal IC1 introns might have invaded or been transferred laterally after the divergence of the four major fungal phyla. Received: 8 February 2001 / Accepted: 1 November 2001     

Phenotypic plasticity of desiccation resistance in Glossina puparia: are there ecotype constraints on acclimation responses?

Phenotypic plasticity allows organisms to cope with environmental variation and may aid in the evolution of novel traits. However, whether phenotypic plasticity is beneficial, or if acclimation responses might be constrained to particular ecotypes is generally poorly explored. Here we test the beneficial acclimation hypothesis (BAH) and its alternatives for desiccation resistance to atmospheric moisture in mesic‐ and xeric‐adapted Glossina species. Highly significant interactions among acclimation and test humidity were detected for water loss rates indicative of significant phenotypic plasticity. Ordered‐factor anova was unable to reject predictions of the ‘drier is better’ acclimation hypothesis in xeric Glossina morsitans and mesic G. austeni. Evidence for the ‘deleterious acclimation hypothesis’ was found for mesic G. palpalis as expected from the moist habitats it typically occupies. By contrast, support for the ‘optimal acclimation hypothesis’ was found in xeric G. pallidipes. Little support for BAH was obtained in the present study, although other hypotheses, which might enhance fitness within the environments these species are typically exposed to, were supported. However, acclimation responses were not necessarily constrained to xeric/mesic ecotypes which might be expected if adaptation to a particular environment arose as a trade‐off between plastic responses and living in a particular habitat. These results highlight the complexity of acclimation responses and suggest an important role for phenotypic plasticity in moderating environmental effects on evolutionary fitness in Glossina.     

Molecular Characterization of the Principal Symbiotic Bacteria of the Weevil Sitophilus oryzae: A Peculiar G + C Content of an Endocytobiotic DNA

The principal intracellular symbiotic bacteria of the cereal weevil Sitophilus oryzae were characterized using the sequence of the 16S rDNA gene (rrs gene) and G + C content analysis. Polymerase chain reaction amplification with universal eubacterial primers of the rrs gene showed a single expected sequence of 1,501 bp. Comparison of this sequence with the available database sequences placed the intracellular bacteria of S. oryzae as members of the Enterobacteriaceae family, closely related to the free-living bacteria, Erwinia herbicola and Escherichia coli, and the endocytobiotic bacteria of the tsetse fly and aphids. Moreover, by high-performance liquid chromatography, we measured the genomic G + C content of the S. oryzae principal endocytobiotes (SOPE) as 54%, while the known genomic G + C content of most intracellular bacteria is about 39.5%. Furthermore, based on the third codon position G + C content and the rrs gene G + C content, we demonstrated that most intracellular bacteria except SOPE are A + T biased irrespective of their phylogenetic position. Finally, using the hsp60 gene sequence, the codon usage of SOPE was compared with that of two phylogenetically closely related bacteria: E. coli, a free-living bacterium, and Buchnera aphidicola, the intracellular symbiotic bacteria of aphids. Taken together, these results show a peculiar and distinctly different DNA composition of SOPE with respect to the other obligate intracellular bacteria, and, combined with biological and biochemical data, they elucidate the evolution of symbiosis in S. oryzae. Received: 8 September 1997 / Accepted: 24 October 1997     

Interspecific Transfer of Bacterial Endosymbionts between Tsetse Fly Species: Infection Establishment and Effect on Host Fitness

Tsetse flies (Glossina spp.) can harbor up to three distinct species of endosymbiotic bacteria that exhibit unique modes of transmission and evolutionary histories with their host. Two mutualist enterics, Wigglesworthia and Sodalis, are transmitted maternally to tsetse flies' intrauterine larvae. The third symbiont, from the genus Wolbachia, parasitizes developing oocytes. In this study, we determined that Sodalis isolates from several tsetse fly species are virtually identical based on a phylogenetic analysis of their ftsZ gene sequences. Furthermore, restriction fragment-length polymorphism analysis revealed little variation in the genomes of Sodalis isolates from tsetse fly species within different subgenera (Glossina fuscipes fuscipes and Glossina morsitans morsitans). We also examined the impact on host fitness of transinfecting G. fuscipes fuscipes and G. morsitans morsitans flies with reciprocal Sodalis strains. Tsetse flies cleared of their native Sodalis symbionts were successfully repopulated with the Sodalis species isolated from a different tsetse fly species. These transinfected flies effectively transmitted the novel symbionts to their offspring and experienced no detrimental fitness effects compared to their wild-type counterparts, as measured by longevity and fecundity. Quantitative PCR analysis revealed that transinfected flies maintained their Sodalis populations at densities comparable to those in flies harboring native symbionts. Our ability to transinfect tsetse flies is indicative of Sodalis ' recent evolutionary history with its tsetse fly host and demonstrates that this procedure may be used as a means of streamlining future paratransgenesis experiments.     

Intraspecies Analysis: Comparison of ITS Sequence Data and Gene Intron Sequence Data with Breeding Data for a Worldwide Collection of Gonium pectorale

The morphologically uniform species Gonium pectorale is a colonial green flagellate of worldwide distribution. The affinities of 25 isolates from 18 sites on five continents were assessed by both DNA sequence comparisons and sexual compatibility. Complete sequences were obtained (i) for the internal transcribed spacer ITS-1 and ITS-2 regions of ribosomal DNA and (ii) for each of three single-copy spliceosomal introns, two in a small G protein and one in the actin gene. ITS sequences appeared to homogenize sufficiently rapidly to behave as a single copy gene. Intron sequence differences between isolates in this species reached nucleotide substitution saturation, while ITS sequences did not. Parsimony and evolutionary distance analysis of the two types of DNA data gave essentially the same tree conformation. By all these criteria, the group of G. pectorale isolates fell into two main clades, A and B. Clade A, with isolates from four continents, was comprised of four subclades of quite closely related isolates, plus one strain of ambiguous affinity. Clade B was comprised of two subclades represented by South African and South American isolates, respectively; thus, only subclades of clade B showed geographical localization. With respect to mating, all isolates except one homothallic strain and one apparently sterile strain fell into either one or the other of two mating types. Pairings in all possible combinations revealed that isolates from the same site formed abundant zygotes, which germinated to produce new, sexually active organisms. Zygotes were also formed in many pairings of other combinations, including crosses of clade A with clade B organisms, but none of the latter produced viable germlings. The ability to mate and produce viable progeny that were themselves capable of sexual reproduction was restricted to members of subclades established on the basis of DNA sequence similarities. Thus, the grades of difference in both nuclear intron sequences and rDNA ITS sequences paralleled those observed in the sexual analysis. Received: 9 March 1998 / Accepted: 1 June 1998     

Origin and Inheritance of Group I Introns in 26S rRNA Genes of Gaeumannomyces graminis

Studies of the distribution of the three group I introns (intron A, intron T, and intron AT) in the 26S rDNA of Gaeumannomyces graminis had suggested that they were transferred to a common ancestor of G. graminis var. avenae and var. tritici after it had branched off from var. graminis. Intron AT and intron A exhibited vertical inheritance and coevolved in concert with their hosts. Intron loss could occur after its acquisition. Loss of any one of the three introns could occur in var. tritici whereas only loss of intron T had been found in the majority of var. avenae isolates. The existence of isolates of var. tritici and var. avenae with three introns suggested that intron loss could be reversed by intron acquisition and that the whole process is a dynamic one. This process of intron acquisition and intron loss reached different equilibrium points for different varieties and subgroups, which explained the irregular distribution of these introns in G. graminis. Each of the three group I introns was more closely related to other intron sequences that share the same insertion point in the 26S rDNA than to each other. These introns in distantly related organisms appeared to have a common ancestry. This system had provided a good model for studies on both the lateral transfer and common ancestry of group I introns in the 26S rRNA genes. Received: 17 May 1996 / Accepted: 14 January 1997     

Mitochondrial DNA Phylogeny and the Evolution of Host-Plant Use in Palearctic Chrysolina (Coleoptera, Chrysomelidae) Leaf Beetles

The genus Chrysolina consists of specialized phytophagous leaf-beetles (Coleoptera, Chrysomelidae) with feed on several plant families. There is no explicit phylogenetic hypothesis available for this genus, which includes 65 subgenera and more than 400 species with a wide distribution. We obtained 839-bp sequence data from the 16S rDNA and cytochrome oxidase subunit I (COI) mitochondrial genes. Thirty Chrysolina taxa representing eight host–plant affiliations, two species of the closely related genus Oreina, and two outgroups were sampled. These data sets were used separately and combined to obtain the mitochondrial cladogram of the group using maximum-parsimony and maximum-likelihood criteria. The results were compared to current proposals for Chrysolina systematics that are based on morphological, ecological, and karyological data. The trees obtained were in the most part congruent with the proposed ancestral association of Chrysolina to Lamiaceae based on chromosome number in several lineages. A minimum of five host-plant switches from the ancestral state inferred at the family level and two at the subclass level suggests the absence of parallel evolution of beetles and their host plants. Another switch leading to oligophagy at the family level was deduced to have occurred in the lineage of the subgenus Chrysolina s.str. Received: 22 May 1998 / Accepted: 16 September 1998     

Polytene chromosome maps in four species of tsetse flies Glossina austeni, G. pallidipes, G. morsitans morsitans and G. m. submorsitans (Diptera: Glossinidae): a comparative analysis

Photographic polytene chromosome maps from pupal trichogen cells of four tsetse species, Glossina austeni, G. pallidipes, G. morsitans morsitans and G. m. submorsitans were constructed and compared. The homology of chromosomal elements between the species was achieved by comparing banding patterns. The telomeric and subtelomeric chromosome regions were found to be identical in all species. The pericentromeric regions were found to be similar in the X chromosome and the left arm of L1 chromosome (L1L) but different in L2 chromosome and the right arm of L1 chromosome (L1R). The L2 chromosome differs by a pericentric inversion that is fixed in the three species, G. pallidipes, G. morsitans morsitans and G. m. submorsitans. Moreover, the two morsitans subspecies appeared to be homosequential and differ only by two paracentric inversions on XL and L2L arm. Although a degree of similarity was observed across the homologous chromosomes in the four species, the relative position of specific chromosome regions was different due to chromosome inversions established during their phylogeny. However, there are regions that show no apparent homology between the species, an observation that may be attributed to the considerable intra—chromosomal rearrangements that have occurred following the species divergence. The results of this comparative analysis support the current phylogenetic relationships of the genus Glossina.     

Evolution of 28S Ribosomal DNA in Chaetognaths: Duplicate Genes and Molecular Phylogeny

The chaetognaths are an extraordinarily homogeneous phylum of animals at the morphological level, with a bauplan that can be traced back to the Cambrian. Despite the attention of zoologists for over two centuries, there is little agreement on classification within the phylum. We have used a molecular biological approach to investigate the phylogeny of extant chaetognaths. A rapidly evolving expansion segment toward the 5′ end of 28S ribosomal DNA (rDNA) was amplified using the polymerase chain reaction (PCR), cloned, and sequenced from 26 chaetognath samples representing 18 species. An unusual finding was the presence of two distinct classes of 28S rDNA gene in chaetognaths; our analyses suggest these arose by a gene (or gene cluster) duplication in a common ancestor of extant chaetognaths. The two classes of chaetognath 28S rDNA have been subject to different rates of molecular evolution; we present evidence that both are expressed and functional. In phylogenetic reconstructions, the two classes of 28S rDNA yield trees that root each other; these clearly demonstrate that the Aphragmophora and Phragmophora are natural groups. Within the Aphragmophora, we find good support for the groupings denoted Solidosagitta, Parasagitta, and Pseudosagitta. The relationships between several well-supported groups within the Aphragmophora are uncertain; we suggest this reflects rapid, recent radiation during chaetognath evolution. Received: 19 March 1996 / Accepted: 5 August 1996     

Complex Patterns of Plastid 16S rRNA Gene Evolution in Nonphotosynthetic Green Algae

This study provides a phylogenetic/comparative approach to deciphering the processes underlying the evolution of plastid rRNA genes in genomes under relaxed functional constraints. Nonphotosynthetic green algal taxa that belong to two distinct classes, Chlorophyceae (Polytoma) and Trebouxiophyceae (Prototheca), were investigated. Similar to the situation described previously for plastid 16S rRNA genes in nonphotosynthetic land plants, nucleotide substitution levels, extent of structural variations, and percentage AT values are increased in nonphotosynthetic green algae compared to their closest photosynthetic relatives. However, the mutational processes appear to be different in many respects. First, with the increase in AT content, more transversions are noted in Polytoma and holoparasite angiosperms, while more transitions characterize the evolution of the 16S rDNA sequences in Prototheca. Second, although structural variations do accumulate in both Polytoma and Prototheca (as well as holoparasitic plastid 16S rRNAs), insertions as large as 1.6 kb characterize the plastid 16S rRNA genes in the former, whereas significantly smaller indels (not exceeding 24 bp) seem to be more prevalent in the latter group. The differences in evolutionary rates and patterns within and between lineages might be due to mutations in replication/repair-related genes; slipped-strand mispairing is likely the mechanism responsible for the expansion of insertions in Polytoma plastid 16S rRNA genes. Received: 29 December 2000 / Accepted: 18 May 2001