Serap Aksoy

Genome sequence of the endocellular obligate symbiont of tsetse flies, Wigglesworthia glossinidia

Many insects that rely on a single food source throughout their developmental cycle harbor beneficial microbes that provide nutrients absent from their restricted diet. Tsetse flies, the vectors of African trypanosomes, feed exclusively on blood and rely on one such intracellular microbe for nutritional provisioning and fecundity. As a result of co-evolution with hosts over millions of years, these mutualists have lost the ability to survive outside the sheltered environment of their host insect cells. We present the complete annotated genome of Wigglesworthia glossinidia brevipalpis, which is composed of one chromosome of 697,724 base pairs (bp) and one small plasmid, called pWig1, of 5,200 bp. Genes involved in the biosynthesis of vitamin metabolites, apparently essential for host nutrition and fecundity, have been retained. Unexpectedly, this obligates genome bears hallmarks of both parasitic and free-living microbes, and the gene encoding the important regulatory protein DnaA is absent.

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

Abstract. 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.

Tsetse immune responses and trypanosome transmission: Implications for the development of tsetse-based strategies to reduce trypanosomiasis

Tsetse flies are the medically and agriculturally important vectors of African trypanosomes. Information on the molecular and biochemical nature of the tsetse/trypanosome interaction is lacking. Here we describe three antimicrobial peptide genes, attacin, defensin, and diptericin, from tsetse fat body tissue obtained by subtractive cloning after immune stimulation with Escherichia coli and trypanosomes. Differential regulation of these genes shows the tsetse immune system can discriminate not only between molecular signals specific for bacteria and trypanosome infections but also between different life stages of trypanosomes. The presence of trypanosomes either in the hemolymph or in the gut early in the infection process does not induce transcription of attacin and defensin significantly. After parasite establishment in the gut, however, both antimicrobial genes are expressed at high levels in the fat body, apparently not affecting the viability of parasites in the midgut. Unlike other insect immune systems, the antimicrobial peptide gene diptericin is constitutively expressed in both fat body and gut tissue of normal and immune stimulated flies, possibly reflecting tsetse immune responses to the multiple Gram-negative symbionts it naturally harbors. When flies were immune stimulated with bacteria before receiving a trypanosome containing bloodmeal, their ability to establish infections was severely blocked, indicating that up-regulation of some immune responsive genes early in infection can act to block parasite transmission. The results are discussed in relation to transgenic approaches proposed for modulating vector competence in tsetse.

Microbiome influences on insect host vector competence

Insect symbioses lack the complexity and diversity of those associated with higher eukaryotic hosts. Symbiotic microbiomes are beneficial to their insect hosts in many ways, including dietary supplementation, tolerance to environmental perturbations and maintenance and/or enhancement of host immune system homeostasis. Recent studies have also highlighted the importance of the microbiome in the context of host pathogen transmission processes. Here we provide an overview of the relationship between insect disease vectors, such as tsetse flies and mosquitoes, and their associated microbiome. Several mechanisms are discussed through which symbiotic microbes can influence the ability of their host to transmit pathogens, as well as potential disease control strategies that harness symbiotic microbes to reduce pathogen transmission through an insect vector.

The Obligate Mutualist Wigglesworthia glossinidia Influences Reproduction, Digestion, and Immunity Processes of Its Host, the Tsetse Fly

ABSTRACT Tsetse flies (Diptera: Glossinidae) are vectors for trypanosome parasites, the agents of the deadly sleeping sickness disease in Africa. Tsetse also harbor two maternally transmitted enteric mutualist endosymbionts: the primary intracellular obligate Wigglesworthia glossinidia and the secondary commensal Sodalis glossinidius. Both endosymbionts are transmitted to the intrauterine progeny through the milk gland secretions of the viviparous female. We administered various antibiotics either continuously by per os supplementation of the host blood meal diet or discretely by hemocoelic injections into fertile females in an effort to selectively eliminate the symbionts to study their individual functions. A symbiont-specific PCR amplification assay and fluorescence in situ hybridization analysis were used to evaluate symbiont infection outcomes. Tetracycline and rifampin treatments eliminated all tsetse symbionts but reduced the fecundity of the treated females. Ampicillin treatments did not affect the intracellular Wigglesworthia localized in the bacteriome organ and retained female fecundity. The resulting progeny of ampicillin-treated females, however, lacked Wigglesworthia but still harbored the commensal Sodalis. Our results confirm the presence of two physiologically distinct Wigglesworthia populations: the bacteriome-localized Wigglesworthia involved with nutritional symbiosis and free-living Wigglesworthia in the milk gland organ responsible for maternal transmission to the progeny. We evaluated the reproductive fitness, longevity, digestion, and vectorial competence of flies that were devoid of Wigglesworthia. The absence of Wigglesworthia completely abolished the fertility of females but not that of males. Both the male and female Wigglesworthia-free adult progeny displayed longevity costs and were significantly compromised in their blood meal digestion ability. Finally, while the vectorial competence of the young newly hatched adults without Wigglesworthia was comparable to that of their wild-type counterparts, older flies displayed higher susceptibility to trypanosome infections, indicating a role for the mutualistic symbiosis in host immunobiology. The ability to rear adult tsetse that lack the obligate Wigglesworthia endosymbionts will now enable functional investigations into this ancient symbiosis.

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.

Interactions between mutualist Wigglesworthia and tsetse peptidoglycan recognition protein (PGRP-LB) influence trypanosome transmission

Tsetse flies, the sole vectors of African trypanosomes, have coevolved with mutualistic endosymbiont Wigglesworthia glossinidiae. Elimination of Wigglesworthia renders tsetse sterile and increases their trypanosome infection susceptibility. We show that a tsetse peptidoglycan recognition protein (PGRP-LB) is crucial for symbiotic tolerance and trypanosome infection processes. Tsetse pgrp-lb is expressed in the Wigglesworthia-harboring organ (bacteriome) in the midgut, and its level of expression correlates with symbiont numbers. Adult tsetse cured of Wigglesworthia infections have significantly lower pgrp-lb levels than corresponding normal adults. RNA interference (RNAi)-mediated depletion of pgrp-lb results in the activation of the immune deficiency (IMD) signaling pathway and leads to the synthesis of antimicrobial peptides (AMPs), which decrease Wigglesworthia density. Depletion of pgrp-lb also increases the hosts susceptibility to trypanosome infections. Finally, parasitized adults have significantly lower pgrp-lb levels than flies, which have successfully eliminated trypanosome infections. When both PGRP-LB and IMD immunity pathway functions are blocked, flies become unusually susceptible to parasitism. Based on the presence of conserved amidase domains, tsetse PGRP-LB may scavenge the peptidoglycan (PGN) released by Wigglesworthia and prevent the activation of symbiont-damaging host immune responses. In addition, tsetse PGRP-LB may have an anti-protozoal activity that confers parasite resistance. The symbiotic adaptations and the limited exposure of tsetse to foreign microbes may have led to the considerable differences in pgrp-lb expression and regulation noted in tsetse from that of closely related Drosophila. A dynamic interplay between Wigglesworthia and host immunity apparently is influential in tsetses ability to transmit trypanosomes.

Wigglesworthia gen. nov. and Wigglesworthia glossinidia sp. nov., taxa consisting of the mycetocyte-associated, primary endosymbionts of tsetse flies

The primary endosymbionts (P-endosymbionts) of tsetse flies (Diptera: Glossinidae) are harbored inside specialized cells (mycetocytes) in the anterior region of the gut, and these specialized cells form a white, U-shaped organelle called mycetome. The P-endosymbionts of five tsetse fly species belonging to the Glossinidae have been characterized morphologically, and their 16S ribosomal DNA sequences have been determined for phylogenetic analysis. These organisms were found to belong to a distinct lineage related to the family Enterobacteriaceae in the gamma subdivision of Proteobacteria, which includes the secondary endosymbionts of various insects and Escherichia coli. These bacteria are also related to the P-endosymbionts of aphids, Buchnera aphidicola. Signature sequences in the 16S ribosomal DNA and genomic organizational differences which distinguish the tsetse fly P-endosymbionts from members of the Enterobacteriaceae and from the genus Buchnera are described in this paper. I propose that the P-endosymbionts of tsetse flies should be classified in a new genus, the genus Wigglesworthia, and a new species, Wigglesworthia glossinidia. The P-endosymbiont found in the mycetocytes of Glossina morsitans morsitans is designated the type strain of this species.

Tsetse – A Haven for Microorganisms

Arthropods are involved in the transmission of parasitic and viral agents that cause devastating diseases in animals and plants. Effective control strategies for many of these diseases still rely on the elimination or reduction of vector insect populations. In addition to these pathogenic organisms, arthropods are rich in microbes that are symbiotic in their associations and are often necessary for the fecundity and viability of their hosts. Because the viability of the host often depends on these obligate symbionts, and because these organisms often live in close proximity to disease-causing pathogens, they have been of interest to applied biologists as a potential means to genetically manipulate populations of pest species. As knowledge on these symbiotic associations accumulates from distantly related insect taxa, conserved mechanisms for their transmission and evolutionary histories are beginning to emerge. Here, Serap Aksoy summarizes current knowledge on the functional and evolutionary biology of the multiple symbionts harbored in the medically and agriculturally important insect group, tsetse, and their potential role in the control of trypanosomiasis.

Phylogenetically distant symbiotic microorganisms reside in Glossina midgut and ovary tissues

Abstract. Many blood‐feeding insects, including tsetse flies (Diptera: Glossinidae), harbour intracellular bacterial symbionts. Using isolates from tissues of several Glossina species and diagnostic DNA oligonucleotide primers, a polymerase chain reaction (PCR) based assay was designed to identify symbiotic bacteria. Those inhabiting the midgut of Glossina were found to belong to the gamma subdivision, whereas ovarian Proteobacteria were of the alpha subdivision ‐ probably genus Wolbachia (Rickettsiaceae). The presence of Wolbachia‐like Rickettsia in the ovaries of G. morsitans subspecies may help to explain the maternally inherited incompatibility of some crosses within this species.