The major protein in the midgut of teneral Glossina morsitans morsitans is a molecular chaperone from the endosymbiotic bacterium Wigglesworthia glossinidia

Molecules in the midgut of the tsetse fly (Diptera: Glossinidiae) are thought to play an important role in the life cycle of African trypanosomes by influencing their initial establishment in the midgut and subsequent differentiation events that ultimately affect parasite transmission. It is thus important to determine the molecular composition of the tsetse midgut to aid in understanding disease transmission by these medically important insect vectors. Here, we report that the most abundant protein in the midguts of teneral (unfed) Glossina morsitans morsitans is a 60 kDa molecular chaperone of bacterial origin. Two species of symbiotic bacteria reside in the tsetse midgut, Sodalis glossinidius and Wigglesworthia glossinidia. To determine the exact origin of the 60 kDa molecule, a protein microchemical approach involving two-dimensional (2-D) gel electrophoresis and mass spectrometry was used. Peptide mass maps were compared to virtual peptide maps predicted for S. glossinidius and W. glossinidia 60 kDa chaperone sequences. Four signature peptides were identified, revealing that the source of the chaperone was W. glossinidia. Comparative 2-D gel electrophoresis and immunoblotting further revealed that this protein was localized to the bacteriome and not the distal portion of the tsetse midgut. The possible function of this highly abundant endosymbiont chaperone in the tsetse midgut is discussed.     

The obligate mutualist Wigglesworthia glossinidia influences reproduction, digestion, and immunity processes of its host, the tsetse fly

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.     

An antimicrobial peptide with trypanocidal activity characterized from Glossina morsitans morsitans

Tsetse flies (Diptera:Glossinidae) are vectors of African trypanosomes, the protozoan agents of devastating diseases in humans and animals. Prior studies in trypanosome infected Glossina morsitans morsitans have shown induced expression and synthesis of several antimicrobial peptides in fat body tissue. Here, we have expressed one of these peptides, Attacin (GmAttA1) in Drosophila (S2) cells in vitro. We show that the purified recombinant protein (recGmAttA1) has strong antimicrobial activity against Escherichia coli-K12, but not against the enteric gram-negative symbiont of tsetse, Sodalis glossinidius. The recGmAttA1 also demonstrated inhibitory effects against both the mammalian bloodstream form and the insect stage Trypanosoma brucei in vitro (minimal inhibitory concentration MIC50 0.075 microM). When blood meals were supplemented with purified recGmAttA1 during the course of parasite infection, the prevalence of trypanosome infections in tsetse midgut was significantly reduced. Feeding fertile females GmAttA1 did not affect the fecundity or the longevity of mothers, nor did it affect the hatchability of their offspring. We discuss a paratransgenic strategy, which involves the expression of trypanocidal molecules such as recGmAttA1 in the midgut symbiont Sodalis in vivo to reduce trypanosome transmission.     

Candidatus Sodalis melophagi sp. nov.: phylogenetically independent comparative model to the tsetse fly symbiont Sodalis glossinidius

Bacteria of the genus Sodalis live in symbiosis with various groups of insects. The best known member of this group, a secondary symbiont of tsetse flies Sodalis glossinidius, has become one of the most important models in investigating establishment and evolution of insect-bacteria symbiosis. It represents a bacterium in the early/intermediate state of the transition towards symbiosis, which allows for exploring such interesting topics as: usage of secretory systems for entering the host cell, tempo of the genome modification, and metabolic interaction with a coexisting primary symbiont. In this study, we describe a new Sodalis species which could provide a useful comparative model to the tsetse symbiont. It lives in association with Melophagus ovinus, an insect related to tsetse flies, and resembles S. glossinidius in several important traits. Similar to S. glossinidius, it cohabits the host with another symbiotic bacterium, the bacteriome-harbored primary symbiont of the genus Arsenophonus. As a typical secondary symbiont, Candidatus Sodalis melophagi infects various host tissues, including bacteriome. We provide basic morphological and molecular characteristics of the symbiont and show that these traits also correspond to the early/intermediate state of the evolution towards symbiosis. Particularly, we demonstrate the ability of the bacterium to live in insect cell culture as well as in cell-free medium. We also provide basic characteristics of type three secretion system and using three reference sequences (16 S rDNA, groEL and spaPQR region) we show that the bacterium branched within the genus Sodalis, but originated independently of the two previously described symbionts of hippoboscoids. We propose the name Candidatus Sodalis melophagi for this new bacterium.     

Increased expression of unusual EP repeat-containing proteins in the midgut of the tsetse fly (Glossina) after bacterial challenge

Proteins containing a glutamic acid-proline (EP) repeat epitope were immunologically detected in midguts from eight species of Glossina (tsetse flies). The molecular masses of the tsetse EP proteins differed among species groups. The amino acid sequence of one of these proteins, from Glossina palpalis palpalis, was determined and compared to the sequence of a homologue, the tsetse midgut EP protein of Glossina m. morsitans. The extended EP repeat domains comprised between 36% (G. m. morsitans) and 46% (G. p. palpalis) of the amino acid residues, but otherwise the two polypeptide chains shared most of their sequences and predicted functional domains. The levels of expression of tsetse EP protein in adult teneral midguts were markedly higher than in midguts from larvae. The EP protein was detected by immunoblotting in the fat body, proventriculus and midgut, the known major immune tissues of tsetse and is likely secreted as it was also detected in hemolymph. The EP protein was not produced by the bacterial symbionts of tsetse midguts as determined by genome analysis of Wigglesworthia glossinidia and immunoblot analysis of Sodalis glossinidius. Bacterial challenge of G. m. morsitans, by injection of live E. coli, induced augmented expression of the tsetse EP protein. The presence of EP proteins in a wide variety of tsetse, their constitutive expression in adult fat body and midguts and their upregulation after immunogen challenge suggest they play an important role as a component of the immune system in tsetse.     

Epidemiological characterization of Streptococcus pyogenes isolated from patients with multiple onsets of pharyngitis

Phylogenetic analyses of 16S rRNA support close relationships between the Gammaproteobacteria Sodalis glossinidius, a tsetse (Diptera: Glossinidae) symbiont, and bacteria infecting diverse insect orders. To further examine the evolutionary relationships of these Sodalis-like symbionts, phylogenetic trees were constructed for a subset of putative surface-encoding genes (i.e. ompA, spr, slyB, rcsF, ycfM, and ompC). The ompA and ompC loci were used toward examining the intra- and interspecific diversity of Sodalis within tsetse, respectively. Intraspecific analyses of ompA support elevated nonsynonymous (dN) polymorphism with an excess of singletons, indicating diversifying selection, specifically within the tsetse Glossina morsitans. Additionally, interspecific ompC comparisons between Sodalis and Escherichia coli demonstrate deviation from neutrality, with higher fixed dN observed at sites associated with extracellular loops. Surface-encoding genes varied in their phylogenetic resolution of Sodalis and related bacteria, suggesting conserved vs. host-specific roles. Moreover, Sodalis and its close relatives exhibit genetic divergence at the rcsF, ompA, and ompC loci, indicative of initial molecular divergence. The application of outer membrane genes as markers for further delineating the systematics of recently diverged bacteria is discussed. These results increase our understanding of insect symbiont evolution, while also identifying early genome alterations occurring upon integration of microorganisms with eukaryotic hosts.     

Tsetse blood-meal sources,endosymbionts and trypanosome-associations in the Maasai Mara National Reserve,a wildlife-human-livestock interface

African trypanosomiasis (AT) is a neglected disease of both humans and animals caused by Trypanosoma parasites, which are transmitted by obligate hematophagous tsetse flies (Glossina spp.). Knowledge on tsetse fly vertebrate hosts and the influence of tsetse endosymbionts on trypanosome presence, especially in wildlife-human-livestock interfaces, is limited. We identified tsetse species, their blood-meal sources, and correlations between endosymbionts and trypanosome presence in tsetse flies from the trypanosome-endemic Maasai Mara National Reserve (MMNR) in Kenya. Among 1167 tsetse flies (1136 Glossina pallidipes, 31 Glossina swynnertoni) collected from 10 sampling sites, 28 (2.4%) were positive by PCR for trypanosome DNA, most (17/28) being of Trypanosoma vivax species. Blood-meal analyses based on high-resolution melting analysis of vertebrate cytochrome c oxidase 1 and cytochrome b gene PCR products (n = 354) identified humans as the most common vertebrate host (37%), followed by hippopotamus (29.1%), African buffalo (26.3%), elephant (3.39%), and giraffe (0.84%). Flies positive for trypanosome DNA had fed on hippopotamus and buffalo. Tsetse flies were more likely to be positive for trypanosomes if they had the Sodalis glossinidius endosymbiont (P = 0.0002). These findings point to complex interactions of tsetse flies with trypanosomes, endosymbionts, and diverse vertebrate hosts in wildlife ecosystems such as in the MMNR, which should be considered in control programs. These interactions may contribute to the maintenance of tsetse populations and/or persistent circulation of African trypanosomes. Although the African buffalo is a key reservoir of AT, the higher proportion of hippopotamus blood-meals in flies with trypanosome DNA indicates that other wildlife species may be important in AT transmission. No trypanosomes associated with human disease were identified, but the high proportion of human blood-meals identified are indicative of human African trypanosomiasis risk. Our results add to existing data suggesting that Sodalis endosymbionts are associated with increased trypanosome presence in tsetse flies.     

Attenuation of the sensing capabilities of PhoQ in transition to obligate insect-bacterial association

Sodalis glossinidius, a maternally inherited endosymbiont of the tsetse fly, maintains genes encoding homologues of the PhoP-PhoQ two-component regulatory system. This two-component system has been extensively studied in facultative bacterial pathogens and is known to serve as an environmental magnesium sensor and a regulator of key virulence determinants. In the current study, we show that the inactivation of the response regulator, phoP, renders S. glossinidius sensitive to insect derived cationic antimicrobial peptides (AMPs). The resulting mutant strain displays reduced expression of genes involved in the structural modification of lipid A that facilitates resistance to AMPs. In addition, the inactivation of phoP alters the expression of type-III secretion system (TTSS) genes encoded within three distinct chromosomal regions, indicating that PhoP-PhoQ also serves as a master regulator of TTSS gene expression. In the absence of phoP, S. glossinidius is unable to superinfect either its natural tsetse fly host or a closely related hippoboscid louse fly. Furthermore, we show that the S. glossinidius PhoQ sensor kinase has undergone functional adaptations that result in a substantially diminished ability to sense ancestral signals. The loss of PhoQ's sensory capability is predicted to represent a novel adaptation to the static symbiotic lifestyle, allowing S. glossinidius to constitutively express genes that facilitate resistance to host derived AMPs.     

Effects of antibiotics on fitness of the B biotype and a non-B biotype of the whitefly Bemisia tabaci

The whitefly, Bemisia tabaci Gennadius (Homoptera: Aleyrodidae), harbors primary and secondary endosymbionts. Previous research showed that the invasive B biotype and an indigenous non‐B biotype (named non‐B ZHJ‐1 population) of B. tabaci from Zhejiang, China, harbored different endosymbionts. To investigate the function of these endosymbionts in the two biotypes of B. tabaci, we fed adult whiteflies with three antibiotics, tetracycline, ampicillin trihydrate, and rifampicin, and evaluated the fitness of their offspring on cotton plants. These three antibiotics did not remove the primary endosymbiont Portiera aleyrodidarum but were capable of eliminating the secondary endosymbionts. In the B biotype, treatments of adults with tetracycline or ampicillin trihydrate accelerated development and increased the survival of their offspring, while treatment of adults with rifampicin significantly retarded the development of their offspring but did not affect their survival. In the non‐B ZHJ‐1 population, treatments of adults with tetracycline or ampicillin trihydrate also accelerated the development of their offspring but did not significantly affect their survival, while treatment of adults with rifampicin significantly retarded development and reduced the survival of their offspring. These results suggest that removal of some secondary endosymbionts and/or reduction of the primary endosymbiont from B. tabaci may produce both favorable and unfavorable effects on the fitness of the host insects.     

Extrachromosomal DNA of the symbiont Sodalis glossinidius

The extrachromosomal DNA of Sodalis glossinidius from two tsetse fly species was sequenced and contained four circular elements: three plasmids, pSG1 (82 kb), pSG2 (27 kb), and pSG4 (11 kb), and a bacteriophage-like pSG3 (19 kb) element. The information suggests S. glossinidius is evolving towards an obligate association with tsetse flies.     

A new Sodalis lineage from bloodsucking fly Craterina melbae (Diptera, Hippoboscoidea) originated independently of the tsetse flies symbiont Sodalis glossinidius

Symbiotic bacterium closely related to the secondary symbiont of tsetse flies, Sodalis glossinidius, has been described from the bloodsucking fly Craterina melbae. Phylogenetic analysis of two genes, 16S rRNA gene and component of type three secretion system, placed the bacterium closer to the Sitophilus-derived branch of Sodalis than to the tsetse symbionts. This indicates that the Craterina-derived lineage of Sodalis originated independent of the tsetse flies symbionts and documents the capability of Sodalis bacteria either to switch between different host groups or to establish the symbiosis by several independent events.     

Interactions among multiple genomes: tsetse, its symbionts and trypanosomes

Insect-borne diseases exact a high public health burden and have a devastating impact on livestock and agriculture. To date, control has proved to be exceedingly difficult. One such disease that has plagued sub-Saharan Africa is caused by the protozoan African trypanosomes (Trypanosoma species) and transmitted by tsetse flies (Diptera: Glossinidae). This presentation describes the biology of the tsetse fly and its interactions with trypanosomes as well as its symbionts. Tsetse can harbor up to three distinct microbial symbionts, including two enterics (Wigglesworthia glossinidia and Sodalis glossinidius) as well as facultative Wolbachia infections, which influence host physiology. Recent investigations into the genome of the obligate symbiont Wigglesworthia have revealed characteristics indicative of its long co-evolutionary history with the tsetse host species. Comparative analysis of the commensal-like Sodalis with free-living enterics provides examples of adaptations to the host environment (physiology and ecology), reflecting genomic tailoring events during the process of transitioning into a symbiotic lifestyle. From an applied perspective, the extensive knowledge accumulated on the genomic and developmental biology of the symbionts coupled with our ability to both express foreign genes in these microbes in vitro and repopulate tsetse midguts with these engineered microbes now provides a means to interfere with the host physiological traits which contribute to vector competence promising a novel tool for disease management.     

Molecular and histological characterization of primary (betaproteobacteria) and secondary (gammaproteobacteria) endosymbionts of three mealybug species

Microscopic localization of endosymbiotic bacteria in three species of mealybug (Pseudococcus longispinus, the long-tailed mealybug; Pseudococcus calceolariae, the citrophilus mealybug; and Pseudococcus viburni, the obscure mealybug) showed these organisms were confined to bacteriocyte cells within a bacteriome centrally located within the hemocoel. Two species of bacteria were present, with the secondary endosymbiont, in all cases, living within the primary endosymbiont. DNA from the dissected bacteriomes of all three species of mealybug was extracted for analysis. Sequence data from selected 16S rRNA genes confirmed identification of the primary endosymbiont as "Candidatus Tremblaya princeps," a betaproteobacterium, and the secondary endosymbionts as gammaproteobacteria closely related to Sodalis glossinidius. A single 16S rRNA sequence of the primary endosymbiont was found in all individuals of each mealybug species. In contrast, the presence of multiple divergent strains of secondary endosymbionts in each individual mealybug suggests different evolutionary and transmission histories of the two endosymbionts. Mealybugs are known vectors of the plant pathogen Grapevine leafroll-associated virus 3. To examine the possible role of either endosymbiont in virus transmission, an extension of the model for interaction of proteins with bacterial chaperonins, i.e., GroEL protein homologs, based on mobile-loop amino acid sequences of their GroES homologs, was developed and used for analyses of viral coat protein interactions. The data from this model are consistent with a role for the primary endosymbiont in mealybug transmission of Grapevine leafroll-associated virus 3.     

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.     

Diversity and infection prevalence of endosymbionts in natural populations of the chestnut weevil: relevance of local climate and host plants

Many insects are ubiquitously associated with multiple endosymbionts, whose infection patterns often exhibit spatial and temporal variations. How such endosymbiont variations are relevant to local adaptation of the host organisms is of ecological interest. Here, we report a comprehensive survey of endosymbionts in natural populations of the chestnut weevil Curculio sikkimensis, whose larvae are notorious pests of cultivated chestnuts and also infest acorns of various wild oaks. From 968 insects representing 55 localities across the Japanese Archipelago and originating from 10 host plant species, we identified six distinct endosymbiont lineages, namely Curculioniphilus, Sodalis, Serratia, Wolbachia, Rickettsia and Spiroplasma, at different infection frequencies (96.7%, 12.8%, 82.3%, 82.5%, 28.2% and 6.8%, respectively) and with different geographical distribution patterns. Multiple endosymbiont infections were very common; 3.18±0.61 (ranging from 1.74 to 5.50) endosymbionts per insect on average in each of the local populations. Five pairs of endosymbionts (Curculioniphilus-Serratia, Curculioniphilus-Wolbachia, Sodalis-Rickettsia, Wolbachia-Rickettsia and Rickettsia-Spiroplasma) co-infected the same host individuals more frequently than expected, while infections with Serratia and Wolbachia were negatively correlated to each other. Infection frequencies of the endosymbionts were significantly correlated with climatic and ecological factors: for example, higher Sodalis, Wolbachia and Rickettsia infections at localities of higher temperature; lower Wolbachia and Rickettsia infections at localities of greater snowfall; and higher Curculioniphilus, Sodalis, Serratia, Wolbachia and Rickettsia infections on acorns than on chestnuts. These patterns are discussed in relation to potential host-endosymbiont co-evolution via local adaptation across geographical populations.     

Implications of microfauna-host interactions for trypanosome transmission dynamics in Glossina fuscipes fuscipes in Uganda

Tsetse flies (Diptera: Glossinidae) are vectors for African trypanosomes (Euglenozoa: kinetoplastida), protozoan parasites that cause African trypanosomiasis in humans (HAT) and nagana in livestock. In addition to trypanosomes, two symbiotic bacteria (Wigglesworthia glossinidia and Sodalis glossinidius) and two parasitic microbes, Wolbachia and a salivary gland hypertrophy virus (SGHV), have been described in tsetse. Here we determined the prevalence of and coinfection dynamics between Wolbachia, trypanosomes, and SGHV in Glossina fuscipes fuscipes in Uganda over a large geographical scale spanning the range of host genetic and spatial diversity. Using a multivariate analysis approach, we uncovered complex coinfection dynamics between the pathogens and statistically significant associations between host genetic groups and pathogen prevalence. It is important to note that these coinfection dynamics and associations with the host were not apparent by univariate analysis. These associations between host genotype and pathogen are particularly evident for Wolbachia and SGHV where host groups are inversely correlated for Wolbachia and SGHV prevalence. On the other hand, trypanosome infection prevalence is more complex and covaries with the presence of the other two pathogens, highlighting the importance of examining multiple pathogens simultaneously before making generalizations about infection and spatial patterns. It is imperative to note that these novel findings would have been missed if we had employed the standard univariate analysis used in previous studies. Our results are discussed in the context of disease epidemiology and vector control.     

Two Tsetse fly species, Glossina palpalis gambiensis and Glossina morsitans morsitans, carry genetically distinct populations of the secondary symbiont Sodalis glossinidius

Genetic diversity among Sodalis glossinidius populations was investigated using amplified fragment length polymorphism markers. Strains collected from Glossina palpalis gambiensis and Glossina morsitans morsitans flies group into separate clusters, being differentially structured. This differential structuring may reflect different host-related selection pressures and may be related to the different vector competences of Glossina spp.     

Vector competence of Glossina palpalis gambiensis for Trypanosoma brucei s.l. and genetic diversity of the symbiont Sodalis glossinidius

Tsetse flies transmit African trypanosomes, responsible for sleeping sickness in humans and nagana in animals. This disease affects many people with considerable impact on public health and economy in sub-Saharan Africa, whereas trypanosomes' resistance to drugs is rising. The symbiont Sodalis glossinidius is considered to play a role in the ability of the fly to acquire trypanosomes. Different species of Glossina were shown to harbor genetically distinct populations of S. glossinidius. We therefore investigated whether vector competence for a given trypanosome species could be linked to the presence of specific genotypes of S. glossinidius. Glossina palpalis gambiensis individuals were fed on blood infected either with Trypanosoma brucei gambiense or Trypanosoma brucei brucei. The genetic diversity of S. glossinidius strains isolated from infected and noninfected dissected flies was investigated using amplified fragment length polymorphism markers. Correspondence between occurrence of these markers and parasite establishment was analyzed using multivariate analysis. Sodalis glossinidius strains isolated from T. brucei gambiense-infected flies clustered differently than that isolated from T. brucei brucei-infected individuals. The ability of T. brucei gambiense and T. brucei brucei to establish in G. palpalis gambiensis insect midgut is statistically linked to the presence of specific genotypes of S. glossinidius. This could explain variations in Glossina vector competence in the wild. Then, assessment of the prevalence of specific S. glossinidius genotypes could lead to novel risk management strategies.