The holobiont transcriptome of teneral tsetse fly species of varying vector competence

Background Tsetse flies are the obligate vectors of African trypanosomes, which cause Human and Animal African Trypanosomiasis. Teneral flies (newly eclosed adults) are especially susceptible to parasite establishment and development, yet our understanding of why remains fragmentary. The tsetse gut microbiome is dominated by two Gammaproteobacteria, an essential and ancient mutualist Wigglesworthia glossinidia and a commensal Sodalis glossinidius. Here, we characterize and compare the metatranscriptome of teneral Glossina morsitans to that of G. brevipalpis and describe unique immunological, physiological, and metabolic landscapes that may impact vector competence differences between these two species. Results An active expression profile was observed for Wigglesworthia immediately following host adult metamorphosis. Specifically, ‘translation, ribosomal structure and biogenesis’ followed by ‘coenzyme transport and metabolism’ were the most enriched clusters of orthologous genes (COGs), highlighting the importance of nutrient transport and metabolism even following host species diversification. Despite the significantly smaller Wigglesworthia genome more differentially expressed genes (DEGs) were identified between interspecific isolates (n = 326, ~ 55% of protein coding genes) than between the corresponding Sodalis isolates (n = 235, ~ 5% of protein coding genes) likely reflecting distinctions in host co-evolution and adaptation. DEGs between Sodalis isolates included genes involved in chitin degradation that may contribute towards trypanosome susceptibility by compromising the immunological protection provided by the peritrophic matrix. Lastly, G. brevipalpis tenerals demonstrate a more immunologically robust background with significant upregulation of IMD and melanization pathways. Conclusions These transcriptomic differences may collectively contribute to vector competence differences between tsetse species and offers translational relevance towards the design of novel vector control strategies.

Sodalis Glossinidius and Wolbachia Infections in Wild Population of Glossina Morsitans Submorsitans Caught in the Area of Lake Iro in the South of Chad

Background: To improve vector control of tsetse transmitted trypanosomiases through a better understanging of the vectorial competence of tsetse flies, investigations have been undertaken on the bacterial fauna of different tsetse species. It is in this light that Wolbachia and Sodalis glossinidius were detected in wild populations of Glossina morsistans submorsistans caught in the area of lake Iro in the south of Chad with the aim of generating data that may help to understand the influence of these symbiotic microorganisms on the vectorial competence of G. m. submorsistans. Methods: Tsetse flies were captured using biconical traps. DNA was extracted from tsetse body using the resin chelex. Sodalis glossinidius and Wolbachia were investigated by PCR using specific primers for pSG 2 and wsp genes. Comparisons between S. glossinidius and Wolbachia infections were performed according to sex and sampling periods and association studies between trypanosome infections and S. glossinidius or Wolbachia were performed. Results: From 345 G. m. submorsitans analyzed, 9.0% and 14.5% were respectively infected with S. glossinidius and Wolbachia. Only 2.31% of all tsetse flies hosted the 2 bacteria. Of all trypanosome-infected flies, 7.1% and 9.8% hosted respectively S. glossinidius and Wolbachia. No association was observed between Wolbachia and trypanosomes while significant association (r = 4.992; P = 0.025) was found between S. glossinidius and trypanosome infections. Although significant association (r = 3.147; P = 0.043) was observed between S. glossinidius and T. simiae, none was found with T. congolense or T. godfreyi. Conclusion: This study revealed S. glossinidius and Wolbachia in wild population of G. m. submorsitans of lake Iro. It showed that few tsetse flies are co-infected by Wolbachia and S. glossinidius. The presence of S. glossinidius seems to favor trypanosome infections while Wolbachia has probably no effect. Decrypting the tripartite association requires to investigate the relationship between haplotypes or genotypes of Wolbachia and/or S. glossiniduis and trypanosome infections.

Paratransgenic manipulation of tsetse miR275 alters the physiological homeostasis of the fly’s midgut environment

Tsetse flies are vectors of parasitic African trypanosomes ( Trypanosoma spp.). Current disease control methods include fly-repelling pesticides, trapping flies, and chemotherapeutic treatment of infected people. Inhibiting tsetse’s ability to transmit trypanosomes by strengthening the fly’s natural barriers can serve as an alternative approach to reduce disease. The peritrophic matrix (PM) is a chitinous and proteinaceous barrier that lines tsetse’s midgut. It protects the epithelial cells from the gut lumen content such as food and invading trypanosomes, which have to overcome this physical barrier to establish an infection. Bloodstream form trypanosomes shed variant surface glycoproteins (VSG) into tsetse’s gut lumen early during the infection establishment. The VSG molecules are internalized by the fly’s PM-producing cardia, which results in a reduction in tsetse miR275 expression and a sequential molecular cascade that compromises the PM integrity. In the present study, we investigated the role(s) of miR275 in tsetse’s midgut physiology and trypanosome infection processes by developing a paratransgenic expression system. We used tsetse’s facultative bacterial endosymbiont Sodalis glossinidius to express tandem antagomir -275 repeats (or miR275 sponge) that constitutively reduce miR275 transcript abundance. This paratransgenic system successfully knocked down miR275 levels in the fly’s midgut, which consequently obstructed blood digestion and modulated infection outcomes with an entomopathogenic bacteria and with trypanosomes. RNA sequencing of cardia and midgut tissues from the paratransgenic tsetse confirmed that miR275 regulates processes related to the expression of PM-associated proteins and digestive enzymes as well as genes that encode abundant secretory proteins. Our study demonstrates that paratransgenesis can be employed to study microRNA- regulated pathways in arthropods housing symbiotic bacteria.

Genetic Modification of Sodalis Species by DNA Transduction

ABSTRACT Bacteriophages (phages) are ubiquitous in nature. These viruses play a number of central roles in microbial ecology and evolution by, for instance, promoting horizontal gene transfer (HGT) among bacterial species. The ability of phages to mediate HGT through transduction has been widely exploited as an experimental tool for the genetic study of bacteria. As such, bacteriophage P1 represents a prototypical generalized transducing phage with a broad host range that has been extensively employed in the genetic manipulation of Escherichia coli and a number of other model bacterial species. Here we demonstrate that P1 is capable of infecting, lysogenizing, and promoting transduction in members of the bacterial genus Sodalis, including the maternally inherited insect endosymbiont Sodalis glossinidius. While establishing new tools for the genetic study of these bacterial species, our results suggest that P1 may be used to deliver DNA to many Gram-negative endosymbionts in their insect host, thereby circumventing a culturing requirement to genetically manipulate these organisms. IMPORTANCE A large number of economically important insects maintain intimate associations with maternally inherited endosymbiotic bacteria. Due to the inherent nature of these associations, insect endosymbionts cannot be usually isolated in pure culture or genetically manipulated. Here we use a broad-host-range bacteriophage to deliver exogenous DNA to an insect endosymbiont and a closely related free-living species. Our results suggest that broad-host-range bacteriophages can be used to genetically alter insect endosymbionts in their insect host and, as a result, bypass a culturing requirement to genetically alter these bacteria.

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.

DNA transduction in Sodalis species: implications for the genetic modification of uncultured endosymbionts of insects

Bacteriophages (phages) are ubiquitous in nature. These viruses play a number of central roles in microbial ecology and evolution by, for instance, promoting horizontal gene transfer (HGT) among bacterial species. The ability of phages to mediate HGT through transduction has been widely exploited as an experimental tool for the genetic study of bacteria. As such, bacteriophage P1 represents a prototypical generalized transducing phage with a broad host range that has been extensively employed in the genetic manipulation of Escherichia coli and a number of other model bacterial species. Here we demonstrate that P1 is capable of infecting, lysogenizing and promoting transduction in members of the bacterial genus Sodalis, including the maternally inherited insect endosymbiont Sodalis glossinidius. While establishing new tools for the genetic study of these bacterial species, our results suggest that P1 may be used to deliver DNA to many Gram negative endosymbionts in their insect host, thereby circumventing a culturing requirement to genetically manipulate these organisms.SummaryA large number of economically important insects maintain intimate associations with maternally inherited endosymbiotic bacteria. Due to the inherit nature of these associations, insect endosymbionts cannot be usually isolated in pure culture nor genetically manipulated. Here we use a broad-host range bacteriophage to deliver exogenous DNA to an insect endosymbiont and a closely related free-living species. Our results suggest that broad host range bacteriophages can be used to genetically alter insect endosymbionts in their insect host and, as a result, bypass a culturing requirement to genetically alter these bacteria.

Conjugal DNA Transfer in Sodalis glossinidius, a Maternally Inherited Symbiont of Tsetse Flies

ABSTRACT Stable associations between insects and bacterial species are widespread in nature. This is the case for many economically important insects, such as tsetse flies. Tsetse flies are the vectors of Trypanosoma brucei, the etiological agent of African trypanosomiasis—a zoonotic disease that incurs a high socioeconomic cost in regions of endemicity. Populations of tsetse flies are often infected with the bacterium Sodalis glossinidius. Following infection, S. glossinidius establishes a chronic, stable association characterized by vertical (maternal) and horizontal (paternal) modes of transmission. Due to the stable nature of this association, S. glossinidius has been long sought as a means for the implementation of anti-Trypanosoma paratransgenesis in tsetse flies. However, the lack of tools for the genetic modification of S. glossinidius has hindered progress in this area. Here, we establish that S. glossinidius is amenable to DNA uptake by conjugation. We show that conjugation can be used as a DNA delivery method to conduct forward and reverse genetic experiments in this bacterium. This study serves as an important step in the development of genetic tools for S. glossinidius. The methods highlighted here should guide the implementation of genetics for the study of the tsetse-Sodalis association and the evaluation of S. glossinidius-based tsetse fly paratransgenesis strategies. IMPORTANCE Tsetse flies are the insect vectors of T. brucei, the causative agent of African sleeping sickness—a zoonotic disease that inflicts a substantial economic cost on a broad region of sub-Saharan Africa. Notably, tsetse flies can be infected with the bacterium S. glossinidius to establish an asymptomatic chronic infection. This infection can be inherited by future generations of tsetse flies, allowing S. glossinidius to spread and persist within populations. To this effect, S. glossinidius has been considered a potential expression platform to create flies which reduce T. brucei stasis and lower overall parasite transmission to humans and animals. However, the efficient genetic manipulation of S. glossinidius has remained a technical challenge due to its complex growth requirements and uncharacterized physiology. Here, we exploit a natural mechanism of DNA transfer among bacteria and develop an efficient technique to genetically manipulate S. glossinidius for future studies in reducing trypanosome transmission.

Conjugal DNA transfer in the maternally inherited symbiont of tsetse flies Sodalis glossinidius

Stable associations between insects and bacterial species are widespread in nature. This is the case for many economically important insects, such as tsetse flies. Tsetse flies are the vectors of Trypanosoma brucei, the etiological agent of African trypanosomiasis—a zoonotic disease that incurs a high socioeconomic cost in endemic regions. Populations of tsetse flies are often infected with the bacterium Sodalis glossinidius. Following infection, S. glossinidius establishes a chronic, stable association characterized by vertical (maternal) and horizontal (paternal) modes of transmission. Due to the stable nature of this association, S. glossinidius has been long sought as a means for the implementation of anti-Trypanosoma paratransgenesis in tsetse flies. However, the lack of tools for the genetic modification of S. glossinidius has hindered progress in this area. Here we establish that S. glossinidius is amenable to DNA uptake by conjugation. We show that conjugation can be used as a DNA delivery method to conduct forward and reverse genetic experiments in this bacterium. This study serves as an important step in the development of genetic tools for S. glossinidius. The methods highlighted here should guide the implementation of genetics for the study of the tsetse-Sodalis association and the evaluation of S. glossinidius-based tsetse fly paratransgenesis strategies.ImportanceTsetse flies are the insect vectors of T. brucei, the causative agent of African sleeping sickness—a zoonotic disease that inflicts a substantial economic cost to a broad region of sub-Saharan Africa. Notably, tsetse flies can be infected with the bacterium S. glossinidius to establish an asymptomatic chronic infection. This infection can be inherited by future generations of tsetse flies allowing S. glossinidius to spread and persist within populations. To this effect, S. glossinidius has been considered as a potential expression platform to create flies which reduce T. brucei stasis and lower overall parasite transmission to humans and animals. However, the efficient genetic manipulation of S. glossinidius has remained a technical challenge due to its complex growth requirements and uncharacterized physiology. Here we exploit a natural mechanism of DNA transfer among bacteria and develop an efficient technique to genetically manipulate S. glossinidius for future studies in reducing trypanosome transmission.