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.     

The host range of the male-killing symbiont Arsenophonus nasoniae in filth fly parasitioids

The Son-killer bacterium, Arsenophonus nasoniae, infects Nasonia vitripennis (Hymenoptera: Pteromalidae), a parasitic wasp that attacks filth flies. This gammaproteobacterium kills a substantial amount of male embryos produced by an infected female. Aside from male death, the bacterium does not measurably affect the host, and how it is maintained in the host population is unknown. Interestingly, this bacterial symbiont can be transmitted both vertically (from mother to offspring) and horizontally (to unrelated Nasonia wasps developing in the same fly host). This latter mode may allow the bacterium to spread throughout the ecological community of filth flies and their parasitoids, and to colonize novel species, as well as permit its long-term persistence.We tested 11 species of filth flies and 25 species of their associated parasitoids (representing 28 populations from 16 countries) using diagnostic PCR to assess the bacterium’s actual host range. In addition to 16S rRNA, two loci were targeted: the housekeeping gene infB, and a sequence with high homology to a DNA polymerase gene from a lysogenic phage previously identified from other insect symbionts. We identified infections of A. nasoniae in four species of parasitoids, representing three taxonomic families. Highly similar phage sequences were also identified in three of the four species. These results identify the symbiont as a generalist, rather than a specialist restricted solely to species of Nasonia, and also that horizontal transmission may play an important role in its maintenance.     

共生菌Tremblaya princeps在扶桑绵粉蚧个体发育中的动态变化

【目的】通过研究共生菌Tremblaya princeps的形态、分布及动态变化,明确该菌在宿主扶桑绵粉蚧Phenacoccus solenopsis体内的基本生物学特性。【方法】通过透射电镜技术观察T.princeps的超微结构;利用实时定量PCR明确T.princeps的分布和数量变化。【结果】扶桑绵粉蚧体内T.princeps的分布因性别而异。在雌虫中,T.princeps集中分布于含菌体,在其他组织器官中无分布;含菌体的大小与雌虫各个阶段的个体大小呈显著线性相关,且临近成虫期,含菌体的膨大速率加快。在雄虫中,T.princeps在个体发育的不同阶段,数量变化明显,在蛹期数量急剧增加,之后相对减少;含菌体结构在预蛹期消解。【结论】共生菌T.princeps的变化因宿主性别而异,且与宿主个体发育存在明显的相关性。     

ALBA proteins are stage regulated during trypanosome development in the tsetse fly and participate in differentiation

The protozoan parasite Trypanosoma brucei is responsible for sleeping sickness and alternates between mammal and tsetse fly hosts, where it has to adapt to different environments. We investigated the role of two members of the ALBA family, which encodes hypothetical RNA-binding proteins conserved in most eukaryotes. We show that ALBA3/4 proteins colocalize with the DHH1 RNA-binding protein and with a subset of poly(A+) RNA in stress granules upon starvation. Depletion of ALBA3/4 proteins by RNA interference in the cultured procyclic stage produces cell modifications mimicking several morphogenetic aspects of trypanosome differentiation that usually take place in the fly midgut. A combination of immunofluorescence data and videomicroscopy analysis of live trypanosomes expressing endogenously ALBA fused with fluorescent proteins revealed that ALBA3/4 are present throughout the development of the parasite in the tsetse fly, with the striking exception of the transition stages found in the proventriculus region. This involves migration of the nucleus toward the posterior end of the cell, a phenomenon that is perturbed upon forced expression of ALBA3 during the differentiation process, showing for the first time the involvement of an RNA-binding protein in trypanosome development in vivo.     

Functional analysis of the twin-arginine translocation pathway in Sodalis glossinidius, a bacterial symbiont of the tsetse fly

This study demonstrates a functional twin-arginine (Tat) translocation pathway present in the tsetse fly symbiont Sodalis glossinidius and its potential to export active heterologous proteins to the periplasm. Functionality was demonstrated using green fluorescent protein (GFP) fused to the Tat signal peptide of Escherichia coli trimethylamine N-oxide reductase (TorA).     

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.     

细菌耐药影响肠道菌群及其宿主免疫调控

抗生素在养殖业、医疗业及制药业的广泛应用导致环境中的细菌耐药性日益严重,环境中的抗生素及耐药细菌一旦进入人体肠道,将破坏肠道菌群稳态,对人体健康造成威胁,而残存于饮食中的环境污染物则加剧了细菌耐药造成的人体健康影响。文中在总结大量文献的基础上,阐述了细菌耐药对人体和动物肠道菌群的影响机制及其相关的机体免疫调控,以环境中影响人体肠道菌群获得耐药性的来源作为切入点,阐述抗生素和耐药细菌进入人体肠道后对人体肠道菌群结构和耐药基因组成的影响,以及与人体免疫和免疫调节相关疾病之间的相关机制,并对今后的研究方向进行了展望。     

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.     

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.     

Dynamics of follicle development and lipid storage in the tsetse fly Glossina palpalis palpalis fed on serum albumin deficient diets

Females of Glossina p. palpalis were fed on a mixture of bovine erythrocytes and a serum substitute which had been lyophilized, for a period of 30 days. The rate of ovarian development and lipid incorporation and the fatty acid composition of whole flies were examined at five-day intervals. Results showed that in flies fed serum albumin-deficient diets, ovarian development was seriously impaired and ovulation did not take place. Flies which were fed on a serum albumin-containing diet ovulated and produced larvae, though at a lower rate than control flies. Compared with controls, lipid incorporation was reduced in experimental flies, but more in the albumin-deficient group than in the albumin-containing group. The diet composition had no marked influence on the relative composition of fatty acids in any one group. It is concluded that, though an essential nutritional element for cow blood fed G. p. palpalis, serum albumin was not the only missing essential factor in the diets that were used in this study.     

The microbial flora of Aphis gossypii: Patterns across host plants and geographical space

The cotton aphid, Aphis gossypii, has a worldwide distribution and causes damage to numerous economically important crops. The bacterial symbionts associated with cotton aphids, sampled mainly from malvaceous and cucurbitaceous plants within Japan and Australia, were characterised using molecular profiling approaches. The goal was to document the aphid symbionts present and determine if patterns of microbial diversity are consistent with the existence of host plant related cryptic species in A. gossypii. The bacterial profiles of the aphids are diverse and reflect local geography more than host plant use.     

Change in levels of cyclic AMP and cyclic GMP during pregnancy and larval development of the tsetse fly, Glossina morsitans

Cyclic AMP and cyclic GMP levels change very little in response to feeding and mating, but during pregnancy and at parturition major changes can be detected in both the mother and larva. In both the female head and larva (whole body) cyclic AMP levels reach a peak at parturition. In the larval brain and ring gland cyclic AMP is at its lowest at parturition but rises sharply, reaching a peak 1.5 hr later at the time of pupariation . Though cyclic AMP levels in the head and thorax are consistently 10-60 times greater than levels of cyclic GMP, both the female abdomen and larva contain high concentrations of cyclic GMP with a ratio of cyclic AMP:cyclic GMP approaching 1:1. In the abdomen, the pattern of high cyclic GMP closely parallels the activity cycle of the female's milk gland.     

The influence of sex and fly species on the development of trypanosomes in tsetse flies

Unlike other dipteran disease vectors, tsetse flies of both sexes feed on blood and transmit pathogenic African trypanosomes. During transmission, Trypanosoma brucei undergoes a complex cycle of proliferation and development inside the tsetse vector, culminating in production of infective forms in the saliva. The insect manifests robust immune defences throughout the alimentary tract, which eliminate many trypanosome infections. Previous work has shown that fly sex influences susceptibility to trypanosome infection as males show higher rates of salivary gland (SG) infection with T. brucei than females. To investigate sex-linked differences in the progression of infection, we compared midgut (MG), proventriculus, foregut and SG infections in male and female Glossina morsitans morsitans. Initially, infections developed in the same way in both sexes: no difference was observed in numbers of MG or proventriculus infections, or in the number and type of developmental forms produced. Female flies tended to produce foregut migratory forms later than males, but this had no detectable impact on the number of SG infections. The sex difference was not apparent until the final stage of SG invasion and colonisation, showing that the SG environment differs between male and female flies. Comparison of G. m. morsitans with G. pallidipes showed a similar, though less pronounced, sex difference in susceptibility, but additionally revealed very different levels of trypanosome resistance in the MG and SG. While G. pallidipes was more refractory to MG infection, a very high proportion of MG infections led to SG infection in both sexes. It appears that the two fly species use different strategies to block trypanosome infection: G. pallidipes heavily defends against initial establishment in the MG, while G. m. morsitans has additional measures to prevent trypanosomes colonising the SG, particularly in female flies. We conclude that the tsetse-trypanosome interface works differently in G. m. morsitans and G. pallidipes.     

Variation of tsetse fly abundance in relation to habitat and host presence in the Maasai Steppe,Tanzania

Human activities modify ecosystem structure and function and can also alter the vital rates of vectors and thus the risk of infection with vector‐borne diseases. In the Maasai Steppe ecosystem of northern Tanzania, local communities depend on livestock and suitable pasture that is shared with wildlife, which can increase tsetse abundance and the risk of trypanosomiasis. We monitored the monthly tsetse fly abundance adjacent to Tarangire National Park in 2014–2015 using geo‐referenced, baited epsilon traps. We examined the effect of habitat types and vegetation greenness (NDVI) on the relative abundance of tsetse fly species. Host availability (livestock and wildlife) was also recorded within 100×100 m of each trap site. The highest tsetse abundance was found in the ecotone between Acacia‐Commiphora woodland and grassland, and the lowest in riverine woodland. Glossina swynnertoni was the most abundant species (68%) trapped throughout the entire study, while G. pallidipes was the least common (4%). Relative species abundance was negatively associated with NDVI, with greatest abundance observed in the dry season. The relationship with the abundance of wildlife and livestock was more complex, as we found positive and negative associations depending on the host and fly species. While habitat is important for tsetse distribution, hosts also play a critical role in affecting fly abundance and, potentially, trypanosomiasis risk.     

Rate of embryonic development in the tsetse fly, Glossina morsitans orientalis

Histological examination of a large sample of the tsetse fly, Glossina morsitans orientalis, killed at timed intervals after emergence, showed that embryogenesis takes about 4 days at 25°C during the first intra-uterine cycle. This is considerably longer than is usually stated. Use of these data to obtain an average rate of development for the sample suggested a new method of age determination for the adult female fly which can be used in conjunction with ovarian determination of age.

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Zusammenfassung Histologische Überprüfung einer großen Stichprobe von Tsetsefliegen, Glossina morsitans orientalis, die in bestimmten Abständen nach dem Schlüpfen getötet worden waren, zeigte, daß die Embryogenese während des ersten intrauterinen Zyklus bei 25° ungefähr 4 Tage dauert. Das ist beträchtlich länger, als gewöhnlich festgestellt wurde. Die Benutzung dieses Zeitabschnitts zur Ermittlung der durchschnittlichen Entwicklungsrate für eine Stichprobe bietet sich als neue Methode der Altersbestimmung erwachsener weiblicher Fliegen an, die in Verbindung mit der ovarialen Altersbestimmung verwendet werden kann.

     

Temperature effects during development on a polyembryonic parasitoid and its host

Ambient temperature can influence development through effects on metabolic rate and by inducing physiological stress. In this study, we assessed temperature effects on a host–parasitoid interaction and on the body size and brood size of emerging wasps. By examining the development at two different temperatures of the koinobiont parasitoid, Copidosoma bakeri, and its host, Agrotis ipsilon, we asked: (1) Does the growth response to temperature by A. ipsilon depend on whether the moth caterpillar is parasitized? (2) Does the allocation pattern of body size and brood size in C. bakeri change with temperature? To answer these questions, we exposed A. ipsilon larvae parasitized by C. bakeri to high or low non-lethal temperatures when A. ipsilon was in early or late larval stages and measured their development time and body mass for all four treatment combinations. We also examined the brood size and body mass of emerging wasps. Whether parasitized or not, A. ipsilon larvae decreased development time, but generally did not decrease final body mass, at the higher temperature. When parasitized A. ipsilon was exposed to the higher temperature only late in their development, enlargement of the host by the parasitoid was reduced. C. bakeri brood size significantly increased when the higher temperature was applied early in host development. We did not detect a shift with temperature in the allocation pattern of the size–number trade-off for wasp offspring, suggesting that this trade-off relationship may be under selection strong enough to yield insensitivity to temperature.