The cotton stainer's gut microbiota suppresses infection of a cotransmitted trypanosomatid parasite

The evolutionary and ecological success of many insects is attributed to mutualistic partnerships with bacteria that confer hosts with novel traits including food digestion, nutrient supplementation, detoxification of harmful compounds and defence against natural enemies. Dysdercus fasciatus firebugs (Hemiptera: Pyrrhocoridae), commonly known as cotton stainers, possess a simple but distinctive gut bacterial community including B vitamin‐supplementing Coriobacteriaceae symbionts. In addition, their guts are often infested with the intestinal trypanosomatid parasite Leptomonas pyrrhocoris (Kinetoplastida: Trypanosomatidae). In this study, using experimental bioassays and fluorescence in situ hybridization (FISH), we report on the protective role of the D. fasciatus gut bacteria against L. pyrrhocoris. We artificially infected 2nd instars of dysbiotic and symbiotic insects with a parasite culture and measured parasite titres, developmental time and survival rates. Our results show that L. pyrrhocoris infection increases developmental time and slightly modifies the quantitative composition of the gut microbiota. More importantly, we found significantly higher parasite titres and a tendency towards lower survival rates in parasite‐infected dysbiotic insects compared to symbiotic controls, indicating that the gut bacteria successfully interfere with the establishment or proliferation of L. pyrrhocoris. The colonization of symbiotic bacteria on the peritrophic matrix along the gut wall, as revealed by FISH, likely acts as a barrier blocking parasite attachment or entry into the hemolymph. Our findings show that in addition to being nutritionally important, D. fasciatus’ gut bacteria complement the host's immune system in preventing parasite invasions and that a stable gut microbial community is integral for the host's health.     

The Bacteriome of Bat Flies (Nycteribiidae) from the Malagasy Region: a Community Shaped by Host Ecology,Bacterial Transmission Mode,and Host-Vector Specificity

The Nycteribiidae are obligate blood-sucking Diptera (Hippoboscoidea) flies that parasitize bats. Depending on species, these wingless flies exhibit either high specialism or generalism toward their hosts, which may in turn have important consequences in terms of their associated microbial community structure. Bats have been hypothesized to be reservoirs of numerous infectious agents, some of which have recently emerged in human populations. Thus, bat flies may be important in the epidemiology and transmission of some of these bat-borne infectious diseases, acting either directly as arthropod vectors or indirectly by shaping pathogen communities among bat populations. In addition, bat flies commonly have associations with heritable bacterial endosymbionts that inhabit insect cells and depend on maternal transmission through egg cytoplasm to ensure their transmission. Some of these heritable bacteria are likely obligate mutualists required to support bat fly development, but others are facultative symbionts with unknown effects. Here, we present bacterial community profiles that were obtained from seven bat fly species, representing five genera, parasitizing bats from the Malagasy region. The observed bacterial diversity includes Rickettsia, Wolbachia, and several Arsenophonus-like organisms, as well as other members of the Enterobacteriales and a widespread association of Bartonella bacteria from bat flies of all five genera. Using the well-described host specificity of these flies and data on community structure from selected bacterial taxa with either vertical or horizontal transmission, we show that host/vector specificity and transmission mode are important drivers of bacterial community structure.     

An out-of-body experience: the extracellular dimension for the transmission of mutualistic bacteria in insects

Across animals and plants, numerous metabolic and defensive adaptations are a direct consequence of symbiotic associations with beneficial microbes. Explaining how these partnerships are maintained through evolutionary time remains one of the central challenges within the field of symbiosis research. While genome erosion and co-cladogenesis with the host are well-established features of symbionts exhibiting intracellular localization and transmission, the ecological and evolutionary consequences of an extracellular lifestyle have received little attention, despite a demonstrated prevalence and functional importance across many host taxa. Using insect–bacteria symbioses as a model, we highlight the diverse routes of extracellular symbiont transfer. Extracellular transmission routes are unified by the common ability of the bacterial partners to survive outside their hosts, thereby imposing different genomic, metabolic and morphological constraints than would be expected from a strictly intracellular lifestyle. We emphasize that the evolutionary implications of symbiont transmission routes (intracellular versus extracellular) do not necessarily correspond to those of the transmission mode (vertical versus horizontal), a distinction of vital significance when addressing the genomic and physiological consequences for both host and symbiont.     

Immune depression induced by acanthocephalan parasites in their intermediate crustacean host: consequences for the risk of super-infection and links with host behavioural manipulation

Parasite survival in hosts mainly depends on the capacity to circumvent the host immune response. Acanthocephalan infections in gammarids are linked with decreased activity of the prophenoloxidase (ProPO) system, suggesting an active immunosuppression process. Nevertheless, experimental evidence for this hypothesis is lacking: whether these parasites affect several immune pathways is unknown and the consequences of such immune change have not been investigated. In particular, the consequences for other pathogens are not known; neither are the links with other parasite-induced manipulations of the host. Firstly, using experimental infections of Pomphorhynchus laevis we confirmed that the lower immune activity in parasitised Gammarus pulex is induced by the parasite infection. Second, using natural infections of three different parasites, P. laevis, Pomphorhynchus tereticollis and Polymorphus minutus, we showed that acanthocephalan infection was associated with reduction of the activity of the ProPO system and the haemocyte concentration (two major parameters of crustacean immunity) suggesting that immune depression is a phenomenon affecting several immunological activities. This was confirmed by the fact that acanthocephalan infection (whatever the parasite species) was linked to a lower efficiency to eliminate a bacterial infection. The result suggests a cost of parasite immune depression. Finally, acanthocephalans are also known to induce behavioural alterations in the intermediate host which favour their transmission to definitive hosts. We did not find any correlation between behavioural and immunological alterations in both experimentally and naturally-infected gammarids. Overall, this study suggests that whilst immune depression might be beneficial to acanthocephalan survival within the intermediate gammarid host, it might also be costly if it increases host mortality to additional infections before transmission of the parasite.     

Sex-specific behavioural symptoms of viral gut infection and Wolbachia in Drosophila melanogaster

All organisms are infected with a range of symbionts spanning the spectrum of beneficial mutualists to detrimental parasites. The fruit fly Drosophila melanogaster is a good example, as both endosymbiotic Wolbachia, and pathogenic Drosophila C Virus (DCV) commonly infect it. While the pathophysiology and immune responses against both symbionts are the focus of intense study, the behavioural effects of these infections have received less attention. Here we report sex-specific behavioural responses to these infections in D. melanogaster. DCV infection caused increased sleep in female flies, but had no detectable effect in male flies. The presence of Wolbachia did not reduce this behavioural response to viral infection. We also found evidence for a sex-specific cost of Wolbachia, as male flies infected with the endosymbiont became more lethargic when awake. We discuss these behavioural symptoms as potentially adaptive sickness behaviours.     

Evolution of conflict and cooperation of nematodes associated with solitary and social sweat bees

Parasites and mutualists can wield great influence on the fitness of social organisms, yet the effect that the host’s social structure has on the evolution of parasites, commensals, and mutualists (collectively referred to here as symbionts) is poorly known. Evolutionary theory suggests that host social structure may select for more cooperative symbiont strains in comparison to symbionts of solitary hosts. We compared the productivity of one social and one solitary bee species (Halictus ligatus and Augochlora pura) in the family Halictidae with and without the presence of their nematode symbionts (Acrostichus halicti and Acrostichus puri, respectively). We measured the number of offspring produced, the number of cells provisioned, and nesting activity (for Au. pura) to test the hypothesis that symbionts specific to a social host exhibit greater cooperation than symbionts specific to a solitary host. Infected and uninfected nests of both species did not differ in any fitness estimates indicating that: (1) Acrostichus species are commensals, or at least lack large fitness effects on their hosts, and (2) the transition from association with a solitary host to association with a social host that lives in small colonies does not have detectable effects on the evolution of conflict and cooperation in this system. This is the first comparative study to test the idea that host social structure may influence the evolution of symbionts; future work should compare closely related mutualists and parasites of more advanced eusocial insects to mutualists and parasites of solitary insects.     

Horizontal Transfer of Bacterial Symbionts: Heritability and Fitness Effects in a Novel Aphid Host

Members of several bacterial lineages are known only as symbionts of insects and move among hosts through maternal transmission. Such vertical transfer promotes strong fidelity within these associations, favoring the evolution of microbially mediated effects that improve host fitness. However, phylogenetic evidence indicates occasional horizontal transfer among different insect species, suggesting that some microbial symbionts retain a generalized ability to infect multiple hosts. Here we examine the abilities of three vertically transmitted bacteria from the Gammaproteobacteria to infect and spread within a novel host species, the pea aphid, Acyrthosiphon pisum. Using microinjection, we transferred symbionts from three species of natural aphid hosts into a common host background, comparing transmission efficiencies between novel symbionts and those naturally infecting A. pisum. We also examined the fitness effects of two novel symbionts to determine whether they should persist under natural selection acting at the host level. Our results reveal that these heritable bacteria vary in their capacities to utilize A. pisum as a host. One of three novel symbionts failed to undergo efficient maternal transmission in A. pisum, and one of the two efficiently transmitted bacteria depressed aphid growth rates. Although these findings reveal that negative fitness effects and low transmission efficiency can prevent the establishment of a new infection following horizontal transmission, they also indicate that some symbionts can overcome these obstacles, accounting for their widespread distributions across aphids and related insects.     

Diversity and stability of egg‐bacterial assemblages: The role of paternal care in the glassfrog Hyalinobatrachium colymbiphyllum

Embryos of oviparous organisms must cope with harsh environments and are especially susceptible to disease, considering that many immune mechanisms do not develop until later in life. Parents may transmit symbiotic microflora to eggs, which can contribute to embryo immune defense. Despite the importance of symbiotic microbes for immune function and survival of adult amphibians, vertical transfer of symbionts in amphibians has received less attention than in other taxa. Here, we test the role of male‐only parental care in establishing and maintaining the diversity of egg‐bacterial assemblages in a Neotropical glassfrog (Centrolenidae). Previous research suggests that father Hyalinobatrachium colymbiphyllum may transfer bacterial symbionts to their eggs. We combined a male‐removal experiment in situ with 16S rRNA gene amplicon sequencing to determine whether egg attendance by father H. colymbiphyllum influences the bacterial community and survival of eggs. We found that eggs harbor a diverse and stable bacterial assemblage. Despite different host environments, we found that adult skin and eggs supported very similar bacterial assemblages—even after removing fathers. While we found overlap in the bacteria present on eggs and their fathers, our experiment reveals that extended male care does not contribute to the maintenance of egg‐bacterial communities, so there may be other potential routes of transfer. This study contributes to our understanding of the diversity and maintenance of egg microbiomes, and motivates further research on how initial bacteria are acquired and the ontogenetic development of host–symbiont communities.     

Effects of bacterial secondary symbionts on host plant use in pea aphids

Aphids possess several facultative bacterial symbionts that have important effects on their hosts'' biology. These have been most closely studied in the pea aphid (Acyrthosiphon pisum), a species that feeds on multiple host plants. Whether secondary symbionts influence host plant utilization is unclear. We report the fitness consequences of introducing different strains of the symbiont Hamiltonella defensa into three aphid clones collected on Lathyrus pratensis that naturally lack symbionts, and of removing symbionts from 20 natural aphid–bacterial associations. Infection decreased fitness on Lathyrus but not on Vicia faba, a plant on which most pea aphids readily feed. This may explain the unusually low prevalence of symbionts in aphids collected on Lathyrus. There was no effect of presence of symbiont on performance of the aphids on the host plants of the clones from which the H. defensa strains were isolated. Removing the symbiont from natural aphid–bacterial associations led to an average approximate 20 per cent reduction in fecundity, both on the natural host plant and on V. faba, suggesting general rather than plant-species-specific effects of the symbiont. Throughout, we find significant genetic variation among aphid clones. The results provide no evidence that secondary symbionts have a major direct role in facilitating aphid utilization of particular host plant species.     

Dietary specialization in mutualistic acacia‐ants affects relative abundance but not identity of host‐associated bacteria

Acacia‐ant mutualists in the genus Pseudomyrmex nest obligately in acacia plants and, as we show through stable isotope analysis, feed at a remarkably low trophic level. Insects with diets such as these sometimes depend on bacterial symbionts for nutritional enrichment. We, therefore, examine the bacterial communities associated with acacia‐ants in order to determine whether they host bacterial partners likely to contribute to their nutrition. Despite large differences in trophic position, acacia‐ants and related species with generalized diets do not host distinct bacterial taxa. However, we find that a small number of previously undescribed bacterial taxa do differ in relative abundance between acacia‐ants and generalists, including several Acetobacteraceae and Nocardiaceae lineages related to common insect associates. Comparisons with an herbivorous generalist, a parasite that feeds on acacias and a mutualistic species with a generalized diet show that trophic level is likely responsible for these small differences in bacterial community structure. While we did not experimentally test for a nutritional benefit to hosts of these bacterial lineages, metagenomic analysis reveals a Bartonella relative with an intact nitrogen‐recycling pathway widespread across Pseudomyrmex mutualists and generalists. This taxon may be contributing to nitrogen enrichment of its ant hosts through urease activity and, concordant with an obligately host‐associated lifestyle, appears to be experiencing genomewide relaxed selection. The lack of distinctiveness in bacterial communities across trophic level in this group of ants shows a remarkable ability to adjust to varied diets, possibly with assistance from these diverse ant‐specific bacterial lineages.     

Symbiont infection affects aphid defensive behaviours

Aphids harbour both an obligate bacterial symbiont, Buchnera aphidicola, and a wide range of facultative ones. Facultative symbionts can modify morphological, developmental and physiological host traits that favour their spread within aphid populations. We experimentally investigated the idea that symbionts may also modify aphid behavioural traits to enhance their transmission. Aphids exhibit many behavioural defences against enemies. Despite their benefits, these behaviours have some associated costs leading to reduction in aphid reproduction. Some aphid individuals harbour a facultative symbiont Hamiltonella defensa that provides protection against parasitoids. By analysing aphid behaviours in the presence of parasitoids, we showed that aphids infected with H. defensa exhibited reduced aggressiveness and escape reactions compared with uninfected aphids. The aphid and the symbiont have both benefited from these behavioural changes: both partners reduced the fitness decrements associated with the behavioural defences. Such symbiont-induced changes of behavioural defences may have consequences for coevolutionary processes between host organisms and their enemies.     

Transitions in symbiosis: evidence for environmental acquisition and social transmission within a clade of heritable symbionts

A dynamic continuum exists from free-living environmental microbes to strict host-associated symbionts that are vertically inherited. However, knowledge of the forces that drive transitions in symbiotic lifestyle and transmission mode is lacking. Arsenophonus is a diverse clade of bacterial symbionts, comprising reproductive parasites to coevolving obligate mutualists, in which the predominant mode of transmission is vertical. We describe a symbiosis between a member of the genus Arsenophonus and the Western honey bee. The symbiont shares common genomic and predicted metabolic properties with the male-killing symbiont Arsenophonus nasoniae, however we present multiple lines of evidence that the bee Arsenophonus deviates from a heritable model of transmission. Field sampling uncovered spatial and seasonal dynamics in symbiont prevalence, and rapid infection loss events were observed in field colonies and laboratory individuals. Fluorescent in situ hybridisation showed Arsenophonus localised in the gut, and detection was rare in screens of early honey bee life stages. We directly show horizontal transmission of Arsenophonus between bees under varying social conditions. We conclude that honey bees acquire Arsenophonus through a combination of environmental exposure and social contacts. These findings uncover a key link in the Arsenophonus clades trajectory from free-living ancestral life to obligate mutualism, and provide a foundation for studying transitions in symbiotic lifestyle.Subject terms: Microbial ecology, Molecular evolution, Bacterial evolution, Bacterial genetics, Phylogenetics     

Easier detection of invertebrate "identification-key characters" with light of different wavelengths

Evolutionary arms-races between avian brood parasites and their hosts have typically resulted in some spectacular adaptations, namely remarkable host ability to recognize and reject alien eggs and, in turn, sophisticated parasite egg mimicry. In a striking contrast to hosts sometimes rejecting even highly mimetic eggs, the same species typically fail to discriminate against highly dissimilar parasite chicks. Understanding of this enigma is still hampered by the rarity of empirical tests - and consequently evidence - for chick discrimination. Recent work on Australian host-parasite systems (Gerygone hosts vs. Chalcites parasites), increased not only the diversity of hosts showing chick discrimination, but also discovered an entirely novel host behavioural adaptation. The hosts do not desert parasite chicks (as in all previously reported empirical work) but physically remove living parasites from their nests. Here, I briefly discuss these exciting findings and put them in the context of recent empirical and theoretical work on parasite chick discrimination. Finally, I review factors responsible for a relatively slow progress in this research area and suggest most promising avenues for future research.     

Female-Specific Specialization of a Posterior End Region of the Midgut Symbiotic Organ in Plautia splendens and Allied Stinkbugs

Many stinkbugs (Insecta: Hemiptera: Heteroptera) are associated with bacterial symbionts in a posterior region of the midgut. In these stinkbugs, adult females excrete symbiont-containing materials from the anus for transmission of the beneficial symbionts to their offspring. For ensuring the vertical symbiont transmission, a variety of female-specific elaborate traits at the cellular, morphological, developmental, and behavioral levels have been reported from diverse stinkbugs of the families Plataspidae, Urostylididae, Parastrachiidae, etc. Meanwhile, such elaborate female-specific traits for vertical symbiont transmission have been poorly characterized for the largest and economically important stinkbug family Pentatomidae. Here, we investigated the midgut symbiotic system of a pentatomid stinkbug, Plautia splendens. A specific gammaproteobacterial symbiont was consistently present extracellularly in the cavity of numerous crypts arranged in four rows on the midgut fourth section. The symbiont was smeared on the egg surface upon oviposition by adult females, orally acquired by newborn nymphs, and thereby transmitted vertically to the next generation and important for growth and survival of the host insects. We found that, specifically in adult females, several rows of crypts at the posterior end region of the symbiotic midgut were morphologically differentiated and conspicuously enlarged, often discharging the symbiotic bacteria from the crypt cavity to the main tract of the symbiotic midgut. The female-specific enlarged end crypts were also found in other pentatomid stinkbugs Plautia stali and Carbula crassiventris. These results suggest that the enlarged end crypts represent a female-specific specialized morphological trait for vertical symbiont transmission commonly found among stinkbugs of the family Pentatomidae.     

Small core communities and high variability in bacteria associated with the introduced ascidian Styela plicata

The solitary ascidian Styela plicata is an introduced species in harbors of temperate and tropical oceans around the world. The invasive potential of this species has been studied through reproductive biology and population genetics but no study has yet examined the microbial diversity associated with this ascidian and its potential role in host ecology and invasiveness. Here, we used 16S rRNA gene tag pyrosequencing and transmission electron microscopy to characterize the abundance, diversity and host-specificity of bacteria associated with 3 Mediterranean individuals of S. plicata. Microscopy revealed low bacterial abundance in the inner tunic and their absence from gonad tissues, while pyrosequencing revealed a high diversity of S. plicata-associated bacteria (284 OTUs from 16 microbial phyla) in the inner tunic. The core symbiont community was small and consisted of 16 OTUs present in all S. plicata hosts. This core community included a recently described ascidian symbiont (Hasllibacter halocynthiae) and several known sponge and coral symbionts, including a strictly anaerobic Chloroflexi lineage. Most recovered bacterial OTUs (79.6 %) were present in single S. plicata individuals and statistical analyses of genetic diversity and community structure confirmed high variability of bacterial communities among host individuals. These results suggest that diverse and variable bacterial communities inhabit the tunic of S. plicata, including environmental and host-associated bacterial lineages that appear to be re-established each host generation. We hypothesize that bacterial communities in S. plicata are dynamic and have the potential to aid host acclimation to new habitats by establishing relationships with beneficial, locally sourced bacteria.     

Host age modulates within-host parasite competition

In many host populations, one of the most striking differences among hosts is their age. While parasite prevalence differences in relation to host age are well known, little is known on how host age impacts ecological and evolutionary dynamics of diseases. Using two clones of the water flea Daphnia magna and two clones of its bacterial parasite Pasteuria ramosa, we examined how host age at exposure influences within-host parasite competition and virulence. We found that multiply-exposed hosts were more susceptible to infection and suffered higher mortality than singly-exposed hosts. Hosts oldest at exposure were least often infected and vice versa. Furthermore, we found that in young multiply-exposed hosts competition was weak, allowing coexistence and transmission of both parasite clones, whereas in older multiply-exposed hosts competitive exclusion was observed. Thus, age-dependent parasite exposure and host demography (age structure) could together play an important role in mediating parasite evolution. At the individual level, our results demonstrate a previously unnoticed interaction of the host''s immune system with host age, suggesting that the specificity of immune function changes as hosts mature. Therefore, evolutionary models of parasite virulence might benefit from incorporating age-dependent epidemiological parameters.     

Evolutionary diversification of the gall midge genus Asteromyia (Cecidomyiidae) in a multitrophic ecological context

Gall-forming insects provide ideal systems to analyze the evolution of host–parasite interactions and understand the ecological interactions that contribute to evolutionary diversification. Flies in the family Cecidomyiidae represent the largest radiation of gall-forming insects and are characterized by complex trophic interactions with plants, fungal symbionts, and predators. We analyzed the phylogenetic history and evolutionary associations of the North American cecidomyiid genus Asteromyia, which is engaged in a complex and perhaps co-evolving community of interactions with host-plants, fungi, and parasitoids. Mitochondrial gene trees generally support current classifications, but reveal extensive cryptic diversity within the eight named species. Asteromyia likely radiated after their associated host-plants in the Astereae, but species groups exhibit strong associations with specific lineages of Astereae. Evolutionary associations with fungal mutualists are dynamic, however, and suggest rapid and perhaps coordinated changes across trophic levels.     

Parasitoids as vectors of facultative bacterial endosymbionts in aphids

Heritable bacterial endosymbionts play an important role in aphid ecology. Sequence-based evidence suggests that facultative symbionts such as Hamiltonella defensa or Regiella insecticola also undergo horizontal transmission. Other than through male-to-female transfer during the sexual generation in autumn, the routes by which this occurs remain largely unknown. Here, we tested if parasitoids or ectoparasitic mites can act as vectors for horizontal transfer of facultative symbionts. Using symbiont-specific primers for diagnostic PCR, we demonstrate for the first time, to our knowledge, that parasitoids can indeed transfer H. defensa and R. insecticola by sequentially stabbing infected and uninfected individuals of their host, Aphis fabae, establishing new, heritable infections. Thus, a natural route of horizontal symbiont transmission is also available during the many clonal generations of the aphid life cycle. No transmissions by ectoparasitic mites were observed, nor did parasitoids that emerged from symbiont-infected aphids transfer any symbionts in our experiments.