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Microbiome reduction prevents lipid accumulation during early diapause in the northern house mosquito, Culex pipiens pipiens
Abstract
The mosquito microbiome is critical to multiple facets of their biology, including larval development and disease transmission. For mosquitoes that reside in temperate regions, periods of diapause are critical to overwintering survival, but how the microbiome impacts this state is unknown. In this study, we compared the midgut microbial communities of diapausing and non-diapausing Culex pipiens and assessed how a reduced midgut microbiome influences diapause preparation. High community variability was found within and between non-diapausing and diapausing individuals, but no specific diapause-based microbiome was noted. Emergence of adult, diapausing mosquitoes under sterile conditions generated low bacterial load (LBL) lines with nearly a 1000-fold reduction in bacteria levels. This reduction in bacterial content resulted in significantly lower survival of diapausing females after two weeks, indicating acquisition of the microbiome in adult females is critical for survival throughout diapause. LBL diapausing females had high carbohydrate levels, but did not accumulate lipid reserves, suggesting an inability to process ingested sugars necessary for lipid accumulation. Expression patterns of select genes associated with mosquito lipid metabolism during diapause showed no significant differences between LBL and control lines, suggesting transcriptional changes may not underlie impaired lipid accumulation. Overall, a diverse, adult-acquired microbiome is critical for diapause in C. pipiens to process sugar reserves and accumulate lipids that are necessary to survive prolonged overwintering.
... Members of the plant and animal kingdoms are not considered to be stand-alone as they are in constant contact with microorganisms including bacteria, fungi, and viruses (Kiers and Denison 2008, Rodriguez et al. 2009, Mcfall-Ngai, 2013, Rey and Schornack 2013. Some organisms are associated with obligate symbionts that are essential for the host's survival; others harbor non-obligate symbionts that nonetheless improve or are critical to their host's status (Marshall et al. 2006, Neufeld et al. 2011, Ridley et al. 2012, Coon et al. 2014, Tapia et al. 2016, Didion et al. 2021. Within mosquitoes, bacterial residents have been implicated as important modulators of various life traits such as vector capability and larval development (Shin et al. 2011, Boissière et al. 2012, Chu et al. 2013, Nieuwdorp et al. 2014, Scully et al. 2014, Coon et al. 2016. ...
... Within mosquitoes, bacterial residents have been implicated as important modulators of various life traits such as vector capability and larval development (Shin et al. 2011, Boissière et al. 2012, Chu et al. 2013, Nieuwdorp et al. 2014, Scully et al. 2014, Coon et al. 2016. Additionally, the microbiota has been linked to nutritional factors including lipid metabolism (Valzania et al. 2018a, Valzania et al. 2018b, Didion et al. 2021. As mosquitoes act as vectors for diseasecausing pathogens, understanding how the microbiota influences their ability to survive, interact with the environment, and transmit these disease-causing agents is of the utmost importance (Cansado-Utrilla et al. 2021, Guégan et al. 2018). ...
... Diapause can be entered during the egg, larval, and adult stages in mosquitoes, though it is typically restrained to one life stage per species (Denlinger and Armbruster 2016). The link between diapause and the microbiota has not been well characterized in mosquitoes, or invertebrates in general, with few studies focusing on this subject and largely investigating early diapause (Almada et al. 2015, Liu et al. 2016, Ferguson et al. 2018, Didion et al. 2021, with the only full surveys of microbial changes during larval dormancy in the parasitoid wasp, Nasonia vitripennis (Hymenoptera: Pteromalidae), and during seasonal changes in adults for the cricket, Gryllus veletis (Orthoptera: Gryllidae) (Ferguson et al. 2018, Dittmer andBrucker 2021). Studies have shown that bacteria can influence host overwintering physiology and bacterial communities can be influenced by cold temperatures, whereas for mosquitoes little is known about how cold or overwintering may impact their microbiome. ...
Diapause is a hormonally driven response which is triggered by environmental cues that signal impending adverse conditions and prompts metabolic, developmental, and behavioral changes to allow survival until the return of favorable conditions. Microbial symbionts have been shown to influence the metabolism, development, and behavior of their host organisms, all of which are common diapause-associated characteristics. Surveys of bacterial components in relation to diapause have been examined in few systems, of which the species are usually inactive during dormancy, such as eggs or pupae. This is specifically intriguing as adult female diapause in Culex pipiens (Diptera: Culicidae) can last between 4 and 7 mo and females remain mobile within their hibernacula. Furthermore, it is unknown how microbiota changes associated with prolonged dormancy are different between the lab and field for insect systems. This study aims to characterize how the microbiota of C. pipiens changes throughout diapause under both field and lab settings when provided identical food and water resources. Based on these studies, C. pipiens microbiota shifts as diapause progresses and there are considerable differences between field and lab individuals even when provided the same carbohydrate and water sources. Specific bacterial communities have more association with different periods of diapause, field and lab rearing conditions, and nutritional reserve levels. These studies highlight that diapausing mosquito microbiota studies ideally should occur in field mesocosms and at multiple locations, to increase applicability to wild C. pipiens as prolonged exposure to artificial rearing conditions could impact metrics related to diapause-microbiome interactions. Additionally, these findings suggest that it would be worthwhile to establish if the microbiota shift during diapause impacts host physiology and whether this shift is critical to diapause success.
... This concurrence suggests there may be predictable or cyclical patterns in the microbiome composition associated with dormancy timing that could be important for coral holobiont health. Additionally, reshuffling or shifts in the microbiome associated with dormancy are common among host-microbes, including diapaused copepods (28), parasitoid wasps (8), mosquitos (29), squirrels (6), and bears (7). ...
... In parasitoid wasps, microbiomes are responsible for synthesizing glucose for nutrition during dormancy (8). In both mosquitos and bears, microbes are suggested to also play a role in host provisioning and lipid storage (7,29). Nutritional provisioning, particularly of nitrogen, during dormancy is potentially a convergent trait across host and microbes that undergo dormant periods. ...
Using a high-resolution sampling time series, this study is the first to demonstrate a persistent microbial community shift with quiescence (dormancy) in a marine organism, the temperate coral Astrangia poculata . Furthermore, during this period of community turnover, there is a shedding of putative pathogens and copiotrophs and an enhancement of the ammonia-oxidizing bacteria ( Nitrosococcales ) and archaea (“ Candidatus Nitrosopumilus”).
... This concurrence suggests there may be predictable or cyclical patterns in the microbiome composition associated with dormancy timing that could be important for coral holobiont health. Additionally, reshuffling or shifts in the microbiome associated with dormancy are common among host-microbes, including diapaused copepods (28), parasitoid wasps (8), mosquitos (29), squirrels (6), and bears (7). ...
... In parasitoid wasps, microbiomes are responsible for synthesizing glucose for nutrition during dormancy (8). In both mosquitos and bears, microbes are suggested to also play a role in host provisioning and lipid storage (7,29). Nutritional provisioning, particularly of nitrogen, during dormancy is potentially a convergent trait across host and microbes that undergo dormant periods. ...
Quiescence, or dormancy, is a response to stressful conditions in which an organism slows or halts physiological functioning. Although most species that undergo dormancy maintain complex microbiomes, there is little known about how dormancy influences and is influenced by the host’s microbiome, including in the temperate coral, Astrangia poculata . Northern populations of A. poculata undergo winter quiescence. Here, we characterized wild A. poculata microbiomes in a high-resolution sampling time series before, during, and after quiescence using 16S ribosomal RNA gene sequencing on active (RNA) and present (DNA) microbiomes. We observed a restructuring of the coral microbiome during quiescence that persisted after re-emergence. Upon entering quiescence, corals shed copiotrophic microbes, including putative pathogens, suggesting removal of these taxa as corals cease normal functioning. During and after quiescence, bacteria and archaea associated with nitrification were enriched, suggesting the quiescent microbiome may replace essential functions through supplying nitrate to corals and/or microbes. Overall, this study demonstrates that key microbial groups related to quiescence in A. poculata may play a role in the onset or emergence from dormancy, and long-term regulation of the microbiome composition. The predictability of dormancy in A. poculata provides an ideal natural manipulation system to further identify factors that regulate host-microbial associations.
Importance
Using a high-resolution sampling time series, we are the first to demonstrate a persistent microbial community shift with quiescence (dormancy) in a marine organism, the temperate coral, Astrangia poculata. Furthermore, during this period of community turnover, there is a shedding of putative pathogens and copiotrophs and an enhancement of the ammonia-oxidizing bacteria (Nitrosococcales) and archaea ( Ca . Nitrosopumilus). Our results suggest that quiescence represents an important period during which the coral microbiome can “reset,” shedding opportunistic microbes and enriching for the re-establishment of beneficial associates, including those that may contribute nitrate while the coral animal is not actively feeding. We suggest this work provides foundational understanding of the interplay of microbes and the host’s dormancy response in marine organisms.
... Indeed, the first papers on this subject are beginning to appear. For example, in the mosquito C. pipiens, the diapauseassociated accumulation of lipids is restricted when the microbiome is altered (Didion et al., 2021), and axenic (see Glossary) diapausing larvae of the parasitoid N. vitripennis fail to generate the high glucose and glycerol levels needed for overwintering (Dittmer and Brucker, 2021). The dramatic differences in gut processes during diapause versus non-diapause also suggest that we might expect distinctions in microbiome composition between these two states, and this appears to be the case, as documented in N. vitripennis (Dittmer and Brucker, 2021). ...
Diapause, a stage-specific developmental arrest, is widely exploited by insects to bridge unfavorable seasons. Considerable progress has been made in understanding the ecology, physiology and evolutionary implications of insect diapause, yet intriguing questions remain. A more complete understanding of diapause processes on Earth requires a better geographic spread of investigations, including more work in the tropics and at high latitudes. Questions surrounding energy management and trade-offs between diapause and non-diapause remain understudied. We know little about how maternal effects direct the diapause response, and regulators of prolonged diapause are also poorly understood. Numerous factors that were recently linked to diapause are still waiting to be placed in the regulatory network leading from photoreception to engagement of the diapause program. These factors include epigenetic processes and small noncoding RNAs, and emerging data also suggest a role for the microbiome in diapause regulation. Another intriguing feature of diapause is the complexity of the response, resulting in a diverse suite of responses that comprise the diapause syndrome. Select transcription factors likely serve as master switches turning on these diverse responses, but we are far from understanding the full complexity. The richness of species displaying diapause offers a platform for seeking common components of a 'diapause toolbox'. Across latitudes, during invasion events and in a changing climate, diapause offers grand opportunities to probe evolutionary change and speciation. At a practical level, diapause responses can be manipulated for insect control and long-term storage. Diapausing insects also contain a treasure trove of pharmacological compounds and offer promising models for human health.
... Similar to the case of glycerophospholipids, the level of most members of carbohydrates (carbs) DAMs was also found to been deeply raised. Increasing carbohydrate content is one of the strategies used by many insects to combat extreme weather conditions in both pupal and larval diapauses [56][57][58]. Combined with the results of this study, the increase of carbohydrate content is an important marker of early diapause maintenance in all stages in insects. Further, several reports have showed the importance of trehalose during diapause in invertebrates [59,60]. ...
Holometabolism is a form of insect development which includes four life stages: egg, larva, pupa, and imago (or adult). The developmental change of whole body in metabolite levels of holometabolous insects are usually ignored and lack study. Diapause is an alternative life-history strategy that can occur during the egg, larval, pupal, and adult stages in holometabolous insects. Kallima inachus (Lepidoptera: Nymphalidae) is a holometabolous and adult diapausing butterfly. This study was intended to analyze metabolic changes in K. inachus during ontogeny and diapause through a non-targeted UPLC-MS/MS (ultra-performance liquid chromatograph coupled with tandem mass spectrometry) based metabolomics analysis. A variety of glycerophospholipids (11), amino acid and its derivatives (16), and fatty acyls (nine) are crucial to the stage development of K. inachus. 2-Keto-6-acetamidocaproate, N-phenylacetylglycine, Cinnabarinic acid, 2-(Formylamino) benzoic acid, L-histidine, L-glutamate, and L-glutamine play a potentially important role in transition of successive stages (larva to pupa and pupa to adult). We observed adjustments associated with active metabolism, including an accumulation of glycerophospholipids and carbohydrates and a degradation of lipids, as well as amino acid and its derivatives shifts, suggesting significantly changed in energy utilization and management when entering into adult diapause. Alpha-linolenic acid metabolism and ferroptosis were first found to be associated with diapause in adults through pathway analyses. Our study lays the foundation for a systematic study of the developmental mechanism of holometabolous insects and metabolic basis of adult diapause in butterflies.
Disease-transmitting vectors are living organisms that pass infectious agents from one animal/human to another. The epidemiologically important vectors are usually hematophagous arthropods, including mosquitoes, ticks, triatome bugs, sand flies, and tsetse flies. All of them harbor an endogenous microbiota that functionally complements their host’s biology. Different arthropod vectors are ecologically and behaviorally distinct, and as such, their relationships with symbiotic microbes vary. In this review, we summarize the recent discoveries that reveal how bacterial metabolic activities influence development, nutrition, and pathogen defense in mosquitoes, ticks, triatome bugs, and sand flies. These studies provide a foundation for a systematic understanding of vector–microbiota interactions and for the development of integrated approaches to control vector-borne diseases.
Ruther et al (2021) evaluated fatty acid synthesis in several parasitic wasp species to test if the general finding that lipogenesis in parasitoids is lacking is upheld (Visser et al 2010 PNAS). As proposed by Visser & Ellers (2008), parasitoids can readily assimilate the triglyceride stores produced by their host. When large triglyceride stores are carried over from larval feeding into adulthood (i.e., up to 30 to 40% of the parasitoid’s dry body weight; Visser et al., 2018, 2021), de novo lipid synthesis from adult feeding is either unnecessary or too costly to maintain, leading to trait loss (Ellers et al., 2012). To test the hypothesis that many parasitoids do not synthesize substantial quantities of fat stores as adults, a previous study used feeding experiments on a wide taxonomic range of insects, including parasitoid wasps, parasitoid flies, a parasitoid beetle, and 65 non-parasitoid species (Visser et al., 2010 and references therein). What is striking is that when compared to non-parasitoid insects, 24 out of 29 evolutionarily distinct parasitoid lineages (Coleoptera, Diptera and Hymenoptera; Visser et al., 2010) did not accumulate significant lipid quantities in adulthood even when fed surplus carbohydrates. When little to no lipids are synthesized de novo by adult parasitoid wasps, this can lead to significant constraints on energy allocation toward key adult functions, such as maintenance, dispersal, and reproduction (Jervis et al., 2008). To our minds, the most important question is ‘why don’t parasitoids accumulate substantial quantities of fat as adults like other insects do, and what does this mean for their life histories?’
Background
The life cycles of many insect species include an obligatory or facultative diapause stage with arrested development and low metabolic activity as an overwintering strategy. Diapause is characterised by profound physiological changes in endocrine activity, cell proliferation and nutrient metabolism. However, little is known regarding host-microbiome interactions during diapause, despite the importance of bacterial symbionts for host nutrition and development. In this work, we investigated (i) the role of the microbiome for host nutrient allocation during diapause and (ii) the impact of larval diapause on microbiome dynamics in the parasitoid wasp Nasonia vitripennis , a model organism for host-microbiome interactions.
Results
Our results demonstrate that the microbiome is essential for host nutrient allocation during diapause in N. vitripennis , as axenic diapausing larvae had consistently lower glucose and glycerol levels than conventional diapausing larvae, especially when exposed to cold temperature. In turn, microbiome composition was altered in diapausing larvae, potentially due to changes in the surrounding temperature, host nutrient levels and a downregulation of host immune genes. Importantly, prolonged larval diapause had a transstadial effect on the adult microbiome, with unknown consequences for host fitness. Notably, the most dominant microbiome member, Providencia sp., was drastically reduced in adults after more than 4 months of larval diapause, while potential bacterial pathogens increased in abundance.
Conclusion
This work investigates host-microbiome interactions during a crucial developmental stage, which challenges both the insect host and its microbial associates. The impact of diapause on the microbiome is likely due to several factors, including altered host regulatory mechanisms and changes in the host environment.
The mosquito microbiota impacts the physiology of its host and is essential for normal larval development, thereby influencing transmission of vector-borne pathogens. Germ-free mosquitoes generated with current methods show larval stunting and developmental deficits. Therefore, functional studies of the mosquito microbiota have so far mostly been limited to antibiotic treatments of emerging adults. In this study, we introduce a method to produce germ-free Aedes aegypti mosquitoes. It is based on reversible colonisation with bacteria genetically modified to allow complete decolonisation at any developmental stage. We show that, unlike germ-free mosquitoes previously produced using sterile diets, reversibly colo-nised mosquitoes show no developmental retardation and reach the same size as control adults. This allows us to uncouple the study of the microbiota in larvae and adults. In adults, we detect no impact of bacterial colonisation on mosquito fecundity or longevity. In larvae, data from our transcriptome analysis and diet supplementation experiments following decolonisation suggest that bacteria support larval development by contributing to folate biosynthesis and by enhancing energy storage. Our study establishes a tool to study the microbiota in insects and deepens our knowledge on the metabolic contribution of bacteria to mosquito development.
The microbiome is an assemblage of microorganisms living in association with a multicellular host. Numerous studies have identified a role for the microbiome in host physiology, development, immunity, and behaviour. The generation of axenic (germ-free) and gnotobiotic model systems has been vital to dissecting the role of the microbiome in host biology. We have previously reported the generation of axenic Aedes aegypti mosquitoes, the primary vector of several human pathogenic viruses, including dengue virus and Zika virus. In order to better understand the influence of the microbiome on mosquitoes, we examined the transcriptomes of axenic and conventionally reared Ae. aegypti before and after a blood meal. Our results suggest that the microbiome has a much lower effect on the mosquito’s gene expression than previously thought with only 170 genes influenced by the axenic state, while in contrast, blood meal status influenced 809 genes. The pattern of expression influenced by the microbiome is consistent with transient changes similar to infection rather than sweeping physiological changes. While the microbiome does seem to affect some pathways such as immune function and metabolism, our data suggest the microbiome is primarily serving a nutritional role in development with only minor effects in the adult.
Western corn rootworm (Diabrotica virgifera virgifera LeConte) is a serious pest of maize (Zea mays L.) in North America and parts of Europe. With most of its life cycle spent in the soil feeding on maize root tissues, this insect is likely to encounter and interact with a wide range of soil and rhizosphere microbes. Our knowledge of the role of microbes in pest management and plant health remains woefully incomplete, yet that knowledge could play an important role in effective pest management strategies. For this study, insects were reared on maize in soils from different locations. Insects from two different laboratory colonies (a diapausing and a non-diapausing colony) were sampled at each life stage to determine the possible core bacteriome. Additionally, soil was sampled at each life stage and resulting bacteria were identified to determine the possible contribution of soil to the rootworm bacteriome, if any. We analyzed the V4 hypervariable region of bacterial 16S rRNA genes with Illumina MiSeq to survey the different species of bacteria associated with the insects and the soils. The bacterial community associated with insects was significantly different from that in the soil. Some differences appear to exist between insects from non-diapausing and diapausing colonies while no significant differences in community composition existed between the insects reared on different soils. Despite differences in the bacteria present in immature stages and in male and female adults, there is a possible core bacteriome of approximately 16 operational taxonomic units (i.e., present across all life stages). This research may provide insights into Bt resistance development, improved nutrition in artificial rearing systems, and new management strategies.
Dormancy and diapause are key adaptations in many organisms, enabling survival of temporarily or seasonally unsuitable environmental conditions. In this review, we examine how our understanding of programmed developmental and metabolic arrest during diapause intersects with the increasing body of knowledge about animal co-development and co-evolution with microorganisms. Host-microbe interactions are increasingly understood to affect a number of metabolic, physiological, developmental, and behavioral traits and to mediate adaptations to various environments. Therefore, it is timely to consider how microbial factors might affect the expression and evolution of diapause in a changing world. We examine how a range of host-microbe interactions, from pathogenic to mutualistic, may have an impact on diapause phenotypes. Conversely, we examine how the discontinuities that diapause introduces into animal host generations can affect the ecology of microbial communities and the evolution of host-microbe interactions. We discuss these issues as they relate to physiology, evolution of development, local adaptation, disease ecology, and environmental change. Finally, we outline research questions that bridge the historically distinct fields of seasonal ecology and host-microbe interactions.
The mosquito gut microbiome plays an important role in mosquito development and fitness, providing a promising avenue for novel mosquito control strategies. Here we present a method for rearing axenic (bacteria free) Aedes aegypti mosquitoes, consisting of feeding sterilized larvae on agar plugs containing a high concentration of liver and yeast extract. This approach allows for the complete development to adulthood while maintaining sterility; however, axenic mosquito’s exhibit delayed development time and stunted growth in comparison to their bacterially colonized cohorts. These data challenge the notion that live microorganisms are required for mosquito development, and suggest that the microbiota’s main role is nutritional. Furthermore, we colonize axenic mosquitoes with simplified microbial communities ranging from a single bacterial species to a three-member community, demonstrating the ability to control the composition of the microbiota. This axenic system will allow the systematic manipulation of the mosquito microbiome for a deeper understanding of microbiota-host interactions.
The Southern green stinkbug (N. viridula) feeds on developing soybean seeds in spite of their strong defenses against herbivory, making this pest one of the most harmful to soybean crops. To test the hypothesis that midgut bacterial community allows stinkbugs to tolerate chemical defenses of soybean developing seeds, we identified and characterized midgut microbiota of stinkbugs collected from soybean crops, different secondary plant hosts or insects at diapause on Eucalyptus trees. Our study demonstrated that while more than 54% of N. viridula adults collected in the field had no detectable bacteria in the V1-V3 midgut ventricles, the guts of the rest of stinkbugs were colonized by non-transient microbiota (NTM) and transient microbiota not present in stinkbugs at diapause. While transient microbiota Bacillus sp., Micrococcus sp., Streptomyces sp., Staphylococcus sp. and others had low abundance, NTM microbiota was represented by Yokenella sp., Pantoea sp. and Enterococcus sp. isolates. We found some isolates that showed in vitro β-glucosidase and raffinase activities plus the ability to degrade isoflavonoids and deactivate soybean protease inhibitors. Our results suggest that the stinkbugs´ NTM microbiota may impact on nutrition, detoxification and deactivation of chemical defenses, and Enterococcus sp., Yokenella sp. and Pantoea sp. strains might help stinkbugs to feed on soybean developing seeds in spite of its chemical defenses.
We recently reported that larval stage Aedes aegypti and several other species of mosquitoes grow when living bacteria are present in the gut but do not grow when living bacteria are absent. We further reported that living bacteria induce a hypoxia signal in the gut, which activates hypoxia-induced transcription factors and other processes larvae require for growth. In this study we assessed whether other types of organisms induce mosquito larvae to grow and asked if the density of non-living microbes or diet larvae are fed obviate the requirement for living organisms prior results indicated are required for growth. Using culture conditions identical to our own prior studies, we determined that inoculation density of living Escherichia coli positively affected growth rates of Ae. aegypti larvae, whereas non-living E. coli had no effect on growth across the same range of inoculation densities. A living yeast, alga, and insect cell line induced axenic Ae. aegypti first instars to grow, and stimulated similar levels of midgut hypoxia, HIF-α stabilization, and neutral lipid accumulation in the fat body as E. coli. However, the same organisms had no effect on larval growth if heat-killed. In addition, no axenic larvae molted when fed two other diets, when fed diets supplemented with heat-killed microbes or lysed and heat-killed microbes. Experiments conducted with An. gambiae yielded similar findings. Taken together, our results indicate that organisms from different prokaryotic and eukaryotic groups induce mosquito larvae to grow, whereas no conditions were identified that stimulated larvae to grow in the absence of living organisms.
Khapra beetle, Trogoderma granarium (Coleoptera: Dermestidae), is among the world’s most invasive and destructive pests of stored agricultural products. Its pest status is enhanced by the ability of the larvae to undergo diapause, which increases their tolerance to adverse conditions including insecticides and extreme temperatures. The ability of insects to tolerate extreme conditions can be influenced by their associated bacterial community (the microbiome). Understanding this relationship may lead to improved methods of pest control, but the microbiome of T. granarium is unknown. Here we use next-generation sequencing to address three main questions: 1) How similar are the microbiomes of the closely-related species T. granarium and T. variabile? 2) How does the microbiome change across life stage and physiological state? 3) How is the microbiome of adult T. granarium affected by extreme temperatures? Our results show that the core microbiomes of T. granarium and T. variabile are similar in composition. However, adults of former species have a microbiome dominated by Spiroplasma bacteria (99% of amplified sequences), whereas Spiroplasma in the latter species is almost absent (< 2%). The microbiome of T. granarium differs across life stage (feeding vs non-feeding life stages); its presence in eggs confirms the vertical transmission of Spiroplasma. High temperatures significantly reduced the relative abundance of Spiroplasma, but an effect of low temperatures on the microbiome of T. granarium was not detected. Given its dominance in a key pest species, further study of the interaction between Spiroplasma and its T. granarium host is warranted.
Current insights into the mosquito dehydration response rely on studies that examine specific responses but ultimately fail to provide an encompassing view of mosquito biology. Here, we examined underlying changes in the biology of mosquitoes associated with dehydration. Specifically, we show that dehydration increases blood feeding in the northern house mosquito, Culex pipiens, which was the result of both higher activity and a greater tendency to land on a host. Similar observations were noted for Aedes aegypti and Anopheles quadrimaculatus. RNA-seq and metabolome analyses in C. pipiens following dehydration revealed that factors associated with carbohydrate metabolism are altered, specifically the breakdown of trehalose. Suppression of trehalose breakdown in C. pipiens by RNA interference reduced phenotypes associated with lower hydration levels. Lastly, mesocosm studies for C. pipiens confirmed that dehydrated mosquitoes were more likely to host feed under ecologically relevant conditions. Disease modeling indicates dehydration bouts will likely enhance viral transmission. This dehydration-induced increase in blood feeding is therefore likely to occur regularly and intensify during periods when availability of water is low.
Koalas ( Phascolarctos cinereus ) are arboreal marsupials native to Australia that eat a specialized diet of almost exclusively eucalyptus leaves. Microbes in koala intestines are known to break down otherwise toxic compounds, such as tannins, in eucalyptus leaves. Infections by Chlamydia , obligate intracellular bacterial pathogens, are highly prevalent in koala populations. If animals with Chlamydia infections are received by wildlife hospitals, a range of antibiotics can be used to treat them. However, previous studies suggested that koalas can suffer adverse side effects during antibiotic treatment. This study aimed to use 16S rRNA gene sequences derived from koala feces to characterize the intestinal microbiome of koalas throughout antibiotic treatment and identify specific taxa associated with koala health after treatment. Although differences in the alpha diversity were observed in the intestinal flora between treated and untreated koalas and between koalas treated with different antibiotics, these differences were not statistically significant. The alpha diversity of microbial communities from koalas that lived through antibiotic treatment versus those who did not was significantly greater, however. Beta diversity analysis largely confirmed the latter observation, revealing that the overall communities were different between koalas on antibiotics that died versus those that survived or never received antibiotics. Using both machine learning and OTU (operational taxonomic unit) co-occurrence network analyses, we found that OTUs that are very closely related to Lonepinella koalarum , a known tannin degrader found by culture-based methods to be present in koala intestines, was correlated with a koala’s health status. This is the first study to characterize the time course of effects of antibiotics on koala intestinal microbiomes. Our results suggest it may be useful to pursue alternative treatments for Chlamydia infections without the use of antibiotics or the development of Chlamydia -specific antimicrobial compounds that do not broadly affect microbial communities.
Nitrogen acquisition is a major challenge for herbivorous animals, and the repeated origins of herbivory across the ants have raised expectations that nutritional symbionts have shaped their diversification. Direct evidence for N-provisioning by internally housed symbionts is rare in animals; among the ants, it has been documented for just one lineage. In this study we dissect functional contributions by bacteria from a conserved, multi-partite gut symbiosis in herbivorous Cephalotes ants through in vivo experiments, metagenomics, and in vitro assays. Gut bacteria recycle urea, and likely uric acid, using recycled N to synthesize essential amino acids that are acquired by hosts in substantial quantities. Specialized core symbionts of 17 studied Cephalotes species encode the pathways directing these activities, and several recycle N in vitro. These findings point to a highly efficient N-economy, and a nutritional mutualism preserved for millions of years through the derived behaviors and gut anatomy of Cephalotes ants.
Significance
Gut microbes positively affect the physiology of many animals, but the molecular mechanisms underlying these benefits remain poorly understood. Recent studies indicate that gut bacteria reduce oxygen levels in the mosquito gut, which serves as a growth signal. Here, we report that transduction of a bacteria-induced low-oxygen signal requires mosquito-encoded hypoxia-inducible transcription factors (HIFs). Our results further indicate that HIFs activate several processes with essential growth and metabolic functions. These findings can potentially be used to disrupt mosquito development into adults that transmit human diseases.
Mosquitoes are insects belonging to the order Diptera and family Culicidae. They are distributed worldwide and include approximately 3500 species, of which about 300 have medical and veterinary importance. The evolutionary success of mosquitoes, in both tropical and temperate regions, is due to the various survival strategies these insects have developed throughout their life histories. Of the many adaptive mechanisms, diapause and quiescence, two different types of dormancy, likely contribute to the establishment, maintenance and spread of natural mosquito populations. This review seeks to objectively and coherently describe the terms diapause and quiescence, which can be confused in the literature because the phenotypic effects of these mechanisms are often similar.
In many regions of the world, mosquito-borne viruses pose a growing threat to human health. As an alternative to traditional control measures, the bacterial symbiont Wolbachia has been transferred from Drosophila into the mosquito Aedes aegypti, where it can block the transmission of dengue and Zika viruses. A recent paper has reported large-scale releases of Wolbachia-infected Ae. aegypti in the city of Cairns, Australia. Wolbachia, which is maternally transmitted, invaded and spread through the populations due to a sperm–egg incompatibility called cytoplasmic incompatibility. Over a period of 2 years, a wave of Wolbachia infection slowly spread out from 2 release sites, demonstrating that it will be possible to deploy this strategy in large urban areas. In line with theoretical predictions, Wolbachia infection at a third, smaller release site collapsed due to the immigration of Wolbachia-free mosquitoes from surrounding areas. This remarkable field experiment has both validated theoretical models of Wolbachia population dynamics and demonstrated that this is a viable strategy to modify mosquito populations.
1.A critical question in symbiosis research is where and how organisms obtain beneficial microbial symbionts in different ecological contexts. Microbiota of juveniles are often derived directly from their mother or from the immediate environment. The origin of beneficial symbionts, however, is less obvious in organisms with diapause and dispersal stages, such as plants with dormant seeds and animals in ephemeral or strongly seasonal habitats. In these cases, parents and offspring are separated in time and space, which may affect opportunities for both vertical and horizontal transmission of symbionts. 2.The planktonic crustacean Daphnia produces long-lasting resting eggs to endure winter freezing and summer droughts and requires microbiota for growth and reproduction. It is unknown how hatchlings from resting stages form associations with microbial consorts after diapause. 3.Using natural samples of D. magna resting eggs after several years of storage, we show that the total bacterial community derived from both the exterior and interior of the eggs' ephippial cases is sufficiently beneficial to ensure normal Daphnia functioning in otherwise bacteria-free conditions. We do not find direct evidence that the required bacteria are of maternal origin, though sequencing reveals that the resting stage is accompanied by bacterial taxa previously found in association with adult animals. 4.These findings suggest that while Daphnia are strongly dependent on environmental bacteria for normal functioning, host-bacteria associations are somewhat general and availability of specific bacteria is not a strong constraint on host ecology. Nevertheless, animals and microbes may be ecologically linked through co-dispersal. This article is protected by copyright. All rights reserved.
Annual killifishes exhibit a number of unique life history characters including the occurrence of embryonic diapause, unique cell movements associated with dispersion and subsequent reaggregation of the embryonic blastomeres, and a short post-embryonic life span. Insulin-like growth factor (IGF) signaling is known to play a role in the regulation of metabolic dormancy in a number of animals but has not been explored in annual killifishes. The abundance of IGF proteins during development, and the developmental effects of blocking IGF signaling by pharmacological inhibition of the insulin-like growth factor I receptor (IGF1R) were explored in embryos of the annual killifish Austrofundulus limnaeus Blocking of IGF signaling in embryos that would normally escape entrance into diapause resulted in a phenotype that was remarkably similar to embryos entering diapause. IGF-I protein abundance spikes during early development in embryos that will not enter diapause. In contrast, IGF-I levels remain low during early development in embryos that will enter diapause II. IGF-II protein levels are packaged at higher levels in escape-bound embryos compared to diapause-bound embryos. However, IGF-II levels quickly decrease and remain low during early development and only increase substantially during late development in both developmental trajectories. Developmental patterns of IGF-I and IGF-II protein abundance under conditions that would either induce or bypass entrance into diapause are consistent with a role for IGF signaling in the regulation of developmental trajectory and entrance into diapause in this species. We propose that IGF signaling may be a unifying regulatory pathway that explains the larger suite of characters that are associated with the complex life history of annual killifishes.
Many studies on the microbiome of animals have been reported but a comprehensive analysis is lacking. Here we present a meta-analysis on the microbiomes of arthropods and their terrestrial habitat, focusing on the functional profile of bacterial communities derived from metabolic traits that are essential for microbial life. We report a detailed analysis of probably the largest set of biochemically defined functional traits ever examined in microbiome studies. This work deals with the phylum proteobacteria, which is usually dominant in marine and terrestrial environments and covers all functions associated with microbiomes. The considerable variation in the distribution and abundance of proteobacteria in microbiomes has remained fundamentally unexplained. This analysis reveals discrete functional groups characteristic for adaptation to anaerobic conditions, which appear to be defined by environmental filtering of taxonomically related taxa. The biochemical diversification of the functional groups suggests an evolutionary trajectory in the structure of arthropods’ microbiome, from metabolically versatile to specialized proteobacterial organisms that are adapted to complex environments such as the gut of social insects. Bacterial distribution in arthropods’ microbiomes also shows taxonomic clusters that do not correspond to functional groups and may derive from other factors, including common contaminants of soil and reagents.
Significance
Honey bees are globally important plant pollinators. Guts of adult workers contain specialized bacteria not found outside bees. Experimental results show that gut bacteria increase weight gain in young adult bees, affect expression of genes governing insulin and vitellogenin levels, and increase sucrose sensitivity. Gut bacteria also shape the physicochemical conditions within the gut, lowering pH and oxygen levels. Peripheral resident bacteria consume oxygen, thus maintaining anoxia, as required for microbial activity. Additionally, gut bacteria produce short-chain fatty acids, with acetate and propionate as the major metabolites, as in guts of human and other animals. This study demonstrates how bacteria in the honey bee gut affect host weight gain and improves our understanding of how gut symbionts influence host health.
Gut microbiota mediate the nutritional metabolism and play important roles in human obesity. Diapausing insects accumulate large fat reserves and develop obese phenotypes in order to survive unfavorable conditions. However, the possibility of an association between gut microbiota and insect diapause has not been investigated. We used the Illumina MiSeq platform to compare gut bacterial community composition in nondiapause- (i.e. reproductive) and diapause-destined female cabbage beetles, Colaphellus bowringi, a serious pest of vegetables in Asia. Based on variation in the V3-V4 hypervariable region of 16S ribosomal RNA gene, we identified 99 operational taxonomic units and 17 core microbiota at the genus level. The relative abundance of the bacterial community differed between reproductive and diapause-destined female adults. Gut microbiota associated with human obesity, including Bacteroidetes, Firmicutes, and Proteobacteria, showed a good correlation with diapause. This association between gut microbiota and diapause in the cabbage beetle may open a new avenue for studying insect diapause, as well as developing a natural insect obesity model with which to explore the mechanisms responsible for human obesity.
The cockroach, Periplaneta americana, is an obnoxious and notorious pest of the world, with a strong ability to adapt to a variety of complex environments. However, the molecular mechanism of this adaptability is mostly unknown. In this study, the genes and microbiota composition associated with the adaptation mechanism were studied by analyzing the transcriptome and 16S rDNA pyrosequencing of the P. americana midgut, respectively. Midgut transcriptome analysis identified 82,905 unigenes, among which 64 genes putatively involved in digestion (11 genes), detoxification (37 genes) and oxidative stress response (16 genes) were found. Evaluation of gene expression following treatment with cycloxaprid further revealed that the selected genes (CYP6J1, CYP4C1, CYP6K1, Delta GST, alpha-amylase, beta-glucosidase and aminopeptidase) were upregulated at least 2.0-fold at the transcriptional level, and four genes were upregulated more than 10.0-fold. An interesting finding was that three digestive enzymes positively responded to cycloxaprid application. Tissue expression profiles further showed that most of the selected genes were midgut-biased, with the exception of CYP6K1. The midgut microbiota composition was obtained via 16S rDNA pyrosequencing and was found to be mainly dominated by organisms from the Firmicutes phylum, among which Clostridiales, Lactobacillales and Burkholderiales were the main orders which might assist the host in the food digestion or detoxification of noxious compounds. The preponderant species, Clostridium cellulovorans, was previously reported to degrade lignocellulose efficiently in insects. The abundance of genes involved in digestion, detoxification and response to oxidative stress, and the diversity of microbiota in the midgut might provide P. americana high capacity to adapt to complex environments.
Diapause is a critical eco-physiological adaptation for winter survival in the West Nile Virus vector, Culex pipiens, but little is known about the molecular mechanisms that distinguish diapause from non-diapause in this important mosquito species. We used Illumina RNA-seq to simultaneously identify and quantify relative transcript levels in diapausing and non-diapausing adult females. Among 65,623,095 read pairs, we identified 41 genes with significantly different transcript abundances between these two groups. Transcriptome divergences between these two phenotypes include genes related to juvenile hormone synthesis, anaerobic metabolism, innate immunity and cold tolerance.
Microbes play key roles in shaping the physiology of insects and can influence behavior, reproduction and susceptibility to pathogens. In Sub-Saharan Africa, two major malaria vectors, Anopheles gambiae and An. coluzzii, breed in distinct larval habitats characterized by different microorganisms that might affect their adult physiology and possibly Plasmodium transmission. We analyzed the reproductive microbiomes of male and female An. gambiae and An. coluzzii couples collected from natural mating swarms in Burkina Faso. 16S rRNA sequencing on dissected tissues revealed that the reproductive tracts harbor a complex microbiome characterized by a large core group of bacteria shared by both species and all reproductive tissues. Interestingly, we detected a significant enrichment of several gender-associated microbial biomarkers in specific tissues, and surprisingly, similar classes of bacteria in males captured from one mating swarm, suggesting that these males originated from the same larval breeding site. Finally, we identified several endosymbiotic bacteria, including Spiroplasma, which have the ability to manipulate insect reproductive success. Our study provides a comprehensive analysis of the reproductive microbiome of important human disease vectors, and identifies a panel of core and endosymbiotic bacteria that can be potentially exploited to interfere with the transmission of malaria parasites by the Anopheles mosquito.
Culex pipiens is the most cosmopolitan mosquito of the Pipiens Assemblage. By studying the nature of interactions between this species and microorganisms common to its breeding environment we can unravel important pitfalls encountered during development. We tested the survival rate of larval stages, pupae and adults of a Cx. pipiens colony exposed to a variety of microorganisms in laboratory conditions and assessed the transmission to offspring (F1) by those organisms that secured development up to adulthood. Three complementary experiments were designed to: 1) explore the nutritional value of yeasts and other microorganisms during Cx. pipiens development; 2) elucidate the transstadial transmission of yeast to the host offspring; and 3) to examine the relevance of all these microorganisms in female choice for oviposition-substratum. The yeast Saccharomyces cerevisiae proved to be the most nutritional diet, but despite showing the highest survival rates, vertical transmission to F1 was never confirmed. In addition, during the oviposition trials, none of the gravid females was attracted to the yeast substratum. Notably, the two native bacterial strains, Klebsiella sp. and Aeromonas sp., were the preferred oviposition media, the same two bacteria that managed to feed neonates until molting into 2nd instar larvae. Our results not only suggest that Klebsiella sp. or Aeromonas sp. serve as attractants for oviposition habitat selection, but also nurture the most fragile instar, L1, to assure molting into a more resilient stage, L2, while yeast proves to be the most supportive diet for completing development. These experiments unearthed survival traits that might be considered in the future development of strategies of Cx. pipiens control. These studies can be extended to other members of the Pipiens Assemblage.
All higher organisms negotiate a truce with their commensal microbes and battle pathogenic microbes on a daily basis. Much attention has been given to the role of the innate immune system in controlling intestinal microbes and to the strategies used by intestinal microbes to overcome the host immune response. However, it is becoming increasingly clear that the metabolisms of intestinal microbes and their hosts are linked and that this interaction is equally important for host health and well-being. For instance, an individual's array of commensal microbes can influence their predisposition to chronic metabolic diseases such as diabetes and obesity. A better understanding of host-microbe metabolic interactions is important in defining the molecular bases of these disorders and could potentially lead to new therapeutic avenues. Key advances in this area have been made using Drosophila melanogaster. Here, we review studies that have explored the impact of both commensal and pathogenic intestinal microbes on Drosophila carbohydrate and lipid metabolism. These studies have helped to elucidate the metabolites produced by intestinal microbes, the intestinal receptors that sense these metabolites, and the signaling pathways through which these metabolites manipulate host metabolism. Furthermore, they suggest that targeting microbial metabolism could represent an effective therapeutic strategy for human metabolic diseases and intestinal infection.
Background:
The native microflora associated with mosquitoes have important roles in mosquito development and vector competence. Sequencing of bacterial V3 region from 16S rRNA genes across the developmental stages of Culex mosquitoes (early and late larval instars, pupae and adults) was used to test the hypothesis that bacteria found in the larval stage of Culex are transstadially transmitted to the adult stage, and to compare the microbiomes of field-collected versus laboratory-reared mosquitoes.
Results:
Beta diversity analysis revealed that bacterial community structure differed among three life stages (larvae, pupae and adults) of Culex tarsalis. Although only ~2% of the total number of bacterial OTUs were found in all stages, sequences from these OTUs accounted for nearly 82% of the total bacterial sequences recovered from all stages. Thorsellia (Gammaproteobacteria) was the most abundant bacterial taxon found across all developmental stages of field-collected Culex mosquitoes, but was rare in mosquitoes from laboratory-reared colonies. The proportion of Thorsellia sequences in the microbiomes of mosquito life stages varied ontogenetically with the greatest proportions recovered from the pupae of C. tarsalis and the lowest from newly emerged adults. The microbiome of field-collected late instar larvae was not influenced significantly by differences in the microbiota of the habitat due to habitat age or biopesticide treatments. The microbiome diversity was the greatest in the early instar larvae and the lowest in laboratory-reared mosquitoes.
Conclusions:
Bacterial communities in early instar C. tarsalis larvae were significantly more diverse when compared to late instar larvae, pupae and newly emerged adults. Some of the bacterial OTUs found in the early instar larvae were also found across developmental stages. Thorsellia dominated the bacterial communities in field-collected immature stages but occurred at much lower relative abundance in adults. Differences in microbiota observed in larval habitats did not influence bacterial community profiles of late instar larvae or adults. However, bacterial communities in laboratory-reared C. tarsalis larvae differed significantly from the field. Determining the role of Thorsellia in mosquitoes and its distribution across different species of mosquitoes warrants further investigation.
During feeding, the gut microbiota contributes to the host energy acquisition and metabolic regulation thereby influencing the development of metabolic disorders such as obesity and diabetes. Short-chain fatty acids (SCFAs) such as acetate, butyrate, and propionate, which are produced by gut microbial fermentation of dietary fiber, are recognized as essential host energy sources and act as signal transduction molecules via G-protein coupled receptors (FFAR2, FFAR3, OLFR78, GPR109A) and as epigenetic regulators of gene expression by the inhibition of histone deacetylase (HDAC). Recent evidence suggests that dietary fiber and the gut microbial-derived SCFAs exert multiple beneficial effects on the host energy metabolism not only by improving the intestinal environment, but also by directly affecting various host peripheral tissues. In this review, we summarize the roles of gut microbial SCFAs in the host energy regulation and present an overview of the current understanding of its physiological functions.
Wood-feeding beetles harbor an ecologically rich and taxonomically diverse assemblage of gut microbes that appear to promote survival in woody tissue, which is devoid of nitrogen and essential nutrients. Nevertheless, the contributions of these apparent symbionts to digestive physiology and nutritional ecology remain uncharacterized in most beetle lineages.
Through parallel transcriptome profiling of beetle- and microbial- derived mRNAs, we demonstrate that the midgut microbiome of the Asian longhorned beetle (Anoplophora glabripennis), a member of the beetle family Cerambycidae, is enriched in biosynthetic pathways for the synthesis of essential amino acids, vitamins, and sterols. Consequently, the midgut microbiome of A. glabripennis can provide essential nutrients that the beetle cannot obtain from its woody diet or synthesize itself. The beetle gut microbiota also produce their own suite of transcripts that can enhance lignin degradation, degrade hemicellulose, and ferment xylose and wood sugars. An abundance of cellulases from several glycoside hydrolase families are expressed endogenously by A. glabripennis, as well as transcripts that allow the beetle to convert microbe-synthesized essential amino acids into non-essential amino acids. A. glabripennis and its gut microbes likely collaborate to digest carbohydrates and convert released sugars and amino acid intermediates into essential nutrients otherwise lacking from their woody host plants.
The nutritional provisioning capabilities of the A. glabripennis gut microbiome may contribute to the beetles' unusually broad host range. The presence of some of the same microbes in the guts of other Cerambycidae and other wood-feeding beetles suggests that partnerships with microbes may be a facilitator of evolutionary radiations in beetles, as in certain other groups of insects, allowing access to novel food sources through enhanced nutritional provisioning.
Non-caloric artificial sweeteners (NAS) are among the most widely used food additives worldwide, regularly consumed by lean and obese individuals alike. NAS consumption is considered safe and beneficial owing to their low caloric content, yet supporting scientific data remain sparse and controversial. Here we demonstrate that consumption of commonly used NAS formulations drives the development of glucose intolerance through induction of compositional and functional alterations to the intestinal microbiota. These NAS-mediated deleterious metabolic effects are abrogated by antibiotic treatment, and are fully transferrable to germ-free mice upon faecal transplantation of microbiota configurations from NAS-consuming mice, or of microbiota anaerobically incubated in the presence of NAS. We identify NAS-altered microbial metabolic pathways that are linked to host susceptibility to metabolic disease, and demonstrate similar NAS-induced dysbiosis and glucose intolerance in healthy human subjects. Collectively, our results link NAS consumption, dysbiosis and metabolic abnormalities, thereby calling for a reassessment of massive NAS usage.
Background and Objectives:
The gut hormones peptide YY (PYY) and glucagon-like peptide 1 (GLP-1) acutely suppress appetite. The short chain fatty acid (SCFA) receptor, free fatty acid receptor 2 (FFA2) is present on colonic enteroendocrine L cells, and a role has been suggested for SCFAs in appetite regulation. Here, we characterise the in vitro and in vivo effects of colonic propionate on PYY and GLP-1 release in rodents, and investigate the role of FFA2 in mediating these effects using FFA2 knockout mice.
Methods:
We used Wistar rats, C57BL6 mice and free fatty acid receptor 2 knockout (FFA−/−) mice on a C57BL6 background to explore the impact of the SCFA propionate on PYY and GLP-1 release. Isolated colonic crypt cultures were used to assess the effects of propionate on gut hormone release in vitro. We subsequently developed an in vivo technique to assess gut hormone release into the portal vein following colonic infusion of propionate.
Results:
Propionate stimulated the secretion of both PYY and GLP-1 from wild-type primary murine colonic crypt cultures. This effect was significantly attenuated in cultures from FFA2−/− mice. Intra-colonic infusion of propionate elevated PYY and GLP-1 levels in jugular vein plasma in rats and in portal vein plasma in both rats and mice. However, propionate did not significantly stimulate gut hormone release in FFA2−/− mice.
Conclusions:
Intra-colonic administration of propionate stimulates the concurrent release of both GLP-1 and PYY in rats and mice. These data demonstrate that FFA2 deficiency impairs SCFA-induced gut hormone secretion both in vitro and in vivo.
A large body of evidence suggests that the regulation of energy balance and glucose homeostasis by fermentable carbohydrates induces specific changes in the gut microbiota. Among the mechanisms, our research group and others have demonstrated that the gut microbiota fermentation (i.e., bacterial digestion of specific compounds) of specific prebiotics or other non-digestible carbohydrates is associated with the secretion of enteroendocrine peptides, such as the glucagon-like peptide-1 (GLP-1) and peptide YY (PYY), produced by L-cells. In this review, we highlight past and recent results describing how dietary manipulation of the gut microbiota, using nutrients or specific microbes, can stimulate GLP-1 secretion in rodents and humans. Furthermore, the purpose of this review is to discuss the putative mechanisms by which specific bacterial metabolites, such as short chain fatty acids, trigger GLP-1 secretion through GPR41/43-dependent mechanisms. Moreover, we conclude by discussing the molecular advance showing that the endocannabinoid system or related bioactive lipids modulated by the gut microbiota may contribute to the regulation of glucose, lipid and energy homeostasis.
Field studies indicate adult mosquitoes (Culicidae) host low diversity communities of bacteria that vary greatly among individuals and species. In contrast, it remains unclear how adult mosquitoes acquire their microbiome, what influences community structure, and whether the microbiome is important for survival. Here we used pyrosequencing of 16S rRNA to characterize the bacterial communities of three mosquito species reared under identical conditions. Two of these species, Aedes aegypti and Anopheles gambiae, are anautogenous and must blood feed to produce eggs, while one, Georgecraigius atropalpus, is autogenous and produces eggs without blood feeding. Each mosquito species contained a low diversity community comprised primarily of aerobic bacteria acquired primarily from the aquatic habitat in which larvae developed. Our results suggested the communities in Ae. aegypti and An. gambiae larvae share more similarities with one another than with Ge. atropalpus. Studies with Ae. aegypti also strongly suggested that adults transstadially acquired several members of the larval bacterial community, but only four genera of bacteria present in blood fed females were detected on eggs. Functional assays showed that axenic larvae of each species failed to develop beyond the first instar. Experiments with Ae. aegypti indicated several members of the microbial community and Escherichia coli successfully colonized axenic larvae and rescued development. Overall, our results provide new insights about the acquisition and structure of bacterial communities in mosquitoes. They also indicate three mosquito species spanning the breadth of the Culicidae depend on their gut microbiome for development.This article is protected by copyright. All rights reserved.
The risks of depletion of energy reserves and encountering lethally low temperatures are considered as two important mortality factors that may limit winter survival of mosquito, Culex pipiens f. pipiens populations. Here we show that the autumn females carry lipid reserves, which are safely sufficient for at least two overwintering periods, provided the females diapausing at temperatures typical for underground spaces (0 °C - 8 °C) would continuously rest at a standard metabolic rate (SMR). The overwintering females, however, switch from SMR to much higher metabolic rate during flight, either seeking for optimal microhabitat within the shelter or in response to disturbances by air current or predator attack. These behaviors result in fast oxidation of lipid reserves and, therefore, the autumn load of energy reserves may actually limit winter survival under specific circumstances. Next, we show that the level of females' cold hardiness is physiologically set relatively weak for overwintering in open field, above-ground habitats, but is ecologically entirely sufficient for overwintering in most underground spaces. The characteristics of suitable overwintering shelters are: no or limited risk of contact with ice crystals, no or limited air movements, winter temperatures relatively stable between +2 and + 6 °C, winter minimum does not drop below −4 °C for longer than one week, or below −8 °C for longer than 1 day.
Recent studies have provided molecular evidence that gut symbiotic bacteria modulate host insect development, fitness and reproduction. However, the molecular mechanisms through which gut symbionts regulate these aspects of host physiology remain elusive. To address these questions, we prepared two different Riptortus-Burkholderia insect models, Burkholderia gut symbiont-colonized (Sym) Riptortus pedestris insects and gut symbiont-noncolonized (Apo) insects. Upon LC-MS analyses, juvenile hormone III skipped bisepoxide (JHSB3) was newly identified from Riptortus Apo- and Sym-female and male adults' insect hemolymph and JHSB3 titer in the Apo- and Sym-female insects were measured because JH is important for regulating reproduction in adult insects. The JHSB3 titer in the Sym-females were consistently higher compared to those of Apo-females. Since previous studies reported that Riptortus hexamerin-α and vitellogenin proteins were upregulated by the topical abdominal application of a JH-analog, chemically synthesized JHSB3 was administered to Apo-females. As expected, the hexamerin-α and vitellogenin proteins were dramatically increased in the hemolymph of JHSB3-treated Apo-females, resulting in increased egg production compared to that in Sym-females. Taken together, these results demonstrate that colonization of Burkholderia gut symbiont in the host insect stimulates biosynthesis of the heteroptera-specific JHSB3, leading to larger number of eggs produced and enhanced fitness in Riptortus host insects.
Marker-gene sequencing is a cost-effective method of taxonomically profiling microbial communities. Unlike metagenomic approaches, marker-gene sequencing does not provide direct information about the functional genes that are present in the genomes of community members. However, by capitalizing on the rapid growth in the number of sequenced genomes, it is possible to infer which functions are likely associated with a marker gene based on its sequence similarity with a reference genome. The PICRUSt tool is based on this idea and can predict functional category abundances based on an input marker gene. In brief, this method requires a reference phylogeny with tips corresponding to taxa with reference genomes as well as taxa lacking sequenced genomes. A modified ancestral state reconstruction (ASR) method is then used to infer counts of functional categories for taxa without reference genomes. The predictions are written to pre-calculated files, which can be cross-referenced with other datasets to quickly generate predictions of functional potential for a community. This chapter will give an in-depth description of these methods and describe how PICRUSt should be used.
1.Seasonal changes in the environment, such as varying temperature, have the potential to change the functional relationship between ectothermic animals, such as insects, and their microbiomes. Our objectives were to determine: a) whether seasonal changes in temperature shift the composition of the insect gut microbiome, and b) if changes in the microbiome are concomitant with changes in the physiology of the host, including the immune system and response to cold. 2.We exposed laboratory populations of the spring field cricket, Gryllus veletis (Orthoptera: Gryllidae), to simulated overwintering conditions in both a laboratory microcosm and a field‐like microcosm containing soil and leaves. In summer, autumn, winter and spring, we extracted and sequenced 16S bacterial genomic DNA from cricket guts, to capture seasonal variation in the composition of the microbiome. 3.The composition of the gut microbiome was similar between microcosms, and overall highly anaerobic. In both microcosms, we captured similar seasonal variation in the composition of the microbiome, where overwintering resulted in permanent changes to these microbial communities. In particular, the abundance of Pseudomonas spp. decreased, and that of Wolbachia spp. increased, during overwintering. 4.Concurrent with overwintering changes in the gut microbiome, G. veletis acquire freeze‐tolerance and immune function shifts temporarily, returning to summer levels of activity in the spring. Specifically, haemocyte concentrations increase but survival of fungal infection decreases in the winter, whereas the ability to clear bacteria from the haemolymph remains unchanged. 5.Overall, we demonstrate that the gut microbiome does shift seasonally, and in concert with other physiological changes. We hypothesize that these changes may be linked, and suggest that it will next be important to determine if these changes in the microbiome contribute to host overwintering success. This article is protected by copyright. All rights reserved.
Copepods harbor diverse bacterial communities, which collectively carry out key biogeochemical transformations in the ocean. However, bulk copepod sampling averages over the variability in their associated bacterial communities, thereby limiting our understanding of the nature and specificity of copepod-bacteria associations. Here, we characterize the bacterial communities associated with nearly 200 individual Calanus finmarchicus copepods transitioning from active growth to diapause. We find that all individual copepods sampled share a small set of "core" operational taxonomic units (OTUs), a subset of which have also been found associated with other marine copepod species in different geographic locations. However, most OTUs are patchily distributed across individual copepods, thereby driving community differences across individuals. Among patchily distributed OTUs, we identified groups of OTUs correlated with common ecological drivers. For instance, a group of OTUs positively correlated with recent copepod feeding served to differentiate largely active growing copepods from those entering diapause. Together, our results underscore the power of individual-level sampling for understanding host-microbiome relationships.
An estimated 3500 species of mosquitoes (family Culicidae) exist worldwide of which several are known vectors of pathogens that cause disease in humans and other vertebrates. Mosquitoes also host communities of microbes in their digestive tract that form a gut microbiota. Recent studies provide important insights on how mosquitoes acquire a gut microbiota and the community of microbes that are present. Results also indicate that the gut microbiota affects several aspects of mosquito biology. Altogether, these effects impact mosquito fitness with potential consequences for disease prevalence.
Animals that undergo seasonal cycles of feeding and fasting have adaptations that maintain integrity of organ systems when dietary nutrients are lacking. Food deprivation also challenges the gut microbiota, which relies heavily on host diet for metabolic substrates and the gastrointestinal tract, which is influenced by enteral nutrients and microbial activity. Winter fasting in hibernators shifts the microbiota to favor taxa with the capacity to degrade and utilize host-derived substrates and disfavor taxa that prefer complex plant polysaccharides. Microbiome alterations may contribute to hibernation-induced changes in the intestinal immune system, epithelial barrier function, and other host features that are affected by microbial short-chain fatty acids and other metabolites. Understanding mechanisms by which the hibernator host and its gut symbionts adapt to the altered nutritional landscape during winter fasting may provide insights into protective mechanisms that are compromised when nonhibernating species, such as humans, undergo long periods of enteral nutrient deprivation. Expected final online publication date for the Annual Review of Nutrition Volume 37 is August 21, 2017. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.
Mosquitoes are insects of interest because several species vector disease-causing pathogens to humans and other vertebrates. We previously reported that mosquitoes from long-term laboratory cultures require living bacteria in their gut to develop, but development does not depend on particular species of bacteria. Here, we focused on three distinct but interrelated areas of study to better understand the role of bacteria in mosquito development by studying field and laboratory populations of Aedes aegypti, Aedes albopictus, and Culex quinquefasciatus from the Southeastern United States. Sequence analysis of bacterial 16S rRNA gene amplicons showed that bacteria community composition differed substantially in larvae from different collection sites, whereas larvae from the same site shared similarities. Although previously unknown to be infected by Wolbachia, results also indicated that Ae. aegypti from one field site hosted a dual infection. Regardless of collection site or factors like Wolbachia infection, however, each mosquito species required living bacteria in their digestive tract to develop. Results also identified several concerns in using antibiotics to eliminate the bacterial community in larvae in order to study its developmental consequences. Altogether, our results indicate that several mosquito species require living bacteria for development. We also hypothesize these species do not rely on particular bacteria because larvae do not reliably encounter the same bacteria in the aquatic habitats they colonize. This article is protected by copyright. All rights reserved.
The dormant state of diapause is exploited by numerous mosquito species to survive seasonal periods of environmental stress. We discuss embryonic, larval and adult diapauses in mosquitoes and probe the molecular and physiological distinctions that comprise the diapause phenotype. Diapause evokes diverse and unique attributes including behavioural changes, arrested development, enhanced stress tolerance, fat accumulation and suppressed metabolic rates. Like most insects, mosquitoes in temperate latitudes precisely monitor daylength as a cue for diapause initiation. We examine the role of circadian clock genes in this response and trace downstream hormonal pathways involved in the diapause response. Insulin and the FOXO transcription factor signalling pathways appear to be keys for generating the diapause phenotype in adult females of Culex pipiens and perhaps other species. Elucidating the molecular regulation of diapause-associated physiology may provide a basis to identify novel targets for the control of mosquito vectors.
When designing a case-control study to investigate differences in microbial composition, it is fundamental to assess the sample sizes needed to detect an hypothesized difference with sufficient statistical power. Our application includes power calculation for (i) a recoded version of the two-sample generalized Wald test of the ‘HMP’ R -package for comparing community composition, and (ii) the Wilcoxon-Mann-Whitney test for comparing operational taxonomic unit-specific abundances between two samples (optional). The simulation-based power calculations make use of the Dirichlet-Multinomial model to describe and generate abundances. The web interface allows for easy specification of sample and effect sizes. As an illustration of our application, we compared the statistical power of the two tests, with and without stratification of samples. We observed that statistical power increases considerably when stratification is employed, meaning that less samples are needed to detect the same effect size with the same power.
Availability and implementation : The web interface is written in R code using Shiny ( RStudio Inc., 2016 ) and it is available at https://fedematt.shinyapps.io/shinyMB . The R code for the recoded generalized Wald test can be found at https://github.com/mafed/msWaldHMP .
Contact:Federico.Mattiello@UGent.be
Lipid decomposition studies in frozen fish have led to the development of a simple and rapid method for the extraction and purification of lipids from biological materials. The entire procedure can be carried out in approximately 10 minutes; it is efficient, reproducible, and free from deleterious manipulations. The wet tissue is homogenized with a mixture of chloroform and methanol in such proportions that a miscible system is formed with the water in the tissue. Dilution with chloroform and water separates the homogenate into two layers, the chloroform layer containing all the lipids and the methanolic layer containing all the non-lipids. A purified lipid extract is obtained merely by isolating the chloroform layer. The method has been applied to fish muscle and may easily be adapted to use with other tissues.
Zooplankton, such as copepods, are highly abundant environmental reservoirs of many bacterial pathogens. Although copepods are known to support diverse and productive bacterial communities, little is understood about whether copepods are affected by bacterial attachment and whether they can regulate these associations through mechanisms such as the innate immune response. This thesis investigates the potential role that copepod physiology may play in regulating Vibrio association and the community structure of its microbiome. To this end, the intrinsic ability of oceanic copepod hosts to transcriptionally respond to mild stressors was first investigated. Specifically, the transcriptional regulation of several heat shock proteins (Hsps), a highly conserved superfamily of molecular chaperones, in the copepod Calanusfinmarchicus was examined and demonstrated that Hsps are a conserved element of the copepod's transcriptional response to stressful conditions and diapause regulation. To then investigate whether copepod hosts respond to and regulate their microbiota, the transcriptomic response of an estuarine copepod Eurytemora affinis to two distinct Vibric species, a free-living strain (V. ordalii 12B09) and a zooplankton specialist (V. sp. F10 9ZB36), was examined with RNA-Seq. Our findings provide evidence that the copepod E. affinis does distinctly recognize and respond to colonizing vibrios via transcriptional regulation of innate immune response elements and transcripts involved in maintaining cuticle integrity. Our work also suggests that association with E. affinis can significantly impact the physiology of Vibrio colonists. Finally, the inter-individual variability of the C.finmarchicus microbiome was examined to identify how specifically and predictably bacterial communities assemble on copepods and whether host physiology influences the bacterial community structure. Our findings suggest that copepods have a predictable "core microbiome" that persists throughout the host's entrance into diapause, a dormancy period characterized by dramatic physiological changes in the host. However, diapausing and active populations harbor distinct flexible microbiomes which may be driven by factors such including the copepod's feeding history, body size, and bacterial interactions. This thesis work highlights the role of copepods as dynamic reservoirs of diverse bacterial communities and implicates copepod host physiology as an important contributor to the activity, abundance, and community structure of its microbiome.
It is hard to assess experimentally the importance of microbial diversity in soil for the functioning of terrestrial ecosystems. An approach that is often used to make such assessment is the so-called dilution method. This method is based on the assumption that the biodiversity of the microbial community is reduced after dilution of a soil suspension and that the reduced diversity persists after incubation of more or less diluted inocula in soil. However, little is known how the communities develop in soil after inoculation. In this study serial dilutions of a soil suspension were made, and re-inoculated into the original soil previously sterilized by γ-irradiation. We determined the structure of the microbial communities in the suspensions and the inoculated soils using 454-pyrosequencing of 16S rDNA. Upon dilution, several diversity indices showed that, indeed, the diversity of the bacterial communities in the suspensions reduced dramatically, with Proteobacteria as the dominant phylum of bacteria detected in all dilutions. The structure of the microbial community was changed considerably in soil with Proteobacteria, Bacteroidetes and Verrucomicrobia as the dominant groups in most diluted samples, indicating the importance of soil related mechanisms operating in the assembly of the communities. We found unique OTUs even in the highest dilution both in the suspensions and in the incubated soil samples. We conclude that the dilution approach does allow for reducing the diversity of microbial communities in soil samples, but it does not allow for accurate predictions on the community assemblage during incubation of (diluted) suspensions in soil.
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Ten years ago next-generation sequencing (NGS) technologies appeared on the market. During the past decade, tremendous progress has been made in terms of speed, read length, and throughput, along with a sharp reduction in per-base cost. Together, these advances democratized NGS and paved the way for the development of a large number of novel NGS applications in basic science as well as in translational research areas such as clinical diagnostics, agrigenomics, and forensic science. Here we provide an overview of the evolution of NGS and discuss the most significant improvements in sequencing technologies and library preparation protocols. We also explore the current landscape of NGS applications and provide a perspective for future developments.
Significance
Factors influencing Wolbachia transfer into new species remain poorly understood. This is important as Wolbachia can influence speciation and is being developed as a novel arthropod-borne disease control approach. We show the native microbiota of Anopheles impede vertical transmission of Wolbachia . Antibiotic microbiome perturbation enables Wolbachia transmission in two Anopheles species. Mosquitoes with altered microbiomes do not exhibit blood meal-induced mortality associated with Wolbachia infection, suggesting that mosquitoes are killed by interactions between Wolbachia and other bacteria present in the mosquito. We identified Asaia as the bacterium responsible for inhibiting Wolbachia transmission, and partially responsible for blood meal-induced mortality. These results suggest that microbial interactions profoundly affect the host, and that microbiome incompatibility may influence distribution of Wolbachia in arthropods.