Testing host‐associated differentiation in a quasi‐endophage and a parthenogen on native trees

Host‐associated differentiation (HAD) is the formation of genetically divergent host‐associated sub‐populations. Evidence of HAD has been reported for multiple insect herbivores to date, but published studies testing more than one herbivore for any given host‐plant species pair is limited to herbivores on goldenrods. This limits the number of pair‐wise comparisons that can be made about insect life‐history traits that might facilitate or inhibit host‐race development in general. Two traits previously proposed to facilitate HAD include endophagy and parthenogenesis. We tested for HAD in two herbivores, a quasi‐endophagous caterpillar and a parthenogenetic aphid, feeding on two closely related species of hickories. We found that the quasi‐endophage is panmictic, whereas the parthenogen exhibits HAD on their sympatric host plants, pecan and water hickory, at a geographic mesoscale. This is an important first step in the characterization of HAD in multiple insect herbivores using North American hickories, a host‐plant system with many shared parthenogens.     

Exploring host‐associated differentiation in the North American native cranberry fruitworm,Acrobasis vaccinii,from blueberries and cranberries

The factors explaining host‐associated differentiation (HAD) have not yet been fully characterized, especially in agricultural systems. It is thought that certain characteristics within a system may increase the probability for HAD to occur. These characteristics include relatively long‐standing evolutionary relationships between insects and their host plants, endophagy, and allochrony in host‐plant phenologies. We assessed the status of these characteristics as well as the presence of HAD in the cranberry fruitworm, Acrobasis vaccinii Riley (Lepidoptera: Pyralidae), a pest associated with blueberry and cranberry in eastern North America. We reveal the occurrence of two distinct populations of A. vaccinii that are allochronically isolated by the phenological stage of their respective host plants (cranberries or blueberries). Laboratory‐reared A. vaccinii adults collected from blueberries emerge at least 1 week earlier than adults from cranberries and the antennal sensitivity of adults to host‐plant volatiles differs between A. vaccinii collected from blueberry and cranberry. Despite finding characteristics indicative of HAD, we did not detect a genetic signature of HAD in A. vaccinii. These findings suggest that HAD may occur through behavioral and phenological mechanisms before there is sufficient genetic variation to be detected.     

Host‐associated differentiation in a highly polyphagous,sexually reproducing insect herbivore

Insect herbivores may undergo genetic divergence on their host plants through host‐associated differentiation (HAD). Much of what we know about HAD involves insect species with narrow host ranges (i.e., specialists) that spend part or all their life cycle inside their hosts, and/or reproduce asexually (e.g., parthenogenetic insects), all of which are thought to facilitate HAD. However, sexually reproducing polyphagous insects can also exhibit HAD. Few sexually reproducing insects have been tested for HAD, and when they have insects from only a handful of potential host‐plant populations have been tested, making it difficult to predict how common HAD is when one considers the entire species' host range. This question is particularly relevant when considering insect pests, as host‐associated populations may differ in traits relevant to their control. Here, we tested for HAD in a cotton (Gossypium hirsutum) pest, the cotton fleahopper (CFH) (Pseudatomoscelis seriatus), a sexually reproducing, highly polyphagous hemipteran insect. A previous study detected one incidence of HAD among three of its host plants. We used Amplified fragment length polymorphism (AFLP) markers to assess HAD in CFH collected from an expanded array of 13 host‐plant species belonging to seven families. Overall, four genetically distinct populations were found. One genetically distinct genotype was exclusively associated with one of the host‐plant species while the other three were observed across more than one host‐plant species. The relatively low degree of HAD in CFH compared to the pea aphid, another hemipteran insect, stresses the likely importance of sexual recombination as a factor increasing the likelihood of HAD.     

Influence of visual cues on host‐searching and learning behaviour of the egg parasitoids Telenomus podisi and Trissolcus basalis

Insect parasitoids use a variety of chemical and physical cues when foraging for hosts and food. Parasitoids can learn cues that lead them to the hosts, thus contributing to better foraging. One of the cues that influence host‐searching behaviour could be colour. In this study, we investigated the ability of females of the parasitoid wasps Telenomus podisi Ashmead and Trissolcus basalis Wollaston (both Hymenoptera: Scelionidae) to respond to colours and to associate the presence of hosts – eggs of Euschistus heros (Fabricius) (Hemiptera: Pentatomidae) – with coloured substrates after training (associative learning). Two sets of experiments were conducted: in one the innate preference for substrate colours was examined, in the other associative learning of substrate colour and host presence was tested in multiple‐choice and dual‐choice experiments. In the associative learning experiments, Te. podisi and Tr. basalis were trained to respond to differently coloured substrates containing hosts in two sessions of 2 h each, with 1‐h intervals. In multiple‐choice experiments, the wasps displayed innate preference for yellow substrates over green, brown, black, or white ones. Even after being trained on substrates of different colours, both parasitoids continued to show preference for yellow substrates. The response to the colours of substrates of both parasitoids was related with the orientation to the plant foliage during the search for hosts.     

Host‐associated genetic differentiation in pecan leaf phylloxera

Host‐associated differentiation (HAD) is the formation of genetically distinct host‐associated populations. One of the genotypic signatures of HAD is that populations exhibit stronger differentiation by host‐plant species than by geographic isolation. HAD, as a mechanism promoting ecological speciation, has been invoked to explain phytophagous insect diversity. Two traits proposed to promote HAD are endophagy and parthenogenesis. Using amplified fragment length polymorphisms (AFLPs), we tested for the presence of HAD in pecan leaf phylloxera, Phylloxera notabilis Pergande (Hemiptera: Phylloxeridae), an endophagous, gall inducing, and cyclically parthenogenetic insect on sympatric pecan and water hickory at a geographic mesoscale. This species shows strong HAD. Whereas the effect of collecting site was significant, accounting for 7.3% of molecular variation, host‐plant species identity accounted for 63.5%. In addition, a choice test indicated that pecan leaf phylloxera originating from water hickory showed weak but significant preference for leaflets of the natal host, whereas pecan leaf phylloxera originating from pecan did not. This is the first such study of a species of arboreal Phylloxeridae, a poorly known insect group. This is also the first endophage and the second parthenogen shared by these two hickory species to show evidence of HAD. This hickory system could be a good parthenogen‐rich counterpoint to the goldenrod system in the study of HAD in insect communities.     

Host‐associated differentiation in a pecan and water hickory Aphidomorpha community

Host‐associated differentiation (HAD) is the formation of genetically distinct, host‐associated populations created and maintained by ecologically mediated reproductive isolation. HAD potentially accounts for species origins in parasites, including herbivorous insects. Although case studies testing the occurrence of HAD are accumulating, it is still unclear how common HAD is and which specific ecological traits explain its occurrence. To address these issues, studies are needed that include negative results (i.e., instances in which parasite populations do not exhibit HAD) and test for HAD across communities (i.e., several parasite species on the same set of host species). In this study, HAD was tested in a community of six species of Aphidomorpha (Hemiptera) that share a host‐plant pair: pecan [Carya illinoinensis (Wangenh.) K.Koch] and water hickory [Carya aquatica (F.Michx) Nutt., both Juglandaceae] trees. All six species are parthenogenetic and three species are endophagous, traits that can exacerbate host‐specific selection. AFLP markers were employed to detect the presence of genetically distinct, host‐associated populations for each insect species. Strict HAD (i.e., the occurrence of genetically distinct pecan‐associated and water hickory‐associated genotypes) was found in Phylloxera notabilis Pergande (Phylloxeridae), Phylloxera devastatrix Pergande, and Monelliopsis pecanis Bissel (Aphididae). Monellia caryella Fitch (Aphididae) displayed a pattern of partial HAD (i.e., the occurrence of only a genetically distinct pecan‐associated genotype). No HAD was found in Melanocallis caryaefoliae Davis (Aphididae) or Phylloxera texana Stoetzel. The pattern of HAD occurrence in the pecan and water hickory Aphidomorpha community indicated that neither parthenogenesis nor endophagy sufficiently explain the occurrence of HAD in this system.     

Effects of quantitative and qualitative differences in volatiles from host‐ and non‐host‐infested maize on the attraction of the larval parasitoid Cotesia kariyai

Cotesia kariyai Watanabe (Hymenoptera: Braconidae) is a specialist larval parasitoid of Mythimna separata Walker (Lepidoptera: Noctuidae). Cotesia kariyai wasps use herbivore‐induced plant volatiles (HIPVs) to locate hosts. However, complex natural habitats are full of volatiles released by both herbivorous host‐ and non‐host‐infested plants at various levels of intensity. Therefore, the presence of non‐hosts may affect parasitoid decisions while foraging. Here, the host‐finding efficiency of naive C. kariyai from HIPVs influenced by host‐ and non‐host‐infested maize [Zea mays L. (Poaceae)] plants was investigated with a four‐arm olfactometer. Ostrinia furnacalis Guenée (Lepidoptera: Crambidae) was selected as a non‐host species. One unit (1 U) of host‐ or non‐host‐infested plant was prepared by infesting a potted plant with five host or seven non‐host larvae. In two‐choice bioassays, host‐infested plants fed upon by different numbers of larvae, and various units of host‐ and non‐host‐infested plants (infestation units; 1 U, 2 U, and 3 U) were arranged to examine the effects of differences in volatile quantity and quality on the olfactory responses of C. kariyai with the assumption that volatile quantity and quality changes with differences in numbers of insects and plants. Cotesia kariyai was found to perceive quantitative differences in volatiles from host‐infested plants, preferring larger quantities of volatiles from larger numbers of larvae or plants. Also, the parasitoids discriminated between healthy plants, host‐infested plants, and non‐host‐infested plants by recognising volatiles released from those plants. Cotesia kariyai showed a reduced preference for host‐induced volatiles, when larger numbers of non‐host‐infested plants were present. Therefore, quantitative and qualitative differences in volatiles from host‐ and non‐host‐infested plants appear to affect the decision of C. kariyai during host‐habitat searching in multiple tritrophic systems.     

Plant‐mediated interactions between two herbivores differentially affect a subsequently arriving third herbivore in populations of wild cabbage

• Plants are part of biodiverse communities and frequently suffer from attack by multiple herbivorous insects. Plant responses to these herbivores are specific for insect feeding guilds: aphids and caterpillars induce different plant phenotypes. Moreover, plants respond differentially to single or dual herbivory, which may cascade into a chain of interactions in terms of resistance to other community members. Whether differential responses to single or dual herbivory have consequences for plant resistance to yet a third herbivore is unknown.
• We assessed the effects of single or dual herbivory by Brevicoryne brassicae aphids and/or Plutella xylostella caterpillars on resistance of plants from three natural populations of wild cabbage to feeding by caterpillars of Mamestra brassicae. We measured plant gene expression and phytohormone concentrations to illustrate mechanisms involved in induced responses.
• Performance of both B. brassicae and P. xylostella was reduced when feeding simultaneously with the other herbivore, compared to feeding alone. Gene expression and phytohormone concentrations in plants exposed to dual herbivory were different from those found in plants exposed to herbivory by either insect alone. Plants previously induced by both P. xylostella and B. brassicae negatively affected growth of the subsequently arriving M. brassicae. Furthermore, induced responses varied between wild cabbage populations.
• Feeding by multiple herbivores differentially activates plant defences, which has plant‐mediated negative consequences for a subsequently arriving herbivore. Plant population‐specific responses suggest that plant populations adapt to the specific communities of insect herbivores. Our study contributes to the understanding of plant defence plasticity in response to multiple insect attacks.

     

Effect of fertilized,unfertilized, and UV‐irradiated hosts on parasitism and suitability for Trichogramma parasitoids

Trichogramma spp. (Hymenoptera: Trichogrammatidae) parasitoids have been commonly used as biological control agents in insect pest management. Host quality is believed to influence parasitism, host preference, and suitability for parasitoids. To date, limited studies have compared the parasitism of Trichogramma parasitoids on fertilized, unfertilized, and sterilized host eggs. Hence, we studied the performance of three Trichogramma egg parasitoids, Trichogramma japonicum Ashmead, Trichogramma chilonis Ishii, and Trichogramma leucaniae Pang & Chen, on fertilized, unfertilized, and ultraviolet (UV)‐irradiated fertilized (UVF) eggs of rice moth, Corcyra cephalonica (Stainton) (Lepidoptera: Pyralidae). In a no‐choice test, T. japonicum and T. leucaniae parasitized significantly more fertilized or UVF than unfertilized hosts, and T. chilonis parasitized significantly more UVF than either fertilized or unfertilized hosts. In a choice test, all three Trichogramma parasitoids parasitized UVF hosts the most, and unfertilized hosts the least. There were similar percentages of adult emergence and female progeny among fertilized, unfertilized, and UVF hosts for all three Trichogramma parasitoids, except that T. japonicum had significantly lower adult emergence on fertilized hosts. We also found that all three Trichogramma parasitoids developed slower on unfertilized hosts. Regardless of host treatments, T. leucaniae had the longest developmental time and T. chilonis had the shortest. We conclude that Trichogramma parasitoids prefer parasitizing UVF eggs of C. cephalonica without negative effects on their emergence and sex allocation.     

Cannibalism of parasitoid‐attacked conspecifics in a non‐carnivorous caterpillar

Cannibalism, the killing and consumption of conspecifics, can even occur in insect species typically considered to be non‐carnivorous. Of particular interest is the cannibalism of parasitoid‐attacked conspecifics, which could reduce parasitism levels in subsequent generations for that conspecific population. This study reports on the occurrence and some of the consequences of cannibalism in parasitoid‐attacked obliquebanded leafroller, Choristoneura rosaceana (Harris) (Lepidoptera: Tortricidae). We show that larvae of C. rosaceana, which is considered to be an herbivorous caterpillar species, did not prey upon live conspecifics, but readily consumed conspecifics attacked by Habrobracon gelechiae Ashmead (Hymenoptera: Braconidae). Further examination found that C. rosaceana larvae feeding on parasitoid‐attacked conspecifics, since their fourth instar, suffered a higher mortality and reduction in body size than those fed on plant material only. The cannibalism of attacked conspecifics did not appear to offer any nutrient benefits for the cannibal. To our best knowledge, this is the first empirical example of the occurrence and some of the consequences of cannibalism by a non‐carnivorous insect on its parasitoid‐attacked conspecifics. We discuss the adaptive significance of such cannibalism on parasitoid‐attacked conspecifics with respect to a trans‐generational fitness gain for the population through the killing of the parasitoids, thereby reducing parasitism in subsequent generations.     

Changes in structure of over‐ and midstory tree species in a Mediterranean‐type forest after an extreme drought‐associated heatwave

Worldwide, extreme climatic events such as drought and heatwaves are associated with forest mortality. However, the precise drivers of tree mortality at individual and stand levels vary considerably, with substantial gaps in knowledge across studies in biomes and continents. In 2010–2011, a drought‐associated heatwave occurred in south‐western Australia and drove sudden and rapid forest canopy collapse. Working in the Northern Jarrah (Eucalyptus marginata) Forest, we quantified the response of key overstory (E. marginata, Corymbia calophylla) and midstory (Banksia grandis, Allocasuarina fraseriana) tree species to the extreme climate event. Using transects spanning a gradient of drought impacts (minimal (50–100 m), transitional (100–150 m) and severe (30–60 m)), tree species mortality in relation to stand characteristics (stand basal area and stem density) and edaphic factors (soil depth) was determined. We show differential mortality between the two overstory species and the two midstory species corresponding to the drought‐associated heatwave. The dominant overstory species, E. marginata, had significantly higher mortality (~19%) than C. calophylla (~7%) in the severe zone. The midstory species, B. grandis, demonstrated substantially higher mortality (~59%) than A. fraseriana (~4%) in the transitional zone. Banksia grandis exhibited a substantial shift in structure in response to the drought‐associated heatwave in relation to tree size, basal area and soil depth. This study illustrates the role of climate extremes in driving ecosystem change and highlights the critical need to identify and quantify the resulting impact to help predict future forest die‐off events and to underpin forest management and conservation.     

Fitness‐related offspring sex allocation of Anastatus disparis,a gypsy moth egg parasitoid,on different‐sized host species

Anastatus disparis (Ruschka) (Hymenoptera: Eupelmidae) is an egg parasitoid and considered a potential biological control agent of the gypsy moth, Lymantria dispar (L.) (Lepidoptera: Lymantriidae). Only male offspring of A. disparis emerge from single eggs of L. dispar in the laboratory, and A. disparis exhibits low parasitism on L. dispar in the field. We therefore selected several lepidopteran species with various body sizes to evaluate the optimal egg size for hosting A. disparis. In addition, we explored whether the nutritional content of a single L. dispar egg influences the sex of A. disparis offspring and why female offspring can be reared from L. dispar eggs in the field. The results indicated that host egg size decisively influenced the body size and sex ratio of the parasitoid offspring. Therefore, larger hosts, especially the largest eggs of Antheraea pernyi Guérin‐Méneville (Saturniidae), might increase the fitness of A. disparis females. Lymantria dispar eggs concealed in the larger egg shell of A. pernyi produced female A. disparis, suggesting that adult A. disparis should prefer hosts with larger bodies and that the nutritional content of L. dispar eggs did not play a decisive role in the sex allocation of A. disparis. The results also indicated that the egg mass and the fur cover of L. dispar egg masses might be the key factors inducing female A. disparis to lay female offspring in L. dispar eggs.     

Growth,development, and behaviour of the parasitised and unparasitised larvae of a shelter‐building moth and consequences for the resulting koinobiont parasitoid

Most attention to size‐time trade‐offs of insects has focused on herbivore risk, with considerably less attention paid to parasitoids. Here, we focus on parasitoid risk, comparing the fates of unparasitised herbivore hosts and parasitised hosts that protect the parasitoids. Success of a koinobiont parasitoid (host grows after parasitisation) depends on maintaining a delicate balance with its host, thereby ensuring its own survival while the host grows. To evaluate growth rate–mortality rate relationships of host and parasitoid, we compared several aspects of the growth, phenology, and behaviour of unparasitised fern moth [Herpetogramma theseusalis (Walker) (Lepidoptera: Crambidae)] larvae and larvae parasitised by Alabagrus texanus (Cresson) (Hymenoptera: Braconidae), a solitary koinobiont (one parasitoid per host) wasp. Host larvae feed and construct shelters on sensitive fern, Onoclea sensibilis L. (Dryopteridaceae). Alabagrus texanus parasitise early‐instar moths in late summer, which overwinter in their host, emerging in mid‐summer to pupate and eclose. During the autumn following hatching and the immediately following spring, parasitised and unparasitised moth larvae did not differ in size, took similar time to choose between satisfactory and unsatisfactory foods, and built similar shelters. Prior to any other changes noted, more parasitised than unparasitised larvae also died when severely starved. Parasitised larvae subsequently grew less and pupated later than unparasitised ones (small size, slow growth), but consumed similar amounts of food. Although the numerically dominant parasitoid of fern moths, we concluded that A. texanus do not efficiently exploit their hosts.     

A novel approach to parasite population genetics: Experimental infection reveals geographic differentiation,recombination and host‐mediated population structure in Pasteuria ramosa,a bacterial parasite of Daphnia

The population structure of parasites is central to the ecology and evolution of host‐parasite systems. Here, we investigate the population genetics of Pasteuria ramosa, a bacterial parasite of Daphnia. We used natural P. ramosa spore banks from the sediments of two geographically well‐separated ponds to experimentally infect a panel of Daphnia magna host clones whose resistance phenotypes were previously known. In this way, we were able to assess the population structure of P. ramosa based on geography, host resistance phenotype and host genotype. Overall, genetic diversity of P. ramosa was high, and nearly all infected D. magna hosted more than one parasite haplotype. On the basis of the observation of recombinant haplotypes and relatively low levels of linkage disequilibrium, we conclude that P. ramosa engages in substantial recombination. Isolates were strongly differentiated by pond, indicating that gene flow is spatially restricted. Pasteuria ramosa isolates within one pond were segregated completely based on the resistance phenotype of the host—a result that, to our knowledge, has not been previously reported for a nonhuman parasite. To assess the comparability of experimental infections with natural P. ramosa isolates, we examined the population structure of naturally infected D. magna native to one of the two source ponds. We found that experimental and natural infections of the same host resistance phenotype from the same source pond were indistinguishable, indicating that experimental infections provide a means to representatively sample the diversity of P. ramosa while reducing the sampling bias often associated with studies of parasite epidemics. These results expand our knowledge of this model parasite, provide important context for the large existing body of research on this system and will guide the design of future studies of this host‐parasite system.     

The role of local ecology during hybridization at the initial stages of ecological speciation in a marine snail

Hybrid zones of ecologically divergent populations are ideal systems to study the interaction between natural selection and gene flow during the initial stages of speciation. Here, we perform an amplified fragment length polymorphism (AFLP) genome scan in parallel hybrid zones between divergent ecotypes of the marine snail Littorina saxatilis, which is considered a model case for the study of ecological speciation. Ridged‐Banded (RB) and Smooth‐Unbanded (SU) ecotypes are adapted to different shore levels and microhabitats, although they present a sympatric distribution at the mid‐shore where they meet and mate (partially assortatively). We used shell morphology, outlier and nonoutlier AFLP loci from RB, SU and hybrid specimens captured in sympatry to determine the level of phenotypic and genetic introgression. We found different levels of introgression at parallel hybrid zones and nonoutlier loci showed more gene flow with greater phenotypic introgression. These results were independent from the phylogeography of the studied populations, but not from the local ecological conditions. Genetic variation at outlier loci was highly correlated with phenotypic variation. In addition, we used the relationship between genetic and phenotypic variation to estimate the heritability of morphological traits and to identify potential Quantitative Trait Loci to be confirmed in future crosses. These results suggest that ecology (exogenous selection) plays an important role in this hybrid zone. Thus, ecologically based divergent natural selection is responsible, simultaneously, for both ecotype divergence and hybridization. On the other hand, genetic introgression occurs only at neutral loci (nonoutliers). In the future, genome‐wide studies and controlled crosses would give more valuable information about this process of speciation in the face of gene flow.     

Limber pine (Pinus flexilis James) genetic map constructed by exome‐seq provides insight into the evolution of disease resistance and a genomic resource for genomics‐based breeding

Limber pine ( Pinus flexilis ) is a keystone species of high‐elevation forest ecosystems of western North America, but some parts of the geographic range have high infection and mortality from the non‐native white pine blister rust caused by Cronartium ribicola . Genetic maps can provide essential knowledge for understanding genetic disease resistance as well as local adaptation to changing climates. Exome‐seq was performed to construct high‐density genetic maps in two seed families. Composite maps positioned 9612 unigenes across 12 linkage groups ( LG s). Syntenic analysis of genome structure revealed that the majority of orthologs were positional orthologous genes ( POG s) with localization on homologous LG s among conifer species. Gene ontology ( GO) enrichment analysis showed relatively fewer constraints for POG s with putative roles in adaptation to environments and relatively more conservation for POG s with roles in basic cell function and maintenance. The mapped genes included 639 nucleotide‐binding site leucine‐rich repeat genes ( NBS ‐ LRR s) , 290 receptor‐like protein kinase genes ( RLK s), and 1014 genes with potential roles in the defense response and induced systemic resistance to attack by pathogens. Orthologous loci for resistance to rust pathogens were identified and were co‐positioned with multiple members of the R gene family, revealing the evolutionary pressure acting upon them. This high‐density genetic map provides a genomic resource and practical tool for breeding and genetic conservation programs, with applications in genome‐wide association studies ( GWASs ), the characterization of functional genes underlying complex traits, and the sequencing and assembly of the full‐length genomes of limber pine and related Pinus species.     

Landscape‐scale genetic differentiation of a mycangial fungus associated with the ambrosia beetle,Xylosandrus germanus (Blandford) (Curculionidae:Scolytinae) in Japan

In this study, we examined the genetic structures of the ambrosia fungus isolated from mycangia of the scolytine beetle, Xylosandrus germanus to understand their co‐evolutionary relationships. We analyzed datasets of three ambrosia fungus loci (18S rDNA, 28S rDNA, and the β‐tubulin gene) and a X. germanus locus dataset (cytochrome c oxidase subunit 1 (COI) mitochondrial DNA). The ambrosia fungi were separated into three cultural morphptypes, and their haplotypes were distinguished by phylogenetic analysis on the basis of the three loci. The COI phylogenetic analysis revealed three distinct genetic lineages (clades A, B, and C) within X. germanus, each of which corresponded to specific ambrosia fungus cultural morphptypes. The fungal symbiont phylogeny was not concordant with that of the beetle. Our results suggest that X. germanus may be unable to exchange its mycangial fungi, but extraordinary horizontal transmission of symbiotic fungi between the beetle's lineages occurred at least once during the evolutionary history of this symbiosis.     

Identification of C‐terminal Hsp70‐interacting protein as a mediator of tumour necrosis factor action in osteoblast differentiation by targeting osterix for degradation

In patients with inflammatory arthritis, tumour necrosis factor (TNF)‐α are overproduced in inflamed joints. This leads to local erosion of cartilage and bone, periarticular osteopenia, as well as osteoporosis. But less is known regarding the molecular mechanisms that mediate the effect of TNF‐α on osteoblast function. The purpose of this study was to test that C terminus of Hsc70‐interacting protein (CHIP) has a specific role in suppressing the osteogenic activity of osteoblasts under inflammatory conditions. C2C12, MC3T3‐E1 and HEK293T cell lines were cultured and cotransfected with related plasmids. After transfection, the cells were cultured further in the presence or absence of murine TNF‐α and subjected to real time RT‐PCR, Western blot, Ubiquitination assay, Co‐immunoprecipitation, Luciferase reporter assay, Small interfering RNAs and Mineralization assay. The expression levels of TNF‐α‐induced CHIP and Osx were examined by RT‐PCR and Western blot analysis. Co‐immunoprecipitation and ubiquitination assays revealed ubiquitinated Osx, confirmed that CHIP indeed interacted with Osx and identified K55 and K386 residues as the ubiquitination sites in Osx, Luciferase reporter assay and Small interfering RNAs examined whether TNF‐α target the bone morphogenetic protein signalling through CHIP. We established stable cell lines with the overexpression of HA‐CHIP, Mineralization assay and CHIP siRNA demonstrated the important roles of CHIP on osteoblast function in conditions in which TNF‐α is overexpressed. We found that the K55 and K386 residues are ubiquitination site(s) in Osx, and that TNF‐α inhibits osteoblast differentiation by promoting Osx degradation through up‐regulation of E3 ubiquitin ligase CHIP in osteoblast. Thus, CHIP targets Osx for ubiquitination and degradation in osteoblasts after chronic exposure to TNF‐α, and inhibition of CHIP expression in osteoblasts may be a new mechanism to limit inflammation‐mediated osteoporosis by promoting their differentiation into osteoblasts.