Role of the ESCRT‐III complex in controlling integrity of the Salmonella‐containing vacuole

Intracellular pathogens need to establish specialised niches for survival and proliferation in host cells. The enteropathogen Salmonella enterica accomplishes this by extensive reorganisation of the host endosomal system deploying the SPI2‐encoded type III secretion system (SPI2‐T3SS). Fusion events of endosomal compartments with the Salmonella‐containing vacuole (SCV) form elaborate membrane networks within host cells enabling intracellular nutrition. However, which host compartments exactly are involved in this process and how the integrity of Salmonella‐modified membranes is accomplished are not fully resolved. An RNA interference knockdown screen of host factors involved in cellular logistics identified the ESCRT (endosomal sorting complex required for transport) system as important for proper formation and integrity of the SCV in infected epithelial cells. We demonstrate that subunits of the ESCRT‐III complex are specifically recruited to the SCV and membrane network. To investigate the role of ESCRT‐III for the intracellular lifestyle of Salmonella, a CHMP3 knockout cell line was generated. Infected CHMP3 knockout cells formed amorphous, bulky SCV. Salmonella within these amorphous SCV were in contact with host cell cytosol, and the attenuation of an SPI2‐T3SS‐deficient mutant strain was partially abrogated. ESCRT‐dependent endolysosomal repair mechanisms have recently been described for other intracellular pathogens, and we hypothesise that minor damages of the SCV during bacterial proliferation are repaired by the action of ESCRT‐III recruitment in Salmonella‐infected host cells.     

Revealing interaction between sulfobutylether‐β‐cyclodextrin and reserpine by chemiluminescence and site‐directed molecular docking

The host–guest interaction between sulfobutylether‐β‐cyclodextrin (SBE‐β‐CD) and reserpine (RSP) is described using flow injection‐chemiluminescence (FI‐CL) and site‐directed molecular docking methods. It was found that RSP could inhibit the CL intensity produced by a luminol/SBE‐β‐CD system. The decrease in CL intensity was logarithmic over an RSP concentration range of 0.03 to 700.0 nM, giving a regression equation of ?I = 107.1lgC_RES + 186.1 with a detection limit of 10 pM (3σ). The CL assay was successfully applied in the determination of RSP in injection, saliva and urine samples with recoveries in the range 93.5–106.1%. Using the proposed CL model, the binding constant (K_CD‐R) and the stoichiometric ratio of SBE‐β‐CD/RSP were calculated to be 7.4 × 10⁶ M^‐1 and 1 : 1, respectively. Using molecular docking, it was confirmed that luminol binds to the small cavity of SBE‐β‐CD with a nonpolar interaction, while RSP targeted the larger cavity of SBE‐β‐CD and formed a 1 : 1 complex with hydrogen bonds. The proposed new CL method has the potential to become a powerful tool for revealing the host–guest interaction between CDs and drugs, as well as monitoring drugs with high sensitivity. Copyright © 2013 John Wiley & Sons, Ltd.     

Host‐specific variation in off‐host performance of a temperate ectoparasite

Antagonistic host–parasite interactions are rarely considered from an ecological perspective of the parasite. We used a blood‐feeding ectoparasite of boreal cervids, the deer ked (Lipoptena cervi L., Hippoboscidae), to study host‐dependent variation in a parasite's ability to cope with an abiotic environment during the free‐living stage(s) in two allopatric Fennoscandian populations. We found that a strongly host‐specific deer ked population in eastern Fennoscandia, exploiting only moose (Alces alces), produced the largest offspring that were the most cold‐tolerant and emerged the earliest as adults, when compared with the western Fennoscandian population that exploited two hosts efficiently. Within the western population, however, offspring produced on roe deer (Capreolus capreolus) were significantly larger, more cold‐tolerant, and had higher survival than those produced on moose in the same area. We discuss potential causes for both host‐specific and geographical differences in off‐host performance: (1) maternal host directly affects the offspring survival prospects; (2) divergent co‐evolution with local main host(s) has shaped the parasite's life history; and/or (3) off‐host performance is shaped by adaptation to the local abiotic environment. In conclusion, this study increases our understanding of the evolution of host–parasite interactions by demonstrating how geographical differences in host exploitation may result in differences in survival prospects outside the host.     

Feeding responses of an oligophagous bean aphid, Megoura crassicauda, to primary and secondary substances in Vicia angustifolia

The feeding behaviour of the aphid Megoura crassicauda Mordivilko (Homoptera: Aphididae), which feeds selectively on plants in the genus Vicia (Fabaceae), was studied. The aphids deposited proteinaceous stylet sheaths intercellularly towards the phloem tissues of host plants. Similar stylet sheaths were formed on a Parafilm membrane when host‐specific acylated flavonoid glycosides [two 2″‐O‐(E)‐p‐coumaroyl esters of quercetin 3‐O‐diglycosides] present in the extracts of the narrow vetch, Vicia angustifolia L., were supplied in the solution covered by the membrane. In contrast, their corresponding deacyl analogues, present more abundantly in the host plant tissues, were not stimulatory, which suggested specificity in the structural requirements of the probing stimulants. While the aphids imbibed an artificial diet composed of primary nutrients (e.g., sucrose and amino acids) and produced a large quantity of honeydew, acylated flavonoids alone and non‐acylated flavonoids supplied with the nutrients more or less suppressed honeydew production. These findings implied that the acylated flavonoids serve as a cue to navigate the stylet sheath towards the phloem prior to sap‐sucking, whereas non‐acylated flavonoids may serve as a negative stimulus to refrain from sucking during tissue penetration before tapping the phloem, although the distribution of these compounds in the plant tissues remains unknown. Thus, the feeding behaviour of M. crassicauda appears to be controlled by multiple chemical stimuli in the process of the settling on its host plant.     

The Actinomyces oris type 2 fimbrial shaft FimA mediates co‐aggregation with oral streptococci,adherence to red blood cells and biofilm development

Interbacterial interactions between oral streptococci and actinomyces and their adherence to tooth surface and the associated host cells are key early events that promote development of the complex oral biofilm referred to as dental plaque. These interactions depend largely on a lectin‐like activity associated with the Actinomyces oris type 2 fimbria, a surface structure assembled by sortase (SrtC2)‐dependent polymerization of the shaft and tip fimbrillins, FimA and FimB respectively. To dissect the function of specific fimbrillins in various adherence processes, we have developed a convenient new technology for generating unmarked deletion mutants of A. oris. Here, we show that the fimB mutant, which produced type 2 fimbriae composed only of FimA, like the wild type co‐aggregated strongly with receptor‐bearing streptococci, agglutinated with sialidase‐treated red blood cells, and formed monospecies biofilm. In contrast, the fimA and srtC2 mutants lacked type 2 fimbriae and were non‐adherent in each of these assays. Plasmid‐based expression of the deleted gene in respective mutants restored adherence to wild‐type levels. These findings uncover the importance of the lectin‐like activity of the polymeric FimA shaft rather than the tip. The multivalent adhesive function of FimA makes it an ideal molecule for exploring novel intervention strategies to control plaque biofilm formation.     

A molecular tool to identify Anastatus parasitoids of the brown marmorated stink bug

Globally, Anastatus species (Hymenoptera: Eupelmidae) are associated with the invasive agricultural pest Halyomorpha halys (Stål) (Hemiptera: Pentatomidae). In Europe, the polyphagous Anastatus bifasciatus (Geoffroy) is the most prevalent native egg parasitoid on H. halys eggs and is currently being tested as a candidate for augmentative biological control. Anastatus bifasciatus frequently displays behavior without oviposition, and induces additional host mortality through oviposition damage and host feeding that is not measured with offspring emergence. This exacerbates accurate assessment of parasitism and host impact, which is crucial for efficacy evaluation as well as for pre‐ and post‐release risk assessment. To address this, a general Anastatus primer set amplifying a 318‐bp fragment within the barcoding region of the cytochrome oxidase I (COI) gene was developed. When challenged with DNA of three Anastatus species —A. bifasciatus, Anastatus japonicus Ashmead, and Anastatus sp.—, five scelionid parasitoid species that might be encountered in the same host environments and 11 pentatomid host species, only Anastatus DNA was successfully amplified. When applied to eggs of the target host, H. halys, and an exemplary non‐target host, Dendrolimus pini L. (Lepidoptera: Lasiocampidae), subjected to host feeding, no Anastatus amplicons were produced. Eggs of the two host species containing A. bifasciatus parasitoid stages, from 1‐h‐old eggs to pupae, and emerged eggs yielded Anastatus fragments. Confirmation of parasitoid presence with dissections and subsequent PCRs with the developed primer pair resulted in 95% success for 1‐h‐old parasitoid eggs. For both host species, field‐exposed sentinel emerged eggs stored dry for 6 months, 100% of the specimens produced Anastatus amplicons. This DNA‐based screening method can be used in combination with conventional methods to better interpret host‐parasitoid and parasitoid‐parasitoid interactions. It will help address ecological questions related to an environmentally friendly approach for the control of H. halys in invaded areas.     

Cellulose production,activated by cyclic di‐GMP through BcsA and BcsZ,is a virulence factor and an essential determinant of the three‐dimensional architectures of biofilms formed by Erwinia amylovora Ea1189

Bacterial biofilms are multicellular aggregates encased in an extracellular matrix mainly composed of exopolysaccharides (EPSs), protein and nucleic acids, which determines the architecture of the biofilm. Erwinia amylovora Ea1189 forms a biofilm inside the xylem of its host, which results in vessel plugging and water transport impairment. The production of the EPSs amylovoran and levan is critical for the formation of a mature biofilm. In addition, cyclic dimeric GMP (c‐di‐GMP) has been reported to positively regulate amylovoran biosynthesis and biofilm formation in E. amylovora Ea1189. In this study, we demonstrate that cellulose is synthesized by E. amylovora Ea1189 and is a major modulator of the three‐dimensional characteristics of biofilms formed by this bacterium, and also contributes to virulence during systemic host invasion. In addition, we demonstrate that the activation of cellulose biosynthesis in E. amylovora is a c‐di‐GMP‐dependent process, through allosteric binding to the cellulose catalytic subunit BcsA. We also report that the endoglucanase BcsZ is a key player in c‐di‐GMP activation of cellulose biosynthesis. Our results provide evidence of the complex composition of the extracellular matrix produced by E. amylovora and the implications of cellulose biosynthesis in shaping the architecture of the biofilm and in the expression of one of the main virulence phenotypes of this pathogen.     

Performance of Sclerodermus brevicornis,a parasitoid of invasive longhorn beetles,when reared on rice moth larvae

Biological control efficiency can be improved by developing effective mass‐rearing systems to produce large numbers of high‐quality parasitoids. This study explored an alternative host for rearing Sclerodermus brevicornis (Kieffer) (Hymenoptera: Bethylidae), a potential biocontrol agent for the suppression of exotic and invasive wood‐boring longhorn beetle (Coleoptera: Cerambycidae) populations in the European agroforestry ecosystems. We tested larvae of the rice moth, Corcyra cephalonica Stainton (Lepidoptera: Pyralidae), as host for the parasitoid. We quantified the probability and timing of host attack and parasitism as well as reproductive success, offspring production, and the characteristics of adult offspring. As S. brevicornis is a quasi‐social species (multiple females, communally produced offspring broods), we also explored the effects of varying the number of females to which individual hosts were presented, with the aim of determining the optimal female‐to‐host ratio. As time to host attack can be a limiting factor in S. brevicornis rearing protocols, we tested the use of adult females of another bethylid species, Goniozus legneri Gordh, to paralyse C. cephalonica larvae prior to presentation. We identified the conditions within our experiment that maximized offspring production per host and offspring production per adult female parasitoid. We found that C. cephalonica is suitable as a factitious host and, as it is considerably more straightforward for laboratory rearing than cerambycid species, it is a good candidate for adoption by future S. brevicornis mass‐rearing and release programmes.     

Serial replacement of a diatom endosymbiont in the marine dinoflagellate Peridinium quinquecorne (Peridiniales, Dinophyceae)

To infer the phylogeny of both the host and the endosymbiont of Peridinium quinquecorne Abé, the small subunit (SSU) ribosomal DNA (rDNA) from the host and two genes of endosymbiont origin (plastid‐encoded rbcL and nuclear‐encoded SSU rDNA) were determined. The phylogenetic analysis of the host revealed that the marine dinoflagellate P. quinquecorne formed a clade with other diatom‐harbouring dinoflagellates, including Kryptoperidinium foliaceum (Stein) Lindeman, Durinskia baltica (Levander) Carty et Cox and Galeidinium rugatum Tamura et Horiguchi, indicating a single endosymbiotic event for this lineage. Phylogenetic analyses of the endosymbiont in these organisms revealed that the endosymbiont of P. quinquecorne formed a clade with a centric diatom (SSU data indicated it to be closely related to Chaetoceros), whereas the endosymbionts of other three dinoflagellates formed a clade with a pennate diatom. The discrepancy between the host and the endosymbiont phylogenies suggests a secondary replacement of the endosymbiont from a pennate to a centric diatom in P. quinquecorne.     

Partial Proteome of the Corynetoxin‐Producing Gram‐Positive Bacterium,Rathayibacter toxicus

Rathayibacter toxicus is a Gram‐positive bacterium that is the causative agent of annual ryegrass toxicity (ARGT), a disease that causes devastating losses in the Australian livestock industry. R. toxicus exhibits a complex life cycle, using the nematode Anguina funesta as a physical vector to carry it up to the seed head of the host plant. ARGT is caused by a tunicamycin‐like corynetoxin that is produced in R. toxicus‐infected seed galls. We analyzed protein expression in R. toxicus under stationary growth phase conditions to obtain a more complete understanding of the biology of this organism and identify potential targets for immunoassay development. A total of 323 unique proteins were identified, including those with putative roles in secondary metabolism and pathogenicity. The proteome analysis for this complex phytopathogenic Gram‐positive bacterium will facilitate in the characterization of proteins necessary for host colonization and toxin production, and assist in the development of diagnostic assays. Data are available via ProteomeXchange with identifier PXD004238.     

Morphology,Molecular Phylogeny,and Ecology of Binucleata daphniae n. g., n. sp. (Fungi: Microsporidia), a Parasite of Daphnia magna Straus, 1820 (Crustacea: Branchiopoda)

ABSTRACT. We describe a new microsporidian species Binucleata daphniae, n. g., n. sp., that infects the integument cells lining the hemocoele cavity of the carapace and the postabdomen of the cladoceran Daphnia magna Straus. Infected cells filled with spores accumulate as large clusters in the carapace cavity and heavily infected hosts are detected by their opaque appearance. Despite the parasite's presence, infected Daphnia grow and molt, but have a reduced fecundity. During the parasite's life cycle, chain‐like meronts with isolated nuclei are formed, giving rise to binucleate presporonts, the most frequently observed, characteristic developmental stage. In sporogony, the nuclei of the presporont separate, divide, and eight spores enclosed in a thin‐walled sporophorous vesicle are formed. Spores are 4.9 × 2.5 μm in size (fresh) and have an anisofilar polar filament with eight coils. DNA sequence analysis places B. daphniae in a clade of microsporidians that parasitize crustaceans and mosquitoes and have assumed complex life cycles. Binucleata daphniae, however, has a simple and direct life cycle and can be transferred to naïve hosts and maintained as persistent infections in populations of its host D. magna. We propose that B. daphniae has simplified its life cycle by losing its secondary host, rendering it unique in this clade.     

Oligomeric states of the Shigella translocator protein IpaB provide structural insights into formation of the type III secretion translocon

The Shigella flexneri Type III secretion system (T3SS) senses contact with human intestinal cells and injects effector proteins that promote pathogen entry as the first step in causing life threatening bacillary dysentery (shigellosis). The Shigella Type III secretion apparatus (T3SA) consists of an anchoring basal body, an exposed needle, and a temporally assembled tip complex. Exposure to environmental small molecules recruits IpaB, the first hydrophobic translocator protein, to the maturing tip complex. IpaB then senses contact with a host cell membrane, forming the translocon pore through which effectors are delivered to the host cytoplasm. Within the bacterium, IpaB exists as a heterodimer with its chaperone IpgC; however, IpaB's structural state following secretion is unknown due to difficulties isolating stable protein. We have overcome this by coexpressing the IpaB/IpgC heterodimer and isolating IpaB by incubating the complex in mild detergents. Interestingly, preparation of IpaB with n‐octyl‐oligo‐oxyethylene (OPOE) results in the assembly of discrete oligomers while purification in N,N‐dimethyldodecylamine N‐oxide (LDAO) maintains IpaB as a monomer. In this study, we demonstrate that IpaB tetramers penetrate phospholipid membranes to allow a size‐dependent release of small molecules, suggesting the formation of discrete pores. Monomeric IpaB also interacts with liposomes but fails to disrupt them. From these and additional findings, we propose that IpaB can exist as a tetramer having inherent flexibility, which allows it to cooperatively interact with and insert into host cell membranes. This event may then lay the foundation for formation of the Shigella T3SS translocon pore.     

Resting complexes of the persistent yeast 20S RNA Narnavirus consist solely of the 20S RNA viral genome and its RNA polymerase p91

The positive strand 20S RNA narnavirus persistently infects Saccharomyces cerevisiae. The 20S RNA genome has a single gene that encodes the RNA‐dependent RNA polymerase (p91). 20S RNA forms ribonucleoprotein resting complexes (RNPs) with p91 and resides in the cytoplasm. Here we found no host proteins stoichiometrically associated with the RNP by pull‐down experiments. Furthermore, 20S RNA, when expressed from a vector in Escherichia coli, formed RNPs with p91 in the absence of yeast proteins. This interaction required the 3′ cis signal for complex formation. Moreover, when 23S RNA, the genome of another narnavirus, was expressed in E. coli, it also formed RNPs with its RNA polymerase p104. Finally, when both RNAs were expressed in the same E. coli cell, they formed RNPs only with their cognate RNA polymerases. These results altogether indicate that narnaviruses RNPs consist of only the viral genomes and their cognate RNA polymerases. Because the copy number of the RNPs can be induced almost equivalent to those of rRNAs in some yeast strains, the absence of host proteins may alleviate the burden on the host by not sequestering proteins into the RNPs. It may also contribute to the persistent infection of narnaviruses by decreasing their visibility.     

Molecular cloning,characterization and expression analysis of MHCI and chemokines CXCR3 and CXCR4 gene from freshwater carp,Catla catla

The immune system with large number of molecules protects the host against a plethora of continuously evolving microbes. Major histocompatibility complex (MHC) molecules serve as cardinal elements of the adaptive immune system responsible for the activation of the adaptive immunity in the host. The present study reports MHCI molecule in freshwater carp, Catla catla, and its differential expression in immunologically relevant tissues post‐infection with Gram‐negative and Gram‐positive bacteria. The MHCI sequence of C. catla had 502 bp nucleotides encoding putative 146 amino acids. The phylogenetic analysis exhibited its evolutionary conservation within the Cyprinidae family and formed a different clade with the higher vertebrates. Simultaneously, CXCR3 and CXCR4 chemokines were cloned and characterized for their expression in infected tissues. Analysis of immunologically relevant tissues of the infected fish exhibited an increase of MHCI gene expression and the down‐regulation of CXCR3 and CXCR4 chemokines, indicating a tricky interaction between the innate and adaptive immune system. It was found that intestine, skin and spleen played a crucial role in the contribution of the defense activity which instigated the self‐immunity. These immune activities can provide useful information to understand the interaction of self and non‐self‐ immune system in freshwater fish, Catla catla.     

Euduboscquella costata n. sp. (Dinoflagellata,Syndinea), an Intracellular Parasite of the Ciliate Schmidingerella arcuata: Morphology,Molecular Phylogeny,Life Cycle,Prevalence, and Infection Intensity

The syndinean dinoflagellate Euduboscquella costata n. sp., an intracellular parasite of the tintinnid ciliate Schmidingerella arcuata, was discovered from Korean coastal water in November of 2013. Euduboscquella costata parasitized in about 62% of the host population, with infection intensity (= number of trophonts in a single host cell) ranging from 1 to 8. Based on morphology and nuclear 18S ribosomal RNA gene sequences, the parasite is new to science. Euduboscquella costata n. sp. had an infection cycle typical of the genus, but had morphological and developmental features that distinguished it from congeneric species. These features include: (1) episome of the trophont with 25–40 grooves converging toward the center of the shield; (2) a narrow, funnel‐shaped lamina pharyngea extending from the margin of the episomal shield to the nucleus; (3) persistence of grooves during extracellular development (sporogenesis); (4) a single food vacuole during sporogenesis; (5) separation of sporocytes early in sporogenesis, regardless of type of spore formed; and (6) dinospore size (ca. 14 μm in length) and shape (bulbous episome with narrower, tapering hyposome). After sporogenesis, E. costata produced four different types of spore that showed completely identical 18S rRNA gene sequences. The gene sequence was completely identical with a previously reported population, Euduboscquella sp. ex S. arcuata, from Assawoman Bay, USA, indicating that the two populations are likely conspecific. Favella ehrenbergii, a widely recorded tintinnid known to host Euduboscquella spp., co‐occurred with S. arcuata, but was not infected by E. costata in field samples or during short‐term, cross‐infection experiments.     

Molecular phylogeny and evolution of host-plant use in conifer seed chalcids in the genus Megastigmus (Hymenoptera: Torymidae)

Abstract. Phylogenetic relationships amongst Megastigmus species (Chalcidoidea: Torymidae) associated with conifer seeds were inferred from DNA sequence data. Twenty‐nine species of seed chalcids were analysed using two different genes, cytochrome b (mitochondrial DNA) and the D2 domain of the 28S ribosomal DNA. Maximum‐parsimony and maximum‐likelihood analyses showed that taxa formed two monophyletic groups, one clade comprising all species associated with Cupressaceae and Taxodiaceae hosts with the exception of Chamaecyparis, and the other clade composed of species associated with Pinaceae. Species infesting Cupressaceae and Taxodiaceae seemed to be specialized to particular host genera or even to be species specific, which was consistent with a taxonomic radiation following initial host adaptation. By contrast, Megastigmus species associated with Pinaceae appeared capable of shifting onto different congeneric species or even onto a new host genus, with their evolution apparently less constrained by plant association. We hypothesized that the Megastigmus group associated with Pinaceae may have a much higher invasive potential than that related to Cupressaceae. The study also confirmed the presence of invasive Nearctic species in the Palaearctic, and demonstrated the existence of a cryptic species complex.     

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.     

Taxonomic identity of a galling adelgid (Hemiptera: Adelgidae) from three spruce species in Central Japan

Gall‐forming insects are commonly highly host‐specific, and galling species once thought to be oligo‐ or polyphagous are often found to represent a complex of host‐specific races or cryptic species. A recent DNA barcoding study documented that an unidentified species of the genus Adelges is a gall‐former associated with four spruce species (Picea bicolor, P. koyamai, P. maximowiczii, P. polita) as the primary hosts, with little genetic differentiation among insects on different host species. In this study, we investigated the morphology of this galling adelgid to determine its taxonomic identity. Morphological inspection of insects collected from three of the spruce species confirmed that this adelgid is a single galling species, and is identified as Adelges (Sacchiphantes) kitamiensis, which was previously known only from the secondary host. We described the gallicola adults of this species, as well as the first‐instar exules which are the offspring of gallicolae. Finally, we verified the taxonomic identity of this species and discuss its life cycle and host distribution.     

Identification and characterisation of a Theileria annulata proline‐rich microtubule and SH3 domain‐interacting protein (TaMISHIP) that forms a complex with CLASP1, EB1, and CD2AP at the schizont surface

Theileria annulata is an apicomplexan parasite that modifies the phenotype of its host cell completely, inducing uncontrolled proliferation, resistance to apoptosis, and increased invasiveness. The infected cell thus resembles a cancer cell, and changes to various host cell signalling pathways accompany transformation. Most of the molecular mechanisms leading to Theileria‐induced immortalization of leukocytes remain unknown. The parasite dissolves the surrounding host cell membrane soon after invasion and starts interacting with host proteins, ensuring its propagation by stably associating with the host cell microtubule network. By using BioID technology together with fluorescence microscopy and co‐immunoprecipitation, we identified a CLASP1/CD2AP/EB1‐containing protein complex that surrounds the schizont throughout the host cell cycle and integrates bovine adaptor proteins (CIN85, 14‐3‐3 epsilon, and ASAP1). This complex also includes the schizont membrane protein Ta‐p104 together with a novel secreted T. annulata protein (encoded by TA20980), which we term microtubule and SH3 domain‐interacting protein (TaMISHIP). TaMISHIP localises to the schizont surface and contains a functional EB1‐binding SxIP motif, as well as functional SH3 domain‐binding Px(P/A)xPR motifs that mediate its interaction with CD2AP. Upon overexpression in non‐infected bovine macrophages, TaMISHIP causes binucleation, potentially indicative of a role in cytokinesis.