The Role of Filippi's Glands in the Silk Moths Cocoon Construction.

Filippi's glands (FGs), formerly also called Lyonet's glands, are accessory secretory structures of the labial (silk) glands of lepidopteran caterpillars, which were implicated to play an important role in the maturation of the silk material and the construction of the cocoon. In our previous study, we have identified several species of giant silk moths that completely lack the FGs. Interestingly, the absence of FGs in these species correlates with the construction of a loose cocoon architecture. We investigated the functions of FGs by their surgical extirpation in the last instar larvae of the silkworm, Bombyx mori. We found that the absence of FGs altered the structure of the resulting cocoon, in which the different layers of silk were separated. In further experiments, we found no effects of the absence of FGs on larval cocoon formation behavior or on changes in cocoon mass or lipid content. Differential proteomic analysis revealed no significant contribution of structural proteins from FGs to silk cocoon material, but we identified several low abundance proteins that may play a role in posttranslational modifications of some silk proteins. Proteomic analysis also revealed a difference in phosphorylation of the N-terminal sequence of fibroin-heavy chain molecule. Thus, FGs appear to affect silk stickiness during spinning by regulating posttranslational modifications. This could also explain the link that exists between the absence of these glands and the formation of loose cocoons in some giant silk moth species.

Cryptochrome 2 mediates directional magnetoreception in cockroaches.

The ability to perceive geomagnetic fields (GMFs) represents a fascinating biological phenomenon. Studies on transgenic flies have provided evidence that photosensitive Cryptochromes (Cry) are involved in the response to magnetic fields (MFs). However, none of the studies tackled the problem of whether the Cry-dependent magnetosensitivity is coupled to the sole MF presence or to the direction of MF vector. In this study, we used gene silencing and a directional MF to show that mammalian-like Cry2 is necessary for a genuine directional response to periodic rotations of the GMF vector in two insect species. Longer wavelengths of light required higher photon fluxes for a detectable behavioral response, and a sharp detection border was present in the cyan/green spectral region. Both observations are consistent with involvement of the FADox, FAD(•-) and FADH(-) redox forms of flavin. The response was lost upon covering the eyes, demonstrating that the signal is perceived in the eye region. Immunohistochemical staining detected Cry2 in the hemispherical layer of laminal glia cells underneath the retina. Together, these findings identified the eye-localized Cry2 as an indispensable component and a likely photoreceptor of the directional GMF response. Our study is thus a clear step forward in deciphering the in vivo effects of GMF and supports the interaction of underlying mechanism with the visual system.

A comprehensive gene expression analysis of the unique three-layered cocoon of the cecropia moth, Hyalophora cecropia.

The larvae of the moth Hyalophora cecropia spin silk cocoons with morphologically distinct layers. We investigated the expression of the individual silk protein components of these cocoons in relation to the morphology of the silk gland and its affiliation to the different layers of the cocoon. The study used transcriptomic and proteomic analyses to identify 91 proteins associated with the silk cocoons, 63 of which have a signal peptide indicating their secretory nature. We checked the specificity of their expression in different parts of the SG and the presence of the corresponding protein products in each cocoon layer. Differences were observed among less abundant proteins with unclear functions. The representation of proteins in the inner envelope and intermediate space was similar, except for a higher proportion of probable contaminating proteins, mostly originating from the gut. On the other hand, the outer envelope contains a number of putative enzymes with unclear function. However, the protein most specific to the outer layer has sequence homology to putative serine/threonine kinase-like proteins and some adhesive proteins, and its closest homolog in Bombyx mori was found in the scaffold silk. This research provides valuable insights into the silk production of the cecropia moth, highlighting both similarities and differences to other moth species.

3D reconstruction of larval and adult brain neuropils of two giant silk moth species: Hyalophora cecropia and Antheraea pernyi.

We present a detailed analysis of the brain anatomy of two saturniid species, the cecropia silk moth, Hyalophora cecropia, and the Chinese oak silk moth, Antheraea pernyi, including 3D reconstructions of the major brain neuropils in the larva and in male and female adults. The 3D reconstructions, prepared from high-resolution optical sections, showed that the corresponding neuropils of these saturniid species are virtually identical. Similarities between the two species include a pronounced sexual dimorphism in the adults in the form of a male-specific assembly of markedly enlarged glomeruli forming the so-called macroglomerular complex. From the reports published to date, it can be concluded that the neuropil architecture of saturniids resembles that of other nocturnal moths, including the sibling family Sphingidae. In addition, compared with previous anatomical data on diurnal lepidopteran species, significant differences were observed in the two saturniid species, which include the thickness of the Y-tract of the mushroom body, the size of the main neuropils of the optic lobes, and the sexual dimorphisms of the antennal lobes.

Knockdown of proteins involved in iron metabolism limits tick reproduction and development.

Ticks are among the most important vectors of a wide range of human and animal diseases. During blood feeding, ticks are exposed to an enormous amount of free iron that must be appropriately used and detoxified. However, the mechanism of iron metabolism in ticks is poorly understood. Here, we show that ticks possess a complex system that efficiently utilizes, stores and transports non-heme iron within the tick body. We have characterized a new secreted ferritin (FER2) and an iron regulatory protein (IRP1) from the sheep tick, Ixodes ricinus, and have demonstrated their relationship to a previously described tick intracellular ferritin (FER1). By using RNA interference-mediated gene silencing in the tick, we show that synthesis of FER1, but not of FER2, is subject to IRP1-mediated translational control. Further, we find that depletion of FER2 from the tick plasma leads to a loss of FER1 expression in the salivary glands and ovaries that normally follows blood ingestion. We therefore suggest that secreted FER2 functions as the primary transporter of non-heme iron between the tick gut and the peripheral tissues. Silencing of the fer1, fer2, and irp1 genes by RNAi has an adverse impact on hatching rate and decreases postbloodmeal weight in tick females. Importantly, knockdown of fer2 dramatically impairs the ability of ticks to feed, thus making FER2 a promising candidate for development of an efficient anti-tick vaccine.

The Exact Timing of Microinjection of Parthenogenetic Silkworm Embryos Is Crucial for Their Successful Transgenesis.

The use of parthenogenetic silkworm (Bombyx mori) strains, which eliminate the problem of recombination, is a useful tool for maintaining transgenic clonal lines. The generation of genetically identical individuals is becoming an important tool in genetic engineering, allowing replication of an existing advantageous trait combination without the mixing that occurs during sexual reproduction. Thus, an animal with a particular genetic modification, such as the ability to produce transgenic proteins, can reproduce more rapidly than by natural mating. One obstacle to the widespread use of parthenogenesis in silkworm genetic engineering is the relatively low efficiency of downstream transgenesis techniques. In this work, we seek to optimize the use of transgenesis in conjunction with the production of parthenogenetic individuals. We found that a very important parameter for the introduction of foreign genes into a parthenogenetic strain is the precise timing of embryo microinjection. Our modification of the original method increased the efficiency of transgene injection as well as the survival rate of injected embryos. We also provide a detailed description of the methodological procedure including a graphical overview of the entire protocol.

Characterization of silk genes in Ephestia kuehniella and Galleria mellonella revealed duplication of sericin genes and highly divergent sequences encoding fibroin heavy chains.

Silk is a secretory product of numerous arthropods with remarkable mechanical properties. In this work, we present the complete sequences of the putative major silk proteins of E. kuehniella and compare them with those of G. mellonella, which belongs to the same moth family Pyralidae. To identify the silk genes of both species, we combined proteomic analysis of cocoon silk with a homology search in transcriptomes and genomic sequences to complement the information on both species. We analyzed structure of the candidate genes obtained, their expression specificity and their evolutionary relationships. We demonstrate that the silks of E. kuehniella and G. mellonella differ in their hydrophobicity and that the silk of E. kuehniella is highly hygroscopic. In our experiments, we show that the number of genes encoding sericins is higher in G. mellonella than in E. kuehniella. By analyzing the synteny of the chromosomal segment encoding sericin genes in both moth species, we found that the region encoding sericins is duplicated in G. mellonella. Finally, we present the complete primary structures of nine fibH genes and proteins from both families of the suborder Pyraloidea and discuss their specific and conserved features. This study provides a foundation for future research on the evolution of silk proteins and lays the groundwork for future detailed functional studies.

Cryptochromes define a novel circadian clock mechanism in monarch butterflies that may underlie sun compass navigation.

The circadian clock plays a vital role in monarch butterfly (Danaus plexippus) migration by providing the timing component of time-compensated sun compass orientation, a process that is important for successful navigation. We therefore evaluated the monarch clockwork by focusing on the functions of a Drosophila-like cryptochrome (cry), designated cry1, and a vertebrate-like cry, designated cry2, that are both expressed in the butterfly and by placing these genes in the context of other relevant clock genes in vivo. We found that similar temporal patterns of clock gene expression and protein levels occur in the heads, as occur in DpN1 cells, of a monarch cell line that contains a light-driven clock. CRY1 mediates TIMELESS degradation by light in DpN1 cells, and a light-induced TIMELESS decrease occurs in putative clock cells in the pars lateralis (PL) in the brain. Moreover, monarch cry1 transgenes partially rescue both biochemical and behavioral light-input defects in cry(b) mutant Drosophila. CRY2 is the major transcriptional repressor of CLOCK:CYCLE-mediated transcription in DpN1 cells, and endogenous CRY2 potently inhibits transcription without involvement of PERIOD. CRY2 is co-localized with clock proteins in the PL, and there it translocates to the nucleus at the appropriate time for transcriptional repression. We also discovered CRY2-positive neural projections that oscillate in the central complex. The results define a novel, CRY-centric clock mechanism in the monarch in which CRY1 likely functions as a blue-light photoreceptor for entrainment, whereas CRY2 functions within the clockwork as the transcriptional repressor of a negative transcriptional feedback loop. Our data further suggest that CRY2 may have a dual role in the monarch butterfly's brain-as a core clock element and as an output that regulates circadian activity in the central complex, the likely site of the sun compass.

The Filippi's Glands of Giant Silk Moths: To Be or Not to Be?

The Filippi's glands (FGs), formerly "Lyonet's glands", are paired accessory organs associated with the silk glands. They are unique to Lepidoptera caterpillars and their exact role is not clear. The FGs are thought to be involved in the construction of a silk cocoon in bombycoid moths. FGs can differ in size and shape, therefore, in this study we attempt to find a correlation between FG morphology and phylogenetic position within the Bombycoidea. We use light and electron microscopy to examine the presence and morphology of FGs in a range of wild (giant) silk moths and several related species. Our results confirm that the majority of studied silk moth species have complex type of FGs that continuously increase in size during larval development. We identified several species of giant silk moths and two hawk moth species that completely lack FGs throughout their larval development. Finally, in several hawk moth species in which FGs are well developed during the first larval stage, these glands do not grow and remain small during later larval growth. Our results suggest that FGs are not critical for spinning and that loss of FGs occurred several times during the evolution of saturniids and sphingids. Comparison of FGs in different moths is an important first step in the elucidation of their physiological significance.

Isolation and Characterization of Highly Pure Type A Spermatogonia From Sterlet (Acipenser ruthenus) Using Flow-Cytometric Cell Sorting.

Sturgeons are among the most ancient linages of actinopterygians. At present, many sturgeon species are critically endangered. Surrogate production could be used as an affordable and a time-efficient method for endangered sturgeons. Our study established a method for identifying and isolating type A spermatogonia from different developmental stages of testes using flow cytometric cell sorting (FCM). Flow cytometric analysis of a whole testicular cell suspension showed several well-distinguished cell populations formed according to different values of light scatter parameters. FCM of these different cell populations was performed directly on glass slides for further immunocytochemistry to identify germ cells. Results showed that the cell population in gate P1 on a flow cytometry plot (with high forward scatter and high side scatter parameter values) contains the highest amount of type A spermatogonia. The sorted cell populations were characterized by expression profiles of 10 germ cell specific genes. The result confirmed that setting up for the P1 gate could precisely sort type A spermatogonia in all tested testicular developmental stages. The P2 gate, which was with lower forward scatter and side scatter values mostly, contained type B spermatogonia at a later maturing stage. Moreover, expressions of plzf, dnd, boule, and kitr were significantly higher in type A spermatogonia than in later developed germ cells. In addition, plzf was firstly found as a reliable marker to identify type A spermatogonia, which filled the gap of identification of spermatogonial stem cells in sterlet. It is expected to increase the efficiency of germ stem cell culture and transplantation with plzf identification. Our study thus first addressed a phenotypic characterization of a pure type A spermatogonia population in sterlet. FCM strategy can improve the production of sturgeons with surrogate broodstock and further the analysis of the cellular and molecular mechanisms of sturgeon germ cell development.

A re-evaluation of silk measurement by the cecropia caterpillar (Hyalophora cecropia) during cocoon construction reveals use of a silk odometer that is temporally regulated.

The late 5th instar caterpillar of the cecropia silk moth (Hyalophora cecropia) spins a silken cocoon with a distinct, multilayered architecture. The cocoon construction program, first described by the seminal work of Van der Kloot and Williams, consists of a highly ordered sequence of events. We perform behavioral experiments to re-evaluate the original cecropia work, which hypothesized that the length of silk that passes through the spinneret controls the orderly execution of each of the discrete events of cocoon spinning. We confirm and extend by three-dimensional scanning and quantitative measurements of silk weights that if cocoon construction is interrupted, upon re-spinning, the caterpillar continues the cocoon program from where it left off. We also confirm and extend by quantitative measurements of silk weights that cecropia caterpillars will not bypass any of the sections of the cocoon during the construction process, even if presented with a pre-spun section of a cocoon spun by another caterpillar. Blocking silk output inhibits caterpillars from performing normal spinning behaviors used for cocoon construction. Surprisingly, unblocking silk output 24-hr later did not restart the cocoon construction program, suggesting the involvement of a temporally-defined interval timer. We confirm with surgical reductions of the silk glands that it is the length of silk itself that matters, rather than the total amount of silk extracted by individuals. We used scanning electron microscopy to directly show that either mono- or dual-filament silk (i.e., equal silk lengths but which vary in their total amount of silk extracted) can be used to construct equivalent cocoons of normal size and that contain the relevant layers. We propose that our findings, taken together with the results of prior studies, strongly support the hypothesis that the caterpillar uses a silk "odometer" to measure the length of silk extracted during cocoon construction but does so in a temporally regulated manner. We further postulate that our examination of the anatomy of the silk spinning apparatus and ablating spinneret sensory output provides evidence that silk length measurement occurs upstream of output from the spinneret.

Functional Analysis of Adipokinetic Hormone Signaling in Bombyx mori.

Insect adipokinetic hormones (AKHs) are short peptides produced in the corpora cardiaca and are responsible for mobilizing energy stores from the fat body to the hemolymph. Three related peptides, AKH1, AKH2, and AKH/corazonin-related peptide (ACP) as well as three AKH receptors have been reported in Bombyx mori. AKH1 and AKH2 are specific for the AKHR1 receptor, whereas ACP interacts with the other two AKHRs. To assess the effect of the two silkworm AKHs and ACP in the regulation of energy homeostasis we examined the expression pattern of the three peptides and their receptors as well as their effect on the level of carbohydrates and lipids in the hemolymph. Our results support the hypothesis that only AKH1 and AKH2 peptides together with the AKHR1 receptor are involved in the maintenance of energy homeostasis. Because Bombyx AKHR1 (BmAKHR1) seems to be a true AKHR we generated its mutation. The BmAKHR1 mutant larvae display significantly lower carbohydrate and lipid levels in the hemolymph and reduced sensitivity to starvation. Our study clarifies the role of BmAKHR1 in energy homeostasis.

Connecting the navigational clock to sun compass input in monarch butterfly brain.

Migratory monarch butterflies (Danaus plexippus) use a time-compensated sun compass to navigate to their overwintering grounds in Mexico. Although polarized light is one of the celestial cues used for orientation, the spectral content (color) of that light has not been fully explored. We cloned the cDNAs of three visual pigment-encoding opsins (ultraviolet [UV], blue, and long wavelength) and found that all three are expressed uniformly in main retina. The photoreceptors of the polarization-specialized dorsal rim area, on the other hand, are monochromatic for the UV opsin. Behavioral studies support the importance of polarized UV light for flight orientation. Next, we used clock protein expression patterns to identify the location of a circadian clock in the dorsolateral protocerebrum of butterfly brain. To provide a link between the clock and the sun compass, we identified a CRYPTOCHROME-staining neural pathway that likely connects the circadian clock to polarized light input entering brain.

Circadian rhythm gene regulation in the housefly Musca domestica.

The circadian mechanism appears remarkably conserved between Drosophila and mammals, with basic underlying negative and positive feedback loops, cycling gene products, and temporally regulated nuclear transport involving a few key proteins. One of these negative regulators is PERIOD, which in Drosophila shows very similar temporal and spatial regulation to TIMELESS. Surprisingly, we observe that in the housefly, Musca domestica, PER does not cycle in Western blots of head extracts, in contrast to the TIM protein. Furthermore, immunocytochemical (ICC) localization using enzymatic staining procedures reveals that PER is not localized to the nucleus of any neurons within the brain at any circadian time, as recently observed for several nondipteran insects. However, with confocal analysis, immunofluorescence reveals a very different picture and provides an initial comparison of PER/TIM-containing cells in Musca and Drosophila, which shows some significant differences, but many similarities. Thus, even in closely related Diptera, there is considerable evolutionary flexibility in the number and spatial organization of clock cells and, indeed, in the expression patterns of clock products in these cells, although the underlying framework is similar.

Photoperiodic and food signals control expression pattern of the clock gene, period, in the linden bug, Pyrrhocoris apterus.

The temporal expression pattern of the circadian clock gene period was compared between heads of the linden bug, Pyrrhocoris apterus , kept under diapause-promoting short days (SD) and diapause-preventing long days (LD) using a real-time PCR quantification. Diapause or reproduction was programmed by photoperiod during the larval stage, but the first difference in per mRNA abundance between SD and LD insects was observed only after adult ecdysis. The expression level of per mRNA was markedly higher, up to more than 10-fold, in the destined-to diapause animals compared with those scheduled for reproduction. Up-regulation of per transcript was restricted to an early diapause peak, with the maximum expression on days 3 to 5 after adult ecdysis. Starvation reduced the peak level of per mRNA to about 50% of the value found in feeding females in the SD conditions, but per mRNA abundance was similarly low in fasting and feeding females in LD. Photoperiodic refractoriness in either wild-type postdiapause adults or in a selected nondiapause variant of P. apterus was associated with reproduction and low, LD-like levels of per mRNA under both SD and LD. Overall, the data suggest that the photoperiodic programming itself has no direct effect on per mRNA abundance, but it does determine the response of per transcript to food signals during subsequent expression of diapause/reproduction physiology.

The tick plasma lectin, Dorin M, is a fibrinogen-related molecule.

A lectin, named Dorin M, previously isolated and characterized from the hemolymph plasma of the soft tick, Ornithodoros moubata, was cloned and sequenced. The immunofluorescence using confocal microscopy revealed that Dorin M is produced in the tick hemocytes. A tryptic cleavage of Dorin M was performed and the resulting peptide fragments were sequenced by Edman degradation and/or mass spectrometry. Two of three internal peptide sequences displayed a significant similarity to the family of fibrinogen-related molecules. Degenerate primers were designed and used for PCR with hemocyte cDNA as a template. The sequence of the whole Dorin M cDNA was completed by the method of RACE. The tissue-specific expression investigated by RT-PCR revealed that Dorin M, in addition to hemocytes, is significantly expressed in salivary glands. The derived amino-acid sequence clearly shows that Dorin M has a fibrinogen-like domain, and exhibited the most significant similarity with tachylectins 5A and 5B from a horseshoe crab, Tachypleus tridentatus. In addition, other protein and binding characteristics suggest that Dorin M is closely related to tachylectins-5. Since these lectins have been reported to function as non-self recognizing molecules, we believe that Dorin M may play a similar role in an innate immunity of the tick and, possibly, also in pathogen transmission by this vector.

Distribution of PER protein, pigment-dispersing hormone, prothoracicotropic hormone, and eclosion hormone in the cephalic nervous system of insects.

Investigations performed on adult insects revealed that putative components of the central pacemaker, the protein Period (PER) and the pigment-dispersing hormone (PDH), are immunocytochemically detectable in discrete sets of brain neurons throughout the class of Insecta, represented by a bristletail, mayfly, damselfly, 2 locust species, stonefly, 2 bug species, goldsmith beetle, caddisfly, honeybee, and 2 blowfly species. The PER-positive cells are localized in the frontal protocerebrum and in most species also in the optic lobes, which are their only location in damselfly and goldsmith beetle. Additional PER-positive cells occur in a few species either in the deuto- and tritocerebrum or in the suboesophageal ganglion. The PER staining was always confined to the cytoplasm. The PDH immunoreactivity consistently occurs in a cluster of perikarya located frontoventrally at the proximal edge of the medulla. The mayfly and both locust species possess additional PDH neurons in 2 posterior cell clusters at the proximal edge of the medulla, and mayfly, waterstrider, and 1 of the blowfly species in the central brain. PDH-positive fibers form a fanlike arrangement over the frontal side of the medulla. Two or just 1 bundle of PDH-positive fibers run from the optic lobe to the protocerebrum, with collaterals passing over to the contralateral optic lobe. Antisera to the prothoracicotropic (PTTH) and the eclosion (EH) hormones, which in some insects regulate the molting and ecdysis rhythms, respectively, typically react with a few neurons in the frontal protocerebrum. However, the PTTH-positive neurons of the mayfly and the damselfly and the EH-positive neurons of the caddisfly are located in the suboesophageal ganglion. No PTTH-like antigen was detected in locusts, and no EH-like antigens were detected in the damselfly, stonefly, locusts, and the honeybee. There are no signs of co-localization of the PER-, PDH-, PTTH-, and EH-like antigens in identical neurons.

Clock gene daily profiles and their phase relationship in the rat suprachiasmatic nucleus are affected by photoperiod.

Rhythmicity of the rat suprachiasmatic nucleus (SCN), a site of the circadian pacemaker, is affected by daylength; that is, by the photoperiod. Whereas various markers of rhythmicity have been followed, so far there have been no studies on the effect of the photoperiod on the expression of the clock genes in the rat SCN. To fill the gap and to better understand the photoperiodic modulation of the SCN state, rats were maintained either under a long photoperiod with 16 h of light and 8 h of darkness per day (LD16:8) or under a short LD8:16 photoperiod, and daily profiles of Per1, Cry1, Bmal1 and Clock mRNA in darkness were assessed by in situ hybridization method. The photoperiod affected phase, waveform, and amplitude of the rhythmic gene expression as well as phase relationship between their profiles. Under the long period, the interval of elevated Per1 mRNA lasted for a longer and that of elevated Bmal1 mRNA for a shorter time than under the short photoperiod. Under both photoperiods, the morning and the daytime Per1 and Cry1 mRNA rise as well as the morning Bmal1 mRNA decline were closely linked to the morning light onset. Amplitude of Per1, Cry1, and Bmal1 mRNA rhythms was larger under the short than under the long photoperiod. Also, under the short photoperiod, the daily Clock mRNA profile exhibited a significant rhythm. Altogether, the data indicate that the whole complex molecular clockwork in the rat SCN is photoperiod dependent and hence may differ according to the season of the year.

Expression of clock genes period and timeless in the central nervous system of the Mediterranean flour moth, Ephestia kuehniella.

Homologous circadian genes are found in all insect clocks, but their contribution to species-specific circadian timing systems differs. The aim of this study was to extend research within Lepidoptera to gain a better understanding of the molecular mechanism underlying circadian clock plasticity and evolution. The Mediterranean flour moth, Ephestia kuehniella (Pyralidae), represents a phylogenetically ancestral lepidopteran species. We have identified circadian rhythms in egg hatching, adult emergence, and adult locomotor activity. Cloning full-length complementary DNAs and further characterization confirmed one copy of period and timeless genes in both sexes. Both per and tim transcripts oscillate in their abundance in E. kuehniella heads under light-dark conditions. PER-like immunoreactivity (PER-lir) was observed in nuclei and cytoplasm of most neurons in the central brain, the ventral part of subesophageal complex, the neurohemal organs, the optic lobes, and eyes. PER-lir in photoreceptor nuclei oscillated during the day with maximal intensity in the light phase of the photoperiodic regime and lack of a signal in the middle of the dark phase. Expression patterns of per and tim messenger RNAs (mRNAs) were revealed in the identical location as the PER-lir was detected. In the photoreceptors, a daily rhythm in the intensity of expression of both per mRNA and tim mRNA was found. These findings suggest E. kuehniella as a potential lepidopteran model for circadian studies.

Thioester-containing proteins of the tick Ixodes ricinus: gene expression, response to microbial challenge and their role in phagocytosis of the yeast Candida albicans.

The ability of ticks to act as vectors for a wide range of serious human and animal infectious diseases is apparently linked to the insufficiency of the tick immune system to effectively eliminate pathogens they transmit. At the tick-pathogen interface, an important role is presumably played by components of an ancient complement system that includes a repertoire of thioester-containing proteins (TEPs), which in Ixodes sp. comprises three α2-macroglobulins (A2M), three C3 complement component-related molecules (C3), two macroglobulin complement-related (Mcr) and one insect-type TEPs (Tep). In order to assess the function of TEPs in tick immunity, a quantitative real-time PCR expression analysis of tick TEPs was performed at various developmental stages of Ixodes ricinus, and in tissues dissected from adult females. Expression of TEP genes was mostly tissue specific; IrA2M1, IrC3-1, IrC3-3 were found to be expressed in cells of tick fat body adjacent to the tracheal trunks, IrA2M2 in hemocytes, IrTep in ovaries, IrMcr1 in salivary glands and only IrA2M3, IrC3-2 and IrMcr2 mRNAs were present in multiple organs. Expression of tick TEPs was further examined in response to injection of model microbes representing Gram-negative, Gram-positive bacteria and yeast. The greatest expression induction was observed for IrA2M1 and IrC3-1 after challenge with the yeast Candida albicans. Phagocytosis of the yeast was strongly dependent on an active thioester bond and the subsequent silencing of individual tick TEPs by RNA interference demonstrated the involvement of IrC3-1 and IrMcr2. This result suggests the existence of a distinct complement-like pathway, different from that leading to phagocytosis of Gram-negative bacteria. Understanding of the tick immune response against model microbes should provide new concepts for investigating interactions between ticks and relevant tick-borne pathogens.