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Arthropods from class Arachnida constitute a large and diverse group with over 100,000 described species, and they are sources of many proteins that have a direct impact on human health. Despite the importance of Arachnida, few proteins originating from these organisms have been characterized in terms of their structure. Here we present a detailed analysis of Arachnida proteins that have their experimental structures determined and deposited to the Protein Data Bank (PDB). Our results indicate that proteins represented in the PDB are derived from a small number of Arachnida families, and two-thirds of Arachnida proteins with experimental structures determined are derived from organisms belonging to Buthidae, Ixodidae, and Theraphosidae families. Moreover, 90% of the deposits come from just a dozen of Arachnida families, and almost half of the deposits represent proteins originating from only fifteen different species. In summary, our analysis shows that the structural analysis of proteins originating from Arachnida is not only limited to a small number of the source species, but also proteins from this group of animals are not extensively studied. However, the interest in Arachnida proteins seems to be increasing, which is reflected by a significant increase in the related PDB deposits during the last ten years.

The Roman Empire expanded through the Mediterranean shores and brought human mobility and cosmopolitanism across this inland sea to an unprecedented scale. However, if this was also common at the Empire frontiers remains undetermined. The Balkans and Danube River were of strategic importance for the Romans acting as an East-West connection and as a defense line against “barbarian” tribes. We generated genome-wide data from 70 ancient individuals from present-day Serbia dated to the first millennium CE; including Viminacium, capital of Moesia Superior province. Our analyses reveal large scale-movements from Anatolia during Imperial rule, similar to the pattern observed in Rome, and cases of individual mobility from as far as East Africa. Between ∼250-500 CE, we detect gene-flow from Central/Northern Europe harboring admixtures of Iron Age steppe groups. Tenth-century CE individuals harbored North-Eastern European-related ancestry likely associated to Slavic-speakers, which contributed >20% of the ancestry of today’s Balkan people.

Arabidopsis (Arabidopsis thaliana) defenses against herbivores are regulated by the jasmonate (JA) hormonal signaling pathway, which leads to the production of a plethora of defense compounds. Arabidopsis defense compounds include tryptophan-derived metabolites, which limit Arabidopsis infestation by the generalist herbivore two-spotted spider mite, Tetranychus urticae. However, the phytochemicals responsible for Arabidopsis protection against T. urticae are unknown. Here, we used Arabidopsis mutants disrupted in the synthesis of tryptophan-derived secondary metabolites to identify phytochemicals involved in the defense against T. urticae. We show that of the three tryptophan-dependent pathways found in Arabidopsis, the indole glucosinolate (IG) pathway is necessary and sufficient to assure tryptophan-mediated defense against T. urticae. We demonstrate that all three IGs can limit T. urticae herbivory, but that they must be processed by myrosinases to hinder T. urticae oviposition. Putative IG breakdown products were detected in mite-infested leaves, suggesting in planta processing by myrosinases. Finally, we demonstrate that besides IGs, there are additional JA-regulated defenses that control T. urticae herbivory. Together, our results reveal the complexity of Arabidopsis defenses against T. urticae that rely on multiple IGs, specific myrosinases, and additional JA-dependent defenses.

Arabidopsis defenses against herbivores are regulated by the jasmonate hormonal signaling pathway, which leads to the production of a plethora of defense compounds, including tryptophan-derived metabolites produced through CYP79B2/CYP79B3. Jasmonate signaling and CYP79B2/CYP79B3 limit Arabidopsis infestation by the generalist herbivore two-spotted spider mite, Tetranychus urticae. However, the phytochemicals responsible for Arabidopsis protection against T. urticae are unknown. Here, using Arabidopsis mutants that disrupt metabolic pathways downstream of CYP79B2/CYP79B3, and synthetic indole glucosinolates, we identified phytochemicals involved in the defense against T. urticae. We show that Trp-derived metabolites depending on CYP71A12 and CYP71A13 are not affecting mite herbivory. Instead, the supplementation of cyp79b2 cyp79b3 mutant leaves with the 3-indolylmethyl glucosinolate and its derived metabolites demonstrated that the indole glucosinolate pathway is sufficient to assure CYP79B2/CYP79B3-mediated defenses against T. urticae. We demonstrate that three indole glucosinolates can limit T. urticae herbivory, but that they have to be processed by the myrosinases to hinder T. urticae oviposition. Finally, the supplementation of the mutant myc2 myc3 myc4 with indole glucosinolates indicated that the transcription factors MYC2/MYC3/MYC4 induce additional indole glucosinolate-independent defenses that control T. urticae herbivory. Together, these results reveal the complexity of Arabidopsis defenses against T. urticae that rely on multiple indole glucosinolates, specific myrosinases, and additional MYC2/MYC3/MYC4-dependent defenses.One sentence summaryThree indole glucosinolates and the myrosinases TGG1/TGG2 help protect Arabidopsis thaliana against the herbivory of the two-spotted spider mite Tetranychus urticae.

Genetic adaptation, occurring over a long evolutionary time, enables host-specialized herbivores to develop novel resistance traits and to efficiently counteract the defenses of a narrow range of host plants. In contrast, physiological acclimation, leading to the suppression and/or detoxification of host defenses, is hypothesized to enable broad generalists to shift between plant hosts. However, the host adaptation mechanisms used by generalists composed of host-adapted populations are not known. Two-spotted spider mite (TSSM; Tetranychus urticae) is an extreme generalist herbivore whose individual populations perform well only on a subset of potential hosts. We combined experimental evolution, Arabidopsis thaliana genetics, mite reverse genetics, and pharmacological approaches to examine mite host adaptation upon the shift of a bean (Phaseolus vulgaris)-adapted population to Arabidopsis. We showed that cytochrome P450 monooxygenases are required for mite adaptation to Arabidopsis. We identified activities of two tiers of P450s: general xenobiotic-responsive P450s that have a limited contribution to mite adaptation to Arabidopsis and adaptation-associated P450s that efficiently counteract Arabidopsis defenses. In approximately 25 generations of mite selection on Arabidopsis plants, mites evolved highly efficient detoxification-based adaptation, characteristic of specialist herbivores. This demonstrates that specialization to plant resistance traits can occur within the ecological timescale, enabling the TSSM to shift to novel plant hosts., Mites can evolve highly efficient detoxification-based adaptation in approximately 25 generations on an initially unfavorable plant host, revealing that specialization can occur within the ecological timescale.

Global viticulture has evolved following market trends, causing loss of cultivar diversity and traditional practices. In Montenegro, modern viticulture co-exists with a traditional viticulture that still maintains ancient practices and exploits local cultivars. As a result, this region provides a unique opportunity to explore processes increasing genetic diversity. To evaluate the diversity of Montenegrin grapevines and the processes involved in their diversification, we collected and analyzed 419 samples in situ across the country (cultivated plants from old orchards and vines growing in the wild), and 57 local varieties preserved in a grapevine collection. We obtained 144 different genetic profiles, more than 100 corresponding to cultivated grapevines, representing a surprising diversity for one of the smallest European countries. Part of this high diversity reflects historical records indicating multiple and intense introduction events from diverse viticultural regions at different times. Another important gene pool includes many autochthonous varieties, some on the edge of extinction, linked in a complex parentage network where two varieties (Razaklija and Kratošija) played a leading role on the generation of indigenous varieties. Finally, analyses of genetic structure unveiled several putative proto-varieties, likely representing the first steps involved in the generation of new cultivars or even secondary domestication events.

Spider mites constitute an assemblage of well-known pests in agriculture, but are less known for their ability to spin silk of nanoscale diameters and high Young’s moduli. Here, we characterize silk of the gorse spider mite Tetranychus lintearius, which produces copious amounts of silk with nano-dimensions. We determined biophysical characteristics of the silk fibres and manufactured nanoparticles and biofilm derived from native silk. We determined silk structure using attenuated total reflectance Fourier transform infrared spectroscopy, and characterized silk nanoparticles using field emission scanning electron microscopy. Comparative studies using T. lintearius and silkworm silk nanoparticles and biofilm demonstrated that spider mite silk supports mammalian cell growth in vitro and that fluorescently labelled nanoparticles can enter cell cytoplasm. The potential for cytocompatibility demonstrated by this study, together with the prospect of recombinant silk production, opens a new avenue for biomedical application of this little-known silk.

Comprehensive understanding of pleiotropic roles of RNAi machinery highlighted the conserved chromosomal functions of RNA interference. The consequences of the evolutionary variation in the core RNAi pathway genes are mostly unknown, but may lead to the species-specific functions associated with gene silencing. The two-spotted spider mite, Tetranychus urticae, is a major polyphagous chelicerate pest capable of feeding on over 1100 plant species and developing resistance to pesticides used for its control. A well annotated genome, susceptibility to RNAi and economic importance, make T. urticae an excellent candidate for development of an RNAi protocol that enables high-throughput genetic screens and RNAi-based pest control. Here, we show that the length of the exogenous dsRNA critically determines its processivity and ability to induce RNAi in vivo. A combination of the long dsRNAs and the use of dye to trace the ingestion of dsRNA enabled the identification of genes involved in membrane transport and 26S proteasome degradation as sensitive RNAi targets. Our data demonstrate that environmental RNAi can be an efficient reverse genetics and pest control tool in T. urticae. In addition, the species-specific properties together with the variation in the components of the RNAi machinery make T. urticae a potent experimental system to study the evolution of RNAi pathways., Tis work was supported by the Government of Canada through the Ontario Research Fund (RE08-067), the Natural Sciences and Engineering Research Council of Canada (NSERC) and by the European Union’s Horizon 2020 research and innovation program (773902-SuperPests) awarded to MG and VG; and by the Japan Society for the Promotion of Science KAKENHI (Grant Nos. 18H02203 to TS, 19K22304 to TF and 19K23674 to MT) and the Institute of Global Innovation Research in TUAT to MT, TS, TF and VG. MA was funded through the Global Tesis program, the University of Bari Aldo Moro, Italy.

GSTs (Glutathione S-transferases) are known to catalyze the nucleophilic attack of the sulfhydryl group of reduced glutathione (GSH) on electrophilic centers of xenobiotic compounds, including insecticides and acaricides. Genome analyses of the polyphagous spider mite herbivore Tetranychus urticae (two-spotted spider mite) revealed the presence of a set of 32 genes that code for secreted proteins belonging to the GST family of enzymes. To better understand the role of these proteins in T. urticae, we have functionally characterized TuGSTd01. Moreover, we have modeled the structure of the enzyme in apo form, as well as in the form with bound inhibitor. We demonstrated that this protein is a glutathione S-transferase that can conjugate glutathione to 1-chloro-2,4-dinitrobenzene (CDNB). We have tested TuGSTd01 activity with a range of potential substrates such as cinnamic acid, cumene hydroperoxide, and allyl isothiocyanate; however, the enzyme was unable to process these compounds. Using mutagenesis, we showed that putative active site variants S11A, E66A, S67A, and R68A mutants, which were residues predicted to interact directly with GSH, have no measurable activity, and these residues are required for the enzymatic activity of TuGSTd01. There are several reports that associate some T. urticae acaricide resistance with increased activity of GSTs . However, we found that TuGSTd01 is not able to detoxify abamectin; in fact, the acaricide inhibits the enzyme with Ki = 101 μM. Therefore, we suggest that the increased GST activity observed in abamectin resistant T. urticae field populations is a part of the compensatory feedback loop. In this case, the increased production of GSTs and relatively high concentration of GSH in cells allow GSTs to maintain physiological functions despite the presence of the acaricide.

The false spider mite Brevipalpus yothersi infests a broad host plant range and has become one of the most economically important species within the genus Brevipalpus. This phytophagous mite inflicts damage by both feeding on plants and transmitting plant viruses., The false spider mite Brevipalpus yothersi infests a broad host plant range and has become one of the most economically important species within the genus Brevipalpus. This phytophagous mite inflicts damage by both feeding on plants and transmitting plant viruses. Here, we report the first draft genome sequence of the false spider mite, which is also the first plant virus mite vector to be sequenced. The ∼72 Mb genome (sequenced at 42× coverage) encodes ∼16,000 predicted protein-coding genes.

The false spider mite Brevipalpus yothersi infests a broad host plant range and has become one of the most economically important species within the genus Brevipalpus. This phytophagous mite inflicts damage by both feeding on plants and transmitting plant viruses. Here, we report the first draft genome sequence of the false spider mite, which is also the first plant virus mite vector to be sequenced. The similar to 72 Mb genome (sequenced at 42x coverage) encodes similar to 16,000 predicted protein-coding genes.

Two-spotted spider mite (TSSM) Tetranychus urticae (Koch) is an important agricultural pest that causes considerable yield losses to over 150 field and greenhouse crops. Mitochondrial electron transport inhibitors (METI) acaricides are commonly used to control mite species in commercial Canadian greenhouses. Development of resistance to METIs in TSSM populations have been reported worldwide, but not until recently in Canada. The objectives of this study were to: 1) monitor the acaricide-susceptibility in greenhouse TSSM populations, and 2) investigate the resistance to pyridaben, a METI acaricide, in greenhouse resistant and pyridaben-selected (SRS) mite strains. The increased mortality to the pyridaben sub-lethal concentration (LC30) when SRS mites were exposed to piperonyl butoxide (PBO), a general cytochrome P450 monooxygenase inhibitor, and higher P450 activity compared to the greenhouse strain (RS) mites, indicated that P450s may be at least partially responsible for the resistance. The molecular mechanisms of target site insensitivity-mediated resistance in the pyridaben resistant strain of TSSM were investigated by comparing the DNA sequence of NADH dehydrogenase subunits TYKY and PSST, NADH-ubiquinone oxidoreductase chain 1 and 5 (ND1, ND5) and the NADH-ubiquinone oxidoreductase subunit 49 kDa from SRS to the reference strain (SS) and RS. Despite a number of nucleotide substitutions, none correlated with the pyridaben resistance. Understanding the underlying mechanisms of TSSM adaptation to acaricides is an essential part of resistance management strategy in any IPM program. The findings of this study will encourage growers to apply acaricides with different modes of action to reduce the rate at which acaricide resistance will occur in greenhouse TSSM populations.

Background The ancestral arthropod is believed to have had a clustered arrangement of ten Hox genes. Within arthropods, Hox gene mutations result in transformation of segment identities. Despite the fact that variation in segment number/character was common in the diversification of arthropods, few examples of Hox gene gains/losses have been correlated with morphological evolution. Furthermore, a full appreciation of the variation in the genomic arrangement of Hox genes in extant arthropods has not been recognized, as genome sequences from each major arthropod clade have not been reported until recently. Initial genomic analysis of the chelicerate Tetranychusurticae suggested that loss of Hox genes and Hox gene clustering might be more common than previously assumed. To further characterize the genomic evolution of arthropod Hox genes, we compared the genomic arrangement and general characteristics of Hox genes from representative taxa from each arthropod subphylum. Results In agreement with others, we find arthropods generally contain ten Hox genes arranged in a common orientation in the genome, with an increasing number of sampled species missing either Hox3 or abdominal-A orthologs. The genomic clustering of Hox genes in species we surveyed varies significantly, ranging from 0.3 to 13.6 Mb. In all species sampled, arthropod Hox genes are dispersed in the genome relative to the vertebrate Mus musculus. Differences in Hox cluster size arise from variation in the number of intervening genes, intergenic spacing, and the size of introns and UTRs. In the arthropods surveyed, Hox gene duplications are rare and four microRNAs are, in general, conserved in similar genomic positions relative to the Hox genes. Conclusions The tightly clustered Hox complexes found in the vertebrates are not evident within arthropods, and differential patterns of Hox gene dispersion are found throughout the arthropods. The comparative genomic data continue to support an ancestral arthropod Hox cluster of ten genes with a shared orientation, with four Hox gene-associated miRNAs, although the degree of dispersion between genes in an ancestral cluster remains uncertain. Hox3 and abdominal-A orthologs have been lost in multiple, independent lineages, and current data support a model in which inversions of the Abdominal-B locus that result in the loss of abdominal-A correlate with reduced trunk segmentation. Electronic supplementary material The online version of this article (doi:10.1186/s13227-016-0048-4) contains supplementary material, which is available to authorized users.

While mechanisms to detoxify plant produced, anti-herbivore compounds have been associated with plant host use by herbivores, less is known about the role of chemosensory perception in their life histories. This is especially true for generalists, including chelicerate herbivores that evolved herbivory independently from the more studied insect lineages. To shed light on chemosensory perception in a generalist herbivore, we characterized the chemosensory receptors (CRs) of the chelicerate two-spotted spider mite, Tetranychus urticae, an extreme generalist. Strikingly, T. urticae has more CRs than reported in any other arthropod to date. Including pseudogenes, 689 gustatory receptors were identified, as were 136 degenerin/Epithelial Na+ Channels (ENaCs) that have also been implicated as CRs in insects. The genomic distribution of T. urticae gustatory receptors indicates recurring bursts of lineage-specific proliferations, with the extent of receptor clusters reminiscent of those observed in the CR-rich genomes of vertebrates or C. elegans. Although pseudogenization of many gustatory receptors within clusters suggests relaxed selection, a subset of receptors is expressed. Consistent with functions as CRs, the genomic distribution and expression of ENaCs in lineage-specific T. urticae expansions mirrors that observed for gustatory receptors. The expansion of ENaCs in T. urticae to > 3-fold that reported in other animals was unexpected, raising the possibility that ENaCs in T. urticae have been co-opted to fulfill a major role performed by unrelated CRs in other animals. More broadly, our findings suggest an elaborate role for chemosensory perception in generalist herbivores that are of key ecological and agricultural importance.

The two-spotted spider mite (TSSM), Tetranychus urticae Koch (Acari: Tetranychidae), is one of the most polyphagous herbivores, feeding on more than 1,100 plant species. Its wide host range suggests that TSSM has an extraordinary ability to modulate its digestive and xenobiotic physiology. The analysis of the TSSM genome revealed the expansion of gene families that encode proteins involved in digestion and detoxification, many of which were associated with mite responses to host shifts. The majority of plant defense compounds that directly impact mite fitness are ingested. They interface mite compounds aimed at counteracting their effect in the gut. Despite several detailed ultrastructural studies, our knowledge of the TSSM digestive tract that is needed to support the functional analysis of digestive and detoxification physiology is lacking. Here, using a variety of histological and microscopy techniques, and a diversity of tracer dyes, we describe the organization and properties of the TSSM alimentary system. We define the cellular nature of floating vesicles in the midgut lumen that are proposed to be the site of intracellular digestion of plant macromolecules. In addition, by following the TSSM's ability to intake compounds of defined sizes, we determine a cut off size for the ingestible particles. Moreover, we demonstrate the existence of a distinct filtering function between midgut compartments which enables separation of molecules by size. Furthermore, we broadly define the spatial distribution of the expression domains of genes involved in digestion and detoxification. Finally, we discuss the relative simplicity of the spider mite digestive system in the context of mite's digestive and xenobiotic physiology, and consequences it has on the effectiveness of plant defenses.

Carotenoids underlie many of the vibrant yellow, orange, and red colors in animals, and are involved in processes ranging from vision to protection from stresses. Most animals acquire carotenoids from their diets because de novo synthesis of carotenoids is primarily limited to plants and some bacteria and fungi. Recently, sequencing projects in aphids and adelgids, spider mites, and gall midges identified genes with homology to fungal sequences encoding de novo carotenoid biosynthetic proteins like phytoene desaturase. The finding of horizontal gene transfers of carotenoid biosynthetic genes to three arthropod lineages was unprecedented; however, the relevance of the transfers for the arthropods that acquired them has remained largely speculative, which is especially true for spider mites that feed on plant cell contents, a known source of carotenoids. Pigmentation in spider mites results solely from carotenoids. Using a combination of genetic approaches, we show that mutations in a single horizontally transferred phytoene desaturase result in complete albinism in the twospotted spider mite, Tetranychus urticae, as well as in the citrus red mite, Panonychus citri. Further, we show that phytoene desaturase activity is essential for photoperiodic induction of diapause in an overwintering strain of T. urticae, consistent with a role for this enzyme in provisioning provitamin A carotenoids required for light perception. Carotenoid biosynthetic genes of fungal origin have therefore enabled some mites to forgo dietary carotenoids, with endogenous synthesis underlying their intense pigmentation and ability to enter diapause, a key to the global distribution of major spider mite pests of agriculture., This work was supported by Research Foundation Flanders (FWO) Grant G009312N (to L.T. and T.V.L.) and Grant G053815N (to L.T., T.V.L., and W.D.); incentive funds from the University of Utah (to R.M.C.); National Science Foundation Grant DEB1457346 (to R.M.C.); Ontario Research Fund–Research 753 Excellence Round 8 Grant RE08-067 (to M.G.); and the Government of Canada through Genome Canada and the Ontario Genomics Institute (Grant OGI-046) (to M.G.). R.G. was funded by National Institutes of Health Genetics Training Grant T32GM007464, and W.D. is a postdoctoral fellow of the Research Foundation Flanders (FWO). M.O. was supported by Japan Society for the Promotion of Science Kakenhi Grant 26292029.

Plant-herbivore interactions evolved over long periods of time, resulting in an elaborate arms race between interacting species. While specialist herbivores evolved specific strategies to cope with the defenses of a limited number of hosts, our understanding of how generalist herbivores deal with the defenses of a plethora of diverse host plants is largely unknown. Understanding the interaction between a plant host and a generalist herbivore requires an understanding of the plant's mechanisms aimed at defending itself and the herbivore's mechanisms intended to counteract diverse defenses. In this review, we use the two-spotted spider mite (TSSM), Tetranychus urticae (Koch) as an example of a generalist herbivore, as this chelicerate pest has a staggering number of plant hosts.We first establish that the ability of TSSM to adapt to marginal hosts underlies its polyphagy and agricultural pest status. We then highlight our understanding of direct plant defenses against spider mite herbivory and review recent advances in uncovering mechanisms of spider mite adaptations to them. Finally, we discuss the adaptation process itself, as it allows TSSM to overcome initially effective plant defenses. A highquality genome sequence and developing genetic tools, coupled with an ease of mite experimental selection to new hosts, make TSSM an outstanding systemto study the evolution of host range, mechanisms of pest xenobiotic resistance and plant-herbivore interactions. In addition, knowledge of plant defense mechanisms that affect mite fitness are of practical importance, as it can lead to development of new control strategies against this important agricultural pest. In parallel, understanding mechanisms of mite counter adaptations to these defenses is required to maintain the efficacy of these control strategies in agricultural practices. © 2017 The American Phytopathological Society.

RNA interference (RNAi) can be used for the protection against agricultural pests through the silencing of genes required for pest fitness. To assess the potential of RNAi approaches in the two-spotted spider mite, Tetranychus urticae, we compared 5 methods for the delivery of double-stranded RNA (dsRNA). These methods include mite feeding on either (i) leaves floating on a dsRNA solution, (ii) dsRNA-expressing plants, (iii) artificial diet supplemented with dsRNA, or (iv) dsRNA-coated leaves, and (v) mite soaking in a dsRNA solution. In all cases, the gene targeted for method validation was the Vacuolar-type H+-ATPase (TuVATPase), encoding a constitutively expressed ATP-driven proton pump located in the membrane. Down-regulation of TuVATPase increased mortality and/or reduced fecundity in all methods, but with variable efficiency. The most efficient methods for dsRNA delivery were direct soaking of mites in the dsRNA solution and mite feeding on dsRNA-coated leaves that mimics dsRNA application as a sprayable pesticide. Both resulted in a dark-body phenotype not observed in mites treated with a control dsRNA. Although with lower efficiency, dsRNA designed for TuVATPase silencing and expressed in transgenic Arabidopsis plants impacted the fitness of mites feeding on these plants. RNAi may thus be a valuable strategy to control spider mite populations, either as a sprayable pesticide or through transgenic crops. This comparative methodological study focusing on the induction of RNAi-based gene silencing in T. urticae paves the way for reverse genetics approaches in this model chelicerate system and prepares large-scale systematic RNAi screens as a first step towards the development of specific RNA-based pesticides. Such alternative molecules may help control spider mites that cause significant damages to crops and ornamental plant species, as well as other chelicerates detrimental to agriculture and health. © 2017 Suzuki et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

The two-spotted spider mite, Tetranychus urticae, is a chelicerate herbivore with an extremely wide host range and an extraordinary ability to develop pesticide resistance. Due to its responsiveness to natural and synthetic xenobiotics, the spider mite is becoming a prime pest herbivore model for studies of the evolution of host range, plant-herbivore interactions and mechanisms of xenobiotic resistance. The spider mite genome has been sequenced and its transcriptional responses to developmental and various biotic and abiotic cues have been documented. However, to identify biological and evolutionary roles of T. urticae genes and proteins, it is necessary to develop methods for the efficient manipulation of mite gene function or protein activity. Here, we describe protocols developed for the delivery of small molecules into spider mites. Starting with mite maintenance and the preparation of the experimental mite populations of developmentally synchronized larvae and adults, we describe 3 methods for delivery of small molecules including artificial diet, leaf coating, and soaking. The presented results define critical steps in these methods and demonstrate that they can successfully deliver tracer dyes into mites. Described protocols provide guidelines for high-throughput setups for delivery of experimental compounds that could be used in reverse genetics platforms to modulate gene expression or protein activity, or for screens focused on discovery of new molecules for mite control. In addition, described protocols could be adapted for other Tetranychidae and related species of economic importance such as Varroa, dust and poultry mites., Correction -- 15 Dec 2017: Suzuki T, España MU, Nunes MA, Zhurov V, Dermauw W, et al. (2017) Correction: Protocols for the delivery of small molecules to the two-spotted spider mite, Tetranychus urticae. PLOS ONE 12(12): e0190025.

Germband size in insects has played a central role in our understanding of insect patterning mechanisms and their evolution. The polarity of evolutionary change in insect patterning has been viewed so far as the unidirectional shift from the ancestral short germband patterning of basal hemimetabolous insects to the long germband patterning observed in most modern Holometabola. However, some orders of holometabolic insects display both short and long germband development, though the absence of a clear phylogenetic context does not permit definite conclusions on the polarity of change. Derived hymenoptera, that is, bees and wasps, represent a classical textbook example of long germband development. Yet, in some wasps putative short germband development has been described correlating with lifestyle changes, namely with evolution of endoparasitism and polyembryony. To address the potential reversion from long to short germband, we focused on the family Braconidae, which displays ancestral long germband development, and examined the derived polyembryonic braconid Macrocentrus cingulum. Using SEM analysis of M. cingulum embryogenesis coupled with analyses of embryonic patterning markers, we show that this wasp evolved short germband embryogenesis secondarily, in a way that is reminiscent of embryogenesis in the beetle Tribolium castaneum. This work shows that the evolution of germband size in insects is a reversible process that may correlate with other life-history traits and suggests broader implications on the mechanisms and evolvability of insect development.

Most molecular-genetic studies of plant defense responses to arthropod herbivores have focused on insects. However, plant-feeding mites are also pests of diverse plants, and mites induce different patterns of damage to plant tissues than do well-studied insects (e.g. lepidopteran larvae or aphids). The two-spotted spider mite (Tetranychus urticae) is among the most significant mite pests in agriculture, feeding on a staggering number of plant hosts. To understand the interactions between spider mite and a plant at the molecular level, we examined reciprocal genome-wide responses of mites and its host Arabidopsis (Arabidopsis thaliana). Despite differences in feeding guilds, we found that transcriptional responses of Arabidopsis to mite herbivory resembled those observed for lepidopteran herbivores. Mutant analysis of induced plant defense pathways showed functionally that only a subset of induced programs, including jasmonic acid signaling and biosynthesis of indole glucosinolates, are central to Arabidopsis’s defense to mite herbivory. On the herbivore side, indole glucosinolates dramatically increased mite mortality and development times. We identified an indole glucosinolate dose-dependent increase in the number of differentially expressed mite genes belonging to pathways associated with detoxification of xenobiotics. This demonstrates that spider mite is sensitive to Arabidopsis defenses that have also been associated with the deterrence of insect herbivores that are very distantly related to chelicerates. Our findings provide molecular insights into the nature of, and response to, herbivory for a representative of a major class of arthropod herbivores.

The genome of the phytophagous two-spotted spider mite Tetranychus urticae was recently sequenced, representing the first complete chelicerate genome, but also the first genome of a highly polyphagous agricultural pest. Genome analysis revealed the presence of an unexpected high number of cases of putative horizontal gene transfers, including a gene that encodes a cyanase or cyanate lyase. In this study we show by recombinant expression that the T. urticae cyanase remained functionally active after horizontal gene transfer and has a high affinity for cyanate. Cyanases were also detected in other plant parasitic spider mites species such as Tetranychus evansi and Panonychus citri, suggesting that an ancient gene transfer occurred before the diversification within the Tetranychidae family. To investigate the potential role of cyanase in the evolution of plant parasitic spider mites, we studied cyanase expression patterns in T. urticae in relation to host plant range and cyanogenesis, a common plant defense mechanism. Spider mites can alter cyanase expression levels after transfer to several new host plants, including the cyanogenic Phaseolus lunatus. However, the role of cyanase is probably not restricted to cyanide response, but likely to the plant nutritional quality as a whole. We finally discuss potential interactions between cyanase activity and pyrimidine and amino acid synthesis. © 2012 Elsevier Ltd.

The two-spotted spider mite, Tetranychus urticae Koch (Acari: Tetranychidae), is one of the most polyphagous herbivores feeding on cell contents of over 1100 plant species including more than 150 crops. It is being established as a model for chelicerate herbivores with tools that enable tracking of reciprocal responses in plant-spider mite interactions. However, despite their important pest status and a growing understanding of the molecular basis of interactions with plant hosts, knowledge of the way mites interface with the plant while feeding and the plant damage directly inflicted by mites is lacking. Here, utilizing histology and microscopy methods, we uncovered several key features of T. urticae feeding. By following the stylet path within the plant tissue, we determined that the stylet penetrates the leaf either in between epidermal pavement cells or through a stomatal opening, without damaging the epidermal cellular layer. Our recordings of mite feeding established that duration of the feeding event ranges from several minutes to more than half an hour, during which time mites consume a single mesophyll cell in a pattern that is common to both bean and Arabidopsis plant hosts. In addition, this study determined that leaf chlorotic spots, a common symptom of mite herbivory, do not form as an immediate consequence of mite feeding. Our results establish a cellular context for the plant-spider mite interaction that will support our understanding of the molecular mechanisms and cell signaling associated with spider mite feeding. © 2016 Bensoussan, Santamaria, Zhurov, Diaz, Grbi and Grbi.

The cys-loop ligand-gated ion channel (cysLGIC) super family of . Tetranychus urticae, the two-spotted spider mite, represents the largest arthropod cysLGIC super family described to date and the first characterised one within the group of chelicerates. Genome annotation, phylogenetic analysis and comparison of the cysLGIC subunits with their counterparts in insects reveals that the . T. urticae genome encodes for a high number of glutamate- and histamine-gated chloride channel genes (GluCl and HisCl) compared to insects. Three orthologues of the insect -aminobutyric acid (GABA)-gated chloride channel gene . Rdl were detected. Other cysLGIC groups, such as the nAChR subunits, are more conserved and have clear insect orthologues. Members of cysLGIC family mediate endogenous chemical neurotransmission and they are prime targets of insecticides. Implications for toxicology associated with the identity and specific features of . T. urticae family members are discussed. We further reveal the accumulation of known and novel mutations in different GluCl channel subunits (Tu_GluCl1 and Tu_GluCl3) associated with abamectin resistance in . T. urticae, and provide genetic evidence for their causality. Our study provides useful toxicological insights for the exploration of the . T. urticae cysLGIC subunits as putative molecular targets for current and future chemical control strategies. © 2012 Elsevier Ltd.

The complete genome of the two-spotted spider mite, Tetranychus urticae, has been reported. This is the first sequenced genome of a highly polyphagous and resistant agricultural pest. The question as to what the genome offers the community working on spider mite control is addressed.

Because of its importance to the arthropod exoskeleton, chitin biogenesis is an attractive target for pest control. This point is demonstrated by the economically important benzoylurea compounds that are in wide use as highly specific agents to control insect populations. Nevertheless, the target sites of compounds that inhibit chitin biogenesis have remained elusive, likely preventing the full exploitation of the underlying mode of action in pest management. Here, we show that the acaricide etoxazole inhibits chitin biogenesis in Tetranychus urticae (the two-spotted spider mite), an economically important pest. We then developed a population-level bulk segregant mapping method, based on high-throughput genome sequencing, to identify a locus for monogenic, recessive resistance to etoxazole in a field-collected population. As supported by additional genetic studies, including sequencing across multiple resistant strains and genetic complementation tests, we associated a nonsynonymous mutation in the major T. urticae chitin synthase ( CHS1 ) with resistance. The change is in a C-terminal transmembrane domain of CHS1 in a highly conserved region that may serve a noncatalytic but essential function. Our finding of a target-site resistance mutation in CHS1 shows that at least one highly specific chitin biosynthesis inhibitor acts directly to inhibit chitin synthase. Our work also raises the possibility that other chitin biogenesis inhibitors, such as the benzoylurea compounds, may also act by inhibition of chitin synthases. More generally, our genetic mapping approach should be powerful for high-resolution mapping of simple traits (resistance or otherwise) in arthropods.

The two-spotted spider mite, Tetranychus urticae, is a chelicerate herbivore with an extremely wide host range and an extraordinary ability to develop pesticide resistance. Due to its responsiveness to natural and synthetic xenobiotics, the spider mite is becoming a prime pest herbivore model for studies of the evolution of host range, plant-herbivore interactions and mechanisms of xenobiotic resistance. The spider mite genome has been sequenced and its transcriptional responses to developmental and various biotic and abiotic cues have been documented. However, to identify biological and evolutionary roles of T. urticae genes and proteins, it is necessary to develop methods for the efficient manipulation of mite gene function or protein activity. Here, we describe protocols developed for the delivery of small molecules into spider mites. Starting with mite maintenance and the preparation of the experimental mite populations of developmentally synchronized larvae and adults, we describe 3 methods for delivery of small molecules including artificial diet, leaf coating, and soaking. The presented results define critical steps in these methods and demonstrate that they can successfully deliver tracer dyes into mites. Described protocols provide guidelines for high-throughput setups for delivery of experimental compounds that could be used in reverse genetics platforms to modulate gene expression or protein activity, or for screens focused on discovery of new molecules for mite control. In addition, described protocols could be adapted for other Tetranychidae and related species of economic importance such as Varroa, dust and poultry mites.

Polyembryonic development, where multiple embryos are formed from a single zygote, evolved at least 15 times in six different phyla in animals. The mechanisms leading to polyembryony and the forces that shaped the evolution of the polyembryonic developmental program have remained poorly understood. Recent studies of the polyembryonic development in the endoparasitic wasp Copidosoma floridanum have revealed that the evolution of polyembryony is associated with the evolution of developmental novelties such as total cleavage, early specification of embryonic and extra-embryonic fates, and a specific cell proliferation phase. These changes cumulatively result in the formation of thousands of embryos from a single egg. Laser ablation studies and analysis of early cell fate specification have revealed that a single blastomere representing the progenitor of the primordial germ cell regulates the proliferation of the embryos. We propose that evolutionary changes in cell cleavage, cell interactions, and the cell-differentiation program, reminiscent of interactions between the germinal stem cell and stem cell niche in fly ovaries, underlies the evolution of polyembryony.

A major prerequisite to understanding the evolution of developmental programs includes an appreciation of gene function in a comparative context. RNA interference (RNAi) represents a powerful method for reverse genetics analysis of gene function. However, RNAi protocols exist for only a handful of arthropod species. To extend functional analysis in basal arthropods, we developed a RNAi protocol for the two-spotted spider mite Tetranychus urticae focusing on Distal-less (Dll), a conserved gene involved in appendage specification in metazoans. First, we describe limb morphogenesis in T. urticae using confocal and scanning electron microscopy. Second, we examine T. urticae Dll (Tu-Dll) mRNA expression patterns and correlate its expression with appendage development. We then show that fluorescently labeled double-stranded RNA (dsRNA) and short interfering RNA (siRNA) molecules injected into the abdomen of adult females are incorporated into the oviposited eggs, suggesting that dsRNA reagents can be systemically distributed in spider mites. Injection of longer dsRNA as well as siRNA induced canonical limb truncation phenotypes as well as the fusion of leg segments. Our data suggest that Dll plays a conserved role in appendage formation in arthropods and that such conserved genes can serve as reliable starting points for the development of functional protocols in nonmodel organisms.

International audience; The two-spotted spider mite Tetranychus urticae is one of the most significant mite pests in agriculture, feeding on more than 1,100 plant hosts, including model plants Arabidopsis thaliana and tomato, Solanum lycopersicum. Here, we describe timecourse tomato transcriptional responses to spider mite feeding and compare them with Arabidopsis in order to determine conserved and divergent defense responses to this pest. To refine the involvement of jasmonic acid (JA) in mite-induced responses and to improve tomato Gene Ontology annotations, we analyzed transcriptional changes in the tomato JA-signaling mutant defenseless1 (def-1) upon JA treatment and spider mite herbivory. Overlay of differentially expressed genes (DEG) identified in def-1 onto those from the timecourse experiment established that JA controls expression of the majority of genes differentially regulated by herbivory. Comparison of defense responses between tomato and Arabidopsis highlighted 96 orthologous genes (of 2,133 DEG) that were recruited for defense against spider mites in both species. These genes, involved in biosynthesis of JA, phenylpropanoids, flavonoids, and terpenoids, represent the conserved core of induced defenses. The remaining tomato DEG support the establishment of tomato-specific defenses, indicating profound divergence of spider mite–induced responses between tomato and Arabidopsis.

© 2014, Springer-Verlag Berlin Heidelberg. Metazoans are known to contain a limited, yet highly conserved, set of signal transduction pathways that instruct early developmental patterning mechanisms. Genomic surveys that have compared gene conservation in signal transduction pathways between various insects and Drosophila support the conclusion that these pathways are conserved in evolution. However, the degree to which individual components of signal transduction pathways vary among more divergent arthropods is not known. Here, we report our results of a survey of the genome of the two-spotted spider mite Tetranychus urticae, using a set of 294 Drosophila orthologs of genes that function in signal transduction. We find a third of all genes surveyed absent from the spider mite genome. We also identify several novel duplications that have not been previously reported for a chelicerate. In comparison with previous insect surveys, Tetranychus contains a decrease in overall gene conservation, as well as an unusual ratio of ligands to receptors and other modifiers. These findings suggest that gene loss and duplication among components of signal transduction pathways are common among arthropods and suggest that signal transduction pathways in arthropods are more evolutionarily labile than previously hypothesized., This research was carried out while R.M.P. and P.C.E was supported through NSF-IGERT grant DGE-0114420. R.M.P. was also supported through NIH Training Grant T32GM008659.

Polyembryonic development is a unique mode of metazoan development in which a single zygote generates multiple embryos by clonal proliferation1. The polyembryonic parasitic insect Copidosoma floridanum shows one of the most extreme cases of polyembryony, producing up to 2,000 embryos from a single egg. In addition, this wasp exhibits an unusual polyphenism, producing two morphologically distinct larval castes, termed precocious and reproductive, that develop clonally from the same zygote2. This form of development seems incompatible with a model of insect development in which maternal pre-patterning of the egg specifies embryonic axial polarity3. Here we show that maternal pre-patterning in the form of germ plasm creates cellular asymmetry at the four-cell stage embryo of Copidosoma that is perpetuated throughout development. Laser ablations of cells show that the cell inheriting the germ plasm regulates both the fate and proliferation of the reproductive caste. Thus, we have uncovered a new mechanism of caste specification, mediated by the regulatory capacity of a single cell. This study shows that the evolution of mammalian-like regulative development of an insect embryo relies on a novel cellular context that might ultimately enhance developmental plasticity.

Embryo segmentation has been studied extensively in the fruit fly,Drosophila. These studies have demonstrated that a mechanism acting with dual segment periodicity is required for correct patterning of the body plan in this insect, but the evolutionary origin of the mechanism, the pair-rule system, is unclear. We have examined the expression of the homologues of two Drosophila pair-rule genes, runt andpaired (Pax Group III), in segmenting embryos of the two-spotted spider mite (Tetranychus urticae Koch). Spider mites are chelicerates, a group of arthropods that diverged from the lineage leading toDrosophila at least 520 million years ago. In T. urticae,the Pax Group III gene Tu-pax3/7 was expressed during patterning of the prosoma, but not the opisthosoma, in a series of stripes which appear first in even numbered segments, and then in odd numbered segments. The miterunt homologue (Tu-run) in contrast was expressed early in a circular domains that resolved into a segmental pattern. The expression patterns of both of these genes also indicated they are regulated very differently from their Drosophila homologues. The expression pattern of Tu-pax3/7 lends support to the possibility that a pair-rule patterning mechanism is active in the segmentation pathways of chelicerates.

The two-spotted spider mite, Tetranychus urticae, is a ubiquitous polyphagous arthropod herbivore that feeds on a remarkably broad array of species, with more than 150 of economic value. It is a major pest of greenhouse crops, especially in Solanaceae and Cucurbitaceae (e.g., tomatoes, eggplants, peppers, cucumbers, zucchini) and greenhouse ornamentals (e.g., roses, chrysanthemum, carnations), annual field crops (such as maize, cotton, soybean, and sugar beet), and in perennial cultures (alfalfa, strawberries, grapes, citruses, and plums)1,2. In addition to the extreme polyphagy that makes it an important agricultural pest, T. urticae has a tendency to develop resistance to a wide array of insecticides and acaricides that are used for its control3-7. T. urticae is an excellent experimental organism, as it has a rapid life cycle (7 days at 27 °C) and can be easily maintained at high density in the laboratory. Methods to assay gene expression (including in situ hybridization and antibody staining) and to inactivate expression of spider mite endogenous genes using RNA interference have been developed8-10. Recently, the whole genome sequence of T. urticae has been reported, creating an opportunity to develop this pest herbivore as a model organism with equivalent genomic resources that already exist in some of its host plants (Arabidopsis thaliana and the tomato Solanum lycopersicum)11. Together, these model organisms could provide insights into molecular bases of plant-pest interactions. Here, an efficient method for quick and easy collection of a large number of adult female mites, their application on an experimental plant host, and the assessment of the plant damage due to spider mite feeding are described. The presented protocol enables fast and efficient collection of hundreds of individuals at any developmental stage (eggs, larvae, nymphs, adult males, and females) that can be used for subsequent experimental application.

The acaricides clofentezine, hexythiazox and etoxazole are commonly referred to as 'mite growth inhibitors', and clofentezine and hexythiazox have been used successfully for the integrated control of plant mite pests for decades. Although they are still important today, their mode of action has remained elusive. Recently, a mutation in chitin synthase 1 (CHS1) was linked to etoxazole resistance. In this study, we identified and investigated a Tetranychus urticae strain (HexR) harboring recessive, monogenic resistance to each of hexythiazox, clofentezine, and etoxazole. To elucidate if there is a common genetic basis for the observed cross-resistance, we adapted a previously developed bulk segregant analysis method to map with high resolution a single, shared resistance locus for all three compounds. This finding indicates that the underlying molecular basis for resistance to all three compounds is identical. This locus is centered on the CHS1 gene, and as supported by additional genetic and biochemical studies, a non-synonymous variant (I1017F) in CHS1 associates with resistance to each of the tested acaricides in HexR. Our findings thus demonstrate a shared molecular mode of action for the chemically diverse mite growth inhibitors clofentezine, hexythiazox and etoxazole as inhibitors of an essential, non-catalytic activity of CHS1. Given the previously documented cross-resistance between clofentezine, hexythiazox and the benzyolphenylurea (BPU) compounds flufenoxuron and cycloxuron, CHS1 should be also considered as a potential target-site of insecticidal BPUs. © 2014 Elsevier Ltd.

Cyanogenic glucosides are among the most widespread defense chemicals of plants. Upon plant tissue disruption, these glucosides are hydrolyzed to a reactive hydroxynitrile that releases toxic hydrogen cyanide (HCN). Yet many mite and lepidopteran species can thrive on plants defended by cyanogenic glucosides. The nature of the enzyme known to detoxify HCN to β-cyanoalanine in arthropods has remained enigmatic. Here we identify this enzyme by transcriptome analysis and functional expression. Phylogenetic analysis showed that the gene is a member of the cysteine synthase family horizontally transferred from bacteria to phytophagous mites and Lepidoptera. The recombinant mite enzyme had both β-cyanoalanine synthase and cysteine synthase activity but enzyme kinetics showed that cyanide detoxification activity was strongly favored. Our results therefore suggest that an ancient horizontal transfer of a gene originally involved in sulfur amino acid biosynthesis in bacteria was co-opted by herbivorous arthropods to detoxify plant produced cyanide. DOI: http://dx.doi.org/10.7554/eLife.02365.001, eLife digest Hydrogen cyanide is a poison that is deadly for most forms of life. Also known as prussic acid, it has killed countless humans throughout history in accidents and during the Holocaust. Hydrogen cyanide is also used by plants to defend themselves against insects and other herbivorous animals. Many plants produce chemicals called cyanogenic glycosides that can be converted into hydrogen cyanide when the plant is eaten. This is an ancient and efficient defense against all sorts of herbivores, including humans. For instance, cassava is a key source of food in sub-Saharan Africa and South America, but it contains cyanogenic glucosides and is highly toxic if eaten in unprocessed form. However, some insects and mites can thrive on cyanogenic plants, often to the extent of becoming pests on these plants. Certain moths, such as burnet moths, have gone further and now depend on cyanogenic glucosides for their own defenses against predators such as birds. How these mites and insects are capable of fending off cyanide toxicity has long remained a mystery. Now Wybouw et al. have identified a mite enzyme that detoxifies hydrogen cyanide to produce a compound called beta-cyanoalanine. Remarkably, the DNA that encodes this enzyme did not evolve in animals but originally belonged to a bacterium. Wybouw et al. show that the gene was transferred to the genome of the spider mite Tetranychus urticae perhaps a few hundred million years ago. An equivalent gene was also found in moths and butterflies, which explains why these insects can thrive on plants that produce hydrogen cyanide. This lateral gene transfer from bacteria to animals is a remarkable coalition of two kingdoms against another, and illustrates a new aspect of the chemical warfare between plants and animals. This study also increases our awareness of the importance of laterally transferred genes in the genomes of higher organisms. DOI: http://dx.doi.org/10.7554/eLife.02365.002

Summary In insects, the regulation of embryonic development has been intensively studied in model species like Drosophila melanogaster. Previous comparative studies have suggested that the developmental processes documented in Drosophila well describe embryogenesis of holometabolous insects generally. However, there have been few attempts to take into account how life history has influenced insect embryogenesis or to characterize early development of species with life histories fundamentally different from flies. Our studies of advanced insects in the order Hymenoptera suggest that punctuated shifts in life history can profoundly influence these events. In particular, alterations associated with the evolution of endoparasitism argue that departures from the fly paradigm may occur commonly among insects that develop under conditions different from typical terrestrial species.

Plants produce a wide range of allelochemicals to defend against herbivore attack, and generalist herbivores have evolved mechanisms to avoid, sequester, or detoxify a broad spectrum of natural defense compounds. Successful arthropod pests have also developed resistance to diverse classes of pesticides and this adaptation is of critical importance to agriculture. To test whether mechanisms to overcome plant defenses predispose the development of pesticide resistance, we examined adaptation of the generalist two-spotted spider mite, Tetranychus urticae, to host plant transfer and pesticides. T. urticae is an extreme polyphagous pest with more than 1,100 documented hosts and has an extraordinary ability to develop pesticide resistance. When mites from a pesticidesusceptible strain propagated on bean were adapted to a challenging host (tomato), transcriptional responses increased over time with ∿7.5% of genes differentially expressed after five generations. Whereas many genes with altered expression belonged to known detoxification families (like P450 monooxygenases), new gene families not previously associated with detoxification in other herbivores showed a striking response, including ring-splitting dioxygenase genes acquired by horizontal gene transfer. Strikingly, transcriptional profiles of tomato-adapted mites resembled those of multipesticide-resistant strains, and adaptation to tomato decreased the susceptibility to unrelated pesticide classes. Our findings suggest key roles for both an expanded environmental response gene repertoire and transcriptional regulation in the life history of generalist herbivores. They also support a model whereby selection for the ability to mount a broad response to the diverse defense chemistry of plants predisposes the evolution of pesticide resistance in generalists., This work was supported by FWO Grants 3G061011 (to T.V.L.) and 3G009312 (to T.V.L. and L.T.), Ghent University Special Research Fund Grant 01J13711 (to T.V.L. and L.T.), the Government of Canada through Genome Canada and the Ontario Genomics Institute Grant OGI-046 (to M.G.), and a University of Utah Funding Incentive Seed Grant (to R.M.C.). N.W. was supported by the Institute for the Promotion of Innovation by Science and Technology in Flanders (IWT, Grant IWT/SB/101451). Part of this research was supported by the “THALIS” project 377301, Operational Programme “Education and Life-Long Learning,” which is cofunded by the European Social Fund and National Resources.

Copidosoma floridanum is a polyembryonic wasp that undergoes total cleavage of the egg followed by proliferation of blastomeres to produce up to 2,000 embryos from a single egg. This unusual mode of development raises several questions about how axial polarity is established in individual embryonic primordia. By examining embryonic development of larvae with duplicated structures (conjoined larvae), we determined that conjoined larvae form by mislocalization of two embryonic primordia to a common chamber of the extraembryonic membrane that surrounds individual embryos. Analysis of an anterior marker, Distalless, in mislocalized early embryos indicated that anterior structures form independently of one another. This suggests each embryonic primordium has some intrinsic polarity. However, during germband extension embryos usually fuse in register with each other, resulting in conjoined larvae with heads facing each other. Analysis of the posterior segmental marker, Engrailed, in conjoined embryos suggested that fusion in register initiates during germband extension. Thus, even though embryonic primordia initially have a random axial orientation, conjoined larvae usually possess a common orientation due to reorientation during germband extension. These observations suggest that differential cellular affinities during segmentation play an important role in embryo fusion.