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Before Darwin many biologists considered organic forms to be immutable natural forms or types which like inorganic forms such as atoms or crystals are part of a changeless world order and determined by physical law. Adaptations were viewed as secondary modifications of these 'crystal like' abstract afunctional 'givens of physics.' We argue here that much of the emerging picture of biological order in the subcellular realm resembles closely the pre-Darwinian conception of nature. We point out that in the subcellular realm, between nano and micrometers, physical law necessarily plays a far more significant role in organizing matter than in the familiar 'Darwinian world' between millimeters and meters (where matter can be arranged into almost any contingent artifactual arrangement we choose, as witness Lego toys, watches or jumbo jets). Consequently, when deploying matter into complex structures in the subcellular realm the cell must necessarily make extensive use of natural forms-such as the protein and RNA folds, microtubular forms and tensegrity structures-which like atoms or crystals self-organize under the direction of physical law into what are essentially 'pre-Darwinian' afunctional abstract molecular architectures in which adaptations are trivial secondary modifications of what are evidently primary givens of physics.

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.

Vasa is a widely conserved germline marker, both in vertebrates and invertebrates. We identify a vasa orthologue, Sgvasa, and use it to study germline development in the grasshopper Schistocerca gregaria, a species in which no germ plasm has been identified. In adults, Sgvasa is specifically expressed in the ovary and testis. It is expressed at high levels during early oogenesis, but no detectable vasa RNA and little Vasa protein are present in mature unlaid eggs. None appears to be localized to any defined region of the egg cortex, suggesting that germline specification may not depend on maternal germ plasm expressing vasa. Vasa protein is expressed in most cleavage energids as they reach the egg surface and persists at high levels in most cells aggregating to form the embryonic primordium. However, after gastrulation, Vasa protein persists only in extraembryonic membranes and in cells at the outer margin of the late heart-stage embryo. In the embryo, it then become restricted to cells at the dorsal margin of the forming abdomen. In older embryos, these Vasa-positive cells move toward the midline; Vasa protein accumulates asymmetrically in their cytoplasm, a pattern closely resembling that of germ cells in late embryonic gonads. Thus, we suggest that the Vasa-stained cells in the abdominal margin are germ cells, as proposed by Nelson (1934), and not cardioblasts, as has been proposed by others.

Honeybees are an important component of modern agricultural systems, and a fascinating and scientifically engrossing insect. Honeybees are not commonly used as model systems for understanding development in insects despite their importance in agriculture. Honeybee embryogenesis, while being superficially similar to Drosophila, is molecularly very different, especially in axis formation and sex determination. In later development, much of honeybee biology is modified by caste development, an as yet poorly understood, but excellent, system to study developmental plasticity. In adult stages, developmental plasticity of the ovaries, related to reproductive constraint exhibits another aspect of plasticity. Here they review the tools, current knowledge and opportunities in honeybee developmental biology, and provide an updated embryonic staging scheme to support future studies.

The possibility that environmentally induced epigenetic marks are passed transgenerationally through the female germline has implications for our understanding of development, disease, and evolution. Although it remains to be seen how widespread and prevalent this mechanism is amongst animal taxa, the handful of studies that we do have provides enough evidence to support the existence of an evolved mechanism for the transmission of environmental information through the maternal germline to offspring.

Background: Female larvae of the honeybee (Apis mellifera) develop into either queens or workers depending on nutrition. This nutritional stimulus triggers different developmental trajectories, resulting in adults that differ from each other in physiology, behaviour and life span. Results: To understand how these trajectories are established we have generated a comprehensive atlas of gene expression throughout larval development. We found substantial differences in gene expression between worker and queen-destined larvae at 6 hours after hatching. Some of these early changes in gene expression are maintained throughout larval development, indicating that caste-specific developmental trajectories are established much earlier than previously thought. Within our gene expression data we identified processes that potentially underlie caste differentiation. Queen-destined larvae have higher expression of genes involved in transcription, translation and protein folding early in development with a later switch to genes involved in energy generation. Using RNA interference, we were able to demonstrate that one of these genes, hexamerin 70b, has a role in caste differentiation. Both queen and worker developmental trajectories are associated with the expression of genes that have alternative splice variants, although only a single variant of a gene tends to be differentially expressed in a given caste. Conclusions: Our data, based on the biases in gene expression early in development together with published data, supports the idea that caste development in the honeybee consists of two phases; an initial biased phase of development, where larvae can still switch to the other caste by differential feeding, followed by commitment to a particular developmental trajectory.

Epigenetic mechanisms are proposed as an important way in which the genome responds to the environment. Epigenetic marks, including DNA methylation and Histone modifications, can be triggered by environmental effects, and lead to permanent changes in gene expression, affecting the phenotype of an organism. Epigenetic mechanisms have been proposed as key in plasticity, allowing environmental exposure to shape future gene expression. While we are beginning to understand how these mechanisms have roles in human biology and disease, we have little understanding of their roles and impacts on ecology and evolution. In this review, we discuss different types of epigenetic marks, their roles in gene expression and plasticity, methods for assaying epigenetic changes, and point out the future advances we require to understand fully the impact of this field.

The C termini of N-methyl-d-aspartate (NMDA) receptor NR2 subunits are thought to play a major role in the molecular establishment of memory across the Bilateria, via the phenomenon known as long-term potentiation (LTP). Despite their long history of use as models in the study of memory, the expression and structure of the NR2 subunit in the Lophotrochozoa has remained uncategorized. Here, we report the phylogenic relationships of NR subunits across the Bilateria, and the cloning and in situ analysis of expression of NMDA NR1 and NR2 subunits in the monogont rotifer Brachionus plicatilis. RNA in situ hybridization suggests expression of NMDA receptor subunits in B. plicatilis is neural, consistent with expression observed in other species, and ours is the first report confirming NR2 expression in the lophotrochozoan clade. However, the single NR2 subunit identified in B. plicatilis was found to lack the long C terminal domain found in vertebrates, which is believed to modulate LTP. Further investigation revealed that mollusc and annelid NR2 subunits possess long intracellular C terminal domains. As data from molluscs (and particularly Aplysia californica) are the basis for much of our understanding of LTP, understanding how these diverse lophotrochozoan C termini function in vivo will have many implications for how we consider the evolution of the molecular control of learning and memory across the Metazoa as a whole and interpret the results of experiments into this vital component of cognition.

International audience; Many genes are differentially regulated by caste development in the honeybee. Identifying and understanding these differences is key to discovering the mechanisms underlying this process. To identify these gene expression differences requires robust methods to measure transcript abundance. RT-qPCR is currently the gold standard to measure gene expression, but requires stable reference genes to compare gene expression changes. Such reference genes have not been established for honeybee caste development. Here, we identify and test potential reference genes that have stable expression throughout larval development between the two female castes. In this study, 15 candidate reference genes were examined to identify the most stable reference genes. Three algorithms (GeNorm, Bestkeeper and NormFinder) were used to rank the candidate reference genes based on their stability between the castes throughout larval development. Of these genes Ndufa8 (the orthologue of a component of complex one of the mitochondrial electron transport chain) and Pros54 (orthologous to a component of the 26S proteasome) were identified as being the most stable. When these two genes were used to normalise expression of two target genes (previously found to be differentially expressed between queen and worker larvae by microarray analysis) they were able to more accurately detect differential expression than two previously used reference genes (awd and RpL12). The identification of these novel reference genes will be of benefit to future studies of caste development in the honeybee.

International audience; The honeybee haplodiploid sex determination system depends on genetic variation at the complementary sex-determiner (csd) locus. In closed populations of honeybees, especially those undergoing selective breeding, the number of csd alleles can drop such that brood viability is affected. Here we present two polymerase chain reaction tests that allow the discrimination of csd alleles. Such tests should find utility in bee breeding programmes allowing the tracking and maintenance of csd alleles through successive generations.

Pair-rule genes are a class of segmentation genes first identified in Drosophila melanogaster. In Drosophila, these genes act to translate non-periodic information produced by the overlapping patterns of gap gene expression into patterns of gene expression in every other segment. While pair-rule genes are, for the most part, conserved in metazoans, their function in pair-rule patterning is not. Many of these genes do, however, regulate segmentation in arthropods and do so with dual-segment periodicity. Here we examine the expression and function of honeybee orthologues of Drosophila pair-rule genes. Knockdown of the expression of these genes leads to extensive patterning defects, implying that they act in early patterning, as well as segmentation in honeybee embryos. We show that these pair-rule gene orthologues indeed regulate the expression of honeybee maternal and gap genes implying roles in maternal patterning of the honeybee embryo.

Axis formation is a key step in development, but studies indicate that genes involved in insect axis formation are relatively fast evolving. Orthodenticle genes have conserved roles, often with hunchback, in maternal anterior patterning in several insect species. We show that two orthodenticle genes, otd1 and otd2, and hunchback act as maternal anterior patterning genes in the honeybee (Apis mellifera) but, unlike other insects, act to pattern the majority of the anteroposterior axis. These genes regulate the expression domains of anterior, central and posterior gap genes and may directly regulate the anterior gap gene giant. We show otd1 and hunchback also influence dorsoventral patterning by regulating zerknült (zen) as they do in Tribolium, but that zen does not regulate the expression of honeybee gap genes. This suggests that interactions between anteroposterior and dorsal-ventral patterning are ancestral in holometabolous insects. Honeybee axis formation, and the function of the conserved anterior patterning gene orthodenticle, displays unique characters that indicate that, even when conserved genes pattern the axis, their regulatory interactions differ within orders of insects, consistent with relatively fast evolution in axis formation pathways.

SUMMARY In Drosophila it is well established that signaling between the germline and surrounding follicle cells establishes the axes of the future embryo and is required for patterning of the eggshell. However, little is known about how this is achieved in other insects. Genome sequencing studies imply that maternal axis determination may be rapidly evolving, as a number of Drosophila maternal patterning genes are absent from the genomes of other insects. We have examined the distribution and function of six developmental signaling pathways present, and active, in honeybee ovarioles. We have confirmed an evolutionarily conserved role for transforming growth factor-α-epidermal growth factor receptor signaling in dorsal-ventral (DV) patterning. We also found evidence for the involvement of Dpp/Mad and JNK-MAPK pathways in DV patterning, unlike Drosophila. Several of these pathways are also active in the germarium, implicating them in germ and somatic stem cell maintenance and proliferation, similar to their activities in Drosophila ovaries.

The Drosophila Enhancer of split complex [E(spl)-C] is a remarkable complex of genes many of which are effectors or modulators of Notch signaling. The complex contains different classes of genes including four bearded genes and seven basic helix-loop-helix (bHLH) genes. We examined the evolution of this unusual complex by identifying bearded and bHLH genes in the genome sequences of Arthropods. We find that a four-gene E(spl)-C, containing three bHLH genes and one bearded gene, is an ancient component of the genomes of Crustacea and Insects. The complex is well conserved in insects but is highly modified in Drosophila, where two of the ancestral genes of the complex are missing, and the remaining two have been duplicated multiple times. Through examining the expression of E(spl)-C genes in honeybees, aphids, and Drosophila, we determined that the complex ancestrally had a role in Notch signaling. The expression patterns of genes found inserted into the complex in some insects, or that of ancestral E(spl)-C genes that have moved out of the complex, imply that the E(spl)-C is a genomic domain regulated as a whole by Notch signaling. We hypothesize that the E(spl)-C is a Notch-regulated genomic domain conserved in Arthropod genomes for around 420 million years. We discuss the consequence of this conserved domain for the recruitment of novel genes into the Notch signaling cascade.

INTRODUCTIONHoneybees (Apis mellifera) are distributed throughout the world and have a critical role in the pollination of crops and pasture. Honeybees have an intricate social structure and display complex behavioral traits such as sociality, language, division of labor, and exceptional feats of learning and memory. Honeybees also exhibit polyphenism, whereby the females can either develop as workers or as morphologically distinct, reproductively active queens. Although historical studies of honeybee biology have been performed, the recent advent of the genome sequence and a number of other modern tools have made dissection of the molecular details of this remarkable insect possible.

INTRODUCTIONThis protocol outlines how to perform in situ hybridization on honeybee embryos, adult brains, or ovaries to detect the expression pattern of a targeted transcript. The tissue is digested by proteinase K to promote probe penetration, and then prehybridized to block nonspecific binding of the probe.

INTRODUCTIONThis protocol describes the preparation and staining of honeybee embryos for immunohistochemistry. An antigen retrieval step is required in order to restore antigens that have become masked by the long fixation step required for honeybee embryos.

In Drosophila, the maternal Torso terminal signaling pathway activates expression of the gene tailless (tll), which is required for the patterning of anterior and posterior termini. We cloned the honeybee orthologue of tll (Am-tll) and found that embryonic expression of Am-tll resembles that of Drosophila, with expression in triangular anterior dorsal–lateral domains and a posterior cap. Functional studies revealed that Am-tll has an essential role in patterning the posterior terminal segments and the brain, similar to the activity of tll in other insects. As the honeybee genome lacks many of the components of the Torso pathway required for terminal patterning, we investigated the regulation of honeybee tailless (Am-tll). Am-tll is expressed maternally and, in the honeybee ovary, Am-tll mRNA becomes localized to the dorsal side of the oocyte, a process requiring the actin cytoskeleton. This RNA becomes redistributed in early embryos to a posterior domain. We also show that the activation of the anterior domain of Am-tll is dependent on honeybee orthodenticle-1. Together these findings indicate major differences in post-transcriptional regulation of tailless in the honeybee compared to other insects but that this regulation leads to a conserved expression pattern. These results provide an example of an early event in development evolving and yet still producing a conserved output for the rest of development to build upon.

Background Gene clusters, such as the Hox gene cluster, are known to have critical roles in development. In eukaryotes gene clusters arise primarily by tandem gene duplication and divergence. Genes within a cluster are often co-regulated, providing selective pressure to maintain the genome organisation, and this co-regulation can result in temporal or spatial co-linearity of gene expression. It has been previously noted that in Drosophila melanogaster, three of the four runt-domain (RD) containing genes are found in a relatively tight cluster on chromosome 1, raising the possibility of a putative functional RD gene cluster in D. melanogaster. Results To investigate the possibility of such a gene cluster, orthologues of the Drosophila melanogaste r RD genes were identified in several endopterygotan insects, two exopterygotan insects and two non-insect arthropods. In all insect species four RD genes were identified and orthology was assigned to the Drosophila sequences by phylogenetic analyses. Although four RD genes were found in the crustacean D. pulex, orthology could not be assigned to the insect sequences, indicating independent gene duplications from a single ancestor following the split of the hexapod lineage from the crustacean lineage. In insects, two chromosomal arrangements of these genes was observed; the first a semi-dispersed cluster, such as in Drosophila, where lozenge is separated from the core cluster of three RD genes often by megabases of DNA. The second arrangement was a tight cluster of the four RD genes, such as in Apis mellifera. This genomic organisation, particularly of the three core RD genes, raises the possibility of shared regulatory elements. In situ hybridisation of embryonic expression of the four RD genes in Drosophila melanogaster and the honeybee A. mellifera shows no evidence for either spatial or temporal co-linearity of expression during embryogenesis. Conclusion All fully sequenced insect genomes contain four RD genes and orthology can be assigned to these genes based on similarity to the D. melanogaster protein sequences. Examination of the genomic organisation of these genes provides evidence for a functional RD gene cluster. RD genes from non-insect arthropods are also clustered, however the lack of orthology between these and insect RD genes suggests this cluster is likely to have resulted from a duplication event independent from that which created the insect RD gene cluster. Analysis of embryonic RD gene expression in two endopterygotan insects, A. mellifera and D. melanogaster, did not show evidence for coordinated gene expression, therefore while the functional significance of this gene cluster remains unknown its maintenance during insect evolution implies some functional significance to the cluster.

Mismatch: Why Our World No Longer Fits Our Bodies by Peter Gluckman & Mark Hanson Oxford University Press, Oxford, UK 272 pp, $30/£17 ISBN 9780192806833 ![][1] In recent years, studies of evolution and development have flourished, providing new insights into the mechanisms of evolution. One area that remains relatively unexplored, however, is the impact of evolution and development on human health. In Mismatch: Why Our World No Longer Fits Our Bodies , Peter Gluckman and Mark Hanson grapple with this idea to provide an ‘evo–devo’ model of many of the problems currently stalking the populations of the developed and developing worlds. Gluckman and Hanson both have distinguished research careers focusing on ‘predictive adaptive response’—the idea that the mammalian fetus uses information from the maternal environment to predict its postnatal environment and then changes its phenotype in response. The ‘mismatch’ of the book title refers to the idea that a mismatch between these predictions and the postnatal environment can lead to disease and other problems. The authors extend this idea and discuss more generally the idea of mismatch between humans and their environment as a … [1]: /embed/graphic-1.gif

The current insect genome sequencing projects provide an opportunity to extend studies of the evolution of developmental genes and pathways in insects. In this paper we examine the conservation and divergence of genes and developmental processes between Drosophila and the honey bee; two holometabolous insects whose lineages separated ∼300 million years ago, by comparing the presence or absence of 308 Drosophila developmental genes in the honey bee. Through examination of the presence or absence of genes involved in conserved pathways (cell signaling, axis formation, segmentation and homeobox transcription factors), we find that the vast majority of genes are conserved. Some genes involved in these processes are, however, missing in the honey bee. We have also examined the orthology of Drosophila genes involved in processes that differ between the honey bee and Drosophila. Many of these genes are preserved in the honey bee despite the process in which they act in Drosophila being different or absent in the honey bee. Many of the missing genes in both situations appear to have arisen recently in the Drosophila lineage, have single known functions in Drosophila, and act early in developmental pathways, while those that are preserved have pleiotropic functions. An evolutionary interpretation of these data is that either genes with multiple functions in a common ancestor are more likely to be preserved in both insect lineages, or genes that are preserved throughout evolution are more likely to co-opt additional functions.

BackgroundStudies of specification of germ-cells in insect embryos has indicated that in many taxa the germ cells form early in development, and their formation is associated with pole plasm, germ plasm or an organelle called the oosome. None of these morphological features associated with germ cell formation have been identified in the HoneybeeApis mellifera. In this study I report the cloning and expression analysis of Honeybee homologues ofvasaandnanos, germ cell markers in insects and other animals.ResultsApis vasaandnanosRNAs are present in early honeybee embryos, but the RNAs clear rapidly, without any cells expressing these germ cell markers past stage 2. These genes are then only expressed in a line of cells in the abdomen from stage 9 onwards. These cells are the developing germ cells that are moved dorsally by dorsal closure and are placed in the genital ridge.ConclusionThis study of the expression of germ cell markers in the honeybee implies that in this species either germ cells are formed by an inductive event, late in embryogenesis, or they are formed early in development in the absence ofvasaandnanosexpression. This contrasts with germ cell development in other members of the Hymenoptera, Diptera and Lepidoptera.

Ce travail résulte de la collaboration de très nombreux chercheurs. Seuls les auteurs de la rubrique Physical and Genetic Mapping sont cités explicitement.; Here we report the genome sequence of the honeybee Apis mellifera, a key model for social behaviour and essential to global ecology through pollination. Compared with other sequenced insect genomes, the A. mellifera genome has high A1T and CpG contents, lacks major transposon families, evolves more slowly, and is more similar to vertebrates for circadian rhythm, RNA interference and DNA methylation genes, among others. Furthermore, A.mellifera has fewer genes for innate immunity, detoxification enzymes, cuticle-forming proteins and gustatory receptors, more genes for odorant receptors, and novel genes for nectar and pollen utilization, consistent with its ecology and social organization. Compared to Drosophila, genes in early developmental pathways differ in Apis, whereas similarities exist for functions that differ markedly, such as sex determination, brain function and behaviour. Population genetics suggests a novel African origin for the species A.mellifera and insights into whether Africanized bees spread throughout the New World via hybridization or displacement

Myriapods (e.g., centipedes and millipedes) display a simple homonomous body plan relative to other arthropods. All members of the class are terrestrial, but they attained terrestriality independently of insects. Myriapoda is the only arthropod class not represented by a sequenced genome. We present an analysis of the genome of the centipede Strigamia maritima. It retains a compact genome that has undergone less gene loss and shuffling than previously sequenced arthropods, and many orthologues of genes conserved from the bilaterian ancestor that have been lost in insects. Our analysis locates many genes in conserved macro-synteny contexts, and many small-scale examples of gene clustering. We describe several examples where S. maritima shows different solutions from insects to similar problems. The insect olfactory receptor gene family is absent from S. maritima, and olfaction in air is likely effected by expansion of other receptor gene families. For some genes S. maritima has evolved paralogues to generate coding sequence diversity, where insects use alternate splicing. This is most striking for the Dscam gene, which in Drosophila generates more than 100,000 alternate splice forms, but in S. maritima is encoded by over 100 paralogues. We see an intriguing linkage between the absence of any known photosensory proteins in a blind organism and the additional absence of canonical circadian clock genes. The phylogenetic position of myriapods allows us to identify where in arthropod phylogeny several particular molecular mechanisms and traits emerged. For example, we conclude that juvenile hormone signalling evolved with the emergence of the exoskeleton in the arthropods and that RR-1 containing cuticle proteins evolved in the lineage leading to Mandibulata. We also identify when various gene expansions and losses occurred. The genome of S. maritima offers us a unique glimpse into the ancestral arthropod genome, while also displaying many adaptations to its specific life history., Author Summary Arthropods are the most abundant animals on earth. Among them, insects clearly dominate on land, whereas crustaceans hold the title for the most diverse invertebrates in the oceans. Much is known about the biology of these groups, not least because of genomic studies of the fruit fly Drosophila, the water flea Daphnia, and other species used in research. Here we report the first genome sequence from a species belonging to a lineage that has previously received very little attention—the myriapods. Myriapods were among the first arthropods to invade the land over 400 million years ago, and survive today as the herbivorous millipedes and venomous centipedes, one of which—Strigamia maritima—we have sequenced here. We find that the genome of this centipede retains more characteristics of the presumed arthropod ancestor than other sequenced insect genomes. The genome provides access to many aspects of myriapod biology that have not been studied before, suggesting, for example, that they have diversified receptors for smell that are quite different from those used by insects. In addition, it shows specific consequences of the largely subterranean life of this particular species, which seems to have lost the genes for all known light-sensing molecules, even though it still avoids light.

Light stimuli produce graded hyperpolarizations of the photoreceptor plasma membrane and an associated decrease in a voltagegated calcium channel conductance that mediates release of glutamate neurotransmitter. The Ca v 1.4 channel is thought to be involved in this process. The CACNA1F gene encodes the poreforming subunit of the Ca v 1.4 channel and various mutations in CACNA1F cause X-linked incomplete congenital stationary night blindness (CSNB2). The molecular mechanism of the pathology underlying the CSNB2 phenotype remains to be established. Recent clinical investigations of a New Zealand family found a severe visual disorder that has some clinical similarities to, but is clearly distinct from, CSNB2. Here, we report investigations into the molecular mechanism of the pathology of this condition. Molecular genetic analyses identified a previously undescribed nucleotide substitution in CACNA1F that is predicted to encode an isoleucine to threonine substitution at CACNA1F residue 745. The I745T CACNA1F allele produced a remarkable approximately –30-mV shift in the voltage dependence of Ca v 1.4 channel activation and significantly slower inactivation kinetics in an expression system. These findings imply that substitution of this wild-type residue in transmembrane segment IIS6 may have decreased the energy required to open the channel. Collectively, these findings suggest that a gain-of-function mechanism involving increased Ca v 1.4 channel activity is likely to cause the unusual phenotype.

Pax group III genes are involved in a number of processes during insect segmentation. In Drosophila melanogaster, three genes, paired, gooseberry and gooseberry-neuro, regulate segmental patterning of the epidermis and nervous system. Paired acts as a pair-rule gene and gooseberry as a segment polarity gene. Studies of Pax group III genes in other insects have indicated that their expression is a good marker for understanding the underlying molecular mechanisms of segmentation. We have cloned three Pax group III genes from the honeybee (Apis mellifera) and examined their relationships to other insect Pax group III genes and their expression patterns during honeybee segmentation. The expression pattern of the honeybee homologue of paired is similar to that of paired in Drosophila, but its expression is modulated by anterior-posterior temporal patterning similar to the expression of Pax group III proteins in Tribolium. The expression of the other two Pax group III genes in the honeybee indicates that they also act in segmentation and nervous system development, as do these genes in other insects.

Background: A key early step in embryogenesis is the establishment of the major body axes; the dorsal-ventral (DV) and anterior-posterior (AP) axes. Determination of these axes in some insects requires the function of different sets of signalling pathways for each axis. Patterning across the DV axis requires interaction between the Toll and Dpp/TGF-β pathways, whereas patterning across the AP axis requires gradients of bicoid/orthodenticle proteins and the actions of a hierarchy of gene transcription factors. We examined the expression and function of Toll and Dpp signalling during honeybee embryogenesis to assess to the role of these genes in DV patterning. Results: Pathway components that are required for dorsal specification in Drosophila are expressed in an AP-restricted pattern in the honeybee embryo, including Dpp and its receptor Tkv. Components of the Toll pathway are expressed in a more conserved pattern along the ventral axis of the embryo. Late-stage embryos from RNA interference (RNAi) knockdown of Toll and Dpp pathways had both DV and AP patterning defects, confirmed by staining with Am-sna, Am-zen, Am-eve ,a ndAm-twi at earlier stages. We also identified two orthologues of dorsal in the honeybee genome, with one being expressed during embryogenesis and having a minor role in axis patterning, as determined by RNAi and the other expressed during oogenesis. Conclusions: We found that early acting pathways (Toll and Dpp) are involved not only in DV patterning but also AP patterning in honeybee embryogenesis. Changes to the expression patterns and function of these genes may reflect evolutionary changes in the placement of the extra-embryonic membranes during embryogenesis with respect to the AP and DV axes.

Although the molecular pathways that pattern the early embryo of Drosophila melanogaster are well understood, how these pathways differ in other types of insect embryo remains largely unknown. We have examined the expression of three markers of early patterning in the embryo of the African plague locust Schistocerca gregaria, an orthopteran insect that displays a mode of embryogenesis very different from that of Drosophila. Transcripts of the caudal gene are expressed maternally and are present in all cells that aggregate to form the early embryonic rudiment. First signs of a posterior-to-anterior gradient in the levels of caudal transcript appear in the early heart-stage embryo, shortly before gastrulation. This gradient rapidly resolves to a defined expression domain marking segment A11. The decapentaplegic (dpp) gene, which encodes a transforming growth factor β family ligand, is first expressed in a circle of cells that delimit the margins of the embryonic primordium, where embryonic and extra-embryonic tissues abut. Patterned transcription of wingless reveals that the first segments are delineated in the Schistocerca embryo substantially earlier than previously thought, at least 14-16 hours before the onset of engrailed expression. By the late heart-stage, gnathal and thoracic segments are all defined. Thus, with respect to the molecular patterning of segments, the short germ Schistocerca embryo differs little from intermediate germ embryos. The expression of these marker genes suggests that embryonic pattern formation in the grasshopper occurs as cells move together to form the blastodisc.

In insects, a key step in the early patterning of the egg is to distinguish the primordium of the embryo proper from those regions that will form extra-embryonic membranes. In Drosophila, where these processes are well understood, the structure of the extra-embryonic membranes is highly derived. The distinct amnion and serosa typical of lower insects is replaced by a single, fused, and much reduced membrane, the amnioserosa, which never secretes an embryonic cuticle. We have used the Zen gene as a marker to study the formation of the extra-embryonic membranes, and other aspects of early embryonic patterning, in the grasshopper Schistocerca gregaria (African Plague Locust). Zen genes are derived from Hox genes, but in Drosophila they appear to have lost any role in patterning the A/P axis of the embryo; instead, they are involved in D/V patterning and the specification of the extra-embryonic membranes. We show that the Schistocerca zen gene is expressed during embryogenesis in three distinct phases. The first of these is during cleavage, when Sgzen is transiently expressed in all energids that reach the cell surface. The second phase of expression initiates in a ring of "necklace cells" that surround the forming embryo, and demarcate the boundary between the amnion and serosa. This leads to expression throughout the serosa. The final phase of expression is in the amnion, after this has separated from the serosa. This complex pattern implies that the role of Sgzen in Schistocerca is not limited solely to the specification of cell identity in the extra-embryonic membranes. We also report that the Schistocerca zen gene is expressed maternally, unlike its Drosophila and Tribolium counterparts. A distinct maternal transcript, and maternal Zen protein, accumulate in the developing oocyte from early post-meiotic stages. They remain uniformly distributed in the oocyte cytoplasm until late vitellogenic stages, when the protein and RNA become somewhat concentrated at the egg cortex and in the posterior polar cap of the oocyte, probably by passive exclusion from the yolk. The cytoplasmic localization of Sgzen protein in the oocyte, and at some stages during embryogenesis, implies that nuclear exclusion of this transcription factor is specifically controlled.

Studies of somitogenesis in vertebrates have identified a number of genes that are regulated by a periodic oscillator that patterns the pre-somitic mesoderm. One of these genes, hairy, is homologous to a Drosophila segmentation gene that also shows periodic spatial expression. This, and the periodic expression of a zebrafish homologue of hairy during somitogenesis, has suggested that insect segmentation and vertebrate somitogenesis may use similar molecular mechanisms and possibly share a common origin. In chicks and mice expression of the lunatic fringe gene also oscillates in the presomitic mesoderm. Fringe encodes an extracellular protein that regulates Notch signalling. This, and the finding that mutations in Notch or its ligands disrupt somite patterning, suggests that Notch signalling plays an important role in vertebrate somitogenesis. Although Notch signalling is not known to play a role in the formation of segments in Drosophila, we reasoned that it might do so in other insects such as the grasshopper, where segment boundaries form between cells, not between syncytial nuclei as they do in Drosophila. Here we report the cloning of a single fringe gene from the grasshopper Schistocerca. We show that it is not detectably expressed in the forming trunk segments of the embryo until after segment boundaries have formed. We conclude that fringe is not part of the mechanism that makes segments in Schistocerca. Thereafter it is expressed in a pattern which shows that it is a downstream target of the segmentation machinery and suggests that it may play a role in segment morphogenesis. Like its Drosophila counterpart, Schistocerca fringe is also expressed in the eye, in rings in the legs, and during oogenesis, in follicle cells.

The Drosophila bicoid gene is well known for encoding a protein that forms a morphogenetic gradient with a key role in anterior patterning of the fruitfly embryo. Recent results suggest the evolution of bicoid might have involved dramatic changes in function – essentially the invention of a new regulatory protein.

A new mathematical biology is emerging. Building on experimental data from developing organisms, it uses the power of computational methods to explore the properties of real gene networks.

INTRODUCTIONThis protocol describes the preparation and fixation of honeybee tissue. Honeybee embryos are very fragile and must be fixed overnight before they can be subjected to downstream applications such as in situ hybridization and immunohistochemistry.

INTRODUCTIONThis protocol outlines how to carry out section in situ hybridization on honeybee larvae to detect expression of a gene of interest in individual cell types. It can also be modified to investigate gene expression in a sectioned honeybee brain. A digoxigenin-labeled probe is hybridized to fixed sections and the probe is detected by 4-nitro blue tetrazolium chloride (NBT)-5-bromo-4-chloro-3-indolyl-phosphate (BCIP) color development.

INTRODUCTIONThis protocol describes RNA interference (RNAi)-mediated knockdown of a target gene in honeybee embryos. dsRNA is directly injected into freshly laid embryos in order to disrupt the function of a specific gene during honeybee embryogenesis.

Purpose Photoreceptor ribbon synapses translate light-dependent changes of membrane potential into graded transmitter release via L-type voltage-dependent calcium channel (VDCC) activity. Functional abnormalities (e.g., a reduced electroretinogram b-wave), arising from mutations of presynaptic proteins, such as Bassoon and the VDCCalpha1 subunit Cacna1f, have been shown to altered transmitter release. L-type VDCCalpha1 subtype expression in wild-type and mutant mice was examined, to investigate the underlying pathologic mechanism. Methods Two antisera against Cacna1f, and a Cacna1f mouse mutant (Cacna1fDeltaEx14-17) were generated. Immunocytochemistry for L-type VDCCalpha1 subunits and additional synaptic marker proteins was performed in wild-type, BassoonDeltaEx4-5 and Cacna1fDeltaEx14-17 mice. Results Active zone staining at photoreceptor ribbon synapses with a panalpha1 antibody colocalized with staining for Cacna1f in wild-type mouse retina. Similarly, in the BassoonDeltaEx4-5 mouse, residual mislocalized staining for panalpha1 and Cacna1f showed colocalization. Unlike the presynaptic location of Cacna1f and panalpha1 antibody staining, the skeletal muscle VDCCalpha1 subunit Cacna1s was present postsynaptically at ON-bipolar cell dendrites, where it colocalized with metabotropic glutamate receptor 6 (mGluR6). Surprisingly, Cacna1s labeling was severely downregulated in the BassoonDeltaEx4-5 and Cacna1fDeltaEx14-17 mutants. Subsequent analyses revealed severely reduced ON-bipolar cell dendritic expression of the sarcoplasmic reticulum Ca(2+) ATPase Serca2 in both mouse mutants and of mGluR6 in the Cacna1fDeltaEx14-17 mutant. Conclusions Presynaptic mutations leading to reduced photoreceptor-to-bipolar cell signaling are associated with disturbances in protein expression within postsynaptic dendrites. Moreover, detection of Cacna1s and Serca2 in ON-bipolar cell dendrites in wild-type animals suggests a putative role in regulation of postsynaptic Ca(2+) flux.

Aphids are the leading pests in agricultural crops. A large-scale sequencing of 40,904 ESTs from the pea aphid Acyrthosiphon pisum was carried out to define a catalog of 12,082 unique transcripts. A strong AT bias was found, indicating a compositional shift between Drosophila melanogaster and A. pisum. An in silico profiling analysis characterized 135 transcripts specific to pea-aphid tissues (relating to bacteriocytes and parthenogenetic embryos). This project is the first to address the genetics of the Hemiptera and of a hemimetabolous insect.

Subcellular localization of RNAs is a critical biological process for generation of cellular asymmetries for many cell types and a critical step in axis determination during the early development of animals. We have identified transcripts localized to the anterior and posterior of honeybee oocyte using laser capture microscopy and microarray analysis. Analysis of orthologous transcripts in Drosophila indicates that many do not show a conserved pattern of localization. By microinjecting fluorescently labeled honeybee transcripts into Drosophila egg chambers we show that these RNAs become localized in a similar manner to their localization in honeybee oocytes, indicating conservation of the localization machinery. Thus while the mechanisms for localizing RNA are conserved, the complement of localized RNAs are not. We propose that this complement of localized RNAs may change relatively rapidly through the loss or evolution of signal sequences detected by the conserved localization machinery, and show this has occurred in one transcript that is localized in a novel way in the honeybee. Our proposal, that the acquisition of novel RNA localization is relatively easy to evolve, has implications for the evolution of symmetry breaking mechanisms that trigger axis formation and development in animal embryos.

Background The Enhancer of split complex is an unusual gene complex found in Arthropod genomes. Where known this complex of genes is often regulated by Notch cell signalling and is critically important for neurogenesis. The Enhancer of split complex is made up of two different classes of genes, basic helix-loop-helix-orange domain transcription factors and bearded class genes. The association of these genes has been detected in the genomes of insects and crustaceans. Results Tracing the evolution of the Enhancer of split complex in recently sequenced Arthropod genomes indicates that enhancer of split basic helix-loop-helix orange domain genes arose before the common ancestor of insects and Crustacea, and before the formation of the complex. Throughout insect and crustacean evolution, a four-gene cluster has been present with lineage specific gene losses and duplications. The complex can be found in the vast majority of genomes, but appears to be missing from the genomes of chalcid wasps, raising questions as to how they carry out neurogenesis in the absence of these crucial genes. Conclusions The enhancer of split complex arose in the common ancestor of Crustacea and insects, probably through the linkage of a basic helix-loop-helix orange domain gene and a bearded class gene. The complex has been maintained, with variations, throughout insect and crustacean evolution indicating some function of the complex, such as coordinate regulation, may maintain its structure through evolutionary time. Electronic supplementary material The online version of this article (doi:10.1186/s12864-015-1926-1) contains supplementary material, which is available to authorized users.

The specification of germ cells is an important process during the development of all animals. Expression of an evolutionarily conserved gene such as vasa can be used as a marker for germ cell fate. We have isolated a vasa-related gene from the two-spotted spider mite (Tetranychus urticae) and used it to examine the segregation of germ cells in this animal. In spider mites, vasa expression first appears in a group of cells that do not join the initial blastoderm surface. Instead, these cells remain in the interior of the blastoderm and then migrate to posterior regions of the embryo, where they form a cluster that appears in regions of the embryo consistent with the gonads. The expression pattern of this spider mite vasa homologue implies a novel process acts to specify germ cells in this species and that the specification of germ cells is an evolutionarily labile process.

Background Recent evidence supports the proposal that the observed diversity of animal body plans has been produced through alterations to the complexity of the regulatory genome rather than increases in the protein-coding content of a genome. One significant form of gene regulation is the contribution made by the non-coding content of the genome. Non-coding RNAs play roles in embryonic development of animals and these functions might be expected to evolve rapidly. Using next-generation sequencing and in situ hybridization, we have examined the miRNA content of early honeybee embryos. Results Through small RNA sequencing we found that 28% of known miRNAs are expressed in the early embryo. We also identified developmentally expressed microRNAs that are unique to the Apoidea clade. Examination of expression patterns implied these miRNAs have roles in patterning the anterior-posterior and dorso-ventral axes as well as the extraembryonic membranes. Knockdown of Dicer, a key component of miRNA processing, confirmed that miRNAs are likely to have a role in patterning these tissues. Conclusions Examination of the expression patterns of novel miRNAs, some unique to the Apis group, indicated that they are likely to play a role in early honeybee development. Known miRNAs that are deeply conserved in animal phyla display differences in expression pattern between honeybee and Drosophila, particularly at early stages of development. This may indicate miRNAs play a rapidly evolving role in regulating developmental pathways, most likely through changes to the way their expression is regulated.

Background The Sox gene family of transcriptional regulators have essential roles during development and have been extensively studied in vertebrates. The mouse, human and fugu genomes contain at least 20 Sox genes, which are subdivided into groups based on sequence similarity of the highly conserved HMG domain. In the well-studied insect Drosophila melanogaster, eight Sox genes have been identified and are involved in processes such as neurogenesis, dorsal-ventral patterning and segmentation. Results We examined the available genome sequences of Apis mellifera, Nasonia vitripennis, Tribolium castaneum, Anopheles gambiae and identified Sox family members which were classified by phylogenetics using the HMG domains. Using in situ hybridisation we determined the expression patterns of eight honeybee Sox genes in honeybee embryo, adult brain and queen ovary. AmSoxB group genes were expressed in the nervous system, brain and Malphigian tubules. The restricted localization of AmSox21b and AmSoxB1 mRNAs within the oocyte, suggested a role in, or that they are regulated by, dorsal-ventral patterning. AmSoxC, D and F were expressed ubiquitously in late embryos and in the follicle cells of the queen ovary. Expression of AmSoxF and two AmSoxE genes was detected in the drone testis. Conclusion Insect genomes contain between eight and nine Sox genes, with at least four members belonging to Sox group B and other Sox subgroups each being represented by a single Sox gene. Hymenopteran insects have an additional SoxE gene, which may have arisen by gene duplication. Expression analyses of honeybee SoxB genes implies that this group of genes may be able to rapidly evolve new functions and expression domains, while the combined expression pattern of all the SoxB genes is maintained.