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Transcriptome analyses of primitively eusocial wasps reveal novel insights into the evolution of sociality and the origin of alternative phenotypes
Genome Biology volume 14, Article number: R20 (2013)
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Understanding how alternative phenotypes arise from the same genome is a major challenge in modern biology. Eusociality in insects requires the evolution of two alternative phenotypes - workers, who sacrifice personal reproduction, and queens, who realize that reproduction. Extensive work on honeybees and ants has revealed the molecular basis of derived queen and worker phenotypes in highly eusocial lineages, but we lack equivalent deep-level analyses of wasps and of primitively eusocial species, the latter of which can reveal how phenotypic decoupling first occurs in the early stages of eusocial evolution.
We sequenced 20 Gbp of transcriptomes derived from brains of different behavioral castes of the primitively eusocial tropical paper wasp Polistes canadensis. Surprisingly, 75% of the 2,442 genes differentially expressed between phenotypes were novel, having no significant homology with described sequences. Moreover, 90% of these novel genes were significantly upregulated in workers relative to queens. Differential expression of novel genes in the early stages of sociality may be important in facilitating the evolution of worker behavioral complexity in eusocial evolution. We also found surprisingly low correlation in the identity and direction of expression of differentially expressed genes across similar phenotypes in different social lineages, supporting the idea that social evolution in different lineages requires substantial de novo rewiring of molecular pathways.
These genomic resources for aculeate wasps and first transcriptome-wide insights into the origin of castes bring us closer to a more general understanding of eusocial evolution and how phenotypic diversity arises from the same genome.
Phenotypic plasticity is a fundamental biological process that allows organisms to adapt to changes in their environment [1, 2]. Examples of plastic phenotypes include insect castes [3, 4], horn-polyphenic beetles [5] and sex differences [6], where they play a crucial role in shaping the ecology and evolution of species and ecosystems. Understanding how alternative phenotypes arise from the same genome is one of the most challenging questions in modern biology [1, 2].
The most impressive examples of phenotypic plasticity are found in the eusocial Hymenoptera (ants, some bees and some aculeate wasps), where specialized reproductive or non-reproductive phenotypes (castes) play a pivotal role in the ecological success of these insects [7]. Eusocial insect castes usually arise through differential expression of shared genes [8, 9], but studies to date have been largely restricted to the highly eusocial bees and ants, where caste phenotypes are evolutionarily derived, being greatly modified from their ancestral state (for example, with morphological adaptations that are established early in development). Thus, some of the molecular processes underlying the origins of castes are likely to differ substantially from those underlying highly eusocial species [10, 11]. Understanding how castes first arise is best studied in primitively eusocial species, but little is known about their molecular evolution, particularly in the aculeate wasps [12–15]. Thus, there is a need for comprehensive gene-level studies on primitively eusocial wasps.
In the evolution of eusociality, behavioral and physiological traits of the non-social ancestor become decoupled into complementary queen and worker phenotypes [16], but we understand little about the molecular patterns and processes by which this originates and to what extent molecular ground plans are re-organized. These questions are of general biological importance in understanding the evolution of phenotypic diversity [1, 2]. Empirical assessment of how gene transcription is decoupled into alternative phenotypes has been difficult to obtain because it requires unbiased quantification of gene expression (of both known and novel (undescribed) genes) across whole genomes [17]. Microarray analyses of the highly eusocial honeybee (Apis mellifera) and fire ant (Solenopsis invicta) suggest marked functional decoupling of molecular processes in queens and workers, involving large numbers of genes [18–21]. Little is known about the patterns and processes of transcriptional decoupling for castes in primitively eusocial insects [12, 14]. A simple prediction is that patterns of transcriptional and functional decoupling should be less pronounced than in highly eusocial species, because phenotypes are in the first stages of merely behavioral decoupling [9, 12].
Processes of phenotypic decoupling at the molecular level may occur via several mechanisms [5, 22]. Conserved toolkits of ancestral molecular processes may be redeployed to regulate similar alternative phenotypes across species and lineages [23–26]. In eusocial insects, conserved toolkit genes appear to be differentially expressed in castes across species, including bees and wasps [12, 27]. An alternative, untested prediction is that at least some of the molecular processes underlying social phenotypes, in different social lineages and among different levels of social complexity, will differ substantially. This is because extensive molecular and developmental re-wiring or new gene evolution may be required for the loss of phenotypic plasticity and elaboration of phenotype-specific traits [10, 28]. There may also be rapid evolution of genes associated with the emergence of alternative phenotypes [6, 17, 29], and there is evidence for this in the highly eusocial honeybees and fire ants [30, 31]. Finally, the importance of novel genes (that is, previously undescribed genes that lack any detectable protein-coding homologues from existing sequence data [32]) in the evolution of phenotypic innovations has recently emerged in animals as varied as yeast [33], hydra, reptiles [34–36], and also honeybees [37]. To date the eusocial insect literature has largely focused on conserved molecular pathways for social behavior [12, 26, 27, 38, 39], and the relative roles of the different mechanisms of phenotypic decoupling are unknown.
We sequenced over 20 Gbp of genome-wide caste-specific transcripts from the primitively eusocial, tropical paper wasp Polistes canadensis using high-throughput sequencing technologies. Castes in Polistes differ only in behavior and are determined through differential gene expression during adulthood [12–14], making Polistes important models for studying the early stage in the evolution of eusociality and phenotypic plasticity [40]. Polistes originated in the tropics, and the secondary adaptations to diapause found in temperate species (for example, [41]) will be absent in P. canadensis [42]. Typically a few foundresses build and provision new colonies (Figure 1). One foundress becomes the primary egg-layer (queen). Emerging adults help raise the offspring of their mother queen, but retain the ability to become egg-layers [43–45]. Thus, Polistes castes represent an early stage of phenotypic decoupling in the evolution of eusociality [16].
Here, we generate a genome-wide catalogue of aculeate wasp genes expressed in adult females to provide a resource for genomic analyses. We then conduct the first RNA-seq analyses of caste-biased expression in a wasp and primitively eusocial insect to test the above hypotheses on the patterns and processes of molecular decoupling of alternative phenotypes at the early stages in the evolution of eusocial behavior. These datasets allow a first simultaneous assessment of the role of conserved genetic toolkits, novel genes and gene evolution when social behavior first evolves. We also use our data to re-assess the phylogenetic relationships between the three aculeate Hymenoptera subfamilies (bees, wasps and ants), which is vital information for interpreting common processes and lineage-specific novelties underlying the evolution of alternative phenotypes. Recently, there has been much debate over the relationships of aculeate bees, wasps and ants, challenging the morphology-based view that Vespoidea (ants plus aculeate wasps) are monophyletic [46, 47]. Genome sequences (or similar catalogue of genes) for aculeate wasps will help resolve this controversy and provide a basis for interpreting comparative data on social evolution.
Results and discussion
Gene assembly for an aculeate wasp
We first generated a reference assembly of the genes expressed by P. canadensis adult female phenotypes (queens, workers, foundresses and callows) (Figure 1; Section 1 in Additional file 1) by sequencing normalized 454 libraries from phenotype-specific pools (37 individuals in total; Section 2 in Additional file 1). Using transcriptome rather than genome sequencing data to provide a gene set is a powerful and accessible approach for initiating genomic analyses on non-model organisms. Library normalization meant that we were able to capture transcripts at all levels of the expression spectrum. The longer read lengths provided by 454 technology and the high coverage of these datasets increase the likelihood of full transcript assembly. The 454 reads were assembled into a reference transcriptome set of 26,284 isogroups - henceforth referred to as 'genes' for simplicity (Section 2 in Additional file 1; Additional file 2). This gene set included 98% of the core eukaryotic genes with equal coverage for each phenotype (Section 3 in Additional file 1). Additionally, sequences were 100% identical to existing overlapping Sanger sequences for this species (n = 36 genes) [14]. Genes were equally spread across honeybee chromosomes and included 94% of publicly available ESTs for the temperate paper wasp Polistes metricus (n = 422 genes [12]; Section 3 in Additional file 1). Although this assembly is restricted to the genes expressed in adult females, it provides an important step forward in generating a comprehensive genome-wide sequence resource for aculeate wasps.
We characterized in detail the sequence and expression features for two groups of genes. The first group corresponds to the set of genes with detectable homology to protein sequences of any species in the comprehensive GenBank non-redundant (NR) database. We identified homologs for 37.4% of the total P. canadensis genes (n = 9,839), which were then annotated using Gene Ontology (GO; Table 1; Section 4 in Additional file 1; Additional files 3 and 4): 71% of best hits were with the honeybee, which reflects the wealth of genomic resources for this species rather than any close phylogenetic relationship (see below). This level of homology is comparable with data for the temperate wasp P. metricus where 39% of the approximately 391K sequence fragments had putative orthologs [13]. In comparison, 60% to 70% of genes in the genomes of other social insects (ants and bees) had homology with sequences in other insects [48, 49].
The second group of genes (n = 16,445; 62%) lacked detectable homology with protein sequences in NR databases, and will be referred to here as putative novel genes as they differ significantly in sequence from any described sequences to date (Section 5 in Additional file 1). These include taxonomically restricted genes, and they may be derived from ancestral coding genes, or arise de novo from noncoding ancestors [32]. Overall, transcripts with homology were longer (with-hits group, 1,718 ± 1,343; no-hits group, 736 ± 768) and had longer ORFs (with-hits group, 256 ± 205; no-hits group, 83 ± 36) than those without (Figure 2a). By scanning the sequences for known protein domains with HMMscan [50] we detected domain homology in 48% of the sequences with NR hits and only 9.9% in the group without hits (Figure 2b). GC content was also lower in the no-hits group (mean GC with-hits, 0.337: no-hits, 0.306; Figure 2c). Analysis of protein coding potential with PORTRAIT [51] revealed a significantly lower potential for protein coding in the group of isotigs without hits (Figure 2d). We then examined whether the novel isotigs could potentially be long non-coding RNAs (lncRNAs). First, we looked more closely at the distribution of isotigs that are longer than 300 bp and have an ORF shorter than 90 amino acids (96% of the A. mellifera transcripts have an ORF greater than 90 amino acids). The vast majority of these isotigs lacked homology (n = 12,751 with no homology; n = 1,162 with homology), suggesting that the putative novel isotigs had shorter ORFs. Next, we imposed a PORTRAIT score lower than 0.5 (at which a sequence is more likely to be non-coding than coding), which yielded more isotigs in the set lacking homology (n = 4,096), than those with (n = 314). Finally, we imposed the condition of no homology with known protein domains, which again yielded more isotigs in the set without homology (n = 3,824) than with (n = 262). These latter two results suggest lower protein coding potential in the putative novel isotigs. The distribution of the median expression across all castes shows that, in general, isotigs with hits have higher expression values than those without (Figure 2e; but see also expression analysis below). The pattern of codon usage was similar in both groups (P = 1, two sample t-test; Figure 2f).
The characteristics of isotigs lacking homology - namely shorter ORFs, lower protein coding potential, lower GC content and lower expression values [52] - suggest that a large proportion of novel isotigs correspond to non-protein coding transcripts. But low sequence conservation across species, lack of genomic information for aculeate wasps and the typical tissue specificity of the non-coding RNAs [52] may also explain the novelty of these genes.
We also verified that the abundance of novel genes was not an artifact resulting from assembly of transcript fragments rather than complete transcript sequences (Section 6 in Additional file 1). Our analyses suggested that less than 10% of genes were affected by this, and thus do not alter significantly our main analyses (below). However, without a genome sequence we cannot exclude the possibility that some genes may represent incomplete assemblies.
Paraphyly of Vespoidea
Long-standing analyses based on the fossil record, cladistics and phylogenetic analyses of the aculeate Hymenoptera place ants and aculeate wasps as a monophyletic clade - the Vespoidea - with the bees as a sister group. This has received mixed support from recent molecular analyses [47, 53–55], but these studies were limited to a maximum of four genes and sparse data matrices. High-throughput transcriptomics data are a reliable source of phylogenetic information [56]. Our transcriptome affords a first assessment of the monophyly of Vespoidea using a large and complete data matrix. We analyzed 93 conserved one-to-one orthologs of P. canadensis genes, derived from 196 single-copy insect phylogeny markers in the eight fully sequenced hymenopteran genomes [57] and nine bee transcriptomes [38] (Additional file 5). A maximum-likelihood (ML) analysis of these genes across all 32 insect species placed Polistes as the basal, sister group to the bees and ants (Figure 3a). This topology was significantly more supported (P < 0.05, Shimodaira-Hasegawa test) than the earlier proposal that Vespoidea (ants and Polistes in our study) are monophyletic. This result is consistent with the levels of gene sharing across aculeates (Table 1), where P. canadensis does not share more genes with ants than bees, as would be expected if wasps and ants were monophyletic.
We further tested the phylogenetic relationships using a Bayesian framework. In contrast, this gave greater support to the classical view of aculeate wasps and ants being monophyletic (Figure 3b; Section 10 in Additional file 1). The reason for the discrepancy in the results of these two methods is likely to be due to the wasp clade being the most poorly represented in our analysis (with only one species), and moreover, it is the clade with the longest branch. Our dataset offers a first chance to examine subfamily relationships across large numbers of genes but, clearly, phylogenomic data on more species of aculeate wasps are required to determine whether the term 'Vespoidea' should be dropped, or reclassified as paraphyletic. This finding would have important general implications for our understanding of eusociality as it would suggest that bees and ants shared an aculeate wasp-like common ancestor, that ants are wingless wasps [58], and that bees are wasps that lost predacious behaviors. Primitively eusocial wasps may therefore hold some important clues to the early stages of eusociality, if they are the basal descendants of non-aculeate Hymenoptera.
Transcriptional and functional decoupling at the origin of alternative phenotypes
We next conducted RNA-seq analyses of phenotype-biased expression to provide a first genome-wide look at molecular decoupling of alternative phenotypes at the early stages of eusocial evolution. We quantified genome-wide transcription in individuals (n = 2 to 12 per phenotype) from each of the four adult female phenotypes by sequencing at least 3 Gbp of Illumina short-reads of brain cDNA per phenotype (Additional files 6, 7, 8 and 9). These samples appeared to capture most transcription across the genome since 99.8% of the 26,284 genes in the reference transcriptome were detected in our pooled Illumina dataset (Section 7 in Additional file 1).
We identified genes that were differentially expressed between phenotypes using NOIseq [59]. Of the 26,284 genes, 2,442 (9.3%) were differentially expressed in any one phenotype (Table 2; Section 8 in Additional file 1; Additional file 10). This suggests that a very small part of the adult female transcriptome is decoupled to produce alternative phenotypes in this species. This is similar to that detected in the temperate wasp P. metricus, where 12% (n = 389) of genes on a microarray were caste-biased [12]. Because RNA-seq is a new methodology, we also confirmed that the number of differentially expressed genes was not artificially elevated due to gene fragmentation (Section 9 in Additional file 1). In a microarray study on whole body samples of the highly eusocial fire ant Solenopsis invicta, the proportion of examined genes that were differentially expressed between adult queens and workers (19% of 14,467 genes) [21] was twice that of Polistes. RNA-seq analyses on highly eusocial species like the fire ant are required to explore these patterns of transcriptional decoupling further.
Workers appear to upregulate significantly more caste-biased genes (n = 2,224; 91% of caste-biased genes) than other phenotypes (callows, foundresses and queens) (Figure 4 and Table 2). This result holds for all different NOIseq [59] selection thresholds (Figure 4) and is independent of the number of biological replicates per phenotype analyzed (Section 8 and Table S2 in Additional file 1). Our original aim was to examine decoupling in queens and workers. We therefore re-analyzed the data using only these two phenotypes and found an even greater asymmetry with workers up-regulating 93.1% (n = 1,797) of 1,909 worker/queen caste-biased genes (queens up-regulate only 112 genes; Table 3 and Figure 5). Upregulation of caste-biased genes in workers, rather than queens, at the early stages of caste evolution may facilitate the expansion of worker trait complexity, typified by workers in the highly eusocial descendants of primitively eusocial species. These results support the emerging picture that much of phenotypic evolution occurs in workers, rather than queens [21, 37], and provide the first suggestion that molecular evolution of the worker caste is in place early in social evolution.
Interestingly, foundresses and callows accounted for the majority (89%) of down-regulated genes. Some of these could be attributed directly to a corresponding up-regulation in workers: specifically, 55% and 75%, respectively, of the down-regulated genes in foundresses and callows. But, the remaining foundress and callow down-regulated genes were not upregulated in other castes. This is also reflected in the GO classification (Additional file 5), where none of the 22 GO terms over-represented among the down-regulated genes in foundresses and only 5 of the 23 terms over-represented among the down-regulated genes in callows were up-regulated in workers. Although preliminary, these results raise some important questions: they suggest that callows are born neither worker- nor queen-like, but perhaps instead as an undifferentiated state, as expected in a species where all females retain full reproductive totipotency; down-regulation in callows may also reflect maturation processes that take place in the first days after emergence as an adult. Foundresses are thought to be hopeful reproductives, co-founding a nest in the hope of becoming the dominant egg-layer (queen); our data suggest that foundresses are quite different from established queens, even though they fit the queen phenotype in all other respects (that is, they are active egglayers, rarely absent from the nest, and are behaviorally dominant). Taken together, these results highlight how the definition of a phenotype in this species is more complex than a simple distinction between queens and workers.
Using the 268 genes differentially expressed between queens and workers that could be annotated (Table 3), we then looked for any signs of functional specialization between these castes. Of the GO terms corresponding to these genes (Additional file 4), 2.4% (53 out of 2,242) were significantly enriched between queens and workers (Additional file 5; 40 terms (1.7%) over-represented in queens, 13 terms (0.57%) over-represented in workers). Although many of our genes did not have any functional information, there are some interesting first insights from these analyses. The putative lack of substantial functional specialization between castes in Polistes contrasts with the marked functional specialization in highly eusocial species, such as honeybees [19]. This may reflect selection on females to retain reproductive totipotency in primitive societies, like P. canadensis, in order that they can exploit alternative reproductive strategies [43], and minimize the risks of specialization associated with small colony size [60]. General terms overrepresented in workers include cellular protein modification processes, membrane and ion transport, which may suggest elevated cellular activity in worker brains. Among the over-represented terms in queens, several candidates associated with cell structure and cytoskeleton were found, such as homologs of myosin chains, actin isoforms and troponin, which may be associated with synaptic plasticity and memory [61]. However, because so few of the genes up-regulated in workers could be annotated, and because of the lack of significant levels of enrichment between castes, these inferred functional differences between castes should be taken with caution and require further analyses once some functionality can be assigned to the unknown genes.
Role of novel genes in the evolution of alternative phenotypes
We explored the hypothesis that novel genes (those lacking homologs in NR databases; see above) may play an important role in the early stages of phenotypic decoupling in P. canadensis. The importance of novel genes was recently highlighted in organisms as varied as Hydra to reptiles for their contribution to phenotypic diversity, through generating both behavioral and morphological variation [34–36]. Of the 2,442 up-regulated caste-biased genes (Table 2), 24.4% (n = 596 genes) had known homologues, indicating that both novel and described genes are important in P. canadensis social behavior. Interestingly, 81.5% of worker-biased genes lacked homologs, compared to only 17.1% of queen-biased genes (Table 2). Foundresses and callows show similar levels of homology to queens. Lack of homology cannot be attributed to fragmentation or other anomalies arising from using transcriptome data (Section 6 in Additional file 1) and they are also valid for the stricter comparison of queens versus workers (Table 3). These results are exactly in line with Johnson and Tsutsui [37] and Barchuk et al. [62], who found an over-representation of novel (taxonomically restricted) genes up-regulated in honeybee workers (adults [37] and brood [62]), and suggested that these novel genes may facilitate expansion of worker behavioral complexity. Our data provide the first suggestion that novel gene transcription may be important at the early stages of worker evolution, confirming the potentially broad role of novel genes in eusocial evolution [37] and adding to the emerging general importance of novel genes in the evolution of phenotypic diversity [34–36]. It is important to note that microarray methods (as used in the honeybee studies) are largely biased towards conserved genes [37], as sequence conservation facilitates gene prediction. Thus, RNA-seq methods as used in our study offer new potential to explore the role of putative novel genes in phenotypic evolution.
Our ability to interpret what these novel genes are is limited without genome and proteome analyses, which is beyond the scope of this study. But, we explored the hypothesis that many of the caste-biased novel genes were in fact non-coding RNAs, as identified from our whole transcriptome analysis (above). Interestingly, we found a significant difference in the proportion of novel transcripts that were putatively lncRNAs, with an over-representation in workers relative to queens (Table 3). lncRNAs are thought to play a role in gene regulation [63], but also may be evidence of de novo gene origin [33]. Future work will determine the significance of these processes in worker, rather than queen, evolution.
Conserved molecular toolkits associated with alternative phenotypes
Social lineages evolve from non-social ancestors [64]. Alternative phenotypes (social insect castes) may therefore evolve through the decoupling of conserved sets of genes that regulated changes in hormone titers underlying the provisioning and reproductive phases of the non-social ancestor [16]. A prevailing hypothesis is that changes in the patterns of expression of these ancestral ground-plan genes could account for caste evolution across social taxa [26, 28]. Using a candidate gene approach, sets of such 'toolkit' genes and molecular processes have been shown to have conserved roles in caste regulation across bees and wasps [12, 27, 38]. We also found some evidence of this in P. canadensis: vitellogenin (four isogroups), insulin (one isogroup) and major royal jelly protein (two isogroups) were queen-biased; juvenile hormone (two isogroups) and methyltransferases (two isogroups) were worker-biased. However, other toolkit genes (hexamerin, Malvolio, Amfor, P450) were present in our dataset, but were not identified as significantly caste-biased (Additional file 4) [12, 27, 65, 66].
As a genome-wide, unbiased representation of genes expressed by alternative phenotypes in wasps, our datasets also offer the opportunity to test to what extent a conserved molecular toolkit in general explains alternative social phenotypes across eusocial taxa [12, 27]. Interestingly, we found little correlation in the general identity and direction of expression of genes underlying castes in other eusocial insects, as identified from microarray analyses. For example, only 6.5% of honeybee (A. mellifera) caste-biased genes [19] were caste-biased in P. canadensis and there was no consistency in the direction of expression. Of 50 cDNAs predictive of honeybee worker behavior [18], only 8 were found in P. canadensis and they were not significantly worker biased (Additional file 5). There was no correlation in direction and identity of gene expression in castes of the highly eusocial fire ant S. invicta (r ~ 0) [21]. Finally, only 22 of the 423 caste-biased genes of the temperate wasp P. metricus [12] were also caste-biased in P. canadensis, and they showed a low correlation in the direction of phenotype-specific expression between species (Pearson correlation of 0.25 for worker, 0.14 for queen, 0.24 for foundress and 0.23 between gyne and callow; Additional file 5). This was unexpected since these two species are close relatives, share a recent common ancestor, and have similar social and behavioral traits. Differences in ecology of tropical and temperate Polistes, and possible secondary adaptation to temperate living [42], may contribute to these differences. These analyses suggest that the social toolkit represents a small portion of the genome involved in phenotype decoupling of P. canadensis, and that there is much lineage and ecology-specific transcription underlying caste differences. This supports the hypothesis that the genes underlying castes in different social lineages will not necessarily be conserved, because extensive molecular and developmental re-wiring may be required in the evolution of caste commitment and eusociality [10, 11].
Insights into gene evolution in alternative phenotypes
Rapid evolution of genes has been associated with alternative phenotypes in a range of animals [6, 17, 29]. Caste-biased genes evolve rapidly in the honeybee and fire ant [30, 31], possibly because fast-evolving genes are preferentially recruited into caste-biased gene expression [30]. Interestingly, we found no evidence of this in P. canadensis (Section 10 in Additional file 1; Additional file 5), suggesting that at the early stages of phenotypic decoupling genes are maintained through purifying selection. This agrees with social evolutionary theory that predicts that females retain the ability to exploit alternative reproductive opportunities (for example, to switch from worker to queen) to maximize individual-level inclusive fitness at the early stages of sociality [43]. Selection may be relaxed after workers commit to a non-reproductive pathway, as found in highly eusocial species [10, 28], resulting in gene evolution, new genes and new gene networks [31, 67–69]. Analyses of incipient phenotypic decoupling in other animals will help determine whether accelerated gene evolution only occurs after key innovations or fundamental shifts in life-history traits.
We next looked for evidence of processes that may have been lost or gained during phenotypic decoupling in eusocial evolution (Section 10 in Additional file 1; Additional file 5). We identified 431 caste-biased genes from across eusocial insect species that are also conserved in the 19 insect species genomes available. From this conserved set of genes, 12 GO terms were over-represented in highly eusocial insects (see Section 10 in Additional file 1 for species list) relative to P. canadensis, and 217 terms were under-represented (Section 10 in Additional file 1; Additional file 5). Under-represented groups (which include regulatory and signaling processes) may be less important in complex eusociality and hence not maintained by stabilizing selection. However, we cannot exclude the possibility that these differences are wasp-specific. Over-represented terms may be important in complex eusocial behavior [30]. These included processes previously highlighted for putative roles in caste determination (for example, oxidation reduction, metabolic processes) [39], suggesting they reflect the level of eusociality rather than differences in ecology or lineage.
Our RNAseq analyses of phenotypic decoupling in P. canadensis provide some intriguing novel insights into the evolution of alternative phenotypes, and the early stages of caste evolution and sociality. First, they highlight the importance of novel genes in phenotypic diversity, emphasizing the need to expand existing genomic datasets beyond established model organisms to include a wider range of taxonomic groups. Secondly, they add important insights to the current emerging picture that much of the molecular changes that accompany social evolution occur predominantly in the worker rather than queen caste, with an over-representation of novel caste-biased genes in workers. These features may be important in facilitating the evolution of behavioral complexity in the worker caste. We show that these patterns are evident even at the early stages of sociality, where castes retain plasticity. Further, contrary to highly eusocial species, genes involved in caste differentiation do not appear to be subject to relaxed selection at the early stages of sociality. Diversity in the molecular regulation of castes across social lineages is expected if many pleiotropic genes involved with sexual conflict are lost in the early stages of social evolution, when ancestral monogamy is required to generate the conditions for the evolution of worker behavior [10]. Subsequent evolution of queen and worker behaviors, therefore, may take very different molecular pathways in different lineages, requiring new gene networks to evolve independently [28]. Further RNA-seq studies on other species (for example, [70]) and also genome sequencing to explore the potential role of alternative splicing (for example, [71]) will help determine the complementary roles of conserved and novel molecular processes in shaping social and other polyphenisms, bringing us closer to understanding how genomes give rise to phenotypic diversity in general.
Materials and methods
Source material
P. canadensis wasps of known behavioral repertoires were collected from wild populations in Panama, in July 2009 (Punta Galeta, Colon). All wasps were collected directly off their nests with forceps around midday during the active periods (that is, sunny weather) and preserved immediately in RNAlater (Ambion, Invitrogen, Applied Biosystems), and stored at -20°C until analysis (Section 1 in Additional file 1).
Transcriptome sequencing, assembly and analyses
454 sequencing of pooled samples of 37 wasps across phenotypes (5 to 18 individuals per phenotype; 2.1 million reads, 80% brain, 10% abdomen, and 10% antennae) was used to generate a reference transcriptome (Section 2 in Additional file 1). Newbler v2.3 was used to generate the final assembled gene set (Table S1 in Additional file 1). Transcripts were annotated using GO categories assigned using BLASTx of GenBank NR databases with a conservative e-value threshold of 10-5, and Blast2Go was used to assess enrichment of GO terms among phenotypes (Section 4 in Additional file 1). Illumina sequencing of 14 biological replicates (>377 million reads) across 5 lanes was conducted to quantify differential gene expression, expressed as RPKM (reads per kilobase per million) values (Section 8 in Additional file 1). We trialed a number of methods for identifying differentially expressed genes and settled on a novel non-parametric method (NOISeq [59]) This method infers the noise distribution from the data and performs pairwise comparison of the samples to identify differentially expressed genes. A variety of probability thresholds were tested (Section 8 in Additional file 1). For the GO analysis we used a q-value >0.6 that represents a 50% chance that the gene is differentially expressed rather than not differentially expressed.
Phylogenetic analyses
Protein sequences were aligned using MUSCLE [72], with default parameters. This is a multiple sequence aligner that includes an iterative alignment refinement phase to overcome known pitfalls of the progressive alignment strategy. Subsequently, poorly aligned regions of the alignment were trimmed with trimAl v1.3. [73] to remove columns with gaps in more than 30% of the sequences. A maximum-likelihood analysis was conducted on the concatenated alignment containing 33,506 sites using PhyML v3.0 [74]. Out of a total of five evolutionary models (LG, JTT, WAG, VT, BLOSUM62), the general replacement model LG (after 'Lee and Gascuel') was found to be the best fitting model using the AIC criterion [75]. In all cases four categories of evolutionary rates were used, estimating the gamma shape parameter and the proportion of invariable sites from the data. Branch supports were obtained using an approximate likelihood ratio test as implemented in PhyML ('Minimum of SH and chi-square' option). The resulting topology was compared to an alternative topology placing Polistes as a sister group to the ants. To do so, the phylogeny was re-computed, fixing the monophyly of Polistes and ants, but allowing the rest of the topology to be optimized. Support for the two topologies were compared using a Shimodaira-Hasegawa test, as implemented in CONSEL [76]. A Bayesian analysis was conducted as implemented in PhyloBayes [77], using the default CAT model and running two independent MCMC runs during 300,000 generations, and sampling every 100 generations. Consensus trees were built after removing the first 20% sampled trees and using a majority consensus rule.
Data access
Raw sequence data are available at the European Read Archive (accession number ERP001342). The Transcriptome Shotgun Assembly project has been deposited at DDBJ/EMBL/GenBank under the accession GAFR01000001-GAFR01045087. The version described in this paper is the first version, GAFR01000000. All data and datasets can also be accessed at [78].
- bp:
base pair
Gene Ontology
- lncRNA:
long non-coding RNA
open reading frame
reads per kilobase per million.
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Data and Datasets. [http://genome.crg.es/~pferreira/pcandata/pcan.htm]
We thank Centre for Genomic Regulation (CRG) (Heinz Himmelbauer and Francisco Câmara), GenePool and the Babraham Institute (Wolf Reik and Simon Andrews) for support and access to their sequencing facilities. We thank RCUK, CONSOLIDER (CSD2007-0005), the Spanish Ministry of Science (BIO2006-03380, BFU2009-09168), GTEX (NIH Project 1RO1MH090941-01), and a Wellcome Trust Senior Investigator Award to Wolf Reik. SS was supported by a UK Research Council (RCUK) fellowship, and PGF by a FCT postdoctoral grant (SFRH/BPD/42003/2007) from the Portuguese Ministry of Science and Technology. We thank the Smithsonian Tropical Research Institute (particularly William Wcislo, Mary Jane West-Eberhard, Jorge Morales and staff at the Galeta Field Station) for hosting the fieldwork, and Stephanie Dreier and Thibault Lengronne for assistance in the field. The fieldwork was conducted under ANAM Permit No. SE/A-33-09.
Additional information
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
SS conceived the idea and designed the research. SP and SS performed the fieldwork, SP performed wet-lab work. RC and Ff-C conducted the MIRA assembly; PGF and RG designed and conducted all other bioinformatics analyses. TG conducted the gene evolution and phylogenetic analyses. SS and PGF wrote the paper, with comments from all authors. All authors read and approved the final manuscript.
Electronic supplementary material
Additional file 1: Sections 1 to 10 and Tables S1 and S2. Additional methods and results referred to in the main text can be found here. Table S1: comparison of transcriptome assemblies. Number of transcripts, genes and respective transcript length statistics for the optimal assemblies generated with Version 2.3 and Version 2.5 of GS Newbler Assembler, and Version 3.0.5 of Mira Assembler and Oases (with Illumina paired-end sequence data). Table S2: numbers of differentially expressed (q > 0.6) genes and transcripts was robust to different numbers of biological replicates. (DOCX 86 KB)
Additional file 2: Read coverage from the 454 transcriptome assembly. (A) Distribution of the number of 454 reads per transcript (truncated to transcripts with less than 500 reads). (B) Distribution weighted by the transcript length (truncated plot). The majority of transcripts had more than 5.19 reads, on average. (TIFF 2 MB)
Additional file 3: Functional groups identified in the P. canadensistranscriptome. Distribution of the number of genes according to different GO terms from the 454 pooled transcriptome assembly. Categories were filtered by a minimum number of sequences: 50 for cellular component; 200 for biological process; and 100 for molecular function. (TIFF 8 MB)
Additional file 4: Information on the best BLAST hits in the NR database for each transcript; noncoding RNA potential for genes with and without hits in NR. Tabs: 'besthits_with_NR', information extracted from the best hits for each transcript; 'PortraitScore_transcriptsWithNOHits', 'PortraitScore_transcriptsWithHits', score from the portrait program. (XLS 8 MB)
Additional file 5: Information on functional analyses and comparison with existing caste expression data from other species. Tabs are as follows. 'Toth2010_Pmetricus_Pcan', mappings between P. metricus and P. canadensis transcripts and their respective expression levels and whether they are significantly differentially expressed in P. canadensis. 'Whitfield2003_50cDNAS', mappings between 50 most discriminative cDNAs for worker behaviors in A. mellifera from Whitfield et al. [18] and P. canadensis, with respective expression values. 'GO-Enrichment Differential Expressed (DE) Genes', list of over/under-represented GO terms for genes significantly up- and down-regulated in each caste comparison. Genes were categorized according to biological processes (P), molecular function (F) and cellular component (C). Most specific terms were retrieved with Blast2GO and correspond to the leaf nodes in the GO tree, this excludes cases where parent and child nodes may be included. Column labels are: Test (number of genes in the test set annotated with this GO term); Ref (number of genes in the ref set annotated with this GO term); notAnnotTest (number of genes in the test set NOT annotated with this GO term); notAnnotRef (number of genes in the reference set NOT annotated with this GO term), where 'test' set is the set of genes up-regulated in queens, and the 'ref' set those up-regulated genes in workers, and vice-versa. 'GO-Enrichment', list of over/under-represented GO terms for the P. canandensis genes that are conserved across the genomes of 6 eusocial insects. 'genes_with_Behavior_GOterm', genes classified with GO term thought to be relevant to behavior, their respective descriptions and direction of differential expression in P. canadensis. 'genes_accelerated_Pol_Bee_Ants', genes found accelerated in Polistes, bee and ants, and direction of differential expression in P. canadensis. 'genes_accelerated_polistes', genes found accelerated only in Polistes; and direction of differential expression in P. canadensis. 'phylogenetic marker identifiers', a list of the 93 marker genes used in the phylogenetic analyses. These correspond to phylomeDB codes in the Aphid phylome used as the reference markers. Sequences for these identifiers can be obtained from Huerta-Cepas et al. [57]. (XLS 353 KB)
Additional file 7: Basic information about the assembled transcripts. Tabs: 'transcript_length', length of each transcript; 'transcript_to_gene', correspondence between transcript and gene. (XLS 5 MB)
Additional file 9: Expression values in RPKM (reads per kilobase per million) for all the individuals, indicating which caste each isotig is up-regulated in. (CSV 10 MB)
Additional file 10: NOISeq probability values for each transcript for the comparison of each caste (that is, W (worker), Fo (foundress), Q (queen), or C (callow)) versus the others, and between queens and workers. Expression values for each caste and comparison, differential expression statistics ('M' and 'D' [12]), probability of differential expression ('prob'); 'ranking', which is a summary statistic of 'M' and 'D'. (CSV 16 MB)
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Ferreira, P.G., Patalano, S., Chauhan, R. et al. Transcriptome analyses of primitively eusocial wasps reveal novel insights into the evolution of sociality and the origin of alternative phenotypes. Genome Biol 14, R20 (2013). https://doi.org/10.1186/gb-2013-14-2-r20
DOI: https://doi.org/10.1186/gb-2013-14-2-r20


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Katherine Gotsick
Submitted on: February 5, 2018
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The Main-Dempster Mile

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A Queen Cersei promotional poster, featuring Lena Headey, and a quote that's not from the books. We have seen the scene in which it appears, and it's one where she's speaking with her son Prince Joffrey.
Because of size limitations, we do not have the highest resolution of this image available here. You can find that at Making Game of Thrones.Copyright ©


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Game On/Scratch Blocks
This is a list of blocks that are useful for Game On.
The block "move ____ steps" causes the sprite to move in the preset direction, a certain number of "steps". A typical noticeable motion is 5-10 steps.
The "turn ___ degrees left/right" blocks cause the sprite's direction to change in either direction, by a certain number of degrees.
Point Toward
The point toward block changes the sprite's direction to point toward a given object, or the mouse.
Point in Direction
This block sets the direction to a given value.
Go To
The "Go To" block sets the position of a sprite in a single block. It is the equivalent of a set X and set Y block combined.
Set X/Y
These blocks set X and Y coordinates individually.
Change X/Y
These blocks increment or decrement the X and Y values. Useful for speed-based advanced motion control.
If On Edge, Bounce
For randomly-moving objects, this block prevents them from collecting on the edges. It does what you would expect.
These blocks change the visibility of a sprite.
Switch Costume
Each sprite can have multiple costumes. This command changes the costume to any costume in the sprite's selection.
Switch Backdrop
The backdrop can be altered from any sprite's code. This block works like Switch Costume.
Change/Set Size
These blocks function to change the size of an object or set it.
Default Sounds
These can be accessed through the "Play Sound" block. There's a variety of soundbites. They must be downloaded from a menu in the Sounds tab.
Recorded Sounds
You can record your own sounds in the Sounds menu. Then, you can play them like default sounds.
Musical Notes and Drums
Scratch offers a musical note system, including volume and tempo controls. There are many drums and instruments which can play any note for any amount of time.
Local and Global Variables
When you first create a variable, you must decide whether it is particular to the sprite in which it is created, or global for the whole program. This cannot be changed!
Showing and Hiding Variables
You can reposition variable displays manually. Moreover, you can change their visibility using these blocks.
Changing and Setting
Scratch offers an assignment block (Set ____ To ____) and an increment/decrement block (Change ____ By ____). Each is fairly self-explanatory.
Flag Event
Any code that has as its header "When <green flag> is clicked" will execute as soon as the program starts.
Click Event
Any code that has as its header "When <this sprite/the backdrop> is clicked" will execute every time the object is clicked by the mouse.
Key Press Event
Any code that has that header will execute when the selected key is pressed.
Message Event and Broadcasts
Sprites can influence each other via broadcasting messages. New messages can be created, and then the sprites will respond the same way no matter who sends a given message.
Control Statements
Scratch offers two wait statements. One sleeps that block of code for a given amount of time. The other waits until a true/false statement becomes true.
Scratch has three loops:
- Its forever loop will run until the program ends or a stop statement is reached.
- Its repeat ___ times loop will run a set number of times.
- Its repeat until loop will run until a certain condition is true.
Scratch offers a simple if-then as well as an if-then-else. These act as in other programming languages.
A stop statement can end all active code in any of the following:
- All sprites and the backdrop
- The current sprite
- All other sprites
There are three clone blocks.
- First, a sprite can create a clone of itself or any other sprite.
- Second, a clone can delete itself, ending a clone block of code.
- Third is the "when I start as a clone" header which is initiated for each new clone.
A sprite can ask if it is touching the mouse, the edge of the screen, or any other sprite (collision handling). It can also ask if it is touching a certain color. Thus, colors can gain significance beyond simple aesthetics.
A sprite can determine whether the mouse is pressed, as well as the mouse's location.
A sprite can determine the distance between it and any object/mouse.
Key Press
A sprite can determine if a given key is being pressed.
Other Sprites
The ____ of ____ offers sprites the opportunity to check on the state of other sprites, such as their position and costume.
Scratch offers a single timer which can be reset to 0 at any time. The sprites can see what time is on the timer.
The backdrop has a block "Ask ____" which gets text input from the user. This input can be accessed via the "answer" block.
Scratch offers the basic arithmetic operators, functions like sine, cosine, square root, and absolute value, boolean operators (and, or, and not), as well as some string functions.
DOs and DON'Ts
Don't: Say/Think
Don't use the say or think blocks on sprites, or the ask block on sprites. The word bubbles produced are unprofessional.
Do: Friction
The following block of code added to a motion loop will create a friction effect: set Speed to (Speed * 0.9)
Advanced Options
Trails with the Pen
Create a trail of any sprite's motion using the pen (green) blocks!
More Blocks
You can define procedures and functions using the Purple section. This can help your code organization.
Lists Mean Levels
Use a list (found under Variables) to define parameters for multiple levels of play.
Scratch can access the current day and time in the Sensing section.
Types of Sprite
The Message
This sprite has text as its costume. It delivers a message, such as winning or losing.
The Player
This sprite uses a forever loop to have continuous motion.
The Autonomous Sprite
This sprite needs to use blocks like If On Edge Bounce to avoid clumping on edges. Use clone to create many of these.
The Button
This sprite uses the Click event and broadcasting to cause major changes in the program.
The Master
An invisible sprite that does heavy processing. This is the core of the program. The backdrop can also take this role.


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Fashion Together: Fashion's Most Extraordinary Duos on the Art of Collaboration
Edited by Lou Stoppard, Foreword by Andrew Bolton
- Publish Date: October 24, 2017
- Format: Hardcover
- Category: Design - Fashion & Accessories
- Publisher: Rizzoli
- Trim Size: 8-1/2 x 11
- Pages: 304
- US Price: $75.00
- CDN Price: $100.00
"Though the myth of the single genius designer looms large in fashion, in practice, the art is often far more collaborative. Fashion Together: Fashion's Most Extravagant Duos on the Art of Collaboration, out October 24, celebrates this dynamic, with editor Lou Stoppard interviewing Marc Jacobs and Katie Grand, Rick Owens and Michèle Lamy, and Inez and Vinoodh, among others."
—Garage / Vice Media
"... stuffed full of photographs, ephemera, and some of the most candid designer interviews you'll read anywhere."
"Collaborations and partnerships are key in fashion, so it's no wonder that "Fashion Together" is the focus of Lou Stoppard's first book. The 304-page tome, published by Rizzoli New York with a foreword by Andrew Bolton explores these alliances. "Fashion Together: Fashion's Most Extraordinary Duos on the Art of Collaboration" includes interviews and unpublished works, photos and drawings as well as notes and sketches. "
—Women's Wear Daily
"The book features never-before-seen photos, original sketches, fashion editorials and handwritten notes that highlight the collaborators' work together. Led by Stoppard herself, their conversations dive into everything from early childhood inspirations to professional advice on how to be successful in today's fashion landscape"
"A book on fashion's most intriguing partnerships . . . "


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Via <setdcgeneratordata> the proprietary data of a dive computer manufacturer are set. The elements inside <setdcgeneratordata> are neglected by other dive computers. All data stored in the <generator> section are transferred to the dive computer.
(see also <setdcdata>)
<setdcdata> <!-- all manufacturer's data are transferred to the dive computer --> <setdcgeneratordata/> </setdcdata>


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Learning Social Skills through Imitation and Dramatic Play
Your child has been gradually learning Self-Control, the ability to control and manage his behavior. One way that he's been learning it is through social interaction and pretend play. By acting out scenarios that he's seen before in real life, he's practicing social rules and roles, developing team work skills, and learning about the concept of give and take. Other ways to help him develop Self-Control are to give him 1-2 step tasks to complete on his own or model positive behavior for him to learn from and follow.
Has your child achieved the following Self-Control developmental milestones yet? If yes, check off all the skill(s) he has already mastered to date!


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Chinese Steam Trains Part 2
Our next stop on the trip was at Mao's birthplace, a sacred shrine for the Chinese. This was interesting as year's later, we visited Fidel Castro's mountain hideout in Cuba. It had a similar feel to it.
I thought I would throw in one of the double QJs we saw at Louyang early to give you a taste for the steam action in this post.
Mao's Birthplace
We begin part 2 in Mao's birthplace, Shaoyang. It is a delightful village, probably typical, if a little more sterile, of the thousands of villages that make up rural China.
It was a real treat to visit this village as for the most part we were barred from leaving the big cities. For someone like me, who loves wandering around the countryside it was a difficult medicine to digest, and my attempts to break free often failed. The couple of hours here were really great and relaxing.
Mao came from humble beginnings in a small cottage in the village. As we walked around the area we saw people tilling the fields with traditional Chinese hoes. There was little mechanisation, except for the small tractors that never seemed to haul a plough. I believe that was so everyone would have a job. The average wage in the countryside was around $1 a day, but this went a long way. Everyone had enough rice to eat and a place to stay.
The farms were communal. Unlike Australia there are no fences, and ducks wandered everywhere. Private ownership was a concept that simply didn't exist. Everything was common land worked by the collective. The village appeared very relaxed. It was a far cry from the huge cities. Naturally, there was no steam around here and the weather was perfect and sunny. Damn!
You can see from the photo how imposing the Chairman is. I am not sure what his position is in China now, as loyalties often change with regimes. The little figure beside the statue is me!
In some ways it is a real shame that the Chinese Government of the time were so paranoid about foreigners talking to their citizens that they tried to keep us apart. The official reason for not letting westerners visit many parts of the country was simply that the facilities were not there. They considered the accommodation for the Chinese would not be good enough for Westerners. I had a more cynical viewpoint. The government only wanted foreigners for their tourist bucks. Like other communist countries, the state did everything it could think of to extract the maximum amount of money from foreign tourists.
Shopping Chinese Style
One other interesting phenomena I witnessed in Shaoyang was the local store. As I said, it was difficult to get exposure to normal Chinese life outside of major cities. Being there for such a long time, I did manage to get a glimpse of things from time to time.
The manager (the State owns everything) of this establishment sits high up in the store on a dias. From here he can watch everything going on and control his staff. You enter the shop and it is quite normal. Glass cases displaying the goods, and attentive Chinese women serving customers. I might add it was very different from the department stores in the cities. More on that later.
The sales clerk helps you pick what you wish to buy. In our case this was done by pointing. They then use an Abacus to add up the total and write that down. Fortunately, Chinese used western numbers or we would have been totally lost. Then they let the manager handle the money side.
If you look closely at the photo you will see that there are a series of wires spreading out from the manager's position. These lead to all of the sales counters. When you make a purchase, a docket is written up and sent through a tube on the wire to the manager with your money. He works out the money, and then sends the tube back for you to receive your change.
This to me is a real insight into old China. I am sure that commerce was carried out in this way for many years. We all know the Abacus is legendary in China, and I am sure there were many shops using the wire and cylinder method of payment.
We Say Goodbye To Shoayang
After a couple of hours, we hopped on another train and headed towards Changsha. This town is famous for a mummy. This is no ordinary mummy like the Egyptian ones, but a 2100 year old Chinese woman so well preserved, you can see her skin. All of her organs are stored in jars around the room, and we were not allowed to take photos, because the flesh would degrade her quickly. If you ever visit China, I would recommend a visit to this to9wn. It is truly amazing and moving to actually see someone who lived over 2,000 years ago in a pretty good state of repair.
Changsha To Wuhan
The final leg of this section is the run to Wuhan. It is a 358 km journey. For us it took 5 hours and 42 minutes. There is a reason. These days the line is serviced by bullet train and the journey is just 1 hour 25 minutes at an average speed of 252 kph. How things have changed!
Anyway, after two days of brilliant sunshine, I was itching to get at least one steam train shot in full sun. As we headed towards Wuhan, we stopped at a major town, probably Yueyang, although I can't be sure. This was not on the official list as acceptable for westerners, but what the heck, I thought I would chance my arm.
I hopped off the train and headed north down the tracks. Pretty soon a freight came by with double QJs at the helm.
This was a busy main line and pretty soon another QJ appeared. Regrettably not far behind were railway officials who escorted us back to the station and put us on the next train north!
By now we were well aware that everyone in China was watching. After all, we did tend to stand out! Many areas we visited rarely saw a westerner, and the Chinese were paranoid about US imperialism to the point that they purchase US equipment from my company in Australia so they wouldn't have to deal with the yanks. That got me a trip to the China Daily in Beijing.
I think you will agree that the last picture is a nice one, if not spectacular. We reached Wuhan without further incident. Still all the expresses were hauled by diesels. The next leg of the journey was from Wuhan to Luoyang, a distance of 703 kms. Unfortunately the express was diesel hauled. Luoyang is the site of the famous Longman Grottos, but I wasn't there for that!
According to the latest information, the rail trip from Wuhan to Luoyang is now just over 5 hours, a distance of 580kms. It was obviously a lot slower back in 1985! We travelled 703 kms according to my notes. They must have straightened the tracks.
We arrived in Luoyang after a 13 ½ hour journey and headed for the local "Hotel Fan Dian" or friendship hotel. It was, of course, bitterly cold at that time of year. The average temperature for January is just 1 degree C. It seemed every journey led us to colder weather. This hotel was one of the old ones. It was large and looking very much run down. Luoyang is a major tourist spot in China, and at least the staff spoke English. There were very few westerners around at the time probably due to its remoteness. As I went to book in, I noticed an English gentleman asking questions. I knew he was English by his accent. Being a friendly Aussie, I asked him what brought him to that part of the world and he was rather cagy. Eventually he said he was on a "special interest" trip. I said I was too, and that I was here to photograph the trains. His eyes lit up and as it turned out he was wandering around China on his own in search of steam too.
Difficulties Getting Trackside
That night we had a banquet. There were at least 7 dishes on the table including some peanuts, rice and various dishes. It was all quite delicious. The next morning my new friend and I headed for the railway station and attempted to hire a cab to go to a nearby good photo spot. The cab driver would have none of it. He wasn't going to risk heading out of town with 2 foreigners on board. There was a trishaw not far away, so we flagged it down and fortunately this driver was far more co-operative. We headed off down the line.
It was a slow trip through the suburbs past the tractors that were just hauling trailers, hoping that we wouldn't encounter any police of military. We knew we would be sent back if anyone saw us, and we did rather stand out!
We picked our spot and had an hour or so with a procession of QJs.
The spot we had chosen was on a bank, so the engines worked their way along. As part of the parade, we witnessed 6 double headed QJs in 75 minutes. This was something I had never seen before!
There were so many trains, we managed to get the above shot with QJ passing QJ. You can see the China haze in this photo. For much of the country it is as close as it gets to sunny at that time of year. The condensation is due to the very cold weather. Yes, rail photography in China is a pretty miserable occupation in winter!
After a while of monotonous QJs, one after the other, we had enough of the cold and decided to return back into town. We arrived without incident, pretty pleased with ourselves.
It seems a little incredible now that there is virtually no mainline steam anywhere in the world, that we would get bored, just because there were thousands of QJs running all over the country. I can imagine a few years later it wouldn't matter where you went in China, all you would see were QJs and JFs. Fortunately for me, it was not all like that back then.
We Move On
The next leg of the journey was on Express 163 to Xian via Baoji. Our destination is one of the more touristy parts of China and home of the Terracotta Warriors.
We travelled 3 hours behind diesel, before we finally attached an RM to the train for the run to Xian. At that time all trains in and out of Xian were steam hauled. It was a 6 ½ hour trip behind steam
I didn't take any log books while in China, but I did record some times. The RM reached sustained speeds of 100 kph. This is pretty good, considering the huge loads they hauled.
The next segment details my time in Xian and then on to Lanzhou, our furthest point west, located on the Yangtze River. As I recall, it was difficult to travel any further West as an individual in those days. For this part of the journey, I am afraid you will have to wait for a little while.


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Below you will find a list of discussions in the Art and beauty in Poltava forums at the Poltava Forum. Photos, postcards, drawings, anything beautiful made in Poltava or related to Poltava
Use this control to limit the display of threads to those newer than the specified time frame.
Allows you to choose the data by which the thread list will be sorted.
Order threads in...
Note: when sorting by date, 'descending order' will show the newest results first.
Forum Rules


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If you've been married for over 5 years, your marriage may have gotten a bit routine. You don't dress up for each other the way you used to or tell each other the little things that mean so much. If you're looking to better your relationship and avoid the so-called "seven-year itch", it's important to remember that a healthy marriage takes work.
Secrets to Avoiding the Seven-Year Itch
- Date Each Other
It's easy to fall into routine when you're both working and taking care of the kids. However, it's just as important to date each other now as it was back when you first met. Why? Because even though you're married, doesn't mean you always know the person the way you think you do. People change and grow during the course of a relationship, giving you an open opportunity to get to know each other over and over again!
Also, going on a once-a-week movie or dinner date gives you a chance to dress up, put on a little perfume or cologne, and remind each other what attracted you in the first place. Intimate conversation and gestures in a new environment can do wonders for keeping your relationship fresh and exciting!
- Write Love Notes
Another way to avoid the seven-year itch is to write love notes to one another. You don't have to spend hours crafting poetry; just slip a Post-It note in his briefcase and tell him you love him and can't wait to see him when he comes home. Or mention something thoughtful and kind she did the other day and slip it into her jacket pocket. Love notes are a way to say, "I still notice the little things and they mean a lot to me."
- Do Things Together
Household chores are inevitable so why tackle them alone? Cooking, cleaning, and doing laundry is a lot more fun when you can do them with a partner. When cooking, try out new recipes together and really get creative. When cleaning, crank up the radio and sing off-key. Why not? A sense of humor is one of the foundations for a strong marriage!
Getting out and active is another great way to better your relationship and your health. Try joining a gym or go hiking together. Make it a point to go for a short walk together after supper. Whatever you and your spouse can do to reconnect will help deepen your bond.
- Give Compliments
In the beginning of your romance, you told her she was beautiful all the time. But with financial worries and getting the kids to the soccer field on time, the feeling is still there but the words don't come as often. It happens in every marriage. We become settled and complacent, believing the other person should just know how we feel.
They do but it's nice to hear it. Pay closer attention to your spouse. Did she recently get a haircut? Is he wearing a new shirt? Look for reasons to compliment your significant other and they'll feel closer to and more appreciated by you.
- Be Thoughtful
If she's running late for work, warm the car up for her. If he's had a stressful day, offer him a relaxing back rub. Avoiding the seven-year itch can be as simple as paying a little more attention to what you can do to improve the marriage without being asked. Thoughtfulness goes a long way in bettering your relationship bond.
- Argue the Right Way
Every couple argues. It's a normal part of being in a marriage. However, it's important even during arguments that you continue to show respect for one another. This means not arguing in public, talking down to your spouse, or resorting to name-calling. When you and your marriage partner are in a heated disagreement, remember to focus on the subject of the argument, not each other's shortcomings.
In other words, focus your irritation on the problem, not each other. The healthiest way to argue is to use "I" statements. Instead of, "You never pay any attention what I'm saying. You're a jerk!" say, "When you continue to watch TV even though I'm trying to tell you something about my day, it makes me feel ignored and unappreciated."
Oftentimes, we are ignorant of the effect our behavior has on others and having it pointed out in a non-confrontational way can go a long way in healing unspoken rifts.
- Give Each Other Space
In any relationship, there's a such thing as too much togetherness. You and your spouse should spend time alone pursuing other hobbies and interests as well as enjoying time with friends. Time apart gives you perspective on your relationship and can help make time together seem that much more precious.
Remember the day you took your vows. It was one of the happiest days of your life. Just because you're coming up on a milestone anniversary doesn't mean things have to get dull. If your sex life isn't what it used to be, improving your communication and trying new things in the bedroom could be just what you need to bring the spark back and keep your marriage fresh!
How do you keep the spark in our marriage?
Click Below To Get Started With Pheromones Today!


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Skip to comments.Almsgiving [Lent]
Posted on 03/07/2007 11:00:08 AM PST by Salvation
Other Articles by Mark Shea
Printer Friendly Version
One of the traditional emphases during Lent is the duty of "almsgiving". The tradition of the Church here comes straight out of ancient Israel and the duty that was enjoined upon the Jews by God to care for the stranger, the orphan, and the widow. In many ways, ancient Israel was commanded to take care of the weakest and least in their midst. For obedience to this command, great mercies and benefits were promised. For disobedience, some of the most strenuous and frightening curses are reserved. The sense that comes across very strongly throughout the Old Testament is that the most helpless are paradoxically the most favored and that God's special tenderness is theirs (and his special fury, ours, if we despise the weak).
This theme becomes even more pronounced in the New Testament as God reveals the extent of his identification with the weak and the poor. For God is born, not in the purple, but in the filth of a stable and grows up among the anawim, the poorest of the poor, in the toolies of Nazareth. He is dirt poor all his life, eventually becoming an itinerant with "no place to lay his head." He is accused by the Refined People of being a glutton and a drunkard. His companions are poor country folk identifiable by their backwoods twang (Matthew 26:73). He is shoved into prison and condemned to death on a trumped up charge, where he dies a slave's death stark naked and in complete humiliation. In every way, he is at one with the most despised people on earth, those whom he himself called "the least of these" and with whom he deliberately identified himself. So the Church has always seen in the poor the face of Christ in his "distressing disguise" as Mother Teresa called it.
What we sometimes forget however, is that poverty can have many kinds of faces besides economic poverty. To be sure, economic poverty is the first and most obvious sort and we do well to care for those who do not have much of the world's goods. But those who will handily forgive a debt of money from a beggar for a cup of coffee can often forget that there are other sorts of debts as well.
In my own life, I've discovered that I often speak like the unmerciful servant in the parable, not about financial debts, but about emotional and spiritual ones. I, who would never think of choking a beggar and demanding he pay me money, have sometimes found myself full of bitterness and resentment when one who is poor in flattery doesn't pay me the compliment I thought they owed me. Never mind that they were too harried by life to notice my wonderfulness and lift their thoughts from their struggles to attend to Me and My Needs. They owed me and I was determined to squeeze what they owed me out of them, even if it meant unforgiveness. Similarly, I have often demanded of the poor in time that they pay me attention. I've demanded of the poor in happiness that they pay me cheerfulness when I am feeling gloomy. I've demanded of the poor in compassion (perhaps because nobody ever gave them any) that they have enough to share with me. There is lots of poverty in the world besides financial poverty, but for a long time I've been stone blind to it.
In all this, I've been no different than the servant in the parable who was forgiven the trillion dollar debt but who throttled the man who owed him a few bucks. This year, I'm going to try seeing poverty and giving alms wherever poverty is: to the poor in flattery, praise; to the poor in time, attentiveness to them; to the poor in happiness, a cheerful heart; to the poor in compassion, love. There's more than one way to give alms.
Prayer Almsgiving Fasting
Disclaimer: Opinions posted on Free Republic are those of the individual posters and do not necessarily represent the opinion of Free Republic or its management. All materials posted herein are protected by copyright law and the exemption for fair use of copyrighted works.


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