Transcriptomic and functional screening of weapon formation genes implies significance of cell adhesion molecules and female-biased genes in broad-horned flour beetle.

For understanding the evolutionary mechanism of sexually selected exaggerated traits, it is essential to uncover its molecular basis. By using broad-horned flour beetle that has male-specific exaggerated structures (mandibular horn, head horn and gena enlargement), we investigated the transcriptomic and functional characters of sex-biased genes. Comparative transcriptome of male vs. female prepupal heads elucidated 673 sex-biased genes. Counter-intuitively, majority of them were female-biased (584 genes), and GO enrichment analysis showed cell-adhesion molecules were frequently female-biased. This pattern motivated us to hypothesize that female-biased transcripts (i.e. the transcripts diminished in males) may play a role in outgrowth formation. Potentially, female-biased genes may act as suppressors of weapon structure. In order to test the functionality of female-biased genes, we performed RNAi-mediated functional screening for top 20 female-biased genes and 3 genes in the most enriched GO term (cell-cell adhesion, fat1/2/3, fat4 and dachsous). Knockdown of one transcription factor, zinc finger protein 608 (zfp608) resulted in the formation of male-like gena in females, supporting the outgrowth suppression function of this gene. Similarly, knockdown of fat4 induced rudimental, abnormal mandibular horn in female. fat1/2/3RNAi, fat4RNAi and dachsousRNAi males exhibited thick and/or short mandibular horns and legs. These cell adhesion molecules are known to regulate tissue growth direction and known to be involved in the weapon formation in Scarabaeoidea beetles. Functional evidence in phylogenetically distant broad-horned flour beetle suggest that cell adhesion genes are repeatedly deployed in the acquisition of outgrowth. In conclusion, this study clarified the overlooked functions of female-biased genes in weapon development.

A specific type of insulin-like peptide regulates the conditional growth of a beetle weapon.

Evolutionarily conserved insulin/insulin-like growth factor (IGF) signaling (IIS) has been identified as a major physiological mechanism underlying the nutrient-dependent regulation of sexually selected weapon growth in animals. However, the molecular mechanisms that couple nutritional state with weapon growth remain largely unknown. Here, we show that one specific subtype of insulin-like peptide (ILP) responds to nutrient status and thereby regulates weapon size in the broad-horned flour beetle Gnatocerus cornutus. By using transcriptome information, we identified five G. cornutus ILP (GcorILP1-5) and two G. cornutus insulin-like receptor (GcorInR1, -2) genes in the G. cornutus genome. RNA interference (RNAi)-mediated gene silencing revealed that a certain subtype of ILP, GcorILP2, specifically regulated weapon size. Importantly, GcorILP2 was highly and specifically expressed in the fat body in a condition-dependent manner. We further found that GcorInR1 and GcorInR2 are functionally redundant but that the latter is partially specialized for regulating weapon growth. These results strongly suggest that GcorILP2 is an important component of the developmental mechanism that couples nutritional state to weapon growth in G. cornutus. We propose that the duplication and subsequent diversification of IIS genes played a pivotal role in the evolution of the complex growth regulation of secondary sexual traits.

Loser-effect duration evolves independently of fighting ability.

Winning or losing contests can impact subsequent competitive behaviour and the duration of these effects can be prolonged. While it is clear effects depend on social and developmental environments, the extent to which they are heritable, and hence evolvable, is less clear and remains untested. Furthermore, theory predicts that winner and loser effects should evolve independently of actual fighting ability, but again tests of this prediction are limited. Here we used artificial selection on replicated beetle populations to show that the duration of loser effects can evolve, with a realized heritability of about 17%. We also find that naive fighting ability does not co-evolve with reductions in the duration of the loser effect. We discuss the implications of these findings and how they corroborate theoretical predictions.

Histone deacetylases control module-specific phenotypic plasticity in beetle weapons.

Nutritional conditions during early development influence the plastic expression of adult phenotypes. Among several body modules of animals, the development of sexually selected exaggerated traits exhibits striking nutrition sensitivity, resulting in positive allometry and hypervariability distinct from other traits. Using de novo RNA sequencing and comprehensive RNA interference (RNAi) for epigenetic modifying factors, we found that histone deacetylases (HDACs) and polycomb group (PcG) proteins preferentially influence the size of mandibles (exaggerated male weapon) and demonstrate nutrition-dependent hypervariability in the broad-horned flour beetle, Gnatocerus cornutus RNAi-mediated HDAC1 knockdown (KD) in G. cornutus larvae caused specific curtailment of mandibles in adults, whereas HDAC3 KD led to hypertrophy. Notably, these KDs conferred opposite effects on wing size, but little effect on the size of the core body and genital modules. PcG RNAi also reduced adult mandible size. These results suggest that the plastic development of exaggerated traits is controlled in a module-specific manner by HDACs.

Social dominance alters nutrition-related gene expression immediately: transcriptomic evidence from a monomorphic queenless ant.

Queen-worker differentiation in eusocial organisms may have originated from decoupling of maternal care and reproductive behaviours. Recent advances in sequencing techniques have begun to elucidate the molecular basis of queen-worker differentiation. However, current knowledge of the molecular basis of caste differentiation is limited, especially to species with morphological castes. It seems likely that at the dawn of eusociality morphologically undifferentiated, monomorphic females underwent physiological differentiation that yielded egg-laying and caretaking castes. The molecular basis of such physiological differentiation may provide evolutionary insight into the emergent state of eusociality. In this study, we identify genes associated with monomorphic caste differentiation, specifically focusing on the onset of queen-worker differentiation, using a monomorphic queenless ant, Diacamma sp., that secondarily lost morphological castes. Using individuals experimentally manipulated to become sterile or reproductive, we identified 1546 caste-biased transcripts in brain and 10 in gaster. Because caste differentiation occurs in Diacamma soon after eclosion via behavioural dominance, identified transcripts are interpreted as molecular agents responding immediately to dominance rank formation. Among identified genes, expression levels of genes involved in nutrition processing and storage, such as insulin signalling genes and hexamerins, were strongly altered soon after dominance rank formation. We conclude that the rapid modification of nutrition-related genes in response to social rank may be the fundamental mechanism underlying caste differentiation in Diacamma. Together with functional evidence from the literature, we show that a specific set of genes frequently plays a role in reproductive differentiation across systems with and without morphological castes.

Ant circadian activity associated with brood care type.

In group-living animals, social interactions influence various traits including circadian activity. Maternal care, in particular, can have a strong effect on the circadian activity of parents or nurses across taxa. In social insects, nest-mates are known to have diverse activity rhythms; however, what kind of social environment is crucial in shaping an individual's rhythm is largely unknown. Here, we show that the focal brood types being taken care of (i.e. egg, larva and pupa) have significant effects on individual activity/rest rhythm, using the monomorphic ant Diacamma (putative species indicum). When isolated from a colony, nurses exhibited a clear circadian rhythm. However, when paired with eggs or larvae, they exhibited around-the-clock activity with no apparent rhythm. In contrast, a clear activity rhythm emerged when nurses were paired with a pupa, requiring little care. Such brood-type-specific changes in circadian activity are considered to arise from the difference in caretaking demands. Our finding may contribute to the understanding of the organization of a colony in the context of behavioural variability under different microenvironments.

Social dominance and reproductive differentiation mediated by dopaminergic signaling in a queenless ant.

In social Hymenoptera with no morphological caste, a dominant female becomes an egg layer, whereas subordinates become sterile helpers. The physiological mechanism that links dominance rank and fecundity is an essential part of the emergence of sterile females, which reflects the primitive phase of eusociality. Recent studies suggest that brain biogenic amines are correlated with the ranks in dominance hierarchy. However, the actual causality between aminergic systems and phenotype (i.e. fecundity and aggressiveness) is largely unknown due to the pleiotropic functions of amines (e.g. age-dependent polyethism) and the scarcity of manipulation experiments. To clarify the causality among dominance ranks, amine levels and phenotypes, we examined the dynamics of the aminergic system during the ontogeny of dominance hierarchy in the queenless ant Diacamma sp., which undergoes rapid physiological differentiation based on dominance interactions. Brain dopamine levels differed between dominants and subordinates at day 7 after eclosion, although they did not differ at day 1, reflecting fecundity but not aggressiveness. Topical applications of dopamine to the subordinate workers induced oocyte growth but did not induce aggressiveness, suggesting the gonadotropic effect of dopamine. Additionally, dopamine receptor transcripts (dopr1 and dopr2) were elevated in the gaster fat body of dominant females, suggesting that the fat body is a potential target of neurohormonal dopamine. Based on this evidence, we suggest that brain dopamine levels are elevated in dominants as a result of hierarchy formation, and differences in dopamine levels cause the reproductive differentiation, probably via stimulation of the fat body.

Environmental Factors Affecting Pupation Decision in the Horned Flour Beetle Gnatocerus cornutus.

Social environments often affect the development of organisms. In Tenebrionidae beetles, larval development can be arrested at the final instar stage in the presence of conspecific larvae. This developmental plasticity is considered to be an anti-cannibalistic strategy but the critical environmental determinants and actual effects remain to be elucidated. In this study, we examined the effects of the heterospecific environment, conspecific sexual environment (i.e., presence of conspecific male or female), and abiotic physical stimulation on the pupation decision of the sexually dimorphic horned-flour beetle Gnatocerus cornutus. Additionally, actual anti-cannibalistic or antipredatory effects of developmental arrest were evaluated by analyzing stage-dependent vulnerabilities. When G. cornutus larvae were maintained with a G. cornutus larva, a G. cornutus adult, or T. castaneum adult, the developmental period up to the prepupal stage was significantly elongated, suggesting that the cue is not species-specific. Sexual environment did not affect the timing of pupation in G. cornutus; however, we found that abiotic tactile stimulations by glass beads could repress pupation. We also discovered that prepupal and pupal stages were more vulnerable to cannibalism and predation than the larval stage. These data suggest that G. cornutus larvae use non-species specific tactile stimulation as a decision cue for pupation and it has broader defensive effects against heterospecific predation as well as conspecific cannibalism.

Morphological and functional analyses for investigation of sexually selected legs in the frog legged beetle Sagra femorata (Coleoptera: Chrysomelidae).

Mate choice and male-male combat over successful mating often cause disproportionate exaggeration of male trait relative to body size. However, the exaggeration is often not the only trait involved with male-male combat and mate choice: suites of co-expressed traits may function together as a coordinated unit. When this occurs, dimorphism may be expected for these additional, non-exaggerated, structures. S. femorata males have disproportionately large hind-legs used in male-male combat over females. During the fights, fore- and mid-legs are used to keep males in positions where advantageous for leverage. Because use of the exaggerated hind-legs is coordinated with the other legs, they will coevolve as a functional unit. Here, we show that 1) S. femorata has sexual size differences in all three legs; 2) males show positive allometry in the relative sizes of all three legs; and 3) microstructures of tarsi on the fore- and mid-legs are also sexually dimorphic. Despite these differences in the tarsal microstructure, 4) adhesion forces of the tarsi had no sexual difference in flat surface. The microstructure would be specialized on attaching elytra surface. These results suggest that the three pairs of legs function together during fighting behavior, with hind-legs employed primarily for fighting, and the fore- and mid-legs functioning to grip females, keeping males positioned on the back of the female during combat.

Soldier morphogenesis in the damp-wood termite is regulated by the insulin signaling pathway.

Eusocial insects exhibit various morphological castes associated with the division of labor within a colony. Termite soldiers possess defensive traits including mandibles that are greatly exaggerated and enlarged, as compared to termite reproductives and workers. The enlarged mandibles of soldiers are known to result from dynamic morphogenesis during soldier differentiation that can be induced by juvenile hormone and its analogs. However, the detailed developmental mechanisms still remain unresolved. Because the insulin/insulin-like growth factor signaling (IIS) pathway has been shown to regulate the relative sizes of organs (i.e., allometry) in other insects, we examined the expression profiles of major IIS factors in the damp-wood termite Hodotermopsis sjostedti, during soldier differentiation. The relative expression patterns of orthologs for termite InR (HsjInR), PKB/Akt (HsjPKB/Akt), and FOXO (HsjFOXO) suggest that HsjInR and HsjPKB/Akt were up-regulated in the period of elongation of mandibles during soldier development. In situ hybridization showed that HsjInR was strongly expressed in the mandibular epithelial tissues, and RNA interference (RNAi) for HsjInR disrupted soldier-specific morphogenesis including mandibular elongation. These results suggest that signaling through the IIS pathway is required for soldier-specific morphogenesis. In addition, up-regulation of the IIS pathway in other body tissues occurred at earlier stages of development, indicating that there is tissue-specific IIS regulation. Because the IIS pathway is generally thought to act upstream of JH in insects, our results suggest the damp-wood termite may have evolved a novel feedback loop between JH and IIS that enables social interactions, rather than nutrition, to regulate caste determination.

Juvenile hormone mediates developmental integration between exaggerated traits and supportive traits in the horned flour beetle Gnatocerus cornutus.

Sexually selected exaggerated traits are often coupled with modifications in other nontarget traits. In insects with weapons, enlargements of nontarget characters that functionally support the weapon often occur (i.e. supportive traits). The support of sexual traits requires developmental coordination among functionally related multiple traits-an explicit example of morphological integration. The genetic theory predicts that developmental integration among different body modules, for which development is regulated via different sets of genes, is likely to be coordinated by pleiotropic factors. However, the developmental backgrounds of morphological integrations are largely unknown. We tested the hypothesis that the juvenile hormone (JH), as a pleiotropic factor, mediates the integration between exaggerated and supportive traits in an armed beetle Gnatocerus cornutus. During combat, males of this beetle use exaggerated mandibles to lift up their opponents with the supportive traits, that is, the head and prothoracic body parts. Application of methoprene, a JH analog (JHA), during the larval to prepupal period, induced the formation of large mandibles relative to the body sizes in males. Morphometric examination of nontarget traits elucidated an increase in the relative sizes of supportive traits, including the head and prothoracic body parts. In addition, reductions in the hind wing area and elytra length, which correspond to flight and reproductive abilities, were detected. Our findings are consistent with the genetic theory and support the idea that JH is a key pleiotropic factor that coordinates the developmental integration of exaggerated traits and supportive characters, as well as resource allocation trade-offs.

Genome-wide deficiency screen for the genomic regions responsible for heat resistance in Drosophila melanogaster.

BACKGROUND: Temperature adaptation is one of the most important determinants of distribution and population size of organisms in nature. Recently, quantitative trait loci (QTL) mapping and gene expression profiling approaches have been used for detecting candidate genes for heat resistance. However, the resolution of QTL mapping is not high enough to examine the individual effects of various genes in each QTL. Heat stress-responsive genes, characterized by gene expression profiling studies, are not necessarily responsible for heat resistance. Some of these genes may be regulated in association with the heat stress response of other genes. RESULTS: To evaluate which heat-responsive genes are potential candidates for heat resistance with higher resolution than previous QTL mapping studies, we performed genome-wide deficiency screen for QTL for heat resistance. We screened 439 isogenic deficiency strains from the DrosDel project, covering 65.6% of the Drosophila melanogaster genome in order to map QTL for thermal resistance. As a result, we found 19 QTL for heat resistance, including 3 novel QTL outside the QTL found in previous studies. CONCLUSION: The QTL found in this study encompassed 19 heat-responsive genes found in the previous gene expression profiling studies, suggesting that they were strong candidates for heat resistance. This result provides new insights into the genetic architecture of heat resistance. It also emphasizes the advantages of genome-wide deficiency screen using isogenic deficiency libraries.

Male aggressive behavior and exaggerated hindlegs of the bean bug Riptortus pedestris.

Males of the bean bug species Riptortus pedestris possess larger hindlegs than females. Observations of male-male interactions showed that the enlarged hindlegs are used as weapons in male fights, and that males with larger hindlegs win fights more frequently. Morphological analysis based on the positive allometry test showed that the femora of larger males are relatively bigger than those of smaller males, but femora of larger females are not relatively larger than those of smaller females. These results suggest that sexual selection in R. pedestris favors larger hindlegs for male fighting. In addition, the thorax and abdomen lengths were larger in the male than in the female. The males often lift their abdomen with their back to the opponent for displays against an opponent. As a result, abdominal size may be under stronger selection in the male than in the female, as for the exaggerated hindlegs.

Genome-wide deficiency mapping of the regions responsible for temporal canalization of the developmental processes of Drosophila melanogaster.

Developmental processes of organisms are programmed to proceed in a finely regulated manner and finish within a certain period of time depending on the ambient environmental conditions. Therefore, variation in the developmental period under controlled genetic and environmental conditions indicates innate instability of the developmental process. In this study, we aimed to determine whether a molecular machinery exists that regulates the canalization of the developmental period and, if so, to test whether the same mechanism also stabilizes a morphological trait. To search for regions that influence the instability of the developmental period, we conducted genome-wide deficiency mapping with 441 isogenic deficiency strains covering 65.5% of the Drosophila melanogaster genome. We found that 11 independent deficiencies significantly increased the instability of the developmental period and 5 of these also significantly increased the fluctuating asymmetry of wing shape although there was no significant correlation between the instabilities of developmental period and wing shape in general. These results suggest that canalization processes of the developmental period and morphological traits are at least partially independent. Our findings emphasize the potential importance of temporal variation in development as an indicator of developmental stability and canalization and provide a novel perspective for understanding the regulation of phenotypic variability.

Bipartite network analysis of ant-task associations reveals task groups and absence of colonial daily activity.

Social insects are one of the best examples of complex self-organized systems exhibiting task allocation. How task allocation is achieved is the most fascinating question in behavioural ecology and complex systems science. However, it is difficult to comprehensively characterize task allocation patterns due to behavioural complexity, such as the individual variation, context dependency and chronological variation. Thus, it is imperative to quantify individual behaviours and integrate them into colony levels. Here, we applied bipartite network analyses to characterize individual-behaviour relationships. We recorded the behaviours of all individuals with verified age in ant colonies and analysed the individual-behaviour relationship at the individual, module and network levels. Bipartite network analysis successfully detected the module structures, illustrating that certain individuals performed a subset of behaviours (i.e. task groups). We confirmed age polyethism by comparing age between modules. Additionally, to test the daily rhythm of the executed tasks, the data were partitioned between daytime and nighttime, and a bipartite network was re-constructed. This analysis supported that there was no daily rhythm in the tasks performed. These findings suggested that bipartite network analyses could untangle complex task allocation patterns and provide insights into understanding the division of labour.

Individual Ants Do Not Show Activity-Rest Rhythms in Nest Conditions.

Circadian rhythms, which respond to the day-night cycle on the earth, arise from the endogenous timekeeping system within organisms, called the "biological clock." For accurate circadian rhythms, daily fluctuations in light and temperature are considered one of the important time cues. In social insects, both abiotic and biotic factors (i.e., social interactions) play a significant role in activity-rest rhythm regulation. However, it is challenging to monitor individual activity-rest rhythms in a colony because of the large group size and small body size. Therefore, it is unclear whether individuals in a colony exhibit activity-rest rhythms and how social interactions regulate their activity-rest rhythms in the colony. This study developed an image-based tracking system using 2D barcodes for Diacamma cf. indicum from Japan (a monomorphic ant) and measured the locomotor activities of all colony members under laboratory colony conditions. We also investigated the effect of broods on activity-rest rhythms by removing all broods under colony conditions. Activity-rest rhythms appeared only in isolated ants, not under colony conditions. In addition, workers showed arrhythmic activities after brood removal. These results suggested that a mixture of social interactions, and not light and temperature, induces the loss of activity-rest rhythms. These results contribute to the knowledge of a diverse pattern of circadian activity rhythms in social insects.

Octopaminergic system orchestrates combat and mating behaviors: A potential regulator of alternative male mating tactics in an armed beetle.

Male-male combats over females and territories are widespread across animal taxa. The winner of a combat gains resources, while the loser suffers significant costs (e.g. time, energy and injury) without gaining resources. Many animals have evolved behavioral flexibility, depending on their nutritional condition and experience, to avoid combat in order to reduce such costs. In these cases, male aggression often correlates with mating behavior changes, that is, the deployment of alternative reproductive tactics. Therefore, uncovering the physiological mechanism that orchestrates combat and mating behaviors is essential to understand the evolution of alternative mating tactics. However, so far, our knowledge is limited to specific behaviors (i.e., fighting or mating) of specific model species. In this study, we used an armed beetle (Gnatocerus cornutus) and hypothesized that one of the key neuromodulators of invertebrate aggression, octopamine (OA), would control male combat and other mating behaviors. Using receptor agonists (chlordimeform and benzimidazole), we showed that the octopaminergic (OAergic) system down-regulated the combat and courtship behaviors, while it up-regulated locomotor activity and sperm size. This suggests that the OAergic system orchestrates a suite of fighting and mating behaviors, thereby implying that correlated behavioral responses to OAergic signaling may have driven the evolution of alternative mating tactics in this beetle.

Natural selection increases female fitness by reversing the exaggeration of a male sexually selected trait.

Theory shows how sexual selection can exaggerate male traits beyond naturally selected optima and also how natural selection can ultimately halt trait elaboration. Empirical evidence supports this theory, but to our knowledge, there have been no experimental evolution studies directly testing this logic, and little examination of possible associated effects on female fitness. Here we use experimental evolution of replicate populations of broad-horned flour beetles to test for effects of sex-specific predation on an exaggerated sexually selected male trait (the mandibles), while also testing for effects on female lifetime reproductive success. We find that populations subjected to male-specific predation evolve smaller sexually selected mandibles and this indirectly increases female fitness, seemingly through intersexual genetic correlations we document. Predation solely on females has no effects. Our findings support fundamental theory, but also reveal unforseen outcomes-the indirect effect on females-when natural selection targets sex-limited sexually selected characters.

Gene up-regulation in response to predator kairomones in the water flea, Daphnia pulex.

BACKGROUND: Numerous cases of predator-induced polyphenisms, in which alternate phenotypes are produced in response to extrinsic stimuli, have been reported in aquatic taxa to date. The genus Daphnia (Branchiopoda, Cladocera) provides a model experimental system for the study of the developmental mechanisms and evolutionary processes associated with predator-induced polyphenisms. In D. pulex, juveniles form neckteeth in response to predatory kairomones released by Chaoborus larvae (Insecta, Diptera). RESULTS: Previous studies suggest that the timing of the sensitivity to kairomones in D. pulex can generally be divided into the embryonic and postembryonic developmental periods. We therefore examined which of the genes in the embryonic and first-instar juvenile stages exhibit different expression levels in the presence or absence of predator kairomones. Employing a candidate gene approach and identifying differentially-expressed genes revealed that the morphogenetic factors, Hox3, extradenticle and escargot, were up-regulated by kairomones in the postembryonic stage and may potentially be responsible for defense morph formation. In addition, the juvenile hormone pathway genes, JHAMT and Met, and the insulin signaling pathway genes, InR and IRS-1, were up-regulated in the first-instar stage. It is well known that these hormonal pathways are involved in physiological regulation following morphogenesis in many insect species. During the embryonic stage when morphotypes were determined, one of the novel genes identified by differential display was up-regulated, suggesting that this gene may be related to morphotype determination. Biological functions of the up-regulated genes are discussed in the context of defense morph formation. CONCLUSIONS: It is suggested that, following the reception of kairomone signals, the identified genes are involved in a series of defensive phenotypic alterations and the production of a defensive phenotype.

Gene expression changes during caste-specific neuronal development in the damp-wood termite Hodotermopsis sjostedti.

BACKGROUND: One of the key characters of social insects is the division of labor, in which different tasks are allocated to various castes. In termites, one of the representative groups of social insects, morphological differences as well as behavioral differences can be recognized among castes. However, very little is known about the neuronal and molecular bases of caste differentiation and caste-specific behavior. In almost all termite species, soldiers play defensive roles in their colonies, and their morphology and behavior are largely different from workers (or pseudergates). Therefore, we predicted that some genes linked to defensive behavior and/or those required for neuronal changes are differentially expressed between workers and soldiers, or during the soldier differentiation, respectively. RESULTS: Using the brain and suboesophageal ganglion (SOG) of the damp-wood termite Hodotermopsis sjostedti, we first screened genes specifically expressed in soldiers or during soldier differentiation by the differential display method, followed by quantitative real-time polymerase chain reaction. No distinctive differences in expression patterns were detected between pseudergates and soldiers. In the course of soldier differentiation, however, five genes were found to be up-regulated in brain and/or SOG: 14-3-3 epsilon, fibrillin2, beta-tubulin, ciboulot, and a hypothetical protein containing a SAP motif. Some of these genes are thought to be associated with cytoskeletal structure or motor-associated proteins in neuronal tissues. CONCLUSION: The identified five genes could be involved in soldier-specific neuronal modifications, resulting in defensive behaviors in termite soldiers. The temporal expression patterns of these genes were consistent with the neuronal changes during soldier differentiation, suggesting that molecular machineries, in which the identified factors would participate, play important roles in behavioral differentiation of termite soldiers.