What happens in the pith stays in the pith: tissue‐localized defense responses facilitate chemical niche differentiation between two spatially separated herbivores

Herbivore attack is known to elicit systemic defense responses that spread throughout the host plant and influence the performance of other herbivores. While these plant‐mediated indirect competitive interactions are well described, and the co‐existence of herbivores from different feeding guilds is common, the mechanisms of co‐existence are poorly understood. In both field and glasshouse experiments with a native tobacco, Nicotiana attenuata, we found no evidence of negative interactions when plants were simultaneously attacked by two spatially separated herbivores: a leaf chewer Manduca sexta and a stem borer Trichobaris mucorea. T. mucorea attack elicited jasmonic acid (JA) and jasmonoyl‐l ‐isoleucine bursts in the pith of attacked stems similar to those that occur in leaves when M. sexta attacks N. attenuata leaves. Pith chlorogenic acid (CGA) levels increased 1000‐fold to levels 6‐fold higher than leaf levels after T. mucorea attack; these increases in pith CGA levels, which did not occur in M. sexta‐attacked leaves, required JA signaling. With plants silenced in CGA biosynthesis (irHQT plants), CGA, as well as other caffeic acid conjugates, was demonstrated in both glasshouse and field experiments to function as a direct defense protecting piths against T. mucorea attack, but not against leaf chewers or sucking insects. T. mucorea attack does not systemically activate JA signaling in leaves, while M. sexta leaf‐attack transiently induces detectable but minor pith JA levels that are dwarfed by local responses. We conclude that tissue‐localized defense responses allow tissue‐specialized herbivores to share the same host and occupy different chemical defense niches in the same hostplant.     

Colony‐age‐dependent variation in cuticular hydrocarbon profiles in subterranean termite colonies

Cuticular hydrocarbons (CHCs) have, in insects, important physiological and ecological functions, such as protection against desiccation and as semiochemicals in social taxa, including termites. CHCs are, in termites, known to vary qualitatively and/or quantitatively among species, populations, castes, or seasons. Changes to hydrocarbon profile composition have been linked to varying degrees of aggression between termite colonies, although the variability of results among studies suggests that additional factors might have been involved. One source of such variability may be colony age, as termite colony demographics significantly change over time, with different caste and instar compositions throughout the life of the colony. We here hypothesize that the intracolonial chemical profile heterogeneity would be high in incipient termite colonies but would homogenize over time as a colony ages and accumulates older workers in improved homeostatic conditions. We studied caste‐specific patterns of CHC profiles in Coptotermes gestroi colonies of four different age classes (6, 18, 30, and 42 months). The CHC profiles were variable among castes in the youngest colonies, but progressively converged toward a colony‐wide homogenized chemical profile. Young colonies had a less‐defined CHC identity, which implies a potentially high acceptance threshold for non‐nestmates conspecifics in young colonies. Our results also suggest that there was no selective pressure for an early‐defined colony CHC profile to evolve in termites, potentially allowing an incipient colony to merge nonagonistically with another conspecific incipient colony, with both colonies indirectly and passively avoiding mutual destruction as a result.     

Host‐associated differentiation in a pecan and water hickory Aphidomorpha community

Host‐associated differentiation (HAD) is the formation of genetically distinct, host‐associated populations created and maintained by ecologically mediated reproductive isolation. HAD potentially accounts for species origins in parasites, including herbivorous insects. Although case studies testing the occurrence of HAD are accumulating, it is still unclear how common HAD is and which specific ecological traits explain its occurrence. To address these issues, studies are needed that include negative results (i.e., instances in which parasite populations do not exhibit HAD) and test for HAD across communities (i.e., several parasite species on the same set of host species). In this study, HAD was tested in a community of six species of Aphidomorpha (Hemiptera) that share a host‐plant pair: pecan [Carya illinoinensis (Wangenh.) K.Koch] and water hickory [Carya aquatica (F.Michx) Nutt., both Juglandaceae] trees. All six species are parthenogenetic and three species are endophagous, traits that can exacerbate host‐specific selection. AFLP markers were employed to detect the presence of genetically distinct, host‐associated populations for each insect species. Strict HAD (i.e., the occurrence of genetically distinct pecan‐associated and water hickory‐associated genotypes) was found in Phylloxera notabilis Pergande (Phylloxeridae), Phylloxera devastatrix Pergande, and Monelliopsis pecanis Bissel (Aphididae). Monellia caryella Fitch (Aphididae) displayed a pattern of partial HAD (i.e., the occurrence of only a genetically distinct pecan‐associated genotype). No HAD was found in Melanocallis caryaefoliae Davis (Aphididae) or Phylloxera texana Stoetzel. The pattern of HAD occurrence in the pecan and water hickory Aphidomorpha community indicated that neither parthenogenesis nor endophagy sufficiently explain the occurrence of HAD in this system.     

Non‐destructive observation of the mycangia of Euwallacea interjectus (Blandford) (Coleoptera: Curculionidae: Scolytinae) using X‐ray computed tomography

X‐ray microtomography has been applied successfully to obtain reliable microstructural information of many insect species. Nonetheless, the technique has not been widely applied to ambrosia beetles. The ambrosia beetle Euwallacea interjectus (Blandford) was first recorded as a vector of plant pathogenic fungus Ceratocystis ficicola Kajitani & Masuya, which has caused serious wilt disease in many fig orchards in Japan since 1999. Previous studies of E. interjectus have not described the mycangia (fungus‐storing organ) in detail. In this study, we non‐destructively examined the internal structure of an adult female of E. interjectus through computed microtomography scans. Paired mycangia were observed on typical computed tomography cross‐sections of the head. Each mycangium, ovoid in shape, was located in tissues just posterior to emarginated notch of eyes, adjacent to pharynx. Three dimensions (length × width × depth) of the mycangia were measured on stereography. We confirmed the absence of mycangia in the other body parts, such as elytra, prothorax and coxa of legs.     

Overexpression of a soybean nuclear localized type–III DnaJ domain‐containing HSP40 reveals its roles in cell death and disease resistance

Heat‐shock proteins such as HSP70 and HSP90 are important molecular chaperones that play critical roles in biotic and abiotic stress responses; however, the involvement of their co‐chaperones in stress biology remains largely uninvestigated. In a screen for candidate genes stimulating cell death in Glycine max (soybean), we transiently overexpressed full‐length cDNAs of soybean genes that are highly induced during soybean rust infection in Nicotiana benthamiana leaves. Overexpression of a type‐III DnaJ domain‐containing HSP40 (GmHSP40.1), a co‐chaperone of HSP70, caused hypersensitive response (HR)‐like cell death. The HR‐like cell death was dependent on MAPKKKα and WIPK, because silencing each of these genes suppressed the HR. Consistent with the presence of a nuclear localization signal (NLS) motif within the GmHSP40.1 coding sequence, GFP‐GmHSP40.1 was exclusively present in nuclear bodies or speckles. Nuclear localization of GmHSP40.1 was necessary for its function, because deletion of the NLS or addition of a nuclear export signal abolished its HR‐inducing ability. GmHSP40.1 co‐localized with HcRed‐SE, a protein involved in pri‐miRNA processing, which has been shown to be co‐localized with SR33‐YFP, a protein involved in pre‐mRNA splicing, suggesting a possible role for GmHSP40.1 in mRNA splicing or miRNA processing, and a link between these processes and cell death. Silencing GmHSP40.1 enhanced the susceptibility of soybean plants to Soybean mosaic virus, confirming its positive role in pathogen defense. Together, the results demonstrate a critical role of a nuclear‐localized DnaJ domain‐containing GmHSP40.1 in cell death and disease resistance in soybean.     

An untargeted global metabolomic analysis reveals the biochemical changes underlying basal resistance and priming in Solanum lycopersicum,and identifies 1‐methyltryptophan as a metabolite involved in plant responses to Botrytis cinerea and Pseudomonas syringae

In this study, we have used untargeted global metabolomic analysis to determine and compare the chemical nature of the metabolites altered during the infection of tomato plants (cv. Ailsa Craig) with Botrytis cinerea (Bot) or Pseudomonas syringae pv. tomato DC3000 (Pst), pathogens that have different invasion mechanisms and lifestyles. We also obtained the metabolome of tomato plants primed using the natural resistance inducer hexanoic acid and then infected with these pathogens. By contrasting the metabolomic profiles of infected, primed, and primed + infected plants, we determined not only the processes or components related directly to plant defense responses, but also inferred the metabolic mechanisms by which pathogen resistance is primed. The data show that basal resistance and hexanoic acid‐induced resistance to Bot and Pst are associated with a marked metabolic reprogramming. This includes significant changes in amino acids, sugars and free fatty acids, and in primary and secondary metabolism. Comparison of the metabolic profiles of the infections indicated clear differences, reflecting the fact that the plant's chemical responses are highly adapted to specific attackers. The data also indicate involvement of signaling molecules, including pipecolic and azelaic acids, in response to Pst and, interestingly, to Bot. The compound 1‐methyltryptophan was shown to be associated with the tomato–Pst and tomato–Bot interactions as well as with hexanoic acid‐induced resistance. Root application of this Trp‐derived metabolite also demonstrated its ability to protect tomato plants against both pathogens.     

A novel approach to parasite population genetics: Experimental infection reveals geographic differentiation,recombination and host‐mediated population structure in Pasteuria ramosa,a bacterial parasite of Daphnia

The population structure of parasites is central to the ecology and evolution of host‐parasite systems. Here, we investigate the population genetics of Pasteuria ramosa, a bacterial parasite of Daphnia. We used natural P. ramosa spore banks from the sediments of two geographically well‐separated ponds to experimentally infect a panel of Daphnia magna host clones whose resistance phenotypes were previously known. In this way, we were able to assess the population structure of P. ramosa based on geography, host resistance phenotype and host genotype. Overall, genetic diversity of P. ramosa was high, and nearly all infected D. magna hosted more than one parasite haplotype. On the basis of the observation of recombinant haplotypes and relatively low levels of linkage disequilibrium, we conclude that P. ramosa engages in substantial recombination. Isolates were strongly differentiated by pond, indicating that gene flow is spatially restricted. Pasteuria ramosa isolates within one pond were segregated completely based on the resistance phenotype of the host—a result that, to our knowledge, has not been previously reported for a nonhuman parasite. To assess the comparability of experimental infections with natural P. ramosa isolates, we examined the population structure of naturally infected D. magna native to one of the two source ponds. We found that experimental and natural infections of the same host resistance phenotype from the same source pond were indistinguishable, indicating that experimental infections provide a means to representatively sample the diversity of P. ramosa while reducing the sampling bias often associated with studies of parasite epidemics. These results expand our knowledge of this model parasite, provide important context for the large existing body of research on this system and will guide the design of future studies of this host‐parasite system.     

X‐ray computed tomography and its potential in ecological research: A review of studies and optimization of specimen preparation

Imaging techniques are a cornerstone of contemporary biology. Over the last decades, advances in microscale imaging techniques have allowed fascinating new insights into cell and tissue morphology and internal anatomy of organisms across kingdoms. However, most studies so far provided snapshots of given reference taxa, describing organs and tissues under “idealized” conditions. Surprisingly, there is an almost complete lack of studies investigating how an organism′s internal morphology changes in response to environmental drivers. Consequently, ecology as a scientific discipline has so far almost neglected the possibilities arising from modern microscale imaging techniques. Here, we provide an overview of recent developments of X‐ray computed tomography as an affordable, simple method of high spatial resolution, allowing insights into three‐dimensional anatomy both in vivo and ex vivo. We review ecological studies using this technique to investigate the three‐dimensional internal structure of organisms. In addition, we provide practical comparisons between different preparation techniques for maximum contrast and tissue differentiation. In particular, we consider the novel modality of phase contrast by self‐interference of the X‐ray wave behind an object (i.e., phase contrast by free space propagation). Using the cricket Acheta domesticus (L.) as model organism, we found that the combination of FAE fixative and iodine staining provided the best results across different tissues. The drying technique also affected contrast and prevented artifacts in specific cases. Overall, we found that for the interests of ecological studies, X‐ray computed tomography is useful when the tissue or structure of interest has sufficient contrast that allows for an automatic or semiautomatic segmentation. In particular, we show that reconstruction schemes which exploit phase contrast can yield enhanced image quality. Combined with suitable specimen preparation and automated analysis, X‐ray CT can therefore become a promising quantitative 3D imaging technique to study organisms′ responses to environmental drivers, in both ecology and evolution.     

Fine‐scale frequency differentiation along a herbivory gradient in the trichome dimorphism of a wild Arabidopsis

Geographic variation is commonly observed in plant resistance traits, where plant species might experience different selection pressure across a heterogeneous landscape. Arabidopsis halleri subsp. gemmifera is dimorphic for trichome production, generating two morphs, trichome‐producing (hairy) and trichomeless (glabrous) plants. Trichomes of A. halleri are known to confer resistance against the white butterfly, cabbage sawfly, and brassica leaf beetle, but not against flea beetles. We combined leaf damage, microclimate, and microsatellite loci data of 26 A. halleri populations in central Japan, to explore factors responsible for fine‐scale geographic variation in the morph frequency. We found that hairy plants were less damaged than glabrous plants within populations, but the among‐site variation was the most significant source of variation in the individual‐level damage. Fixation index () of a putative trichome locus exhibited a significant divergence along population‐level damage with an exception of an outlier population, inferring the local adaptation to herbivory. Notably, this outlier was a population wherein our previous study reported a balancing role of the brassica leaf beetle Phaedon brassicae on the morph frequency. This differentiation of the trichome locus was unrelated to neutral genetic differentiation (evaluated by of microsatellite loci) and meteorological factors (including temperature and solar radiation). The present findings, combined with those of our previous work, provide suggestive evidence that herbivore‐driven divergence and occasional outbreak of a specific herbivore have jointly contributed to the ecogeographic pattern in the frequency of two morphs.     

Testing for host‐associated differentiation in two egg parasitoids of a forest herbivore

Several studies underline the importance of ecological barriers and differential selection in driving sympatric speciation. Host‐associated differentiation (HAD) has been proposed as one of the mechanisms leading to sympatric speciation. However, it is still unclear how common HAD is or which are the factors that could promote it. In particular, not much is known about HAD in predators and parasitoids of herbivorous insects. One of the characteristics postulated to pre‐dispose insects to HAD is parthenogenesis as it may favour adaptive responses to particular environments, amplifying selected gene complexes. In this study, we used amplified fragment length polymorphism (AFLP) markers to determine whether HAD is present in two parthenogenetic egg parasitoids attacking the same herbivore species – the pine processionary moth, Thaumetopoea pityocampa (Denis & Schiffermüller) (Lepidoptera: Notodontidae) – on two host Pinus species. A total of 100 loci for 59 individuals sampled in four populations of Baryscapus servadeii (Domenichini) (Hymenoptera: Eulophidae), a specialist parasitoid, and 106 loci for 117 individuals sampled in six populations of Ooencyrtus pityocampae Mercet (Hymenoptera: Encyrtidae), a generalist parasitoid, were analysed. Levels of genetic differentiation were also assessed with an outlier analysis, checking for alleles associated to host plants. No evidence of HAD was detected in any of the two parasitoid species. We hypothesize that both the lack of strict parthenogenetic reproduction and the ectophagous nature of the insect host could explain the absence of HAD. The genetic variation observed in the generalist parasitoid responded to a pattern of local adaptation, whereas no relationship with either host or geography was found in the specialist parasitoid.     

Population‐level manipulations of field vole densities induce subsequent changes in plant quality but no impacts on vole demography

Grazing‐induced changes in plant quality have been suggested to drive the negative delayed density dependence exhibited by many herbivore species, but little field evidence exists to support this hypothesis. We tested a key premise of the hypothesis that reciprocal feedback between vole grazing pressure and the induction of anti‐herbivore silicon defenses in grasses drives observed population cycles in a large‐scale field experiment in northern England. We repeatedly reduced population densities of field voles (Microtus agrestis) on replicated 1‐ha grassland plots at Kielder Forest, northern England, over a period of 1 year. Subsequently, we tested for the impact of past density on vole life history traits in spring, and whether these effects were driven by induced silicon defenses in the voles’ major over‐winter food, the grass Deschampsia caespitosa. After several months of density manipulation, leaf silicon concentrations diverged and averaged 22% lower on sites where vole density had been reduced, but this difference did not persist beyond the period of the density manipulations. There were no significant effects of our density manipulations on vole body mass, spring population growth rate, or mean date for the onset of spring reproduction the following year. These findings show that grazing by field voles does induce increased silicon defenses in grasses at a landscape scale. However, at the vole densities encountered, levels of plant damage appear to be below those needed to induce changes in silicon levels large and persistent enough to affect vole performance, confirming the threshold effects we have previously observed in laboratory‐based studies. Our findings do not support the plant quality hypothesis for observed vole population cycles in northern England, at least over the range of vole densities that now prevail here.     

Population genomics reveals multiple drivers of population differentiation in a sex‐role‐reversed pipefish

A major goal of molecular ecology is to identify the causes of genetic and phenotypic differentiation among populations. Population genomics is suitably poised to tackle these key questions by diagnosing the evolutionary mechanisms driving divergence in nature. Here, we set out to investigate the evolutionary processes underlying population differentiation in the Gulf pipefish, Syngnathus scovelli. We sampled approximately 50 fish from each of 12 populations distributed from the Gulf coast of Texas to the Atlantic coast of Florida and performed restriction‐site‐associated DNA sequencing to identify SNPs throughout the genome. After imposing quality and stringency filters, we selected a panel of 6348 SNPs present in all 12 populations, 1753 of which were not physically linked. We identified a genome‐wide pattern of isolation by distance, in addition to a more substantial genetic break separating populations in the Gulf of Mexico from those in the Atlantic. We also used several divergence outlier approaches and tests for genotype–environment correlations to identify 400 SNPs putatively involved in local adaptation. Patterns of phenotypic differentiation and variation diverged from the overall genomic pattern, suggesting that selection, phenotypic plasticity or demographic factors may be shaping phenotypes in distinct populations. Overall, our results suggest that population divergence is driven by a variety of factors in S. scovelli, including neutral processes and selection on multiple traits.     

Collective decision‐making promotes fitness loss in a fusion‐fission society

While collective decision‐making is recognised as a significant contributor to fitness in social species, the opposite outcome is also logically possible. We show that collective movement decisions guided by individual bison sharing faulty information about habitat quality promoted the use of ecological traps. The frequent, but short‐lived, associations of bison with different spatial knowledge led to a population‐wide shift from avoidance to selection of agricultural patches over 9 years in and around Prince Albert National Park, Canada. Bison were more likely to travel to an agricultural patch for the first time by following conspecifics already familiar with agricultural patches. Annual adult mortality increased by 12% due to hunting of bison on agricultural lands. Maladaptive social behaviour accordingly was a major force that contributed to a ~50% population decline in less than a decade. In human‐altered landscapes, social learning by group‐living species can lead to fitness losses, particularly in fusion‐fission societies.     

Insulin induces proliferation and cardiac differentiation of P19CL6 cells in a dose‐dependent manner

Insulin is a peptide hormone produced by beta cells of the pancreas. The roles of insulin in energy metabolism have been well studied, with most of the attention focused on glucose utilization, but the roles of insulin in cell proliferation and differentiation remain unclear. In this study, we observed for the first time that 10 nmol/L insulin treatment induces cell proliferation and cardiac differentiation of P19CL6 cells, whereas 50 and 100 nmol/L insulin treatment induces P19CL6 cell apoptosis and blocks cardiac differentiation of P19CL6 cells. By using real‐time polymerase chain reaction (PCR) and Western blotting analysis, we found that the mRNA levels of cyclin D1 and α myosin heavy chain (α‐MHC) are induced upon 10 nmol/L insulin stimulation and inhibited upon 50/100 nmol/L insulin treatment, whereas the mRNA levels of BCL‐2‐antagonist of cell death (BAD) exists a reverse trend. The similar results were observed in P19CL6 cells expressing GATA‐6 or peroxisome proliferator‐activated receptor α (PPARα). Our results identified the downstream targets of insulin, cyclin D1, BAD, α‐MHC, and GATA‐4, elucidate a novel molecular mechanism of insulin in promoting cell proliferation and differentiation.