The highly divergent Jekyll genes,required for sexual reproduction,are lineage specific for the related grass tribes Triticeae and Bromeae

Phylogenetically related groups of species contain lineage‐specific genes that exhibit no sequence similarity to any genes outside the lineage. We describe here that the Jekyll gene, required for sexual reproduction, exists in two much diverged allelic variants, Jek1 and Jek3. Despite low similarity, the Jek1 and Jek3 proteins share identical signal peptides, conserved cysteine positions and direct repeats. The Jek1/Jek3 sequences are located at the same chromosomal locus and inherited in a monogenic Mendelian fashion. Jek3 has a similar expression as Jek1 and complements the Jek1 function in Jek1‐deficient plants. Jek1 and Jek3 allelic variants were almost equally distributed in a collection of 485 wild and domesticated barley accessions. All domesticated barleys harboring the Jek1 allele belong to single haplotype J1‐H1 indicating a genetic bottleneck during domestication. Domesticated barleys harboring the Jek3 allele consisted of three haplotypes. Jekyll‐like sequences were found only in species of the closely related tribes Bromeae and Triticeae but not in other Poaceae. Non‐invasive magnetic resonance imaging revealed intrinsic grain structure in Triticeae and Bromeae, associated with the Jekyll function. The emergence of Jekyll suggests its role in the separation of the Bromeae and Triticeae lineages within the Poaceae and identifies the Jekyll genes as lineage‐specific.     

Functional specialization of duplicated AP3‐like genes in Medicago truncatula

The B–class of MADS box genes has been studied in a wide range of plant species, but has remained largely uncharacterized in legumes. Here we investigate the evolutionary fate of the duplicated AP3‐like genes of a legume species. To obtain insight into the extent to which B‐class MADS box gene functions are conserved or have diversified in legumes, we isolated and characterized the two members of the AP3 lineage in Medicago truncatula: MtNMH7 and MtTM6 (euAP3 and paleoAP3 genes, respectively). A non‐overlapping and complementary expression pattern of both genes was observed in petals and stamens. MtTM6 was expressed predominantly in the outer cell layers of both floral organs, and MtNMH7 in the inner cell layers of petals and stamens. Functional analyses by reverse genetics approaches (RNAi and Tnt1 mutagenesis) showed that the contribution of MtNMH7 to petal identity is more important than that of MtTM6, whereas MtTM6 plays a more important role in stamen identity than its paralog MtNMH7. Our results suggest that the M. truncatula AP3‐like genes have undergone a functional specialization process associated with complete partitioning of gene expression patterns of the ancestral gene lineage. We provide information regarding the similarities and differences in petal and stamen development among core eudicots.     

Impact of field‐realistic doses of glyphosate and nutritional stress on mosquito life history traits and susceptibility to malaria parasite infection

Glyphosate is the world's most widely used herbicide. The commercial success of this molecule is due to its nonselectivity and its action, which would supposedly target specific biosynthetic pathways found mainly in plants. Multiple studies have however provided evidence for high sensitivity of many nontarget species to glyphosate and/or to formulations (glyphosate mixed with surfactants). This herbicide, found at significant levels in aquatic systems through surface runoffs, impacts life history traits and immune parameters of several aquatic invertebrates' species, including disease‐vector mosquitoes. Mosquitoes, from hatching to emergence, are exposed to aquatic chemical contaminants. In this study, we first compared the toxicity of pure glyphosate to the toxicity of glyphosate‐based formulations for the main vector of avian malaria in Europe, Culex pipiens mosquito. Then we evaluated, for the first time, how field‐realistic dose of glyphosate interacts with larval nutritional stress to alter mosquito life history traits and susceptibility to avian malaria parasite infection. Our results show that exposure of larvae to field‐realistic doses of glyphosate, pure or in formulation, did not affect larval survival rate, adult size, and female fecundity. One of our two experimental blocks showed, however, that exposure to glyphosate decreased development time and reduced mosquito infection probability by malaria parasite. Interestingly, the effect on malaria infection was lost when the larvae were also subjected to a nutritional stress, probably due to a lower ingestion of glyphosate.     

Discovery of rice essential genes by characterizing a CRISPR‐edited mutation of closely related rice MAP kinase genes

The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR‐associated protein 9 nuclease (Cas9) system depends on a guide RNA (gRNA) to specify its target. By efficiently co‐expressing multiple gRNAs that target different genomic sites, the polycistronic tRNA‐gRNA gene (PTG) strategy enables multiplex gene editing in the family of closely related mitogen‐activated protein kinase (MPK) genes in Oryza sativa (rice). In this study, we identified MPK1 and MPK6 (Arabidopsis AtMPK6 and AtMPK4 orthologs, respectively) as essential genes for rice development by finding the preservation of MPK functional alleles and normal phenotypes in CRISPR‐edited mutants. The true knock‐out mutants of MPK1 were severely dwarfed and sterile, and homozygous mpk1 seeds from heterozygous parents were defective in embryo development. By contrast, heterozygous mpk6 mutant plants completely failed to produce homozygous mpk6 seeds. In addition, the functional importance of specific MPK features could be evaluated by characterizing CRISPR‐induced allelic variation in the conserved kinase domain of MPK6. By simultaneously targeting between two and eight genomic sites in the closely related MPK genes, we demonstrated 45–86% frequency of biallelic mutations and the successful creation of single, double and quadruple gene mutants. Indels and fragment deletion were both stably inherited to the next generations, and transgene‐free mutants of rice MPK genes were readily obtained via genetic segregation, thereby eliminating any positional effects of transgene insertions. Taken together, our study reveals the essentiality of MPK1 and MPK6 in rice development, and enables the functional discovery of previously inaccessible genes or domains with phenotypes masked by lethality or redundancy.     

Development of Asian parasitoids in larvae Of Drosophila Suzukii feeding on blueberry and artificial diet

In just a few years, the Asian fly Drosophila suzukii has invaded several continents and has become a very serious pest of many fruit crops worldwide. Current control methods rely on chemical insecticides or expensive and labour‐intensive cultural practices. Classical biological control through the introduction of Asian parasitoids that have co‐evolved with the pest may provide a sustainable solution on condition that they are sufficiently specific to avoid non‐target effects on local biodiversity. Here, we present the first study on the development of three larval parasitoids from China and Japan, the Braconidae Asobara japonica and the Figitidae Leptopilina japonica and Ganaspis sp., on D. suzukii. The Asian parasitoids were compared with Leptopilina heterotoma, a common parasitoid of several Drosophilidae worldwide. The three Asian species were successfully reared on D. suzukii larvae in both, blueberry and artificial diet, in contrast to L. heterotoma whose eggs and larvae were encapsulated by the host larvae. All parasitoids were able to oviposit one day after emergence. Asobara japonica laid as many eggs in larvae feeding in blueberry as in artificial diet, whereas L. heterotoma oviposited more in larvae on the artificial diet and the Asian Figitidae oviposited more in larvae feeding on blueberry. Ganaspis sp. laid very few eggs in larvae in the artificial diet, suggesting that it may be specialized in Drosophila species living in fresh fruits. These data will be used for the development of a host range testing to assess the suitability of Asian parasitoids as biological control agents in invaded regions.     

Physio‐biochemical assessment and expression analysis of genes associated with drought tolerance in sugarcane (Saccharum spp. hybrids) exposed to GA3 at grand growth stage

• Drought is one of the most serious environmental factors limiting production of sugarcane worldwide. In order to assess the influence of gibberellins (GA₃) on drought and plant growth, along with associated physio‐biochemical attributes, expression of eight drought‐responsive genes were quantified and analysed.
• At grand growth stage (120 DAP) two sugarcane varieties (CoLk94184, CoPK05191) were exposed to drought by withholding irrigation. GA₃ (35 ppm) was applied using battery‐operated uniform controlled dispensing sprayer twice at 1‐week intervals on 2‐week drought‐stressed plants. Physio‐biochemical attributes including antioxidant enzyme activities were estimated following standard protocols. RT‐PCR was performed to visualise the drought‐associated gene expression patterns.
• Drought triggered a reduction in RWC and chlorophyll content but these recovered when droughted plants were exposed to GA₃. Proline content increased many fold in both varieties under stress, but decreased under the influence of GA₃. There was a mixed response of antioxidant enzyme activity, which distinctly declined after GA₃ exposure, together with a lesser reduction in dry matter content over that of control plants. With increasing stress, expression of pyrroline‐5‐carboxylase synthetase (P5CS) and betaine‐aldehyde dehydrogenase genes was observed, selectively up‐regulated in CoPK05191. Expression of proline oxidase/transporter was high in CoPK05191 but diminished along with proline content after exposure to GA_3. CoLk94184 showed no significant difference in P5CS gene expression under stress condition, whereas expression of betaine‐aldehyde dehydrogenase gene was unchanged in response to stress.
• Results demonstrated that exposure of droughted plants to GA₃ not only led to recovery of activity of drought‐associated physio‐biochemical attributes, but also minimised impact on cane dry weight and quality. Further, GA₃ application caused differential gene expression that possibly triggers increased responsiveness towards drought tolerance in sugarcane.

     

Wnt signaling loss accelerates the appearance of neuropathological hallmarks of Alzheimer's disease in J20‐APP transgenic and wild‐type mice

Alzheimer's disease (AD ) is a neurodegenerative pathology characterized by aggregates of amyloid‐β (Aβ) and phosphorylated tau protein, synaptic dysfunction, and spatial memory impairment. The Wnt signaling pathway has several key functions in the adult brain and has been associated with AD , mainly as a neuroprotective factor against Aβ toxicity and tau phosphorylation. However, dysfunction of Wnt/β‐catenin signaling might also play a role in the onset and development of the disease. J20 APP swInd transgenic (Tg) mouse model of AD was treated i.p. with various Wnt signaling inhibitors for 10 weeks during pre‐symptomatic stages. Then, cognitive, biochemical and histochemical analyses were performed. Wnt signaling inhibitors induced severe changes in the hippocampus, including alterations in Wnt pathway components and loss of Wnt signaling function, severe cognitive deficits, increased tau phosphorylation and Aβ_1–42 peptide levels, decreased Aβ42/Aβ40 ratio and Aβ_1–42 concentration in the cerebral spinal fluid, and high levels of soluble Aβ species and synaptotoxic oligomers in the hippocampus, together with changes in the amount and size of senile plaques. More important, we also observed severe alterations in treated wild‐type (WT ) mice, including behavioral impairment, tau phosphorylation, increased Aβ_1–42 in the hippocampus, decreased Aβ_1–42 in the cerebral spinal fluid, and hippocampal dysfunction. Wnt inhibition accelerated the development of the pathology in a Tg AD mouse model and contributed to the development of Alzheimer's‐like changes in WT mice. These results indicate that Wnt signaling plays important roles in the structure and function of the adult hippocampus and suggest that inhibition of the Wnt signaling pathway is an important factor in the pathogenesis of AD .

Read the Editorial Highlight for this article on page 356 .

     

Identification and validation of reference genes for RT‐qPCR analysis in banana (Musa spp.) under Fusarium wilt resistance induction conditions

Banana (Musa spp.) is severely damaged by Fusarium wilt caused by Fusarium oxysporum f. sp. cubense (Foc). Biocontrol by inducing systemic resistance has been considered as one of the most important strategies to improve plant health. Very few studies have investigated appropriate reference gene selection for RT‐qPCR (quantitative real‐time polymerase chain reaction) analysis suitable for conditions of systemic activated resistance. In this study, we assessed over a time‐course the expression of seven candidate reference genes (EF1, TUB, ACT1, ACT2, L2, RPS2 and RAN) for Cavendish cultivar Brazilian (Musa spp. AAA) and dwarf banana cultivar Guangfen No. 1 (Musa spp. ABB) that were inoculated by Bacillus subtilis strain TR21 and Foc. We choose these plants because they are commonly planted in Southern China. Expression stability of the candidate genes was evaluated using various software packages (GeNorm, NormFinder and BestKeeper). L2 and TUB genes displayed maximum stability in Guangfen No. 1. In Brazilian, ACT1 and TUB were the most stable genes. To further validate the suitability of the reference genes identified in this study, the expression of pathogenesis‐related 1 (PR1) gene under TR21 and Foc strains Foc004/Foc009 treatments was also studied. Identified reference genes in this work that are most suitable for normalizing gene expression data in banana under Fusarium wilt resistance induction conditions will contribute to the understanding of disease resistance mechanisms induced by biocontrol strains in banana.     

Spatiotemporal and demographic variation in the diet of New Zealand lesser short‐tailed bats (Mystacina tuberculata)

Variation in the diet of generalist insectivores can be affected by site‐specific traits including weather, habitat, and season, as well as demographic traits such as reproductive status and age. We used molecular methods to compare diets of three distinct New Zealand populations of lesser short‐tailed bats, Mystacina tuberculata. Summer diets were compared between a southern cold‐temperate (Eglinton) and a northern population (Puroera). Winter diets were compared between Pureora and a subtropical offshore island population (Hauturu). This also permitted seasonal diet comparisons within the Pureora population. Lepidoptera and Diptera accounted for >80% of MOTUs identified from fecal matter at each site/season. The proportion of orders represented within prey and the Simpson diversity index, differed between sites and seasons within the Pureora population. For the Pureora population, the value of the Simpson diversity index was higher in summer than winter and was higher in Pureora compared to Eglinton. Summer Eglinton samples revealed that juvenile diets appeared to be more diverse than other demographic groups. Lactating females had the lowest dietary diversity during summer in Pureora. In Hauturu, we found a significant negative relationship between mean ambient temperature and prey richness. Our data suggest that M. tuberculata incorporate a narrower diversity of terrestrial insects than previously reported. This provides novel insights into foraging behavior and ecological interactions within different habitats. Our study is the first from the Southern Hemisphere to use molecular techniques to examine spatiotemporal variation in the diet of a generalist insectivore that inhabits a contiguous range with several habitat types and climates.     

Pythons,parasites, and pests: anthropogenic impacts on Sarcocystis (Sarcocystidae) transmission in a multi‐host system

Parasites are essential components of ecosystems and can be instrumental in maintaining host diversity and populations; however, their role in trophic interactions has often been overlooked. Three apicomplexan parasite species of Sarcocystis (S. singaporensis, S. zamani, and S. villivillosi) use the reticulated python as their definitive hosts and several species within the Rattus genus as intermediate hosts, and they form a system useful for studying interactions between host–parasite and predator–prey relationships, as well as anthropogenic impacts on parasite transmission. Based on predictions from a 1998 survey, which detected an inverse relationship between urban development and Sarcocystis infection in Rattus, we tested the hypothesis that Sarcocystis transmission in Singapore will decrease over time due to anthropogenic activities. Despite a large proportion of the reticulated python diet consisting of Rattus species at all sizes of pythons, Sarcocystis infection rates decreased from 1998 to 2010. Pythons found in industrial areas had lower Sarcocystis infection rates, particularly in the western industrial area of Singapore Island. Average python size also decreased, with implications that we predict may disrupt host–parasite relationships. Anthropogenic activities such as habitat modification, fragmentation, and systematic removal and translocation of pythons have negative impacts on Sarcocystis transmission in Singapore, which in turn may augment pest rat populations. Trends observed may ultimately have negative impacts on human health and biodiversity in the region.     

Effects of inbreeding on behavioural plasticity of parent–offspring interactions in a burying beetle

Inbreeding depression is defined as a fitness decline in progeny resulting from mating between related individuals, the severity of which may vary across environmental conditions. Such inbreeding‐by‐environment interactions might reflect that inbred individuals have a lower capacity for adjusting their phenotype to match different environmental conditions better, as shown in prior studies on developmental plasticity. Behavioural plasticity is more flexible than developmental plasticity because it is reversible and relatively quick, but little is known about its sensitivity to inbreeding. Here, we investigate effects of inbreeding on behavioural plasticity in the context of parent–offspring interactions in the burying beetle Nicrophorus vespilloides. Larvae increase begging with the level of hunger, and parents increase their level of care when brood sizes increase. Here, we find that inbreeding increased behavioural plasticity in larvae: inbred larvae reduced their time spent associating with a parent in response to the length of food deprivation more than outbred larvae. However, inbreeding had no effect on the behavioural plasticity of offspring begging or any parental behaviour. Overall, our results show that inbreeding can increase behavioural plasticity. We suggest that inbreeding‐by‐environment interactions might arise when inbreeding is associated with too little or too much plasticity in response to changing environmental conditions.     

Organization of the TC and TE cellular T‐DNA regions in Nicotiana otophora and functional analysis of three diverged TE‐6b genes

Nicotiana otophora contains Agrobacterium‐derived T‐DNA sequences introduced by horizontal gene transfer (Chen et al., 2014). Sixty‐nine contigs were assembled into four different cellular T‐DNAs (cT‐DNAs) totalling 83 kb. TC and TE result from two successive transformation events, each followed by duplication, yielding two TC and two TE inserts. TC is also found in other Nicotiana species, whereas TE is unique to N. otophora. Both cT‐DNA regions are partially duplicated inverted repeats. Analysis of the cT‐DNA divergence patterns allowed reconstruction of the evolution of the TC and TE regions. TC and TE carry 10 intact open reading frames. Three of these are TE‐6b genes, derived from a single 6b gene carried by the Agrobacterium strain which inserted TE in the N. otophora ancestor. 6b genes have so far only been found in Agrobacterium tumefaciens or Agrobacterium vitis T‐DNAs and strongly modify plant growth (Chen and Otten, 2016). The TE‐6b genes were expressed in Nicotiana tabacum under the constitutive 2 × 35S promoter. TE‐1‐6b‐R and TE‐2‐6b led to shorter plants, dark‐green leaves, a strong increase in leaf vein development and modified petiole wings. TE‐1‐6b‐L expression led to a similar phenotype, but in addition leaves show outgrowths at the margins, flowers were modified and plants became viviparous, i.e. embryos germinated in the capsules at an early stage of their development. Embryos could be rescued by culture in vitro. The TE‐6b phenotypes are very different from the earlier described 6b phenotypes and could provide new insight into the mode of action of the 6b genes.