Masculinizer gene controls male sex determination in the codling moth, Cydia pomonella.

The molecular mechanisms of sex determination in moths and butterflies (Lepidoptera) with female heterogamety (WZ/ZZ) are poorly understood, except in the silkworm Bombyx mori. However, the Masculinizer (Masc) gene that controls male development and dosage compensation in B. mori, appears to be conserved in Lepidoptera, as its masculinizing function was recently confirmed in several moth species. In this work, we investigated the role of the Masc gene in sex determination of the codling moth Cydia pomonella (Tortricidae), a globally important pest of pome fruits and walnuts. The gene structure of the C. pomonella Masc ortholog, CpMasc, is similar to B. mori Masc. However, unlike B. mori, we identified 14 splice variants of CpMasc in the available transcriptomes. Subsequent screening for sex specificity and genetic variation using publicly available data and RT-PCR revealed three male-specific splice variants. Then qPCR analysis of these variants revealed sex-biased expression showing a peak only in early male embryos. Knockdown of CpMasc by RNAi during early embryogenesis resulted in a shift from male-to female-specific splicing of the C. pomonella doublesex (Cpdsx) gene, its downstream effector, in ZZ embryos, leading to a strongly female-biased sex ratio. These data clearly demonstrate that CpMasc functions as a masculinizing gene in the sex-determining cascade of C. pomonella. Our study also showed that CpMasc transcripts are provided maternally, as they were detected in unfertilized eggs after oviposition and in mature eggs dissected from virgin females. This finding is unique, as maternal provision of mRNA has rarely been studied in Lepidoptera.

A conserved role of the duplicated Masculinizer gene in sex determination of the Mediterranean flour moth, Ephestia kuehniella.

Sex determination in the silkworm, Bombyx mori, is based on Feminizer (Fem), a W-linked Fem piRNA that triggers female development in WZ individuals, and the Z-linked Masculinizer (Masc), which initiates male development and dosage compensation in ZZ individuals. While Fem piRNA is missing in a close relative of B. mori, Masc determines sex in several representatives of distant lepidopteran lineages. We studied the molecular mechanisms of sex determination in the Mediterranean flour moth, Ephestia kuehniella (Pyralidae). We identified an E. kuehniella Masc ortholog, EkMasc, and its paralog resulting from a recent duplication, EkMascB. Both genes are located on the Z chromosome and encode a similar Masc protein that contains two conserved domains but has lost the conserved double zinc finger domain. We developed PCR-based genetic sexing and demonstrated a peak in the expression of EkMasc and EkMascB genes only in early male embryos. Simultaneous knock-down experiments of both EkMasc and EkMascB using RNAi during early embryogenesis led to a shift from male- to female-specific splicing of the E. kuehniella doublesex gene (Ekdsx), their downstream effector, in ZZ embryos and resulted in a strong female-biased sex-ratio. Our results thus confirmed the conserved role of EkMasc and/or EkMascB in masculinization. We suggest that the C-terminal proline-rich domain, we have identified in all functionally confirmed Masc proteins, in conjunction with the masculinizing domain, is important for transcriptional regulation of sex determination in Lepidoptera. The function of the Masc double zinc finger domain is still unknown, but appears to have been lost in E. kuehniella.

Large-scale comparative analysis of cytogenetic markers across Lepidoptera.

Fluorescence in situ hybridization (FISH) allows identification of particular chromosomes and their rearrangements. Using FISH with signal enhancement via antibody amplification and enzymatically catalysed reporter deposition, we evaluated applicability of universal cytogenetic markers, namely 18S and 5S rDNA genes, U1 and U2 snRNA genes, and histone H3 genes, in the study of the karyotype evolution in moths and butterflies. Major rDNA underwent rather erratic evolution, which does not always reflect chromosomal changes. In contrast, the hybridization pattern of histone H3 genes was well conserved, reflecting the stable organisation of lepidopteran genomes. Unlike 5S rDNA and U1 and U2 snRNA genes which we failed to detect, except for 5S rDNA in a few representatives of early diverging lepidopteran lineages. To explain the negative FISH results, we used quantitative PCR and Southern hybridization to estimate the copy number and organization of the studied genes in selected species. The results suggested that their detection was hampered by long spacers between the genes and/or their scattered distribution. Our results question homology of 5S rDNA and U1 and U2 snRNA loci in comparative studies. We recommend the use of histone H3 in studies of karyotype evolution.

Next-generation biological control: the need for integrating genetics and genomics.

Biological control is widely successful at controlling pests, but effective biocontrol agents are now more difficult to import from countries of origin due to more restrictive international trade laws (the Nagoya Protocol). Coupled with increasing demand, the efficacy of existing and new biocontrol agents needs to be improved with genetic and genomic approaches. Although they have been underutilised in the past, application of genetic and genomic techniques is becoming more feasible from both technological and economic perspectives. We review current methods and provide a framework for using them. First, it is necessary to identify which biocontrol trait to select and in what direction. Next, the genes or markers linked to these traits need be determined, including how to implement this information into a selective breeding program. Choosing a trait can be assisted by modelling to account for the proper agro-ecological context, and by knowing which traits have sufficiently high heritability values. We provide guidelines for designing genomic strategies in biocontrol programs, which depend on the organism, budget, and desired objective. Genomic approaches start with genome sequencing and assembly. We provide a guide for deciding the most successful sequencing strategy for biocontrol agents. Gene discovery involves quantitative trait loci analyses, transcriptomic and proteomic studies, and gene editing. Improving biocontrol practices includes marker-assisted selection, genomic selection and microbiome manipulation of biocontrol agents, and monitoring for genetic variation during rearing and post-release. We conclude by identifying the most promising applications of genetic and genomic methods to improve biological control efficacy.

Absence of W Chromosome in Psychidae Moths and Implications for the Theory of Sex Chromosome Evolution in Lepidoptera.

Moths and butterflies (Lepidoptera) are the largest group with heterogametic females. Although the ancestral sex chromosome system is probably Z0/ZZ, most lepidopteran species have the W chromosome. When and how the W chromosome arose remains elusive. Existing hypotheses place the W origin either at the common ancestor of Ditrysia and Tischeriidae, or prefer independent origins of W chromosomes in these two groups. Due to their phylogenetic position at the base of Ditrysia, bagworms (Psychidae) play an important role in investigating the W chromosome origin. Therefore, we examined the W chromosome status in three Psychidae species, namely Proutiabetulina, Taleporiatubulosa, and Diplodomalaichartingella, using both classical and molecular cytogenetic methods such as sex chromatin assay, comparative genomic hybridization (CGH), and male vs. female genome size comparison by flow cytometry. In females of all three species, no sex chromatin was found, no female-specific chromosome regions were revealed by CGH, and a Z-chromosome univalent was observed in pachytene oocytes. In addition, the genome size of females was significantly smaller than males. Overall, our study provides strong evidence for the absence of the W chromosome in Psychidae, thus supporting the hypothesis of two independent W chromosome origins in Tischeriidae and in advanced Ditrysia.

Cure for increasing health care costs: The Bernhoven case as driver of new standards of appropriate care.

Containing costs is a major challenge in health care. Cost and quality are often seen as trade-offs, but high quality and low costs can go hand-in-hand as waste exists in unnecessary and unfounded care. In the Netherlands, two healthcare insurers and a hospital collaborate to improve quality of care and decrease healthcare costs. Their aim is to reduce unnecessary care by shifting the business model and culture from a focus on volume to a focus on quality. Key drivers to support this are taking time for integrated diagnosis ('first time right'), the right care at the right place and shared decision making between doctor and patient. Conditions to realize this are 1) contract innovation between the hospital and insurers to move away from fee-for-service reimbursement, 2) a culture change within the organization with emphasis on collaboration and empowerment of medical leadership and physicians to change daily practice, and 3) a reorganization of the hospital organization structure from a large number of medical departments to four business units related to the fundamental underlying patient need (acute care, solution shop, intervention unit and chronic care). Results from this whole-system-approach experiment show it is possible to provide better care (as experienced by patients) with lower volumes (16% lower DRG claims after 3 years) and provides valuable lessons for further healthcare reform.

The arduous quest for translating health care productivity gains into cost savings. Lessons from their evolution at economic scoring agencies in the Netherlands and the US.

We analyze the assessments of recent health reforms by the Congressional Budget Office (CBO) in the United States and the Bureau for Economic Policy Analysis (CPB) in the Netherlands. Both reforms aim to capitalize on productivity gains, which is appealing for policymakers because of the potential for cost savings while maintaining - or enhancing - quality and access. These measures however generally translate into more health care, rather than care that is affordable and appropriate. Scoring agencies therefore have rightfully been reluctant to assign significant savings to these measures. Thus with regard to cost savings, both agencies instead have favored more traditional policy measures in the past. They are however increasingly mapping out loose ends and dilemmas for payers, including information asymmetries, reputation issues and provider business models that contradict the goals of policymakers. This calls for further exploring this avenue and the development of more integrated agendas that might commit actors and the spread of best practices.

IncP-1 and PromA group plasmids are major providers of horizontal gene transfer capacities across bacteria in the mycosphere of different soil fungi.

Plasmids of the IncP-1ß group have been found to be important carriers of accessory genes that enhance the ecological fitness of bacteria, whereas plasmids of the PromA group are key agents of horizontal gene transfer in particular soil settings. However, there is still a paucity of knowledge with respect to the diversity, abundance, and involvement in horizontal gene transfer of plasmids of both groups in the mycosphere. Using triparental exogenous isolation based on the IncQ tracer plasmid pSUP104 as well as direct molecular detection, we analyzed the pool of mobilizer and self-transferable plasmids in mycosphere soil. Replicate mushroom types that were related to Russula, Inocybe, Ampulloclitocybe, and Galerina spp. were sampled from a forest soil area, and bulk soil was used as the control. The data showed that the levels of IncP-1ß plasmids are significantly raised across several of the mycospheres analyzed, whereas those of PromA group plasmids were similar across the mycospheres and corresponding bulk soil. Moreover, the frequencies of triparental exogenous isolation of mobilizer plasmids into a Pseudomonas fluorescens recipient strain were significantly elevated in communities from several mycospheres as compared with those from bulk soil. Molecular analysis of selected transconjugants, as well as from directly isolated strains, revealed the presence of plasmids of three size groups, i.e., (1) 40-45, (2) 50-60, and (3) ≥60 kb, across all isolations. Replicon typing using IncN, IncW and IncA/C proxies revealed no positive signals. In contrast, a suite of plasmids produced signals with IncP-1ß as well as PromA type replicon typing systems. Moreover, a selected subset of plasmids, obtained from the Inocybe and Galerina isolates, was transferred out further, revealing their capacities to transfer and mobilize across a broad host range.