Trait Variation and Spatiotemporal Dynamics across Avian Secondary Contact Zones.

A secondary contact zone (SCZ) is an area where incipient species or divergent populations may meet, mate, and hybridize. Due to the diverse patterns of interspecific hybridization, SCZs function as field labs for illuminating the on-going evolutionary processes of speciation and the establishment of reproductive isolation. Interspecific hybridization is widely present in avian populations, making them an ideal system for SCZ studies. This review exhaustively summarizes the variations in unique traits within avian SCZs (vocalization, plumage, beak, and migratory traits) and the various movement patterns of SCZs observed in previous publications. It also highlights several potential future research directions in the genomic era, such as the relationship between phenotypic and genomic differentiation in SCZs, the genomic basis of trait differentiation, SCZs shared by multiple species, and accurate predictive models for forecasting future movements under climate change and human disturbances. This review aims to provide a more comprehensive understanding of speciation processes and offers a theoretical foundation for species conservation.

PE (Prickly Eggplant) encoding a cytokinin-activating enzyme responsible for the formation of prickles in eggplant.

Eggplant is one of the most important vegetables worldwide, with some varieties displaying prickles. These prickles, present on the leaves, stems, and fruit calyxes, posing challenges during cultivation, harvesting, and transportation, making them an undesirable agronomic trait. However, the genetic mechanisms underlying prickle morphogenesis in eggplant remain poorly understood, impeding genetic improvements. In this study, genetic analyses revealed that prickle morphogenesis is governed by a single dominant nuclear gene, termed PE (Prickly Eggplant). Subsequent bulk segregant RNA-sequencing (BSR-seq) and linkage analysis preliminarily mapped PE to chromosome 6. This locus was then fine mapped to a 9233 bp interval in a segregating population of 1109 plants, harboring only one candidate gene, SmLOG1, which encodes a LONELY GUY (LOG)-family cytokinin biosynthetic enzyme. Expression analyses via transcriptome and qRT-PCR demonstrate that SmLOG1 is predominantly expressed in immature prickles. CRISPR-Cas9 knockout experiments targeting SmLOG1 in prickly parental line 'PI 381159' abolished prickles across all tissues, confirming its critical role in prickle morphogenesis. Sequence analysis of SmLOG1 pinpointed variations solely within the non-coding region. We developed a cleaved amplified polymorphic sequences (CAPS) marker from a distinct SNP located at -735-bp within the SmLOG1 promoter, finding significant association with prickle variation in 190 eggplant germplasms. These findings enhance our understanding of the molecular mechanisms governing prickle development in eggplant and facilitate the use of marker-assisted selection (MAS) for breeding prickleless cultivars.

Delimiting species, revealing cryptic diversity, and population divergence in Qinghai-Tibet Plateau weevils through DNA barcoding.

The Leptomias group represents one of the most diverse taxonomic group of weevils in the Qinghai-Tibet Plateau and its adjacent areas. Despite the potential of hidden diversity, relatively few comprehensive studies have been conducted on species diversity in this taxonomic group. In this study, we performed DNA barcoding analysis for species of the Leptomias group using a comprehensive DNA barcode dataset that included 476 sequences representing 54 morphospecies. Within the dataset, our laboratory contributed 474 sequences, and 390 sequences were newly generated for this study. The average Kimura 2-parameter distances among morphospecies and genera were 0.76% and 19.15%, respectively. In 94.4% of the species, the minimum interspecific distances exceeded the maximum intraspecific distances, indicating the presence of barcode gaps in most species of Leptomias group. The application of Automatic Barcode Gap Discovery, Assemble Species by Automatic Partitioning, Barcode Index Number, Bayesian Poisson tree processes, jMOTU, and Neighbor-joining tree methods revealed 45, 45, 63, 54, and 55 distinct clusters representing single species, respectively. Additionally, a total of four morphospecies, Leptomias kangmarensis, L. midlineatus, L. siahus, and L. sp.9RL, were found to be assigned to multiple subclade each, indicating the geographical divergences and the presence of cryptic diversity. Our findings of this study demonstrate that Qinghai-Tibet Plateau exhibits a higher species diversity of the Leptomias group, and it is imperative to investigate cryptic species within certain morphospecies using integrative taxonomic approaches in future studies. Moreover, the construction of a DNA barcode reference library presented herein establishes a robust foundational dataset to support forthcoming research on weevil taxonomy, phylogenetics, ecology, and evolution.

Ink-based additive manufacturing for electrochemical applications.

Additive manufacturing (AM), commonly known as three-dimensional (3D) printing, has drawn substantial attention in recent decades due to its efficiency and precise control in part fabrication. The limitations of conventional fabrication processes, especially regarding geometry complexity, supply chain, and environmental impact, have prompted the exploration of diverse AM technologies in electrochemistry. Especially, three ink-based AM techniques, binder jet printing (BJP), direct ink writing (DIW), and Inkjet Printing (IJP), have been extensively applied by numerous research teams to produce electrodes, catalyst scaffolds, supercapacitors, batteries, etc. BJP's versatility in utilizing a wide range of materials as powder feedstock promotes its potential for various electrode and battery applications. DIW and IJP stand out for their ability to handle multi-material manufacturing tasks and deliver high printing resolution. To capture recent advancements in this field, we present a comprehensive review of the applications of BJP, DIW, and IJP techniques in fabricating electrochemical devices and components. This review intends to provide an overview of the process-structure-property relationship in electrochemical materials and components across diverse applications manufactured using AM techniques. We delve into how the significantly improved design freedom over the structure offered by these ink-based AM techniques highlights the performance of electrochemical products. Moreover, we highlight their advantages in terms of material compatibility, geometry control, and cost-effectiveness. In specific cases, we also compare the performance of electrochemical components fabricated using AM and conventional manufacturing methods. Finally, we conclude this review article by offering some insights into the future development in this research field.

Association of computed tomography scan-assessed body composition with immune and PI3K/AKT pathway proteins in distinct breast cancer tumor components.

This hypothesis-generating study aims to examine the extent to which computed tomography-assessed body composition phenotypes are associated with immune and PI3K/AKT signaling pathways in breast tumors. A total of 52 patients with newly diagnosed breast cancer were classified into four body composition types: adequate (lowest two tertiles of total adipose tissue [TAT]) and highest two tertiles of total skeletal muscle [TSM] areas); high adiposity (highest tertile of TAT and highest two tertiles of TSM); low muscle (lowest tertile of TSM and lowest two tertiles of TAT); and high adiposity with low muscle (highest tertile of TAT and lowest tertile of TSM). Immune and PI3K/AKT pathway proteins were profiled in tumor epithelium and the leukocyte-enriched stromal microenvironment using GeoMx (NanoString). Linear mixed models were used to compare log2-transformed protein levels. Compared with the normal type, the low muscle type was associated with higher expression of INPP4B (log2-fold change = 1.14, p = 0.0003, false discovery rate = 0.028). Other significant associations included low muscle type with increased CTLA4 and decreased pan-AKT expression in tumor epithelium, and high adiposity with increased CD3, CD8, CD20, and CD45RO expression in stroma (P<0.05; false discovery rate >0.2). With confirmation, body composition can be associated with signaling pathways in distinct components of breast tumors, highlighting the potential utility of body composition in informing tumor biology and therapy efficacies.

Proactive resistance management for sustaining the efficacy of RNA interference for pest control.

Biopesticides based on RNA interference (RNAi) took a major step forward with the first registration of a sprayable RNAi product, which targets the world's most damaging potato pest. Proactive resistance management is needed to delay the evolution of resistance by pests and sustain the efficacy of RNAi biopesticides.

Revision of the Chinese Pachynotus Kollar & L. Redtenbacher, 1844 (Coleoptera, Curculionidae), with descriptions of two new species.

The Chinese species of the highland weevil genus Pachynotus is revised, including a single known species, P.lampoglobus Chao & Y.-Q. Chen, 1980, and the descriptions of two new species, P.pilosussp. nov. and P.arcuatussp. nov. All Chinese Pachynotus species occur in Xizang (Tibet), China, and a key to these species is presented. Additionally, the COI sequences of two species, P.lampoglobus and P.pilosussp. nov., are provided, with details of the genetic distance.

Functional Response and Intraspecific Competition of Three Ladybird Species Feeding on Aphids on Goji Berry Plants in Laboratory and Semi-Field Conditions.

The aphid, Aphis gossypii Glover, is identified as a significant pest that causes severe damage to goji berries in China. To analyze the ladybird consumption of aphids, the functional responses of three ladybird species, Harmonia axyridis, Coccinella septempunctata, and Hippodamia variegata, and intraspecific competition among ladybird individuals were evaluated under laboratory conditions. Moreover, the practical impact of ladybirds on aphid population reduction was investigated in semi-field conditions. We found that all adult ladybirds of the three species exhibited a type II functional response toward aphids. According to Holling's disc equation, H. axyridis exhibited the highest searching efficiency (a = 0.79), while C. septempunctata had the shortest handling time (Th = 5.07 min) among the three ladybird species studied. Additionally, intraspecific competition had a greater impact on H. variegata (m = 0.41) compared to the other two ladybird species. The semi-field study demonstrated that H. axyridis (83.9% reduction) and C. septempunctata (78.7% reduction) exhibited higher efficacy in reducing aphid populations compared to H. variegata (27.3% reduction). This study suggests that H. axyridis and C. septempunctata exhibit potential as effective biological control agents against aphids on goji berry plants and highlights the importance of considering intraspecific competition. However, the results obtained from laboratory and semi-field studies cannot be directly extrapolated to field conditions due to the simplification of these experimental systems. Future field studies are crucial in ensuring the effective implementation of a biological control program.

Adaptive Sparse Multi-Block PLS Discriminant Analysis: An Integrative Method for Identifying Key Biomarkers from Multi-Omics Data.

With the growing use of high-throughput technologies, multi-omics data containing various types of high-dimensional omics data is increasingly being generated to explore the association between the molecular mechanism of the host and diseases. In this study, we present an adaptive sparse multi-block partial least square discriminant analysis (asmbPLS-DA), an extension of our previous work, asmbPLS. This integrative approach identifies the most relevant features across different types of omics data while discriminating multiple disease outcome groups. We used simulation data with various scenarios and a real dataset from the TCGA project to demonstrate that asmbPLS-DA can identify key biomarkers from each type of omics data with better biological relevance than existing competitive methods. Moreover, asmbPLS-DA showed comparable performance in the classification of subjects in terms of disease status or phenotypes using integrated multi-omics molecular profiles, especially when combined with other classification algorithms, such as linear discriminant analysis and random forest. We have made the R package called asmbPLS that implements this method publicly available on GitHub. Overall, asmbPLS-DA achieved competitive performance in terms of feature selection and classification. We believe that asmbPLS-DA can be a valuable tool for multi-omics research.

asmbPLS: Adaptive Sparse Multi-block Partial Least Square for Survival Prediction using Multi-Omics Data.

Background: As high-throughput studies advance, more and more high-dimensional multi-omics data are available and collected from the same patient cohort. Using multi-omics data as predictors to predict survival outcomes is challenging due to the complex structure of such data. Results: In this article, we introduce an adaptive sparse multi-block partial least square (asmbPLS) regression method by assigning different penalty factors to different blocks in different PLS components for feature selection and prediction. We compared the proposed method with several competitive algorithms in many aspects including prediction performance, feature selection and computation efficiency. The performance and the efficiency of our method were demonstrated using both the simulated and the real data. Conclusions: In summary, asmbPLS achieved a competitive performance in prediction, feature selection, and computation efficiency. We anticipate asmbPLS to be a valuable tool for multi-omics research. An R package called asmbPLS implementing this method is made publicly available on GitHub.

Association Between Recreational Physical Activity and mTOR Signaling Pathway Protein Expression in Breast Tumor Tissue.

Physical activity (PA) is associated with decreased signaling in the mTOR pathway in animal models of mammary cancer, which may indicate favorable outcomes. We examined the association between PA and protein expression in the mTOR signaling pathway in breast tumor tissue. Data on 739 patients with breast cancer, among which 125 patients had adjacent-normal tissue, with tumor expression for mTOR, phosphorylated (p)-mTOR, p-AKT, and p-P70S6K were analyzed. Self-reported recreational PA levels during the year prior to diagnosis were classified using the Centers for Disease Control and Prevention guideline as sufficient (for moderate or vigorous) PA or insufficient PA (any PA but not meeting the guideline) or no PA. We performed linear models for mTOR protein and two-part gamma hurdle models for phosphorylated proteins. Overall, 34.8% of women reported sufficient PA; 14.2%, insufficient PA; 51.0%, no PA. Sufficient (vs. no) PA was associated with higher expression for p-P70S6K [35.8% increase; 95% confidence interval (CI), 2.6-80.2] and total phosphoprotein (28.5% increase; 95% CI, 5.8-56.3) among tumors with positive expression. In analyses stratified by PA intensity, sufficient versus no vigorous PA was also associated with higher expression levels of mTOR (beta = 17.7; 95% CI, 1.1-34.3) and total phosphoprotein (28.6% higher; 95% CI, 1.4-65.0 among women with positive expression) in tumors. The study found that guideline-concordant PA levels were associated with increased mTOR signaling pathway activity in breast tumors. Studying PA in relation to mTOR signaling in humans may need to consider the complexity of the behavioral and biological factors. Significance: PA increases energy expenditure and limits energy utilization in the cell, which can influence the mTOR pathway that is central to sensing energy influx and regulating cell growth. We studied exercise-mediated mTOR pathway activities in breast tumor and adjacent-normal tissue. Despite the discrepancies between animal and human data and the limitations of our approach, the findings provide a foundation to study the mechanisms of PA and their clinical implications.

Soft Robot Proprioception Using Unified Soft Body Encoding and Recurrent Neural Network.

Compared with rigid robots, soft robots are inherently compliant and have advantages in the tasks requiring flexibility and safety. But sensing the high dimensional body deformation of soft robots is a challenge. Encasing soft strain sensors into the internal body of soft robots is the most popular solution to address this challenge. But most of them usually suffer from problems like nonlinearity, hysteresis, and fabrication complexity. To endow the soft robots with body movement awareness, this work presents a bioinspired architecture by taking cues from human proprioception system. Differing from the popular usage of smart material-based sensors embedded in soft actuators, we created a synthetic analog to the human muscle system, using paralleled soft pneumatic chambers to serve as receptors for sensing body deformation. We proposed to build the system with redundant receptors and explored deep learning tools for generating the kinematic model. Based on the proposed methodology, we demonstrated the design of three degrees of freedom continuum joint and how its kinematic model was learned from the unified pressure information of the actuators and receptors. In addition, we investigated the response of the soft system to receptor failures and presented both hardware and software level solutions for achieving graceful degradation. This approach offers an alternative to enable soft robots with proprioception capability, which will be useful for closed-loop control and interaction with environment.

Investment Trade-Off between Mating Behavior and Tonic Immobility in the Sweetpotato Weevil Cylas formicarius (Coleoptera: Brentidae).

Numerous studies have confirmed that the trade-off between anti-predator behavior and mating behavior occurs in certain insect species. This suggests that insects invest more in anti-predator behavior, and fewer resources or time can be used in mating behavior. However, few studies focus on tonic immobility, an important anti-predator behavior in nature, and different stages in mating behavior. Tonic immobility (TI) is considered to be an important anti-predator behavior. Herein, we investigated the relationship between TI and mating behavior in the sweetpotato weevil (SPW), Cylas formicarius. As the first step, we artificially selected SPWs for the longer duration of TI (L-strain) and the shorter duration of TI (S-strain). The effect of courtship and copulation on the duration of TI in two artificial selection strains was tested. Furthermore, we compared the frequency and duration of two mating behaviors in four kinds of pairs (LF×LM, LF×SM, SF×LM, and SF×SM: LM-L-strain male; SM-S-strain male; LF-L-strain female; SF-S-strain female). Finally, we tested insemination success in four kinds of pairs (male and female SPWs from the L-strain or the S-strain). The courtship and copulation significantly reduced the duration of TI. Pairs with males from the L-strain showed lower frequency and longer duration of courtship than pairs with males from the S-strain. Similarly, males from L-strain pairs showed a longer period of copulation than pairs with males from the S-strain. However, there is no significant difference in the frequency of copulation and the success of insemination. These results support that there was a significant trade-off between TI and courtship as well as copulation in the SPW.

Chromosome-level genome assembly of the Colorado potato beetle, Leptinotarsa decemlineata.

The Colorado potato beetle (Leptinotarsa decemlineata) is one of the most notorious insect pests of potatoes globally. Here, we generated a high-quality chromosome-level genome assembly of L. decemlineata using a combination of the PacBio HiFi sequencing and Hi-C scaffolding technologies. The genome assembly (-1,008 Mb) is anchored to 18 chromosomes (17 + XO), with a scaffold N50 of 58.32 Mb. It contains 676 Mb repeat sequences and 29,606 protein-coding genes. The chromosome-level genome assembly of L. decemlineata provides in-depth knowledge and will be a helpful resource for the beetle and invasive biology research communities.

Comparison of Predatory Performance among Three Ladybird Species, Harmonia axyridis, Coccinella septempunctata and Hippodamia variegata, Feeding on Goji Berry Psyllid, Bactericera gobica.

The psyllid Bactericera gobica is a serious pest in goji berry orchards. The current primary psyllid control methods involve chemical pesticides, which pose potential risks to human health and the environment. The implementation and promotion of biological control agents should receive increased attention as an alternative approach to safeguarding goji berry orchards. To compare the predatory performance of three potential biocontrol agents of psyllids, including Harmonia axyridis, Coccinella septempunctata and Hippodamia variegata, the functional response and intraspecific interactions of adult ladybirds were studied under laboratory conditions. We observed a significantly higher searching efficiency (0.84 ± 0.09) in H. axyridis when preying on psyllids compared to H. variegata (0.55 ± 0.05), whereas the handling time for psyllids was considerably longer in H. axyridis (7.33 ± 0.83 min) than in H. variegata (5.67 ± 0.97 min). The impact of intraspecific interactions on H. variegata (0.44 ± 0.04) was significantly greater than that on C. septempunctata (0.29 ± 0.03), whereas the maximum consumption by C. septempunctata (223.35 ± 41.3) significantly exceeded that of H. variegata (133.4 ± 26.9). Our study suggests that each of these three ladybird species possesses distinct advantages as a potential predator of psyllids. However, further field studies are required to determine the most promising ladybird species for rapid impact through inundative biological control, taking into consideration the specific environmental adaptability of each ladybird species. The present study is expected to provide evidence that supports the potential of incorporating promising ladybird species as an effective biological control agent in goji berry orchard management programs.

Review of Chinese species of genus Phlogothamnus Ishihara (Hemiptera: Cicadellidae: Deltocephalinae) with description of a new species.

The Chinese leafhopper species of the genus Phlogothamnus Ishihara,1961 are reviewed, and a new species Phlogothamnus circinatus sp. nov., is described and illustrated from Guizhou Province, China. And the species Phlogothamnus fanjingshanensis Li, 2011 treated as Phlogotettix fanjingshanensis (Li, 2011) comb. nov.. A key is given to separate all species of the genus from China. The type specimens of the new species are deposited in the Institute of Entomology, Guizhou University, Guiyang, China (GUGC).

Three new and two newly recorded species of the water mite genus Arrenurus Dugs, 1834 (Acari, Hydrachnidia: Arrenuridae) from China.

This paper deals with five species of Arrenurus Dugs, 1834 from P. R. China. Arrenurus (Truncaturus) linguaus sp. nov., Arrenurus (Arrenurus) pseudodistinctus sp. nov. and Arrenurus (Arrenurus) yanchengensis sp. nov, are new to science; Arrenurus (Arrenurus) crassicaudatus Kramer, 1875 and Arrenurus (Megaluracarus) ussuriensis Sokolow, 1931 are recorded for the first time for the Chinese fauna. Arrenurus (Truncaturus) linguaus sp. nov. can be distinguished by tongue-shaped hyaline petiole, trapezoid hyaline membrane, and more or less triangular acetabular plates. Arrenurus (Arrenurus) pseudodistinctus sp. nov. can be distinguished by the median of petiole enlarged, obviously wider than the front and rear ends; ligulate process well-developed and fishtail-shaped; hyaline membrane trapezoid. Arrenurus (Arrenurus) yanchengensis sp. nov can be distinguished by a short hyaline membrane and extending to the base of the well-developed pygal lobes; petiole lobes have the same length as the petiole. Detailed descriptions and illustrations of the new and newly recorded species are given in this paper.

Polygenic adaptation contributes to the invasive success of the Colorado potato beetle.

How invasive species cope with novel selective pressures with limited genetic variation is a fundamental question in molecular ecology. Several mechanisms have been proposed, but they can lack generality. Here, we addressed an alternative solution, polygenic adaptation, wherein traits that arise from multiple combinations of loci may be less sensitive to loss of variation during invasion. We tested the polygenic signal of environmental adaptation of Colorado potato beetle (CPB) introduced in Eurasia. Population genomic analyses showed declining genetic diversity in the eastward expansion of Eurasian populations, and weak population genetic structure (except for the invasion fronts in Asia). Demographic history showed that all populations shared a strong bottleneck about 100 years ago when CPB was introduced to Europe. Genome scans revealed a suite of genes involved in activity regulation functions that are plausibly related to cold stress, including some well-founded functions (e.g., the activity of phosphodiesterase, the G-protein regulator) and discrete functions. Such polygenic architecture supports the hypothesis that polygenic adaptation and potentially genetic redundancy can fuel the adaptation of CPB despite strong genetic depletion, thus representing a promising general mechanism for resolving the genetic paradox of invasion. More broadly, most complex traits based on polygenes may be less sensitive to invasive bottlenecks, thus ensuring the evolutionary success of invasive species in novel environments.

Positive Interactions between Aceria pallida and Bactericera gobica on Goji Berry Plants.

The gall mite Aceria pallida and the psyllid Bactericera gobica are serious Goji berry pests. The mite can be phoretic on the psyllid to overwinter, but it is unclear whether the vector can obtain benefits from the phoront during the growing season. After detachment, the mite shares the same habitat with its vector, so there are very likely to be interspecific interactions. To better understand whether the interactions are positive or negative, information on relationships between abundances of A. pallida and B. gobica on leaves is needed. Here, B. gobica abundance was represented by the egg abundance because the inactive nymphs develop on the same sites after hatching. (1) We found a positive linear relationship between the gall diameter and the mite abundance in the gall (one more millimeter on gall diameter for every 30 mites increase), which provided a way to rapidly estimate mite abundances in the field by measuring gall diameters. (2) There was a positive relationship between the abundance of mites and psyllid eggs on leaves. (3) Both species had positive effects on each other's habitat selections. More importantly, the interactions of the two species prevented leaf abscission induced by B. gobica (leaf lifespan increased by 62.9%), increasing the continuation of the psyllid population. Our study suggests positive interactions between two pests during the growing season. The positive relationship between A. pallida and B. gobica egg abundances highlights the increasing need for novel methods for Goji berry pest management. In practice, A. pallida control can be efficient by eliminating its vector B. gobica. Both pests can be controlled together, which reduces chemical usage.

Unraveling T Cell Responses for Long Term Protection of SARS-CoV-2 Infection.

Due to the COVID-19 pandemic, the global need for vaccines to prevent the disease is imperative. To date, several manufacturers have made efforts to develop vaccines against SARS-CoV-2. In spite of the success of developing many useful vaccines so far, it will be helpful for future vaccine designs, targetting long-term disease protection. For this, we need to know more details of the mechanism of T cell responses to SARS-CoV-2. In this study, we first detected pairwise differentially expressed genes among the healthy, mild, and severe COVID-19 groups of patients based on the expression of CD4+ T cells and CD8+ T cells, respectively. The CD4+ T cells dataset contains 6 mild COVID-19 patients, 8 severe COVID-19 patients, and 6 healthy donors, while the CD8+ T cells dataset has 15 mild COVID-19 patients, 22 severe COVID-19 patients, and 4 healthy donors. Furthermore, we utilized the deep learning algorithm to investigate the potential of differentially expressed genes in distinguishing different disease states. Finally, we built co-expression networks among those genes separately. For CD4+ T cells, we identified 6 modules for the healthy network, 4 modules for the mild network, and 1 module for the severe network; for CD8+ T cells, we detected 6 modules for the healthy network, 4 modules for the mild network, and 3 modules for the severe network. We also obtained hub genes for each module and evaluated the differential connectivity of each gene between pairs of networks constructed on different disease states. Summarizing the results, we find that the following genes TNF, CCL4, XCL1, and IFITM1 can be highly identified with SARS-CoV-2. It is interesting to see that IFITM1 has already been known to inhibit multiple infections with other enveloped viruses, including coronavirus. In addition, our networks show some specific patterns of connectivity among genes and some meaningful clusters related to COVID-19. The results might improve the insight of gene expression mechanisms associated with both CD4+ and CD8+ T cells, expand our understanding of COVID-19 and help develop vaccines with long-term protection.