Investigating the effects of a cryptic splice site in the En2 splice acceptor sequence used in the IKMC knockout-first alleles.

Targeted mouse mutants are a common tool used to investigate gene function. The International Knockout Mouse Consortium undertook a large-scale screen of mouse mutants, making use of the knockout-first allele design that contains the En2 splice acceptor sequence coupled to the lacZ reporter gene. Although the knockout-first allele was designed to interfere with splicing and thus disrupt gene function, the En2 sequence has been reported to be transcribed within the host gene mRNA due to a cryptic splice site within the En2 sequence which allows splicing to the next exon of the host gene. In some circumstances, this has the potential to permit translation of a mutant protein. Here, we describe our computational analysis of all the mouse protein-coding genes with established knockout-first embryonic stem cell lines, and our predictions of their transcription outcome should the En2 sequence be included. As part of the large-scale mutagenesis program, mutant mice underwent a broad phenotyping screen, and their phenotypes are available. No wide-scale effects on mouse phenotypes reported were found as a result of the predicted En2 insertion. However, the En2 insertion was found experimentally in the transcripts of 24 of 35 mutant alleles examined, including the five already described, two with evidence of readthrough. Splicing from the cryptic splice site also has the potential to disrupt expression of the lacZ reporter gene. It is recommended that mutant transcripts be checked for this insertion as well as for leaky transcription in studies involving knockout-first alleles.

Back together: Over 1000 single-copy nuclear loci and reproductive features support the holoendoparasitic Apodanthaceae as sister to the Rafflesiaceae in the order Malpighiales.

The systematics the holoendoparasitic flowering plant families Apodanthaceae and Rafflesiaceae has been discussed for over two centuries. The morphological reduction of roots, shoots and leaves in all members of both families, resulting in a cryptic mycelium-like vegetative body, has been interpreted either as a key common feature, or as a result of convergent evolution due to full dependence upon their hosts. Historically, the two families have been placed together due to similar morphological features, but recent analyses based on few mitochondrial and ribosomal gene markers placed them in the distantly related orders Cucurbitales and Malpighiales. Here we reevaluate the affinities of the Apodanthaceae and the Rafflesiaceae using a phylogenomic approach. We present (1) a historical account on their phylogenetic affinities over the last 200 years; (2) phylogenetic analyses reinstating their sister group relationship as part of the order Malpighiales, based on over 1000 single-copy nuclear protein-coding loci; and (3) a comprehensive list of putative morphoanatomical and developmental synapomorphies in light of the phylogenomic results, with emphasis on shared reproductive traits regardless of dramatic differences in floral size.

DNA barcoding and cryptic diversity in fishes from the Ili River Valley in China, Xinjiang.

The Ili River Valley, located in the northwest of China, serves as a vital repository for fish genetic resources. Its extensive water network and diverse climate have given rise to a unique fish composition and endemic species. In this study, we collected the cytochrome c oxidase subunit I (COI) sequences from 660 fish specimens in the Ili River Valley. The effectiveness of DNA barcoding in identifying fish species in the area was assessed by examining genetic distances, constructing phylogenetic trees, and performing ABGD (Automatic Barcode Gap Discovery) analyses, among other methods. In total, 20 species were identified, including one unidentified species (Silurus sp.). Except for Silurus asotus and Hypophthalmichthys molitrix (only one sample), the maximum intraspecific genetic distance among the remaining species was smaller than the minimum interspecific distance, which proves that the species exhibit obvious barcode gaps. In the Neighbor-Joining trees, 20 species formed separate monophyletic branches. According to ABGD analysis, 660 sequences were categorized into 19 Operational Taxonomic Units, with Silurus sp. and S. asotus grouped into a single OTU. The Silurus in this study exhibits shared haplotypes and significant genetic divergence, suggesting the potential presence of cryptic species. Furthermore, the nucleotide diversity across all species fell below the threshold level, indicating that the local fish population is gradually declining. In conclusion, this study has demonstrated the effectiveness of DNA barcoding in identifying fish species in the Ili River Valley, providing valuable data to support the conservation of local fish resources.

A shift in substrate requirement might cause speciation of the lichenized fungi, Varicellaria hemisphaerica and V. lactea (Pertusariales, Ascomycota).

Proper species recognition is required to correctly estimate species preferences and their vulnerability or for eco-evolutionary inference. Varicellaria hemisphaerica and Varicellaria lactea are almost completely morphologically homogeneous species with unclear identification features. To evaluate the importance of morphological, chemical, and ecological characteristics used in recognition of these species, we tested 670 specimens, of which 42 were analyzed phylogenetically using nucITS rDNA, SSU rDNA, and LSU rDNA markers. This integrated taxonomical approach showed that V. hemisphaerica is distinct from V. lactea, and that substrate requirements, together with phylogenetic differences and the size of soredia, differentiate these species. The chemical composition of secondary lichen metabolites in both analyzed species showed similar variation and, therefore, this feature is not diagnostic in species recognition, although suggested by previous studies. The potential speciation of the two species seems to be caused by the shift in the substrate requirements.

Widespread occurrence of dissolved oxygen anomalies, aerobic microbes, and oxygen-producing metabolic pathways in apparently anoxic environments.

Nearly all molecular oxygen (O2) on Earth is produced via oxygenic photosynthesis by plants or photosynthetically active microorganisms. Light-independent O2 production, which occurs both abiotically, e.g., through water radiolysis, or biotically, e.g., through the dismutation of nitric oxide or chlorite, has been thought to be negligible to the Earth system. However, recent work indicates that O2 is produced and consumed in dark and apparently anoxic environments at a much larger scale than assumed. Studies have shown that isotopically light O2 can accumulate in old groundwaters; that strictly aerobic microorganisms are present in many apparently anoxic habitats; and that microbes and metabolisms that can produce O2 without light are widespread and abundant in diverse ecosystems. Analysis of published metagenomic data reveals that the enzyme putatively capable of nitric oxide dismutation (NOD) forms four major phylogenetic clusters and occurs in at least 16 bacterial phyla most notably the Bacteroidota. Similarly, a re-analysis of published isotopic signatures of dissolved O2 in groundwater suggests in-situ production in up to half of the studied ecosystems. Geochemical and microbiological data supports the conclusion that "dark oxygen" production is an important and widespread yet overlooked process in apparently anoxic environments with far-reaching implications for subsurface biogeochemistry and ecology.

Identification of non-model mammal species using the MinION DNA sequencer from Oxford Nanopore.

Background: The Neotropics harbors the largest species richness of the planet; however, even in well-studied groups, there are potentially hundreds of species that lack a formal description, and likewise, many already described taxa are difficult to identify using morphology. Specifically in small mammals, complex morphological diagnoses have been facilitated by the use of molecular data, particularly from mitochondrial sequences, to obtain accurate species identifications. Obtaining mitochondrial markers implies the use of PCR and specific primers, which are largely absent for non-model organisms. Oxford Nanopore Technologies (ONT) is a new alternative for sequencing the entire mitochondrial genome without the need for specific primers. Only a limited number of studies have employed exclusively ONT long-reads to assemble mitochondrial genomes, and few studies have yet evaluated the usefulness of such reads in multiple non-model organisms. Methods: We implemented fieldwork to collect small mammals, including rodents, bats, and marsupials, in five localities in the northern extreme of the Cordillera Central of Colombia. DNA samples were sequenced using the MinION device and Flongle flow cells. Shotgun-sequenced data was used to reconstruct the mitochondrial genome of all the samples. In parallel, using a customized computational pipeline, species-level identifications were obtained based on sequencing raw reads (Whole Genome Sequencing). ONT-based identifications were corroborated using traditional morphological characters and phylogenetic analyses. Results: A total of 24 individuals from 18 species were collected, morphologically identified, and deposited in the biological collection of Universidad EAFIT. Our different computational pipelines were able to reconstruct mitochondrial genomes from exclusively ONT reads. We obtained three new mitochondrial genomes and eight new molecular mitochondrial sequences for six species. Our species identification pipeline was able to obtain accurate species identifications for up to 75% of the individuals in as little as 5 s. Finally, our phylogenetic analyses corroborated the identifications from our automated species identification pipeline and revealed important contributions to the knowledge of the diversity of Neotropical small mammals. Discussion: This study was able to evaluate different pipelines to reconstruct mitochondrial genomes from non-model organisms, using exclusively ONT reads, benchmarking these protocols on a multi-species dataset. The proposed methodology can be applied by non-expert taxonomists and has the potential to be implemented in real-time, without the need to euthanize the organisms and under field conditions. Therefore, it stands as a relevant tool to help increase the available data for non-model organisms, and the rate at which researchers can characterize life specially in highly biodiverse places as the Neotropics.

Cryptic transmission and novel introduction of Dengue 1 and 2 genotypes in Colombia.

Dengue fever remains as a public health challenge in Colombia, standing as the most prevalent infectious disease in the country. The cyclic nature of dengue epidemics, occurring approximately every 3 years, is intricately linked to meteorological events like El Niño Southern Oscillation (ENSO). Therefore, the Colombian system faces challenges in genomic surveillance. This study aimed to evaluate local dengue virus (DENV) transmission and genetic diversity in four Colombian departments with heterogeneous incidence patterns (department is first-level territorial units in Colombia). For this study, we processed 266 serum samples to identify DENV. Subsequently, we obtained 118 genome sequences by sequencing DENV genomes from serum samples of 134 patients infected with DENV-1 and DENV-2 serotypes. The predominant serotype was DENV-2 (108/143), with the Asian-American (AA) genotype (91/118) being the most prevalent one. Phylogenetic analysis revealed concurrent circulation of two lineages of both DENV-2 AA and DENV-1 V, suggesting ongoing genetic exchange with sequences from Venezuela and Cuba. The continuous migration of Venezuelan citizens into Colombia can contribute to this exchange, emphasizing the need for strengthened prevention measures in border areas. Notably, the time to most recent common ancestor analysis identified cryptic transmission of DENV-2 AA since approximately 2015, leading to the recent epidemic. This challenges the notion that major outbreaks are solely triggered by recent virus introductions, emphasizing the importance of active genomic surveillance. The study also highlighted the contrasting selection pressures on DENV-1 V and DENV-2 AA, with the latter experiencing positive selection, possibly influencing its transmissibility. The presence of a cosmopolitan genotype in Colombia, previously reported in Brazil and Peru, raises concerns about transmission routes, emphasizing the necessity for thorough DENV evolution studies. Despite limitations, the study underscores genomic epidemiology's crucial role in early detection and comprehension of DENV genotypes, recommending the use of advanced sequencing techniques as an early warning system to help prevent and control dengue outbreaks in Colombia and worldwide.

Stimulatory Functions of Male Genitalia in Tipula (Triplicitipula) colei Alexander and Tipula (Lunatipula) translucida Doane (Diptera: Tipulidae) and Implications for Theories of Genital Evolution.

Male genitalia have been hypothesized to function as courtship devices during copulation, but it is difficult to use behavioral observations to test this hypothesis because male genitalia are usually hidden inside the female during copulation. In tipuloid flies, however, nearly all of the male's complex genital structures remain outside the female. Copulation behavior and genital morphology in Tipula (Triplicitipula) colei and T. (Lunatipula) translucida suggest that some male genital structures function to stimulate the female: male structures that contact the female bear tufts or dense arrays of modified setae on precisely the surfaces that contact the female; contact involves repeated, stereotyped rhythmic movements that include brushing, vibrating, scraping, and tapping; the movements are appropriately designed to utilize the morphology of the modified setae to stimulate the female; and the movements have little or no other perceptible mechanical effects on the female. The female structures contacted by these male genital movements fail to show the defensive designs predicted by the theories of genital evolution that are based on morphological species isolation or male-female morphological conflicts of interest; also unexplained by the conflict of interest hypothesis are female movements that seem designed to increase rather than avoid stimulation by the male.

A Sister Species for the Blue Crab, Callinectes sapidus? A Tale Revealed by Mitochondrial DNA.

The Atlantic blue crab, Callinectes sapidus, is acknowledged as one of the worst invasive alien species in the Mediterranean, impacting biodiversity and human activities. Native to the western Atlantic, it has expanded to European coastal waters since the early 1900s. Despite its ecological and commercial importance, genetic research on this species is limited. Here we show a comprehensive investigation of the genetic variation and evolutionary history in Callinectes sapidus using 667 mitochondrial COI gene sequences. Our dataset encompasses 36 newly generated sequences from previously understudied Mediterranean sites and 631 from worldwide locations obtained from the GenBank public database. Our findings reveal two distinct, but closely related, genetic groups within the species' distribution range, suggesting the occurrence of a potential species complex. Furthermore, in the Mediterranean, low levels of genetic variability were observed except for substantial haplotypic differentiation in Turkish samples. This study depicts the global genetic diversity and evolutionary patterns of Callinectes sapidus, offering new insights into the taxonomic status of the species.

Comparative transcriptome analysis of whiteflies raised on cotton leaf curl Multan virus-infected cotton plants.

Cotton leaf curl Multan virus (CLCuMuV), a serious viral disease causative agent in cotton plants in South Asia, is transmitted by the Bemisia tabaci cryptic species complex in a persistent circulative manner. A previous study indicated that Asia II-7 whiteflies could transmit CLCuMuV, while Mediterranean (MED) whiteflies failed to transmit CLCuMuV. However, little is known about the genes involved in this process. In this study, Asia II-7 and MED B. tabaci were utilized to determine transcriptomic responses after 48 h of acquisition access periods (AAPs). Result of Illumina sequencing revealed that, 14,213 and 8,986 differentially expressed genes (DEGs) were identified. Furthermore, DEGs related to the immune system and metabolism of Asia II-7 and MED in response to CLCuMuV-infected plants were identified and analyzed using Gene Ontologies (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG), and the number of related DEGs in MED was lower than that of Asia II-7. The most abundant groups of DEGs between both viruliferous and aviruliferous whitefly species were the zf-C2H2 family of transcription factors (TFs). Notably, in comparison to viruliferous MED, Asia II-7 exhibited more DEGs related to cathepsin biosynthesis. Overall, this study provides the basic information for investigating the molecular mechanism of how begomoviruses affect B. tabaci metabolism and immune response either as vector cryptic species or non-vector species.

In vivo DNA replication dynamics unveil aging-dependent replication stress.

The genome duplication program is affected by multiple factors in vivo, including developmental cues, genotoxic stress, and aging. Here, we monitored DNA replication initiation dynamics in regenerating livers of young and old mice after partial hepatectomy to investigate the impact of aging. In young mice, the origin firing sites were well defined; the majority were located 10-50 kb upstream or downstream of expressed genes, and their position on the genome was conserved in human cells. Old mice displayed the same replication initiation sites, but origin firing was inefficient and accompanied by a replication stress response. Inhibitors of the ATR checkpoint kinase fully restored origin firing efficiency in the old mice but at the expense of an inflammatory response and without significantly enhancing the fraction of hepatocytes entering the cell cycle. These findings unveil aging-dependent replication stress and a crucial role of ATR in mitigating the stress-associated inflammation, a hallmark of aging.

Phylogenetic analyses show the Select Agent Coniothyrium glycines represents a single species that has significant morphological and genetic variation.

Soybean red leaf blotch (RLB), caused by the fungus Coniothyrium glycines, represents a foliar disease of soybean that is thus far restricted to Africa. The fungus is listed as a Select Agent by the Federal Select Agent Program because it could pose a severe threat to plant health were it to establish in the United States. Previous work uncovered tremendous molecular diversity at the internal transcribed spacer region, suggesting that there may be multiple species causing RLB. To determine whether multiple species cause RLB, we reconstructed the phylogeny of C. glycines and taxonomic allies using sequence data from four genes. We included 33 C. glycines isolates collected from six African countries and determined that all isolates form a well-supported, monophyletic lineage. Within this lineage there are at least six well-supported clades that largely correspond to geography, with one clade exclusively composed of isolates from Ethiopia, another exclusively composed of isolates from Uganda, and four composed of isolates from southern Africa. However, we did not detect any concordance for these clades between the four genes, indicating that all isolates included in this analysis are representative of a single species. Isolates in the Ethiopia clade are morphologically distinct from isolates in the other clades, as they produce larger sclerotia and smaller pycnida and more sclerotia in planta. Additionally, ancestral range estimations suggest that the C. glycines lineage emerged in southern Africa. These results show that there is significantly more genetic and morphological diversity than was initially suspected with this high-consequence fungal plant pathogen.

Is mating failure caused by cryptic male choice in the seed bug Lygaeus simulans?

One yet unresolved question in the study of mating system evolution is the occurrence of mating failure, when individuals go through their lives without successfully mating. This includes the failure to produce offspring even following copulation, for instance due to insemination or fertilisation failure. Copulations are costly in a variety of ways, but also a fundamental route to fitness in sexual species, and so we should expect that engaging in copulations that generate no offspring should be strongly selected against. Nonetheless, it has become apparent that mating failure is quite common in nature. Here we consider post-copulatory sexual selection in Lygaeus simulans seed bugs to test the hypothesis that the high levels of mating failure found in this species (approximately 40%-60%) are caused by cryptic male choice (i.e. males choosing not to inseminate a female during copulation). In our first experiment, we found that mating failure depended on female size, but not male size, with smaller females experiencing mating failure more frequently. Mechanistically this is likely to be due to copulation duration, as shorter copulations were more likely to lead to mating failure. Likewise, copulations with smaller females were shorter. In our second and third experiments, rates of mating failure decreased when pairs were allowed to repeatedly interact with the same partner over longer durations (hours through to days), implying that mating failure is not primarily caused by infertility or chronic mechanical failure. Instead, our results strongly suggest cryptic male choice as the cause of mating failure in this species.

Characterization of cryptic complex chromosome rearrangements in balanced chromosomal rearrangement carriers and their PGT-SR clinical outcome assessments.

Several reports have presented that balanced chromosomal rearrangements (BCRs) carriers with normal phenotypes may be carriers of complex rearrangements. However, the incidence and PGT clinical outcomes of cryptic complex chromosome rearrangements (CCCRs) in individuals with BCRs is remain unknown. We recruited a cohort of 1,264 individuals with BCR carriers from 2016 to 2021 at the Reproductive and Genetic Hospital of CITIC Xiangya. Peripheral blood was collected for karyotyping and genomic DNA extraction and the PGT-SR clinical outcomes of CCCRs carriers were analyzed and compared with those of BCR carriers. Our findings revealed that 3.6% (45/1,264) of BCR carriers had CCCRs, involving 3-25 breakpoints on 1-3 chromosomes. Furthermore, when mate-pair sequencing was employed, 63.3% (19/30) of CCCR carriers were found to have chromosome rearrangements that were different from those identified by the MicroSeq technique. And the transferable embryo rate of CCCR carriers with 3 chromosomes was significantly lower than that of CCCR carriers with only 1-2 chromosomes. In this research, we revealed that some of the BCR carriers were actually CCCR carriers, and the prognosis of PGT in CCCR carriers with one or two chromosomes is better than that of CCCR carriers with three chromosomes.

The role of condition on sexual selection in the seed bug Lygaeus simulans.

Organism condition plays an important role in sexual selection. Sexual ornaments and displays can be condition-dependent, reflecting either underlying genetic quality, experience of environmental stressors, or both. As such, the phenotypic expression of such traits, and the resulting patterns of mate choice acting on them, may be shaped by intrinsic genetic quality and the environment. Moreover, condition may also influence the choosing individual in mate choice, influencing their ability to invest in mate discrimination, or changing what traits of the chosen, including resources, are most preferred. Here we consider sexual selection and condition in the seed bug Lygaeus simulans, a species characterised by strong post-copulatory sexual selection, but rather limited pre-copulatory discrimination. We manipulated short-term condition in both males and females by restricting access to water for 24 h. Water is particularly important in these bugs, given their feeding ecology and physiology. We found that water-deprived males proved less likely to mate, while copulation duration with water-deprived females was significantly reduced. Given the importance of copulation duration for the successful transfer of sperm by males to females, the data suggest cryptic male choice acting against water-deprived females. These data add to those suggesting that cryptic male choice for fecund females plays an important role in sexual selection in this species. More generally, our results support the widespread importance of condition in terms of mating dynamics and sexual selection.

Harnessing native-cryptic plasmids for stable overexpression of heterologous genes in Clostridium butyricum DSM 10702 for industrial and medical applications.

Clostridium butyricum has emerged as a promising candidate for both industrial and medical biotechnologies, underscoring the key pursuit of stable gene overexpression in engineering C. butyricum. Unlike antibiotic-selective vectors, native-cryptic plasmids can be utilized for antibiotic-free expression systems in bacteria but have not been effectively exploited in C. butyricum to date. This study focuses on leveraging these plasmids, pCB101 and pCB102, in C. butyricum DSM10702 for stable gene overexpression without antibiotic selection via efficient gene integration using the SacB-based allelic exchange method. Integration of reporter IFP2.0 and glucuronidase generated sustained near-infrared fluorescence and robust enzyme activity across successive subcultures. Furthermore, successful secretion of a cellulase, Cel9M, and the human interleukin 10 from pCB102 highlights native-cryptic plasmids' potential in conferring stable gene products for industrial and medical applications in C. butyricum. This work appears to be the first study to harness the Clostridium native-cryptic plasmid for stable gene overexpression without antibiotics, thereby advancing the biotechnological prospects of C. butyricum.

A new discovery of cryptic species in barred sand burrower Limnichthys fasciatus (Pisces: Creediidae) from the western Pacific, with evolutionary perspectives of anti-equatorial species.

Although the barred sand burrower, Limnichthys fasciatus, is widely distributed throughout the western Pacific, including Japan, Taiwan, and Australia, its morphology and genetics are poorly known. We discovered four cryptic species of Limnichthys from the western Pacific based on mtDNA cytochrome c oxidase subunit I (COI) and 16S ribosomal RNA (rRNA) sequences. Genetic distances showed remarkably large differences (12.7%-24.3% in COI and 7.9%-19.6% in 16S rRNA) between true L. fasciatus (type locality: southeastern Australia) and the others. A relaxed clock model with optimized selected substitution models showed that their deep divergence began in the middle Miocene epoch and subsequently diverged into the current cryptic species in the Plio-Pleistocene. A eurythermal common ancestor may have evolved independently in each region due to geographical events and paleoclimatic fluctuations, which made it possible for L. fasciatus complex to be an anti-equatorial species. Despite their deep genetic divergence, they showed marked phenotypic similarity, suggesting that they have experienced similar selective pressures related to their specific behavior.

Decrypting the phylogenetics history of EGF-CFC proteins Cripto and Cryptic.

EGF-CFC proteins are obligate coreceptors for Nodal signaling and are thus required for gastrulation and left-right patterning. Species with multiple family members show evidence of specialization. For example, mouse Cripto is required for gastrulation, whereas Cryptic is involved in left-right patterning. However, the members of the family across model organisms have little sequence conservation beyond the EGF-CFC domain, posing challenges for determining their evolutionary history and functional conservation. In this study we outline the evolutionary history of the EGF-CFC family of proteins. We traced the EGF-CFC gene family from a single gene in the deuterostome ancestor through its expansion and functional specialization in tetrapods, and subsequent gene loss and translocation in eutherian mammals. Mouse Cripto and Cryptic, zebrafish Tdgf1, and all three Xenopus EGF-CFC genes (Tdgf1, Tdgf1.2 and Cripto.3) and are all descendants of the ancestral Tdgf1 gene. We propose that subsequent to the family expansion in tetrapods, Tdgf1B (Xenopus Tdgf1.2) acquired specialization in the left-right patterning cascade, and after its translocation in eutherians to a different chromosomal location, Cfc1/Cryptic has maintained that specialization.

Neurobiology of egg-laying behavior in Drosophila: neural control of the female reproductive system.

Egg-laying is one of the key aspects of female reproductive behavior in insects. Egg-laying has been studied since the dawn of Drosophila melanogaster as a model organism. The female's internal state, hormones, and external factors, such as nutrition, light, and social environment, affect egg-laying output. However, only recently, neurobiological features of egg-laying behavior have been studied in detail. fruitless and doublesex, two key players in the sex determination pathway, have become focal points in identifying neurons of reproductive significance in both central and peripheral nervous systems. The reproductive tract and external terminalia house sensory neurons that carry the sensory information of egg maturation, mating and egg-laying. These sensory signals include the presence of male accessory gland products and mechanical stimuli. The abdominal neuromere houses neurons that receive information from the reproductive tract, including sex peptide abdominal ganglion neurons (SAGs), and send their information to the brain. In the brain, neuronal groups like aDNs and pC1 clusters modulate egg-laying decision-making, and other neurons like oviINs and oviDNs are necessary for egg-laying itself. Lastly, motor neurons involved in egg-laying, which are mostly octopaminergic, reside in the abdominal neuromere and orchestrate the muscle movements required for laying the egg. Egg-laying neuronal control is important in various evolutionary processes like cryptic female choice, and using different Drosophila species can provide intriguing avenues for the future of the field.