In the medico-legal application of forensic entomology, estimating the time of death is critical and traditionally relies on changes in observable traits of carrion feeding insect larvae. Traits such as size, weight, and morphology can be used to predict the insect specimen age and help define the minimum time since death. The blowfly Phormia regina Meigen (Diptera: Calliphoridae) is a key forensic insect, yet age estimation for older maggots in this and other carrion-feeding species is particularly challenging due to the limited morphological changes in the late-stage larvae. To enhance age-estimation precision, we employed transcriptomic profiling on blowfly maggots, aiming to identify genes as markers for time of death estimation. Our study characterized maggot development, reinforcing that weight and behavior cannot precisely determine age between 100 and 130 hours at 27.5 °C. We built a chromosomal scale annotated genome, establishing a reliable database for uncovering transcriptomic signatures during larval development. Applying differential gene expression analyses, weighted gene co-expression network analysis, and the generalized linear model, we identified nine candidate genes (y5078, y5076, agt2, ech1, dhb4, asm, gabd, acohc, ivd) that delineate the age of otherwise indeterminate maggots. This research introduces a molecular approach to address a longstanding problem in forensic entomology and promises to increase precision in determining the time of death at a crime scene.

Cryptococcus deneoformans is a human fungal pathogen capable of both a-α and α-α mating and sexual reproduction in laboratory settings. However, the extent of a-α and α-α sexual reproductions in natural populations remain unexplored. Here we analyzed the whole-genome sequences of 24 environmental strains of C. deneoformans from western Saudi Arabia, including one MATa and 23 MATα isolates, with 15 MATα isolates belonging to multi-locus sequence type ST160 as defined by their combined DNA sequences at seven loci. To identify signatures for a-α and α-α reproduction, three samples were analyzed: total, MATα, and ST160. For each subpopulation, single nucleotide polymorphisms (SNPs) were identified for both the nuclear and mitochondrial genomes and subjected to four-gamete tests. In the total population and the MATα subpopulation, variable proportions of SNP pairs within as well as between the nuclear and the mitochondrial genomes showed evidence for recombination. Though no mitogenome SNPs were found among the 15 strains of ST160, the nuclear genome showed clear evidence for recombination, including among SNPs within the MATα mating type locus. In addition, the nuclear genome SNP pairs located further apart on the same chromosome showed a greater frequency of recombination in all three sample types. In contrast, mitogenome SNPs involved in recombination were mainly located in two large genomic regions. Together, these results provide robust evidence for both a-α and α-α sexual reproduction within this environmental population of C. deneoformans.

Long non-coding RNAs (lncRNAs) play crucial roles in regulating gene expression. Some are essential for organismal development and physiology, and they can contribute to diseases including cancer. Whilst most lncRNAs exhibit little sequence similarity, conservation of lncRNA transcription relative to neighbouring protein-coding genes suggests potential functional significance. Most positionally equivalent lncRNAs are uncharacterized and it remains unclear whether they exert similar roles in distant species. Here, we identified melanoma-associated lncRNAs predicted to be components of the MITF gene regulatory network in human melanoma that have positionally equivalent transcripts in zebrafish. We prioritized the cancer-associated lncRNA Differentiation Antagonizing Non-Protein Coding RNA (DANCR) as an exemplar for functional investigation. DANCR is a multi-exonic, cytoplasmically-enriched lncRNA and small RNA host gene transcribed from syntenic regions in the human and zebrafish genomes. MITF and c-MYC, key melanoma transcription factors, regulate human DANCR expression and melanoma patients with high DANCR display significantly decreased survival. DANCR is a melanoma oncogene that controls cancer-associated gene expression networks to promote human melanoma cell proliferation and migration. Zebrafish dancr is essential for embryonic development. It is dynamically expressed across multiple different cell types in the developing embryo, transcriptionally activated by mitfa during early zebrafish development and it regulates genes involved in cell death. Our work suggests that cancer-critical lncRNAs such as DANCR, expressed from similar regions in vertebrate genomes, may control related genes and processes involved in both embryonic development and tumorigenesis across species.

Axon injury initiates transcriptional reprogramming that in competent cells leads to regeneration. In vertebrate neurons, DLK acts upstream of Jun, STAT and Atf3, core transcription factors that mediate regeneration. It is unclear whether these three proteins are activated independently, or whether they function in a linear cascade. To investigate relationships between these transcription factors we wished to use Drosophila as a model system as it has one ortholog of each. However, the only transcription factor linked to DLK-mediated axon regeneration (AR) in flies was Fos. Using loss of function approaches we demonstrate that Jun, STAT and Atf3 are required for Drosophila sensory axon regeneration, indicating transcriptional control of axon regeneration is broadly conserved. We next investigated temporal roles for Fos, Jun, STAT and Atf3. Only Fos is required for the early transcriptional response, which coincides with neuroprotection, and its nuclear entry and homodimerization coincide with this phase. Reduction of Jun homodimerization occurs after axon injury downstream of DLK/JNK, but independently from Fos, at a later stage associated with axon regrowth. STAT nuclear entry occurs downstream of Jun as part of this stage, is inhibited by Fos, and does not require JAK, which is dispensable for axon regeneration. Atf3 nuclear exit is in turn downstream of Fos, Jun, and STAT. Our results suggest that DLK/JNK separately activates Fos and Jun, and that Jun initiates a transcriptional cascade that includes STAT and Atf3. These two transcriptional modules control separate steps of the injury response that culminates in axon regeneration.

Genetic selection occurs at multiple stages before and during pregnancy. While parental genomes influence the probability of fertilization, the fetal genome, once established, plays a critical role in early fetal survival. However, when estimated separately, parental and fetal genetic effects may confound each other. To address this, we developed an extension of the case-parent triad design to jointly estimate the genetic contributions of the parents and the fetus. Our approach considers all offspring as carriers of the trait "fetal survival". As use of assisted reproductive technology (ART) usually reflects fertility issues, we performed separate analyses on non-ART and ART family units, hypothesizing that parental and fetal effects differ between these groups. In the Norwegian Mother, Father, and Child Cohort Study, we had access to genotypes for approximately 43,000 family triads and dyads, including 1,336 offspring conceived through ART. In the non-ART sample, we identified genome-wide significant fetal effects on fetal survival for SNPs within regions harboring genes relevant to infertility and fetal development, such as MDC1, MICB, HCP5, and NOTCH4. These effects remained significant after adjusting for parental interaction effects, confirming their origin as fetal effects. When we replicated the analysis in the ART sample, we observed partial overlap in fetal effects with those identified in the non-ART sample. Parental interaction effects were observed in both the non-ART and ART samples, but the specific genetic associations differed between the groups. Notably, several SNPs associated with parental interaction effects in the ART sample mapped to genes previously implicated in male infertility, including ACTB, FSCN1, and RNF216. Our findings have broad implications for understanding the genetic architecture of infertility and fetal development. To support the interpretation of our results, we provide detailed descriptions of the models, highlighting their strengths and limitations.

Obligate host-associated bacteria with reduced genomes, such as phytoplasmas, face strong evolutionary constraints, including metabolic dependence on hosts, limited opportunities for horizontal gene transfer (HGT), and frequent population bottlenecks. Despite these limitations, phytoplasmas, which are parasitic, insect-transmitted plant pathogens, maintain a diverse arsenal of secreted effectors that manipulate both plant and insect hosts to promote infection and transmission. These effectors can suppress immunity and reprogram plant development, inducing alterations such as witch's broom and leaf-like flowers, through ubiquitin-independent degradation of key transcription factors. However, how phytoplasmas diversify and maintain these effectors in the absence of frequent genetic exchange remains unclear. To address this, we analysed the effectoromes of 239 phytoplasma genomes and identified a diverse set of secreted proteins, which we designated as putative Phytoplasma Effectors (PhAMEs). We found that PhAMEs targeting evolutionarily conserved and structurally constrained surfaces of host proteins are widespread across phytoplasmas. These effectors adopt compact, efficient folds. They often function as molecular scaffolds with dual interaction surfaces capable of linking host proteins or integrating signalling pathways. Such scaffolding PhAMEs have evolved multiple times independently, providing clear evidence of convergent evolution. Despite severe genomic constrains imposed by genome reduction and limited HGT, gene duplications, interface variations, domain fusions, and repeat expansions have helped the shaping effector fold and diversity. While the overall effector repertoire of phytoplasmas appeared largely unique, some PhAME domains share similarities with proteins from other mollicutes and pathogens. Collectively, our findings shed light on how genome-reduced bacteria innovate molecular functions and offer insights into phytoplasma biology, effector evolution, and host-pathogen dynamics. They also lay the groundwork for protein engineering approaches aimed at discovering or designing novel biomolecules with biotechnological potential.

Genetic exchange is a cornerstone of evolutionary biology and genomics, driving adaptation and enabling the identification of genetic determinants underlying phenotypic traits. In Escherichia coli, horizontal gene transfer via conjugation and transduction not only promotes diversification and adaptation but has also been instrumental in mapping genetic traits. However, the dynamics and variability of bacterial recombination remain poorly understood, particularly concerning the patterns of recombined DNA fragments. To elucidate these patterns and simultaneously develop a tool for trait mapping, we designed a high-throughput conjugation method to generate recombinant libraries. Recombination profiles were inferred through whole-genome sequencing of individual clones and populations after selection of a marker from the donor strain in the recipient. This analysis revealed an extraordinary range of recombined fragment sizes, spanning less than ten kilobases to over a megabase—a pattern that varied across the three tested strains. Mathematical modelling indicated that this diversity in recombined fragment size enables precise identification of selected loci following genetic crosses. Consistently, population sequencing pinpointed a selected marker at kilobase-scale accuracy, offering a robust tool for identifying subtle genetic determinants that could include point mutations in core genes. These findings challenge the conventional view that conjugation always transfers large fragments, suggesting that even short recombined segments, traditionally attributed to transduction, may originate from conjugation.

Telomerase Reverse Transcriptase (TERT), in addition to its well-known role in telomere lengthening, also has non-canonical functions, including gene regulation and protection against apoptosis. Beyond its nuclear functions, it is now recognized for its presence inside mitochondria. However, the biological role of TERT in mitochondrial physiological activity, with its specific mechanism of action, still needs to be clarified. This work clearly demonstrates the presence of TERT inside the mitochondrion under physiological conditions, in different cellular contexts, both with endogenous and ectopic TERT expression, and regardless of the presence of telomerase RNA counterpart TERC. TERT was shown to bind mitochondrial DNA, influencing mitochondrial replication and transcription. Furthermore, electron microscopy analysis of morphology revealed TERT-induced fragmentation of the mitochondrial network. Collectively, our findings suggest that TERT may play a role in regulating mitochondrial biogenesis and dynamics, and influencing processes such as fission and mitophagy, essential for maintaining mitochondrial homeostasis and closely connected to cellular states.

Sperm deliver male genomic DNA to the ovum, playing a pivotal role in sexual reproduction across the animal kingdom. The molecular regulation of sperm morphogenesis has consequently become a focal point of genetic research, with dual implications for both reproductive medicine and sustainable agriculture. Here, we characterize the functional role of the testis-specific serine/threonine protein kinase-like (TSSKL) gene in the model lepidopteran insect Bombyx mori and the globally destructive crop pest Plutella xylostella. RNA-seq and qPCR analyses revealed TSSKL's testis-specific expression pattern. Using CRISPR/Cas9-mediated mutagenesis, we demonstrate that TSSKLknockout induces complete male sterility, while female fertility remains unaffected compared to wild-type. Fluorescence microscopy and ultrastructural analyses revealed that TSSKL deletion leads to severe morphological defects in both eupyrene and apyrene sperm, accompanied by impaired mitochondrial dynamics and aberrant autophagy. Comparative transcriptome and functional analyses linked these phenotypes to dysregulated energy metabolism pathways. Crucially, this sterility phenotype is conserved in P. xylostella, recapitulating the findings in B. mori. Our study demonstrates that TSSKL is crucial for male fertility, coordinating both structural and metabolic aspects of sperm development. Beyond advancing fundamental knowledge of insect reproductive biology, this work also identifies TSSKL as an ideal target for lepidopteran pest control through precision sterility induction.

Mitochondria and plastids of various lineages exhibit genetic code alterations. However, the knowledge of the diversity and occurrence, mechanistic underpinnings, and evolutionary origins of codon reassignments in organelles remains incomplete. To address this gap, we focused on organelles of the neglected green algal class Pedinophyceae, as well as pedinophyte-derived secondary plastids of green-coloured dinoflagellates (peDinoflagellates). We isolated and characterized a novel pedinophyte, herein formally described as Oistococcus okinawensis gen. et sp. nov., and phenotypically documented the previously sequenced but morphologically uncharacterized strain YPF-701, herein described as Akinorimonas japonica gen. et sp. nov. Based on phylogenetic analyses, both new taxa were classified into the expanded family Resultomonadaceae. We sequenced the organellar genomes of O. okinawensis, and utilizing existing raw (meta)genomic data we assembled organellar genome sequences from other previously unexplored pedinophyte lineages. Bioinformatic analyses of the expanded set of pedinophyte organellar genomes painted a complex picture of their genetic code landscape. Concerning mitochondria, the stop-to-Trp reassignment of the UGA codon turned out to have evolved multiple times in pedinophytes, and the Arg-to-Ala reassignment of AGA/AGG codons was shown to be apomorphic for the whole order Marsupiomonadales. The latter has additionally converted UUA and UUG into termination codons, relying on specific mutations in the mtRF1a protein. All pedinophyte mitochondria seem to decode AUA as methionine rather than the standard isoleucine, and an analogous reassignment seems to be evolving also in plastids of two separate pedinophyte lineages. Finally, apart from the previously reported Ile-to-Met AUA reassignment, peDinoflagellate plastids have switched the meaning of the AGA/AGG codons from arginine to another amino acid (most likely alanine), and have modified their pRF2 protein to mediate translation termination at UUA/UCA codons. Pedinophyte(-derived) organelles present a broad spectrum of codon reassignments and provide important insights into the emergence and mechanisms of non-standard codon translation.