Biology subjects

Deep, A.

Publications and source records attributed to Deep, A..

At least 19 recordsLinked to original sources

bioRxiv2026

Uncovering moonlighting role of mitochondrial presequence translocase machinery in SOD1-mediated ALS pathogenesis

Familial Amyotrophic Lateral Sclerosis (fALS) is a fatal neurodegenerative disease, mainly caused by mutations in the superoxide dismutase 1 (SOD1) protein. Mitochondrial dysfunction is a primary hallmark of ALS pathogenesis. However, the molecular mechanism by which SOD1 mutants impair organellar health remains enigmatic. This study demonstrates that mutant SOD1 associates with the TIM23 complex in Saccharomyces cerevisiae via its intermembrane space (IMS) domain. In ALS-associated SOD1 mutants, both binding and expression of TIM23 complex proteins were downregulated, leading to altered translocation of the substrate protein Sdh3, a component of the electron transport chain (ETC) complex II. Disrupted Sdh3 translocation leads to mitochondrial dysfunction, evidenced by decreased ETC complex II activity, reduced functional mass, and compromised organelle integrity. Overexpression of Tim23 partially rescued mitochondrial integrity by increasing ETC complex activity and functional mass and restoring reticular morphology. Strikingly, the improved mitochondrial homeostasis in Tim23-overexpressing cells partially rescued the growth defects caused by mutant SOD1. Collectively, these findings reveal a previously unrecognized regulatory axis between mutant SOD1 and the mitochondrial pre-sequence translocase machinery, highlighting this pathway as a promising target for future ALS therapies and opening new avenues for mechanistic and translational research. Author SummaryFamilial Amyotrophic Lateral Sclerosis (fALS) is a progressive, fatal neuromuscular disorder marked by motor neuron degeneration. The exact cause of ALS remains unclear. Previous research links familial ALS to mutations in the superoxide dismutase 1 (SOD1) gene. SOD1 mutants in ALS disrupt mitochondrial protein translocation, a key mitochondrial process. The mechanism by which SOD1 mutants affect mitochondrial function and integrity by modulating presequence translocase (TIM23 complex) import is not yet understood. The current study addresses a critical gap in ALS research by demonstrating a novel, direct interaction between SOD1 and Tim23 that regulates mitochondrial function in yeast. We found that SOD1 binds Tim23 via Tim23 IMS domain, stabilizes the Tim23CORE complex, enabling Sdh3 import. Loss of SOD1, Tim23, or Tim50 destabilizes the TIM23CORE complex, leading to impaired Sdh3 import and decreased ETC complex-II activity. These changes disrupt mitochondrial structure, causing fragmentation and a loss of functional mitochondrial mass in {Delta}sod1. ALS-linked SOD1 mutants show similar effects: they diminish Sdh3 import by weakening SOD1-Tim23 interaction and lowering TIM23 complex stability, resulting in punctate mitochondria and reduced mitochondrial mass. Collectively, our study identifies the SOD1-TIM23 interaction as a key regulator of mitochondrial health through Sdh3 import via the TIM23CORE complex and indicates this pathway as a potential early intervention target for ALS therapy.

genetics↗

bioRxiv2024

A bacterial NLR-related protein recognizes multiple unrelated phage triggers to sense infection

Immune systems must rapidly sense viral infections to initiate antiviral signaling, but sensing poses a unique biochemical challenge because viruses rapidly evolve to escape detection. Immune receptors must therefore detect conserved components or activities that are crucial to the viral lifecycle and cannot easily be altered. Here, we show that a bacterial NLR-related protein, bNACHT11, senses viral (phage) infection via direct interactions with multiple phage proteins that are unrelated in their sequences, structures, and functions, thereby limiting viral escape. A conserved surface on the bNACHT11 C-terminal sensor domain binds at least five distinct activators, and a cryo-electron microscopy structure reveals sensing of the protein backbone through {beta}-augmentation. Activator protein binding to bNACHT11 induced oligomerization and effector domain clustering, which limited phage infection by initiating programmed cell death through plasmolysis. These findings reveal a sophisticated immune strategy that counters the rapid evolution of viruses, with parallels to human and plant immune signaling.

microbiology↗

bioRxiv2024

TRIM37 employs peptide motif recognition and substrate-dependent oligomerization to prevent ectopic spindle pole assembly

Tightly controlled duplication of centrosomes, the major microtubule-organizing centers of animal cells, ensures bipolarity of the mitotic spindle and accurate chromosome segregation. The RBCC (RING-B-box-coiled coil) ubiquitin ligase TRIM37, whose loss is associated with elevated chromosome missegregation and the tumor-prone developmental human disorder Mulibrey nanism, prevents the formation of ectopic spindle poles that assemble around structured condensates containing the centrosomal protein centrobin. Here, we show that TRIM37s TRAF domain, unique in the extended TRIM family, engages peptide motifs in centrobin to suppress condensate formation. TRIM proteins form anti-parallel coiled-coil dimers with RING-B-box domains on each end. Oligomerization due to RING-RING interactions and conformational regulation by B-box-2-B-box-2 interfaces are critical for TRIM37 to suppress centrobin condensate formation. These results indicate that, analogous to anti-viral TRIM ligases, TRIM37 activation is linked to the detection of oligomerized substrates. Thus, TRIM37 couples peptide motif recognition and substrate-dependent oligomerization to effect ubiquitination-mediated clearance of ectopic centrosomal protein assemblies.

cell biology↗

bioRxiv2024

Symmetry of loop extrusion by dimeric SMC complexes is DNA-tension-dependent

Structural maintenance of chromosome (SMC) complexes organize and regulate genomes by extruding DNA loops. During loop extrusion, DNA can be reeled into the growing loop from one or both sides, generating distinct extrusion directionality states whose physical basis remains unclear. Here, we combine single-molecule analysis and molecular dynamics simulations to investigate loop extrusion directionality across SMC complexes. We show that dimeric Smc5/6 and Wadjet predominantly perform two-sided loop extrusion during initial loop growth, whereas monomeric condensin exhibits one-sided extrusion, consistent with a relationship between stoichiometry and extrusion directionality. Surprisingly, however, cohesin predominantly exhibits one-sided extrusion despite functioning as a dimeric complex. Notably, dimeric Smc5/6 and Wadjet progressively transition from two-sided to one-sided extrusion as loop growth matures. Simulations and force-dependent analysis reveal that loop extrusion directionality is governed by a tension-dependent transition in dimeric motors. Increasing DNA tension erodes two-sided extrusion near the motor stalling force, driving a tug-of-war regime in which competing motors transiently dominate one another. This transition promotes one-sided extrusion during mature Smc5/6 and Wadjet-mediated loop extrusion and places cohesin, which has a comparatively low stalling force ([~]0.1 pN), constitutively near this regime. Together, our findings establish loop extrusion directionality as a dynamic emergent property governed by SMC stoichiometry, DNA tension, and stochastic motor competition.

biophysics↗

bioRxiv2024

Complex compositional and cellular response of river sediment microbiomes to multiple anthropogenic stressors

Rivers face constant anthropogenic stressors, resulting in significant changes in microbial community composition. What remains unclear is whether these changes render the microbiome better adapted to the stressed environment. Here, by subjecting 64 river-connected mesocosms to multiple stressors, we show that sediment microbiomes of small lowland rivers are highly sensitive to lowered flow velocity resulting in substantially altered community compositions not fully capable of compensating for the stressor effect within two weeks albeit having stable functions encoded in metagenomes. Transcriptomics revealed a systematic heat shock response in the community and a highly active, previously unknown anaerobic key stone species with great metabolic versatility. Increases in temperature (+3.5 {degrees}C) or salinity (+0.5 mS/cm) were outcompeted by lowered flow and elicited only minor responses at community or transcriptomic level with, e.g., upregulation of the photosystem of chloroplasts. Following a two-week recovery period, transcriptomic stress responses vanished completely compared to control mesocosms, exemplifying the river microbiomes resilience. We conclude that, given the complex community responses at both the cellular and compositional level, maintaining natural river flow is vital to preventing energy loss and reduced microbiome activity in river sediments.

ecology↗

bioRxiv2024

Loop extrusion-mediated plasmid DNA cleavage by the bacterial SMC Wadjet complex

Structural maintenance of chromosomes (SMC) protein complexes play pivotal roles in genome organization and maintenance across all domains of life. In prokaryotes, SMC family Wadjet complexes structurally resemble the widespread Muk-BEF genome-organizing complexes but serve a defensive role by inhibiting plasmid transformation. We previously showed that Wadjet specifically cleaves circular DNA; however, the molecular mechanism underlying DNA substrate recognition remains unclear. Here, we use in vitro single-molecule imaging to directly visualize DNA loop extrusion and plasmid cleavage by Wadjet. We find that Wadjet is a symmetric DNA loop extruder that simultaneously reels in DNA from both sides of a growing loop and that this activity requires a dimeric JetABC supercomplex containing two dimers of the JetC motor subunit. On surface-anchored plasmid DNAs, Wadjet extrudes the full length of a 44 kilobase pair plasmid, stalls, and then cleaves DNA. Our findings reveal the role of loop extrusion in the specific recognition and elimination of plasmids by Wadjet, and establish loop extrusion as an evolutionarily conserved mechanism among SMC complexes across kingdoms of life.

molecular biology↗

bioRxiv2024

Phospho-KNL-1 recognition by a TPR domain targets the BUB-1/BUB-3 complex to C. elegans kinetochores

During mitosis, the Bub1-Bub3 complex concentrates at kinetochores, the microtubule-coupling interfaces on chromosomes, where it contributes to spindle checkpoint activation, kinetochore-spindle microtubule interactions, and protection of centromeric cohesion. Bub1 has a conserved N-terminal tetratricopeptide (TPR) domain followed by a binding motif for its conserved interactor Bub3. The current model for Bub1-Bub3 localization to kinetochores is that Bub3, along with its bound motif from Bub1, recognizes phosphorylated "MELT" motifs in the kinetochore scaffold protein Knl1. Motivated by the greater phenotypic severity of BUB-1 versus BUB-3 loss in C. elegans, we show that the BUB-1 TPR domain directly recognizes a distinct class of phosphorylated motifs in KNL-1 and that this interaction is essential for BUB-1-BUB-3 localization and function. BUB-3 recognition of phospho-MELT motifs additively contributes to drive super-stoichiometric accumulation of BUB-1-BUB-3 on its KNL-1 scaffold during mitotic entry. Bub1s TPR domain interacts with Knl1 in other species, suggesting that collaboration of TPR-dependent and Bub3-dependent interfaces in Bub1-Bub3 localization and functions may be conserved.

cell biology↗

bioRxiv2024

Architecture and infection-sensing mechanism of the bacterial PARIS defense system

Bacteria and the viruses that infect them (bacteriophages or phages) are engaged in an evolutionary arms race that has resulted in the development of hundreds of bacterial defense systems and myriad phage-encoded counterdefenses1-5. While the mechanisms of many bacterial defense systems are known1, how these systems avoid toxicity outside infection yet activate quickly upon sensing phage infection is less well understood. Here, we show that the bacterial Phage Anti-Restriction-Induced System (PARIS) operates as a toxin-antitoxin system, in which the antitoxin AriA sequesters and inactivates the toxin AriB until triggered by the T7 phage counterdefense protein Ocr. Using cryoelectron microscopy (cryoEM), we show that AriA is structurally similar to dimeric SMC-family ATPases but assembles into a distinctive homohexameric complex through two distinct oligomerization interfaces. In the absence of infection, the AriA hexamer binds up to three monomers of AriB, maintaining them in an inactive state. Ocr binding to the AriA-AriB complex triggers rearrangement of the AriA hexamer, releasing AriB and allowing it to dimerize and activate. AriB is a toprim/OLD-family nuclease whose activation arrests cell growth and inhibits phage propagation by globally inhibiting protein translation. Collectively, our findings reveal the intricate molecular mechanisms of a bacterial defense system that evolved in response to a phage counterdefense protein, and highlight how an SMC-family ATPase has been adapted as a bacterial infection sensor.

biochemistry↗

bioRxiv2023

Phosphorylation regulates tau's phase separation behavior and interactions with chromatin

Tau is a microtubule-associated protein often found in neurofibrillary tangles (NFTs) in the brains of patients with Alzheimers disease (AD). Beyond this context, mounting evidence suggests that tau localizes into the nucleus, where it may play a role in DNA protection and heterochromatin regulation. Models of tau depletion or pathology show loss of genetically silent heterochromatin, aberrant expression of heterochromatic genes, and transposable element activation. The molecular mechanisms behind these observations are currently unclear. Using in vitro biophysical experiments, here we demonstrate that tau can undergo liquid-liquid phase separation (LLPS) with DNA, mononucleosomes, and reconstituted nucleosome arrays under low salt conditions. Low concentrations of tau promote chromatin compaction and protect DNA from digestion. While the material state of samples at physiological salt is dominated by chromatin oligomerization, tau can still associate strongly and reversibly with nucleosome arrays. These properties are driven by taus strong interactions with linker and nucleosomal DNA, while magic angle spinning (MAS) solid-state NMR experiments show that tau binding does not drastically alter nucleosome structure and dynamics. In addition, tau co-localizes into droplets formed by nucleosome arrays and phosphorylated HP1, a key heterochromatin constituent thought to function through an LLPS mechanism. Importantly, LLPS and chromatin interactions are disrupted by aberrant tau hyperphosphorylation. These biophysical properties suggest that tau may directly impact DNA and chromatin accessibility and that loss of these interactions could contribute to the aberrant nuclear effects seen in tau pathology.

biophysics↗

bioRxiv2023

A phage nucleus-associated RNA-binding protein is required for jumbo phage infection

Large-genome bacteriophages (jumbo phages) of the Chimalliviridae family assemble a nucleus-like compartment bounded by a protein shell that protects the replicating phage genome from host-encoded restriction enzymes and CRISPR/Cas nucleases. While the nuclear shell provides broad protection against host nucleases, it necessitates transport of mRNA out of the nucleus-like compartment for translation by host ribosomes, and transport of specific proteins into the nucleus-like compartment to support DNA replication and mRNA transcription. Here we identify a conserved phage nuclear shell-associated protein that we term Chimallin C (ChmC), which adopts a nucleic acid-binding fold, binds RNA with high affinity in vitro, and binds phage mRNAs in infected cells. ChmC also forms phase-separated condensates with RNA in vitro. Targeted knockdown of ChmC using mRNA-targeting dCas13d halts infections at an early stage. Taken together, our data suggest that the conserved ChmC protein acts as a chaperone for phage mRNAs, potentially stabilizing these mRNAs and driving their translocation through the nuclear shell to promote translation and infection progression.

microbiology↗

bioRxiv2023

Sequential membrane- and protein-bound organelles compartmentalize genomes during phage infection

Many eukaryotic viruses require membrane-bound compartments for replication, but no such organelles are known to be formed by prokaryotic viruses1-3. Bacteriophages of the Chimalliviridae family sequester their genomes within a phage-generated organelle, the phage nucleus, which is enclosed by a lattice of the viral protein ChmA4-10. Previously, we observed lipid membrane-bound vesicles in cells infected by Chimalliviridae, but due to the paucity of genetics tools for these viruses it was unknown if these vesicles represented unproductive, abortive infections or a bona fide stage in the phage life cycle. Using the recently-developed dRfxCas13d-based knockdown system CRISPRi-ART11 in combination with fluorescence microscopy and cryo-electron tomography, we show that inhibiting phage nucleus formation arrests infections at an early stage in which the injected phage genome is enclosed within a membrane-bound early phage infection (EPI) vesicle. We demonstrate that early phage genes are transcribed by the virion-associated RNA polymerase from the genome within the compartment, making the EPI vesicle the first known example of a lipid membrane-bound organelle that separates transcription from translation in prokaryotes. Further, we show that the phage nucleus is essential for the phage life cycle, with genome replication only beginning after the injected DNA is transferred from the EPI vesicle to the newly assembled phage nucleus. Our results show that Chimalliviridae require two sophisticated subcellular compartments of distinct compositions and functions that facilitate successive stages of the viral life cycle.

cell biology↗

bioRxiv2023

Bacterial Shedu immune nucleases share a common enzymatic core regulated by diverse sensor domains

Prokaryotes encode diverse anti-bacteriophage immune systems, including the single-protein Shedu nuclease. Here we reveal the structural basis for activation of Bacillus cereus Shedu. In the inactive homotetramer, a key catalytic residue in Shedus nuclease domain is sequestered away from the catalytic site. Activation involves a conformational change that completes the active site and promotes assembly of a homo-octamer for coordinated double-strand DNA cleavage. Removal of Shedus N-terminal domain ectopically activates the enzyme, suggesting that this domain allosterically inhibits Shedu in the absence of infection. Bioinformatic analysis of nearly 8,000 Shedu homologs reveals remarkable diversity in their N-terminal regulatory domains: we identify 79 domain families falling into eight functional classes, including diverse nucleic acid binding, enzymatic, and other domains. Together, these data reveal Shedu as a broad family of immune nucleases with a common nuclease core regulated by diverse N-terminal domains that likely respond to a range of infection-related signals.

microbiology↗

bioRxiv2023

The role of greater competitive ability in countering age disadvantages in larval competition in Drosophila melanogaster

BackgroundPopulations of Drosophila melanogaster adapted to high larval densities evolve increased larval competitive ability compared to low density controls. However, traits contributing to greater competitive ability can differ across such populations, depending on the exact details of high-density conditions imposed. In the current study, we consider three sets of D. melanogaster populations adapted to three different kinds of high-density scenarios. These population sets have evolved different degrees of increases in egg size and decreases in egg hatching time as correlates of increased larval competitive ability. QuestionWe asked two related questions: O_LIDo populations adapted to larval crowding counter an imposed initial age disadvantage in larval competition, better than their controls? C_LIO_LIDo differences in egg size and egg hatching time among crowding-adapted populations matter in competitive performance when suffering age disadvantage? C_LI Study systemWe used three sets of outbred laboratory Drosophila melanogaster populations selected for larval crowding with different egg number, food volume and vial type combinations (named MCU, CCU and LCU). We also used one set of low-density ancestral control populations (named MB). MethodsWe implemented high density cultures with half the eggs from one of the study (focal) populations, and the other half from a common marked competitor population (Orange Eye: OE). We provided head start durations of 0, 3, 5, or 7 hours to the eggs of the common competitor. This imposed the respective age disadvantage to the larvae of the focal population. Pre-adult development time of survivors was the indicator of competitive performance used. ResultsAll crowding-adapted populations countered an initial age disadvantage better than the control populations. We did not see any differences among crowding adapted populations regarding their performance in countering the age disadvantage. The common competitors showed the best competitive performance against the populations with the greatest egg size and shortest hatching time. ConclusionsAdaptation to crowding leads to significantly better chances against age disadvantages in larval competition. Temporal head starts need not be of overwhelming advantage in larval competition against superior competitors in Drosophila. Among crowding adapted populations, evolving greater egg size and shorter hatching time does not seem to better offset the effects of age disadvantage to larvae. Differences in larval effectiveness and tolerance of the populations are likely to explain these results.

evolutionary biology↗

bioRxiv2023

Identification of the bacteriophage nucleus protein interaction network

In the arms race between bacteria and bacteriophages (phages), some large-genome jumbo phages have evolved a protein shell that encloses their replicating genome to protect it against DNA-targeting immune factors. By segregating the genome from the host cytoplasm, however, the "phage nucleus" introduces the need to specifically transport mRNA and proteins through the nuclear shell, and to dock capsids on the shell for genome packaging. Here, we use proximity labeling and localization mapping to systematically identify proteins associated with the major nuclear shell protein chimallin (ChmA) and other distinctive structures assembled by these phages. We identify six uncharacterized nuclear shell-associated proteins, one of which directly interacts with self-assembled ChmA. The structure and protein-protein interaction network of this protein, which we term ChmB, suggests that it forms pores in the ChmA lattice that serve as docking sites for capsid genome packaging, and may also participate in mRNA and/or protein transport.

biochemistry↗

bioRxiv2023

The small mycobacterial ribosomal protein, bS22, modulates aminoglycoside accessibility to its 16S rRNA helix-44 binding site

Treatment of tuberculosis continues to be challenging due to the widespread latent form of the disease and the emergence of antibiotic-resistant strains of the pathogen, Mycobacterium tuberculosis. Bacterial ribosomes are a common and effective target for antibiotics. Several second line anti-tuberculosis drugs, e.g. kanamycin, amikacin, and capreomycin, target ribosomal RNA to inhibit protein synthesis. However, M. tuberculosis can acquire resistance to these drugs, emphasizing the need to identify new drug targets. Previous cryo-EM structures of the M. tuberculosis and M. smegmatis ribosomes identified two novel ribosomal proteins, bS22 and bL37, in the vicinity of two crucial drug-binding sites: the mRNA-decoding center on the small (30S), and the peptidyl-transferase center on the large (50S) ribosomal subunits, respectively. The functional significance of these two small proteins is unknown. In this study, we observe that an M. smegmatis strain lacking the bs22 gene shows enhanced susceptibility to kanamycin compared to the wild-type strain. Cryo-EM structures of the ribosomes lacking bS22 in the presence and absence of kanamycin suggest a direct role of bS22 in modulating the 16S rRNA kanamycin-binding site. Our structures suggest that amino-acid residue Lys-16 of bS22 interacts directly with the phosphate backbone of helix 44 of 16S rRNA to influence the micro-configuration of the kanamycin-binding pocket. Our analysis shows that similar interactions occur between eukaryotic homologues of bS22, and their corresponding rRNAs, pointing to a common mechanism of aminoglycoside resistance in higher organisms.

biochemistry↗

bioRxiv2022

PLK-1 tethered on BUB-1 directs CDC-20 kinetochore recruitment to ensure timelyembryonic mitoses

During mitosis chromosomes assemble kinetochores in order to dynamically couple with spindle microtubules (Cheeseman, 2014; Musacchio & Desai, 2017). Kinetochores also function as signaling hubs directing mitotic progression by recruiting and controlling the fate of the Anaphase Promoting Complex/Cyclosome (APC/C) activator CDC-20 (Lara-Gonzalez et al., 2017; Lara-Gonzalez, Pines, et al., 2021; Musacchio, 2015). Kinetochores either incorporate CDC-20 into checkpoint complexes that inhibit the APC/C or dephosphorylate CDC-20, which allows it to interact with and activate the APC/C (Kim et al., 2017; Lara-Gonzalez et al., 2017). The importance of these two CDC-20 fates likely depends on biological context. In somatic cells the major mechanism controlling mitotic progression is the spindle checkpoint. By contrast, progression through mitosis during the cell cycles of early embryos is largely checkpoint-independent (Clute & Masui, 1995; Duro & Nilsson, 2021; Gerhart et al., 1984; Zhang et al., 2015). Here, by manipulating CDC-20 phosphorylation status, we show that CDC-20 phosphoregulation controls mitotic duration in the C. elegans embryo and defines a checkpoint-independent temporal mitotic optimum for robust embryogenesis. Flux of CDC-20 through kinetochores for local dephosphorylation requires an ABBA motif on BUB-1 that directly interfaces with the structured WD40 domain of CDC-20 (Di Fiore et al., 2015; Diaz-Martinez et al., 2015; He et al., 2013; Kim et al., 2017). We show that a conserved "STP" motif in BUB-1 that docks the mitotic kinase PLK-1 (Qi et al., 2006) is also necessary to recruit CDC-20 to kinetochores and for timely mitotic progression. The kinase activity of PLK-1 is required for CDC-20 to localize to kinetochores and targets a site within the CDC-20-binding ABBA motif of BUB-1; phosphorylation of this site promotes BUB-1-CDC-20 interaction and mitotic progression. Thus, the BUB-1-bound pool of PLK-1 ensures timely mitosis during embryonic cell cycles by promoting CDC-20 recruitment to the vicinity of kinetochore-localized phosphatase activity.

cell biology↗

bioRxiv2022

Architecture and self-assembly of the jumbo bacteriophage nuclear shell

Bacteria encode myriad defenses that target the genomes of infecting bacteriophage, including restriction-modification and CRISPR/Cas systems. In response, one family of large bacteriophage employs a nucleus-like compartment to protect their replicating genomes by excluding host defense factors. However, the principle composition and structure of this compartment remain unknown. Here, we find that the bacteriophage nuclear shell assembles primarily from one protein, termed chimallin. Combining cryo-electron tomography of nuclear shells in bacteriophage-infected cells and cryo-electron microscopy of a minimal chimallin compartment in vitro, we show that chimallin cooperatively self-assembles as a flexible sheet into closed micron-scale compartments. The architecture and assembly dynamics of the chimallin shell suggest mechanisms for its nucleation and growth, and its role as a scaffold for phage-encoded factors mediating macromolecular transport, cytoskeletal interactions, and viral maturation.

biophysics↗

bioRxiv2021

Density-dependent selection in Drosophila: evolution of egg size and hatching time

Many different laboratory studies of adaptation to larval crowding in Drosophila spp. have all yielded the evolution of pre-adult competitive ability, even though the ecological context in which crowding was experienced varied across studies. However, the evolution of competitive ability was achieved through different suites of traits in studies wherein crowding was imposed in slightly different ways. Earlier studies showed the evolution of increased competitive ability via increased larval feeding rate and tolerance to nitrogenous waste, at the cost of food to biomass conversion efficiency. However, more recent studies, with crowding imposed at relatively low food levels, showed the evolution of competitive ability via decreased larval development time and body size, and an increase in the time efficiency of conversion of food to biomass, with no change in larval feeding rate or waste tolerance. Taken together, these studies have led to a more nuanced understanding of how the specific details of larval numbers, food amounts etc. can affect which traits evolve to confer increased competitive ability. Here, we report results from a study in which egg size and hatching time were assayed on three sets of populations adapted to larval crowding experienced in slightly different ways, as well as their low density ancestral control populations. Egg size and hatching time are traits that may provide larvae with initial advantages under crowding through increased starting larval size and a temporal head-start, respectively. In each set of populations adapted to some form of larval crowding, the evolution of longer and wider eggs was seen, compared to controls, thus making egg size the first consistent correlate of the evolution of increased larval competitive ability across Drosophila populations experiencing crowding in slightly different ways. Among the crowding-adapted populations, those crowded at the lowest overall eggs/food density, but the highest density of larvae in the feeding band, showed the largest eggs, on an average. All three sets of crowding-adapted populations showed shorter average egg hatching time than controls, but the difference was significant only in the case of populations experiencing the highest feeding band density. Our results underscore the importance of considering factors other than just eggs/food density when studying the evolution of competitive ability, as also the advantages of having multiple selection regimes within one experimental set up, allowing for a more nuanced understanding of the subtlety with which adaptive evolutionary trajectories can vary across even fairly similar selection regimes.

evolutionary biology↗