Biology subjects

Crawford, S.

Publications and source records attributed to Crawford, S..

13 recordsLinked to original sources

bioRxiv2025

Size-stabilized, Hypoallometric, Genitalia Determined for Male Black Soldier Fly, Hermetia illucens (Diptera: Stratiomyidae)

The black soldier fly, Hermetia illucens, is mass-reared throughout the world to convert organic waste into ingredients for pet and livestock feed, as well as frass and other biological products. To promote the optimization of breeding regimes carried out by industrial black soldier fly operations, it is critical to better understand adult reproductive anatomy and its potential relationship with fertile egg production. However, in this species, little is known about how reproductive anatomy changes with respect to increases in body size. Hence, this study investigated the nutritional static allometric relationship between the external genitalia of adult male black soldier flies and their body size. Size differences were induced top-down by increasing the larval rearing density, which has a negative effect on adult body size. For each of 3 rearing densities, a random sample of 30 adults was selected, and measurements taken three times each for the head, thorax, parameral sheath, and gonostylus. Fitting a generalized linear log-log model to the data revealed that for every 10.0% increase in body size (thorax length), genital length (parameral shealth) only increased by 1.8%. The resulting allometric slope of genitalia to thorax size was 0.195, indicating a pattern of hypoallometry. The presence of hypoallometric genitalia in a domestic population suggests individuals should be able to copulate regardless of differences in body size, which is consistent with most other insects. Moreover, this finding implies that black soldier fly genitalia were hypoallometric prior to their domestication and continues to persist within captive black soldier fly populations. To confirm, future work should investigate the direct impacts of hypoallometric genitalia on fitness, especially in flies which have been genetically edited or artificially selected to be increasingly large. LAY SUMMARYAdult male black soldier fly shown to have similar sized genitals despite differences in body size. SHORT SUMMARYThe black soldier fly, Hermetia illucens, is an economically important insect mass-reared throughout the globe; however, a large knowledge gap exists in terms of its reproductive anatomy and physiology. This study examined the relationship of male genitalia to body size, finding a 10% increase in body size corresponded with a 1.8% increase in genitalia size, meaning the structures are hypoallometric. This finding is important because it indicates large and small flies have similar sized genitalia, which may allow differently-sized individuals to copulate.

zoology↗

bioRxiv2025

Scalable human neuronal models of tauopathy producing endogenous seed-competent 4R tau

The accumulation of pathological four-repeat (4R) tau is central to several frontotemporal dementia (FTD) subtypes, but human neuronal models amenable to high-throughput screening of 4R tau-targeting therapies remain very limited. To address this, we developed iPSC-derived i3Neuron (i3N) lines expressing >75% 4R tau, driven by FTD splice-shifting mutations (S305N or S305N/IVS10+3). These neurons develop hyperphosphorylated tau and demonstrate somatodendritic mis-localization. Unlike other stem cell models of tauopathy, these i3N neurons develop endogenous seed-competent tau and present pFTAA-positive tau assemblies after 28 days in culture. For scalable screening, we CRISPR-engineered a HiBiT luminescence tag at the endogenous MAPT locus into the S305N/IVS10+3 iPSC line, enabling precise quantification of tau levels and pharmacological responses. The model responded predictably to compounds affecting tau clearance, demonstrating its suitability for drug discovery. Overall, this i3N platform recapitulates key features of 4R tauopathy and provides a robust system to identify therapeutic modulators of pathological tau.

neuroscience↗

bioRxiv2025

Bacterial species and surface structures shape gene transfer and the transcriptional landscape during early conjugation

Conjugative plasmids drive bacterial evolution and niche adaptation, yet how their active acquisition reshapes host transcription remains poorly understood. Most studies focus on stable plasmid carriage, overlooking the dynamic transcriptional changes during conjugation itself. Here, we show that active RP4 conjugation triggers an immediate, host- and surface factor-dependent transcriptional response. This includes activation of non-SOS stress pathways, motility, exopolysaccharide production, anaerobic respiration, and metabolic adaptation. These responses do not inhibit conjugation, suggesting they serve to maintain host homeostasis. Capsule expression blocks these responses by preventing conjugation, and also inhibits RP4 activation by physically blocking donor-recipient contact. Unexpectedly, RP4 overcomes this barrier by exploiting single-cell variation in recipient capsule thickness, successfully conjugating with thin-capsulated recipients. These findings reveal a striking interplay between plasmid, host, and surface architecture in shaping the conjugation transcriptional landscape, with broad implications for plasmid dissemination and bacterial evolution.

microbiology↗

bioRxiv2024

Immune cell single-cell RNA sequencing analyses link an age-associated T cell subset to symptomatic benign prostatic hyperplasia

Benign prostatic hyperplasia (BPH) is among the most common age-associated diseases in men; however, the contribution of age-related changes in immune cells to BPH is not clear. The current study determined that an age-associated CD8+ T cell subset (Taa) with high Granzyme K (GZMKhi) and low Granzyme B (GZMBlow) gene expression infiltrate aged human prostates and positively correlate with International Prostate Symptom Score (IPSS). A velocity analysis indicated that CD8+ T cell differentiation is altered in large BPH prostates compared to small age-matched prostates, favoring Taa accumulation. In vitro granzyme K treatment of human BPH patient-derived large prostate fibroblasts increased secretion of pro-inflammatory senescence-associated secretory phenotype (SASP)-associated cytokines. These data suggest that granzyme K-mediated stimulation of prostate stromal fibroblast SASP cytokine and chemokine production promotes prostate immune cell recruitment and activation. Overall, these results connect symptomatic BPH with immune aging.

immunology↗

bioRxiv2024

Heterogeny in cages: Age-structure and timing of attractant availability impacts fertile egg production in the black soldier fly, Hermetia illucens

Adult behavior is a growing area of interest for those researching the black soldier fly, Hermetia illucens (L.) (Diptera: Stratiomyidae), which is affected by underlying demography and spatiotemporal patterns. This greenhouse experiment examined the interaction of age-related effects that can accrue within heterogeneous breeding populations and the potential benefits of delaying an oviposition attractant in concert with restricting mean cohort age. The impetus for this investigation was because if flies are introduced into a mating-cage before old flies are removed or culled, this creates a population of mixed-age and mating-status. We hypothesized this potentially reduces quality among available mate choices, especially in small cages where flies might not be able to spatially segregate. Metrics for fitness included copulation frequency, oviposition frequency, weight of eggs produced, and hatch percentage. "Same"-aged cohorts (maximum 4-d-old at introduction) performed better than highly heterogeneous (1-16-d-old) "mixed" cohorts by mating 2.32-times more frequently and laying 6.58-times more eggs that were 1.17-times more fertile, despite 1.41 fewer observed oviposition events. Delaying the attractant had a significant effect on egg collection weight and led to 1.25-times higher egg yields for same- age populations compared to mixed-aged cohorts where the delaying the attractant had no significant effect. These results are likely due in part to an immediate desire to lay eggs by older females as well as haphazard egg laying in cage material, which was 2.09-times higher for mixed cohorts. The results highlight the importance of constraining the age of breeding populations and removing old adults from cages to improve yields and better manipulate behavior. For those for whom this is logistically unfeasible, providing an attractant box initially and continuously may be the preferred method to trap more eggs from heterogeneous populations.

ecology↗

bioRxiv2024

Domesticated cannabinoid synthases amid a wild mosaic cannabis pangenome

Cannabis sativa is a globally significant seed-oil, fiber, and drug-producing plant species. However, a century of prohibition has severely restricted legal breeding and germplasm resource development, leaving potential hemp-based nutritional and fiber applications unrealized. Existing cultivars are highly heterozygous and lack competitiveness in the overall fiber and grain markets, relegating hemp to less than 200,000 hectares globally1. The relaxation of drug laws in recent decades has generated widespread interest in expanding and reincorporating cannabis into agricultural systems, but progress has been impeded by the limited understanding of genomics and breeding potential. No studies to date have examined the genomic diversity and evolution of cannabis populations using haplotype-resolved, chromosome-scale assemblies from publicly available germplasm. Here we present a cannabis pangenome, constructed with 181 new and 12 previously released genomes from a total of 156 biological samples from both male (XY) and female (XX) plants, including 42 trio phased and 36 haplotype-resolved, chromosome-scale assemblies. We discovered widespread regions of the cannabis pangenome that are surprisingly diverse for a single species, with high levels of genetic and structural variation, and propose a novel population structure and hybridization history. Conversely, the cannabinoid synthase genes contain very low levels of diversity, despite being embedded within a variable region containing multiple pseudogenized paralogs and distinct transposable element arrangements. Additionally, we identified variants of acyl-lipid thioesterase (ALT) genes2 that are associated with fatty acid chain length variation and the production of the rare cannabinoids, tetrahydrocannabinol varin (THCV) and cannabidiol varin (CBDV). We conclude the Cannabis sativa gene pool has only been partially characterized, and that the existence of wild relatives in Asia remains likely, while its potential as a crop species remains largely unrealized.

plant biology↗

bioRxiv2023

RNA-dependent RNA polymerase of predominant human norovirus forms liquid-liquid phase condensates as platforms for viral replication

Many viral proteins form biomolecular condensates via liquid-liquid phase separation (LLPS) to support viral replication and evade host antiviral responses, and thus, they are potential targets for designing antivirals. In the case of non-enveloped positive-sense RNA viruses, forming such condensates for viral replication is unclear and less understood. Human noroviruses (HuNoV) are positive-sense RNA viruses that cause epidemic and sporadic gastroenteritis worldwide. Here, we show that the RNA-dependent-RNA polymerase (RdRp) of pandemic GII.4 HuNoV forms distinct condensates that exhibit all the signature properties of LLPS with sustained polymerase activity and the capability of recruiting components essential for viral replication. We show that such condensates are formed in HuNoV-infected human intestinal enteroid cultures and are the sites for genome replication. Our studies demonstrate the formation of phase separated condensates as replication factories in a positive-sense RNA virus, which plausibly is an effective mechanism to dynamically isolate RdRp replicating the genomic RNA from interfering with the ribosomal translation of the same RNA. TeaserPolymerase of a positive-sense RNA virus forms LLPS to regulate replication as an elegant solution for an enigmatic question.

biochemistry↗

bioRxiv2023

Microglia produce the amyloidogenic ABri peptide in familial British dementia

Mutations in ITM2B cause familial British, Danish, Chinese and Korean dementias. In familial British dementia (FBD) a mutation in the stop codon of the ITM2B gene (also known as BRI2) causes a C-terminal cleavage fragment of the ITM2B/BRI2 protein to be extended by 11 amino acids. This fragment, termed amyloid-Bri (ABri), is highly insoluble and forms extracellular plaques in the brain. ABri plaques are accompanied by tau pathology, neuronal cell death and progressive dementia, with striking parallels to the aetiology and pathogenesis of Alzheimers disease. The molecular mechanisms underpinning FBD are ill-defined. Using patient-derived induced pluripotent stem cells, we show that expression of ITM2B/BRI2 is 34-fold higher in microglia than neurons, and 15-fold higher in microglia compared with astrocytes. This cell-specific enrichment is supported by expression data from both mouse and human brain tissue. ITM2B/BRI2 protein levels are higher in iPSC-microglia compared with neurons and astrocytes. Consequently, the ABri peptide was detected in patient iPSC-derived microglial lysates and conditioned media but was undetectable in patient-derived neurons and control microglia. Pathological examination of post-mortem tissue support ABri expression in microglia that are in proximity to pre-amyloid deposits. Finally, gene co-expression analysis supports a role for ITM2B/BRI2 in disease-associated microglial responses. These data demonstrate that microglia are the major contributors to the production of amyloid forming peptides in FBD, potentially acting as instigators of neurodegeneration. Additionally, these data also suggest ITM2B/BRI2 may be part of a microglial response to disease, motivating further investigations of its role in microglial activation. This has implications for our understanding of the role of microglia and the innate immune response in the pathogenesis of FBD and other neurodegenerative dementias including Alzheimers disease.

neuroscience↗

bioRxiv2023

Genetic Mapping of SNP Markers and Candidate Genes Associated with Day-Neutral Flowering in Cannabis sativa L.

Hemp (Cannabis sativa L.) is a primarily short-day crop grown for grain, fiber, and secondary metabolites. Although terminal flowering of most non-domesticated hemp is regarded as photoperiod sensitive, limited germplasm is available for the development of day-neutral hemp. Day-neutral plants experience flower maturation independently of the short-day photoperiod cue which is typically triggered by photoperiods of less than 14 hours. The day-neutral trait is the subject of increased commercial interest for the purpose of breeding varieties with accelerated flowering time or cultivars that do not require labor- and cost-intensive light deprivation production systems. Genetic markers for the photoperiodic response would help breeders make early selection in the design of suitable cultivars for specific environments and cultivation calendars. Limited refereed information has been produced regarding the genetic regulation of photoperiodism in C. sativa. A population of 318 F2 individuals segregating for day-neutral flowering was developed, phenotyped, and genotyped. Genome-wide association analysis identified markers associated with the day-neutral trait indicating that a single recessive gene controlling photoperiod sensitivity is located within a large region of Chromosome 1. Flanking region sequence data of linked SNP markers was used in the development and validation of a TaqMan-based qPCR assay for the day-neutral trait. A genetic linkage map was produced, and QTL mapping identified two additional markers on Chromosome 1. Candidate genes, TARGET OF EARLY ACTIVATION TAGGED (TOE)/APETALA2 (AP2), and PSEUDO-RESPONSE REGULATOR 3 (PRR3), may work together to impact phase transition and photoperiodic flowering respectively and have key domains that are disrupted in day-neutral plants. The day-neutral flowering (a.k.a. "autoflower") phenotype is a valuable and understudied trait in Cannabis sativa The phenotype may result from a conserved linkage block of phase transition and photoperiodic flowering genes A segregating population was developed, genome wide analyses identified markers linked to the day-neutral trait Correlated markers were mapped to Chromosome 1 across a sizable region that appears to be largely non-recombining

plant biology↗

bioRxiv2019

Injection molded open microfluidic well plate inserts for user-friendly coculture and microscopy

Open microfluidic cell culture systems are powerful tools for interrogating biological mechanisms. We have previously presented a microscale cell culture system, based on spontaneous capillary flow of biocompatible hydrogels, that is integrated into a standard cell culture well plate, with flexible cell compartment geometries and easy pipet access. Here, we present two new injection molded open microfluidic devices that also easily insert into standard cell culture well plates and standard culture workflows, allowing seamless adoption by biomedical researchers. These platforms allow culture and study of soluble factor communication among multiple cell types, and the microscale dimensions are well-suited for rare primary cells. Unique advances include optimized evaporation control within the well, manufacture with reproducible and cost-effective rapid injection molding, and compatibility with sample preparation workflows for high resolution microscopy (following well-established coverslip mounting procedures). In this work, we present several use cases that highlight the usability and widespread utility of our platform including culture of limited primary testis cells from surgical patients, microscopy readouts including immunocytochemistry and single molecule fluorescence in situ hybridization (smFISH), and coculture to study interactions between adipocytes and prostate cancer cells.

bioengineering↗

bioRxiv2019

High-throughput propagation of human prostate tissue from induced-pluripotent stem cells

Primary culture of human prostate organoids is slow, inefficient and laborious. To overcome this, we demonstrate a new high-throughput model where rapidly proliferating and easily handled induced pluripotent stem cells, for the first time, enable generation of human prostate tissue in vivo and in vitro. Using a co-culture technique with urogenital sinus mesenchyme, we recapitulated the in situ prostate histology, including the stromal compartment and the full spectrum of epithelial differentiation. This approach overcomes major limitations in primary cultures of human prostate stem, luminal and neuroendocrine cells, as well as the stromal microenvironment. These models provide new opportunities to study prostate development, homeostasis and disease.

developmental biology↗

bioRxiv2017

The Anorexia Nervosa Genetics Initiative: Overview and Methods

BackgroundGenetic factors contribute to anorexia nervosa (AN); and the first genome-wide significant locus has been identified. We describe methods and procedures for the Anorexia Nervosa Genetics Initiative (ANGI), an international collaboration designed to rapidly recruit 13000 individuals with AN as well as ancestrally matched controls. We present sample characteristics and the utility of an online eating disorder diagnostic questionnaire suitable for large-scale genetic and population research.\n\nMethodsANGI recruited from the United States (US), Australia/New Zealand (ANZ), Sweden (SE), and Denmark (DK). Recruitment was via national registers (SE, DK); treatment centers (US, ANZ, SE, DK); and social and traditional media (US, ANZ, SE). All cases had a lifetime AN diagnosis based on DSM-IV or ICD-10 criteria (excluding amenorrhea). Recruited controls had no lifetime history of disordered eating behaviors. To assess the positive and negative predictive validity of the online eating disorder questionnaire (ED100K-v1), 109 women also completed the Structured Clinical Interview for DSM-IV (SCID), Module H.\n\nResultsBlood samples and clinical information were collected from 13,364 individuals with lifetime AN and from controls. Online diagnostic phenotyping was effective and efficient; the validity of the questionnaire was acceptable.\n\nConclusionsOur multipronged recruitment approach was highly effective for rapid recruitment and can be used as a model for efforts by other groups. High online presence of individuals with AN rendered the Internet/social media a remarkably effective recruitment tool in some countries. ANGI has substantially augmented Psychiatric Genomics Consortium AN sample collection. ANGI is a registered clinical trial: clinicaltrials.gov NCT01916538; https://clinicaltrials.gov/ct2/show/NCT01916538?cond=Anorexia+Nervosa&draw=1&rank=3.

genetics↗

bioRxiv2016

Genome-Wide Association Study Reveals First Locus for Anorexia Nervosa and Metabolic Correlations

Anorexia nervosa (AN) is a serious eating disorder characterized by restriction of energy intake relative to requirements, resulting in abnormally low body weight. It has a lifetime prevalence of approximately 1%, disproportionately affects females1,2, and has no well replicated evidence of effective pharmacological or psychological treatments despite high morbidity and mortality2. Twin studies support a genetic basis for the observed aggregation of AN in families3, with heritability estimates of 48%-74%4. Although initial genome-wide association studies (GWASs) were underpowered5,6, evidence suggested that signals for AN would be detected with increased power5. We present a GWAS of 3,495 AN cases and 10,982 controls with one genome-wide significant locus (index variant rs4622308, p=4.3x10-9) in a region (chr12:56,372,585-56,482,185) which includes six genes. The SNP-chip heritability [Formula] of AN from these data is 0.20 (SE=0.02), suggesting that a substantial fraction of the twin-based heritability stems from common genetic variation. Using these GWAS results, we also find significant positive genetic correlations with schizophrenia, neuroticism, educational attainment, and HDL cholesterol, and significant negative genetic correlations with body mass, insulin, glucose, and lipid phenotypes. Our results support the reconceptualization of AN as a disorder with both psychiatric and metabolic components.

genomics↗