Biology subjects

Holford, M.

Publications and source records attributed to Holford, M..

9 recordsLinked to original sources

bioRxiv2026

A Primary Cell Culture Platform for Studying Venom Gland and Brain Tissue in Octopus

Coleoid cephalopods, squids, cuttlefish, and octopuses, have emerged as powerful model organisms for studying neurobiology, development, and behavior, however, cellular tools for investigating their specialized tissues remain limited. In particular, their venom producing gland, the posterior salivary gland (PSG), has been extensively described anatomically and histologically, yet remains largely inaccessible to experimental investigation at the cellular level. Here, we report the first establishment of primary cell cultures derived from both the optical lobe and PSG tissues of Octopus bimaculoides. Building on recent advances in cephalopod brain cultures, we adapted and optimized dissociation and culture conditions to support short-term survival and attachment of cells in vitro. We show that passive cell release during tissue handling, rather than enzymatic treatment, yields viable cultures from both tissues, and poly-D-lysine markedly improves the adherence of PSG-derived cells. Morphological analyses and fluorescent staining confirm the presence and viability of distinct cell populations, while cell cycle analysis indicates that the majority of cells reside in G0/G1 phase. Notably, O. bimaculoides brain cultures exhibit features comparable to those previously described in squid, suggesting conserved aspects of coleoid cellular physiology. Together, our findings establish a foundational in vitro platform for studying octopus PSG and neural cell biology, providing a tractable system for probing venom biosynthesis, secretion, and neural regulation in coleoid cephalopods.

cell biology↗

bioRxiv2026

Lineage-Specific Venom Gene Expression Shapes Chemical Diversity in Cephalopods

Animal venoms represent a major source of chemical novelty, yet how venom compounds originate, diversify, and are maintained across deep evolutionary timescales remains poorly understood. This gap is especially pronounced in cephalopods, which evolved venom systems used in predation, defense, and sexual competition, but whose venom genetic architectures, secretory cell types, and venom-producing glands remain largely unexplored. To date, only a single cephalopod venom compound with confirmed paralytic activity and a known primary sequence, SE-CTX from the golden cuttlefish Acanthosepion esculentum, has been described. Here, we reconstruct the evolutionary history, molecular diversity, and glandular localization of SE-CTX-like proteins using a multimodal approach. We identify 29 homologs across 20 squid and cuttlefish species and define a previously unrecognized venom gene family, which we name deca-ctx, specific to decapodiform cephalopods (squids and cuttlefish). Phylogenetic analyses reveal a single origin of deca-ctx followed by gene duplication and lineage-specific diversification, indicating long-term retention of this venom gene. Predicted DECA-CTX protein structures were separated into two clusters and 20 singletons highlighting potentially extensive structural diversity within a single cephalopod venom gene family. Proteomic analysis confirms expression of five DECA-CTX proteins across three species. Our imaging and histological analyses localize deca-ctx expression to specialized secretory cells within squid and cuttlefish venom glands. Together, these findings reposition SE-CTX as part of an evolutionarily and chemically diverse venom system, rather than an isolated venom protein, and establish cephalopods as a key lineage for investigating how new venom genes arise, diversify, and are integrated into functional venom arsenals.

evolutionary biology↗

bioRxiv2025

Neurons, Muscles, and Venom: Identifying Drivers of Cephalopod Predation

Venom plays a central role in the predatory ecology of coleoid cephalopods (octopuses, squids, and cuttlefish), yet the mechanisms governing venom release from the posterior salivary gland (PSG) are unknown. Using a multimodal approach combining X-ray micro-histotomography, histological stainings, in situ hybridization, comparative phylogenetics, and ex vivo imaging across multiple coleoid species, we characterize the structural and neuronal regulatory organization of the PSG. We verified that the gland comprises two distinct tubular systems: secretory tubules specialized for venom production and smooth-striated tubules positioned to facilitate venom transport toward the beak for injection into its prey. Molecular localization of filamentous and -actin confirms a circular smooth muscle layer surrounding the tubules. Mapping of six neuronal markers, including neurofilament (NF-H), synapsin, and muscle-type nicotinic acetylcholine receptors, reveals dense and stereotyped neural innervation closely associated with the muscular compartments. Comparative phylogenetic analyses of cys-loop ligand-gated ion channel sequences indicate a predominancy of excitatory acetylcholine- and dopamine-gated receptors in coleoid venom glands, implicating potential molecular agents involved in neural control of venom release. Consistent with neural regulation, ex vivo stimulation of the PSG elicits calcium signaling throughout the gland. Together, our results reveal a conserved venom gland structure among octopuses, squids, and cuttlefish, with a spatially distinct neuromuscular tissue organization indicating venom production and release sites modulated by a network of neuronal agents. This work provides a mechanistic framework into venom gland organization and molecular regulation of venom release in one of the oldest venomous lineages.

neuroscience↗

bioRxiv2024

Towards the identification of the molecular toolkit involved in scale worm bioluminescence (Polinoidae, Annelida)

BackgroundBioluminescence, or the ability of a living organism to produce light, has evolved independently in numerous taxa inhabiting a panoply of ecosystems, although it is more frequent among marine animals. Scale worms are a group of marine polynoid annelids characterized by having dorsal scales, known as elytra, capable of emitting bioluminescent light by a mostly unknown molecular mechanism that may involve a photoprotein called polynoidin. Here, we used RNA-seq data to characterize the expression of genes potentially involved in light production in the polynoid species Harmothoe imbricata (Linnaeus, 1767) and Harmothoe areolata (Grube, 1860) across tissues of the specimens. We also compared the transcriptomes of the selected species with other bioluminescent and non-bioluminescent polynoids, to identify shared orthologous genes potentially involved in light production. In addition, we investigated the disposition of the photocytes on the elytra using confocal microscopy and histological analyses. ResultsOur results showed a total of 16 candidate genes, 15 orthologous genes and 12 enriched GO terms potentially involved in bioluminescence, including genes related with oxidative stress, cytoskeleton, nervous system, stress response, wounding response, eye constituents and metabolic pathways. We also confirmed the presence of photocytes in both species, which appeared distributed around the elytrophore. ConclusionsAmong the genes found potentially implicated in bioluminescence we suggest that the oxidoreductase protein, peroxidasin, could be a polynoidin candidate since it appears overexpressed in the elytra of both species and it is located in the endoplasmic reticulum, where this photoprotein has been described to be found.

evolutionary biology↗

bioRxiv2022

Comparative Mucomic Analysis of Three Functionally Distinct Cornu aspersum Secretions

Every animal secretes mucus, placing them among the most diverse biological materials. Mucus hydrogels are complex mixtures of water, ions, carbohydrates, and proteins. Uncertainty surrounding their composition and how interactions between components contribute to mucus function complicates efforts to exploit their properties. There is substantial interest in commercializing mucus from the garden snail, Cornu aspersum, for skincare, drug delivery, tissue engineering, and composite materials. C. asperum secretes three mucus -- one shielding the animal from environmental threats, one adhesive mucus from the pedal surface of the foot, and another pedal mucus that is lubricating. It remains a mystery how compositional differences account for their substantially different properties. Here, we characterize mucus proteins, glycosylation, ion content, and mechanical properties to understand structure-function relationships through an integrative "mucomics" approach. We identify new macromolecular components of these hydrogels, including a novel protein class termed Conserved Anterior Mollusk Proteins (CAMPs). Revealing differences between C. aspersum mucus shows how considering structure at all levels can inform the design of mucus-inspired materials.

biochemistry↗

bioRxiv2020

Induced Disassembly of a Virus-Like Particle under Physiological Conditions for Venom Peptide Delivery

Virus-like nanoparticles (VLPs) show considerable promise for the in vivo delivery of therapeutic compounds such as bioactive venom peptides. While loading and targeting protocols have been developed for numerous VLP prototypes, induced disassembly under physiological conditions of neutral pH, moderate temperature, and aqueous medium, remain a challenge. Here, we implement and evaluate a ring-opening metathesis polymerization (ROMP) general mechanism for controllable VLP disassembly that is independent of cell-specific factors or the manipulation environmental conditions such as pH and temperature that cannot be readily controlled in vivo. The ROMP substrate norbornene is covalently conjugated to surface-exposed lysine residues of a P22 bacteriophage-derived VLP, and ROMP is induced by treatment of water-soluble ruthenium catalyst AquaMet. Disruption of the P22 shell and release of a GFP reporter is confirmed via native agarose electrophoresis and quantitative microscopy and light scattering analyses. Our ROMP disassembly strategy does not depend on the particular structure or morphology of the P22 nanocontainer and is adaptable to other VLP prototypes for the potential delivery of venom peptides for pharmacological applications. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=90 SRC="FIGDIR/small/278598v1_ufig1.gif" ALT="Figure 1"> View larger version (19K): org.highwire.dtl.DTLVardef@1d24020org.highwire.dtl.DTLVardef@702e3aorg.highwire.dtl.DTLVardef@eb0d25org.highwire.dtl.DTLVardef@1ac00d0_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2019

Antitumor effects of Tv1 venom peptide in liver cancer

A strategy for treating the most common type of liver cancer, hepatocellular carcinoma (HCC) applies a targeted therapy using venom peptides that are selective for ion channels and transporters overexpressed in tumor cells. Here, we report selective anti-HCC properties of Tv1, a venom peptide from the predatory marine terebrid snail, Terebra variegata. Tv1 was applied in vitro to liver cancer cells and administered in vivo to allograft tumor mouse models. Tv1 inhibited the proliferation of murine HCC cells via calcium dependent apoptosis resulting from down-regulation of the cyclooxygenase-2 (COX-2) pathway. Additionally, tumor sizes were significantly reduced in Tv1-treated syngeneic tumor-bearing mice. Tv1s mechanism of action involves binding to specific transient receptor potential (TRP) cation channels that are overexpressed in HCC cell models. Our findings demonstrate the unique potential of venom peptides to function as tumor specific ligands in the quest for targeted cancer therapies.

cancer biology↗

bioRxiv2018

Using Drosophila behavioral assays to characterize terebrid venom-peptide bioactivity

The number of newly discovered peptides from the transcriptomes and proteomes of animal venom arsenals is rapidly increasing, resulting in an abundance of uncharacterized peptides. There is a pressing need for a systematic, cost effective, and scalable approach to identify physiological effects of venom peptides. To address this discovery-to-function gap, we developed a sequence driven:activity-based hybrid approach for screening venom peptides that is amenable to large-venom peptide libraries with minimal amounts of peptide. Using this approach, we characterized the physiological and behavioral phenotypes of two peptides from the venom of predatory terebrid marine snails, teretoxins Tv1 from Terebra variegata and Tsu1.1 from Terebra subulata. Our results indicate that Tv1 and Tsu1.1 have distinct bioactivity. Tv1 (100 M) had an antinociceptive effect in adult Drosophila using a thermal nociception assay to measure heat avoidance. Alternatively, Tsu1.1 (100 M) increased food intake. These findings describe the first functional bioactivity of terebrid venom peptides in relation to pain and diet and indicate that Tv1 and Tsu1.1 may, respectively, act as antinociceptive and orexigenic agents. Tv1 and Tsu1.1 are distinct from previously identified venom peptides, expanding the toolkit of peptides that can potentially be used to investigate the physiological mechanisms of pain and diet.

animal behavior and cognition↗

bioRxiv2018

Molecular phylogeny of Odontosyllis (Annelida, Syllidae): A recent and rapid radiation of marine bioluminescent worms.

Marine worms of the genus Odontosyllis (Syllidae, Annelida) are well known for their spectacular bioluminescent courtship rituals. During the reproductive period, the benthic marine worms leave the ocean floor and swim to the surface to spawn, using bioluminescent light for mate attraction. The behavioral aspects of the courtship ritual have been extensively investigated, but little is known about the origin and evolution of light production in Odontosyllis, which might in fact be a key factor shaping the natural history of the group, as bioluminescent courtship might promote speciation. To investigate the speciation patterns and evolutionary history of Odontosyllis and to trace the origin of bioluminescence within the group, we inferred phylogenies using both gene concatenation and multispecies coalescent species-tree approaches with a multilocus molecular dataset (18S rRNA, 16S rRNA and COI). We also used this dataset to estimate divergence times and diversification rates in a relaxed molecular clock Bayesian framework. Our results suggest that Odontosyllis has undergone a recent rapid radiation, possibly triggered by the origin of bioluminescent courtship, which might have increased speciation rates and lineage divergence through sexual selection. Additionally, our analyses reveal that the genus Odontosyllis as currently delineated is a paraphyletic group that needs to be reorganized to reflect evolutionary relationships.

evolutionary biology↗