Steep Hierarchies without Skew? Modeling How Ecology and Decision-Making Shape Despotism of Relationships.

AbstractAnimals can form dominance relationships that vary from highly unequal, or despotic, to egalitarian, and this variation likely impacts the fitness of individuals. How and why these differences in relationships and fitness exist among groups, populations, and species has been the subject of much debate. Here, we investigated the influence of two major factors: (1) spatial resource distribution and (2) the presence or absence of winner-loser effects. To determine the effects of these factors, we built an agent-based model that represented 10 agents directly competing over food resources on a simple landscape. By varying the food distribution and using either asymmetry of strength or experience, we contrasted four scenarios from which we recorded attack decisions, fight outcomes, and individual energy intake to calculate dominance hierarchy steepness and energetic skew. Surprisingly, resource distribution and winner-loser effects did not have the predicted effects on hierarchy steepness. However, skew in energy intake arose when resources were distributed heterogeneously, despite hierarchy steepness frequently being higher in the homogeneous resource scenarios. Thus, this study confirms some decades-old predictions about feeding competition but also casts doubt on the ability to infer energetic consequences from observations of agonistic interactions.

Shell disease syndromes of decapod crustaceans.

The term shell disease subsumes a number of debilitating conditions affecting the outer integument (the carapace) of decapod crustaceans, such as lobsters and crabs. Herein, we seek to find commonality in the aetiology and pathology of such conditions, and those cases that result in the progressive erosion of the cuticle through to the visceral tissues by a cocktail of microbial-derived enzymes including lipases, proteases and chitinases. Aquimarina spp. are involved in shell disease in many different crustaceans across a wide geographical area, but the overall view is that the condition is polymicrobial in nature leading to dysbiosis within the microbial consortium of the damaged cuticle. The role of environment, decapod behaviour and physiology in triggering this disease is also reviewed. Finally, we provide a conceptual model for disease aetiology and suggest several avenues for future research that could improve our understanding of how such factors trigger, or exacerbate, this condition.

Virulent and necrotrophic strategies of Bacillus thuringiensis in susceptible and resistant insects, Galleria mellonella.

Bacillus thuringiensis (Bt) is one of the most common entomopathogenic bacteria used as a biopesticide, and source of endotoxin genes for generating insect-resistant transgenic plants. The mechanisms underpinning an insect's susceptibility or resistance to B. thuringiensis are diverse. The bacterial lifecycle does not end with the death of a host, they continue to exploit the cadaver to reproduce and sporulate. Herein, we studied the progression of B. thuringiensis subsp. galleriae infection in two populations of wax moth larvae (Galleria mellonella) to gain further insight into the "arms race" between B. thuringiensis virulence and insect defences. Two doses of B. thuringiensis subsp. galleriae (spore and crystalline toxin mixtures) were administered orally to compare the responses of susceptible (S) and resistant (R) populations at ∼30% mortality each. To investigate B. thuringiensis-insect antibiosis, we used a combination of in vivo infection trials, bacterial microbiome analysis, and RNAi targeting the antibacterial peptide gloverin. Within 48 h post-inoculation, B. thuringiensis-resistant insects purged the midgut of bacteria, i.e., colony forming unit numbers fell below detectable levels. Second, B. thuringiensis rapidly modulated gene expression to initiate sporulation (linked to quorum sensing) when exposed to resistant insects in contrast to susceptible G. mellonella. We reinforce earlier findings that elevated levels of antimicrobial peptides, specifically gloverin, are found in the midgut of resistant insects, which is an evolutionary strategy to combat B. thuringiensis infection via its main portal of entry. A sub-population of highly virulent B. thuringiensis can survive the enhanced immune defences of resistant G. mellonella by disrupting the midgut microbiome and switching rapidly to a necrotrophic strategy, prior to sporulation in the cadaver.

Effect of acute ultraviolet radiation on Galleria mellonella health and immunity.

For humans, acute and chronic overexposure to ultraviolet (UV) radiation can cause tissue damage in the form of sunburn and promote cancer(s). The immune-modulating properties of UV radiation and health-related consequences are not well known. Herein, we used the larvae of the wax moth Galleria mellonella, to determine UV-driven changes in cellular components of innate immunity. From immune cell (haemocyte) reactivity and the production of antimicrobial factors, these insects share many functional similarities with mammalian cellular innate immunity. After exposing insects to UVA or UVB for up to two hours, we monitored larval viability, susceptibility to infection, haemolymph (blood) physiology and faecal discharge. Prolonged exposure of larvae to UVB coincided with decreased survival, enhanced susceptibility to bacterial challenge, melanin synthesis in the haemolymph, compromised haemocyte functionality and changes in faecal (bacterial) content. We contend G. mellonella is a reliable in vivo model for assessing the impact of UV exposure at the whole organism and cellular levels.

Histopathological survey of putative parasites and pathogens in non-native slipper limpets Crepidula fornicata.

Two populations of the invasive slipper limpet Crepidula fornicata were sampled in Swansea Bay and Milford Haven, Wales, UK, to determine the presence of putative pathogens and parasites known to affect co-located commercially important shellfish (e.g. oysters). A multi-resource screen, including molecular and histological diagnoses, was used to assess 1800 individuals over 12 mo for microparasites, notably haplosporidians, microsporidians and paramyxids. Although initial PCR-based methods suggested the presence of these microparasites, there was no evidence of infection when assessed histologically, or when all PCR amplicons (n = 294) were sequenced. Whole tissue histology of 305 individuals revealed turbellarians in the lumen of the alimentary canal, in addition to unusual cells of unknown origin in the epithelial lining. In total, 6% of C. fornicata screened histologically harboured turbellarians, and approximately 33% contained the abnormal cells-so named due to their altered cytoplasm and condensed chromatin. A small number of limpets (~1%) also had pathologies in the digestive gland including tubule necrosis, haemocytic infiltration and sloughed cells in the tubule lumen. Overall, these data suggest that C. fornicata are not susceptible to substantive infections by microparasites outside of their native range, which may contribute in part to their invasion success.

The genetic basis of neurocranial size and shape across varied lab mouse populations.

Brain and skull tissues interact through molecular signalling and mechanical forces during head development, leading to a strong correlation between the neurocranium and the external brain surface. Therefore, when brain tissue is unavailable, neurocranial endocasts are often used to approximate brain size and shape. Evolutionary changes in brain morphology may have resulted in secondary changes to neurocranial morphology, but the developmental and genetic processes underlying this relationship are not well understood. Using automated phenotyping methods, we quantified the genetic basis of endocast variation across large genetically varied populations of laboratory mice in two ways: (1) to determine the contributions of various genetic factors to neurocranial form and (2) to help clarify whether a neurocranial variation is based on genetic variation that primarily impacts bone development or on genetic variation that primarily impacts brain development, leading to secondary changes in bone morphology. Our results indicate that endocast size is highly heritable and is primarily determined by additive genetic factors. In addition, a non-additive inbreeding effect led to founder strains with lower neurocranial size, but relatively large brains compared to skull size; suggesting stronger canalization of brain size and/or a general allometric effect. Within an outbred sample of mice, we identified a locus on mouse chromosome 1 that is significantly associated with variation in several positively correlated endocast size measures. Because the protein-coding genes at this locus have been previously associated with brain development and not with bone development, we propose that genetic variation at this locus leads primarily to variation in brain volume that secondarily leads to changes in neurocranial globularity. We identify a strain-specific missense mutation within Akt3 that is a strong causal candidate for this genetic effect. Whilst it is not appropriate to generalize our hypothesis for this single locus to all other loci that also contribute to the complex trait of neurocranial skull morphology, our results further reveal the genetic basis of neurocranial variation and highlight the importance of the mechanical influence of brain growth in determining skull morphology.

Invasive slipper limpets Crepidula fornicata are hosts for sterilizing digenean parasites.

Invasion and spread of alien species can drive ecosystem changes, such as, the dynamics of infectious diseases. The non-native, marine gastropod Crepidula fornicata has become established across European coastlines over the last century, but there remains little insight into its disease carrying capacity and potential role as a source/sink of parasites. To address this knowledge gap, we surveyed limpets from two sites in South Wales, UK for signatures of disease/pathology using polymerase chain reaction-based methods (haemolymph) and histology (solid tissue). We encountered trematode-like parasites in ~1% individuals (5 out of 462). Three limpets displayed gross damage in the gonad, i.e. castration, and encysted metacercariae were found in the muscle of two other individuals. On the basis of 28S rDNA and internal transcribed spacer 2 genomic targets, we identified the gonad-infecting trematodes as members of the family Microphallidae ­ putative novel species related to the genus Longiductotrema. Earlier reports suggest that C. fornicata is not a host for trematode parasites in either its native or alien range but may act as a sink due to its filter feeding lifestyle. We provide clear evidence that C. fornicata is parasitized by at least one trematode species at two sites in Wales, UK, and likely act as a spillback or accidental host among native littorinids.

The diarrhetic shellfish-poisoning toxin, okadaic acid, provokes gastropathy, dysbiosis and susceptibility to bacterial infection in a non-rodent bioassay, Galleria mellonella.

Diarrhetic shellfish-poisoning (DSP) toxins such as okadaic acid and dinophysistoxins harm the human gastrointestinal tract, and therefore, their levels are regulated to an upper limit of 160 µg per kg tissue to protect consumers. Rodents are used routinely for risk assessment and studies concerning mechanisms of toxicity, but there is a general move toward reducing and replacing vertebrates for these bioassays. We have adopted insect larvae of the wax moth Galleria mellonella as a surrogate toxicology model. We treated larvae with environmentally relevant doses of okadaic acid (80-400 µg/kg) via intrahaemocoelic injection or gavage to determine marine toxin-related health decline: (1) whether pre-exposure to a sub-lethal dose of toxin (80 µg/kg) enhances susceptibility to bacterial infection, or (2) alters tissue pathology and bacterial community (microbiome) composition of the midgut. A sub-lethal dose of okadaic acid (80 µg/kg) followed 24 h later by bacterial inoculation (2 × 105 Escherichia coli) reduced larval survival levels to 47%, when compared to toxin (90%) or microbial challenge (73%) alone. Histological analysis of the midgut depicted varying levels of tissue disruption, including nuclear aberrations associated with cell death (karyorrhexis, pyknosis), loss of organ architecture, and gross epithelial displacement into the lumen. Moreover, okadaic acid presence in the midgut coincided with a shift in the resident bacterial population over time in that substantial reductions in diversity (Shannon) and richness (Chao-1) indices were observed at 240 µg toxin per kg. Okadaic acid-induced deterioration of the insect alimentary canal resembles those changes reported for rodent bioassays.

Multifunctional Roles of Hemocyanins.

The copper-containing hemocyanins are proteins responsible for the binding, transportation and storage of dioxygen within the blood (hemolymph) of many invertebrates. Several additional functions have been attributed to both arthropod and molluscan hemocyanins, including (but not limited to) enzymatic activity (namely phenoloxidase), hormone transport, homeostasis (ecdysis) and hemostasis (clot formation). An important secondary function of hemocyanin involves aspects of innate immunity-such as acting as a precursor of broad-spectrum antimicrobial peptides and microbial/viral agglutination. In this chapter, we present the reader with an up-to-date synthesis of the known functions of hemocyanins and the structural features that facilitate such activities.

Recent Insights into the Diversity and Evolution of Invertebrate Hemerythrins and Extracellular Globins.

There are three broad groups of oxygen-transport proteins found in the haemolymph (blood) of invertebrates, namely the hemocyanins, the hemerythrins and the globins. Both hemerythrins and extracellular globins are iron-based proteins that are understudied when compared to the copper-containing hemocyanins. Recent evidence suggests that hemerythrins and (giant) extracellular globins (and their linker chains) are more widely distributed than previously thought and may have biological functions beyond oxygen transport and storage. Herein, we review contemporary literature of these often-neglected proteins with respect to their structural configurations on formation and ancestral states.

The stress-immunity axis in shellfish.

It is a difficult task to describe what constitutes a 'healthy' shellfish (e.g., crustacean, bivalve). Visible defects such as discolouration, missing limbs or spines, fouling, lesions, and exoskeletal fractures can be indicative of underlying issues, senescence, or a 'stressed' animal. The absence of such symptoms is not evidence of a disease-free or a stress-free state. Now, more than ever, aquatic invertebrates must cope with acute and chronic environmental perturbations, such as, heatwaves and cold shocks, xenobiotic contaminants, intoxication events, and promiscuous pathogens expanding their host and geographic ranges. With that in mind, how does one determine the extent to which shellfish become stressed in situ (natural) or in cultured (artificial) settings to enhance disease susceptibility? Many biomarkers - predominantly biochemical and cellular measures of shellfish blood (haemolymph) - are considered to gauge immunosuppression and immunocompetence. Such measures range from immune cell (haemocyte) counts to enzymic activities and metabolite quantitation. Stressed invertebrates often reflect degraded conditions of their ecosystems, referred to as environmental indicators. We audit briefly the broad immune functions of shellfish, how they are modulated by known and emerging stressors, and discuss these concepts with respect to neuroendocrinology and immunotoxicology. We assert that chronic stress, alone or in combination with microbial, chemical and abiotic factors, increases the risk of infectious disease in shellfish, exacerbates idiopathic morbidity, and reduces the likelihood of recovery. Acute stress events can lead to immunomodulation, but these effects are largely transient. Enhancing our understanding of shellfish health and immunity is imperative for tackling losses at each stage of the aquatic food cycle and disease outbreaks in the wild.

Characterizing the Mechanisms of Nonopsonic Uptake of Cryptococci by Macrophages.

The pathogenic fungus Cryptococcus enters the human host via inhalation into the lung and is able to reside in a niche environment that is serum- (opsonin) limiting. Little is known about the mechanism by which nonopsonic phagocytosis occurs via phagocytes in such situations. Using a combination of soluble inhibitors of phagocytic receptors and macrophages derived from knockout mice and human volunteers, we show that uptake of nonopsonized Cryptococcus neoformans and C. gattii via the mannose receptor is dependent on macrophage activation by cytokines. However, although uptake of C. neoformans is via both dectin-1 and dectin-2, C. gattii uptake occurs largely via dectin-1. Interestingly, dectin inhibitors also blocked phagocytosis of unopsonized Cryptococci in wax moth (Galleria mellonella) larvae and partially protected the larvae from infection by both fungi, supporting a key role for host phagocytes in augmenting early disease establishment. Finally, we demonstrated that internalization of nonopsonized Cryptococci is not accompanied by the nuclear translocation of NF-κB or its concomitant production of proinflammatory cytokines such as TNF-α. Thus, nonopsonized Cryptococci are recognized by mammalian phagocytes in a manner that minimizes proinflammatory cytokine production and potentially facilitates fungal pathogenesis.

Diagnosis and prevalence of two new species of haplosporidians infecting shore crabs Carcinus maenas: Haplosporidium carcini n. sp., and H. cranc n. sp.

This study provides a morphological and phylogenetic characterization of two novel species of the order Haplosporida (Haplosporidium carcini n. sp., and H. cranc n. sp.) infecting the common shore crab Carcinus maenas collected at one location in Swansea Bay, South Wales, UK. Both parasites were observed in the haemolymph, gills and hepatopancreas. The prevalence of clinical infections (i.e. parasites seen directly in fresh haemolymph preparations) was low, at ~1%, whereas subclinical levels, detected by polymerase chain reaction, were slightly higher at ~2%. Although no spores were found in any of the infected crabs examined histologically (n = 334), the morphology of monokaryotic and dikaryotic unicellular stages of the parasites enabled differentiation between the two new species. Phylogenetic analyses of the new species based on the small subunit (SSU) rDNA gene placed H. cranc in a clade of otherwise uncharacterized environmental sequences from marine samples, and H. carcini in a clade with other crustacean-associated lineages.

Prevalence and histopathology of the parasitic barnacle, Sacculina carcini in shore crabs, Carcinus maenas.

Sacculina carcini is a common parasite of the European shore crab, Carcinus maenas. Following successful penetration of the host, numerous rootlets are formed that permeate through the hosts' tissues. Ultimately, these form an externa that houses the developing nauplii larvae of the parasite. Most studies have quantified levels of infection by counting the presence of reproductive externae and their breakdown structures, called scars. However, the diagnosis of the disease based only on external features may lead to underreporting the prevalence of the parasite. In the current study, we examined the presence and severity of S. carcini in C. maenas (n = 221) in the Prince of Wales Dock, South Wales, U.K. using a range of diagnostic approaches to give an accurate representation of temporal dynamics of infection. Parasitized crabs were found with a mean prevalence of 24% as determined by histological examination of the hepatopancreas. However, the prevalence of S. carcini based on the presence of externae and scars was only 6.3% and 1.8%, respectively. Overall, parasitism was associated with smaller crabs, crabs later in the moulting cycle that were orange in colour (as opposed to green or yellow), and those with a higher number of bacteria in the haemolymph. Interestingly, only 7.5% of infected crabs showed evidence of distinct host (cellular) response to the presence of rootlets in the hepatopancreas.

The insect, Galleria mellonella, is a compatible model for evaluating the toxicology of okadaic acid.

The polyether toxin, okadaic acid, causes diarrhetic shellfish poisoning in humans. Despite extensive research into its cellular targets using rodent models, we know little about its putative effect(s) on innate immunity. We inoculated larvae of the greater wax moth, Galleria mellonella, with physiologically relevant doses of okadaic acid by direct injection into the haemocoel (body cavity) and/or gavage (force-feeding). We monitored larval survival and employed a range of cellular and biochemical assays to assess the potential harmful effects of okadaic acid. Okadaic acid at concentrations ≥ 75 ng/larva (≥ 242 µg/kg) led to significant reductions in larval survival (> 65%) and circulating haemocyte (blood cell) numbers (> 50%) within 24 h post-inoculation. In the haemolymph, okadaic acid reduced haemocyte viability and increased phenoloxidase activities. In the midgut, okadaic acid induced oxidative damage as determined by increases in superoxide dismutase activity and levels of malondialdehyde (i.e. lipid peroxidation). Our observations of insect larvae correspond broadly to data published using rodent models of shellfish-poisoning toxidrome, including complementary LD50 values: 206-242 µg/kg in mice, ~ 239 µg/kg in G. mellonella. These data support the use of this insect as a surrogate model for the investigation of marine toxins, which offers distinct ethical and financial incentives.

Indomethacin-induced gut damage in a surrogate insect model, Galleria mellonella.

Indomethacin is a non-steroidal anti-inflammatory drug that causes gastric ulceration and increased 'leakiness' in rat models, and is used routinely as a toxicology assay to screen novel compounds for repair and restitution properties. We set out to establish conditions for indomethacin-induced gut damage in wax-moth (Galleria mellonella) larvae with a view to reducing the need for rodents in such experimentation. We administered indomethacin (0.5-7.5 µg/larva; 2-30 mg/kg) to G. mellonella via intrahaemocoelic injection and gavage (force-feeding) and monitored survival and development, blood cell (haemocyte) numbers, and changes in gut permeability. Increased levels of gut leakiness were observed within the first 4- to 24 h by tracking fluorescent microspheres in the faeces and haemolymph (blood equivalent). Additionally, we recorded varying levels of tissue damage in histological sections of the insect midgut, including epithelial sloughing and cell necrosis. Degeneration of the midgut was accompanied by significant increases in detoxification-associated activities (superoxide dismutase and glutathione-S-transferase). Herein, we present the first evidence that G. mellonella larvae force-fed indomethacin display broad symptoms of gastric damage similar to their rodent counterparts.

Immunological properties of oxygen-transport proteins: hemoglobin, hemocyanin and hemerythrin.

It is now well documented that peptides with enhanced or alternative functionality (termed cryptides) can be liberated from larger, and sometimes inactive, proteins. A primary example of this phenomenon is the oxygen-transport protein hemoglobin. Aside from respiration, hemoglobin and hemoglobin-derived peptides have been associated with immune modulation, hematopoiesis, signal transduction and microbicidal activities in metazoans. Likewise, the functional equivalents to hemoglobin in invertebrates, namely hemocyanin and hemerythrin, act as potent immune effectors under certain physiological conditions. The purpose of this review is to evaluate the true extent of oxygen-transport protein dynamics in innate immunity, and to impress upon the reader the multi-functionality of these ancient proteins on the basis of their structures. In this context, erythrocyte-pathogen antibiosis and the immune competences of various erythroid cells are compared across diverse taxa.

A denitrifying community associated with a major, marine nitrogen fixer.

The diazotrophic cyanobacterium, Trichodesmium, is an integral component of the marine nitrogen cycle and contributes significant amounts of new nitrogen to oligotrophic, tropical/subtropical ocean surface waters. Trichodesmium forms macroscopic, fusiform (tufts), spherical (puffs) and raft-like colonies that provide a pseudobenthic habitat for a host of other organisms including marine invertebrates, microeukaryotes and numerous other microbes. The diversity and activity of denitrifying bacteria found in association with the colonies was interrogated using a series of molecular-based methodologies targeting the gene encoding the terminal step in the denitrification pathway, nitrous oxide reductase (nosZ). Trichodesmium spp. sampled from geographically isolated ocean provinces (the Atlantic Ocean, the Red Sea and the Indian Ocean) were shown to harbor highly similar, taxonomically related communities of denitrifiers whose members are affiliated with the Roseobacter clade within the Rhodobacteraceae (Alphaproteobacteria). These organisms were actively expressing nosZ in samples taken from the mid-Atlantic Ocean and Red Sea implying that Trichodesmium colonies are potential sites of nitrous oxide consumption and perhaps earlier steps in the denitrification pathway also. It is proposed that coupled nitrification of newly fixed N is the most likely source of nitrogen oxides supporting nitrous oxide cycling within Trichodesmium colonies.

Zoothamnium duplicatum infestation of cultured horseshoe crabs (Limulus polyphemus).

An outbreak of the sessile peritrich Zoothamnium duplicatum in a pilot, commercial-scale Limulus polyphemus hatchery resulted in the loss of ∼96% (40,000) second/third instar larvae over a 61day period. peritrich growth was heavy, leading to mechanical obstruction of the gills and physical damage. The peritrichs were controlled without resultant loss of juvenile crabs by administering 10ppm chlorine in freshwater for 1h and the addition of aquarium grade sand; a medium into which the crabs could burrow and facilitate cleaning of the carapace. Peritrich identity was confirmed from a partial SSU rDNA contiguous sequence of 1343bp (99.7% similarity to Z. duplicatum).

Infection of Norway lobster (Nephrops norvegicus) by the parasite Hematodinium sp.: insights from 30 years of field observations.

The Norway lobster, Nephrops norvegicus, is an important representative of the benthos and also supports valuable fisheries across Europe. Nephrops are susceptible to infection by Hematodinium sp., an endoparasitic dinoflagellate that causes morbidity and mortality. From an epizootiological perspective, the Clyde Sea Area (CSA; west of Scotland) is the best-studied Hematodinium-Nephrops pathosystem, with historical data available between 1988 and 2008. We have revisited this pathosystem by curating and updating prevalence values, differentiating host traits associated with disease exposure and progression, and comparing Hematodinium sp. disease dynamics in the CSA to other locations and to other decapod hosts (Cancer pagurus, Carcinus maenas). Prevalence from a 2018/2019 survey (involving 1739 lobsters) revealed Hematodinium sp. still mounts a synchronized patent infection in the CSA; hence this pathogen can be considered as enzootic in this location. We highlight for the first time that Nephrops size is associated with high severity infection, while females are more exposed to Hematodinium sp. More generally, regardless of the host (Norway lobster, brown and shore crabs) or the geographical area (Ireland, Wales, Scotland), Hematodinium sp. patent infections peak in spring/summer and reach their nadir during autumn. We contend that Hematodinium must be considered one of the most important pathogens of decapod crustaceans in temperate waters.