Transgenesis enables mapping of segmental ganglia in the leech Helobdella austinensis.

The analysis of how neural circuits function in individuals and change during evolution is simplified by the existence of neurons identified as homologous within and across species. Invertebrates, including leeches, have been used for these purposes in part because their nervous systems comprise a high proportion of identified neurons, but technical limitations make it challenging to assess the full extent to which assumptions of stereotypy hold true. Here, we introduce Minos plasmid-mediated transgenesis as a tool for introducing transgenes into the embryos of the leech Helobdella austinensis (Spiralia; Lophotrochozoa; Annelida; Clitellata; Hirudinida; Glossiphoniidae). We identified an enhancer driving pan-neuronal expression of markers, including histone2B:mCherry, which allowed us to enumerate neurons in segmental ganglia. Unexpectedly, we found that the segmental ganglia of adult transgenic H. austinensis contain fewer and more variable numbers of neurons than in previously examined leech species.

Zooplankton-based adverse outcome pathways: A tool for assessing endocrine disrupting compounds in aquatic environments.

Endocrine disrupting compounds (EDCs) pose a significant ecological risk, particularly in aquatic ecosystems. EDCs have become a focal point in ecotoxicology, and their identification and regulation have become a priority. Zooplankton have gained global recognition as bioindicators, benefiting from rigorous standardization and regulatory validation processes. This review aims to provide a comprehensive summary of zooplankton-based adverse outcome pathways (AOPs) with a focus on EDCs as toxicants and the utilisation of freshwater zooplankton as bioindicators in ecotoxicological assessments. This review presents case studies in which zooplankton have been used in the development of AOPs, emphasizing the identification of molecular initiating events (MIEs) and key events (KEs) specific to zooplankton exposed to EDCs. Zooplankton-based AOPs may become an important resource for understanding the intricate processes by which EDCs impair the endocrine system. Furthermore, the data sources, experimental approaches, advantages, and challenges associated with zooplankton-based AOPs are discussed. Zooplankton-based AOPs framework can provide vital tools for consolidating toxicological knowledge into a structured toxicity pathway of EDCs, offering a transformative platform for facilitating enhanced risk assessment and chemical regulation.

A simple and rapid pre-clinical in vivo model reveals comparative cardiotoxicity profiles of kinase inhibitors.

Despite significant success, targeted therapeutics such as kinase inhibitors (KIs) still pose adverse events such as the cardiotoxicity. There is a lot of variation in the type and intensity of cardiotoxicity caused by different KIs and current pre-clinical models are inadequate to predict it. Thus, there is a need to develop more simple and rapid models for screening of novel KIs at the pre-clinical step itself. We thus aimed to establish a rapid and robust pre-clinical animal model for predicting cardiotoxicity of KIs and identify comparative cardiotoxicity profiles of a panel of FDA-approved KIs. Heart rate measurement and survival analysis of Daphnia was performed at regular intervals following treatment with ten KIs that were approved for the treatment of various cancers. The heart rates of Daphnia as well as the survival varied between KIs in a dose and time dependent manner suggesting differential cardiotoxicity profiles of various KIs. Further, the correlation between the cardiotoxicity and survival also varied among the ten KIs. Importantly, sorafenib and vemurafenib displayed maximum and least cardiotoxicity, respectively. The comparative cardiotoxicity profiles also are in conformity with the previous studies indicating the utility of Daphnia as a valuable and relevant animal model to rapidly predict the cardiotoxicity of novel KIs at a pre-clinical stage.

Is Galleria mellonella model a good alternative to study virulence in Staphylococcus aureus from bovine mastitis?

Staphylococcus aureus is one of the agents of bovine mastitis of hardest control due to a complex pathogenesis comprising a variety of virulence factors, which ensures its persistence in the mammary gland, causing significant health and economic losses. Therefore, understanding the pathogenesis of this agent is imperative. Galleria mellonella has stood out as an invertebrate animal model for the study of infectious diseases that affect several hosts. This work aimed to evaluate G. mellonella larvae as an experimental model for the study of virulence phenotypes in an S. aureus population isolated from bovine mastitis. Thirty genetically divergent S. aureus strains were chosen based on PFGE analysis. After experimental infection, larvae survival rates, bacterial growth in hemolymph, melanization intensity of the dorsal vessel, and histological characteristics of the infected tissues were evaluated. The G. mellonella model showed a clear diversity in the S. aureus pathogenicity pattern, allowing the differentiation of strains with virulence phenotypes ranging from high to low degrees. Histological analysis confirmed that the strains tested were capable of inducing the formation of nodules and melanization spots in the dorsal vessels of the larvae in different magnitudes. The strains 16S-717, 19C-828, and 31S-1443 presented the highest virulence intensity among the bacteria tested and will be used further for the generation of S. aureus mutant populations to prospect genetic targets aimed to develop control strategies of bovine mastitis. Altogether, our results suggest that G. mellonella is an attractive and low-cost animal model for characterizing virulence phenotypes of large S. aureus populations.

Assessing the Effects of Ozonation on the Concentrations of Personal Care Products and Acute Toxicity in Sludges of Wastewater Treatment Plants.

The aim of this study was to understand the distribution of the personal care products nonylphenol (NP), triclosan (TCS), benzophenone-3 (BP-3), and caffeine in the sludges from three wastewater treatment plants (WWTP-A, -B, and -C) in southern Taiwan. The four compounds were analyzed from activated sludge and dewatered sludge samples, and then the samples were treated with pressure-assisted ozonation under different conditions and removal efficiencies. All four target compounds were detected, especially NP, which was detected in the highest concentrations in the activated sludges of WWTP-A and dewatered sludges of WWTP-C at 17.19 ± 4.10 and 2.41 ± 1.93 µg/g, respectively. TCS was dominant in dewatered sludges from WWTP-B, and the highest detected concentration was 13.29 ± 6.36 µg/g. Removals of 70% and 90% were attained under 150 psi at 40 cycles for NP and TCS, respectively, with 5 min of ozonation reaction time, a solid/water ratio of 1:20, and 2% ozone concentration. Ecological risk quotients (RQs) were calculated by the ratios of the 10-day Hyalella azteca (freshwater amphipod) LC50 to the environmental concentrations of the target compounds. High RQs were found to be >10 for NP, TCS, and BP-3 in untreated sludges, resulting in significant ecological risks to aquatic organisms when the sludges are arbitrarily disposed. However, the toxic effects on Hyalella azteca were not significantly different among ozone sludge treatments. The reason for this may be related to the formation of toxic oxidation by-products and incomplete mineralization of organic compounds. This could also be true for unknown intermediates. The relatively high detection frequencies of these emerging compounds in WWTP sludges requires further applications and treatments.

Exercise and the Brain: Lessons From Invertebrate Studies.

Benefits of physical exercise for brain functions are well documented in mammals, including humans. In this review, we will summarize recent research on the effects of species-specific intense locomotion on behavior and brain functions of different invertebrates. Special emphasis is made on understanding the biological significance of these effects as well as underlying cellular and molecular mechanisms. The results obtained in three distantly related clades of protostomes, Nematodes, Molluscs and Artropods, suggest that influence of intense locomotion on the brain could have deep roots in evolution and wide adaptive significance. In C. elegans, improved learning, nerve regeneration, resistance to neurodegenerative processes were detected after physical activity; in L. stagnalis-facilitation of decision making in the novel environment, in Drosophila-increased endurance, improved sleep and feeding behavior, in G. bimaculatus-improved orientation in conspecific phonotaxis, enhanced aggressiveness, higher mating success, resistance to some disturbing stimuli. Many of these effects have previously been described in mammals as beneficial results of running, suggesting certain similarity between distantly-related species. Our hypothesis posits that the above modulation of cognitive functions results from changes in the organism's predictive model. Intense movement is interpreted by the organism as predictive of change, in anticipation of which adjustments need to be made. Identifying the physiological and molecular mechanisms behind these adjustments is easier in experiments in invertebrates and may lead to the discovery of novel neurobiological mechanisms for regulation and correction of cognitive and emotional status.

Experimental models for pharmacokinetic and pharmacodynamic studies of antifungals used in cryptococcosis treatment.

Studies on cryptococcosis in the mammal animal model have demonstrated the occurrence of central nervous system infection and similarities in fungal pathogenicity with clinical and immunological features of the human infection. Although there is still a lack of studies involving pharmacokinetics (PK) and pharmacodynamics (PD) in animal models of cryptococcosis in the literature, these experimental models are useful for understanding this mycosis and antifungal effectiveness in improving the therapeutic schemes. The scope of this review is to describe and discuss the main mammal animal models for PK and PD studies of antifungals used in cryptococcosis treatment. Alternative models and computational methods are also addressed. All approaches for PK/PD studies are relevant to investigating drug-infection interaction and improving cryptococcosis therapy.

Methods for Using the Galleria mellonella Invertebrate Model to Probe Enterococcus faecalis Pathogenicity.

The Enterococci, mainly Enterococcus faecalis and E. faecium, are ubiquitous members of the human gastrointestinal tract consortia but also a leading cause of opportunistic infections. The global rise in human-associated enterococcal infections, often caused by multidrug resistant strains, highlights an urgent need to identify the bacterial factors contributing to its pathogenicity such that new therapies can be devised. The use of the Galleria mellonella (greater wax moth) larvae, commonly known as wax worm, as a model to study host-pathogen interactions has allowed the identification and characterization of numerous bacterial factors that contribute to disease in humans, serving both as an alternative and complementary approach to mammalian models. Here, we describe the methods for using G. mellonella to characterize the virulence factors of E. faecalis.

Current Models to Study the Sporothrix-Host Interaction.

Sporotrichosis is a worldwide distributed subcutaneous mycosis that affects mammals, including human beings. The infection is caused by members of the Sporothrix pathogenic clade, which includes Sporothrix schenckii, Sporothrix brasiliensis, and Sporothrix globosa. The fungus can be acquired through traumatic inoculation of conidia growing in vegetal debris or by zoonotic transmission from sick animals. Although is not considered a life-threatening disease, it is an emergent health problem that affects mostly immunocompromised patients. The sporotrichosis causative agents differ in their virulence, host range, and sensitivity to antifungal drugs; therefore, it is relevant to understand the molecular bases of their pathogenesis, interaction with immune effectors, and mechanisms to acquired resistance to antifungal compounds. Murine models are considered the gold standard to address these questions; however, some alternative hosts offer numerous advantages over mammalian models, such as invertebrates like Galleria mellonella and Tenebrio molitor, or ex vivo models, which are useful tools to approach questions beyond virulence, without the ethical or budgetary features associated with the use of animal models. In this review, we analyze the different models currently used to study the host-Sporothrix interaction.

Repurposing the PDMA-approved drugs in Japan using an insect model of staphylococcal infection.

A total of 1253 compounds approved as therapeutic drugs in Japan (Pharmaceuticals and Medical Devices Agency (PMDA)-approved compounds) were screened for their therapeutic effects against Staphylococcus aureus infection using the silkworm infection model. In the first stage of screening with an index of prolonged survival, 80 compounds were identified as hits. Of these, 64 compounds were clinically used as antimicrobial agents, and the remaining 16 compounds were not. The 16 compounds were examined for their dose-dependent therapeutic effects on the silkworm model as a second screening step, and we obtained five compounds as a result. One of the compounds (capecitabine) had no documented in vitro minimum inhibitory concentration (MIC) value against S. aureus. The MIC value of capecitabine against S. aureus strains ranged from 125 to 250 µg/ml, and capecitabine was therapeutically effective at a dose of 200 mg/kg in a murine model of S. aureus infection. These results suggest that silkworm-based drug repositioning studies are of potential value. Furthermore, the therapeutic effects of capecitabine demonstrated in this study provide an important scientific rationale for clinical observational studies examining the association between staphylococcal infection events and capecitabine administration in cancer chemotherapy patients.

Aplysia Neurons as a Model of Alzheimer's Disease: Shared Genes and Differential Expression.

Although Alzheimer's disease (AD) is the most common form of dementia in the United States, development of therapeutics has proven difficult. Invertebrate alternatives to current mammalian AD models have been successfully employed to study the etiology of the molecular hallmarks of AD. The marine snail Aplysia californica offers a unique and underutilized system in which to study the physiological, behavioral, and molecular impacts of AD. Mapping of the Aplysia proteome to humans and cross-referencing with two databases of genes of interest in AD research identified 898 potential orthologs of interest in Aplysia. Included among these orthologs were alpha, beta and gamma secretases, amyloid-beta, and tau. Comparison of age-associated differential expression in Aplysia sensory neurons with that of late-onset AD in the frontal lobe identified 59 ortholog with concordant differential expression across data sets. The 21 concordantly upregulated genes suggested increased cellular stress and protein dyshomeostasis. The 47 concordantly downregulated genes included important components of diverse neuronal processes, including energy metabolism, mitochondrial homeostasis, synaptic signaling, Ca++ regulation, and cellular cargo transport. Compromised functions in these processes are known hallmarks of both human aging and AD, the ramifications of which are suggested to underpin cognitive declines in aging and neurodegenerative disease.

Reflections on the Use of an Invertebrate Chordate Model System for Studies of Gut Microbial Immune Interactions.

The functional ecology of the gastrointestinal tract impacts host physiology, and its dysregulation is at the center of various diseases. The immune system, and specifically innate immunity, plays a fundamental role in modulating the interface of host and microbes in the gut. While humans remain a primary focus of research in this field, the use of diverse model systems help inform us of the fundamental principles legislating homeostasis in the gut. Invertebrates, which lack vertebrate-style adaptive immunity, can help define conserved features of innate immunity that shape the gut ecosystem. In this context, we previously proposed the use of a marine invertebrate, the protochordate Ciona robusta, as a novel tractable model system for studies of host-microbiome interactions. Significant progress, reviewed herein, has been made to fulfill that vision. We examine and review discoveries from Ciona that include roles for a secreted immune effector interacting with elements of the microbiota, as well as chitin-rich mucus lining the gut epithelium, the gut-associated microbiome of adults, and the establishment of a large catalog of cultured isolates with which juveniles can be colonized. Also discussed is the establishment of methods to rear the animals germ-free, an essential technology for dissecting the symbiotic interactions at play. As the foundation is now set to extend these studies into the future, broadening our comprehension of how host effectors shape the ecology of these microbial communities in ways that establish and maintain homeostasis will require full utilization of "multi-omics" approaches to merge computational sciences, modeling, and experimental biology in hypothesis-driven investigations.

The Aquatic Invertebrate Hydra vulgaris Releases Molecular Messages Through Extracellular Vesicles.

Recent body of evidence demonstrates that extracellular vesicles (EVs) represent the first language of cell-cell communication emerged during evolution. In aquatic environments, transferring signals between cells by EVs offers protection against degradation, allowing delivering of chemical information in high local concentrations to the target cells. The packaging of multiple signals, including those of hydrophobic nature, ensures target cells to receive the same EV-conveyed messages, and the coordination of a variety of physiological processes across cells of a single organisms, or at the population level, i.e., mediating the population's response to changing environmental conditions. Here, we purified EVs from the medium of the freshwater invertebrate Hydra vulgaris, and the molecular profiling by proteomic and transcriptomic analyses revealed multiple markers of the exosome EV subtype, from structural proteins to stress induced messages promoting cell survival. Moreover, positive and negative regulators of the Wnt/ß-catenin signaling pathway, the major developmental pathway acting in body axial patterning, were identified. Functional analysis on amputated polyps revealed EV ability to modulate both head and foot regeneration, suggesting bioactivity of the EV cargo and opening new perspectives on the mechanisms of developmental signalling. Our results open the path to unravel EV biogenesis and function in all cnidarian species, tracing back the origin of the cell-cell, cross-species or cross-kingdom communication in aquatic ecosystems.

A locust embryo as predictive developmental neurotoxicity testing system for pioneer axon pathway formation.

Exposure to environmental chemicals during in utero and early postnatal development can cause a wide range of neurological defects. Since current guidelines for identifying developmental neurotoxic chemicals depend on the use of large numbers of rodents in animal experiments, it has been proposed to design rapid and cost-efficient in vitro screening test batteries that are mainly based on mixed neuronal/glial cultures. However, cell culture tests do not assay correct wiring of neuronal circuits. The establishment of precise anatomical connectivity is a key event in the development of a functional brain. Here, we expose intact embryos of the locust (Locusta migratoria) in serum-free culture to test chemicals and visualize correct navigation of identified pioneer axons by fluorescence microscopy. We define separate toxicological endpoints for axonal elongation and navigation along a stereotyped pathway. To distinguish developmental neurotoxicity (DNT) from general toxicity, we quantify defects in axonal elongation and navigation in concentration-response curves and compare it to the biochemically determined viability of the embryo. The investigation of a panel of recognized DNT-positive and -negative test compounds supports a rather high predictability of this invertebrate embryo assay. Similar to the semaphorin-mediated guidance of neurites in mammalian cortex, correct axonal navigation of the locust pioneer axons relies on steering cues from members of this family of cell recognition molecules. Due to the evolutionary conserved mechanisms of neurite guidance, we suggest that our pioneer axon paradigm might provide mechanistically relevant information on the DNT potential of chemical agents on the processes of axon elongation, navigation, and fasciculation.

Acute toxicity of Zinc Oxide nanoparticles to silkworm (Bombyx mori L.).

Zinc Oxide nanoparticles (ZnO NPs) has been heavily used in the industry, and increasing concerns on the ecotoxicity has arisen due to the risk of release into the environment. In this work, silkworm was used here as a model organism to study the toxicity of ZnO NPs, due to the presence of a conserved immune response as well as a pharmacokinetics similar to mammals. Zn accumulation, biodistribution and toxicity in silkworms were monitored at different time points after a subcutaneous injection. The highest cumulative content of ZnO NPs was detected in the midgut. The results of catalytic activity studies confirmed that the antioxidant enzymes (SOD, CAT, GSH-PX) in midgut cells were expressed in response to ZnO NPs. The expression of genes (Dronc and Caspase-1) related to apoptosis was increased, while the Trt gene was down-regulated. A possible mechanism was proposed for toxicity of ZnO NPs to silkworms.

Cryptic species of Aspergillus section Terrei display essential physiological features to cause infection and are similar in their virulence potential in Galleria mellonella.

Aspergillus species account for the majority of invasive mold infections in immunocompromised patients. Most commonly, members of the Aspergillus section Fumigati are isolated from clinical material, followed by isolates belonging to section Terrei. The section Terrei contains 16 accepted species. Six species were found to be of clinical relevance and studied for differences in growth adaptability and virulence potential. Therefore, a set of 73 isolates (22 A. terreus s.s., 8 A. alabamensis, 27 A. citrinoterreus, 2 A. floccosus, 13 A. hortai, and 1 A. neoafricanus) was studied to determine differences in (a) germination kinetics, (b) temperature tolerance, (c) oxygen stress tolerance (1% O2), and (d) a combination of the latter two. Virulence potential of phialidic (PC) and accessory conidia (AC) was studied in G. mellonella larvae, using survival as read out. Further, the formation of AC was evaluated in larval tissue. All isolates were able to grow at elevated temperature and hypoxia, with highest growth and germination rates at 37°C. A. terreus s.s., A. citrinoterreus, and A. hortai exhibited highest growth rates. Virulence potential in larvae was inoculum and temperature dependent. All species except A. floccosus formed AC and germination kinetics of AC was variable. Significantly higher virulence potential of AC was found for one A. hortai isolate. AC could be detected in larval tissue 96 h post infection. Based on these findings, cryptic species of section Terrei are well adapted to the host environment and have similar potential to cause infections.

Methods and Challenges of Using the Greater Wax Moth (Galleria mellonella) as a Model Organism in Antimicrobial Compound Discovery.

Among non-mammalian infection model organisms, the larvae of the greater wax moth Galleria mellonella have seen increasing popularity in recent years. Unlike other invertebrate models, these larvae can be incubated at 37 °C and can be dosed relatively precisely. Despite the increasing number of publications describing the use of this model organism, there is a high variability with regard to how the model is produced in different laboratories, with respect to larva size, age, origin, storage, and rest periods, as well as dosing for infection and treatment. Here, we provide suggestions regarding how some of these factors can be approached, to facilitate the comparability of studies between different laboratories. We introduce a linear regression curve correlating the total larva weight to the liquid volume in order to estimate the in vivo concentration of pathogens and the administered drug concentration. Finally, we discuss several other aspects, including in vivo antibiotic stability in larvae, the infection doses for different pathogens and suggest guidelines for larvae selection.

Characterization of locomotor response to psychostimulants in the parthenogenetic marbled crayfish (Procambarus fallax forma virginalis): A promising model for studying the neural and molecular mechanisms of drug addiction.

Although scientific research using mammalian models has made great strides in uncovering the enigmatic neural and molecular mechanisms orchestrating the state of drug addiction, a complete understanding has thus far eluded researchers. The complexity of the task has led to the use of invertebrate model systems to complement the research of drug-induced reward in mammalian systems. Invertebrates, such as crayfish, offer excellent model systems to help reveal the underlying mechanisms of drug addiction as they retain the ancestral neural reward circuit that is evolutionarily conserved across taxa, and they possess relatively few, large neurons, laid out in an accessible, modularly organized nervous system. Crayfish offer the benefits of delineated developmental life stages, a large body size suitable for a variety of experimental methods, and stereotyped behaviors. Unique among crayfish is the parthenogenetic marbled crayfish (Procambarus fallax forma virginalis), a species of asexually reproducing, genetically identical clones. With the benefits of reduced individual variation, high fecundity, and easy lab husbandry, the marbled crayfish would make a particularly powerful addition to the animal model repertoire. Here we characterize the locomotor response of juvenile P. f. f. virginalis exposed to the psychostimulant, d-amphetamine sulfate. Custom video-tracking software was used to record the movement patterns of juveniles exposed to water infused with varying concentrations of d-amphetamine sulfate. ANOVA demonstrated that crayfish locomotion was significantly impacted by drug concentration. These psychostimulant effects provide the foundation of P. f. f. virginalis as a model for parsing the neural and molecular mechanisms of drug addiction.

In vivo activity of fluconazole/tetracycline combinations in Galleria mellonella with resistant Candida albicans infection.

OBJECTIVES: Treatment of azole-resistant Candida albicans infections continues to pose significant challenges. With limited options of licensed agents, drug combinations may be a practical treatment alternative. In our previous studies, the combinations minocycline/fluconazole (MINO/FLC) and doxycycline/fluconazole (DOXY/FLC) shown synergistic effects in vitro. It is necessary to explore their appropriate dosage, potential toxicity and in vivo efficacy. METHODS: The Galleria mellonella infection model was employed to study the in vivo efficacy of MINO/FLC and DOXY/FLC by survival analysis, quantification of C. albicans fungal burden and histological studies. RESULTS: The survival rates of G. mellonella larvae infected with lethal doses of resistant C. albicans CA10 increased significantly when treated with the drug combinations compared with FLC treatment alone, and the fungal burden was reduced by almost four-fold. The histopathological study showed that fewer infected areas in larvae were observed and the destructive degree was less when larvae were exposed to the drug combinations. CONCLUSIONS: These findings suggest that combination of a tetracycline antibiotic (MINO or DOXY) with FLC has antifungal activity against azole-resistant C. albicans in vivo. This is in agreement with several previous in vitro studies and provides preliminary in vivo evidence that such a combination might be useful therapeutically.

Activity of levofloxacin in combination with colistin against Acinetobacter baumannii: In vitro and in a Galleria mellonella model.

BACKGROUND/PURPOSE: Treatment of Acinetobacter baumannii infections is challenging owing to widespread multidrug-resistant A. baumannii (MDR-AB) and the lack of novel agents. Although recent data suggest that levofloxacin (LVX) may have unique activity against MDR-AB in combination with colistin (CST), further preclinical work is needed. METHODS: We used a A. baumannii type strain ATCC19606, a CST-resistant strain AB19606R, and two clinical isolates (GN0624 and GN1115) of MDR-AB to investigate the in vitro and in vivo efficacy of LVX-CST combination. Synergy studies were performed using the microtiter plate chequerboard assay and time-kill methodology. Inhibitory activity of antibiotics against biofilms and the mutant prevention concentrations were also studied in vitro. A simple invertebrate model (Galleria mellonella) has been used to assess the in vivo activity of antimicrobial therapies. RESULTS: The LVX-CST combination was bactericidal against the CST-susceptible clinical isolate (GN0624). In checkerboard assays, synergy (defined as a fractional inhibitory concentration index of < 0.5) was observed between CST and LVX in GN0624. The combination had antibiofilm properties on the preformed biofilms of four tested strains and could prevent the emergence of CST-resistant A. baumanni. Treatment of G. mellonella larvae infected with lethal doses of A. baumannii resulted in significantly enhanced survival rates when LVX was given with CST compared with CST treatment alone (p < 0.05). CONCLUSION: In summary, a synergistic or additive effect between CST and LVX was observed in vitro and in vivo against CST-susceptible A. baumannii strains, although not against CST-resistant ones.