Injured Larvae Research Articles

Tri-n-butyl phosphate delays tissue repair by dysregulating neutrophil function in zebrafish

Tri-n-butyl phosphate (TnBP) is a widely used organophosphate ester, but its effects on the regenerative process under damaging circumstances remain unknown. In the present study, zebrafish larvae were exposed to 0, 50, 100, 200 and 1000 μg/L TnBP, and the caudal fins were cut at 72 hours post fertilization (hpf). First, after exposure to TnBP, the number of total neutrophils decreased together with decreased neutrophils in the tail, and TnBP inhibited chemotaxis. Second, reactive oxygen species (ROS) levels in the zebrafish decreased greatly. Following exposure to TnBP, transcription levels of many genes regulating fin regeneration, such as fgf20a, fgfr1a, bmp2a and bmp4, were significantly downregulated, while inflammatory factors such as cxcl8a, cxcl18b, il-6, and tnfa were abnormally upregulated. In addition, TnBP inhibited the regenerative area after caudal fin amputation. The inflammatory state was adverse during the regenerative process. In summary, TnBP exposure is immunotoxic and decreases oxidative stress in injured zebrafish larvae.

Transgenic zebrafish larvae as a non-rodent alternative model to assess pro-inflammatory (neutrophil) responses to nanomaterials

Hazard studies for nanomaterials (NMs) commonly assess whether they activate an inflammatory response. Such assessments often rely on rodents, but alternative models are needed to support the implementation of the 3Rs principles. Zebrafish (Danio rerio) offer a viable alternative for screening NM toxicity by investigating inflammatory responses. Here, we used non-protected life stages of transgenic zebrafish (Tg(mpx:GFP)i114) with fluorescently-labeled neutrophils to assess inflammatory responses to silver (Ag) and zinc oxide (ZnO) NMs using two approaches. Zebrafish were exposed to NMs via water following a tail fin injury, or NMs were microinjected into the otic vesicle. Zebrafish were exposed to NMs at 3 days post-fertilization (dpf) and neutrophil accumulation at the injury or injection site was quantified at 0, 4, 6, 8, 24, and 48 h post-exposure. Zebrafish larvae were also exposed to fMLF, LTB4, CXCL-8, C5a, and LPS to identify a suitable positive control for inflammation induction. Aqueous exposure to Ag and ZnO NMs stimulated an enhanced and sustained neutrophilic inflammatory response in injured zebrafish larvae, with a greater response observed for Ag NMs. Following microinjection, Ag NMs stimulated a time-dependent neutrophil accumulation in the otic vesicle which peaked at 48 h. LTB4 was identified as a positive control for studies investigating inflammatory responses in injured zebrafish following aqueous exposure, and CXCL-8 for microinjection studies that assess responses in the otic vesicle. Our findings support the use of transgenic zebrafish to rapidly screen the pro-inflammatory effects of NMs, with potential for wider application in assessing chemical safety (e.g. pharmaceuticals).

A nontoxic fungal natural product modulates fin regeneration in zebrafish larvae upstream of FGF-WNT developmental signaling.

The regeneration of larvae zebrafish fin emerged as a new model of regeneration in the last decade. In contrast to genetic tools to study fin regeneration, chemical probes to modulate and interrogate regeneration processes are not well developed. We set up a zebrafish larvae fin regeneration assay system and tested activities of natural product compounds and extracts, prepared from various microbes. Colomitide C, a recently isolated product from a fungus obtained from Antarctica, inhibited larvae fin regeneration. Using fluorescent reporter transgenic lines, we show that colomitide C inhibited fibroblast growth factor (FGF) signaling and WNT/β-catenin signaling, which were activated after larvae fin amputation. By using the endothelial cell reporter line and immunofluorescence, we showed that colomitide C did not affect migration of the blood vessel and nerve into the injured larvae fin. Colomitide C did not show any cytotoxic activities when tested against FGF receptor-amplified human cancer cell lines. Colomitide C, a natural product, modulated larvae fin regeneration likely acting upstream of FGF and WNT signaling. Colomitide C may serve as a template for developing new chemical probes to study regeneration and other biological processes.

Correspondence Between Behavioral, Physiological, and Anatomical Measurements of Visual Function in Inhibitory Neuron–Ablated Zebrafish

To compare the effects of reduced inhibitory neuron function in the retina across behavioral, physiological, and anatomical levels. Inhibitory neurons were ablated in larval zebrafish retina. The Ptf1a gene, which determines inhibitory neuron fate in developing vertebrates, was used to express nitroreductase. By exposing larvae to the prodrug metronidazole, cytotoxicity was selectively induced in inhibitory neurons. Visual phenotypes were characterized at behavioral, physiological, and anatomical levels using an optomotor response (OMR) assay, electroretinography (ERG), and routine histology, respectively. Nonvisual locomotion was also assessed to reveal any general behavioral effects due to ablation of other nonvisual neurons that also express Ptf1a. Injured larvae showed severely reduced OMR relative to controls. Locomotor assessment showed unaltered swimming ability, indicating that reduced OMR was due to visual deficits. For ERG, injured larvae manifested either reduced (type-I) or absent (type-II) b-wave signals originating from bipolar interneurons in the retina. Histologic analysis showed altered retinal morphology in injured larvae, with reductions in synaptic inner plexiform layer (IPL) thickness and synaptic density more pronounced in type-II than type-I larvae; type-II larvae also had smaller retinae overall. The consequences of inhibitory neuron ablation corresponded closely across behavioral, physiological, and anatomical levels. Inhibitory neuron loss likely increases the ratio of neural excitation to inhibition, leading to hyperexcitability. In addition to modulating visual signals, inhibitory neurons may be critical for maintaining retinal structure and organization. This study highlights the utility of a multidisciplinary approach and provides a template for characterizing other zebrafish models of neurological disease.

Stealing from the Future: Injured Larvae Spend Stem Cell Deposits

Because Drosophila larvae do not possess intestinal stem cells, it is unknown how damaged gut cells are replenished. In this issue of Cell Host & Microbe, Houtz etal. (2019) show that larvae have a unique gut repair mechanism that involves borrowing stem cells originally reserved for adult gut formation.

Nanoliposomes encapsulating immunostimulants modulate the innate immune system and elicit protection in zebrafish larvae

Here we present immunostimulant-loaded nanoliposomes (NLc) as a strategy to protect zebrafish larvae against bacterial infection. The NLc encapsulate crude lipopolysaccharide (LPS) from E. coli and polyinosinic:polycytidylic acid (Poly I:C), a synthetic analogue of viral dsRNA. Fluorescently-labeled NLc were ingested by zebrafish larvae 4 days post fertilization, when administrated by bath immersion, and accumulated in the intestine. RT-qPCR analysis showed the expression of innate immune related genes (tnfα, il1β, nos2a, irf1a and ptgs2a) was significantly upregulated at 48 h post NLc treatment. A zebrafish larvae infection model for Aeromonas hydrophila was set up by bath immersion, achieving bacterial-dose-dependent significant differences in survival at day 5 post infection in both injured and non-injured larvae. Using this model, NLc protected non-injured zebrafish larvae against an A. hydrophila lethal infection. In contrast, neither the empty nanoliposomes nor the mixture of immunostimulants could protect larvae against lethal challenges. Our results demonstrate that nanoliposomes could be further developed as an efficient carrier, widening the scope for delivery of other immunostimulants in aquaculture.

Transstadial immune activation in a mosquito: Adults that emerge from infected larvae have stronger antibacterial activity in their hemocoel yet increased susceptibility to malaria infection.

Larval and adult mosquitoes mount immune responses against pathogens that invade their hemocoel. Although it has been suggested that a correlation exists between immune processes across insect life stages, the influence that an infection in the hemocoel of a larva has on the immune system of the eclosed adult remains unknown. Here, we used Anopheles gambiae to test whether a larval infection influences the adult response to a subsequent bacterial or malaria parasite infection. We found that for both female and male mosquitoes, a larval infection enhances the efficiency of bacterial clearance following a secondary infection in the hemocoel of adults. The adults that emerge from infected larvae have more hemocytes than adults that emerge from naive or injured larvae, and individual hemocytes have greater phagocytic activity. Furthermore, mRNA abundance of immune genes—such as cecropin A, Lysozyme C1, Stat‐A, and Tep1—is higher in adults that emerge from infected larvae. A larval infection, however, does not have a meaningful effect on the probability that female adults will survive a systemic bacterial infection, and increases the susceptibility of females to Plasmodium yoelii, as measured by oocyst prevalence and intensity in the midgut. Finally, immune proficiency varies by sex; females exhibit increased bacterial killing, have twice as many hemocytes, and more highly express immune genes. Together, these results show that a larval hemocoelic infection induces transstadial immune activation—possibly via transstadial immune priming—but that it confers both costs and benefits to the emerged adults.

Properties of induced antimicrobial activity in Musca domestica larvae.

Insects produce antimicrobial molecules that contribute to their innate immune responses to eliminate invading microorganisms. To explore the potential utility of these antimicrobial molecules, we focused on larvae of the house fly Musca domestica, which is an efficient processor of organic waste and a good resource of protein and oil for animal feeding. The induction of hemagglutinating activity, which is usually accompanied by activation of innate immune responses in fly larvae, was observed in the hemolymph following needle injury. Hemolymph collected from injured larvae demonstrated potent antimicrobial activities against both Gram-positive and Gram-negative bacteria, including Staphylococcus aureus and Pseudomonas aeruginosa. Furthermore, the antimicrobial activity was significantly retained in hemolymph after heat-treatments, suggesting that pasteurization of animal feed prepared from fly larvae would be a useful sterilization method. These observations indicate that injured Musca domestica larvae are a source of antimicrobial agents, and highlight the utility of preparing animal feed from these larvae.

The Animal Model Determines the Results of Aeromonas Virulence Factors.

The selection of an experimental animal model is of great importance in the study of bacterial virulence factors. Here, a bath infection of zebrafish larvae is proposed as an alternative model to study the virulence factors of Aeromonas hydrophila. Intraperitoneal infections in mice and trout were compared with bath infections in zebrafish larvae using specific mutants. The great advantage of this model is that bath immersion mimics the natural route of infection, and injury to the tail also provides a natural portal of entry for the bacteria. The implication of T3SS in the virulence of A. hydrophila was analyzed using the AH-1::aopB mutant. This mutant was less virulent than the wild-type strain when inoculated into zebrafish larvae, as described in other vertebrates. However, the zebrafish model exhibited slight differences in mortality kinetics only observed using invertebrate models. Infections using the mutant AH-1ΔvapA lacking the gene coding for the surface S-layer suggested that this protein was not totally necessary to the bacteria once it was inside the host, but it contributed to the inflammatory response. Only when healthy zebrafish larvae were infected did the mutant produce less mortality than the wild-type. Variations between models were evidenced using the AH-1ΔrmlB, which lacks the O-antigen lipopolysaccharide (LPS), and the AH-1ΔwahD, which lacks the O-antigen LPS and part of the LPS outer-core. Both mutants showed decreased mortality in all of the animal models, but the differences between them were only observed in injured zebrafish larvae, suggesting that residues from the LPS outer core must be important for virulence. The greatest differences were observed using the AH-1ΔFlaB-J (lacking polar flagella and unable to swim) and the AH-1::motX (non-motile but producing flagella). They were as pathogenic as the wild-type strain when injected into mice and trout, but no mortalities were registered in zebrafish larvae. This study demonstrates that zebrafish larvae can be used as a host model to assess the virulence factors of A. hydrophila. This model revealed more differences in pathogenicity than the in vitro models and enabled the detection of slight variations in pathogenesis not observed using intraperitoneal injections of mice or fish.

Establishment of Infection Models in Zebrafish Larvae (Danio rerio) to Study the Pathogenesis of Aeromonas hydrophila.

Aeromonas hydrophila is a Gram-negative opportunistic pathogen of fish and terrestrial animals. In humans, A. hydrophila mainly causes gastroenteritis, septicaemia, and tissue infections. The mechanisms of infection, the main virulence factors and the host immune response triggered by A. hydrophila have been studied in detail using murine models and adult fish. However, the great limitation of studying adult animals is that the animal must be sacrificed and its tissues/organs extracted, which prevents the study of the infectious processes in the whole living animal. Zebrafish larvae are being used for the analysis of several infectious diseases, but their use for studying the pathogenesis of A. hydrophila has never been explored. The great advantage of zebrafish larvae is their transparency during the first week after fertilization, which allows detailed descriptions of the infectious processes using in vivo imaging techniques such as differential interferential contrast (DIC) and fluorescence microscopy. Moreover, the availability of fluorescent pathogens and transgenic reporter zebrafish lines expressing fluorescent immune cells, immune marker genes or cytokines/chemokines allows the host–pathogen interactions to be characterized. The present study explores the suitability of zebrafish larvae to study the pathogenesis of A. hydrophila and the interaction mechanisms between the bacterium and the innate immune responses through an infection model using different routes for infection. We used an early-embryo infection model at 3 days post-fertilization (dpf) through the microinjection of A. hydrophila into the duct of Cuvier, caudal vein, notochord, or muscle and two bath infection models using 4 dpf healthy and injured larvae. The latter resembled the natural conditions under which A. hydrophila produces infectious diseases in animals. We compared the cellular processes after infection in each anatomical site by confocal fluorescence imaging and determined the implication of inflammatory immune genes by measuring gene expression by qPCR.

Induced Cannibalism in Experimental Populations of the Forensic Indicator Chrysomya putoria Wiedemann (Diptera: Calliphoridae).

We analyzed the behavior of third-instars of Chrysomya putoria as potential cannibals in experimental populations. Cannibalism rates were evaluated in three settings observed for 3, 6, 9, and 24 h, placing injured and uninjured larvae of C. putoria together. Our data heavily support that C. putoria larvae behave as cannibals when induced by a wound in another larva, and also after starving for 24 h. The probability of cannibalism increased as a function of time, both in no-choice and in choice experiments evidencing that time is a determining factor for cannibalism induction in C. putoria. However, the treatment combining injured with uninjured larvae showed the highest probability of cannibalism. These results suggest that C. putoria larvae may cannibalize under scarcity of food over long time or the presence of injured larvae. This study is useful to understand the behavior of C. putoria feeding on ephemeral substrates such as carrion or corpses and brings relevant and significant contribution to population ecology of blowflies and also forensic entomology.

Induction of a gloverin-like antimicrobial polypeptide in the sugarcane borer Diatraea saccharalis challenged by septic injury

Diatraea saccharalis (Fabricius, 1794) (Lepidoptera: Crambidae) is an important pest for Brazilian sugarcane. In the present study, we detected two distinct spots in hemolymph from septic injured larvae (HDs1 and HDs2), which are separated by 2DE gel electrophoresis. Both spots were subjected to in-gel tryptic digestion and MALDI-TOF/TOF analysis, which revealed the sequence VFGTLGSDDSGLFGK present in both HDs1 and HDs2. This sequence had homology and 80% identity with specific Lepidoptera antimicrobial peptides called gloverins. Analyses using the ImageMaster 2D software showed pI 8.94 of the HDs1 spot, which is similar to that described to Hyalophora gloveri gloverin (pI 8.5). Moreover, the 14-kDa molecular mass of the spot HDs1 is compatible to that of gloverins isolated from the hemolymph of Trichoplusia ni, Helicoverpa armigera and H. gloveri. Antimicrobial assays with partially purified fractions containing the HDs1 and HDs2 polypeptides demonstrated activity against Escherichia coli. This is the first report of antimicrobial polypeptides in D. saccharalis, and the identification of these peptides may help in the generation of new strategies to control this pest.

Larval alarm pheromones as a potential control for invasive cane toads (Bufo marinus) in tropical Australia

Novel approaches to control invasive species are urgently needed. Cane toads (Bufo marinus) are large, highly toxic anurans that are spreading rapidly through tropical Australia. Injured toad larvae produce an alarm pheromone that elicits rapid avoidance by conspecifics but not by frog larvae. Experiments in outdoor ponds show that repeated exposure to the pheromone reduced toad tadpole survival rates (by >50%) and body mass at metamorphosis (by 20%). The alarm pheromone did not induce tadpoles to seek shelter, but accelerated ontogenetic differentiation. Perhaps reflecting mortality of weaker individuals during larval life, growth rates post-metamorphosis were higher in animals emerging from the pheromone exposure treatment than from the control treatment. Nonetheless, body size differentials established at metamorphosis persisted through the first 8 days of post-metamorphic life. We will need substantial additional research before evaluating whether the alarm pheromone provides a way to reduce cane toad recruitment in nature, but our field trials are encouraging in this respect.

A monoclonal antibody as a tool to study the subcommissural organ and Reissner's fibre of the sea lamprey: An immunofluorescence study before and after a spinal cord transection

Lampreys are vertebrate animal models in spinal cord regeneration studies. In order to gain knowledge on the mechanisms that provide to the lamprey spinal cord its capacity of regeneration we decided to compare the expression patterns of the growth-associated protein 43 (GAP-43) in the CNS of the sea lamprey before and after a complete spinal transection by immunocytochemical methods using an anti-GAP-43 antibody. Surprisingly, in the brain/spinal cord of both normal and injured animals, anti-GAP-43-like labeling was only observed in the subcommissural organ (SCO) and Reissner's fibre (RF). In injured larvae, a dotted labeling was also observed in the meninges and in the blood the vessels of the neighbouring tissues at the site of lesion. The experiments in injured animals showed that after complete spinal cord transection the SCO seems to continue to produce the Reissner's substance (RS), which is accumulated at the proximal site of spinal transection. The dotted labeling observed in the neighbouring tissues could correspond to RS that was released from the site of injury. In Western blot experiments done using protein extracts of the lamprey brain, the anti-GAP-43 antibody did not recognize any protein band of the expected GAP-43 molecular weight, indicating that the secreted material is not this protein. An anti-serotonin antibody was also used as a marker of some brain structures. Serotonergic afferent fibres innervated the SCO. Here we show a new tool that can be used as a highly specific marker in further studies of the SCO/RF system of lampreys.

Influence of Body Size on the Responses of Fathead Minnows, Pimephales promelas, to Damselfly Alarm Cues

AbstractA wide diversity of aquatic organisms release chemical alarm cues upon encountering or being attacked by a predator. These alarm cues can be used by nearby individuals to assess local predation risk. Receivers warned by chemical alarm cues gain a survival benefit when encountering predators. Animals that are in the same prey guild (i.e. that co‐occur and share the same predators) may learn to recognize each others’ chemical alarm cues. This ability may confer an adaptive advantage if the prey animals are vulnerable to the same predators. However, if the prey grow to different sizes and as a consequence are no longer vulnerable to the same suite of predators, then there should no longer be an advantage for the prey to respond to each others’ alarm cues. In this study, we exposed small and large fathead minnows (Pimephales promelas) to cues from syntopic injured damselfly larvae (Enallagma boreale), cues from injured mealworm larvae (Tenebrio molitor) and to distilled water. Small minnows exhibited antipredatory behaviour and increased shelter use in response to injured damselfly cues but not to the controls of injured mealworm or distilled water. On the contrary, large minnows exhibited no significant change in shelter use in response to any of the injured cues. These data demonstrate that fathead minnows exhibit an antipredator response to damselfly alarm cues, but only when minnows are small and members of the same prey guild as damselfly larvae. These results demonstrate the considerable flexibility in the responses to heterospecific alarm cues.

Effects of juvenile hormones and precocenes on the immune response of Spodoptera littoralis larvae to supernumerary larvae of the solitary parasitoid, Microplitis rufiventris Kok.

The study represents one of the first direct tests of juvenile hormones (JHs) on the cellular immune response of a lepidopteran insect, Spodoptera littoralis (Boisd.), superparasitized by a solitary parasitoid, Microplitis rufiventris Kok.. JHII-treated S. littoralis larvae exhibited a significant reduction in encapsulation response to the supernumerary M. rufiventris larvae at different developmental ages (0–4, 10–14 and 24–26 h) of supernumerary M. rufiventris embryos within 48 or 144 h after parasitization than either JHI-treated or control hosts. The reduction in encapsulation response was significantly greater in hosts which received higher JHII dose (5 μg) than those treated with lower one (1 μg). The progression of the encapsulation, culminating in melanization in the capsules occurred more frequently in precocene-treated hosts than either JH-treatment or control ones. Application of the anti-juvenile hormones (PI or PII) caused stronger encapsulation reaction to the injured supernumerary parasitoid larvae than JHI or JHII. In PI-treatments, the reaction was rapid and dramatic by lower (25 μg) dose treatments than higher ones (70 μg). The capsules formed around the supernumerary larvae of M. rufiventris showed signs of melanization within 48 h in PI-treated hosts. Melanization was more pronounced in either PI- or PII-treated hosts prior emergence of successful parasitoid larvae. Generally, the injured supernumerary larvae were seen within melanin-rich capsules in PI- and PII-treated hosts. Melanization was not observed in most capsules of JHI- and JHII-treated hosts. Additional physiological effects were observed: (1) in some cases, application of JHI to parasitized S. littoralis larvae inhibited subsequent emergence of its wasp, M. rufiventris, and (2) teratocytes (cells of wasp origin) from PI or PII-treated hosts were smaller in size than those observed in the JH-treated or control hosts. Finally, the dramatic increase in encapsulation responses of precocene-treated S. littoralis larvae of the supernumerary of M. rufiventris and definitely opposite reactions in typical hosts but JHII-treated hosts suggested that the cellular defense reaction may be under inhibitory hormonal control. The results of the study have implications for medically important insects such as mosquitoes and their interactions with parasites, as well as having significance for biocontrol and mass rearing programs using parasitoids.

Molecular cloning of cDNAs coding Sarcophaga diptericin family

We purified an antibacterial protein named sarcotoxin III from the hemolymph of injured larvae of Sarcophaga peregrina (Baba K., et al., J. Biochem., 102, 69-74 (1987)). In this report we determined the partial amino acid sequence of sarcotoxin III and tried to conduct the cloning of sarcotoxin III cDNA. As a result we obtained two clones coding a protein similar to sarcotoxin III in sequence. Their primary structures are highly homologous to N-terminal sequences of an antibacterial protein from Phormia, diptericin, and another 8 kD antibacterial protein purified from Sarcophaga peregrina. These results suggested that sarcotoxin III is one of the proteins forming a diptericin family in Sarcophaga peregrina as well as newly obtained clone-coded products and 8 kD protein.

Combined effects of temperature and salinity on development and survival of Atlantic halibut larvae

The effects of different combinations of temperature and salinity on development and survival of Atlantic halibut, Hippoglossus hippoglossus (L.), larvae were studied in two experiments. In the period from 57 to 120 d° post hatching, approximately 38% of the larvae died: in both experiments, mortality was significantly related to temperature, but there was no relationship to salinity. Cumulative, chronic mortality during the yolk-sac stage (until 210 d°) was related to salinity. High mortality was observed in both experiments when high salinity (>34%) was combined with incubation at either high (9 °C) or low (3 °C) temperature. The development of head lesions was significantly related to temperature, and for larvae kept at high salinity, increased mortality may have been a result of salinity stress in the injured larvae. Abnormal development of the caudal notochord, sometimes resulting in a 90-degree bend of the tail, occurred during incubation of early yolk-sac larvae in high-salinity water. The occurrence of larvae with locked jaws, and larvae with oedema in the yolk-sac sinus and pericardium, was significantly related to temperature. © Rapid Science Ltd. 1998

Purification and characterization of a diptericin homologue from Sarcophaga peregrina (flesh fly).

A protein with a molecular mass of 8 kDa was found to be synthesized specifically when the fat-body from injured Sarcophaga peregrina larvae was cultured in vitro. This protein was purified from the haemolymph of the injured larvae to near-homogeneity. Partial amino acid sequencing revealed that this protein is a diptericin homologue. It showed bactericidal activity on growing, but not resting Escherichia coli cells. E. coli cells become elongated on treatment with this protein.

Cloning and sequencing of cDNA of Sarcophaga peregrina humoral lectin induced on injury of the body wall.

A previous paper described the purification of a lectin induced in the hemolymph of larvae of Sarcophaga peregrina (flesh-fly) on injury of their body wall (Komano, H., Mizuno, D., and Natori, S. (1980) J. Biol. Chem. 255, 2919-2924). This paper describes cDNA cloning and the complete nucleotide sequence of the gene for Sarcophaga lectin. Although active lectin consists of alpha and beta subunits in a molar ratio of 2:1, the fat body of injured larvae was found to contain only mRNA for the alpha subunit, suggesting that these two subunits are derived from a common gene and that the alpha subunit is converted to the beta subunit post-translationally. The alpha subunit was found to consist of 260 amino acid residues with an additional signal sequence of 19 or 23 amino acid residues.