Open field behavior in the house cricket (Acheta domesticus): effect of illumination, sex differences and individual consistency

Edible insects are worldwide promoted as an alternative protein, trace mineral and lipid source in animal feed and human food. The house cricket (Acheta domesticus) is already being reared at an industrial scale, yet current mass-rearing practices and facility design may still leave room for improvement. Behavioral tests have been suggested as an important assessment tool at the whole-organism level that can be used to find optimal housing conditions (e.g. density, diet, temperature). Here, we adapt the widely used open field test to the house cricket. We tested 16 male and 16 female house crickets four times under two different light intensities. Videos were analysed with Ethovision™ tracking software and variables distance moved, velocity, and duration and frequency in zone were extracted. Results showed that house crickets, like vertebrate model species, spent most time close to the walls of the arena, and crossed the center zone with high velocity. Brighter illumination was associated with increased velocity, in particular in the center zone during the first test occasion, suggesting avoidance of this zone. Male crickets had higher locomotory activity than females. Consistency repeatabilities and intraclass correlation coefficients (ICCs) were moderate to high, and the correlation between subsequent occasions became stronger over the four occasions at day 1, 2, 3 and 7. The first test occasion differed from subsequent occasions, therefore repeated testing may be necessary when analysing experimental manipulations of small effect size. Overall, the results are promising for use of the open field test as a precise phenotyping tool

The zebrafish Multivariate Concentric Square Field: A Standardized Test for Behavioral Profiling of Zebrafish (Danio rerio)

The zebrafish (Danio rerio) is an important model organism in the study of the neurobiological basis of human mental disorders. Yet the utility of this species is limited by the quality of the phenotypical characterization tools available. Here, we present a complex testing environment for the quantification of explorative behavior in adult zebrafish, the zebrafish Multivariate Concentric Square Field (TM) (zMCSF), adapted from the rodent equivalent that has been used in > 40 studies. The apparatus consists of a central open area which is surrounded by a dark corner with a roof (DCR), corridors, and an inclined ramp. These areas differ in illumination, water depth, and are sheltered or exposed to different degrees. We quantified behavior of male and female wild-caught and AB strain zebrafish in the zMCSF (day 1) and cross-validated these results using the novel tank diving test (NTDT) (day 2). To assess the effect of repeated testing, AB zebrafish we tested a second time in both tests 1 week later (on days 7 and 8). We detected strong differences between the strains, with wild zebrafish swimming faster and spending more time in the corridors and on the ramp, while they avoided the open area in the center. AB zebrafish were less hesitant to enter the center but avoided the ramp, and often left one or more zones unexplored. No major sex differences in exploratory behavior were detected in either strain, except for a slightly higher velocity of AB males which has been reported before. Importantly, the zMCSF was largely resilient to repeated testing. The diving test revealed only one difference confined to one sex; wild females paid more visits to the top third than AB females. In isolation, this finding could lead to the conclusion that wild zebrafish are more risk-taking, which is incorrect given this strain's avoidance of open areas. To conclude, our results suggest that the zMCSF presents a sophisticated behavioral tool that can distinguish between different magnitudes and types of risk, allowing the user to create an intricate behavioral profile of individual adult zebrafish

Clostridioides difficile infection with isolates of cryptic clade C-II: a genomic analysis of polymerase chain reaction ribotype 151

Objectives: We report a patient case of pseudomembranous colitis associated with a monotoxin-producing Clostridioides difficile belonging to the very rarely diagnosed polymerase chain reaction (PCR) ribotype (RT) 151. To understand why this isolate was not identified using a routine commercial test, we performed a genomic analysis of RT151. Methods: Illumina short-read sequencing was performed on n = 11 RT151s from various geographical regions to study their genomic characteristics and relatedness. Subsequently, we used PacBio circular consensus sequencing to determine the complete genome sequence of isolates belonging to cryptic clades C–I and C-II, which includes the patient isolate. Results: We found that 1) RT151s are polyphyletic with isolates falling into clades 1 and cryptic clades C–I and C-II; 2) RT151 contains both nontoxigenic and toxigenic isolates and 3) RT151 C-II isolates contained monotoxin pathogenicity loci. The isolate from our patient case report contains a novel-pathogenicity loci insertion site, lacked tcdA and had a divergent tcdB sequence that might explain the failure of the diagnostic test. Discussion: This study shows that RT151 encompasses both typical and cryptic clades and provides conclusive evidence for C. difficile infection due to clade C-II isolates that was hitherto lacking. Vigilance towards C. difficile infection as a result of cryptic clade isolates is warranted

A Facile, One-Pot, Surfactant-Free Nanoprecipitation Method for the Preparation of Nanogels from Polyglycerol–Drug Conjugates that Can Be Freely Assembled for Combination Therapy Applications

A well-established strategy to treat drug resistance is the use of multiple therapeutics. Polymer-based drug delivery systems (DDS) can facilitate a simultaneous delivery of two or more drugs. In this study, we developed and synthesized a dendritic polyglycerol (PG) nanogel (NG) system that allows for free combination of different fixed ratios of active compound conjugates within a single NG particle. As a proof of concept, we synthesized NGs bearing the chemotherapeutic agent doxorubicin (DOX) and paclitaxel (PTX) in different ratios, as well as conjugated dye molecules. Our combination PG NGs were formed by simply mixing PG–drug/dye conjugates bearing free thiol groups with PG-acrylate in an inverse surfactant-free nanoprecipitation method. With this method we obtained PG-NGs in the size range of 110–165 nm with low polydispersity indices. Solubility of hydrophobic PTX was improved without the need for additional solubilizing agents such as polyethylene glycol (PEG). Interestingly, we found that our NGs made from PG-DOX conjugates have a high quenching efficiency for DOX, which could be interesting for theranostic purposes

Boldness, activity, and aggression: Insights from a large-scale study in Baltic salmon (Salmo salar L)

Atlantic salmon (Salmo salar) display high levels of agonistic behavior in aquaculture farms, resulting in fin damage and chronic stress. Aggression affects fish growth and performance negatively, and presents a serious welfare problem. Indeed, it would be beneficial to identify, separate or exclude overly aggressive individuals. Research on behavioral syndromes suggests that aggressive behavior may correlate with other behavioral traits, such as boldness and locomotory activity. We aimed to develop a high-throughput method to quantify and predict aggressive behavior of individual parr in hatchery-reared Baltic salmon (Salmo salar L.). We screened approximately 2000 parr in open field (OF) and mirror image stimulation (MIS) tests. We extracted seven variables from video tracking software for each minute of the tests; distance moved and duration moving (activity), the duration in and number of entries to the center of the arena (boldness), the distance moved in and duration spent in the area adjacent to the mirror during the MIS test (aggressiveness) and head direction (lateralization). To investigate the relationship between activity, boldness and aggression we first correlated the first six variables to one another. Second, we assigned individuals to high, medium, low or zero aggression groups based on the MIS test and quantified activity and boldness in each group. Third, we analyzed whether the fish viewed the mirror with the left or right eye. Our results show that medium and low aggressive fish were the most active, while highly aggressive fish showed average activity. Aggressive groups did not differ in boldness. Activity and boldness were positively correlated. Finally, we detected a preference for fish to view the mirror with the left eye. We conclude that aggressiveness cannot be predicted from the results of the OF test alone but that the MIS test can be used for large-scale individual aggression profiling of juvenile salmon

Effect of elevated pCO2 and environmental oxazepam on the behavior and physiology of teleost fish

This doctoral thesis investigated the effect of two aquatic pollutants on the behavior and physiology of teleost fish: i) elevated concentrations of carbon dioxide (CO2) in the context of ocean acidification and ii) low concentrations of the anxiolytic pharmaceutical oxazepam in the context of pharmaceutical pollution. Anthropogenic emissions of CO2 are lowering the pH of the oceans. Studies on coral reef fish exposed to CO2 concentrations projected for the year 2100 (~1000 μatm) reported alarming behavioral effects, of which attraction to predator odor was the most surprising. To explain this behavioral reversal, it was hypothesized that ion-regulatory adjustments to compensate for the decrease in blood pH would result in altered transmembrane gradients of chloride ions, rendering the major inhibitory neurotransmitter in the brain, γ-aminobutyric acid (GABA), excitatory. We investigated whether zebrafish (Danio rerio), an often used model species, showed similar behavioral disruptions in elevated CO2. Zebrafish behavior was however largely unaffected by an approximately month long exposure to ~1600 μatm CO2. We continued by investigating the reproductive, anxiety-related behavior and aggression in another model species, the three-spined stickleback (Gasterosteus aculeatus). However, also stickleback behavior and responses to social subordination were not affected by CO2, in contrast to earlier findings. We concluded that CO2 had no major effect on the behavior of zebrafish and three-spined stickleback. In the second part of this thesis, I investigated behavioral effects of oxazepam, an anxiolytic pharmaceutical (benzodiazepine) acting on the GABA system. Studies on perch (Perca fluviatilis) have shown that exposure to dilute concentrations of oxazepam (1.8 μg L-1), close to those found outside the municipal sewage treatment plant in Uppsala (0.58 μg L-1), can increase activity, decrease sociality and increase feeding rates. I show that similar oxazepam concentrations can also affect zebrafish. Moreover, I show that females are more sensitive to oxazepam showing reduced anti-predator responses at 0.57 μg L-1 while in males this effect was observed first at 60 μg L-1. Furthermore, and in contrast to wild-caught zebrafish, laboratory zebrafish did not show any effect of the oxazepam exposure. This finding has implications for the use of laboratory zebrafish in ecotoxicological and pharmacological studies, as results might not translate to wild fish. Finally, I show that zebrafish can develop tolerance to the anxiolytic effects of oxazepam during chronic (28 days) exposure. This is an important discovery that could mitigate the effects of this form of pharmaceutical pollution on wild fish.Doktorsavhandlingen undersöker effekter av två olika vattenföroreningar på beteende och fysiologi hos benfisk, nämligen: i) förhöjda koncentrationer av koldioxid (CO2) i relation till havsförsurning och ii) låga koncentrationer av den ångestdämpande farmakologiska substansen oxazepam i samband med förorening av vatten med läkemedel. Utsläpp av CO2 sänker havets pH-värde och studier där korallrevsfiskar exponerats för CO2-koncentrationer i nivå med vad som beräknats för år 2100 (~1000 μatm) har rapporterat om alarmerande beteendestörningar, såsom att försöksfiskar inte längre undvek lukt av rovfisk utan istället attraherades till den. En hypotes är att detta omvända beteende orsakas av att de mekanismer som är nödvändiga för att återställa fiskens syra/bas-balans resulterar i att hjärnans främsta inhibitoriska signalsubstans, γ-aminobuturatsyra (GABA), istället får en excitatorisk effekt. Vi har undersökt om sebrafisk (Danio rerio), som är en vanlig modellorganism, visar liknande beteendeförändringar vid förhöjd CO2. Sebrafiskens beteende var emellertid i stort sett opåverkat efter ungefär en månads exponering för ~1600 μatm CO2. Vi fortsatte med att undersöka beteendeeffekter av förhöjda halter av CO2 hos storspigg (Gasterosteus aculeatus), en annan vanlig modellorganism. Inte heller hos denna art kunde vi påvisa några tydliga beteendeeffekter av CO2 varför vi drog slutsatsen att CO2 inte har några påtaglig beteendeeffekter, vare sig hos sebrafisk eller storspigg. I den andra delen av doktorsavhandlingen undersökte vi hur miljörelevanta koncentrationer av det ångesthämmande läkemedlet oxazepam påverkar GABA systemet och fiskars beteende. Tidigare studier på abborre (Perca fluviatilis) har visat att 1.8 μg L-1oxazepam, vilket är i nivå med vad som påträffas utanför Uppsala avloppsreningsverk (0.58 μg L-1), ger ökad simaktivitet, minskad attraktion till stimmet och ökat födointag. I doktorsavhandlingen visar jag att liknande koncentrationer av oxazepam också påverkar sebrafisk och att honor är mer känsliga för oxazepam då dessa visar minskat anti-predator beteende redan vid 0.57 μg L-1 medan denna effekt inte uppträder hos hanar förrän vid koncentrationen 60 μg L-1. Dessutom fann vi att laboratoriestammar i motsats till vildfångade sebrafiskar inte visade några av dessa oxazepam-inducerade beteendeeffekter, en upptäckt som kan få konsekvenser för användandet av sebrafisk i ekotoxikologiska och farmakologiska studier, eftersom resultat som erhålls på labstammar av sebrafisk inte är direkt överförbara till vild fisk. Slutligen visar min studie att sebrafisk kan utveckla tolerans för de ångesthämmande effekterna av oxazepam vid kronisk (28 dygn) exponering. Detta är ett viktigt resultat som kan tyda på att effekterna av läkemedelsföreningar på vild fisk, åtminstone i detta fall, kan vara mindre än befarat

Mannose-Decorated Dendritic Polyglycerol Nanocarriers Drive Antiparasitic Drugs To Leishmania infantum-Infected Macrophages

Macrophages are hosts for intracellular pathogens involved in numerous diseases including leishmaniasis. They express surface receptors that may be exploited for specific drug-targeting. Recently, we developed a PEGylated dendritic polyglycerol-based conjugate (PG–PEG) that colocalizes with intracellular parasite. We hereby study the effect of surface decoration with mannose units on the conjugates’ targeting ability toward leishmania intracellular parasites. Murine and human macrophages were exposed to fluorescently labeled mannosylated PG–PEG and uptake was quantified by flow cytometry analysis. Nanocarriers bearing five mannose units showed the highest uptake, which varied between 30 and 88% in the population in human and murine macrophages, respectively. The uptake was found to be dependent on phagocytosis and pinocytosis (80%), as well as clathrin-mediated endocytosis (79%). Confocal microscopy showed that mannosylated PG–PEGs target acidic compartments in macrophages. In addition, when both murine and human macrophages were infected and treated, colocalization between parasites and mannosylated nanoconjugates was observed. Leishmania-infected bone marrow-derived macrophages (BMM) showed avidity by mannosylated PG–PEG whereas non-infected macrophages rarely accumulated conjugates. Moreover, the antileishmanial activity of Amphotericin B was kept upon conjugation to mannosylated PG–PEG through a pH-labile linker. This study demonstrates that leishmania infected macrophages are selectively targeted by mannosylated PEGylated dendritic conjugates

Effect of elevated pCO2 and environmental oxazepam on the behavior and physiology of teleost fish

This doctoral thesis investigated the effect of two aquatic pollutants on the behavior and physiology of teleost fish: i) elevated concentrations of carbon dioxide (CO2) in the context of ocean acidification and ii) low concentrations of the anxiolytic pharmaceutical oxazepam in the context of pharmaceutical pollution. Anthropogenic emissions of CO2 are lowering the pH of the oceans. Studies on coral reef fish exposed to CO2 concentrations projected for the year 2100 (~1000 μatm) reported alarming behavioral effects, of which attraction to predator odor was the most surprising. To explain this behavioral reversal, it was hypothesized that ion-regulatory adjustments to compensate for the decrease in blood pH would result in altered transmembrane gradients of chloride ions, rendering the major inhibitory neurotransmitter in the brain, γ-aminobutyric acid (GABA), excitatory. We investigated whether zebrafish (Danio rerio), an often used model species, showed similar behavioral disruptions in elevated CO2. Zebrafish behavior was however largely unaffected by an approximately month long exposure to ~1600 μatm CO2. We continued by investigating the reproductive, anxiety-related behavior and aggression in another model species, the three-spined stickleback (Gasterosteus aculeatus). However, also stickleback behavior and responses to social subordination were not affected by CO2, in contrast to earlier findings. We concluded that CO2 had no major effect on the behavior of zebrafish and three-spined stickleback. In the second part of this thesis, I investigated behavioral effects of oxazepam, an anxiolytic pharmaceutical (benzodiazepine) acting on the GABA system. Studies on perch (Perca fluviatilis) have shown that exposure to dilute concentrations of oxazepam (1.8 μg L-1), close to those found outside the municipal sewage treatment plant in Uppsala (0.58 μg L-1), can increase activity, decrease sociality and increase feeding rates. I show that similar oxazepam concentrations can also affect zebrafish. Moreover, I show that females are more sensitive to oxazepam showing reduced anti-predator responses at 0.57 μg L-1 while in males this effect was observed first at 60 μg L-1. Furthermore, and in contrast to wild-caught zebrafish, laboratory zebrafish did not show any effect of the oxazepam exposure. This finding has implications for the use of laboratory zebrafish in ecotoxicological and pharmacological studies, as results might not translate to wild fish. Finally, I show that zebrafish can develop tolerance to the anxiolytic effects of oxazepam during chronic (28 days) exposure. This is an important discovery that could mitigate the effects of this form of pharmaceutical pollution on wild fish.Doktorsavhandlingen undersöker effekter av två olika vattenföroreningar på beteende och fysiologi hos benfisk, nämligen: i) förhöjda koncentrationer av koldioxid (CO2) i relation till havsförsurning och ii) låga koncentrationer av den ångestdämpande farmakologiska substansen oxazepam i samband med förorening av vatten med läkemedel. Utsläpp av CO2 sänker havets pH-värde och studier där korallrevsfiskar exponerats för CO2-koncentrationer i nivå med vad som beräknats för år 2100 (~1000 μatm) har rapporterat om alarmerande beteendestörningar, såsom att försöksfiskar inte längre undvek lukt av rovfisk utan istället attraherades till den. En hypotes är att detta omvända beteende orsakas av att de mekanismer som är nödvändiga för att återställa fiskens syra/bas-balans resulterar i att hjärnans främsta inhibitoriska signalsubstans, γ-aminobuturatsyra (GABA), istället får en excitatorisk effekt. Vi har undersökt om sebrafisk (Danio rerio), som är en vanlig modellorganism, visar liknande beteendeförändringar vid förhöjd CO2. Sebrafiskens beteende var emellertid i stort sett opåverkat efter ungefär en månads exponering för ~1600 μatm CO2. Vi fortsatte med att undersöka beteendeeffekter av förhöjda halter av CO2 hos storspigg (Gasterosteus aculeatus), en annan vanlig modellorganism. Inte heller hos denna art kunde vi påvisa några tydliga beteendeeffekter av CO2 varför vi drog slutsatsen att CO2 inte har några påtaglig beteendeeffekter, vare sig hos sebrafisk eller storspigg. I den andra delen av doktorsavhandlingen undersökte vi hur miljörelevanta koncentrationer av det ångesthämmande läkemedlet oxazepam påverkar GABA systemet och fiskars beteende. Tidigare studier på abborre (Perca fluviatilis) har visat att 1.8 μg L-1oxazepam, vilket är i nivå med vad som påträffas utanför Uppsala avloppsreningsverk (0.58 μg L-1), ger ökad simaktivitet, minskad attraktion till stimmet och ökat födointag. I doktorsavhandlingen visar jag att liknande koncentrationer av oxazepam också påverkar sebrafisk och att honor är mer känsliga för oxazepam då dessa visar minskat anti-predator beteende redan vid 0.57 μg L-1 medan denna effekt inte uppträder hos hanar förrän vid koncentrationen 60 μg L-1. Dessutom fann vi att laboratoriestammar i motsats till vildfångade sebrafiskar inte visade några av dessa oxazepam-inducerade beteendeeffekter, en upptäckt som kan få konsekvenser för användandet av sebrafisk i ekotoxikologiska och farmakologiska studier, eftersom resultat som erhålls på labstammar av sebrafisk inte är direkt överförbara till vild fisk. Slutligen visar min studie att sebrafisk kan utveckla tolerans för de ångesthämmande effekterna av oxazepam vid kronisk (28 dygn) exponering. Detta är ett viktigt resultat som kan tyda på att effekterna av läkemedelsföreningar på vild fisk, åtminstone i detta fall, kan vara mindre än befarat

A Facile, One-Pot, Surfactant-Free Nanoprecipitation Method for the Preparation of Nanogels from Polyglycerol–Drug Conjugates that Can Be Freely Assembled for Combination Therapy Applications

A well-established strategy to treat drug resistance is the use of multiple therapeutics. Polymer-based drug delivery systems (DDS) can facilitate a simultaneous delivery of two or more drugs. In this study, we developed and synthesized a dendritic polyglycerol (PG) nanogel (NG) system that allows for free combination of different fixed ratios of active compound conjugates within a single NG particle. As a proof of concept, we synthesized NGs bearing the chemotherapeutic agent doxorubicin (DOX) and paclitaxel (PTX) in different ratios, as well as conjugated dye molecules. Our combination PG NGs were formed by simply mixing PG–drug/dye conjugates bearing free thiol groups with PG-acrylate in an inverse surfactant-free nanoprecipitation method. With this method we obtained PG-NGs in the size range of 110–165 nm with low polydispersity indices. Solubility of hydrophobic PTX was improved without the need for additional solubilizing agents such as polyethylene glycol (PEG). Interestingly, we found that our NGs made from PG-DOX conjugates have a high quenching efficiency for DOX, which could be interesting for theranostic purposes